UNIT 2

Cell culture

Stem Cells
Stem cells are unique cells with the ability to develop into various cell types in the body
during early life and growth. They function as an internal repair system in many tissues,
capable of dividing indefinitely to replenish other cells for the lifespan of the person or
animal. When stem cells divide, the resulting cells can either remain as stem cells or
differentiate into specialized cells such as muscle cells, red blood cells, or brain cells.
Progenitor cell
A progenitor cell is a biological cell that can differentiate into a specific cell type. While both
progenitor cells and stem cells share the ability to differentiate, progenitor cells are more
specified than stem cells. Progenitor cells can only differentiate into their designated "target"
cell type and can divide only a limited number of times, unlike stem cells which can replicate
indefinitely. The precise definition of progenitor cells is still debated and evolving.

Stem Cells Progenitor Cells

1. Can differentiate into many different cell 1. Can only differentiate into a specific cell
types (pluripotent) or a limited range of cell type (unipotent or oligopotent)
types (multipotent)
2. Less specified compared to progenitor cells. 2.More specified than stem cells

3.Can replicate indefinitely. 3.Can divide only a limited number of times

4.Found in early development (Embryonic 4.Typically found in specific tissues and

stage) and various tissues in adults. stages of development.

5.Function of stem cells is the Development of 5.Function of progenitor cell is to Replenish

tissues and organs, repair of damaged tissues specific cell types in adult tissues

Cell-Cell Interactions
Cell-cell interactions refer to direct interactions between the surfaces of adjacent cells. These
interactions are essential for various biological processes in multicellular organisms,
including development, tissue organization, and immune responses. Cell-cell interactions
enable cells to communicate with each other, allowing them to respond to changes in their
environment and coordinate their activities. These interactions can be stable, such as those
formed by cell junctions that facilitate strong adhesion and communication between cells
within tissues.
Tight Junctions
1. Tight junctions are multi-protein complexes that hold cells together and prevent the
movement of water and water-soluble molecules between cells.
2. In epithelial cells, they separate the extracellular fluid around the apical and
basolateral membranes, forming a continuous band just below the apical surface.
3. Tight junctions align on adjacent cells to seal different tissues and body cavities; the
apical surface of gastrointestinal epithelial cells acts as a selective permeable barrier
separating the external environment from the body.
4. The permeability of tight junctions depends on factors such as protein makeup, tissue
type, and cell signaling.
5. Tight junctions are composed of various proteins, with four main transmembrane
proteins: occludin, claudin, junctional adhesion molecules (JAMs), and tricellulins.
6. The extracellular domains form the tight junction barrier through homophilic and
heterophilic interactions with adjacent cells, while cytoplasmic domains anchor them
to the cell cytoskeleton.

Gap junctions
1. Gap junctions are the main site of cell-cell signalling or communication that
allow small molecules to diffuse between adjacent cells.
2. In vertebrates, gap junctions are composed of transmembrane proteins called
connexins.
3. They form hexagonal pores or channels through which ions, sugars,
and other small molecules can pass.
4. Each pore is made of 12 connexin molecules; 6 form a hemichannel on one cell
membrane and interact with a hemichannel on an adjacent cell membrane.
5. The permeability of these junctions is regulated by many factors
including pH and Ca2+ concentration.
Anchoring junctions
1. Of the three types of anchoring junctions, only adherens junctions and desmosomes
are involved in cell-cell interactions.
2. Both are found in many types of cells.
3. Adjacent epithelial cells are connected by adherens junctions on their lateral
membranes, located just below tight junctions.
4. Adherens junctions give shape and tension to cells and tissues and are also sites of
cell-cell signaling.
5. Adherens junctions are made of cell adhesion molecules from the cadherin family,
with over 100 types corresponding to different cells and tissues.
6. The most common cadherins are E-, N-, and P-cadherins, with E-cadherin being the
most abundant in epithelial cells.
Sterilization:

Sterilization is the process of removing, killing, or deactivating all forms of life and
biological agents on a specific surface, object, or fluid. This can be achieved through
heat, chemicals, irradiation, high pressure, and filtration. Sterilization eliminates all
forms of microorganisms and biological agents. After sterilization, an object is
considered sterile or aseptic.it is used in various fields the medicine, research, and
food processing to prevent contamination.

Methods of Sterilization

Heat

Heat sterilization is a method of eliminating all forms of microbial life, including

bacteria, viruses, fungi, and spores, by exposing them to high temperatures. This
process is widely used in medical, laboratory, and food industries to ensure the
sterility of instruments, equipment, and products.

Types of Heat Sterilization:

1. Steam sterilization
Steam sterilization also known as moist heat sterilization, is a method that uses
steam under pressure to kill microorganisms. It is performed using an
autoclave at high temperature for a specific time. This process is faster than
dry heat sterilization. Typical cycles take 3-30 minutes at 121-134°C.The
steam condenses on the surfaces of objects being sterilized, transferring heat
and destroying bacteria, viruses, and fungi.

2. Dry heat sterilization

Dry heat sterilization, the earliest method, is slower than moist heat
sterilization and gradually kills microorganisms with prolonged exposure to
lethal temperatures. Forced hot air can accelerate heat transfer, reducing the
temperature and time needed for sterility. Standard settings are 160 °C for two
hours or a rapid method of 190 °C for 6-12 minutes, depending on item
wrapping. This method is suitable for powders and heat-stable items, avoiding
issues like rusting in steel objects.

Chemical Sterilization
Chemical sterilization is used for heat-sensitive materials, employing gases or liquids
to avoid heat damage. It requires compatibility between the sterilant and materials and
ensuring full surface contact. However, these chemicals can be harmful to humans,
necessitating careful handling and residue removal.
 Types of chemical Sterilization
1. Ethylene oxide
Ethylene oxide (EO, EtO) gas is widely used for sterilization due to its
compatibility with diverse materials, including heat-sensitive items,
making it the predominant method for sterilizing over 50% of disposable
medical devices and 70% of total sterilizations.

2. Hydrogen peroxide
Hydrogen peroxide, in liquid and vapor form (VHP), is a potent oxidizing
agent used for sterilizing heat-sensitive medical tools like endoscopes. It
offers rapid cycles, though limitations in material compatibility and
penetration exist, posing health risks such as skin irritation and respiratory
hazards.

3. Glutaraldehyde and formaldehyde

Glutaraldehyde and formaldehyde are liquid sterilizing agents requiring
long immersion times, up to 22 hours for spores, and are volatile and toxic.
Glutaraldehyde has a short shelf-life and is costly, while formaldehyde,
also used as a gas, is less expensive but highly volatile and used in vaccine
sterilization.

4. Peracetic acid
Peracetic acid (0.2%), FDA-approved for sterilizing medical devices like
endoscopes, is a disinfectant produced from acetic acid and hydrogen
peroxide. It's environmentally friendly, widely used in sanitizers, and
increasingly employed globally for surface fumigation to combat COVID-
19 and other diseases.

Radiation sterilization
Radiation sterilization is a method that uses electromagnetic radiation (such as UV
light, X-rays, and gamma rays) or subatomic particles (such as electron beams) to
eliminate all forms of microorganisms from materials, including bacteria, viruses,
fungi, and spores. This technique can involve ionizing radiation, which has
enough energy to ionize atoms and molecules, or non-ionizing radiation, which is
less energetic.

Types of Radiation Sterilization:

1. Non-ionizing radiation sterilization

Non-ionizing radiation sterilization uses lower-energy electromagnetic
waves, such as ultraviolet (UV) light, to disinfect surfaces and transparent
objects by damaging the DNA of microorganisms. It is effective for surface
sterilization but limited by its inability to penetrate shaded areas or dirt.
 2. Ionizing radiation sterilization
Ionizing radiation sterilization uses high-energy radiation, such as gamma
rays, X-rays, or electron beams, to eliminate microorganisms by damaging
their DNA. This method is effective for sterilizing medical devices,
pharmaceuticals, and food products.

Autoclave
An autoclave is a machine that uses high temperatures and pressure to sterilize items by
killing harmful microorganisms like bacteria, viruses, fungi, and spores. Autoclaves are often
used in healthcare, laboratory, food, and pharmaceutical industries. They can also be used for
industrial applications like curing composite resins and material testing in the aeronautical
industry.

There are different types of autoclaves, including horizontal, gravity displacement, and
vertical:
1. Horizontal autoclaves
These are floor-mounted units that are often used for medical, industrial,
research, and education purposes. They have rectangular pressure vessels that
are good for sterilizing flat trays of surgical instruments.
2. Gravity displacement autoclaves
Also known as gravity autoclaves, these are the most basic type of autoclave
and are commonly used in research laboratories. They use steam to displace
air in the chamber by gravity through a drain port.
3. Vertical autoclaves
Also known as top loading autoclaves, these generate steam to create pressure
and heat for sterilizing medical tools like flasks and beakers.
Bioreactor
A bioreactor is a type of fermentation vessel used to generate a variety of chemicals and
biological reactions. This is a closed container with aeration, agitation, temperature and pH
control, and well-placed drainage or overflow openings to remove waste biomass with its
products from cultured microorganisms’. enzymes, antibodies, vaccines, etc.

Different Types of Bioreactors for animal cell culture:

1. Stirred Tank Bioreactors:
A stirred tank bioreactor is a cylindrical vessel equipped with a motor-driven central shaft
that supports agitators (impellers) to mix the contents and enhance gas distribution. It is
widely used in industry due to its ease of operation, cost-effectiveness, simple cleaning
process, and efficient temperature control, making it ideal for fermentation and cell culture
processes.

2. Airlift Bioreactors:
An airlift bioreactor is a special tank that uses bubbles to move liquids around. It's split into
two parts: one where the liquid goes up with the bubbles, and one where it comes back down.
This helps mix everything evenly and is good for growing cells and making stuff like
medicine.
 3. Packed bed Bioreactors:
Packed bed bioreactors are like tall columns filled with small particles where microorganisms
or cells grow. Liquid passes through the packed particles, providing nutrients and removing
waste. They're used for various biological processes like fermentation or water treatment,
offering high surface area for efficient reactions and easy control over conditions.

4. Photobioreactors:
Photobioreactors are specialized tanks or containers where microorganisms like algae or
cyanobacteria grow using sunlight as their energy source. These reactors are designed to
optimize light exposure, temperature, and nutrient supply for efficient growth. They're used in
industries like biofuel production, wastewater treatment, and food supplements.
 5. Fluidize bed bioreactors:
A fluidized-bed bioreactor is a type of vessel where solid particles are suspended and behave
like a fluid when liquid passes through them. Microorganisms attached to these particles
carry out biological processes such as fermentation or waste degradation. These bioreactors
offer efficient mixing and increased surface area for microbial activity, making them useful in
various industries for tasks like wastewater treatment and biofuel production.

Microfluidics
Microfluidics in tissue engineering involves the use of tiny fluid channels to precisely control
the cellular microenvironment, enhancing tissue growth and organization. This technology
enables the creation of complex tissue structures by mimicking physiological conditions. It
allows for high-throughput screening and real-time monitoring of cellular responses.
Microfluidics thus plays a crucial role in advancing regenerative medicine and developing
personalized medical treatments.

Applications of microfluidics
1. Point-of-Care Testing (POCT):
Provides rapid, on-site diagnostic results without the need for centralized laboratories,
enhancing immediate patient care. These devices are compact, user-friendly, and designed for
quick analysis of biological samples.
2. Lab-on-a-Chip (LOC):
Miniaturizes and integrates multiple laboratory processes onto a single chip for efficient and
comprehensive analysis. This technology allows complex biochemical reactions and analyses
to be conducted on a tiny, portable platform.
3. High-Throughput Screening (HTS):
Facilitates the rapid screening of large libraries of drug compounds against target molecules
for therapeutic efficacy. Microfluidic HTS systems are efficient and cost-effective,
significantly accelerating the drug discovery process.
4. Single-Cell Analysis:
Allows for the detailed examination of individual cells, aiding in understanding cellular
behavior, genetics, and disease mechanisms. This application is crucial for research in cancer,
immunology, and personalized medicine.

5. Microfluidic Organ-on-a-Chip:
Mimics the physiological responses of human organs on a chip to study disease mechanisms
and drug responses. These models provide a more accurate representation of human biology
than traditional cell cultures or animal models.
6. PCR and DNA Analysis:
Conducts rapid and precise genetic analysis and amplification in a compact format,
enhancing molecular diagnostics. Microfluidic PCR devices offer high sensitivity and
specificity for detecting genetic material.
7. Cell Sorting:
Isolates and sorts cells using precise fluid control mechanisms for research and therapeutic
applications. This technique is essential for stem cell research, cancer treatment, and the
study of rare cell populations.
8. Drug Delivery Systems:
Enables controlled and targeted delivery of drugs to specific sites in the body, improving
therapeutic outcomes. Microfluidic devices can release drugs at a controlled rate and target
specific tissues, minimizing side effects.
UNIT 3

Hormonal Growth Factors in animal tissue culture

Hormonal growth factors are crucial in animal tissue culture to promote cell growth,
differentiation, and survival. These factors are proteins or steroid hormones that interact with
specific cell surface receptors to trigger intracellular signaling pathways. Here are some
commonly used hormonal growth factors in animal tissue culture:
1. Epidermal Growth Factor (EGF):
Epidermal growth factor (EGF) is a protein that stimulates cell growth and
differentiation by binding to its receptor, EGFR. EGF is used to modify synthetic
scaffolds for manufacturing of bioengineered grafts by emulsion electrospinning or
surface modification methods. Stimulates cell growth and differentiation by binding
to its receptor, EGFR.Commonly used in epithelial cell cultures.

2. Fibroblast Growth Factor (FGF):

Fibroblast growth factor (FGF) is a representative growth factor which has shown the
potential effects on the repair and regeneration of tissues. These growth factors
typically act as systemic or locally circulating molecules of extracellular origin that
activate cell surface receptors. It Promotes proliferation and differentiation in various
cells including fibroblasts, endothelial cells, and neurons. There are multiple types,
including FGF-1 (acidic FGF) and FGF-2 (basic FGF).

3. Insulin-like Growth Factor (IGF):

 The insulin-like growth factors (IGFs) are proteins with high sequence similarity to
insulin. IGFs are part of a complex system that cells use to communicate with their
physiologic environment. This complex system consists of two cell-surface receptors
(IGF1R and IGF2R). IGF-1 and IGF-2 play roles in cell growth and development.
They are Often used in combination with insulin in serum-free media to promote
growth.

4. Vascular Endothelial Growth Factor (VEGF):

Vascular endothelial growth factor (VEGF) is a signal protein produced by many cells
that stimulates the formation of blood vessels. VEGF is a sub-family of growth
factors, the platelet-derived growth factor family of cystine-knot growth factors. They
are important signaling proteins involved in both and angiogenesis it Stimulates the
formation of blood vessels.

5. Platelet-Derived Growth Factor (PDGF):

Platelet-derived growth factor (PDGF) regulates cell growth and division, crucial for
blood vessel formation, and the proliferation and migration of mesenchymal cells like
fibroblasts and osteoblasts. It stimulates cell proliferation in connective tissue, smooth
muscle cells, and glial cells, playing a vital role in wound healing and tissue repair.

6. Transforming Growth Factor-beta (TGF-β):

Transforming growth factor beta (TGF-β) is a multifunctional cytokine belonging to
the transforming growth factor and many other signaling proteins. TGFB proteins are
produced by all white blood cell lineages.it Regulates cell growth, differentiation, and
apoptosis.it Has a dual role, being both a growth inhibitor and promoter depending on
the context and cell type.

7. Nerve Growth Factor (NGF):

Nerve growth factor (NGF) is a neurotrophic factor and neuropeptide primarily
involved in the regulation of growth, maintenance, proliferation, and survival of
certain target neurons.it Promotes the survival, development, and function of neurons.
It is Essential in neural tissue culture.

Cell Signalling
Cell signaling is the process by which cells communicate with each other to coordinate their
actions. This involves the transmission of signals, such as hormones or neurotransmitters,
from one cell to another, allowing cells to respond appropriately to various stimuli and
maintain proper functioning within the body.
Methods of cell signalling
1. Paracrine signaling
Often, cells that are near one another communicate through the release of chemical
messengers (ligands that can diffuse through the space between the cells). This type of
signaling, in which cells communicate over relatively short distances, is known as
paracrine signaling.
Paracrine signaling allows cells to locally coordinate activities with their neighbors.
Although they're used in many different tissues and contexts, paracrine signals are
especially important during development, when they allow one group of cells to tell a
neighboring group of cells what cellular identity to take on.

2. Synaptic Signaling
In synaptic signaling, nerve cells transmit signals via a junction called a synapse.
When a neuron fires, an electrical impulse travels down the axon. Upon reaching the
synapse, it releases neurotransmitters that cross the gap between nerve cells. These
neurotransmitters bind to receptors on the receiving cell, causing a chemical change,
often by opening ion channels and altering the cell's electrical potential.

3. Autocrine signalling
Autocrine signaling is a form of cell signaling in which a cell secretes a hormone or
chemical messenger (called the autocrine agent) that binds to autocrine receptors on
 that same cell, leading to changes in the cell.[1] This can be contrasted with paracrine
signaling, intracrine signaling, or classical endocrine signaling.

4. Endocrine signalling
Endocrine signaling is a type of signaling. In this type of signaling, one cell secretes
the chemical which will affect other cells. These cells are located far from those cells
releasing the chemicals. Therefore, to transport the chemical to the target cells,
various means are used which highlight the critical role of this signaling function.
Endocrine signaling is linked to the body’s endocrine glands. It influences the
secretion of hormones, which travel through the body to reach their target cells. Upon
reaching the target cells, these chemicals initiate the hormone secretion process.

Cell surface markers

Cell surface markers are special proteins expressed on the surface of cells or
carbohydrates attached to the cell membrane that often conveniently serve as markers
of specific cell types. Cell surface markers are used to classify cells according to their
markers. Beyond the mere identification of cells, cell surface markers also hold the
potential to aid in drug discovery and especially personalized medicine.

Importance of cell surface marker:

1. Identification and Classification:

Cell surface markers identify and classify different cell types.

2. Cell Communication:
Facilitate communication between cells and their environment.

3. Immune Response Regulation:

Help distinguish between self and non-self-cells, aiding immune responses.

4. Disease Diagnosis and Monitoring:

Indicate the presence of specific cell types or disease states for early detection and
treatment.
5. Targeted Therapies:
Enable precise drug delivery to diseased cells, minimizing side effects.

6. Stem Cell Research and Therapy:

Isolate and identify different stem cell populations.

7. Tissue and Organ Transplantation:

Important in tissue typing to reduce the risk of rejection and improve transplant
outcomes.