Jaber, N. (2024) - Toxicology in Vitro

Toxicology in Vitro 94 (2024) 105718
Contents lists available at ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
Review
How to use an in vitro approach to characterize the toxicity of

airborne compounds
Nour Jaber, Sylvain Billet *
UR4492, Unité de Chimie Environnementale et Interactions sur le Vivant, Université du Littoral Côte d’Opale, Dunkerque, France
A R T I C L E I N F O A B S T R A C T
Editor: Maxime Culot As part of the development of new approach methodologies (NAMs), numerous in vitro methods are being
developed to characterize the potential toxicity of inhalable xenobiotics (gases, volatile organic compounds,
Keywords: polycyclic aromatic hydrocarbons, particulate matter, nanoparticles). However, the materials and methods
Respiratory in vitro model employed are extremely diverse, and no single method is currently in use. Method standardization and validation
Method of exposure
would raise trust in the results and enable them to be compared.
Airborne compounds
This four-part review lists and compares biological models and exposure methodologies before describing
Air-liquid interface
Organotypic model measurable biomarkers of exposure or effect. The first section emphasizes the importance of developing alter
native methods to reduce, if not replace, animal testing (3R principle). The biological models presented are
mostly to cultures of epithelial cells from the respiratory system, as the lungs are the first organ to come into
contact with air pollutants. Monocultures or cocultures of primary cells or cell lines, as well as 3D organotypic
cultures such as organoids, spheroids and reconstituted tissues, but also the organ(s) model on a chip are ex
amples. The exposure methods for these biological models applicable to airborne compounds are submerged,
intermittent, continuous either static or dynamic. Finally, within the restrictions of these models (i.e. relative tiny
quantities, adhering cells), the mechanisms of toxicity and the phenotypic markers most commonly examined in
models exposed at the air-liquid interface (ALI) are outlined.
Abbreviations: 8-OHdG, 8-hydroxy-2′-deoxyguanosine; ABC, ATP Binding Cassette transporter; ADME, Absorption, Distribution, Metabolism, Excretion; AECI,
Alveolar Epithelial Cell type I; AECII, Alveolar Epithelial Cell type II; ALI, Air-Liquid Interface; ALICE, Air-Liquid Interface Cell Exposure; AOP, Adverse Outcome
Pathway; BCRP, Breast Cancer Resistance Protein; CBF, Ciliary Beating Frequency; CCL22, Chemokine Ligand 22; COPD, Chronic Obstructive Pulmonary Disease;
CYP, Cytochrome P450; DMSO, Dimethyl Sulfoxide; EAVES, Electrostatic Aerosol in vitro Exposure System; ELISA, Enzyme-linked immunoassay; ELLA, Enzyme-
linked Lectin Assay; EMT, Epithelial-mesenchymal Transition; ENM, Engineered Nanomaterial; EPA, Environmental Protection Agency; EPHX1, Epoxide Hydrolase 1;
FACS, Fluorescence-Activated Cell Sorting; FBS, Fetal Bovine Serum; FDA, Food and Drug Administration; GSTM1, Glutathione-S-transferase μ 1; H2AX, Histone H2A
variant X; hAECB, human Airway Epithelial Cell of Bronchial origin; hAECN, human Airway Epithelial Cell of Nasal origin; hAELVi, human Alveolar Epithelial
Lentivirus immortalized; HD, Hanging Drop; HMOX-1, Heme Oxygenase 1; HNE, Human Nasal Epithelial; huAEC, human Airway Epithelial Cell; huBroBEC, human
Bronchial Basal Epithelial Cell; ICP-MS, Inductively Coupled Plasma Mass Spectrometry; ICRP, International Commission on Radiological Protection; IFN, Interferon;
IL, Interleukin; iTRAQ, isobaric Tag for Relative and Absolute Quantitation; LC-MS/MS, Liquid Chromatography - Tandem Mass Spectrometry; LDH, Lactate De
hydrogenase; LPS, Lipopolysaccharide; LY, Lucifer Yellow; MCP-1, Monocyte Chemoattractant Protein 1; MDA, Malondialdehyde; MIE, Molecular Initiating Event;
MPPD, Multi-Path Particle Dosimetry; MRP1–7, Multidrug Resistance-associated Proteins; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide;
NAMs, New Approach Methodologies; NACIVT, Nano Aerosol Chamber In-Vitro Toxicity; NF-κB, Nuclear Factor-kappa B; NHBE, Normal Human Bronchial Epithelial
Cell; NLRP3, NLR family Pyrin domain containing 3; NP, Nanoparticle; NQO1, NAD(P)H Quinone Oxidoreductase 1; PAH, Polycyclic Aromatic Hydrocarbon; PBPK,
Physiologically Based Pharmacokinetic; P-gp, P-glycoprotein; PM, Particulate Matter; QSAR, Quantitative Structure-Activity Relationship; RNA-Seq, RNA
Sequencing; ROS, Reactive Oxygen Species; SOD2, Superoxide Dismutase 2; TBARS, Thiobarbituric Acid Reactive Substances; TEER, Trans-Epithelial Electrical
Resistance; TGF-β, Transforming Growth Factor-β; TJ, Tight Junctions; TLR4, Toll Like Receptor 4; TNF-α, Tumor Necrosis Factor-alpha; TSCA, Toxic Substances
Control Act; TXNIP, Thioredoxin-Interacting Protein; VOC, Volatile Organic Compound; WST-1, Water-Soluble Tetrazolium salt 1; WTC, World Trade Center; XME,
Xenobiotics Metabolizing Enzyme; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; ZO-1, Zona Occludens 1.
* Corresponding author.
E-mail address: [email protected] (S. Billet).
https://doi.org/10.1016/j.tiv.2023.105718
Received 22 June 2023; Received in revised form 13 September 2023; Accepted 21 September 2023
Available online 21 October 2023
0887-2333/© 2023 Published by Elsevier Ltd.
N. Jaber and S. Billet Toxicology in Vitro 94 (2024) 105718
1. Introduction the main harmful signaling pathways, focusing on the specific bio
markers that are most often measured, will be presented and their
Humans are exposed to a wide range of toxic compounds in their relevance discussed in relation to the literature and research objectives.
daily lives via three major routes of exposure: inhalation, ingestion and
dermal contact. According to a recent survey by the French National 2. In vitro biological models from the human respiratory system
Health and Safety Agency, adults in France consume on average around
3 kg of food per day, half of which is drinks, and breathe around 15 m3 of 2.1. Structure and morphology of the respiratory system
air per day (Dubuisson et al., 2019). The chemical compounds present in
our food and the air we breathe penetrate our body and can interact with The respiratory system is normally separated into two levels: the
their biological targets. In the case of airborne compounds, the respi upper and lower respiratory tracts. The nasal passages, pharynx, and
ratory system is the first to come into contact, although their toxic ef larynx comprise the upper respiratory system, whereas the trachea,
fects also affect other systems such as the cardiovascular and nervous bronchi, bronchioles, and alveoli comprise the lower respiratory tract
systems (WHO, 2016). For example, air pollution and particulate matter (Ball et al., 2023) (Fig. 1). The respiratory system can also be divided
(PM) have been classified as lung carcinogenic to humans (Loomis et al., into the conducting zone and the respiratory zone. The nasal passage,
2013). Many volatile organic compounds and polycyclic aromatic hy trachea, main bronchi, intra-pulmonary bronchi, and bronchioles are all
drocarbons are well-known for their toxicity, in terms of genotoxicity located in the conducting zone and do not participate in gas exchange.
and leukemia, pulmonary effects such as asthma, and reproductive The respiratory bronchioles, alveolar ducts, and alveolar sacs comprise
toxicity such as low birth weight (Billet et al., 2010; Liu et al., 2022; the respiratory zone. The respiratory zone is where gas exchange occurs
Paget et al., 2012). Airborne compounds can also be absorbed through between the air in the lung and the blood in the pulmonary capillaries
the skin by direct contact with the air, and through ingestion in the case (Hou, 2015). From the nasal cavity to the bronchi, the majority of the
of large particles, which are inhaled and then cleared from the respi respiratory tree is lined by pseudostratified columnar ciliated epithe
ratory system and swallowed. To assess the risks associated with expo lium. Simple columnar to cuboidal epithelium lines the bronchioles,
sure to these xenobiotics, a global approach integrating all three routes while thin squamous epithelium lines the alveoli, enabling gas ex
may be necessary. change. The respiratory system consists of the respiratory mucosa,
Therefore, toxicological investigations are essential for detecting which is made up of the epithelium and supporting lamina propria, the
dangerous compounds and devising effective risk management mea submucosa, the cartilage and/or muscle layer, and the adventitia (Kia’i
sures. Such researchs include establishing the hazardous characteristics and Bajaj, 2023). The respiratory epithelium has a wide variety of cell
of substances, defining their modes of action, and evaluating the dose- types. Ciliated, basal, goblet, and other secretory cells, including Clara
response relationship and exposure kinetics. There are now three toxi cells, make up the majority of the pseudo-stratified epithelium. Ciliated
cological approaches in use: in vivo, in vitro, and in silico. Each has cells are the most numerous epithelial cells in the respiratory tract.
benefits and drawbacks that must be considered when studying airborne Mucus secretion from the respiratory tract is provided by goblet cells
chemicals, whether gaseous, particulate, or representing a heteroge (mucosa). Mucus cells create and exude a rich mucus, which traps the
neous combination. majority of inhaled particles, viruses and bacteria. These are finally
As far as in vitro methods are concerned, it is important first of all to eliminated from the body by muco-ciliary clearance, thanks to the
determine the biological model to be used, depending on the applica beating of the cilia. Mucus is propelled up the airway by ciliated
tion. Monocultures derived from cancerous lung biopsies or immortali epithelial cells, which removes PM. The peripheral lung is lined with
zation of normal cells have enabled many discoveries to be made, but cuboidal type II and squamous type I cells. During the respiratory cycle,
their suitability for representing a system as complex as the respiratory type II cells produce and secrete the pulmonary surfactant required to
system, which has around forty different cell types, is now being keep the lung inflated. Type II cells largely gave rise to squamous type I
increasingly questioned. This improvement in the extrapolation of in cells. The respiratory epithelium plays a key role in pulmonary immu
vitro effects to human health requires suitable culture conditions, such as nity. It forms a physical barrier against infection, lining the airways from
air/liquid interface (ALI) culture, as well as the use of co-cultures and 3D the nose to the alveoli with a wide range of immune cell types. Mast
models (Silva et al., 2023). cells, neutrophils, eosinophils, basophils and resident dendritic cells are
The next step is to expose these biological models to airborne com present in the mucosa of the pulmonary airways and are considered
pounds in the most relevant way possible, either alone or in combina effector cells that play a role in umminity by secreting a variety of in
tion. To do that, the cultured cells should be exposed to the compounds flammatory mediators. They have distinct functions in broncho-
in the same physical and chemical form as humans. This poses a number constriction, mucus secretion and airway remodeling (Huang et al.,
of challenges. For example, we can need to generate the test atmosphere. 2011; Plopper and Harkema, 2005).
The compounds tested are also often modified by interactions with the The nasal and alveolar epithelia are the major deposition sites and
culture medium. These modifications can lead to a chemical trans the largest barriers for drug absorption following drug delivery through
formation (reactivity) or a physical transformation (agglomeration, ag the airway route. The respiratory epithelium of the nasal cavity is the
gregation) which will then modify the toxic properties of the primary site for systemic xenobiotic absorption, due to its high degree of
compounds. vascularization and plays a crucial role in physiological functions,
Finally, following the exposure of the cellular models, the in vitro including the regulation of temperature and humidity, and the filtration
toxicological evaluation is generally divided into two phases: assessing of particles (Silva et al., 2023). The olfactory mucosa, on the other hand,
the cytotoxicity of airborne contaminants and discovering the underly provides a direct connection to the brain, either through olfactory sen
ing mechanisms of action in several pathways, such as oxidative stress, sory neurons or because there are multiple terminal branches of the
inflammation, DNA damage, epigenetic changes but also changes in trigeminal nerve present. These attributes make intranasal administra
phenotypic properties. tion possible (Giunchedi et al., 2020).
This literature review aims to support researchers in assessing the
toxicity of gaseous, volatile, or airborne chemical compounds in vitro. It 2.2. Penetration of airborne compounds into the respiratory system
covers the physicochemical properties of compounds, available cellular
models and exposure methods for characterizing dose-response re Airborne chemicals are exogenous substances present in indoor or
lationships and assessing biological effects using specific toxicological outdoor air, comprising both particles and gaseous compounds, which
parameters. Available biological models of the human respiratory sys can have adverse health effects through their interaction with biological
tem, followed by methods for achieving cellular exposure, and finally systems. Based on their physical properties airborne chemicals can be
2
Fig. 1. Anatomy of the human respiratory system.
classified into two main types. The first category includes gases and delivered throughout the body via the pulmonary blood supply system
vapors, existing as distinct molecules which dissolve and form true so (Bakand and Hayes, 2010; Klaassen and Rozman, 1991). The most
lutions in air and follow the fundamental gas laws. The second category important factor affecting how aerosols behave in the air and how they
is aerosols or suspended air pollutants and refers to both solid particles deposit in the respiratory system is their particle size distribution.
and liquid droplets that may vary in size, composition and origin (Marx Impaction, sedimentation, interception, and diffusion mechanisms are
and Sandig, 2006). As well as olfactory, gas exchange and blood principally responsible for controlling the transport and deposition of
oxygenation function the human respiratory system has evolved to deal particles in the respiratory system. By inertial impaction, larger particles
with xenobiotics and airborne materials that usually occur in the at are often deposited in the nasopharyngeal area (5–30 μm). Aerosols that
mosphere (Bakand et al., 2005). However, the respiratory system can are not eliminated by mucociliary clearance in the upper airway region
not always deal adequately with the wide range of airborne pollutants will be deposited by sedimentation in the tracheobronchial region (1–5
occurring in urban or occupational atmospheres. According to their μm), where they may then be further absorbed or ejected. The remaining
physicochemical characteristics airborne chemicals may enter different particles with the smallest size distribution (1 μm) will enter the alveolar
regions of the respiratory tract. An understanding of airflow structures region deeply since there are insufficient clearance mechanisms there.
of the human respiratory tract is essential for analyzing transportation For particles smaller than 0.5 μm, diffusion is a key deposition mecha
and deposition of airborne materials (Zhang and Kleinstreuer, 2004). nism (Bakand and Hayes, 2010).
Insoluble gaseous compounds may cross the membrane of the pulmo Human cell models have been established for the evaluation of res
nary region efficiently and enter into the blood supply system. There piratory toxicity studies across all areas of the respiratory system,
fore, the respiratory system is considered both as a target organ for including the nasal region, proximal region (trachea and bronchi), and
pulmonary toxicants, and also a pathway for inhaled chemicals to reach distal region (alveoli) (Al-Rekabi et al., 2023). These models can be
other organs distant from the lungs and elicit their effects at these classified into two categories: primary cell cultures and cell lines.
extrapulmonary sites (Bakand and Hayes, 2010). To examine the toxicity of airborne substances, several kinds of
The most significant route through which humans are exposed to human respiratory tract cells are used in toxicology. The most common
chemicals in the atmosphere is inhalation. Physicochemical properties are epithelial cells, such as nasal, bronchial, small airway, and alveolar
and aerodynamic performance of airborne chemicals are crucial in type II epithelial cells, although they can also be lung fibroblasts or
regulating the site of deposition or penetration of inhaled materials, in immune system cells. The respiratory tract contains macrophages,
addition to physiological and anatomical features of the respiratory dendritic cells, and lymphocytes, which can interact with airborne
tract. Chemicals that are inhaled may enter the tracheobronchial, pul substances that enter the body by inhalation (Gordon and Read, 2002).
monary, and nasopharyngeal sections of the respiratory system. The By producing inflammatory mediators and cytokines, these immune
final reaction of the respiratory system to substances inhaled is sub cells can identify and respond to external molecules, including harmful
stantially influenced by the place of deposition or penetration. Water compounds. In vitro research with immune cells can give essential in
solubility is the significant factor which determines the penetration site sights into the processes by which airborne pollutants impact the im
of a gas in the respiratory tract. For example, the gases extremely soluble mune response, as well as aid in the identification of novel therapeutic
in water and tend to be absorbed nearly completely within the nose and targets for treating respiratory toxicity (Tuazon et al., 2022). However,
upper airways. Gases with low solubility penetrate further into the it is essential to note that the in vitro immune response may not fully
pulmonary region and exert their toxicity in the distal lung. Ultimately, represent the complex interactions that occur in human. These models
very insoluble gases pass through the respiratory tract efficiently and are are less commonly used than respiratory system models, which are the
3
major target organ for many airborne contaminants. genes regulated by arsenic exposure in BEAS-2B were also modified
(Zhao and Klimecki, 2015).
2.3. The in vitro approach within new approach methodologies Despite the fact that in vitro toxicology cannot fully reflect the in
tricacy of interactions that occur in the human body, it can give useful
A range of alternative approaches to animal testing has emerged to insights into the potential toxicity of substances and how they influence
overcome the limitations of animal experimentation and to ensure that cells in a simplified environment. It is crucial to remember that the re
ethical standards are met. By adopting the “3Rs principle” and exploring sults gained from in vitro models may not always correspond to the ef
alternative approaches, known as NAMs (New approach methodolo fects found in humans. In vitro models often involve exposing cells or
gies), researchers can minimize the use of animals in testing while still tissues to a fixed concentration of a chemical, which may not fully reflect
ensuring the safety of humans and the environment. In vitro (test tube) the complexities of human exposures. In real life, humans are frequently
and in silico (computer-based) models, as well as other non-animal exposed to a mix of chemicals (e.g. cocktail effect). As a result, in vitro
techniques such as biomarkers, high-throughput screening, and read- tests are increasingly being designed to mimic real-world exposure cir
across methodologies, are used in these procedures. Compared to cumstances, such as varied doses or exposure periods, which can help in
traditional animal-based testing procedures, NAMs can be quicker, better understanding the harmful consequences of substances (Luo et al.,
cheaper, and more dependable, as well as give more human-relevant 2022; Tralau et al., 2021).
data. Furthermore, NAMs can assist to lessen animal suffering as well The in vitro approach offers certain advantages over the in vivo
as resolve ethical and regulatory issues about animal experimentation. method. In vitro models may be employed as a high throughput approach
The in silico approach is based on two main methodologies: quanti to examine a wide range of chemicals concurrently, helping for the
tative structure-activity relationship (QSAR) and absorption, distribu identification of potential toxins and the prioritization of molecules for
tion, metabolism, and excretion (ADME) modeling, specifically subsequent in vivo testing. Furthermore, employing human cell models
physiologically based pharmacokinetic (PBPK) modeling. In these ap can reduce the impact of species-specific differences in how drugs are
proaches, computer simulations are used to predict the behavior of a metabolized or eliminated, which may affect their toxicity in humans vs
substance in the body, its internal dose and potential adverse conse animals in vivo (Botham et al., 2023). Choosing a relevant cell model is
quences. The adverse outcome pathway (AOP) concept was established crucial to ensure the in vitro system accurately represents the target
lately to characterize the causal relationship between a molecular organ. Moreover, the selected cellular model should be sensitive to the
initiating event (MIE) and an adverse outcome. This framework is toxic effects of the compound and replicate the in vivo response as closely
intended to create an organized and transparent knowledge of the as possible. Population-specific considerations, such as age (Fougère
toxicity pathways and biological mechanisms that result in negative et al., 2018), sex (Kiris, 2022), genetic background, and pre-existing
health impacts (Clippinger et al., 2018). Researchers can improve the health conditions, such as asthma (Despréaux et al., 2023) may also
accuracy and reliability of toxicity predictions by combining in vitro and be important to reflect interindividual differences of susceptibility.
in silico approaches, as in vitro tests can give experimental data to
confirm and update computer models (Jin et al., 2021). In vitro experi 2.4. Culture conditions
ments, for example, can be used to provide data on a chemical’s toxicity,
such as its effects on cell viability, gene expression, or protein function. Cell cultures have become a standard method to advance the un
These data may then be used to construct and refine in silico models for derstanding of the mechanisms underlying cell behavior in vivo. Cell
predicting the toxicity of related substances, such as QSAR or PBPK differentiation, migration, growth, and signalization pathways are all
models (Ruiz et al., 2020). Furthermore, in vitro testing can help over examples of such mechanisms, which are influenced by biochemical and
come some of the limitations of in silico technologies, such as a lack of biomechanical cues from the microenvironment. Cell culture provides a
knowledge of complex biological processes and intercellular connec better understanding of the in vivo processes that lead to the formation
tions. In vitro experiments can provide more precise information on the and function of tissues and organs. However, creating a tissue-tissue
molecular and cellular causes of toxicity, as well as help identify critical interface, customizing the microenvironmental elements, and
events and biomarkers to be used in developing more accurate and providing adequate nutrition and waste removal are challenges in
predictive in silico models. developing tissue models that accurately mimic in vivo processes (Duval
The term “in vitro” is derived from the Latin expression meaning “in et al., 2017). Depending on the objective of the study, cells are grown in
glass”, and refers to the technique of performing a given procedure in an mono- or co-culture, either in a basic manner or in a complex approach
artificial environment outside of a living organism. In biological and called organotypic cultures to mimic the tissue architectures and/or
medical research, in vitro models are a good place to start when looking behavior in vivo, such as organoids, spheroids and organotypic tissue
at different biological processes (Doke and Dhawale, 2015). In vitro models (Fig. 2). They can be cultivated in either a liquid culture or at the
procedures in toxicology entail the use of cells that are maintained or ALI. Recently, microfluidic lung-on-a-chip models have also been
cultivated under controlled conditions to investigate the hazardous developed (McMillan, 2020).
characteristics of various substances and combinations. Because of the The traditional approach, submerged culture, induces hypoxic con
diverse interests that will be explored, in vitro toxicology is a critical tool ditions. In the case of bronchial epithelial cell, such environment re
for investigating toxicity processes and screening potential harmful stricts cell growth by suppressing the expression of ciliogenesis-related
compounds. genes and producing a pro-inflammatory response (Silva et al., 2023). In
In vitro models have the advantage of enabling researchers to control vivo, the airways are covered with mucus or surfactant, which is not
environmental conditions and direct exposure to harmful compounds, possible under several millimeters of culture medium. More physiolog
enabling them to study the precise effects on cell development, differ ical growing trials, such as ALI, were first published almost half a cen
entiation, and shape in cells that can proliferate sufficiently (Harris, tury ago (Michalopoulos and Pitot, 1975). These were immersed
2012). As long as the model is appropriately set up to focus on the primary cultures of adult rat liver parenchymal cells on plates covered
specific mechanism of interest, in vitro research offers a considerable with a collagen gel. After 6 h, enabling the cells to adhere to the collagen
benefit in understanding the molecular pathways underlying toxico gel, the latter was removed with a pasteur pipette to form what the
logical processes in a simplified and direct biological model system. On authors called floating collagen membranes on the surface of the culture
the other hand, cells are extremely sensitive to culture conditions and medium. This first culture published at the ALI showed a longer main
can produce inconsistent results. The administration of fetal calf serum, tenance of cell viability than submerged cultures, as well as morpho
for example, can occasionally significantly alter the biological response. logical and functional characteristics reminiscent of the liver in vivo
For example, when fetal bovine serum (FBS) was introduced, 43% of the (structures resembling bile canaliculi, microvilli and junctional
4
Fig. 2. Biological models of the human respiratory system, from the least to the most representative of human physiology (created with BioRender.com)
complexes of tight junctions and desmosomes, etc.). Pruniéras et al. media. It is critical to maintain appropriate phenotypic characterization
shown 40 years ago, based on these research, that cell differentiation, in and function of airway epithelial cells during primary cell passage
this example of keratinocytes, was also connected to the flexibility of the (Rayner et al., 2019). Long-term culture and a high number of passages
membranes on which the cells were cultivated at the ALI (Pruniéras can although affect the phenotype of cells that may even acquire weak
et al., 1983). The culture at the ALI enables to obtain a differentiated tumorigenicity (Ohnuki et al., 1996; Peters-Hall et al., 2018). Culture
epithelium with morphological and functional characteristics similar to conditions can also have a profound impact on cell phenotype. In
those of the human epithelium. Airway cells are first seeded on a semi- addition, the cell lines generally show genome modifications due either
permeable support. Following cell adhesion, the medium from the apical to the immortalization process of normal cells or to the cancerous origin.
side is withdraw. The ALI environment increases the amount of oxygen They may have an altered genome and an accumulation of genetic ab
available and transforms cellular respiration into a more aerobic state. errations, as well as an abnormal karyotype. For a more detailed com
As a result, oxidative cellular metabolism is enhanced, promoting parison of cell lines versus primary cultures, see a recent publication
cellular differentiation, tight epithelial barrier, mucus secretion, recep (Richter et al., 2021)
tor expression, and cytokine production (Sanchez-Guzman et al., 2021;
Silva et al., 2023). Culture at the ALI presently forms the majority of 2.5.1. Nasal epithelium
research aiming at better understanding the physiology of respiratory To our knowledge, RPMI 2650 is the only cell line available derived
cells by placing them in culture settings that match reality, most typi from human nasal epithelium. The cells were cultured from a pleural
cally on permeable supports with microporous membranes (Lu and effusion of a 52-year-old, male patient with metastatic anaplastic
Kolls, 2023). An even more realistic and dynamic model such as cyclic squamous cell carcinoma of the nasal septum (Moore and Sandberg,
mechanical strain (where cyclic stretch plays an imminent role in 1964). RPMI 2650 exhibit a quasi-diploid karyotype that is highly stable
alveolar cell differentiation and lung disease progression), perfusion and throughout long continued cultivation in vitro (Moorhead, 1965). This
inflammatory or thrombotic pathomechanisms was recently constructed nearly normal karyotype is quite an exception when cultivating
by combining ALI culture with lung-on-a-chip technology to examine cancerous cell lines. RPMI 2650 can grow under submerged or ALI
the impact of nanoparticles (NPs) on the respiratory system and the conditions to form a polarized epithelium resembling nasal mucosa
blood-alveolar capillary barrier (Sengupta et al., 2023; Sengupta et al., (Kreft et al., 2015). The mode of culture impacts the phenotype of the
2022). cells, such as barrier integrity and gene expression. Thus, ALI grown
cultures express occludin, claudin-1 and E-cadherin, and have a higher
TEER (Kreft et al., 2015). Two significant drawbacks of RPMI 2650 are
2.5. Cell lines the absence of goblet and ciliated cells on the one hand, and the diffi
culties in forming cell monolayers, on the other hand. As a result, RPMI
In this review, we will focus on the toxicological consequences of 2650 has limited mucin production, unlike the primary nasal model
airborne pollutants, particularly respiratory toxicity. As a result, a (Silva et al., 2023). This model is well characterized for the assessment
thorough description of the numerous human cellular models important of nasal formulations (Sibinovska et al., 2022), but it serves sometimes
to respiratory toxicity studies is required. This part will focus solely on to assess the pro-inflammatory and pro-oxidant properties of environ
respiratory epithelial cells, although certain immune cells, such as mental particles (Lindbom et al., 2006; Zhao et al., 2018).
alveolar macrophages, are resident cells in the lung, and monocyte-
derived macrophages are quite often used in monoculture or coculture 2.5.2. Bronchial epithelium
exposed to airborne compounds. To overcome the disadvantages and Three main cell lines derived from human bronchi. BEAS-2B and
difficulties of supplying and maintaining primary cells in culture, 16HBE14o- cell lines were immortalized by transfection of normal
numerous cell lines have been established using the human respiratory human bronchial epithelium from non-cancerous individuals with a
system. Cell lines are the most widely used in vitro model for under hybrid adenovirus 12-SV40 (Ad12SV40) (Cozens et al., 1994; Reddel
standing the underlying mechanisms of lung toxicity. They are the et al., 1988). These two cell lines must be cultured in a defined medium
easiest to manipulate. They have no variability because they come from without FBS to maintain their initial phenotype. BEAS-2B cell line re
a single donor, which makes it easier to compare toxicity studies. They sembles basal airway epithelial cells, but does not differentiate and does
are characterized by their long lifespan and therefore increased avail not form a strong barrier. This cell line has been widely used in a diverse
ability. They are grown in relatively simple and inexpensive culture
5
variety of respiratory disease investigations, including lung carcino cancer resistance protein (BCRP), and multidrug resistance-associated
genesis. BEAS-2B has been extensively employed to investigate the proteins (MRP1–7) found in primary cultures are expressed in this cell
cellular and molecular processes involved in lung carcinogenesis, line (Endter et al., 2009; Silva et al., 2023). Unlike most lung cell lines,
particularly the involvement of epithelial-mesenchymal transition Cytochrome P450 (CYP) expression by A549 cells is not restricted to the
(EMT) in lung cancer. It should be noted, however, that a recent review CYP1 family (i.e. CYP1A1 and CYP1B1) but concerns also other
questions the culture methods used in most studies, such as the presence biotransformation enzymes, such as CYP2B6, CYP2Cs, CYP2E1,
of FBS, which induces EMT in BEAS-2B (Cochard et al., 2020). CYP2F1, CYP2S1, CYP3A5, CYP3A7, EPHX1, NQO1, and GSTM1 (Al
Furthermore, the BEAS-2B cell line has been used as an in vitro cell Zallouha et al., 2017; Billet et al., 2007; Castell et al., 2005; Hukkanen
model for testing or screening a variety of drugs and biological agents et al., 2000). The A549 cell line has therefore been widely used in the
with potential pulmonary toxicity or lung carcinogenicity (Han et al., study of human lung damage caused by toxic agents such as PM (Billet
2020). 16HBE14o- cell line seems to be closer to the in vivo phenotype: et al., 2018; Kouassi et al., 2010), NPs (Leibrock et al., 2019; Radiom
barrier function with tight junctions and cilia, mucus production via the et al., 2020), and VOCs such as toluene in exposure at the ALI (Al Zal
airway specific mucin protein MUC5AC. Monolayers generate trans louha et al., 2017) and formaldehyde in a microfluidic lung chip (Su
epithelial resistance, as measured in Ussing chambers (Cozens et al., et al., 2023).
1994). 16HBE14o- can be used as a model for etiology and therapy of The NCI-H441 cell line has been derived from the pericardial fluid of
cystic fibrosis, COPD, asthma, and lung cancer (Callaghan et al., 2020). a male patient with papillary adenocarcinoma of the lung in 1982. In
As non-cancerous human bronchial epithelial cells, BEAS-2B and addition to secreting mucins such as MUC5AC, NCl-H441 cells represent
16HBE14o- are often used in studies on the toxicity of solid or gaseous the first cell line derived from the distal lung epithelium of humans that
airborne pollutants (Billet et al., 2018; Méausoone et al., 2021), tobacco can form monolayers with significant barrier properties (Radiom et al.,
(Malyla et al., 2023) and nanomaterials (Zhang et al., 2023). 16HBE14o- 2020; Salomon et al., 2014). These cells form tight junctions and have
has been also used to understand the role of exosomal secretion in the been shown to develop a significant increase in transepithelial electrical
adaption of its source cells to the stimuli of environmental chemicals (He resistance as well as strong, well-organized peripheral expression of
et al., 2021b). zonula occludens-1 (ZO-1) when cultured to confluence, making them
The third human bronchial cell line often used in toxicity studies is suitable as an epithelial barrier model (Jacob and Gaver, 2012; Radiom
the Calu-3 cell line established in 1975 from a metastatic site of bron et al., 2020). They do, however, express less drug transporter proteins
chial adenocarcinoma in a 25-year-old Caucasian male (Fogh et al., than the A549 cells (Sakamoto et al., 2015). When cultivated at the ALI
1977). Calu-3 cells form cell layers with tight intracellular junctions and on transwell inserts using specific medium, the NCI-H441 can be
contain secretory granules, express mucus genes MUC1 and MUC5AC, polarized in a surfactant ALI model (SALI) stable from day 7 to at least
and secrete mucins MUC1/5 AC (Roggen et al., 2006). Additionally, the day 28. SALI cultures have good barrier properties (TEER >300 Ω.cm2)
Calu-3 cells can develop cilia and cilia-like structures as well as grow and produces surfactant proteins like the AECIIs (Munis et al., 2020).
mucus when cultivated for a longer period of time at the ALI (Braakhuis SALI culture is an appropriate non-diseased human lung alveolar cell
et al., 2020). Compared with 16HBE14o-, NCI-H292 and BEAS-2B cells, model.
Calu-3 cultures at the ALI showed the most suitable characteristics for hAELVi (human Alveolar Epithelial Lentivirus immortalized) cells
exposure at the ALI: no obvious multilayer structure, good TEER values have been more recently developed as a human alveolar type I cell line
and relatively lower LDH levels (He et al., 2021a). Calu-3 culture model (Kuehn et al., 2016). This new model exhibit AECI characteristics and
should therefore be preferred for such experiments. For this reason, the functional tight junctions. hAELVi cells can grow as monolayers on
Calu-3 cell line has been used to study a wide range of bronchial per permeable filter supports. They maintain the capacity to form tight
turbations, such as the impact of supplemental oxygen administration intercellular junctions, with high TEER (> 1000 Ω.cm2). hAELVI cells
and positive pressure ventilation, which are strategy commonly could be kept in culture up to passage 75, under liquid-liquid as well as
employed in respiratory failure (Zhu et al., 2010), the assessment of ALI conditions. This model seems to be a promising model of air-blood
nasal formulations (Sibinovska et al., 2022) and of gene carriers (), but barrier of the peripheral lung, since it has already been used for study
also the impact of exposure to airborne compounds, such as gas mixtures ing phenotype of alveolar cells, such as ABC transporters (Visigalli et al.,
(Kastner et al., 2013) and NPs (George et al., 2015). It is also worth 2022), exposed to NPs (Leibrock et al., 2019) and cultivated in co
noting that this model shows good viability and functionality when cultures with Tohoku Hospital Pediatrics-1 (THP-1) derived macro
cultured at the ALI with minimal FBS supplementation for 21 days phages (Kletting et al., 2018). However, as these cells have a phenotype
without subculturing. Calu-3 cells can therefore be exposed over the similar to that of AECI, the barrier and absorption properties of inhaled
long term, which is one of the limitations of the usual in vitro models. drugs have been investigated, rather than the mechanisms of toxic ac
They are a relevant model for chronic toxicity studies of airborne tion such as oxidative stress and metabolic activation.
compounds (Sanchez-Guzman et al., 2021).
Recently, Inscreenex developed some novel immortal human lung 2.6. Primary cells
epithelial cell line displaying a primary like phenotype. Human airway
epithelial cells (huAEC) and human bronchial basal epithelial cells Primary human epithelial cells are normal cells obtained from the
(huBroBEC). These models are not yet widely published but have some lung system and cultured, retaining similar phenotypic and metabolic
interesting features, such as expression of epithelial cell markers (e.g. E- features to living cells. They can be obtained from donors who have
cadherin, ZO-1), maintained polarity, basal epithelial phenotype in 2D respiratory conditions such chronic obstructive pulmonary disease
then differentiation in ALI, and formation of a tight barrier (COPD) or asthma by bronchial biopsies, bronchoscopy brushes, or
(TEER>1000 Ω.cm2) (Leibrock et al., 2019). resected lung tissue. These cultures can be derived from nasal, proximal
(trachea and bronchi), or distal (alveoli) regions of the pulmonary sys
2.5.3. Alveolar epithelium tem. Primary cells can also be obtained from commercial sources
The A549 cell line was derived from a lung carcinoma of a 58-year- including Lonza, MatTek, and Epithelix.. Primary cells retain their
old man in 1972 (Giard et al., 1973). This line has several characteristics physiological function better than continuous cell lines, although they
of AECIIs and is used in the literature to evaluate alveolar reactivity. have a shorter life span than continuous cell lines. When selective media
They grow in monolayers and do not form tight junctions. The culture of and culture at the ALI are used, primary cultures of human lung
the A549 cell line is less problematic than that of other cell lines due to epithelial cell create resistant monolayers, can differentiate, and display
the cells’ relatively stable and long-term proliferative capacity. The certain lineage markers specific to lung epithelial cells. These models
alveolar epithelial cell markers MUC1, P-glycoprotein (P-gp), breast demonstrate the ability to develop ciliated cells, tight junctions, and
6
mucus production in culture. However, the primary cells are unable to recycle it such as surfactant protein A, C, and D, while also generating a
preserve the trans-epithelial electrical resistance (TEER) when grown in diverse range of cytokines and chemokines (Corbière et al., 2011;
conditions that support the growth of distal/alveolar cells. (Wang et al., Woischnik et al., 2010). After isolation, they spontaneously undergo
2020). While a high TEER suggests epithelial barrier coherence, a low morphological and histochemical changes, differentiating into AECI
resistance implies a lack of functional tight junction formation, along (Hiemstra et al., 2018; Rothen-Rutishauser et al., 2008). It has been
with the absence or uneven distribution of crucial proteins such clau shown that AECs from primary cultures form a tight epithelial barrier
dins, occludin, and ZO-1.(Jacob and Gaver, 2012). Primary cultures that resembles the lung barrier in vivo, monolayers with high TEER
have also other limitations including the low availability of normal (>1000 Ω.cm2) can be generated (Rothen-Rutishauser et al., 2008). The
human respiratory tissue. (Rothen-Rutishauser et al., 2008). In addition, isolation and culture of human alveolar epithelial cells is difficult due to
self-renewal ability in the culturing of airway epithelial cells declines the limited availability of lung tissue and the isolation procedure
after a few passes. Additionally, it is difficult to compare results between requiring enzymatic digestion of the tissue and a combination of dif
studies due to the genetic variation among cell batches. ferential attachment, density gradient centrifugation, magnetic cell
sorting and/or FACS. In addition, AECs have a very limited lifespan in
2.6.1. Nasal epithelium culture. Because of these limitations, relatively few toxicity studies have
The airway epithelium in the nasal cavity humidifies, warms and used AECs. AECIIs from three volunteers were exposed to PM of different
filters inhaled air. It is considered to be a key regulator of inflammatory, fractions collected in New York City a few days after the World Trade
immunological, and regenerative responses to allergens, viruses, and Center (WTC) disaster in order to assess, in particular, the induction of a
contaminants (Proud and Leigh, 2011). After surgery for a septal devi pro-inflammatory response (Payne et al., 2004).
ation or chronic sinusitis, or through the superficial scraping of biopsy
samples taken from human volunteers, human nasal epithelial (HNE) 2.6.4. Endothelium
cells are obtained. Human airway epithelial cells of nasal origin Endothelial cells are responsible for lung perfusion and, along with
(hAECN) are also commercialized (Bardet et al., 2014). After confluence type I alveolar cells, for gas diffusion between alveoli and blood (Mor
on tissue culture inserts, the cells are grown, yielding cultures of both issette et al., 2009). The close association between alveolar epithelial
ciliated and non-ciliated cells. HNE cells have historically been used cells and microvascular endothelial cells is essential for efficient gas
extensively to research toxicity, pharmaceutical delivery, histological exchange between blood and alveolar space via vascular endothelial
and morphological comparisons, and the evolution of cell culture con growth factor (VEGF) (Signorelli et al., 2009). Endothelial cells were
ditions due to their ease of access. These cells were particularly created identified by Cldn5, Pecam1 and Cdh5 expression and subclustered
to research the biological consequences of exposure to indoor gases, (Gillich et al., 2020). In view of the toxic effects of air pollution on the
environmental pollutants, or partial or whole microorganisms, such as cardiovascular system, several studies have demonstrated the changes
the secretome. (Bianchi et al., 2021; Kim et al., 2022a). undergone by endothelial cells, either primary or derived from lines,
when directly exposed to pollutants, or when brought into contact with
2.6.2. Bronchial epithelium extracellular vesicles secreted by exposed epithelial cells (Brinchmann
Primary airway epithelial cells can be isolated from bronchial bi et al., 2018; Rota et al., 2020).
opsies, bronchial brushes obtained during bronchoscopy, or resected
lung tissue. For an overview of the procedure of airway epithelial cell 2.7. Stem cell-derived in vitro models
isolation, expansion, and culture at the ALI, see (Jiang et al., 2018).
Normal human bronchial epithelial cells (NHBE) (Lonza™ CC-2450) The interest in the generation of airway epithelial cells has expanded
and human airway epithelial cells of bronchial origin (hAECB) (Epi as a result of cell differentiation, whether using human embryonic stem
thelix™) are commercialized primary bronchial epithelial cells. Even if cells (hESC) or induced pluripotent stem cells (iPSC)(Silva et al., 2023).
the isolation of cells is difficult, the availability of commercial cells and These models have drawbacks, including the expensive acquisition,
the phenotypic relevance make primary bronchial cells often used. In maintenance, and differentiation of stem cells, which supports the few
fact, these cells represent a very good lung model since they can be used studies of scientific research that are now available, in particular in
as a model for influenza A virus infection (Davis et al., 2015), but also for toxicology. However, Van Haute et al. demonstrated that undifferenti
toxicity studies such as exposure to PM (Fujii et al., 2002; Ghio et al., ated hESC grown for 4 days in both hESC medium and differentiation
2013), cigarette smoke (Fields et al., 2005), electronic cigarette (Pearce medium, followed by 20 days of ALI, resulted in high expression of a
et al., 2020), e-liquid, nicotine and cannabidiol (Johne et al., 2023). number of lung markers, including Clara, type I and type II cell markers,
as well as ciliated cell markers (Van Haute et al., 2009). Comparing
2.6.3. Alveolar epithelium primary cells with iPSC-derived alveolar type II cells grown under sub
The large alveolar epithelial surface is composed mainly of three cell merged circumstances, similar morphological traits were discovered
types, the squamous type I alveolar epithelial cell (AECI),the cuboidal (Tamò et al., 2018).
type II (AECII) and the endothelial cells. In addition to these three cell
types, the alveolus is also composed of multiple types of immune cells 2.8. Cocultures
such as monocytes and macrophages (Travaglini et al., 2020).Both
alveolar epithelial cells and microvascular endothelial cells are highly Traditional two-dimensional monocultures, in which respiratory
sensitive to hypoxia, and together orchestrate a rapid and sustained cells grow in monolayers on plastic surfaces, do not always preserve the
adaptive response (Signorelli et al., 2009). Alveolar epithelial cells are in vivo properties essential for toxicological study. Awareness of this
subjected to mechanical stress during respiration, while vascular endo problem has grown as a result of the creation of more complex 3D
thelial cells are mainly affected by shear stress, deformation and hy models that more accurately reflect cell interactions in their real in vivo
drostatic pressure (Lin et al., 2022). AECIs, which are less abundant but environment. (Harris, 2012). The respiratory system is made up of
larger, enable gas exchange and also acts as a barrier, limiting the around forty distinct cell types. Numerous 3D co-culture models have
movement of solutes between the alveolar airspace and the capillary been developed to simulate the complexity and physiology of the res
blood. This cell type can perform a wide range of functions, including piratory system. In general, the objective is to simulate the lung-blood
solute transporter activity, ion transport, and fluid homeostasis, as well barrier or to take into consideration the interactions between the im
as the uptake of nutrients (Campbell et al., 2003). mune system and the lung.
AECIIs have long been acknowledged as significant contributors to Since the recent creation of an AECI model, a coculture model can
the innate immune system, as they release antimicrobial proteins and represent the alveolar compartment. The coculture of the cell lines from
7
AECI (hAELVi) and AECII (NCI-H441) shows an organization of cell induced pluripotent stem cells or adult tissue stem cells possess cell
layers in the two lines. The TEER stability in the cocultures showed that polarity and consist of basal cells, functional multiciliated cells, mucus-
barrier properties might last for 30 days.. The coculture model estab producing secretory cells and club cells. Organoids are more and more
lished a persistent barrier comparable to that reported in hAELVi, while used in disease modeling and drug discovery optimization (Nadkarni
expressing surfactant protein C and demonstrating a claudin and aqua et al., 2016; Svensson and Chen, 2018). For instance, because they may
porin expression profile suited for the distal lung. In the coculture, show the mutational fingerprints of certain cancers, organoids are
cavities were found that showing the polarization of the cells. (Brookes extensively used in cancer research. It takes a long time and effort to
et al., 2021). convert human iPS cells into lung epithelial cells. Starting with primary
To model the alveolocapillary barrier, respiratory cells and endo human airway cells that have already undergone differentiation is
thelial cells are cocultured. Lung epithelial cells (A549) and endothelial another approach. To create structures resembling airways, primary
cells (EA.hy926), for example, were exposed to gaseous or particle airway cells are currently isolated, grown in vitro as a monolayer, and
pollutants. According to the type of cell, this coculture model demon then differentiated (in 2D and 3D). (Paschini and Kim, 2019). Long-term
strated various levels of oxidative stress, a pro-inflammatory response, cultivation of organoids is effective (Sachs et al., 2019). It also has a
and DNA damage. (Offer et al., 2022). In another study, primary human proven track record of reproducibility (Hui et al., 2018). Airway orga
small airway epithelial cells and lung microvascular endothelial cells noids have been touted as a useful toxicological model, particularly for
were cocultured in a multilayered microfluidic device for up to 18 days drug testing. However, due to their sphere-like shape, lumens inside,
to reproduce the parenchymal-vascular interface in the distal lung and lack of an air interface, lung organoids cannot be directly exposed to
(Sakolish et al., 2022). While coculture models are mainly used to airborne compound.. Since organoids are a relatively new model in
investigate intercellular communication pathways, barrier models can respiratory toxicity, upcoming advancements, in particular those made
help in the representation of toxicokinetic. The translocation of NPs possible by organ-on-a-chip technology, may offer a solution. Therefore,
across the bronchial barrier can be tracked using a two-chamber co- a brief description of a few organoids developed from the human res
culture model of bronchial epithelial cells (Calu-3) and endothelial cells piratory system will be provided.
(EA.hy926) (Zhang et al., 2019).
In order to reproduce the relationship between lung cells and the 2.9.2. Nasal epithelium
immune system in response to exposure to airborne pollutants, co Directly from patient material, a well-differentiated organoid model
cultures have often been performed between epithelial cells and mac of the human nasal epithelium can be established. As early as day 3 of
rophages. The seeding ratio of macrophages to epithelial cells ranges culture, when nasal epithelial cells with visible lumens began to grow,
from 1:1 to 1:5, equating to about 104 macrophages/cm2, which is organoids were produced. A circular culture with the apical lumen in
slightly lower than that of the human airway lining from lung tissue side, thick and thin epithelial walls, cilia-forming cells, and mucus
sections (≈3.5 × 104 macrophages/cm2)(He et al., 2021a). Most studies expression can be seen in the fixed cross sections of the organoid. (Liu
use THP-1- or monocyte-derived macrophages. When exposed at the ALI et al., 2020).
to lipopolysaccharide (LPS) aerosol, the coculture of Calu-3 and mac
rophages derived from primary human CD14+ monocytes isolated from 2.9.3. Bronchial epithelium
buffy coats produced more pro-inflammatory cytokines (i.e. IL-1β, IL-10 Human pluripotent stem cells (hPSCs) can be used to create lung bud
and TNF-α) than the coculture of Calu-3 and THP-1-derived macro progenitor organoids starting at day 22 and human lung organoids be
phages (He et al., 2021a). A few studies have been able to work with tween days 50 and 85. In addition to use in developmental biology,
alveolar macrophages collected by bronchoalveolar lavage from pa human lung organoids and bud tip progenitor organoids can be used in
tients (Abbas et al., 2013). This co-culture model has shown its relevance tissue engineering, regenerative medicine, drug safety and efficacy
because one of the most interesting results is the occurrence of DNA testing, and more (Miller et al., 2019). These organoids have mucus-
damage in epithelial cells exposed indirectly via macrophages to fine air secreting goblet cells, club cells, and ciliated epithelial cells with
pollution particles with an aerodynamic diameter lower than 2.5 μm active ciliary activity and mucus flow on the epithelial surface, as well as
(PM2.5). a transcriptional profile, very similar to that of the embryonic lung.
Finally, some co-cultures include more than two cell types and may
therefore seek to model both the role of the barrier and interactions with 2.9.4. Alveolar epithelium
the immune system. To reproduce a lung-blood barrier, anticipate lung AECII isolated by specific antibodies were used to create respiratory
absorption, and explore lung cell changes, a coculture model of organoids (Gonzalez et al., 2010). In the presence of supportive stromal
16HBE14o- cells, monocytic THP-1 cells, and human lung microvascular cells, AECII multiply and develop into AECI, forming organoids known
endothelial cells was constructed (Luyts et al., 2015). This model re as alveolospheres (van der Vaart and Clevers, 2020).
sponds to stimuli such as LPS. It is somewhat challenging to cultivate
due to its need for successive culturing. It doesn’t appear to have 2.10. Spheroids
recently been exposed to chemicals in the air because of this..
2.10.1. Definition
2.9. Organoids Spheroids consist of one or different cells types which have the ca
pacity to expand in three dimensions and develop a cell- or tissue-
2.9.1. Definition specific architecture (Cerimi et al., 2022). Cell lines and primary respi
Organoids are miniature biological systems with a self-organizing 3D ratory epithelial cells can be used to create spheroids with high repro
structure that closely resemble the lowest functional unit of the corre ducibility.Spheroids with polarized, ciliated bronchial epithelium, tight
sponding tissue. The first lung organoids were produced by the differ junctions, ciliary beats, basal cells, and functional secretory cells can
entiation of induced pluripotent stem (iPS) cells from human fetal also be produced using undissociated bronchial epithelium obtained
tissues (Zimmermann, 1987). The most used environment for organoid through bronchial brushing. Spheroids can be produced using methods
formation is hydrogels like Matrigel, which include gelatinous mixtures like low attachment U-plates, the hanging drop method, or the magnetic
of extracellular matrix elements including laminin (60%) and collagen method. The formation of multicellular spheroids begins with loose cell
IV (30%). The development of sophisticated in vitro models based on aggregates under low attachment conditions, followed by compaction
human pulmonary stem cells could contribute in a better understanding due to cell-cell connections mediated by integrin-CEM binding. Spher
of the basic causes of human pulmonary toxicity (Varghese et al., 2022). oids assembled on a chitosan membrane exhibit distinct characteristics
Pseudostratified human airway organoids developed from human compared to those in suspension or on non-adherent polymer surfaces
8
(Cesarz and Tamama, 2016). However, long-term cultivation of spher tumor biology and act as a preclinical model for drug discovery (Salas
oids is challenging (Zscheppang et al., 2018). It should be noted that et al., 2020). Organotypic culture is an effective method that enables the
spheroids lack other cell types like fibroblasts, which have significant examination of tissue behavior. It was initially developed as an alter
effects on epithelial cell function and behavior (Svensson and Chen, native to 2D in vitro culture of neurons to maintain living neurons
2018). Spheroids are widely used in oncology to gain insights into the in outside the body (Schwerdtfeger and Tobet, 2019). They may result
vivo microenvironments of tumors and predict therapeutic efficacy in from the differentiation of a primary cell type or from the reconstitution
cancer research. Tumoroids, which are fairly close to organoids in that of an airway epithelium from already differentiated primary cells. These
they are made directly from patient tumor tissue, maintain the entire latest airway models, which combine a 3D tissue culture approach with
architecture of the extracellular matrix and the tumor microenviron the use of primary cells, are manufactured by Epithelix™ (Geneva,
ment. They have been applied to research a variety of aspects of cancer Switzerland) and MatTek™ (Ashland, USA).
biology, including tumor mutational signatures and interactions be
tween cancer and stromal cells during carcinogenesis and cancer pro 2.11.2. 3D cultures developed from the differentiation of primary cells
gression. Additionally, they are used to create living biobanks of tumors, Primary normal human bronchial epithelial cells (NHBE) isolated
run drugs screens, and conduct pharmacogenetic investigations. Very from the tracheobronchial region of the airway are undifferentiated cells
few publications to date has shown exposure of spheroids of respiratory when propagated as a monolayer in submerged culture. The organotypic
origin, unlike in other organs such as the liver (Bovard et al., 2022; ALI airway model is then generated in situ by differentiation of NHBE
Bovard et al., 2018; Cerimi et al., 2022; Schimek et al., 2020). cells at the ALI. The apical surface of the ALI culture is exposed to
ambient air, which oxygenates the epithelial cells and promotes cell
2.10.2. Nasal epithelium differentiation into a polarized pseudostratified structure resembling
The generation of 3D explant spheroids from a nasal brush biopsy is a that of the airway epithelium, with ciliated cells interspersed with goblet
quick and easy ex vivo method to investigate ciliary activity and path cells on the apical side and basal cells extending along the basolateral
ological characteristics of the ciliated epithelium in the airway epithe membrane. In addition, during differentiation, extracellular matrix is
lium. (Marthin et al., 2017). secreted and deposited on the basal side of the epithelium. 3D cultures
express higher levels of genes involved in cell cycle and proliferation
2.10.3. Bronchial epithelium than 2D cultures (Cao et al., 2020). In addition, a large panel of xeno
The production of tiny connecting ducts between these spheres that biotics metabolizing enzymes (XME) of phase I and II, including CYPs,
mimic acini in vivo, the formation of lumens in sectioned spheres, and glutathione S-transferases (GST) is significantly upregulated during
mucus expression are only a few of the characteristics of the airways that maturation of ALI cultures and their expression remains high over a
are displayed in 3D HBEC cell culture. The developed spheroids thus period of 6 months in culture (Boei et al., 2017).
mimic a number of bronchial architectural features in vivo, including the
development of polarized, acini-like growth arrested spheroids (Fessart 2.11.3. Nasal model
et al., 2013). Three-dimensional epithelia cultured at the ALI are available and
Using 3D bioprinting technique using biocompatible NanoShuttle™ consist of human epithelial cells freshly isolated from biopsies by nasal
(Greiner Bio-one) to magnetize the BEAS-2B cells, 3D spheroid cultures cavity polypectomy or by nasal scraping (Müller et al., 2013). Muci
were obtained then exposed to reference diesel exhaust particles lAir™ is a 3D in vitro model composed of human basal, goblet and cili
(Baarsma et al., 2022). The authors showed an increase in mesenchymal ated cells cultured at the ALI. It comes from different anatomical sites
markers (i.e. collagen 1A1 and β-catenin) and a decrease in epithelial (nasal, tracheal or bronchial). In addition, MucilAir™ is available from
markers (i.e. ZO-1 and E-cadherin). However, as previously discussed, single donor or donor pool, which can be healthy or with pathology (e.g.
the presence of SVF in their culture medium is also able to induce MET asthma, COPD, cystic fibrosis, allergic rhinitis) or smoker. Working with
(Zhao and Klimecki, 2015). nasal pools reconstituted from samples taken from 14 donors should
eliminate the bias of collecting data from a single individual. It is
2.11. Organotypic airway tissue model characterized by its quality and reproducibility for the tissues prepared
from one donor. It is fully differentiated and functional for more than a
2.11.1. Definition year in culture as shown after a targeted proteomic analysis (Baxter
At first, organotypic cultures consisted of small slices of sectioned et al., 2015). MucilAir™ model comprises a tight, polarized, pseudos
tumor tissue placed on porous membranes for mechanical support and tratified nasal epithelium composed of fully differentiated ciliated,
grown in a controlled environment. Now, organotypic tissue models can goblet and basal cells. In addition, the cell membranes express ABC
be established using primary, tissue-specific cells or cell lines grown on transporters that were functional. MucilAir™ exhibits in vivo charac
porous membrane. Once seeded, the cells are usually maintained under teristics such as stratification, tight junctions, cilia beating and the
immersion conditions, and when the epithelial cells reach confluence, production of mucus, cytokines, chemokines and metalloproteinases. As
the culture medium is replaced and only returned to the basolateral a result, these models accurately recreate the physiology of the nasal
compartment. The apical side of the epithelial cells is thus exposed to epithelium, including mucociliary function and mucus secretion in a
air. The use of primary epithelial cells is more advantageous than homeostatic condition. A literature review showed that the MucilAir™
immortalized epithelial cell lines because the cell lines generally fail to Nasal model represents a powerful tool to evaluate the interaction of
differentiate into cell populations composed of polarized, mucus- nasally administered drugs with ABC transporters (Mercier et al., 2019).
producing or ciliated cells (Svensson and Chen, 2018). In addition, This in vitro model of differentiated human nasal epithelial cells enable
primary airway epithelial cells closely recapitulate the transcriptional the study of treatment with pharmacological agents and of the toxicity of
profile of airway epithelia in vivo (Pezzulo et al., 2011). 3D cell cultures airborne compounds (Balogh Sivars et al., 2018; Müller et al., 2013).
present cell-to-cell and cell-to-matrix interactions, which are crucial for The lifespan of this tissue model is up to one year after seeding, enabling
differentiation, proliferation, and cellular activities in vivo (Jensen and long-term exposure (Cervena et al., 2019). MatTek sells the EpiNasal™
Teng, 2020; Ravi et al., 2015). Organotypic tissue models exhibit com model, which appears to be very similar to the MucilAir Nasal. This
plete structural and functional differentiation. They maintained the model is currently only accessible in congress communications. How
metabolic capacity, intercellular and extracellular connections, cell ever, it exhibits pseudostratified morphology, a ciliated apical surface,
matrix components, and 3D histological structure. (Hayden and Harbell, mucin production, and an in vivo-like barrier. When EpiNasal™ comes is
2021). exposed to an irritant, its TEER value plummets dramatically.
Organotypic tissue models have been used to better understand
9
2.11.4. Bronchial model those observed in vivo.

Reconstituted human bronchial tissues (EpiAirwayFT and Muci
lAir™) are available, as are nasal models. As for the nasal models, it is 2.11.7. Air-blood barrier models
possible to keep bronchial 3D models functional for one year without Until recently, alveolar models were difficult to maintain due to the
altering the tissue because of continual cell renewal. A functioning rapid differentiation of AECII in AECI. AlveolAir™ is reconstituted using
mucociliary phenotype with tight junctions may be shown in this model. primary human pneumocytes (AECI and AECII) and endothelial cells.
In addition to basal cells, it has goblet-shaped ciliated columnar cells. EpiAlveolar™ tissues contain fibroblasts in addition. The epithelium
They might also be exposed repeatedly to airborne compounds. Studies exhibits functional tight junctions and a good barrier function that re
have already been experimented with gaseous contaminants (Cervena mains stable for studies lasting >30 days. Surfactant, cytokines and
et al., 2019; Rossner et al., 2019). In fact, the integrity of the tissues and chemokines are produced as expected. Alveolar models are used for
the tight junctions were measured before and after the exposure, and the toxicology of inhaled products such as carbon nanotubes (Meindl et al.,
TEER values revealed that there were no negative biological effects from 2023) 1,3-dichloropropene (Moreau et al., 2022), drug transport and
the exposure to the emissions, indicating that this model may be used for drug development research (Visigalli et al., 2022), and disease modeling
long-term exposures. upon repeated subchronic exposures to toxics (e.g., fibrosis)(Barosova
Models reconstituted from bronchial biopsies have features that are et al., 2020).
quite comparable to models obtained from nasal samples. Because of the
significantly lower rate of proliferation and differentiation of goblet cells 2.12. Lung-on-a-chip models
and ciliated cells, nasal epithelial cells would not be a perfect match for
bronchial models from a morphological standpoint. However, on a A lung-on-a-chip is a sophisticated 3D representation of a living,
physiological level, the models appear to be extremely similar. For breathing human lung on a microchip. This new model enables re
example, nasal and bronchial epithelial cells responded similarly to searchers to reproduce the structure and functions of the human lung,
cytokine stimulation, the expression of key surface receptors was iden making it easier to assess the pulmonary toxicity of contaminants,
tical, and there were strong correlations between mediator release by particularly in the case of airborne NP absorption (Rothbauer et al.,
nasal and bronchial cells (McDougall et al., 2008; Thavagnanam et al., 2021). The first chips were fabricated with polydiméthylsiloxane cast on
2014). Furthermore, xenobiotic reactions in smokers’ bronchial and a photolithographically prepared master that contains a positive relief of
nasal epithelial cells were identical to those shown in their respective parallel photoresist microchannels (Huh et al., 2010). The cross-
organotypic models exposed to cigarette smoke (Iskandar et al., 2015). sectional size of the microchannels was 400 or 200 μm (width) x 70
Because of this, and because bronchial biopsies are invasive, the nasal μm (height). The device is usually made using human lung and endo
model could be employed as a physiological alternative and it should be thelial cells in order to predict absorption of airborne compounds and
the most extensively used in the next future. mimic the underlying mechanisms of action. For example, a human
small airway on a chip that may be employed in medium/high-
2.11.5. Bronchial models with fibroblasts throughput research of the subacute effects of inhalation toxicants was
Both Epithelix and MatTek make tissue models that combine human- constituted by primary human small airway epithelial cells and lung
derived tracheal/bronchial epithelial cells with fibroblasts (Epi microvascular endothelial cells cocultured in a multilayered micro
AirwayFT and MucilAir™-HF). These devices are being proposed for fluidic device for up to 18 days to reproduce the parenchymal-vascular
research into lung inflammation and fibrosis, wound healing, and cell- interface in the distal lung (Sakolish et al., 2022). This biomimetic
cell communication. These two models were exposed to poly microsystem recreates the essential functional alveolar-capillary inter
hexamethyleneguanidine phosphate and bleomycin to characterize the face of the human lung. The essential anatomical, functional, and me
fibrotic responses (Kim et al., 2022b). In the two 3D airway models, the chanical features of the human alveolar-capillary interface, which is the
fibrogenic agents elicited different responses. While the EpiAirwayFT core functional unit of the living lung, are reproduced in this lung mime.
model was related with inflammatory and EMT responses, the Muci The lung mimic device, in particular, recreates the microarchitecture of
lAir™-HF model was associated with fibroblast responses. This is the the alveolar-capillary unit, maintains AECs at the ALI, exerts physio
first study comparing the two models, so it should help in the future logically relevant mechanical forces on the entire structure. It enables
identification of appropriate 3D airway models for inhalation toxicity the analysis of the influence of these forces on various physiological
studies. Two MucilAir™-HF models, reconstituted from healthy and factors and pathological lung functions, such as interactions with im
asthmatic donors, have been exposed twice a week for three consecutive mune cells and pathogens, epithelial and endothelial barrier functions,
weeks to PM of industrial or traffic origins. The authors discovered and NP toxicity and uptake across this critical tissue-tissue interface.
changes in the XME and inflammatory pathways after measuring 33 Furthermore, in this biomimetic microsystem, epithelial cells not only
genes implicated in inflammation, oxidative stress, cell development, remained viable, but also increased surfactant production, improved
autophagy, mucus production, metabolism, and epigenetic mechanisms structural integrity and restored normal barrier permeability (Huh et al.,
(Despréaux et al., 2023). However, the interest of the coculture with 2010).
fibroblasts was not shown in this study.
3. Methods of exposure to airborne compounds
2.11.6. Small airways models
SmallAir™, produced by Epithelix is the only in vitro cell model of 3.1. Requirements
the human small airway epithelium cultivated at the ALI. It was
reconstituted from primary small airway cells. After 3 weeks of incu In vitro experiments aim to represent exposure conditions as close as
bation, the epithelium had fully differentiated, with columnar epithelial possible to the real world. When designing a toxicological test, several
morphology. Most notably, CC-10, a particular marker of Club cells, was factors must be carefully considered: (i) culture conditions that do not
found to be substantially expressed in SmallAir™ but not in MucilAir™. disturb cell physiology and maintain the most sterile conditions
SmallAir™ contained less Muc5-Ac positive cells (i.e. goblet cells) than possible; (ii) determination, production and maintenance of appropriate
MucilAir™. The tissue remains fully differentiated and functional for concentrations of xenobiotics during exposure; (iii) a real contact be
more than a year (Huang et al., 2017). This model has already been tween the pollutant and the cells enabling the bioavailability of the
exposed to gaseous compounds alone (i.e. 1,3-dichloropropene)(Moreau compound; (iv) an exposure period based on the compound’s kinetics (i.
et al., 2022), as a mixture (i.e. e-vapor products)(Johne et al., 2023) or to e. half-life) and the expected real exposure time; and (v) an exposure
NPs (i.e. silver)(Guo et al., 2018) mainly to compare toxicity levels with period sufficient to induce relevant biomarkers of exposure/effect
10
(Rasmussen, 1984). serum proteins, rendering them less accessible to the cells. These alter
Various in vitro exposure techniques have thus been developed. ations are especially important for chemically reactive substances and
These techniques include submerged cell exposure, intermittent pro particles. With specific regard to NPs, exposure studies under immersion
cedures, and ALI methods. The primary objective of these exposure conditions have shown interactions between the medium and the NPs, a
systems is to achieve a homogeneous and uniform distribution of the corona formation, agglomeration or dissolution of NPs and other
airborne compounds across the cells. This is mainly challenging for changes in physicochemical properties (Latvala et al., 2017; Loret et al.,
particles. For such xenobiotics, the exposure method should minimize 2016). The presence of a protein corona was shown to elicit an immune
particle agglomeration and preserve the size distribution and chemical response and affect NP toxicity (Fasoli, 2021). Moreover, sonication is
composition of the aerosol. In addition, these techniques aim to repro frequently employed to prepare the particles suspension, and the
duce in vivo exposure conditions as closely as possible. In the case of method used, as well as the usage of serum, will impact the NP char
airborne compounds, the use of dedicated exposure chambers makes it acteristics. Furthermore, in submerged conditions, NPs may adhere to
possible to approach in vivo conditions, ensuring intimate contact be the cell exposure plate’s wall, as well as remain in the liquid, preventing
tween the cells and the pollutants while minimizing potential in them from reaching the cells (Cappellini et al., 2020; Lovén et al., 2021;
teractions. The case of airborne compounds is special because exposure Watson et al., 2016). The sedimentation of NP in vitro is a continuous
metrics are highly variable, particularly for particles, since concentra process, and determining the actual deposited cellular dose remains
tions can be expressed in mass, number or specific surface per unit of cell difficult because the cellular internal dose varies with time (Leroux
surface, liquid or gaseous volume or number of cells (Loret et al., 2016). et al., 2020). In addition, because it does not accurately represent the
Concerning the exposure period, two aspects need to be considered: lung and is unrealistic in terms of physiology and exposure environment
the contact time of the cells with the toxicant and any recovery time (s), cellular exposure in submerged conditions is unreal. Lung cells are
after exposure. These times are determined according to the research covered by a layer of surfactant that is exposed to air and do not exist in
hypothesis and the temporality of the corresponding human exposure. submerged conditions. As a result, this kind of exposure has the potential
Human exposure may be continuous or discontinuous, acute, repeated to change the way engineered nanomaterials (ENM) affect cells when
short-term or long-term. Some toxic substances will act rapidly, while they are exposed to them (Meldrum et al., 2022).
others will require a latency period before triggering adverse effects. In Secondly, cell exposure to PM, PM extracts, or chemicals usually
experimental toxicology, exposure times are precisely regulated and can requires the use of dimethyl sulfoxide (DMSO) or cell culture media
range from one minute to several days for continuous exposure(Billet (Billet et al., 2018). However, about 10% of the compounds of interest,
et al., 2018; Dong et al., 2019; Ji et al., 2019). Most repeated exposure as stated by the Toxic substances control act (TSCA) of the United States
experiments last from 15 min to 60 min, sometimes 6 h, repeated daily Environmental Protection Agency (EPA), are insoluble in DMSO or
or regularly for 3, 5 or 90 days (Bardet et al., 2014; Boublil et al., 2013; culture media, or are volatile. This means that their toxicity cannot be
García-Salvador et al., 2021; Kastner et al., 2013; Méausoone et al., effectively investigated.
2021; Mistry et al., 2020). Short exposure times and repeated exposures Thirdly, as the cells are maintained at 37 ◦ C, the xenobiotics may
are particularly suited to cell models grown in ALI, and to the more evaporate, especially if they are highly volatile compounds such as
complex way in which exposure atmospheres are generated than in benzene or toluene. Exposure to gaseous chemicals can be achieved by
submerged exposures. In addition, a post-processing time of several placing them in vacuum test tubes containing a cell solution. Despite the
hours to several weeks can be used to monitor the development of the fact that this method enables some interaction between the target cells
cellular response to exposure. Here again, numerous possibilities have and the gaseous chemical of interest, the exposure pattern in such a
been published. Recovery or post-treatment times can range from a few constantly submerged system does not mirror what happens in vivo
hours to several weeks (Boublil et al., 2013; Juarez-Facio et al., 2022; (Bakand and Hayes, 2010; Zavala et al., 2018).
Méausoone et al., 2019). When several post-treatment times are applied,
kinetics in the reaction can be demonstrated. In the case of comparisons 3.3. Air-liquid interface exposure
between several atmospheres of different composition, this can show
that the molecules do not activate the cellular defense mechanisms at 3.3.1. Production of the test atmosphere
the same time (Juarez-Facio et al., 2022). To achieve a more realistic exposure of the cells corresponding to the
conditions found in the lung, improved test models for respiratory ex
3.2. Submerged exposure posures were created and required the generation of test atmospheres
enabling the cell exposure at the air-liquid interface (ALI). Several
3.2.1. Advantages methods can be used to produce an atmosphere containing the airborne
The majority of exposure approaches, particularly those used in PM compound to be tested in the required concentration. In the case of a gas
investigations, limit cell exposure to particles solubilized or suspended or volatile compound, the usual methods are based on the dilution of the
in culture medium. Cells, whether adherent or not, develop in culture pure compound with a carrier gas to achieve the desired concentration:
media and are exposed to toxicants spiked in the medium (Baulig et al., (i) direct injection of the gas or vapor of interest into the exposure
2004; Billet et al., 2007; Kouassi et al., 2010). This procedure is simple chamber using calibrated syringes or specialized injection systems
and inexpensive. A gaseous test compound can be conveniently pumped (Persoz et al., 2010); (ii) dynamic flow systems: a controlled flow of the
through the cell solution using various laboratory settings to achieve gas or vapor is generated using mass flow controllers (Al Zallouha et al.,
continually submerged exposure conditions (Bakand and Hayes, 2010). 2017; Méausoone et al., 2021); and (iii) permeation chambers: the gas or
For a long time, submerged exposure was the only option for repeated vapor permeate through a permeable membrane (Bardet et al., 2014).
exposure even for volatile compounds, whereas exposure to gas-phase The last two methods provide continuous exposure and precise control
substances was confined to single short-term exposures (Kastner et al., over the concentration and flow rate a continuous and controlled release
2013). of the gas or vapor.
With regard to the generation of an atmosphere containing particles,
3.2.2. Disdvantages aerosols can be first produced from pure test materials in three ways: by
However, there are a number of drawbacks to submerged exposure. dispersion of liquids containing particles, by dispersion of powders or
Firstly, it can lead to mistakes in calculating the toxicity of pollutants by ablation from solid bulk materials, or by particle formation by gas to
overestimating or underestimating the degree of exposure. Concentra particle formation (i.e. nucleation, condensation and coagulation). It is
tions of exposure are frequently far higher than those found in ambient also possible to use a combustion source to produce specific aerosols
air, and they may interact with molecules in the culture media, such as such as cigarette smoke (i.e. smoking machine)(Bardet et al., 2016), the
11
fine and ultrafine particles produced by the combustion of propane, the culture medium. It is evident that by carefully controlling these
gasoline and biofuels (i.e. soot generator miniCAST)(Juárez-Facio et al., parameters, effective exposure conditions can be achieved while opti
2022), or the exhaust of diesel combustion (i.e. engine with or without mizing cell viability preservation (Lacroix et al., 2018; Meldrum et al.,
catalytic after-treatment)(Barraud et al., 2017). Finally, cells can also be 2022).
exposed to real mixtures sampled outdoor, in an occupational environ The primary concept underlying exposure at the ALI is to grow cell
ment, close to a specific source of pollution, or to secondary organic models on porous membranes and then directly expose the apical side of
aerosols produced in atmospheric simulation chambers. It is also critical the cells to the test atmosphere while the culture media is on the basal
to have appropriate in situ exposure systems in the event of highly side to supply nutrients essential for viability (Zavala et al., 2018). All
reactive pollutant mixtures, such as the primary pollutants produced by lung cell models that can be cultured at the ALI should be able to be then
motor traffic. Chen et al. developed a mobile platform outfitted with an exposed at the ALI. However, certain types of cultures, particularly cell
on-board diagnostic system, a custom-made portable emission mea lines with a fast rate of division, such as A549, BEAS-2B, and 16HBE14o-
surement system, and an electrostatic ALI exposure system with human , are not suitable for long-term exposure (i.e. more than one week).
monocytic THP-1 cells to characterize on-road tailpipe emissions under Primary epithelial cells, which may be cultured for weeks at the ALI,
real-world driving conditions to study the health impact of primary may be a viable alternative for ALI exposure. The ability of primary
pollutants produced by the combustion of petrol in internal combustion cultures in ALI to produce mucus or surfactant protects them from the
engines (Chen et al., 2023). Two additional research have employed drying effects of airflow (Abdullah et al., 2012; de Bruijne et al., 2009).
Cultex® or Vitrocell® technologies to expose cultivated lung cells in situ The Calu-3 cell line also meets all criteria needed for realistic, repeated
to examine the toxicity of more complicated atmospheres under urban ALI exposure when compared to other cell lines because they remain
or industrial influences (Augustin et al., 2020; Gualtieri et al., 2018). viable for weeks while cultured at the ALI, provide high barrier integrity,
do not overgrow, and are easy to culture and maintain (Sanchez-Guz
3.3.2. Intermittent exposure man et al., 2021). When examining the toxicity of any particle at ALI, it
When exposing to gaseous pollutants, an intermittent exposure is also crucial to use a positive control that must be carefully selected to
strategy might be used. It is accomplished using rotating bottles or examine exposure to DQ 12 the engineered nanomaterials (ENMs). Due
oscillating trays. The cell culture dishes are kept in chambers or circular to the increase in IL-6 concentrations found, the study determined that
platforms that are inclined at particular angles and are regularly exposed the A549 + dTHP-1 model was the most effective model for establishing
to gaseous compounds. In this type of exposure, cells grow on collagen potential pro-inflammatory responses in vitro (Meldrum et al., 2022).
gels in submerged conditions. Before the cells are exposed, the con In the case of exposure of the cell to an atmosphere, some re
tainers are inverted and the medium is aspirated, leaving the cells linked quirements have to be considered: (i) atmosphere testing with controlled
to an air/collagen interface (Aufderheide, 2005). Although this method production and monitoring to investigate dose-dependent effects; (ii)
provides more direct cell exposure than submerged approaches and a maximizing proximity between cells and substances of interest to pre
greater interface between the gaseous chemical and the target cells, the vent interactions (for complex mixtures of gaseous compounds, direct
cells are still covered by the medium for half of the experiment, which contact between cells and gas phase components should be ensured);
can compromise precision and reproducibility. As a result, these devices and (iii) enabling long exposure durations using properly tuned expo
are not widely used at the time because they have the same drawbacks as sure systems. Cells exposed to dry air at room temperature tend to
submerged exposure such as interactions between the xenobiotics and deactivate rapidly. Therefore, strategies for maintaining humidification
the culture medium (Bakand and Hayes, 2010). have been developed and implemented (Rasmussen, 1984).
For ALI exposure, both static and dynamic direct exposure ap
3.3.3. Continuous direct exposure proaches have been established. In static exposure, cells are exposed to a
To address the challenges associated with existing exposure predetermined volume of gaseous chemicals pumped into a closed
methods, new devices were developed at the ALI. As described previ container. For the in vitro investigation of volatile test compounds, this
ously, cultures at the ALI represent a promising approach for faithfully static technique is similar and repeatable. Compared to the submerged
replicating the in vivo biological structure of the respiratory system in methods, it better mimic in vivo exposure patterns, and it can overcome
vitro. Unlike submerged cultures, ALI accelerates cell differentiation, issues that arise during VOC in vitro testing, such as low water solubility
increases the establishment of tight junctions. Furthermore, ALI systems and excessive volatilization of certain VOCs (Bakand and Hayes, 2010).
also offer distinct advantages in terms of exposure relevance. These As part of a dynamic exposure, the airborne compound is diluted in a
technologies enable exposure to real-world situations by eliminating continuous stream of carrier gas (e.g. purified air). The dynamic tech
direct interaction between the exposure agent and the cell culture me nique has the advantage of enabling the concentration of chemicals in
dium. Consequently, ALI devices serve as a valuable experimental the test atmosphere to be modified by adjusting the dilution, thereby
foundation for incorporating crucial factors such as the physicochemical recreating a pollution peak (Bakand and Hayes, 2010).
properties of the test atmosphere, diverse methods for generating and In ALI exposure setups, the flow can be either horizontal or vertical.
characterizing test aerosols, and establishing defined and unobstructed In the incubator/box structure, cultures are placed inside a box, which
contact between the biological test system and the airborne test mate may also be a cell culture incubator. After that, the box is filled with the
rial. Growing rat lung epithelial cells of Type II origin on hydrated test atmosphere, creating a horizontal exposure flow above the cultures’
collagen gels and then maintaining the cultures at the ALI to expose lung inserts. The stagnation flow setup a more sophisticated setup is repre
cells directly to NO2 was the first described exposure at the ALI (Zamora sented by where the exposure flow is directed toward the ALI surface.
et al., 1986). Using this flow alignment, an individual exposure of single cultures can
In the ALI exposure condition, both the mass flow from the liquid be achieved. Moreover, an optimization of fluid dynamics is possible
culture medium through the membrane to the cells and the fluid flow leading to a better-defined dosimetry and a higher exposure efficiency.
necessary to deliver the airborne test material to the surface of the Exposure setups based on these basic ALI flow alignments are
biological test system are essential. When using 24- or 6-well cell culture commercially available (e.g. TSE Systems, Vitrocell® and Cultex®)
inserts, the flow rate applied is usually a few ml per minute. Addition (Fig. 3)(Lacroix et al., 2018).
ally, exposure outcomes can be influenced by facility-specific variables, A variety of systems enabling exposure at the ALI have been devel
including specific cell properties such as epithelial barrier activity oped over the past 20 years. Two commercial devices developed in
facilitated by cell-to-cell connections, membrane pore size and density, Germany are frequently used: Vitrocell® and Cultex® devices (Gualtieri
membrane surface area, gas flow rate, test atmosphere humidification, et al., 2018; Leroux et al., 2022). They work in much the same way (i.e.
culture medium and test atmosphere pressures, and characteristics of distribution to the cells cultured on a porous membrane of the test
12
Fig. 3. Main ALI exposure setups. a. “Incubator/box type” setup featuring a horizontal flow and often using standard commercial culture plates. b. “Stagnation point
flow” setup. Inspired by (Lacroix et al., 2018).
atmosphere through inlet tubes called “trumpets” using a negative future HARN studies. Cellulose nanocrystals (CNCs) from cotton and
pressure)(Aufderheide, 2005). The Vitrocell® device has been used to tunicates were used as model HARN samples, with varying aspect ratios.
investigate the toxicity of gases, complex mixtures, and NPs (Augustin Specifically, well-dispersed and characterized CNC suspensions were
et al., 2020; Brunet et al., 2015; Loret et al., 2016). The Vitrocell com aerosolised using an ALICE at realistic, cell-delivered concentrations
pany also develops systems for generating test atmospheres, either by ranging from 0.14 to 1.57 μg/cm2. The biological impact of each sample
continuous flow, nebulization of a toxic substance suspended in a liquid was assessed using a 3D in vitro model of the human epithelial airway
(i.e. Vitrocell® Cloud) or aerosolization of a powder (Vitrocell® Pow barrier. The testing strategy was validated using crystalline quartz
derX) (Ding et al., 2020). The Continuous Flow Exposure System consists (DQ12) and long fibre amosite asbestos (LFA). No significant cytotox
of stainless-steel modules that ensure durability and high temperature icity, oxidative stress, or pro-inflammatory responses were observed up
resistance. The base section contains wells for culture media and inserts, to the highest concentration, but both DQ12 and LFA elicited a signifi
while the upper section distributes the gaseous component to the apical cant pro-inflammatory response (Endes et al., 2014).
portion of the cells via three trumpets. The temperature within the unit is The “Hanging Drop (HD)” method involves inoculating cells in suf
maintained at 37 ◦ C using a heated water circuit. Using a vacuum pump, ficiently small drops of culture (approximately 20 μL), enabling the cells
the test atmosphere passes through the modules at a regulated rate, to remain on the dish when it is flipped upside down and placed on a
coming into contact with the cells grown on the inserts in the wells. The sticky surface of the upper half of a module containing the air and
system is available for use with 6-, 12-, 24- and 96-well sized, cell culture gaseous chemicals of interest (Fig. 4). When the drop is flipped over,
inserts, suspension cells and Petri dishes. gravity collects the cells at the bottom corresponding to the ALI. This
For the past 5 years, a team from Karolinska Institutet (Sweden) has simulates the effects of direct air contact on cells and enables long-term
been regularly publishing research using the XposeALI® system devel exposure. This type of air exposure was employed to study the direct
oped and marketed by Inhalation sciences. Operating on a similar interaction of volatilized benzene with cells (Liu et al., 2013).
principle to the Vitrocell® and Cultex® models, XposeALI® appears to A new cell culture exposure system (CCES) was developed to test
be dedicated to aerosol exposure when coupled with the PreciseInhale® volatile compounds. The foundation module of this system consists of a
system, an aerosol generator fitted with a dosing system. In this system, 24-well tissue culture plate with Transwell inserts for ALI exposure. Two
aerosolization is achieved under pressure (i.e. 160 bar) to produce well- manifolds on the base module’s lid are in charge of parallel gas distri
dispersed particles via dry powder deagglomeration (Cappellini et al., bution. This system is controlled and managed by a vacuum pump and
2020; Ji et al., 2019; Ji et al., 2018; Ji et al., 2017). To show the full precision valves, and it is kept at 37 ◦ C with the assistance of a hu
range of possibilities, the Table S1 (supplementary information) sum midification system to keep the cell in good form. Most systems require
marizes the toxicological studies made using these tree commercial ALI intensive handling of the porous membrane inserts, changes of cell
exposure devices: Vitrocell®, Cultex® and XposeALI®. culture media from multi-well culture plates to the exposure system then
Numerous non-commercial ALI exposure systems have been devel back to the multi-well plate after exposures, and cleaning/maintenance
oped, although with limited user availability. Among these systems, the between experiments, whereas this type of exposure uses 24-well plates
ALICE (Air-Liquid Interface Cell Exposure) method stands out as a reli with Transwell inserts, which require no maintenance or cleaning be
able approach for controlled exposure of cells to aerosolized NPs. The tween experiments (Zavala et al., 2018).
ALICE system is made up of a temperature and humidity-controlled cell As a complement to the exposure systems, the Electrostatic aerosol in
exposure chamber that is coupled to a continuous filtered air flow to vitro exposure system (EAVES) was designed to improve particle depo
create a physiological environment. A vibrating membrane nebulizer on sition on epithelial cells’ surfaces. This method efficiently deposited
the side of the exposure chamber creates a thick cloud of aerosol par particles directly onto the cells from an air stream (de Bruijne et al.,
ticles that is deposited on the ALI cells. At the ALI, this exposure method 2009). Electrostatic deposition is a very effective, quick, and simple
enables the uniform, efficient, and dosed deposition of NP suspensions method of determining the toxicity of fine and ultrafine particle depo
on cell surfaces. This technique has gained particular prominence in sition. An electric field is used to generate a net neutral aerosol charge
studying the effects of such NPs (Lenz et al., 2013; Lenz et al., 2009). The on the cultures in this system, which is made up of tissue culture plates.
ALICE nebulisation platform was successfully used for the aerosolisation Diffusion, sedimentation, and electrostatic force are three processes
of airbone HARN, with CNCs isolated from cotton or tunicates(Endes used in this sort of direct air-cell exposure system. Using EAVES to
et al., 2014). The system met all cornerstones of controllability, dose expose NHBE cells to nebulized diesel particles did not increase the
dependency, spatial homogeneity, and deposition efficiency. The QCM cellular response measured elsewhere without the application of an
was used as a real-time monitoring tool, indicating its applicability for electric field (Hawley et al., 2014). The concept of an adjustable electric
13
Fig. 4. “Hanging Drop” (HD) air exposure method.
field between the aerosol inlet and the cell culture to significantly in cells decreased by >80% (Dong et al., 2019). A device equipped with a
crease particle deposition on cell cultures has been implemented to the porous membrane on which A549 cells are grown enables the cells to be
Vitrocell® system (Juarez-Facio et al., 2022). The deposit of soot par exposed both apically with a gas or a volatile compound (e.g. formal
ticles generated by Cast was multiplied by 5.5 to 7.7 using an electro dehyde) and in the basal liquid medium. Depending on the study, the
static field. The efficiency of this technology is determined by the type of cells can be exposed on one or both sides at the same time (Su et al.,
particles analyzed. Organic carbon-rich particles deposit more than 2023).
organic carbon-deficient ones (Juarez-Facio et al., 2022). The amount
deposited appears to be related to toxicity which is in line with other
work using similar technology (Latvala et al., 2016). 3.5. Comparisons between submerged and continuous direct exposure
ALI systems enable direct contact between the cell and the toxic
chemical, but also with an airflow that simulates that of the human 3.5.1. Comparison studies
respiratory system. Exposure times can exceed 4 h (Cultex®) or even 6 h Due to the expanding advancement of ALI exposure methods
(Vitrocell®). After 2 h of exposure, it is important to humidify the test throughout the last ten years, several researchers have compared the
atmosphere, as the cells do not survive and dry out after this time biological responses of human lung cell cultures to exposure in liquid
(Gałęzowska et al., 2016). Temperature control at 37 ◦ C and a relative medium or at the ALI (Table 1).
humidity of at least 75% are essential for adequate exposure at the ALI Cell cultures were exposed to aerosols in two conditions: submerged
(Zavala et al., 2017). and ALI, according to Cappellini et al. In comparison to the cultivation in
submerged circumstances (no impact or very modest effects max 10 μg/
cm2), this study found an increase in inflammatory potential (IL-1, IL-6
3.4. Lung-on-a-chip exposure for the dosage 3 μg/cm2) in ALI conditions (Cappellini et al., 2020). ALI
grown primary airway epithelial cell cultures were exposed to cigarette
This kind of system is booming and several devices are being smoke. Toxicological tests revealed that the cells decreased cilia beating
developed and tested. A few examples of devices exposed to solid or activity and hindered barrier formation. Exposure caused dysregulation
gaseous airborne compounds will be presented. According to both the of basal and secretory cell marker genes, altering the development of
set-up of the chip and the nature of the biological model, the exposure small airway basal cells in vitro, according to RNA sequencing studies
can correspond to submerged or to ALI. For a complete and recent re (Gindele et al., 2020). Also, exposure of NHBE cells to e-cigarette smoke
view of microfluidic lung chips for assessing cell exposure to airborne caused mucin production, altered ciliary beat frequency and hyperse
compounds, see (Wang et al., 2023). A lung-on-a-chip can be for cretion of interleukin IL-6 and IL-8 (Garcia-Arcos et al., 2016).
example made up of three parallel channels for co-culture of human Two studies comparing cellular responses following exposure to ZnO
vascular endothelial cells and human alveolar epithelial cells sand NPs at the ALI (ALICE) or submerged conditions revealed a higher gene
wiched between layers of Matrigel membrane that mimic the major induction of pro-inflammatory cytokines in ALI conditions, which more
features of the human lung’s alveolar capillary barrier (Zhang et al., closely resemble realistic inhalation scenarios. This finding emphasizes
2018). This device enables cell-cell interaction, cell-matrix interaction, the importance of adopting ALI techniques over submerged conditions
and vascular mechanical cues to work together. Among the culture for avoiding the interactions between cell culture medium and NPs in
conditions tested, perfusion of a culture medium through the chip is the toxicological studies (Lenz et al., 2013; Lenz et al., 2009). These results
one that best enables the establishment of an effective barrier with were recently confirmed by another study which showed some differ
limited permeability. The exposure of the device to TiO2 and ZnO NPs ences in the toxic effects of ZnO NPs between exposure at the ALI and
induces ROS generation and increases the permeability of the mem submerged exposure (Lovén et al., 2021). Dry aerosol exposure of ZnO
brane. In another device, bronchial epithelial cells (Calu-3), endothelial NPs at 1.0 μg/cm2 via the nano aerosol chamber in-vitro toxicity
cells (EA.hy926) and Thp-1-derived macrophage are cultured in a two- (NACIVT) system induced significant increases in metabolic activity and
chamber system (Zhang et al., 2019). Silver NPs exposure causes the release of IL-8 and MCP-1, whereas submerged exposure did not
cellular uptake and translocation of AgNPs over the modelled barrier, induce toxicity. Factors associated with the exposure method, such as
modest cytotoxicity, and a decrease in the release of interleukins IL-6 the different aggregation of NPs on the one hand, and a relatively high
and IL-8, and tumor necrosis factor-alpha (TNF-α). BEAS-2B cells solubility of ZnO in the cell culture medium on the other hand, may
grown on ALI porous membranes in a PDMS microfluidic chip were explain the different cellular responses observed. These results
exposed to PM2.5 using a nebulizer. RNA-Seq, two-dimensional LC-MS/ encourage the use of more physiologically realistic exposure systems to
MS coupled with an iTRAQ labeling, flow cytometry and western-blot assess the toxicity of airborne ENMs. Another study obtained similar
analyses were used to identify the activation of a number of signaling results in human alveolar epithelial cells exposed to other metal oxides
pathways such as inflammation and apoptosis (Dong et al., 2019). NPs (Bessa et al., 2021). After 24 h of exposure to engineered nano
Briefly, ALI exposure causes a deregulation of proinflammatory cyto particles (ENP) liquid suspensions, no substantial cytotoxicity was
kines (e.g. TNF-α, TGF-β, NF-κB). In addition, the viability of BEAS-2B observed, while aerosolized ENP clearly reduced cell survival and LDH
cells was over 98% within 21 days. >21 days later, the viability of the release.
14
Table 1
Comparative study of submerged and continuous (ALI) direct exposure.
Studies Biomarkers of exposure Submerged versus Air-liquid interface Reference
A549 Viability Assay: WST-1 Submerged > ALI: cell viability affected, HMOX-1 (Lenz et al., 2013)
Exposure: 0.7 and 2.5 μg/cm2 ZnO for 1 h Expression of pro-inflammatory gene: IL-8, IL-6, ALI > Submerged: IL-8, IL-6, and GM-CSF
and GM-CSF
NHBE and BEAS-2B: grown submerged in media Expression of pro-inflammatory gene: IL-8, IL-6 - both cell types undifferentiated: Submerged > (Ghio et al., 2013)
and differentiated at ALI Expression of MOX-1 and COX2 ALI: IL-8, IL-6, HOX1 and COX2 for
- NHBE cells differentiated at ALI over 3 to 21 days:
Submerged > ALI: IL-8, IL-6, MOX-1, and COX2
NHBE Expression of pro-inflammatory gene: IL-8, IL-6 ↘ CBF (Garcia-Arcos
- Exposure 36 puffs to 36 mg/mL nicotine e- ciliary beat frequency (CBF) No cytotoxicity et al., 2016)
cigarette vapors Cytotoxicity: LDH Exposure basolateral: ↗ IL-6 and IL-8
- Exposure to nicotine in ASL: 100 nM
- Exposure to nicotine in basolateral medium: 1
and 10 μM for 12 h and 5 days.
BEAS-2B Viability assay (cultivation at ALI microfluidics Viability: was above 98% within 21 days on this (Dong et al.,
Exposure: 5, 10 and 15 μg/cm2 to PM for 24 h system at 0, 3, 6, 9, 12, 15, 18, 21, and 24 days for system. 2019)
cell) ↗ TNF, IL6, and TGF-β
Expression of cytokine: HO-1, JNK, IL6, TNF, ↗ apoptosis (10 μg/cm2)
Csap3, NF-κB, FGF14, and FGFR ↗ HO-1, JNK, IL6, TNF, NF-κB, and FGF14
Apoptosis assay
A549 ROS production Submerged > ALI: cell arrest at G2/M phase (Tollstadius et al.,
Exposure: 4.09, 8.17, 16.35, 32.7, 65.4, 130.8, Cell cycle progression ALI > Submerged: ROS generation, Nrf2 nucleus 2019)
261.6, 523.2, 1046.4 μM to Carbendazim for 24 Expression of Nrf2 nucleus, Caspase-3 and expression, Caspase-3 and α-tubulin
h α-tubulin
LPS stimulated A549 cells expression of pro-inflammatory cytokines: IL-6, IL- Submerged > ALI: IL-6, IL-8 (Hu et al., 2020)
Exposure 24 h to curcumin at ALI (10–100 μM) 8 ALI = Submerged: cell viability and cytotoxicity
and submerged cell culture (1–20 μM). cell viability and cytotoxicity
A549 and THP-1 (co-culture) Cytotoxicity: LDH Submerged > ALI: TNFα at 22 μg/cm2 (Cappellini et al.,
Exposure to CeO2 NPs: Expression of pro-inflammatory cytokines: IL-1β, ALI > Submerged: at 5 μg/cm2 2020)
- submerged: 1, 5, 9,15, and 22 μg/cm2 for 24 h IL-6, TNFα, MCP-1 Submerged = ALI: IL-1β, IL-6, and MCP-1
- ALI: 0.5, 1, 2 and 5 μg/cm2 for 5–20 min
A549 Cytotoxicity: LDH Submerged = ALI: Cytotoxicity and DNA damage (Medina-Reyes
Exposure to 2, 10 and 30 μg/cm2 TiO2 DNA damage ↗ ROS production et al., 2020)
nanofibers and nanoparticles for 24, 48 and 72 ROS production just in submerged method
h
Human Small Airway Epithelial Cells (SAEC) Cytotoxicity: LDH No cytotoxicity (Gindele et al.,
Exposure to 9 puffs of whole cigarette smoke, 3 Transepithelial Electrical Resistance (TEER) deregulation of marker genes for basal and 2020)
times a week during 28 days RNA sequencing secretory cells
↘ TEER
A549 Expression of pro-inflammatory gene: IL-8 and ALI > Submerged: metabolic activity, IL-8 and (Lovén et al.,
Exposure: 1.0 μg/cm2 ZnO for 1 h MCP-1 MCP-1 2021)
Metabolic activity Submerged = ALI: no cytotoxicity
Cytotoxicity: LDH
A549 Cytotoxicity: LDH ALI > Submerged: cytotoxicity (Bessa et al.,
Exposure: Genotoxicity: strand breaks Submerged = ALI: Genotoxicity at high 2021)
- submerged: to engineered nanoparticles: concentration
5–150 μg/cm2 for 24 h
- ALI: ATO: 6 and 12 μg/cm2, CeO2 17 and 34
μg/cm2, ZrO2 46 and 91 μg/cm2 for 2 or 4 h
A549, THP-1 (mono- and co-cultures) Expression of pro-inflammatory gene: IL‑1α, IL-1β, Submerged > ALI: cytotoxicity, IL‑1α, IL-1β, IL-6, (Friesen et al.,
Exposure: 15 to 66 μg/cm2 Min-U-Sil5 particles IL-6, IL-8, CCL22 IL-8, CCL22, SOD2 in A549 monocultures 2022)
for 10 min Expression of oxidative stress gene: SOD
A549 and THP-1 (co-culture) Gene expression of pro-inflammatory cytokine Submerged > ALI: CYP1A1 (Kaur et al., 2022)
Exposure to flame-generated particles for 30 or TNFα and CYP1A1 ALI > Submerged: TNFα
60 min:
- submerged: 2 and 6 μg/cm2
- ALI: 2 and ~ 4 μg/cm2
Finally, Kaur et al. compared three exposure methods of cocultures of potential mechanisms of action for the toxicity of air pollutants,
A549 and differentiated THP-1 to flame-generated particles: submerged, particularly particle ones, have been disclosed through submerged
pseudo-ALI, and ALI exposure (Kaur et al., 2022). For all exposure exposure, which has been the most often used method. In addition,
methods an enhanced pro-inflammatory response (TNFα) and CYP1A1 submerged exposure is a simple, inexpensive and reproducible proced
gene expression was observed. ALI exposure induced a significantly ure. However, the exposure concentrations used, particularly for short-
greater TNFα response compared to submerged exposure. The sub term exposure, have often been much higher than the actual exposure
merged exposures exhibited greater but not statistically significant in doses (Upadhyay and Palmberg, 2018). In addition, as submerged cells
duction of CYP1A1 than other exposure methods. are rarely able to differentiate, cellular and molecular processes,
including cilia development, mucus and surfactant secretion, trans
3.5.2. Criteria to consider before exposing porter and protein expression, may be impacted. As a consequence, in
To guide the reader between the different exposure methods, Table 2 vitro lung cell models established under submerged conditions do not
summarizes the criteria to be consider when choosing the most appro faithfully mimic the cellular and physiological properties seen in vivo or
priate exposure mode. Fig. 5 recapitulates the advantages and limita display the physiological characteristics of the airways. The limitation of
tions of submerged and at ALI exposure methods. The majority of the physiological relevance of the human respiratory mucosa was
15
Table 2
Advantages and limitations of submerged and ALI exposure.
Exposure methods Advantages Limitations References
Submerged - Easy to use - May not accurately reflect the cellular and (Bessa et al., 2023; Lacroix et al.,
exposure - Fast physiological characteristics observed in vivo: 2018; Rothen-Rutishauser et al., 2008;
- Low cost - Cells are unable to differentiate Upadhyay and Palmberg, 2018)
- Reproducible - Ineffective cell-cell and cell-stimulant interaction.
- Effective for soluble compounds and particles - Cilia development, mucus and surfactant secretion,
transporter and protein expression may be impacted.
- Controlling the time and dose deposition in cells is a
challenge due to potential particle suspension or
interaction, which can affect the actual exposure dose:
- No direct contact between cell surface and the test
atmosphere
- Difficult to determine the real concentration for
gaseous compounds
- Particle properties change in solution and depend on
the composition of the cell culture medium
- Chronic exposure is difficult due to the limited lifetime
or to the unlimited proliferation of the cultured cells.
Exposure at the - Potential differenciation of the cells: Expensive (Barosova et al., 2020; Upadhyay and
Air-Liquid - Polarization, production of mucus or surfactant Generation of stable exposure atmosphere is difficult Palmberg, 2018)
Interface - Similarity to the in vivo environment Need of specific exposure devices
- Feasible approach to implement the “3 R principle”
replacement, reduction, and refinement of animal
usage in pulmonary toxicity studies
- Real gas exchange between the environment and the
cells
- No interaction of the airborne compound with the cell
culture medium: no changes in characteristics
(concentration, dissolution and agglomeration).
- Effective for both hydro- and lipophilic compounds
- Repeated and chronic exposures are possible thanks
to the high lifetime of some 3D models.
overcome by using more sophisticated models with cell cultures at the Upadhyay and Palmberg, 2018). Additionally, the real concentration of
ALI. These can enable cells to differentiate and sometimes form complex exposure needs to be monitored.
tissues.
The choice of exposure method also depends on the properties of the
3.6. Assessment of the exposure
toxicant to be tested. A water-soluble compound is perfectly suited to
submerged exposure, whereas a gaseous, volatile or particulate com
The dose, concentration, duration, and route of exposure to the
pound will entail a number of difficulties (Bessa et al., 2023; Lacroix
airborne compound are important factors to consider when character
et al., 2018; Rothen-Rutishauser et al., 2008; Upadhyay and Palmberg,
izing its toxicity. Exposure assessment can be done by measuring the
2018). For instance, it may be challenging to estimate the real dose of
concentration of the compound in the air or in biological samples, and
gaseous compound that interact with a submerged culture. In contrary,
by assessing the frequency and duration of exposure.
gas exchange between the environment and the cells is made possible
For the interpretation of observed biological effects, the dose of
during exposure at ALI continuously or using rotating bottles (Aufder
exposure must be determined. For gaseous compounds, exposure con
heide, 2005).
centrations are generally monitored by mass spectrometry, with or
When exposing cells to particulate compounds, attention must be
without chromatography. When particles are present in the exposure
paid to maintaining particle properties during exposure. This includes
atmosphere, the quantification of the dose of exposure becomes more
particle size, surface chemistry and the presence or absence of aggre
difficult. Accurate dosimetry is one of the most critical criteria. While
gates. Indeed, it is well known that the characteristics of individual NPs,
most studies provide accurate data on PM exposure concentration, i.e.
such as their agglomeration state, are modified in the presence of pro
mass of particles per volume air (g/m3) or - for in vitro submerged cell-
teins such as in cell culture media. Culture medium components can
based assays - mass of particles per volume cell culture medium (g/cm3),
form a medium-specific corona around the NPs. Controlling the actual
the critical role of dose delivered to the site of exposure has been widely
deposition time and dose in the cells is thus difficult due to the possi
overlooked. In hazard and risk assessment research, exposure is
bility of particle suspension, dissolution or interaction with both me
frequently regarded an acceptable dose measure. Unlike exposure con
dium and culture materials, which may change the actual dose of
centration, delivered dose is frequently difficult to detect, and real-time
exposure. Determining the precise dose of particles that are available for
dose monitoring instruments are not always available. The dosimetry
interaction with the cells is thus one of the main drawbacks of sub
considerations are often ignored in the literature. Instead of comparing
merged grown cells. Compared to the submerged cultures, cellular
the actual dose of particles that come into contact with the cells during
models cultured at the ALI conditions, where aerosol particles are
the exposure period, administered dose metrics, such as the mass con
delivered directly to the cell surface, are thought to provide a more
centration of suspended particles, are typically used. The determination
relevant and realistic exposure system. Furthermore, the effective po
of the effective dose delivered to cells in vitro will be made by the
larization and ability to produce surfactants in ALI-exposed or ALI-
adoption of dosimetric methodologies. A critical review has emphasized
grown cells increases the similarity to in vivo environments. That’s
the need of using adapted methodologies for in vitro dosimetry (Cohen
why exposure at the ALI might be the best option for determining the
et al., 2015). Briefly, ENM dispersion preparation is critical to generate
toxicity in respiratory model systems under realistic exposure condi
reproducible particle exposures, accurate characterization of delivered
tions. However, creating a stable exposure atmosphere is challenging,
to cell doses in vitro and limit interactions between the particles and the
which makes ALI exposure systems expensive (Barosova et al., 2020;
culture medium. Integrated in vitro dosimetry platform using numerical
16
Fig. 5. Methods of in vitro exposure to airborne compounds
models or microbalances may be used (Loret et al., 2016). The properties model that may be used from enaloscloud.novamechanics.com/riskgone
of ENM can also affect their delivery to the cells by sedimentation or .html. The Harvard DG model developed by Deloid et al. serves as the
diffusion. These properties include agglomerate diameter and effective foundation for this model. The Harvard DG models are PBFTPK (phys
density (Cohen et al., 2015). iologically based finite time pharmacokinetic) models used to analyze
Particle deposition in the lung in vivo is a complicated process that is oral pharmacokinetic data. These models are an effective tool for
heavily influenced by the test aerosol’s physicochemical characteristics analyzing oral pharmacokinetic data since they are founded on the
such as size, shape, density, and solvability. This results in particle physiologically sound principle of finite absorption time, which will
deposition rates that vary depending on the attributes evaluated, as well enhance knowledge of oral drug absorption phenomenon. By using
as the concerned lung area (Lacroix et al., 2018). A computer model of PBFTPK model to reanalyze oral data, it is possible to obtain input rate
solution particle kinetics (sedimentation, diffusion) and dosimetry for estimates that are unquestionably connected to the parameter(s) regu
non-interacting spherical particles and their agglomerates in monolayer lating absorption rate, solubility, and/or permeability. The development
cell culture systems has been developed. The In vitro Sedimentation, of models based on multiple drug oral administration, the creation of
Diffusion, and Dosimetry model (ISDD) for inhaled particles is an in vitro percentage absorbed vs. time diagrams, and the use of in vitro-in vivo
analogue to the Multipath Particle Deposition Model (MPPD). The correlations (IVIVC) with a view to the concept of Finite absorption time
model is based on fundamental principles–Stokes sedimentation and (FAT) are some additional applications of PBFTPK models that can be
Stokes-Einstein diffusion–and has been validated against experimentally envisaged. Other applications include the extension of modeling work to
determined rates of nano- and microparticle transport across a variety of population studies and the coupling of PBFTPK modes with pharmaco
particle sizes, densities, and agglomerates. ISDD gives an excellent dynamic models (Chryssafidis et al., 2022). For cells exposed in vitro at
approximation of the expected cellular dose as a function of particle size the ALI to particles, the dose can be monitored by a quartz crystal bal
and density, and permits reasonable estimations of the range of errors ance located in an insert or by analyzing the material obtained from the
induced by exposure metrics, as well as predictions of particle transport insert chemically and fluoroscopically or predicted by computational or
trends (Hinderliter et al., 2010). Consequently, a new in vitro dosimetry semi-empirical aerosol deposition models that take into account all
model has been developed, called ISD3 – the in vitro sedimentation, relevant aerosol properties as well as the experimental characteristics of
diffusion, dissolution, and dosimetry model. This model combines the each exposure system (Schmid and Cassee, 2017; Taterra et al., 2021).
effect of particle dissolution kinetics with effects of sedimentation and The Vitrocell® system can be fitted with a quartz crystal microbalance
diffusion, to compute the number of particles and ions delivered to cells. for (quasi-) real-time (nano)particle dosimetry (Ding et al., 2020;
The model accounts for simultaneous changes in both the number and Juarez-Facio et al., 2022). Submerged exposures, on the other hand, are
size of particles in the liquid media, by solving for the number density of quite simple to accomplish by simply adding a specified amount of PM to
particles as a function of size and spatial location, based on a population cell growth medium. However, this is misleading because a significant
balance formalism. ISD3 is modular, enabling adaptation by inclusion of amount of work must be expended on preparing stable particle sus
alternative boundary conditions, models of uptake, dissolution, or pensions and determining the fraction of the administered dose (dose
sedimentation of agglomerates (Thomas et al., 2018). ISD3 is a valuable added to the medium) that is finally delivered to the cells. However,
extension of ISDD to describe the influence of dissolution on the cellular validation of these dosimetry models is hindered by confusion over
dosimetry of soluble NPs such as silver (Thomas et al., 2018). Another whether cellular absorption of PM is required for toxicological reaction
model also published online is RiskGone. RiskGone is a dosimetric or whether contact between the cell membrane and PM is sufficient to
17
stipulate toxicological response (Schmid and Cassee, 2017). inflammation, DNA damage, and epigenetic changes.
The deposited mass can be assessed using several methods. In a study
of the toxicity of Ag NPs using an electric field, the cell-free inserts were - Oxidative stress corresponds to an imbalance between pro-oxidants
analyzed gravimetrically, whereas the cell-containing inserts were and antioxidants that favors pro-oxidants. This causes a build-up of
analyzed chemically with ICP-MS. The mass of Ag deposited in cell-free reactive oxygen species (ROS), which can harm biomolecules, such
membranes was often higher (about 2.5–3 fold) than the mass of Ag as DNA, proteins, and lipids (Gualtieri et al., 2017). Because free
deposited on cells (Latvala et al., 2016). This result was even more radicals have such a short half-life, quantifying them is difficult.
(about 15 fold) with Ni NPs (Latvala et al., 2017). These findings imply Recently, it has been done in bronchial cells exposed at the ALI to
that cellular deposition cannot be determined exclusively from deposi heated tobacco products by flow cytometry using CellROX green
tion investigations performed under cell-free conditions. In a subsequent reagent (Rahman et al., 2022). ROS induce damages such as lipid
study of the toxicity of CeO2 NPs, the team quantified the exposure doses peroxidation, protein oxidation, and DNA mutations. These damages
solely on the basis of the cells exposed (Cappellini et al., 2020). are more often measured than ROS (Czerska et al., 2015).
- Xenobiotic metabolizing enzymes, including cytochrome P450 (CYP)
4. Biomarkers of exposure/effect enzymes like CYP1A1, CYP1A2, and CYP2E1, play a crucial role in
biotransforming exogenous substances, including airborne toxins.
4.1. Global cytotoxicity Phase 1 enzymes like CYPs are often measured in cells exposed to ALI
(Al Zallouha et al., 2017; Méausoone et al., 2021).
To evaluate cell viability, growth, and death when cells are exposed - Inflammation is a complex immune response very often assessed
to airborne chemicals, multiple strategies are typically combined for a using ELISA to quantify cytokines released by cells exposed at the
comprehensive assessment (Table S2, supplementary information). ALI. Cytokines, including chemokines, interleukins, growth factors,
Various tests like dye release, dye absorption, and metabolic markers and interferons, play a key role in regulating the immune response.
indirectly assess cell viability by examining cell number, cell membrane Activation of receptors and transcription factors, such as TLR4 and
integrity, metabolic activity, lysosome activity, nuclear structure, and NF-κB, can trigger the release of pro-inflammatory cytokines (Bardet
total cellular proteins: et al., 2016; Gualtieri et al., 2018; Kastner et al., 2013; Sanchez-
Guzman et al., 2021).
- Cell Metabolism assays provide information on cell viability and - Exposure to airborne compounds can also lead to genotoxicity,
cytotoxicity by measuring ongoing biological processes. Assays like which refers to permanent DNA alterations, including chromosomal
AlamarBlue, MTT, XTT, and WST-1 measure mitochondrial succinate breaks. Various in vitro methods are employed to test mutagenicity or
dehydrogenase activity (Dusautoir et al., 2021; Kastner et al., 2013). genotoxicity, such as the micronucleus test, comet assay, and
Non-Lethal AlamarBlue is more and more preferred, allowing cell detection of histone H2AX phosphorylation (Gminski et al., 2010).
cultures to be recovered for further analysis (Méausoone et al.,
2019). In addition to these classical toxicity markers, the use of complex 3D
- Cytolysis/membrane leakage tests detect cell membrane damage biological models enables to assess the cellular phenotype and its per
through either enzyme leakage or staining with membrane- turbations in the event of exposure to airborne compounds. This in
impermeable dyes. Trypan blue, propidium iodide, and methylene cludes evaluating barrier properties through parameters like Trans-
blue are used to detect dead cells, while neutral red dye quantifies Epithelial Electrical Resistance (TEER) and membrane integrity using
absorption by living cells. LDH Assay measures the release out of the fluorescent molecules like lucifer yellow (LY)(Despréaux et al., 2023;
cells of a cytosolic enzyme, lactate dehydrogenase, indicating cell Kastner et al., 2013; Sanchez-Guzman et al., 2021). LY which does not
damage. It is non-destructive because it can be quantified in culture interact with cells, has been developed as a quantitative indicator of the
medium and apical surface layer (wash fluid). paracellular pathway of cell membrane permeabilization (Pires et al.,
- Cell proliferation and cell cycle assays track cell population expan 2021; Pourmirjafari Firoozabadi et al., 2015). Ciliary beat frequency
sion, daughter cell creation, and cell cycle states. Techniques include (CBF) is measured as the respiratory epithelium consists of ciliated cells.
DNA staining dyes, dye dilution assays, and nucleoside analog This non-destructive measurement is conducted using high-speed cam
incorporation. eras and has been employed in various studies to assess the impact of
- Cell death and apoptosis tests identify specific markers associated exposures on ciliary function (Baxter et al., 2015). Histological analyses
with different forms of cell death, such as annexin V binding, DNA are used to describe cell phenotypes before and after ALI exposure.
condensation, caspase activation, mitochondrial membrane poten Moreover, these analyses are typically destructive and have been
tial, cytochrome C release, and assays for necrosis, anoikis, and employed to assess structural properties, pharmacological research, and
autophagy (Dong et al., 2019; Tollstadius et al., 2019). the impact of exposure to pollutants. Markers like ZO-1, Occludin, and
E-cadherin are often studied in ALI cultured cells (Kreft et al., 2015).
It should be noted that the cells exposed at the ALI are grown on Finally, fluorescent staining can be used to detect the production of
inserts. So, it can be difficult to use a number of methods, particularly secretory components, such as mucins. The enzyme-linked lectin assay
flow cytometry and microscopy. Moreover, cytotoxicity tests should be (ELLA) is used to quantify these glycoproteins in the apical secretome of
rapid, simple, and amenable to automation for high-throughput ALI-exposed cells ().
screening. Finally, toxicants can sometimes interact with or degrade
the reagents used in the assays, which must be considered when 5. Conclusion and recomendations
designing experiments.
Because respiratory exposures have a significant impact on human
4.2. Mechanisms of action health and illness, there has been a lot of interest in building accurate
and predictive in vitro model systems for respiratory toxicology and
Researchers face challenges when studying the underlying mecha fundamental research for a comprehensive understanding of the bio
nisms of action in cells exposed at the AL due to the limited number of logical mechanisms of airborne compounds. To guide the reader in using
cells available for analysis. Scientists often focus on single toxicity an in vitro approach to characterize the toxicity of airborne compounds,
pathways or employ global approaches using omics techniques. the Table 3 summarizes the relevance of each biological model and
Different mechanisms of action are often described to characterize exposure method according to the literature analyzed in this review.
the toxicity of airborne chemicals, including oxidative stress, There are numerous lung cell culture models, ranging from 2D
18
culture primary cell models, cell lines up to complex models. Mono genotoxicology, which can require a lot of biological material. However,
culture of primary cells or immortal cell lines are easy to use, quite low lung physiology is not respected and cells are undifferentiated. Some
expensive. These models are frequently used to assess the acute toxicity interactions with the culture medium can modify the airborne com
of airborne compounds, i.e. after a single or repeated exposure over a pound either gaseous or particulate, and the real level of exposure is
short period of less than a week. Simple models can be used for ab unknown. Intermittent procedures enable the cells to be partly in the
sorption studies or to investigate underlying mechanisms of toxicity. atmospheric phase containing the pollutant. However, the medium
However, 2D models do not display all the differentiated and functional regularly covers the cells, which makes it impossible to maintain a
characteristics of the respiratory epithelium. To improve their rele differentiated state. In addition, the compound can interact with the
vance, monoculture models can be grown at the ALI instead of sub medium. Sophisticated methods of exposure at the ALI paired with
merged. When at the ALI, certain cells produce mucus or surfactant and complex and differentiated cell models can provide accurate data for a
form a barrier. Some primary cultures have cilia whose beating fre large range of airborne compounds (e.g. dry powders, liquid or gas
quency can be monitored. To get closer to the in vivo physiology, 3D aerosols, complex mixes, NPs, and fibers). Cells are indeed exposed
culture models have been developed such as cocultures and organotypic directly and in a dynamic way to the airborne compounds at a realistic
models (organoids, spheroids, and reconstituted tissue models). Com concentration. PBPK modeling can even calculate the amount of
plex models contain many cell types, making them a good representa chemicals present in lung tissue using real exposure concentrations as a
tion of airway physiology. The pulmonary architecture is maintained starting point, bringing us even closer to real exposure scenarios.
with tight junctions, the apical surface exposed to air and nourished by However, using ALI exposure devices is expensive and needs a lot of
the basal surface, and in some cases several cellular layers. The pul optimization. There are no established recommendations outlining how
monary phenotype is respected, with intercellular interactions, func they should be used (i.e. flow rate, exposure time, temperature and
tional barrier, cilia beating, secretome and mucociliary clearance relative humidity of the test atmosphere). Most research are indeed
function. Thanks to these characteristics, these organotypic models are results-oriented, with little explanation of the experimental setup, issues
often used to study pathophysiology and drug development, as well as to encountered, and any system adjustments required. Test atmospheres
study the repeated and long-term toxicity of substances until at least 3 need to be generated at a correct and stable concentration, and then
months. chemically characterized. According to the nature of the airborne
Exposure to airborne compounds should achieve a homogeneous and compound, producing test atmosphere at the correct concentration
uniform distribution of the airborne compounds across the cells. Various could be difficult. Furthermore, exposure at the ALI can induce physical
in vitro exposure techniques have been developed: submerged cell stress and dry out the cells. Humidification of test atmospheres (i.e. RH
exposure, intermittent procedures, and ALI methods. Submerged > 75%) is often necessary for exposures of more than one hour. For these
method is easy, fast and has been used for decades. It also enables us to reasons, cell cultures exposed to purified air and others maintained at
have large surface cultures, i.e. a large number of cells, which means we the ALI in the incubator are mandatory as controls.
can perform a wide set of analyses on the same sample, particularly in Moreover, understanding the toxic mechanisms of airborne
Table 3
Except for parameters where the + sign is specifically defined, the legend is as follows: ++: very appropriate, +: possible, =: depends on biological models, − : known as
impossible, empty box: information not available.
Biological model Exposure method
Cell line Cell line Primary Stem Organoids Spheroids Organotypic Submerged Exposure at
monoculture coculture cells cells tissue model exposure ALI
Objectives:
Characterization of the + + + + + ++ + ++
mechanisms of action
Dose-response relationship ++ + − − + ++ ++
Exposure:
Way of exposure (+: + ++ ++ + ++ ++ ++ + ++
relevance)
Duration (+: long) + + + ++ ++ ++ +
Concentration (+: close to − ++
reality)
Delivered dose (+: known) − ++
Acute ++ ++ ++ + + + + + +
Repeated (+: long term) + + + ++ − ++ + +
Markers of toxicity:
Cytotoxicity ++ + + + + + ++ +
Oxidative stress (ROS) + + + + ++ ++
Oxidative stress (damages) + + + + ++ ++
Inflammation cytokines + ++ + + ++ + ++
TEER + + + + ++ + ++
CBF − − + + + + ++ − ++
Mucins = = + + + ++ − ++
Cell proliferation + + + − − − + =
Genotoxicity markers + + + + + + + ++ +
General comments:
Model physiology (+: − + + ++ ++ ++ + ++
relevance)
Number of replicates ++ + − − + ++ +
Operating costs (+: − − + + + + ++ − +
expensive)
Repetability ++ + + + ++ ++
Replicability + + − − + +
Reproducibility − − − − + −
19
compounds requires targeted and non-targeted in vitro studies of toxicity Declaration of Competing Interest
biomarkers to identify the molecular initiating events and key events
(KEs). These biomarkers originate from databases such as the compar The authors declare the following financial interests/personal re
ative toxicogenomics database (CTD) and the Kyoto encyclopaedia of lationships which may be considered as potential competing interests:
genes and genomes (KEGG) or include the research of conventional Billet Sylvain reports financial support was provided by National
mechanisms of action (e.g., oxidative stress, inflammation, genotox Agency for Food Environmental and Occupational Health and Safety.
icity). One of the advantages of differentiated biological models such as
those presented in this review is the possibility of integrating phenotypic Data availability
markers.
That can enrich existing AOPs and provide additional information to No data was used for the research described in the article.
give a complete picture of the biological response to a chemical stressor.
Thus, to characterize the mechanisms of toxic action in a way that is Acknowledgments
most predictive of the effects observed in humans, exposure of organo
typic tissue models at the ALI is the most relevant. In contrast, when a The authors would like to thank the French National Research Pro
mechanism is determined, due to the high cost of these 3D models, gram for Environmental and Occupational Health of Anses (2020/01/
monocultures of cell lines can be used to precisely determine a dose- 208). Nour Jaber’s PhD is funded by the French Environment and En
response relationship, using a wider range of concentrations and a ergy Management Agency (ADEME) and the University of the Littoral
larger number of replicates. In this case, exposure to ALI is preferable for Opal Coast. The “Unité de Chimie Environnementale et Interactions sur
the majority of airborne compounds, if the production of a range of le Vivant” (UCEIV, UR4492) participates in the ECRIN and MOSOPS
concentrations is experimentally feasible and with a monitoring of the projects, which are financially supported by the Hauts-de-France Region
generated concentrations. However, submerged exposure is still possible Council, the French Ministry of Higher Education and Research, and the
for soluble and non-volatile compounds. European Regional Development Funds. The authors also thank Perrine
Finally, in the context of the search for greater reproducibility for the J. Martin for helpful and critical comments.
publication of reliable results, it is useful to compare the models and
methods proposed in terms of repeatability, replicability and repro
Appendix A. Supplementary data
ducibility. Repeatability means that many trials using the same mea
surement process, measuring system, operational circumstances by the
Supplementary data to this article can be found online at https://doi.
same team in one location give the same results. This should be the case
org/10.1016/j.tiv.2023.105718.
for monocultures of cell lines, whatever the exposure method, once it
has been optimized. This can be more difficult for coculture or primary
cells that may differ from one experiment to another. Replicability is References
when another team apply the same experimental setup. There are dif
Abbas, I., Garçon, G., Saint-Georges, F., Andre, V., Gosset, P., Billet, S., Le Goff, J.,
ferences between materials, both for culture (e.g. cell culture room, Verdin, A., Mulliez, P., Sichel, F., Shirali, P., 2013. Polycyclic aromatic hydrocarbons
culture supports) and exposure (e.g. NP solubilization, test atmosphere within airborne particulate matter (PM(2.5)) produced DNA bulky stable adducts in
generation). There do not appear to be any differences between expo a human lung cell coculture model. J. Appl. Toxicol. JAT 33, 109–119. https://doi.
org/10.1002/jat.1722.
sure methods in terms of their influence on the reliability of results. On Abdullah, L.H., Wolber, C., Kesimer, M., Sheehan, J.K., Davis, C.W., 2012. Studying
the contrary, the choice of biological model has an influence, and only mucin secretion from human bronchial epithelial cell primary cultures. Methods
cell lines should be used for reproducibility studies. Finally, repeat Mol. Biol. Clifton NJ 842, 259–277. https://doi.org/10.1007/978-1-61779-513-8_
16.
ability denotes the ability of multiple teams employing different
Al Zallouha, M., Landkocz, Y., Brunet, J., Cousin, R., Genty, E., Courcot, D., Siffert, S.,
experimental setups to produce the same results. This is not sound in Shirali, P., Billet, S., 2017. Usefulness of toxicological validation of VOCs catalytic
toxicology. Only submerged exposure can provide the same results in degradation by air-liquid interface exposure system. Environ. Res. 152, 328–335.
https://doi.org/10.1016/j.envres.2016.10.027.
some circumstances. However, many factors influence the final results,
Al-Rekabi, Z., Dondi, C., Faruqui, N., Siddiqui, N.S., Elowsson, L., Rissler, J., Kåredal, M.,
including culture media, the number of cell passages, test material Mudway, I., Larsson-Callerfelt, A.-K., Shaw, M., 2023. Uncovering the cytotoxic
preparation, air temperature, and relative humidity. These parameters effects of air pollution with multi-modal imaging of in vitro respiratory models.
need to be standardized. R. Soc. Open Sci. 10, 221426 https://doi.org/10.1098/rsos.221426.
Aufderheide, M., 2005. Direct exposure methods for testing native atmospheres. Exp.
Toxicol. Pathol. Off. J. Ges. Für Toxikol. Pathol. 57 Suppl 1, 213–226.
Author contributions Augustin, P., Billet, S., Crumeyrolle, S., Deboudt, K., Dieudonné, E., Flament, P.,
Fourmentin, M., Guilbaud, S., Hanoune, B., Landkocz, Y., Méausoone, C., Roy, S.,
Schmitt, F.G., Sentchev, A., Sokolov, A., 2020. Impact of sea breeze dynamics on
NJ contributed to the literature review, first draft, tables and illus atmospheric pollutants and their toxicity in industrial and urban coastal
trations. SB contributed to the design of the review, literature review environments. Remote Sens. 12, 648. https://doi.org/10.3390/rs12040648.
and writing of the manuscript. All authors have read, revised and agreed Baarsma, H.A., Van der Veen, C.H.T.J., Lobee, D., Mones, N., Oosterhout, E., Cattani-
Cavalieri, I., Schmidt, M., 2022. Epithelial 3D-spheroids as a tool to study air
to the manuscript. pollutant-induced lung pathology. SLAS Discov Adv. Life Sci. R D 27, 185–190.
https://doi.org/10.1016/j.slasd.2022.02.001.
Informed consent statement Bakand, S., Hayes, A., 2010. Troubleshooting methods for toxicity testing of airborne
chemicals in vitro. J. Pharmacol Toxicol. Methods, Troubleshoot. Meth. Pharmacol.
Toxicol. 61, 76–85. https://doi.org/10.1016/j.vascn.2010.01.010.
Not applicable. Bakand, S., Winder, C., Khalil, C., Hayes, A., 2005. Toxicity assessment of industrial
chemicals and airborne contaminants: transition from in vivo to in vitro test methods:
a review. Inhal. Toxicol. 17, 775–787. https://doi.org/10.1080/
Declaration of generative AI and AI-assisted technologies in the
08958370500225240.
writing process Ball, M., Hossain, M., Padalia, D., 2023. Anatomy, Airway, in: StatPearls. StatPearls
Publishing, Treasure Island (FL).
During the preparation of this work the authors used OpenAI Balogh Sivars, K., Sivars, U., Hornberg, E., Zhang, H., Brändén, L., Bonfante, R.,
Huang, S., Constant, S., Robinson, I., Betts, C.J., Åberg, P.M., 2018. A 3D human
ChatGPT in order to improve language and readability of the manu airway model enables prediction of respiratory toxicity of inhaled drugs in vitro.
script. After using this tool, the authors reviewed and edited the content Toxicol. Sci. 162, 301–308. https://doi.org/10.1093/toxsci/kfx255.
as needed and take full responsibility for the content of the publication. Bardet, G., Achard, S., Loret, T., Desauziers, V., Momas, I., Seta, N., 2014. A model of
human nasal epithelial cells adapted for direct and repeated exposure to airborne
pollutants. Toxicol. Lett. 229, 144–149. https://doi.org/10.1016/j.
toxlet.2014.05.023.
20
Bardet, G., Mignon, V., Momas, I., Achard, S., Seta, N., 2016. Human reconstituted nasal epithelium. PLoS One 16, e0248798. https://doi.org/10.1371/journal.
epithelium, a promising in vitro model to assess impacts of environmental complex pone.0248798.
mixtures. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 32, 55–62. https://doi.org/ Brunet, J., Genty, E., Landkocz, Y., Al Zallouha, M., Billet, S., Courcot, D., Siffert, S.,
10.1016/j.tiv.2015.11.019. Thomas, D., De Weireld, G., Cousin, R., 2015. Identification of by-products issued
Barosova, H., Maione, A.G., Septiadi, D., Sharma, M., Haeni, L., Balog, S., O’Connell, O., from the catalytic oxidation of toluene by chemical and biological methods. In:
Jackson, G.R., Brown, D., Clippinger, A.J., Hayden, P., Petri-Fink, A., Stone, V., Comptes Rendus Chim., International Symposium on Air & Water Pollution
Rothen-Rutishauser, B., 2020. Use of EpiAlveolar lung model to predict fibrotic Abatement Catalysis (AWPAC)Voume 1 – Catalytic Pollution Control for Stationary
potential of multiwalled carbon nanotubes. ACS Nano 14, 3941–3956. https://doi. and Mobile Sources, 18, pp. 1084–1093. https://doi.org/10.1016/j.
org/10.1021/acsnano.9b06860. crci.2015.09.001.
Barraud, C., Corbière, C., Pottier, I., Estace, E., Blanchard, K., Logie, C., Lagadu, S., Callaghan, P.J., Ferrick, B., Rybakovsky, E., Thomas, S., Mullin, J.M., 2020. Epithelial
Kéravec, V., Pottier, D., Dionnet, F., Morin, J.P., Préterre, D., André, V., Monteil, C., barrier function properties of the 16HBE14o- human bronchial epithelial cell culture
Sichel, F., 2017. Impact of after-treatment devices and biofuels on diesel exhausts model. Biosci. Rep. 40 https://doi.org/10.1042/BSR20201532.
genotoxicity in A549 cells exposed at air-liquid interface. Toxicol. Vitro Int. J. Publ. Campbell, L., Abulrob, A.-N.G., Kandalaft, L.E., Plummer, S., Hollins, A.J., Gibbs, A.,
Assoc. BIBRA 45, 426–433. https://doi.org/10.1016/j.tiv.2017.04.025. Gumbleton, M., 2003. Constitutive expression of P-glycoprotein in Normal lung
Baulig, A., Poirault, J.J., Ausset, P., Schins, R., Shi, T., Baralle, D., Dorlhene, P., alveolar epithelium and functionality in primary alveolar epithelial cultures.
Meyer, M., Lefevre, R., Baeza-Squiban, A., Marano, F., 2004. Physicochemical J. Pharmacol. Exp. Ther. 304, 441–452. https://doi.org/10.1124/jpet.102.042994.
characteristics and biological activities of seasonal atmospheric particulate matter Cao, X., Coyle, J.P., Xiong, R., Wang, Y., Heflich, R.H., Ren, B., Gwinn, W.M., Hayden, P.,
sampling in two locations of Paris. Environ. Sci Technol 38, 5985–5992. Rojanasakul, L., 2020. Invited review: human air-liquid-interface organotypic
Baxter, A., Thain, S., Banerjee, A., Haswell, L., Parmar, A., Phillips, G., Minet, E., 2015. airway tissue models derived from primary tracheobronchial epithelial
Targeted omics analyses, and metabolic enzyme activity assays demonstrate cells—overview and perspectives. In Vitro Cell. Dev. Biol. Anim. 1–29 https://doi.
maintenance of key mucociliary characteristics in long term cultures of reconstituted org/10.1007/s11626-020-00517-7.
human airway epithelia. Toxicol. in Vitro 29, 864–875. https://doi.org/10.1016/j. Cappellini, F., Di Bucchianico, S., Karri, V., Latvala, S., Malmlöf, M., Kippler, M.,
tiv.2015.03.004. Elihn, K., Hedberg, J., Odnevall Wallinder, I., Gerde, P., Karlsson, H.L., 2020. Dry
Bessa, M.J., Brandão, F., Fokkens, P.H.B., Leseman, D.L.A.C., Boere, A.J.F., Cassee, F.R., generation of CeO2 nanoparticles and deposition onto a co-culture of A549 and THP-
Salmatonidis, A., Viana, M., Vulpoi, A., Simon, S., Monfort, E., Teixeira, J.P., 1 cells in air-liquid Interface-dosimetry considerations and comparison to submerged
Fraga, S., 2021. In vitro toxicity of industrially relevant engineered nanoparticles in exposure. Nanomater. Basel Switz. 10, 618. https://doi.org/10.3390/
human alveolar epithelial cells: air–liquid interface versus submerged cultures. nano10040618.
Nanomaterials 11, 3225. https://doi.org/10.3390/nano11123225. Castell, J.V., Teresa Donato, M., Gómez-Lechón, M.J., 2005. Metabolism and
Bessa, M.J., Brandão, F., Rosário, F., Moreira, L., Reis, A.T., Valdiglesias, V., Laffon, B., bioactivation of toxicants in the lung. The in vitro cellular approach. Exp. Toxicol.
Fraga, S., Teixeira, J.P., 2023. Assessing the in vitro toxicity of airborne (nano) Pathol. 57 (Supplement 1), 189–204. https://doi.org/10.1016/j.etp.2005.05.008.
particles to the human respiratory system: from basic to advanced models. Cerimi, K., Jäckel, U., Meyer, V., Daher, U., Reinert, J., Klar, S., 2022. In vitro systems for
J. Toxicol. Environ. Health B Crit. Rev. 26, 67–96. https://doi.org/10.1080/ toxicity evaluation of microbial volatile organic compounds on humans: current
10937404.2023.2166638. status and trends. J. Fungi Basel Switz. 8, 75. https://doi.org/10.3390/jof8010075.
Bianchi, M., Sivarajan, R., Walles, T., Hackenberg, S., Steinke, M., 2021. Susceptibility of Cervena, T., Vrbova, K., Rossnerova, A., Topinka, J., Rossner, P., 2019. Short-term and
primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin long-term exposure of the MucilAirTM model to polycyclic aromatic hydrocarbons.
in two- and three-dimensional culture conditions. Innate Immun. 27, 89–98. https:// Altern. Lab. Anim 47, 9–18. https://doi.org/10.1177/0261192919841484.
doi.org/10.1177/1753425920979354. Cesarz, Z., Tamama, K., 2016. Spheroid culture of mesenchymal stem cells. Stem Cells
Billet, S., Garçon, G., Dagher, Z., Verdin, A., Ledoux, F., Cazier, F., Courcot, D., Int. 2016 https://doi.org/10.1155/2016/9176357.
Aboukais, A., Shirali, P., 2007. Ambient particulate matter (PM2.5): Chen, T.-L., Hsiao, T.-C., Chuang, H.-C., Ting, Y.-C., Wang, C.-H., 2023. A mobile
physicochemical characterization and metabolic activation of the organic fraction in platform for characterizing on-road tailpipe emissions and toxicity of ultrafine
human lung epithelial cells (A549). Environ. Res. 105, 212–223. https://doi.org/ particles under real driving conditions. Environ. Res. 216, 114523 https://doi.org/
10.1016/j.envres.2007.03.001. 10.1016/j.envres.2022.114523.
Billet, S., Paget, V., Garçon, G., Heutte, N., André, V., Shirali, P., Sichel, F., 2010. Chryssafidis, P., Tsekouras, A.A., Macheras, P., 2022. Re-writing oral pharmacokinetics
Benzene-induced mutational pattern in the tumour suppressor gene TP53 analysed using physiologically based finite time pharmacokinetic (PBFTPK) models. Pharm.
by use of a functional assay, the functional analysis of separated alleles in yeast, in Res. 39, 691–701. https://doi.org/10.1007/s11095-022-03230-0.
human lung cells. Arch. Toxicol. 84, 99–107. https://doi.org/10.1007/s00204-009- Clippinger, A.J., Allen, D., Behrsing, H., BéruBé, K.A., Bolger, M.B., Casey, W.,
0478-z. DeLorme, M., Gaça, M., Gehen, S.C., Glover, K., Hayden, P., Hinderliter, P.,
Billet, S., Landkocz, Y., Martin, P.J., Verdin, A., Ledoux, F., Lepers, C., André, V., Hotchkiss, J.A., Iskandar, A., Keyser, B., Luettich, K., Ma-Hock, L., Maione, A.G.,
Cazier, F., Sichel, F., Shirali, P., Gosset, P., Courcot, D., 2018. Chemical Makena, P., Melbourne, J., Milchak, L., Ng, S.P., Paini, A., Page, K., Patlewicz, G.,
characterization of fine and ultrafine PM, direct and indirect genotoxicity of PM and Prieto, P., Raabe, H., Reinke, E.N., Roper, C., Rose, J., Sharma, M., Spoo, W.,
their organic extracts on pulmonary cells. J. Environ. Sci. 71, 168–178. https://doi. Thorne, P.S., Wilson, D.M., Jarabek, A.M., 2018. Pathway-based predictive
org/10.1016/j.jes.2018.04.022. approaches for non-animal assessment of acute inhalation toxicity. Toxicol. in Vitro
Boei, J.J.W.A., Vermeulen, S., Klein, B., Hiemstra, P.S., Verhoosel, R.M., Jennen, D.G.J., 52, 131–145. https://doi.org/10.1016/j.tiv.2018.06.009.
Lahoz, A., Gmuender, H., Vrieling, H., 2017. Xenobiotic metabolism in differentiated Cochard, M., Ledoux, F., Landkocz, Y., 2020. Atmospheric fine particulate matter and
human bronchial epithelial cells. Arch. Toxicol. 91, 2093–2105. https://doi.org/ epithelial mesenchymal transition in pulmonary cells: state of the art and critical
10.1007/s00204-016-1868-7. review of the in vitro studies. J. Toxicol. Environ. Health B Crit. Rev. 23, 293–318.
Botham, J., Lewis, R.W., Travis, K.Z., Baze, A., Richert, L., Codrea, E., Semino https://doi.org/10.1080/10937404.2020.1816238.
Beninel, G., Garcin, J.-C., Strupp, C., 2023. Species differences and human relevance Cohen, J.M., DeLoid, G.M., Demokritou, P., 2015. A critical review of in vitro dosimetry
of the toxicity of 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors and a for engineered nanomaterials. Nanomed. 10, 3015–3032. https://doi.org/10.2217/
new approach method in vitro for investigation. Arch. Toxicol. 97, 991–999. https:// nnm.15.129.
doi.org/10.1007/s00204-023-03458-8. Corbière, V., Dirix, V., Norrenberg, S., Cappello, M., Remmelink, M., Mascart, F., 2011.
Boublil, L., Assémat, E., Borot, M.-C., Boland, S., Martinon, L., Sciare, J., Baeza- Phenotypic characteristics of human type II alveolar epithelial cells suitable for
Squiban, A., 2013. Development of a repeated exposure protocol of human bronchial antigen presentation to T lymphocytes. Respir. Res. 12, 15. https://doi.org/
epithelium in vitro to study the long-term effects of atmospheric particles. Toxicol. in 10.1186/1465-9921-12-15.
Vitro 27, 533–542. https://doi.org/10.1016/j.tiv.2012.11.008. Cozens, A.L., Yezzi, M.J., Kunzelmann, K., Ohrui, T., Chin, L., Eng, K., Finkbeiner, W.E.,
Bovard, D., Sandoz, A., Luettich, K., Frentzel, S., Iskandar, A., Marescotti, D., Trivedi, K., Widdicombe, J.H., Gruenert, D.C., 1994. CFTR expression and chloride secretion in
Guedj, E., Dutertre, Q., Peitsch, M.C., Hoeng, J., 2018. A lung/liver-on-a-chip polarized immortal human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol.
platform for acute and chronic toxicity studies. Lab Chip 18, 3814–3829. https://doi. 10, 38–47. https://doi.org/10.1165/ajrcmb.10.1.7507342.
org/10.1039/c8lc01029c. Czerska, M., Mikołajewska, K., Zieliński, M., Gromadzińska, J., Wąsowicz, W., 2015.
Bovard, D., Renggli, K., Marescotti, D., Sandoz, A., Majeed, S., Pinard, L., Ferreira, S., Today’s oxidative stress markers. Med. Pr. 66, 393–405. https://doi.org/10.13075/
Pak, C., Barbier, A., Beguin, A., Iskandar, A., Frentzel, S., Hoeng, J., Peitsch, M.C., mp.5893.00137.
2022. Impact of aerosols on liver xenobiotic metabolism: a comparison of two Davis, A.S., Chertow, D.S., Moyer, J.E., Suzich, J., Sandouk, A., Dorward, D.W.,
methods of exposure. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 79, 105277. https:// Logun, C., Shelhamer, J.H., Taubenberger, J.K., 2015. Validation of normal human
doi.org/10.1016/j.tiv.2021.105277. bronchial epithelial cells as a model for influenza a infections in human distal
Braakhuis, H.M., He, R., Vandebriel, R.J., Gremmer, E.R., Zwart, E., Vermeulen, J.P., Trachea. J. Histochem. Cytochem. 63, 312. https://doi.org/10.1369/
Fokkens, P., Boere, J., Gosens, I., Cassee, F.R., 2020. An air-liquid Interface bronchial 0022155415570968.
epithelial model for realistic, repeated inhalation exposure to airborne particles for de Bruijne, K., Ebersviller, S., Sexton, K.G., Lake, S., Leith, D., Goodman, R., Jetters, J.,
toxicity testing. JoVE J. Vis. Exp. e61210 https://doi.org/10.3791/61210. Walters, G.W., Doyle-Eisele, M., Woodside, R., Jeffries, H.E., Jaspers, I., 2009.
Brinchmann, B.C., Skuland, T., Rambøl, M.H., Szoke, K., Brinchmann, J.E., Gutleb, A.C., Design and testing of electrostatic aerosol in vitro exposure system (EAVES): an
Moschini, E., Kubátová, A., Kukowski, K., Le Ferrec, E., Lagadic-Gossmann, D., alternative exposure system for particles. Inhal. Toxicol. 21, 91–101. https://doi.
Schwarze, P.E., Låg, M., Refsnes, M., Øvrevik, J., Holme, J.A., 2018. Lipophilic org/10.1080/08958370802166035.
components of diesel exhaust particles induce pro-inflammatory responses in human Despréaux, P., Jeanton, C., Desaulle, D., Al Zallouha, M., Verdin, A., Momas, I.,
endothelial cells through AhR dependent pathway(s). Part. Fibre Toxicol. 15, 21. Achard, S., 2023. Innovative graph analysis method to assess gene expression
https://doi.org/10.1186/s12989-018-0257-1. modulation after fine particles exposures of 3D human airway epithelia. Environ.
Brookes, O., Boland, S., Lai Kuen, R., Miremont, D., Movassat, J., Baeza-Squiban, A., Res. 221, 115296 https://doi.org/10.1016/j.envres.2023.115296.
2021. Co-culture of type I and type II pneumocytes as a model of alveolar
21
Ding, Y., Weindl, P., Lenz, A.-G., Mayer, P., Krebs, T., Schmid, O., 2020. Quartz crystal Gillich, A., Zhang, F., Farmer, C.G., Travaglini, K.J., Tan, S.Y., Gu, M., Zhou, B.,
microbalances (QCM) are suitable for real-time dosimetry in nanotoxicological Feinstein, J.A., Krasnow, M.A., Metzger, R.J., 2020. Capillary cell-type specialization
studies using VITROCELL®cloud cell exposure systems. Part. Fibre Toxicol. 17, 44. in the alveolus. Nature 586, 785–789. https://doi.org/10.1038/s41586-020-2822-7.
https://doi.org/10.1186/s12989-020-00376-w. Gindele, J.A., Kiechle, T., Benediktus, K., Birk, G., Brendel, M., Heinemann, F.,
Doke, S.K., Dhawale, S.C., 2015. Alternatives to animal testing: a review. Saudi Pharm J. Wohnhaas, C.T., LeBlanc, M., Zhang, H., Strulovici-Barel, Y., Crystal, R.G.,
SPJ Off. Publ. Saudi Pharm. Soc. 23, 223–229. https://doi.org/10.1016/j. Thomas, M.J., Stierstorfer, B., Quast, K., Schymeinsky, J., 2020. Intermittent
jsps.2013.11.002. exposure to whole cigarette smoke alters the differentiation of primary small airway
Dong, H., Zheng, L., Duan, X., Zhao, W., Chen, J., Liu, S., Sui, G., 2019. Cytotoxicity epithelial cells in the air-liquid interface culture. Sci. Rep. 10, 6257. https://doi.org/
analysis of ambient fine particle in BEAS-2B cells on an air-liquid interface (ALI) 10.1038/s41598-020-63345-5.
microfluidics system. Sci. Total Environ. 677, 108–119. https://doi.org/10.1016/j. Giunchedi, P., Gavini, E., Bonferoni, M.C., 2020. Nose-to-brain delivery. Pharmaceutics
scitotenv.2019.04.203. 12, 138. https://doi.org/10.3390/pharmaceutics12020138.
Dubuisson, C., Dufour, A., Carrillo, S., Drouillet-Pinard, P., Havard, S., Volatier, J.-L., Gminski, R., Tang, T., Mersch-Sundermann, V., 2010. Cytotoxicity and genotoxicity in
2019. The third French individual and national food consumption (INCA3) survey human lung epithelial A549 cells caused by airborne volatile organic compounds
2014-2015: method, design and participation rate in the framework of a European emitted from pine wood and oriented strand boards. Toxicol. Lett. 196, 33–41.
harmonization process. Public Health Nutr. 22, 584–600. https://doi.org/10.1017/ https://doi.org/10.1016/j.toxlet.2010.03.015.
S1368980018002896. Gonzalez, R.F., Allen, L., Gonzales, L., Ballard, P.L., Dobbs, L.G., 2010. HTII-280, a
Dusautoir, R., Zarcone, G., Verriele, M., Garçon, G., Fronval, I., Beauval, N., Allorge, D., biomarker specific to the apical plasma membrane of human lung alveolar type II
Riffault, V., Locoge, N., Lo-Guidice, J.-M., Anthérieu, S., 2021. Comparison of the cells. J. Histochem. Cytochem. 58, 891–901. https://doi.org/10.1369/
chemical composition of aerosols from heated tobacco products, electronic cigarettes jhc.2010.956433.
and tobacco cigarettes and their toxic impacts on the human bronchial epithelial Gordon, S.B., Read, R.C., 2002. Macrophage defences against respiratory tract infections:
BEAS-2B cells. J. Hazard. Mater. 401, 123417 https://doi.org/10.1016/j. the immunology of childhood respiratory infections. Br. Med. Bull. 61, 45–61.
jhazmat.2020.123417. https://doi.org/10.1093/bmb/61.1.45.
Duval, K., Grover, H., Han, L.-H., Mou, Y., Pegoraro, A.F., Fredberg, J., Chen, Z., 2017. Gualtieri, M., Ledoux, F., Verdin, A., Billet, S., Martin, P.J., Courcot, D., 2017. Particulate
Modeling physiological events in 2D vs. 3D cell culture. Physiology 32, 266–277. matter Physico-chemical characterization and in vitro toxicological effects. In:
https://doi.org/10.1152/physiol.00036.2016. Airborne Particles: Origin, Emissions and Health Impacts, Environmental
Endes, C., Schmid, O., Kinnear, C., Mueller, S., Camarero-Espinosa, S., Vanhecke, D., Remediation Technologies, Regulations and Safety. Prashant Kumar.
Foster, E.J., Petri-Fink, A., Rothen-Rutishauser, B., Weder, C., Clift, M.J., 2014. An in Gualtieri, M., Grollino, M.G., Consales, C., Costabile, F., Manigrasso, M., Avino, P.,
vitro testing strategy towards mimicking the inhalation of high aspect ratio Aufderheide, M., Cordelli, E., Di Liberto, L., Petralia, E., Raschellà, G.,
nanoparticles. Part. Fibre Toxicol. 11, 40. https://doi.org/10.1186/s12989-014- Stracquadanio, M., Wiedensohler, A., Pacchierotti, F., Zanini, G., 2018. Is it the time
0040-x. to study air pollution effects under environmental conditions? A case study to
Endter, S., Francombe, D., Ehrhardt, C., Gumbleton, M., 2009. RT-PCR analysis of ABC, support the shift of in vitro toxicology from the bench to the field. Chemosphere 207,
SLC and SLCO drug transporters in human lung epithelial cell models. J. Pharm. 552–564. https://doi.org/10.1016/j.chemosphere.2018.05.130.
Pharmacol. 61, 583–591. https://doi.org/10.1211/jpp.61.05.0006. Guo, C., Buckley, A., Marczylo, T., Seiffert, J., Römer, I., Warren, J., Hodgson, A.,
Fasoli, E., 2021. Protein corona: Dr. Jekyll and Mr. Hyde of nanomedicine. Biotechnol. Chung, K.F., Gant, T.W., Smith, R., Leonard, M.O., 2018. The small airway
Appl. Biochem. 68, 1139–1152. https://doi.org/10.1002/bab.2035. epithelium as a target for the adverse pulmonary effects of silver nanoparticle
Fessart, D., Begueret, H., Delom, F., 2013. Three-dimensional culture model to inhalation. Nanotoxicology 12, 539–553. https://doi.org/10.1080/
distinguish normal from malignant human bronchial epithelial cells. Eur. Respir. J. 17435390.2018.1465140.
42, 1345–1356. https://doi.org/10.1183/09031936.00118812. Han, X., Na, T., Wu, T., Yuan, B.-Z., 2020. Human lung epithelial BEAS-2B cells exhibit
Fields, W.R., Leonard, R.M., Odom, P.S., Nordskog, B.K., Ogden, M.W., Doolittle, D.J., characteristics of mesenchymal stem cells. PLoS One 15, e0227174. https://doi.org/
2005. Gene expression in normal human bronchial epithelial (NHBE) cells following 10.1371/journal.pone.0227174.
in vitro exposure to cigarette smoke condensate. Toxicol. Sci. Off. J. Soc. Toxicol. 86, Harris, C., 2012. Overview of in vitro models in developmental toxicology. Methods Mol.
84–91. https://doi.org/10.1093/toxsci/kfi179. Biol. Clifton NJ 889, 105–113. https://doi.org/10.1007/978-1-61779-867-2_8.
Fogh, J., Fogh, J.M., Orfeo, T., 1977. One hundred and twenty-seven cultured human Hawley, B., McKenna, D., Marchese, A., Volckens, J., 2014. Time course of bronchial cell
tumor cell lines producing tumors in nude mice. J. Natl. Cancer Inst. 59, 221–226. inflammation following exposure to diesel particulate matter using a modified
https://doi.org/10.1093/jnci/59.1.221. EAVES. Toxicol. in Vitro 28, 829–837. https://doi.org/10.1016/j.tiv.2014.03.001.
Fougère, B., Landkocz, Y., Lepers, C., Martin, P.J., Armand, L., Grossin, N., Verdin, A., Hayden, P.J., Harbell, J.W., 2021. Special review series on 3D organotypic culture
Boulanger, E., Gosset, P., Sichel, F., Shirali, P., Billet, S., 2018. Influence of aging in models: introduction and historical perspective. In Vitro Cell. Dev. Biol. Anim. 57,
the modulation of epigenetic biomarkers of carcinogenesis after exposure to air 95–103. https://doi.org/10.1007/s11626-020-00500-2.
pollution. Exp. Gerontol. 110, 125–132. https://doi.org/10.1016/j. He, R.-W., Braakhuis, H.M., Vandebriel, R.J., Staal, Y.C.M., Gremmer, E.R., Fokkens, P.H.
exger.2018.05.018. B., Kemp, C., Vermeulen, J., Westerink, R.H.S., Cassee, F.R., 2021a. Optimization of
Friesen, A., Fritsch-Decker, S., Hufnagel, M., Mülhopt, S., Stapf, D., Weiss, C., an air-liquid interface in vitro cell co-culture model to estimate the hazard of aerosol
Hartwig, A., 2022. Gene expression profiling of mono- and co-culture models of the exposures. J. Aerosol Sci. 153, 105703 https://doi.org/10.1016/j.
respiratory tract exposed to crystalline quartz under submerged and air-liquid jaerosci.2020.105703.
interface conditions. Int. J. Mol. Sci. 23, 7773. https://doi.org/10.3390/ He, Z., Li, Y., Lian, Z., Liu, J., Xian, H., Jiang, R., Hu, Z., Fang, D., Hu, D., 2021b.
ijms23147773. Exosomal secretion may be a self-protective mechanism of its source cells under
Fujii, T., Hayashi, S., Hogg, J.C., Mukae, H., Suwa, T., Goto, Y., Vincent, R., van Eeden, S. environmental stress: a study on human bronchial epithelial cells treated with
F., 2002. Interaction of alveolar macrophages and airway epithelial cells following hydroquinone. J. Appl. Toxicol. JAT 41, 265–275. https://doi.org/10.1002/
exposure to particulate matter produces mediators that stimulate the bone marrow. jat.4043.
Am. J. Respir. Cell Mol. Biol. 27, 34–41. https://doi.org/10.1165/ajrcmb.27.1.4787. Hiemstra, P.S., Grootaers, G., van der Does, A.M., Krul, C.A.M., Kooter, I.M., 2018.
Gałęzowska, G., Chraniuk, M., Wolska, L., 2016. In vitro assays as a tool for Human lung epithelial cell cultures for analysis of inhaled toxicants: lessons learned
determination of VOCs toxic effect on respiratory system: a critical review. TrAC and future directions. Toxicol. in Vitro 47, 137–146. https://doi.org/10.1016/j.
Trends Anal. Chem. 77, 14–22. https://doi.org/10.1016/j.trac.2015.10.012. tiv.2017.11.005.
Garcia-Arcos, I., Geraghty, P., Baumlin, N., Campos, M., Dabo, A.J., Jundi, B., Hinderliter, P.M., Minard, K.R., Orr, G., Chrisler, W.B., Thrall, B.D., Pounds, J.G.,
Cummins, N., Eden, E., Grosche, A., Salathe, M., Foronjy, R., 2016. Chronic Teeguarden, J.G., 2010. ISDD: a computational model of particle sedimentation,
electronic cigarette exposure in mice induces features of COPD in a nicotine- diffusion and target cell dosimetry for in vitro toxicity studies. Part. Fibre Toxicol. 7,
dependent manner. Thorax 71, 1119–1129. https://doi.org/10.1136/thoraxjnl- 36. https://doi.org/10.1186/1743-8977-7-36.
2015-208039. Hou, J., 2015. Airway Epithelium and its Region- Specific Stem and Progenitor Cells.
García-Salvador, A., Katsumiti, A., Rojas, E., Aristimuño, C., Betanzos, M., Martínez- Hu, Y., Sheng, Y., Ji, X., Liu, P., Tang, L., Chen, Gang, Chen, Guiliang, 2020. Comparative
Moro, M., Moya, S.E., Goñi-de-Cerio, F., 2021. A complete in vitro toxicological anti-inflammatory effect of curcumin at air-liquid interface and submerged
assessment of the biological effects of cerium oxide nanoparticles: from acute conditions using lipopolysaccharide stimulated human lung epithelial A549 cells.
toxicity to multi-dose subchronic cytotoxicity study. Nanomater. Basel Switz. 11, Pulm. Pharmacol. Ther. 63, 101939 https://doi.org/10.1016/j.pupt.2020.101939.
1577. https://doi.org/10.3390/nano11061577. Huang, S., Wiszniewski, L., Constant, S., 2011. The use of in vitro 3D cell models in drug
George, I., Vranic, S., Boland, S., Courtois, A., Baeza-Squiban, A., 2015. Development of development for respiratory diseases. In: Drug Discovery and Development - Present
an in vitro model of human bronchial epithelial barrier to study nanoparticle and Future. IntechOpen. https://doi.org/10.5772/28132.
translocation. Toxicol. in Vitro 29, 51–58. https://doi.org/10.1016/j. Huang, S., Boda, B., Vernaz, J., Ferreira, E., Wiszniewski, L., Constant, S., 2017.
tiv.2014.08.003. Establishment and characterization of an in vitro human small airway model
Ghio, A.J., Dailey, L.A., Soukup, J.M., Stonehuerner, J., Richards, J.H., Devlin, R.B., (SmallAirTM). Eur. J. Pharm. Biopharm Off. J. Arbeitsgemeinschaft Pharm.
2013. Growth of human bronchial epithelial cells at an air-liquid interface alters the Verfahrenstechnik EV 118, 68–72. https://doi.org/10.1016/j.ejpb.2016.12.006.
response to particle exposure. Part. Fibre Toxicol. 10, 25. https://doi.org/10.1186/ Huh, D., Matthews, B.D., Mammoto, A., Montoya-Zavala, M., Hsin, H.Y., Ingber, D.E.,
1743-8977-10-25. 2010. Reconstituting organ-level lung functions on a Chip. Science 328, 1662–1668.
Giard, D.J., Aaronson, S.A., Todaro, G.J., Arnstein, P., Kersey, J.H., Dosik, H., Parks, W. https://doi.org/10.1126/science.1188302.
P., 1973. In vitro cultivation of human tumors: establishment of cell lines derived Hui, K.P.Y., Ching, R.H.H., Chan, S.K.H., Nicholls, J.M., Sachs, N., Clevers, H., Peiris, J.S.
from a series of solid tumors. J. Natl. Cancer Inst. 51, 1417–1423. https://doi.org/ M., Chan, M.C.W., 2018. Tropism, replication competence, and innate immune
10.1093/jnci/51.5.1417. responses of influenza virus: an analysis of human airway organoids and ex-vivo
bronchus cultures. Lancet Respir. Med. 6, 846–854. https://doi.org/10.1016/S2213-
2600(18)30236-4.
22
Hukkanen, J., Lassila, A., Paivarinta, K., Valanne, S., Sarpo, S., Hakkola, J., Pelkonen, O., conditions and its use as an optimized in vitro model to investigate bronchial
Raunio, H., 2000. Induction and regulation of xenobiotic-metabolizing cytochrome epithelial function. Eur. J. Pharm. Sci. 69, 1–9. https://doi.org/10.1016/j.
P450s in the human A549 lung adenocarcinoma cell line. Am. J. Respir. Cell Mol. ejps.2014.12.017.
Biol. 22, 360–366. Kuehn, A., Kletting, S., de Souza Carvalho-Wodarz, C., Repnik, U., Griffiths, G.,
Iskandar, A.R., Xiang, Y., Frentzel, S., Talikka, M., Leroy, P., Kuehn, D., Guedj, E., Fischer, U., Meese, E., Huwer, H., Wirth, D., May, T., Schneider-Daum, N., Lehr, C.-
Martin, F., Mathis, C., Ivanov, N.V., Peitsch, M.C., Hoeng, J., 2015. Impact M., 2016. Human alveolar epithelial cells expressing tight junctions to model the air-
assessment of cigarette smoke exposure on organotypic bronchial epithelial tissue blood barrier. ALTEX 33, 251–260. https://doi.org/10.14573/altex.1511131.
cultures: a comparison of mono-culture and coculture model containing fibroblasts. Lacroix, G., Koch, W., Ritter, D., Gutleb, A.C., Larsen, S.T., Loret, T., Zanetti, F.,
Toxicol. Sci. 147, 207–221. https://doi.org/10.1093/toxsci/kfv122. Constant, S., Chortarea, S., Rothen-Rutishauser, B., Hiemstra, P.S., Frejafon, E.,
Jacob, A.-M., Gaver, D.P., 2012. Atelectrauma disrupts pulmonary epithelial barrier Hubert, P., Gribaldo, L., Kearns, P., Aublant, J.-M., Diabaté, S., Weiss, C., de
integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4. Groot, A., Kooter, I., 2018. Air–liquid interface in vitro models for respiratory
J. Appl. Physiol. Bethesda Md 1985 (113), 1377–1387. https://doi.org/10.1152/ toxicology research: consensus workshop and recommendations. Appl. Vitro Toxicol.
japplphysiol.01432.2011. 4, 91–106. https://doi.org/10.1089/aivt.2017.0034.
Jensen, C., Teng, Y., 2020. Is it time to start transitioning from 2D to 3D cell culture? Latvala, S., Hedberg, J., Möller, L., Odnevall Wallinder, I., Karlsson, H.L., Elihn, K., 2016.
Front. Mol. Biosci. 7 https://doi.org/10.3389/fmolb.2020.00033. Optimization of an air–liquid interface exposure system for assessing toxicity of
Ji, J., Hedelin, A., Malmlöf, M., Kessler, V., Seisenbaeva, G., Gerde, P., Palmberg, L., airborne nanoparticles. J. Appl. Toxicol. 36, 1294–1301. https://doi.org/10.1002/
2017. Development of combining of human bronchial mucosa models with jat.3304.
XposeALI® for exposure of air pollution nanoparticles. PLoS One 12, e0170428. Latvala, S., Vare, D., Karlsson, H.L., Elihn, K., 2017. In vitro genotoxicity of airborne Ni-
https://doi.org/10.1371/journal.pone.0170428. NP in air-liquid interface. J. Appl. Toxicol. JAT 37, 1420–1427. https://doi.org/
Ji, J., Upadhyay, S., Xiong, X., Malmlöf, M., Sandström, T., Gerde, P., Palmberg, L., 2018. 10.1002/jat.3510.
Multi-cellular human bronchial models exposed to diesel exhaust particles: Leibrock, L., Wagener, S., Singh, A.V., Laux, P., Luch, A., 2019. Nanoparticle induced
assessment of inflammation, oxidative stress and macrophage polarization. Part. barrier function assessment at liquid-liquid and air-liquid interface in novel human
Fibre Toxicol. 15, 19. https://doi.org/10.1186/s12989-018-0256-2. lung epithelia cell lines. Toxicol. Res. 8, 1016–1027. https://doi.org/10.1039/
Ji, J., Ganguly, K., Mihai, X., Sun, J., Malmlöf, M., Gerde, P., Upadhyay, S., Palmberg, L., c9tx00179d.
2019. Exposure of normal and chronic bronchitis-like mucosa models to aerosolized Lenz, A.G., Karg, E., Lentner, B., Dittrich, V., Brandenberger, C., Rothen-Rutishauser, B.,
carbon nanoparticles: comparison of pro-inflammatory oxidative stress and tissue Schulz, H., Ferron, G.A., Schmid, O., 2009. A dose-controlled system for air-liquid
injury/repair responses. Nanotoxicology 13, 1362–1379. https://doi.org/10.1080/ interface cell exposure and application to zinc oxide nanoparticles. Part. Fibre
17435390.2019.1655600. Toxicol. 6, 32. https://doi.org/10.1186/1743-8977-6-32.
Jiang, D., Schaefer, N., Chu, H.W., 2018. Air-liquid Interface culture of human and Lenz, A.-G., Karg, E., Brendel, E., Hinze-Heyn, H., Maier, K.L., Eickelberg, O., Stoeger, T.,
mouse airway epithelial cells. Methods Mol. Biol. Clifton NJ 1809, 91–109. https:// Schmid, O., 2013. Inflammatory and oxidative stress responses of an alveolar
doi.org/10.1007/978-1-4939-8570-8_8. epithelial cell line to airborne zinc oxide nanoparticles at the air-liquid interface: a
Jin, Y., Feng, M., Ma, W., Wei, Y., Qi, G., Luo, J., Xu, L., Li, X., Li, C., Wang, Y., Li, D., comparison with conventional, submerged cell-culture conditions. Biomed. Res. Int.
Chen, J., Zhao, Y., Hou, Y., Zhao, Q., Jiang, L., Xie, M., Zheng, Y., Yu, D., 2021. 2013, 652632 https://doi.org/10.1155/2013/652632.
A toxicity pathway-oriented approach to develop adverse outcome pathway: AHR Leroux, M.M., Doumandji, Z., Chezeau, L., Hocquel, R., Ferrari, L., Joubert, O., Rihn, P.,
activation as a case study. Environ. Pollut. Barking Essex 1987 (268), 115733. Rihn, B.H., 2020. Validation of an air/liquid interface device for TiO2 nanoparticle
https://doi.org/10.1016/j.envpol.2020.115733. toxicity assessment on NR8383 cells: preliminary results. Cell. Mol. Biol. Noisy–Gd.
Johne, S., van der Toorn, M., Iskandar, A.R., Majeed, S., Torres, L.O., Hoeng, J., Fr. 66, 112–116.
Peitsch, M.C., 2023. An in vitro evaluation of e-vapor products: the contributions of Leroux, M.M., Hocquel, R., Bourge, K., Kokot, B., Kokot, H., Koklič, T., Štrancar, J.,
chemical adulteration, concentration, and device power. Food Chem. Toxicol. Int. J. Ding, Y., Kumar, P., Schmid, O., Rihn, B.H., Ferrari, L., Joubert, O., 2022. Aerosol-
Publ. Br. Ind. Biol. Res. Assoc. 175, 113708 https://doi.org/10.1016/j. cell exposure system applied to semi-adherent cells for Aerosolization of lung
fct.2023.113708. surfactant and nanoparticles followed by high quality RNA extraction. Nanomater.
Juarez-Facio, A.T., Castilla, C., Corbière, C., Lavanant, H., Afonso, C., Morin, C., Merlet- Basel Switz. 12, 1362. https://doi.org/10.3390/nano12081362.
Machour, N., Chevalier, L., Vaugeois, J.-M., Yon, J., Monteil, C., 2022. Development Lin, C., Zheng, X., Lin, S., Zhang, Y., Wu, J., Li, Y., 2022. Mechanotransduction regulates
of a standardized in vitro approach to evaluate microphysical, chemical, and the interplays between alveolar epithelial and vascular endothelial cells in lung.
toxicological properties of combustion-derived fine and ultrafine particles. Front. Physiol. 13.
J. Environ. Sci. (China) 113, 104–117. https://doi.org/10.1016/j.jes.2021.06.001. Lindbom, J., Gustafsson, M., Blomqvist, G., Dahl, A., Gudmundsson, A., Swietlicki, E.,
Juárez-Facio, A.T., Rogez-Florent, T., Méausoone, C., Castilla, C., Mignot, M., Devouge- Ljungman, A.G., 2006. Exposure to wear particles generated from studded tires and
Boyer, C., Lavanant, H., Afonso, C., Morin, C., Merlet-Machour, N., Chevalier, L., pavement induces inflammatory cytokine release from human macrophages. Chem.
Ouf, F.-X., Corbière, C., Yon, J., Vaugeois, J.-M., Monteil, C., 2022. Ultrafine Res. Toxicol. 19, 521–530. https://doi.org/10.1021/tx0503101.
particles issued from gasoline-fuels and biofuel surrogates combustion: a Liu, F.F., Peng, C., Escher, B.I., Fantino, E., Giles, C., Were, S., Duffy, L., Ng, J.C., 2013.
comparative study of the physicochemical and in vitro toxicological effects. Toxics Hanging drop: an in vitro air toxic exposure model using human lung cells in 2D and
11, 21. https://doi.org/10.3390/toxics11010021. 3D structures. J. Hazard. Mater. 261, 701–710. https://doi.org/10.1016/j.
Kastner, P.E., Le Calvé, S., Zheng, W., Casset, A., Pons, F., 2013. A dynamic system for jhazmat.2013.01.027.
single and repeated exposure of airway epithelial cells to gaseous pollutants. Toxicol. Liu, Z., Anderson, J.D., Deng, L., Mackay, S., Bailey, J., Kersh, L., Rowe, S.M.,
Vitro Int. J. Publ. Assoc. BIBRA 27, 632–640. https://doi.org/10.1016/j. Guimbellot, J.S., 2020. Human nasal epithelial organoids for therapeutic
tiv.2012.11.011. development in cystic fibrosis. Genes 11. https://doi.org/10.3390/genes11060603.
Kaur, K., Mohammadpour, R., Sturrock, A., Ghandehari, H., Reilly, C., Paine, R., Kelly, K. Liu, N., Bu, Z., Liu, W., Kan, H., Zhao, Z., Deng, F., Huang, C., Zhao, B., Zeng, X., Sun, Y.,
E., 2022. Comparison of biological responses between submerged, pseudo-air-liquid Qian, H., Mo, J., Sun, C., Guo, J., Zheng, X., Weschler, L.B., Zhang, Y., 2022. Health
interface, and air-liquid interface exposure of A549 and differentiated THP-1 co- effects of exposure to indoor volatile organic compounds from 1980 to 2017: a
cultures to combustion-derived particles. J. Environ. Sci. Health Part A Tox Hazard. systematic review and meta-analysis. Indoor Air 32, e13038. https://doi.org/
Subst. Environ. Eng. 57, 540–551. https://doi.org/10.1080/ 10.1111/ina.13038.
10934529.2022.2083429. Loomis, D., Grosse, Y., Lauby-Secretan, B., Ghissassi, F.E., Bouvard, V., Benbrahim-
Kia’i, N., Bajaj, T., 2023. Histology, respiratory epithelium. In: StatPearls. StatPearls Tallaa, L., Guha, N., Baan, R., Mattock, H., Straif, K., 2013. The carcinogenicity of
Publishing, Treasure Island (FL). outdoor air pollution. Lancet Oncol. 14, 1262–1263. https://doi.org/10.1016/
Kim, N., Han, D., Wang, I.J., Han, D.H., Suh, M.-W., Lee, J.H., Oh, S.-H., Park, M.K., S1470-2045(13)70487-X.
2022a. Altered secretome by diesel exhaust particles and lipopolysaccharide in Loret, T., Peyret, E., Dubreuil, M., Aguerre-Chariol, O., Bressot, C., le Bihan, O.,
primary human nasal epithelium. J. Allergy Clin. Immunol. 149, 2126–2138. Amodeo, T., Trouiller, B., Braun, A., Egles, C., Lacroix, G., 2016. Air-liquid interface
https://doi.org/10.1016/j.jaci.2021.12.793. exposure to aerosols of poorly soluble nanomaterials induces different biological
Kim, J.W., Jeong, M.H., Kim, G.E., Han, Y.B., Park, Y.J., Chung, K.H., Kim, H.R., 2022b. activation levels compared to exposure to suspensions. Part. Fibre Toxicol. 13, 58.
Comparison of 3D airway models for the assessment of fibrogenic chemicals. Toxicol. https://doi.org/10.1186/s12989-016-0171-3.
Lett. 356, 100–109. https://doi.org/10.1016/j.toxlet.2021.12.007. Lovén, K., Dobric, J., Bölükbas, D.A., Kåredal, M., Tas, S., Rissler, J., Wagner, D.E.,
Kiris, E., 2022. Human-induced pluripotent stem cell-based models for studying sex- Isaxon, C., 2021. Toxicological effects of zinc oxide nanoparticle exposure: an in
specific differences in neurodegenerative diseases. Adv. Exp. Med. Biol. 1387, 57–88. vitro comparison between dry aerosol air-liquid interface and submerged exposure
https://doi.org/10.1007/5584_2021_683. systems. Nanotoxicology 15, 494–510. https://doi.org/10.1080/
Klaassen, C.D., Rozman, K., 1991. Absorption, distribution, and excretion of toxicants. In: 17435390.2021.1884301.
Casarett and Doull’s Toxicology: The Basic Science of Poisons, pp. 50–87. Lu, S., Kolls, J.K., 2023. Multi-omic comparisons between CFBE41o- cells stably
Kletting, S., Barthold, S., Repnik, U., Griffiths, G., Loretz, B., Schneider-Daum, N., de expressing wild-type CFTR and F508del-mutant CFTR. J. Cyst. Fibros. 22, 146–155.
Souza Carvalho-Wodarz, C., Lehr, C.-M., 2018. Co-culture of human alveolar https://doi.org/10.1016/j.jcf.2022.06.010.
epithelial (hAELVi) and macrophage (THP-1) cell lines. ALTEX 35, 211–222. https:// Luo, Y.-S., Chen, Z., Hsieh, N.-H., Lin, T.-E., 2022. Chemical and biological assessments
doi.org/10.14573/altex.1607191. of environmental mixtures: a review of current trends, advances, and future
Kouassi, K.S., Billet, S., Garçon, G., Verdin, A., Diouf, A., Cazier, F., Djaman, J., perspectives. J. Hazard. Mater. 432, 128658 https://doi.org/10.1016/j.
Courcot, D., Shirali, P., 2010. Oxidative damage induced in A549 cells by physically jhazmat.2022.128658.
and chemically characterized air particulate matter (PM2.5) collected in Abidjan. Luyts, K., Napierska, D., Dinsdale, D., Klein, S.G., Serchi, T., Hoet, P.H.M., 2015.
Côte d’Ivoire. J. Appl. Toxicol. 30, 310–320. https://doi.org/10.1002/jat.1496. A coculture model of the lung–blood barrier: the role of activated phagocytic cells.
Kreft, M.E., Jerman, U.D., Lasič, E., Hevir-Kene, N., Rižner, T.L., Peternel, L., Kristan, K., Toxicol. in Vitro 29, 234–241. https://doi.org/10.1016/j.tiv.2014.10.024.
2015. The characterization of the human cell line Calu-3 under different culture
23
Malyla, V., Paudel, K.R., De Rubis, G., Hansbro, N.G., Hansbro, P.M., Dua, K., 2023. Paget, V., Lechevrel, M., André, V., Goff, J.L., Pottier, D., Billet, S., Garçon, G., Shirali, P.,
Cigarette smoking induces lung cancer tumorigenesis via upregulation of the WNT/ Sichel, F., 2012. Benzo[a]pyrene, aflatoxine B₁ and acetaldehyde mutational
β-catenin signaling pathway. Life Sci. 326, 121787 https://doi.org/10.1016/j. patterns in TP53 gene using a functional assay: relevance to human cancer aetiology.
lfs.2023.121787. PLoS One 7, e30921. https://doi.org/10.1371/journal.pone.0030921.
Marthin, J.K., Stevens, E.M., Larsen, L.A., Christensen, S.T., Nielsen, K.G., 2017. Patient- Paschini, M., Kim, C.F., 2019. An airway organoid is forever. EMBO J. 38, e101526
specific three-dimensional explant spheroids derived from human nasal airway https://doi.org/10.15252/embj.2019101526.
epithelium: a simple methodological approach for ex vivo studies of primary ciliary Payne, J.P., Kemp, S.J., Dewar, A., Goldstraw, P., Kendall, M., Chen, L.C., Tetley, T.D.,
dyskinesia. Cilia 6. https://doi.org/10.1186/s13630-017-0049-5. 2004. Effects of airborne world trade center dust on cytokine release by primary
Marx, U., Sandig, V. (Eds.), 2006. Drug Testing In Vitro: Breakthroughs and Trends in human lung cells in vitro. J. Occup. Environ. Med. 46, 420–427. https://doi.org/
Cell Culture Technology, 1st ed. Wiley. https://doi.org/10.1002/9783527609611. 10.1097/01.jom.0000126021.25149.64.
McDougall, C.M., Blaylock, M.G., Douglas, J.G., Brooker, R.J., Helms, P.J., Walsh, G.M., Pearce, K., Gray, N., Gaur, P., Jeon, J., Suarez, A., Shannahan, J., Pappas, R.S.,
2008. Nasal epithelial cells as surrogates for bronchial epithelial cells in airway Wright, C., 2020. Toxicological analysis of aerosols derived from three electronic
inflammation studies. Am. J. Respir. Cell Mol. Biol. 39, 560–568. https://doi.org/ nicotine delivery systems using normal human bronchial epithelial cells. Toxicol.
10.1165/rcmb.2007-0325OC. Vitro Int. J. Publ. Assoc. BIBRA 69, 104997. https://doi.org/10.1016/j.
McMillan, A., 2020. Lung-on-a-Chip: History, Origins and Development. Elveflow. tiv.2020.104997.
Méausoone, C., El Khawaja, R., Tremolet, G., Siffert, S., Cousin, R., Cazier, F., Billet, S., Persoz, C., Achard, S., Leleu, C., Momas, I., Seta, N., 2010. An in vitro model to evaluate
Courcot, D., Landkocz, Y., 2019. In vitro toxicological evaluation of emissions from the inflammatory response after gaseous formaldehyde exposure of lung epithelial
catalytic oxidation removal of industrial VOCs by air/liquid interface (ALI) exposure cells. Toxicol. Lett. 195, 99–105. https://doi.org/10.1016/j.toxlet.2010.03.003.
system in repeated mode. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 58, 110–117. Peters-Hall, J.R., Coquelin, M.L., Torres, M.J., LaRanger, R., Alabi, B.R., Sho, S., Calva-
https://doi.org/10.1016/j.tiv.2019.03.030. Moreno, J.F., Thomas, P.J., Shay, J.W., 2018. Long-term culture and cloning of
Méausoone, C., Landkocz, Y., Cazier, F., Seigneur, M., Courcot, D., Billet, S., 2021. primary human bronchial basal cells that maintain multipotent differentiation
Toxicological responses of BEAS-2B cells to repeated exposures to benzene, toluene, capacity and CFTR channel function. Am. J. Phys. Lung Cell. Mol. Phys. 315,
m-xylene, and mesitylene using air-liquid interface method. J. Appl. Toxicol. JAT 41, L313–L327. https://doi.org/10.1152/ajplung.00355.2017.
1262–1274. https://doi.org/10.1002/jat.4113. Pezzulo, A.A., Starner, T.D., Scheetz, T.E., Traver, G.L., Tilley, A.E., Harvey, B.-G.,
Medina-Reyes, E.I., Delgado-Buenrostro, N.L., Leseman, D.L., Déciga-Alcaraz, A., He, R., Crystal, R.G., McCray, P.B., Zabner, J., 2011. The air-liquid interface and use of
Gremmer, E.R., Fokkens, P.H.B., Flores-Flores, J.O., Cassee, F.R., Chirino, Y.I., 2020. primary cell cultures are important to recapitulate the transcriptional profile of in
Differences in cytotoxicity of lung epithelial cells exposed to titanium dioxide vivo airway epithelia. Am. J. Phys. Lung Cell. Mol. Phys. 300, L25–L31. https://doi.
nanofibers and nanoparticles: comparison of air-liquid interface and submerged cell org/10.1152/ajplung.00256.2010.
cultures. Toxicol. in Vitro 65, 104798. https://doi.org/10.1016/j.tiv.2020.104798. Pires, C.L., Praça, C., Martins, P.A.T., Batista de Carvalho, A.L.M., Ferreira, L.,
Meindl, C., Absenger-Novak, M., Jeitler, R., Roblegg, E., Fröhlich, E., 2023. Assessment Marques, M.P.M., Moreno, M.J., 2021. Re-use of Caco-2 monolayers in permeability
of carbon nanotubes on barrier function, ciliary beating frequency and cytokine assays—validation regarding cell monolayer integrity. Pharmaceutics 13, 1563.
release in in vitro models of the respiratory tract. Nanomater. Basel Switz. 13, 682. https://doi.org/10.3390/pharmaceutics13101563.
https://doi.org/10.3390/nano13040682. Plopper, C., Harkema, J., 2005. The respiratory system and its use in research. In: The
Meldrum, K., Evans, S.J., Vogel, U., Tran, L., Doak, S.H., Clift, M.J.D., 2022. The Laboratory Primate, pp. 503–526. https://doi.org/10.1016/B978-012080261-6/
influence of exposure approaches to in vitro lung epithelial barrier models to assess 50030-1.
engineered nanomaterial hazard. Nanotoxicology 16, 114–134. https://doi.org/ Pourmirjafari Firoozabadi, T., Shankayi, Z., Izadi, A., Pourmirjafari Firoozabadi, S.M.,
10.1080/17435390.2022.2051627. 2015. Can Lucifer yellow indicate correct permeability of biological cell membrane
Mercier, C., Jacqueroux, E., He, Z., Hodin, S., Constant, S., Perek, N., Boudard, D., under an electric and magnetic field? Cell. J. Yakhteh 16, 560–563.
Delavenne, X., 2019. Pharmacological characterization of the 3D MucilAirTM nasal Proud, D., Leigh, R., 2011. Epithelial cells and airway diseases. Immunol. Rev. 242,
model. Eur. J. Pharm. Biopharm. 139, 186–196. https://doi.org/10.1016/j. 186–204. https://doi.org/10.1111/j.1600-065X.2011.01033.x.
ejpb.2019.04.002. Pruniéras, M., Régnier, M., Woodley, D., 1983. Methods for cultivation of keratinocytes
Michalopoulos, G., Pitot, H.C., 1975. Primary culture of parenchymal liver cells on with an air-liquid interface. J. Invest. Dermatol. 81, 28s–33s. https://doi.org/
collagen membranes: morphological and biochemical observations. Exp. Cell Res. 10.1111/1523-1747.ep12540324.
94, 70–78. https://doi.org/10.1016/0014-4827(75)90532-7. Radiom, M., Sarkis, M., Brookes, O., Oikonomou, E.K., Baeza-Squiban, A., Berret, J.-F.,
Miller, A.J., Dye, B.R., Ferrer-Torres, D., Hill, D.R., Overeem, A.W., Shea, L.D., Spence, J. 2020. Pulmonary surfactant inhibition of nanoparticle uptake by alveolar epithelial
R., 2019. Generation of lung organoids from human pluripotent stem cells in vitro. cells. Sci. Rep. 10, 19436. https://doi.org/10.1038/s41598-020-76332-7.
Nat. Protoc. 14, 518–540. https://doi.org/10.1038/s41596-018-0104-8. Rahman, M., Irmler, M., Introna, M., Beckers, J., Palmberg, L., Johanson, G.,
Mistry, A., Bowen, L.E., Dzierlenga, M.W., Hartman, J.K., Slattery, S.D., 2020. Upadhyay, S., Ganguly, K., 2022. Insight into the pulmonary molecular toxicity of
Development of an in vitro approach to point-of-contact inhalation toxicity testing of heated tobacco products using human bronchial and alveolar mucosa models at air-
volatile compounds, using organotypic culture and air-liquid interface exposure. liquid interface. Sci. Rep. 12, 16396. https://doi.org/10.1038/s41598-022-20657-y.
Toxicol. in Vitro 69, 104968. https://doi.org/10.1016/j.tiv.2020.104968. Rasmussen, R.E., 1984. In vitro systems for exposure of lung cells to NO2 and O3.
Moore, G.E., Sandberg, A.A., 1964. Studies of a human tumor cell line with a diploid J. Toxicol. Environ. Health 13, 397–411. https://doi.org/10.1080/
karyotype. Cancer 17, 170–175. https://doi.org/10.1002/1097-0142. 15287398409530506.
Moorhead, P.S., 1965. Human tumor cell line with a quasi-diploid karyotype (RPMI Ravi, M., Paramesh, V., Kaviya, S.R., Anuradha, E., Solomon, F.D.P., 2015. 3D cell
2650). Exp. Cell Res. 39, 190–196. https://doi.org/10.1016/0014-4827(65)90022- culture systems: advantages and applications: 3D cell culture systems. J. Cell.
4. Physiol. 230, 16–26. https://doi.org/10.1002/jcp.24683.
Moreau, M., Fisher, J., Andersen, M.E., Barnwell, A., Corzine, S., Ranade, A., Rayner, R.E., Makena, P., Prasad, G.L., Cormet-Boyaka, E., 2019. Optimization of normal
McMullen, P.D., Slattery, S.D., 2022. NAM-based prediction of point-of-contact human bronchial epithelial (NHBE) cell 3D cultures for in vitro lung model studies.
toxicity in the lung: a case example with 1,3-dichloropropene. Toxicology 481, Sci. Rep. 9, 1–10. https://doi.org/10.1038/s41598-018-36735-z.
153340. https://doi.org/10.1016/j.tox.2022.153340. Reddel, R.R., Ke, Y., Gerwin, B.I., McMenamin, M.G., Lechner, J.F., Su, R.T., Brash, D.E.,
Morissette, M.C., Parent, J., Milot, J., 2009. Alveolar epithelial and endothelial cell Park, J.-B., Rhim, J.S., Harris, C.C., 1988. Transformation of human bronchial
apoptosis in emphysema: what we know and what we need to know. Int. J. Chron. epithelial cells by infection with SV40 or Adenovirus-12 SV40 hybrid virus, or
Obstruct. Pulmon. Dis. 4, 19–31. transfection via strontium phosphate Coprecipitation with a plasmid containing
Müller, L., Brighton, L.E., Carson, J.L., Fischer, W.A., Jaspers, I., 2013. Culturing of SV40 early region genes. Cancer Res. 48, 1904–1909.
human nasal epithelial cells at the air liquid Interface. J. Vis. Exp. 50646 https://doi. Richter, M., Piwocka, O., Musielak, M., Piotrowski, I., Suchorska, W.M., Trzeciak, T.,
org/10.3791/50646. 2021. From donor to the lab: a fascinating journey of primary cell lines. Front. Cell
Munis, A.M., Hyde, S.C., Gill, D.R., 2020. A human surfactant B deficiency air-liquid Dev. Biol. 9, 711381 https://doi.org/10.3389/fcell.2021.711381.
interface cell culture model suitable for gene therapy applications. Mol. Ther. Roggen, E.L., Soni, N.K., Verheyen, G.R., 2006. Respiratory immunotoxicity: an in vitro
Methods Clin. Dev. 20, 237–246. https://doi.org/10.1016/j.omtm.2020.11.013. assessment. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 20, 1249–1264. https://doi.
Nadkarni, R.R., Abed, S., Draper, J.S., 2016. Organoids as a model system for studying org/10.1016/j.tiv.2006.03.009.
human lung development and disease. Biochem. Biophys. Res. Commun Special Rossner, P., Cervena, T., Vojtisek-Lom, M., Vrbova, K., Ambroz, A., Novakova, Z.,
Issue: Stem Cells 473, 675–682. https://doi.org/10.1016/j.bbrc.2015.12.091. Elzeinova, F., Margaryan, H., Beranek, V., Pechout, M., Macoun, D., Klema, J.,
Offer, S., Hartner, E., Di Bucchianico, S., Bisig, C., Bauer, S., Pantzke, J., Zimmermann, E. Rossnerova, A., Ciganek, M., Topinka, J., 2019. The biological effects of complete
J., Cao, X., Binder, S., Kuhn, E., Huber, A., Jeong, S., Käfer, U., Martens, P., gasoline engine emissions exposure in a 3D human airway model (MucilAirTM) and
Mesceriakovas, A., Bendl, J., Brejcha, R., Buchholz, A., Gat, D., Hohaus, T., in human bronchial epithelial cells (BEAS-2B). Int. J. Mol. Sci. 20 https://doi.org/
Rastak, N., Jakobi, G., Kalberer, M., Kanashova, T., Hu, Y., Ogris, C., Marsico, A., 10.3390/ijms20225710.
Theis, F., Pardo, M., Gröger, T., Oeder, S., Orasche, J., Paul, A., Ziehm, T., Zhang, Z.- Rota, F., Ferrari, L., Hoxha, M., Favero, C., Antonioli, R., Pergoli, L., Greco, M.F.,
H., Adam, T., Sippula, O., Sklorz, M., Schnelle-Kreis, J., Czech, H., Kiendler- Mariani, J., Lazzari, L., Bollati, V., 2020. Blood-derived extracellular vesicles isolated
Scharr, A., Rudich, Y., Zimmermann, R., 2022. Effect of atmospheric aging on soot from healthy donors exposed to air pollution modulate in vitro endothelial cells
particle toxicity in lung cell models at the air-liquid Interface: differential behavior. Sci. Rep. 10, 20138. https://doi.org/10.1038/s41598-020-77097-9.
toxicological impacts of biogenic and anthropogenic secondary organic aerosols Rothbauer, M., Bachmann, B.E.M., Eilenberger, C., Kratz, S.R.A., Spitz, S., Höll, G.,
(SOAs). Environ. Health Perspect. 130, 27003. https://doi.org/10.1289/EHP9413. Ertl, P., 2021. A decade of organs-on-a-chip emulating human physiology at the
Ohnuki, Y., Reddel, R.R., Bates, S.E., Lehman, T.A., Lechner, J.F., Harris, C.C., 1996. microscale: a critical status report on Progress in toxicology and pharmacology.
Chromosomal changes and progressive tumorigenesis of human bronchial epithelial Micromachines 12, 470. https://doi.org/10.3390/mi12050470.
cell lines. Cancer Genet. Cytogenet. 92, 99–110. https://doi.org/10.1016/s0165- Rothen-Rutishauser, B., Blank, F., Mühlfeld, C., Gehr, P., 2008. In vitro models of the
4608(96)00156-2. human epithelial airway barrier to study the toxic potential of particulate matter.
24
Expert Opin. Drug Metab. Toxicol. 4, 1075–1089. https://doi.org/10.1517/ on human platelet function. Nanotoxicology 15, 52–65. https://doi.org/10.1080/
17425255.4.8.1075. 17435390.2020.1841845.
Ruiz, P., Emond, C., McLanahan, E.D., Joshi-Barr, S., Mumtaz, M., 2020. Exploring Thavagnanam, S., Parker, J.C., McBrien, M.E., Skibinski, G., Shields, M.D., Heaney, L.G.,
mechanistic toxicity of mixtures using PBPK modeling and computational systems 2014. Nasal epithelial cells can act as a physiological surrogate for paediatric asthma
biology. Toxicol. Sci. Off. J. Soc. Toxicol. 174, 38–50. https://doi.org/10.1093/ studies. PLoS One 9, e85802. https://doi.org/10.1371/journal.pone.0085802.
toxsci/kfz243. Thomas, D.G., Smith, J.N., Thrall, B.D., Baer, D.R., Jolley, H., Munusamy, P., Kodali, V.,
Sachs, N., Papaspyropoulos, A., Zomer-van Ommen, D.D., Heo, I., Böttinger, L., Klay, D., Demokritou, P., Cohen, J., Teeguarden, J.G., 2018. ISD3: a particokinetic model for
Weeber, F., Huelsz-Prince, G., Iakobachvili, N., Amatngalim, G.D., de Ligt, J., van predicting the combined effects of particle sedimentation, diffusion and dissolution
Hoeck, A., Proost, N., Viveen, M.C., Lyubimova, A., Teeven, L., Derakhshan, S., on cellular dosimetry for in vitro systems. Part. Fibre Toxicol. 15, 6. https://doi.org/
Korving, J., Begthel, H., Dekkers, J.F., Kumawat, K., Ramos, E., van Oosterhout, M. 10.1186/s12989-018-0243-7.
F., Offerhaus, G.J., Wiener, D.J., Olimpio, E.P., Dijkstra, K.K., Smit, E.F., van der Tollstadius, B.F., da Silva, A.C.G., Pedralli, B.C.O., Valadares, M.C., 2019. Carbendazim
Linden, M., Jaksani, S., van de Ven, M., Jonkers, J., Rios, A.C., Voest, E.E., van induces death in alveolar epithelial cells: a comparison between submerged and at
Moorsel, C.H., van der Ent, C.K., Cuppen, E., van Oudenaarden, A., Coenjaerts, F.E., the air-liquid interface cell culture. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 58,
Meyaard, L., Bont, L.J., Peters, P.J., Tans, S.J., van Zon, J.S., Boj, S.F., Vries, R.G., 78–85. https://doi.org/10.1016/j.tiv.2019.03.004.
Beekman, J.M., Clevers, H., 2019. Long-term expanding human airway organoids for Tralau, T., Oelgeschläger, M., Kugler, J., Bloch, D., Braeuning, A., Burgdorf, T., Marx-
disease modeling. EMBO J. 38 https://doi.org/10.15252/embj.2018100300. Stoelting, P., Ritz, V., Schmeisser, S., Trubiroha, A., Zellmer, S., Luch, A.,
Sakamoto, A., Matsumaru, T., Yamamura, N., Suzuki, S., Uchida, Y., Tachikawa, M., Schönfelder, G., Solecki, R., Hensel, A., 2021. A prospective whole-mixture approach
Terasaki, T., 2015. Drug transporter protein quantification of immortalized human to assess risk of the food and chemical exposome. Nat. Food 2, 463–468. https://doi.
lung cell lines derived from tracheobronchial epithelial cells (Calu-3 and BEAS2-B), org/10.1038/s43016-021-00316-7.
bronchiolar–alveolar Cells (NCI-H292 and NCI-H441), and alveolar type II-like Cells Travaglini, K.J., Nabhan, A.N., Penland, L., Sinha, R., Gillich, A., Sit, R.V., Chang, S.,
(A549) by liquid chromatography–tandem mass spectrometry. J. Pharm. Sci. 104, Conley, S.D., Mori, Y., Seita, J., Berry, G.J., Shrager, J.B., Metzger, R.J., Kuo, C.S.,
3029–3038. https://doi.org/10.1002/jps.24381. Neff, N., Weissman, I.L., Quake, S.R., Krasnow, M.A., 2020. A molecular cell atlas of
Sakolish, C., Georgescu, A., Huh, D.D., Rusyn, I., 2022. A model of human small airway the human lung from single-cell RNA sequencing. Nature 587, 619–625. https://doi.
on a chip for studies of subacute effects of inhalation toxicants. Toxicol. Sci. Off. J. org/10.1038/s41586-020-2922-4.
Soc. Toxicol. 187, 267–278. https://doi.org/10.1093/toxsci/kfac036. Tuazon, J.A., Kilburg-Basnyat, B., Oldfield, L.M., Wiscovitch-Russo, R., Dunigan-
Salas, A., López, J., Reyes, R., Évora, C., de Oca, F.M., Báez, D., Delgado, A., Almeida, T. Russell, K., Fedulov, A.V., Oestreich, K.J., Gowdy, K.M., 2022. Emerging insights
A., 2020. Organotypic culture as a research and preclinical model to study uterine into the impact of air pollution on immune-mediated asthma pathogenesis. Curr
leiomyomas. Sci. Rep. 10, 5212. https://doi.org/10.1038/s41598-020-62158-w. Allergy Asthma Rep 22, 77–92. https://doi.org/10.1007/s11882-022-01034-1.
Salomon, J., Muchitsch, V., Gausterer, J., Schwagerus, E., Huwer, H., Schneider- Upadhyay, S., Palmberg, L., 2018. Air-liquid interface: relevant in vitro models for
Daum, N., Lehr, C.-M., Ehrhardt, C., 2014. The cell line NCl-H441 is a useful in vitro investigating air pollutant-induced pulmonary toxicity. Toxicol. Sci. Off. J. Soc.
model for transport studies of human distal lung epithelial barrier. Mol. Pharm. 11 Toxicol. 164, 21–30. https://doi.org/10.1093/toxsci/kfy053.
https://doi.org/10.1021/mp4006535. van der Vaart, J., Clevers, H., 2020. Liu. J. Intern. Med.. https://doi.org/10.1111/
Sanchez-Guzman, D., Boland, S., Brookes, O., Mc Cord, C., Lai Kuen, R., Sirri, V., Baeza joim.13075.
Squiban, A., Devineau, S., 2021. Long-term evolution of the epithelial cell secretome Van Haute, L., De Block, G., Liebaers, I., Sermon, K., De Rycke, M., 2009. Generation of
in preclinical 3D models of the human bronchial epithelium. Sci. Rep. 11, 6621. lung epithelial-like tissue from human embryonic stem cells. Respir. Res. 10, 105.
https://doi.org/10.1038/s41598-021-86037-0. https://doi.org/10.1186/1465-9921-10-105.
Schimek, K., Frentzel, S., Luettich, K., Bovard, D., Rütschle, I., Boden, L., Rambo, F., Varghese, B., Ling, Z., Ren, X., 2022. Reconstructing the pulmonary niche with stem
Erfurth, H., Dehne, E.-M., Winter, A., Marx, U., Hoeng, J., 2020. Human multi-organ cells: a lung story. Stem Cell Res Ther 13, 161. https://doi.org/10.1186/s13287-
chip co-culture of bronchial lung culture and liver spheroids for substance exposure 022-02830-2.
studies. Sci. Rep. 10, 7865. https://doi.org/10.1038/s41598-020-64219-6. Visigalli, R., Rotoli, B.M., Ferrari, F., Di Lascia, M., Riccardi, B., Puccini, P., Dall’Asta, V.,
Schmid, O., Cassee, F.R., 2017. On the pivotal role of dose for particle toxicology and risk Barilli, A., 2022. Expression and function of ABC transporters in human alveolar
assessment: exposure is a poor surrogate for delivered dose. Part. Fibre Toxicol. 14, epithelial cells. Biomolecules 12, 1260. https://doi.org/10.3390/biom12091260.
52. https://doi.org/10.1186/s12989-017-0233-1. Wang, Q., Bhattacharya, S., Mereness, J.A., Anderson, C., Lillis, J.A., Misra, R.S.,
Schwerdtfeger, L.A., Tobet, S.A., 2019. From organotypic culture to body-on-a-chip: A Romas, S., Huyck, H., Howell, A., Bandyopadhyay, G., Donlon, K., Myers, J.R.,
neuroendocrine perspective. J. Neuroendocrinol. 31, e12650 https://doi.org/ Ashton, J., Pryhuber, G.S., Mariani, T.J., 2020. A novel in vitro model of primary
10.1111/jne.12650. human pediatric lung epithelial cells. Pediatr. Res. 87, 511–517. https://doi.org/
Sengupta, A., Roldan, N., Kiener, M., Froment, L., Raggi, G., Imler, T., de Maddalena, L., 10.1038/s41390-019-0340-9.
Rapet, A., May, T., Carius, P., Schneider-Daum, N., Lehr, C.-M., Kruithof-de Wang, H., Yin, F., Li, Z., Su, W., Li, D., 2023. Advances of microfluidic lung chips for
Julio, M., Geiser, T., Marti, T.M., Stucki, J.D., Hobi, N., Guenat, O.T., 2022. A new assessing atmospheric pollutants exposure. Environ. Int. 172, 107801 https://doi.
immortalized human alveolar epithelial cell model to study lung injury and toxicity org/10.1016/j.envint.2023.107801.
on a breathing lung-on-chip system. Front. Toxicol. 4, 840606 https://doi.org/ Watson, C.Y., DeLoid, G.M., Pal, A., Demokritou, P., 2016. Buoyant nanoparticles:
10.3389/ftox.2022.840606. implications for nano-biointeractions in cellular studies. Small Weinh. Bergstr. Ger.
Sengupta, A., Dorn, A., Jamshidi, M., Schwob, M., Hassan, W., De Maddalena, L.L., 12, 3172–3180. https://doi.org/10.1002/smll.201600314.
Hugi, A., Stucki, A.O., Dorn, P., Marti, T.M., Wisser, O., Stucki, J.D., Krebs, T., WHO, 2016. Ambient Air Pollution: A Global Assessment of Exposure and Burden of
Hobi, N., Guenat, O.T., 2023. A multiplex inhalation platform to model in situ like Disease. World Health Organization.
aerosol delivery in a breathing lung-on-chip. Front. Pharmacol. 14, 1114739. Woischnik, M., Sparr, C., Kern, S., Thurm, T., Hector, A., Hartl, D., Liebisch, G.,
https://doi.org/10.3389/fphar.2023.1114739. Mulugeta, S., Beers, M.F., Schmitz, G., Griese, M., 2010. A non-BRICHOS surfactant
Sibinovska, N., Žakelj, S., Trontelj, J., Kristan, K., 2022. Applicability of RPMI 2650 and protein c mutation disrupts epithelial cell function and intercellular signaling. BMC
Calu-3 cell models for evaluation of nasal formulations. Pharmaceutics 14, 369. Cell Biol. 11, 88. https://doi.org/10.1186/1471-2121-11-88.
https://doi.org/10.3390/pharmaceutics14020369. Zamora, P.O., Gregory, R.E., Li, A.P., Brooks, A.L., 1986. An in vitro model for the
Signorelli, S., Jennings, P., Leonard, M., Pfaller, W., 2009. Differential effects of hypoxic exposure of lung alveolar epithelial cells to toxic gases. J. Environ. Pathol. Toxicol.
stress in alveolar epithelial cells and microvascular endothelial cells. Cell. Physiol. Oncol Off. Organ Int. Soc. Environ. Toxicol. Cancer 7, 159–168.
Biochem. 25, 135–144. https://doi.org/10.1159/000272066. Zavala, J., Greenan, R., Krantz, Q.T., DeMarini, D.M., Higuchi, M., Gilmour, M.I.,
Silva, S., Bicker, J., Falcão, A., Fortuna, A., 2023. Air-liquid interface (ALI) impact on White, P.A., 2017. Regulating temperature and relative humidity in air–liquid
different respiratory cell cultures. Eur. J. Pharm. Biopharm Off. J. interface in vitro systems eliminates cytotoxicity resulting from control air
Arbeitsgemeinschaft Pharm. Verfahrenstechnik EV 184, 62–82. https://doi.org/ exposures. Toxicol. Res. 6, 448–459. https://doi.org/10.1039/c7tx00109f.
10.1016/j.ejpb.2023.01.013. Zavala, J., Ledbetter, A.D., Morgan, D.S., Dailey, L.A., Puckett, E., McCullough, S.D.,
Su, M., Li, X., Li, Z., Hua, C., Shang, P., Zhao, J., Liu, K., Xie, F., 2023. Design of a Higuchi, M., 2018. A new cell culture exposure system for studying the toxicity of
microfluidic lung chip and its application in assessing the toxicity of formaldehyde. volatile chemicals at the air-liquid interface. Inhal. Toxicol. 30, 169–177. https://
Toxicol. Mech. Methods 33, 427–436. https://doi.org/10.1080/ doi.org/10.1080/08958378.2018.1483983.
15376516.2022.2159903. Zhang, Z., Kleinstreuer, C., 2004. Airflow structures and nano-particle deposition in a
Svensson, M., Chen, P., 2018. Human organotypic respiratory models. In: Current Topics human upper airway model. J. Comput. Phys. 198, 178–210. https://doi.org/
in Microbiology and Immunology. Springer, Berlin Heidelberg, Berlin, Heidelberg. 10.1016/j.jcp.2003.11.034.
https://doi.org/10.1007/82_2018_91. Zhang, M., Xu, C., Jiang, L., Qin, J., 2018. A 3D human lung-on-a-chip model for
Tamò, L., Hibaoui, Y., Kallol, S., Alves, M.P., Albrecht, C., Hostettler, K.E., Feki, A., nanotoxicity testing. Toxicol. Res. 7, 1048–1060. https://doi.org/10.1039/
Rougier, J.-S., Abriel, H., Knudsen, L., Gazdhar, A., Geiser, T., 2018. Generation of c8tx00156a.
an alveolar epithelial type II cell line from induced pluripotent stem cells. APSselect Zhang, F., Aquino, G.V., Dabi, A., Bruce, E.D., 2019. Assessing the translocation of silver
5, L921–L932. https://doi.org/10.1152/[email protected]. nanoparticles using an in vitro co-culture model of human airway barrier. Toxicol.
issue-11. Vitro Int. J. Publ. Assoc. BIBRA 56, 1–9. https://doi.org/10.1016/j.tiv.2018.12.013.
Taterra, D., Skinningsrud, B., Pękala, P.A., Tomaszewska, I.M., Marycz, K., Radomski, M. Zhang, H., Zhou, H., Zhang, N., Jia, Y., Qiu, M., Yao, S., Chen, X., Qiu, L., Li, S., Jiang, Y.,
W., Tomaszewski, K.A., 2021. In vitro effects of cobalt and chromium nanoparticles Zhou, Y., 2023. Circ_0089282 inhibits carbon black nanoparticle-induced DNA
25
damage by promoting DNA repair protein in the lung. Toxicol. Sci. Off. J. Soc. Zhu, Y., Chidekel, A., Shaffer, T.H., 2010. Cultured human airway epithelial cells (Calu-
Toxicol. 192, 71–82. https://doi.org/10.1093/toxsci/kfad002. 3): a model of human respiratory function, structure, and inflammatory responses.
Zhao, F., Klimecki, W.T., 2015. Culture conditions profoundly impact phenotype in Crit. Care Res. Pract. 2010, 394578 https://doi.org/10.1155/2010/394578.
BEAS-2B, a human pulmonary epithelial model. J. Appl. Toxicol. JAT 35, 945–951. Zimmermann, B., 1987. Lung organoid culture. Differentiation 36, 86–109. https://doi.
https://doi.org/10.1002/jat.3094. org/10.1111/j.1432-0436.1987.tb00183.x.
Zhao, R., Guo, Z., Zhang, R., Deng, C., Xu, J., Dong, W., Hong, Z., Yu, H., Situ, H., Liu, C., Zscheppang, K., Berg, J., Hedtrich, S., Verheyen, L., Wagner, D.E., Suttorp, N.,
Zhuang, G., 2018. Nasal epithelial barrier disruption by particulate matter ≤2.5 μm Hippenstiel, S., Hocke, A.C., 2018. Human pulmonary 3D models for translational
via tight junction protein degradation. J. Appl. Toxicol. JAT 38, 678–687. https:// research. Biotechnol. J. 13 https://doi.org/10.1002/biot.201700341.
doi.org/10.1002/jat.3573.
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Jaber, N. (2024) - Toxicology in Vitro

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Toxicology in Vitro 94 (2024) 105718

Contents lists available at ScienceDirect

How to use an in vitro approach to characterize the toxicity of

Fig. 1. Anatomy of the human respiratory system.

2.11.4. Bronchial model those observed in vivo.

Fig. 4. “Hanging Drop” (HD) air exposure method.

Fig. 5. Methods of in vitro exposure to airborne compounds

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