Patient-Derived and Mouse Endo-Ectocervical Organoid Generation, Genetic Manipulation and Applications To Model Infection

PROTOCOL
https://doi.org/10.1038/s41596-022-00695-6
Patient-derived and mouse endo-ectocervical

organoid generation, genetic manipulation and
applications to model infection
Rajendra Kumar Gurumurthy1, Stefanie Koster 1
, Naveen Kumar 2
, Thomas F. Meyer 1,3
and
Cindrilla Chumduri 1,2 ✉
The cervix is the gateway to the upper female reproductive tract, connecting the uterus and vagina. It plays crucial roles in
fertility and pregnancy maintenance from onset until delivery of the fetus, and prevents pathogen ascension.
Compromised functionality of the cervix can lead to disorders, including infertility, chronic infections and cancers. The
cervix comprises two regions: columnar epithelium-lined endocervix and stratified squamous epithelium-lined ectocervix,
meeting at the squamocolumnar transition zone. So far, two-dimensional cultures of genetically unstable immortalized or
cancer cell lines have been primarily used to study cervix biology in vitro. The lack of an in vitro system that reflects the
cellular, physiological and functional properties of the two epithelial types has hampered the study of normal physiology,
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disease development and infection processes. Here we describe a protocol for cell isolation, establishment, long-term
culture and expansion of adult epithelial stem cell-derived endocervical and ectocervical organoids from human biopsies
and mouse tissue. These two organoid types require unique combinations of growth factors reminiscent of their in vivo
tissue niches and different culturing procedures. They recapitulate native three-dimensional tissue architecture and
patterning. The protocol to generate these organoids takes 4–6 weeks. We also describe procedures to introduce human
papillomavirus oncogenes into the cervical stem cells by genetic manipulation to model cervical cancer and infection of the
organoids with the highly prevalent sexually transmitted bacterial pathogen Chlamydia trachomatis. These organoid
systems open new possibilities to study cervix biology, infections and cancer evolution, and have potential applications in
personalized medicine, drug screening, genome editing and disease modeling.
Introduction
The female reproductive tract (FRT) comprises ovaries, fallopian tubes, uterus, cervix and vagina1.
Each FRT region has distinct tissue microenvironments, including epithelial, immune and stromal
compartments, which undergo specific changes during different stages of women’s reproductive
health, such as menstruation, pregnancy and postpartum1–3. These distinct microenvironments
control the homeostasis of the epithelial cells lining the mucosal surfaces that are morphologically
unique to each region of the FRT. The epithelial cells have evolved as sentinels and adapted to
perform specialized functions locally, such as transporting sperm and ova to the site of fertilization
and implantation and supporting the fetus through gestation and childbirth.
The cervix links the uterine cavity to the vagina. It comprises the endocervix, which is contiguous
with the uterus, and the ectocervix, projecting into the vagina. The endocervix is lined by columnar
epithelium comprising secretary and ciliated cells, while the ectocervix is lined by multilayered
squamous epithelium comprising basal, parabasal and differentiated superficial layers. These two
epithelia meet at the squamocolumnar transition zone (TZ). Recently we showed that the regen-
eration of the columnar and squamous epithelia and homeostasis of the TZ is controlled by distinct
WNT signaling from the stromal compartments1,4. The primary functions of the cervix include
facilitating the passage of sperm into the uterine cavity for fertilization and protecting the upper FRT
and the fetus from ascending infections. However, at the end of pregnancy, the cervix softens and
dilates to allow delivery of the fetus before reverting to its normal shape and consistency after the
delivery5. As an FRT gatekeeper, the cervical epithelium continually encounters bacterial, viral and
1
Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany. 2Chair of Microbiology, University of Würzburg,
Würzburg, Germany. 3Present address: Laboratory of Infection Oncology, Institute of Clinical Molecular Biology (IKMB), Christian–Albrechts
University of Kiel, Kiel, Germany. ✉e-mail: [email protected]
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NATURE PROTOCOLS PROTOCOL
fungal pathogens and responds with a robust innate immune response leading to pathogen clearance.
However, many pathogens have developed strategies to avoid or suppress innate defense, leading to
cervix and upper FRT pathologies, including cervicitis, polyps, condylomas, pelvic inflammatory
diseases, dysplasia and cancers. Cancers occurring in the cervical part of the FRT are the fourth
most common cancer and the second leading cause of death worldwide in women6. Cervical
cancer development is associated with high-risk human papillomavirus (HPV) and Chlamydia
trachomatis infections7,8.
The lack of in vitro systems that recapitulate the in vivo endocervical and ectocervical epithelium
has been a bottleneck to systematically deciphering the mechanisms underlying cervical pathologies,
including carcinogenesis and infection processes with viruses and bacteria such as human immu-
nodeficiency virus (HIV), HPV, Herpes simplex virus (HSV), Chlamydia trachomatis and Neisseria
gonorrhoeae9,10. Recent developments in adult stem cell-derived 3D organoid systems have made it
possible to generate organoids from various tissues11,12. Here we describe in detail our recently
published method to generate 3D organoids from columnar endocervical and squamous stratified
ectocervix-derived stem cells of human and mouse tissue4. These culture conditions can also be
employed to propagate cervical cancer organoids derived from adeno and squamous cell carcinomas.
These organoids recapitulate the architecture, cellular composition and functional properties of the
native tissue and can be maintained long term. In addition, we provide our newly established
methods for the genetic manipulation of ectocervical organoids and application of ectocervical and
endocervical organoids for Chlamydia trachomatis infection studies13. We also detail how these
organoid cultures can be used to study cervix biology and model host–pathogen interactions and
disease development.
Development of human and mouse endocervix and ectocervix organoid culture protocol
Despite intense research in cervix biology, the molecular features of the cell types that make up the
endocervix lined with simple columnar epithelium and adjoining ectocervix lined by stratified
squamous epithelium have been unclear. Thus, to define the basic cellular features of the endocervix
and ectocervix, we performed single-cell RNA sequencing of the mouse tissue. We defined the
columnar and squamous epithelial subpopulations and demonstrated that these two epithelial
lineages arise from distinct cytokeratin 8 (KRT8)+ and KRT5+ lineage-specific stem cells4. We
identified that the endocervical and ectocervical stroma comprises unique stromal cell subpopulations
that provide distinct signals to their epithelial counterparts.
Applying single-molecule RNA in situ hybridization analysis, we revealed a distinct spatial dis-
tribution of Wnt agonists and antagonists in the underlying stroma of the endocervix and ectocervix,
which defined the borders between these two epithelia at the TZ. The Wnt signaling agonists Rspo1
and its downstream target Axin2 were highly expressed in the stroma beneath the columnar epi-
thelium, and Rspo3 was expressed in the endocervical muscularis. Interestingly, Wnt antagonist Dkk2
expression was restricted to the stroma proximal to the ectocervical basal stem cells. Together, these
data revealed that, in vivo, the differential proliferation of endocervical and ectocervical epithelial
stem cell populations is maintained by opposing Wnt-signaling microenvironments controlled by the
tight interaction of epithelial and stromal cell compartments4. On the basis of our insights into the
spatial organization of the niche of endocervical and ectocervical epithelia and knowledge from
developmental studies and various factors known to play a role in the regulation of adult stem cells
in diverse tissues14–17, we developed culture conditions that support the formation of adult stem
cell-derived endocervical and ectocervical organoids and their long-term expansion.
We used endocervical or ectocervical biopsies of healthy cervices from women undergoing
standard surgical procedures or from mice to develop cervical organoids. We used Matrigel as an
extracellular matrix substitute. We tested various growth factor cocktails that mimic the cervix tissue-
specific stem-cell niches, including the canonical Wnt agonists WNT3A and R-spondin-1 (RSPO1),
fibroblast growth factor 10 (FGF-10), epidermal growth factor (EGF), hydrocortisone, the cyclic
adenosine monophosphate (cAMP) pathway agonist forskolin (FSK), the bone morphogenetic
protein (BMP) signaling inhibitor noggin, nicotinamide, ROCK inhibitor (Y-27632) and the trans-
forming growth factor-β (TGF-β) pathway inhibitor A83-01.
Our analysis revealed that the efficient growth and long-term expansion of ectocervical squamous
stratified organoids depend on EGF, FGF-10, TGF-β inhibitor and active BMP signaling. Unlike
many other adult stem cell-derived organoids, the addition of WNT3A and R-spondin-1 to the
ectocervical organoid medium inhibited the growth and long-term expansion. Furthermore, FSK,
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PROTOCOL NATURE PROTOCOLS
which synergizes the EGF pathway through increased cAMP levels, enhanced the ectocervical
organoid’s growth rate. In line with our transcriptomic analysis, squamous epithelial stem cells
mainly depend on the active EGF signaling pathway. Notably, our analysis revealed that the Notch
pathway is essential for the differentiation and stratification of the squamous epithelial layers. Basal
stem cells were found to express a high level of Notch ligands, and parabasal cells destined to
form differentiated cells express a high level of Notch receptors. Inhibition of Notch activation by
a γ-secretase inhibitor (dibenzazepine) resulted in organoids without stratified layers.
While we were able to generate human ectocervical organoids directly from the isolated stem cells,
we observed enrichment and long-term propagation of squamous epithelial stem cells when cultured
in 2D along with lethally irradiated mitotically inactive 3T3-J2 fibroblast feeder cells18. These ecto-
cervical epithelial stem cells could be directly biobanked or used for generating 3D organoids by
placing them in Matrigel with the ectocervical organoid medium. Mature ectocervical organoids
resembled the original tissue architecture. They consist of the multilayered KRT5+, KRT17+ and
cystatin A (CSTA)+ epithelium with proliferative P63+ basal stem cells, differentiated parabasal,
intermediate cell layers and enucleated superficial cells residing in the lumen of organoids. Ecto-
cervical organoids from mice were obtained by directly culturing the stem cells isolated from tissue in
Matrigel in mouse ectocervical organoid medium. Similar to human ectocervical organoids, mice
organoids resemble the native tissue.
In contrast to ectocervical organoids, generation and maintenance of endocervical organoid cul-
tures require WNT3A and RSPO1 in addition to EGF, FGF-10, active BMP signaling, TGF-β and
ROCK inhibitors4. The endocervical organoids are hollow structures composed of a monolayer of
columnar, polarized cells with sporadically proliferating cells recapitulating endocervix epithelium
in vivo. The endocervical organoids express characteristic markers of their parent tissue, including
KRT8, KRT7, KRT19, AGR2 and GDA. Using these culture conditions and the defined medium, we
could propagate human and mouse endocervical and ectocervical organoids for more than 6 months.
Experimental design
This detailed protocol describes the recently established methods for generation and long-term
expansion of human and mouse endocervical and ectocervix 3D organoids, their genetic manip-
ulation, and infection with Chlamydia trachomatis. The workflow of the experimental protocol is
depicted in Figs. 1a–e, 3a and 4a,b. This protocol is separated into six separate procedures, covering:
the generation of mouse endocervical and ectocervical organoids from cervical tissue (Procedure 1),
the generation of human endocervical organoids from tissue biopsies (Procedure 2), the culturing of
the human ectocervical organoids from tissue biopsies and epithelial stem cells in 2D in the presence
of 3T3-J2 feeder cells and subsequent generation of 3D organoids (Procedure 3), the genetic
manipulation of human ectocervical stem cells (Procedure 4) and the infection of human endo-
cervical and ectocervical organoids with Chlamydia trachomatis (Procedures 5 and 6, respectively).
This section describes the experimental aspects that should be considered before adopting the pro-
tocol. Before following this protocol, it is essential to obtain institutional human ethics permission
and informed patient consent to use the tissue for scientific use. Necessary approvals from national,
institutional and local authorities must be obtained before conducting animal experiments. For
Chlamydia trachomatis infection experiments and genetic manipulation of primary cells, it is essential
to obtain approval from the institutional genetic engineering committee.
Starting material
Humans have a single simplex uterus, while mice have a bicornuate uterus (uterine horns)
(Fig. 1a–d). Both species have ectocervix, endocervix and squamocolumnar TZ with a single vagina
(Fig. 1a–d). Tissue biopsies for human endocervix and ectocervix organoid cultures were obtained
from anonymous donors from standard surgical procedures. The current protocol tested biopsies
derived from donors aged between 35 and 75 years. Although not tested in our studies, the age and
menstrual status of the donor might influence the organoid-forming efficiency. The primary epithelial
cells were isolated from cervical tissue biopsies within 2–3 h of surgical removal. Parts of the tissue
were preserved for histological analysis, while DNA and RNA were isolated for validation purposes.
HPV status of patients was determined by subjecting the DNA samples to PCR amplification with
HPV type-specific primers targeting carcinogenic HPVs (16, 18, 31, 33, 35, 45, 51, 52 and 58),
probably carcinogenic HPVs (53 and 66) and noncarcinogenic HPVs (6, 11, 40, 42, 43, 44 and 54).
Isolation of epithelial cells from both endocervix and ectocervix was performed using collagenase II

a b c d
Fallopian tube Ovary Cervix
Ovary
Fallopian
tube
Uterine horn Endocervix
Endocervix Uterine corpus Endometrium
Cervix
TZ TZ
Endocervix
Ectocervix
Cervix TZ
Ectocervix
Ectocervix
Vagina
Vagina
e
Tissue Single cells
Immunohistochemistry
Endocervical cells Columnar Transcriptome/genomic

organoid analysis
Matrigel
Ectocervical cells Protein analysis
Host–pathogen interaction
Epithelial Stratified Genetical manipulation

Epithelial organoid
cells cells
Matrigel
Collagen
Biobank
Irradiated feeder cells
Fig. 1 | Schematic representation of endo-ectocervical stem cell isolation, organoid generation, cultivation and characterization. a,b, Schematic
representation of mouse FRT (a) and representation of endocervix, ectocervix and TZ regions of mouse FRT for dissection (b). c,d, Schematic
representation of human FRT (c) and cervix tissue cut into pieces according to the endocervix, ectocervix and TZ region (d). e, Single cells isolated
from ectocervical tissue can be cultured in 2D to enrich stem cells and propagate on feeder cultures and as organoids, while cells isolated from
endocervix are directly used to culture organoids. The established cultures can be characterized by various molecular techniques, genetically modified,
used to study host–pathogen interactions, and cryopreserved.
and TrypLE treatment. Specimens obtained from either punch biopsy or surgically removed tissue
were suitable for isolating primary cells and generating organoids.
Mouse FRT was obtained from euthanized 5- to 20-week-old female wild-type C57BL/6J mice
(Fig. 1a,b). Although we have not tested this, we speculate that the procedure can be extended to
other age groups of mice. We separate the ectocervix, TZ and endocervix from the FRT on the basis
of the physical characteristics of the tissue as follows. The ectocervix is first located on the basis of its
proximity to the uterine horn. The tissue texture of the ectocervix is hard, dense and fibrous, unlike
the columnar endocervix and uterine horns. In mice, there is no clear anatomical definition of the
endocervix and uterine epithelial borders. Therefore, we chose the region close to uterine horn
bifurcation and dissected endocervical tissue. Distal to the ectocervix lies the vagina, which is hollow.
We dissect an area that physically overlaps with the defined ectocervix and the endocervix extending
toward the uterine horns. Thus, the adjoined TZ is included within the obtained tissue sample.
Further, the vaginal part is left out from the ectocervical bulb region. These tissues are minced, and
epithelial cells are isolated by collagenase II and TrypLE treatments.
Maintenance of organoid cultures

We successfully generated organoids from the endocervix and ectocervix of humans and mice
(Fig. 2a–e) using single cells isolated from the tissue combined with organoid medium optimized for
each epithelial type. In human ectocervix, expansion of stem cells was also optimized in 2D culture
with lethally irradiated 3T3-J2 mouse fibroblasts (Fig. 2d). Three-dimensional ectocervical organoids
were generated by embedding cells isolated from the tissues or the stem cells grown on feeder cultures
in 2D. Early passages of each tissue-specific organoid and 2D stem cell cultures were biobanked for
future study purposes. In contrast to ectocervix, organoids from endocervix were generated directly
from cells isolated from tissue biopsies without prior expansion with feeder cells in 2D (Fig. 1e).
Similarly, the mouse endocervix and ectocervix organoids were established from cells isolated from
the tissues, and these were cultured for the long term.

Mouse Human
Endocervical organoids Ectocervical organoids Endocervical organoids Ectocervical stem cells Ectocervical organoids
a b c d e
200 µm 200 µm 600 µm 150 µm 150 µm
f Endocervical organoids g Ectocervical organoids h i

KRT5
Ectocervical
Ectocervical
organoids
organoids
200 µm 200 µm
KRT8
j k
Endocervical
Endocervical
organoids
organoids
DNA
25 µm 25 µm
500 µm 500 µm
Fig. 2 | Human and mouse endo-ectocervical organoids. a,b, Phase-contrast images of mouse endocervical (a) and ectocervical (b) organoid cultures.
c–e, Phase-contrast images of human endocervix organoids (c) and of ectocervical stem cells maintained on 3T3-J2 cells (d) and as organoids (e).
f,g, Representative confocal images of human endocervical (f) and ectocervical (g) organoids stained for columnar epithelial marker KRT8 (abcam cat.
no. ab59400; RRID: AB_942041) and stratified squamous lineage marker KRT5 (abcam cat. no. ab193894; RRID: AB_2893023), and nuclei (Hoechst).
h,i, Phase-contrast images of human ectocervical organoids representing 3D-to-2D formation in Matrigel (h) and seeding at higher density (i).
j,k, Phase-contrast images of human endocervical organoids representing 3D-to-2D formation in Matrigel (j) and seeding at higher density (k).
The yellow arrows point to the 2D epithelial cells.
Validation
Upon establishing organoids, part of the organoid cultures should be biobanked at early passages for
future use. Further, part of the organoids should be used for DNA and RNA extraction, fixation and
embedding for later microscopic evaluation. We recommend the following validation experiments for
the generated organoids.
● HPV testing: to determine the HPV status of human donor tissue specimens, we recommend isolating
DNA from all samples and subjecting the DNA to PCR amplification with HPV type-specific primers
targeting carcinogenic HPVs (16, 18, 31, 33, 35, 45, 51, 52 and 58), probably carcinogenic HPVs
(53 and 66) and noncarcinogenic HPVs (6, 11, 40, 42, 43, 44 and 54). Primer sequences are listed in
Herfs et al.19. We perform PCR as described in the procedure step describing validation of
oncogene integration.
● Lineage validation: the endocervix and ectocervix organoids have distinct morphology under a bright-
field microscope. Ectocervical organoids are dense and multilayered, while the endocervix organoids
are hollow, single-layered, and relatively larger than the ectocervical organoids grown for a similar
time. However, we recommend confirmation of the organoid lineage by immunohistochemistry (IHC)
using lineage-specific KRT5 and KRT8 markers for the stratified and columnar epithelia, respectively.
For this, we fix 14-day-old organoids derived from the endocervix and ectocervix with 4%
paraformaldehyde (PFA), paraffinize, cut into thin sections and immunostain for KRT8 (endocervix)
and KRT5 (ectocervix) (Fig. 2f–g)4.
Genetic manipulation of organoids

Genetic manipulation of cells is one of the fundamental techniques to understand the biological
function of a gene. It provides important insights into the function of a gene or mutation and how it
contributes to disease development and affects human physiology. Commonly liposomal transfection,
viral gene transduction, RNA interference or CRISPR/Cas-mediated gene editing are employed for
genetic manipulations to study the influence of a specific gene or its downstream target genes. Here
we describe the lentivirus-mediated transduction and integration of HPV16 E6E7 into healthy
ectocervical epithelial stem cells to mimic persistent or latent HPV infection (Fig. 3a,b). For genetic
manipulation of ectocervical epithelial cells, dissociation into single cells is necessary. Therefore,
primary cells are separated by digestion with TrypLE and seeded on a collagen-coated cell culture
plate. Cells should be seeded at a density to reach at least 60% confluency at the time of transduction.

a
pLXSN E6E7 Lentiviral particles
psPAX2
pMD.2G (VSVG)
293T cells
Selection
Epithelial
cells
Collagen
Validation
b c d
hCEcto hCEctoE6E7 End1 Water

– + + – HPV16 E6E7
HPV16
GAPDH
150 µm 150 µm
Fig. 3 | Genetic manipulation of human ectocervical stem cells and organoid generation. a, Schematic representation for lentivirus-mediated genetic
manipulation of patient-derived ectocervical stem cells. b, Validation by PCR for HPV16 and GAPDH of hCEcto (human ectocervical stem cells),
hCEctoE6E7 (HPV16 E6E7 transduced hCEcto cells), End1 (End1 cell line is a positive control for HPV16 E6E7) and water (a negative control).
c,d, Representative phase-contrast images of genetically modified ectocervical stem cells positive for HPV16 E6E7 grown on 3T3-J2 cells (c) and 10 d
organoids (d) grown from these stem cells in Matrigel.
We show that the genetically manipulated HPV16 E6E7 transgene expressing ectocervical stem cells
retain organoid formation capacity (Fig. 3d) and can be maintained in 2D feeder culture in the long
term (Fig. 3c).
It is strongly recommended to include controls for transfection and transduction efficiency, such
as GFP-expressing plasmids, before genetic manipulation. We also recommend including an
untreated control and a control treated only with the selection marker beside the transgene-carrying
lentiviruses. If chemical components such as polybrene are used to enhance transduction efficiency,
control treatment with the chemical should also be included. Human ectocervical organoids
expressing HPV16 E6E7 allow us to study the long-term effects of virus infection on the cervical
squamous epithelium and the contribution of co-infections with other pathogens, such as Chlamydia
trachomatis, toward the initiation and progression of epithelial cell transformations.
Chlamydia trachomatis infection of organoids

We describe our newly established protocols to infect the human endocervical and ectocervical
organoids with Chlamydia trachomatis (Fig. 4a–d)13. The Gram-negative obligate intracellular
bacterium Chlamydia trachomatis is one of the most prevalent sexually transmitted infections,
causing several diseases, including infertility, ectopic pregnancy, pelvic inflammatory disease and
cervicitis, and has been identified to play a role in cervical and ovarian carcinogenesis20,21. The
established cervical organoid cultures provide an excellent opportunity to study infection processes
and their effect on the cervical epithelium in near-physiological conditions. We found that efficient
infection of endocervical organoids is obtained by incubating organoid fragments with Chlamydia
trachomatis. Therefore, before performing the infection, endocervical organoids are fragmented when
they have reached the appropriate size for expansion. Infection of ectocervical organoids is different
and requires the single cells to be embedded in Matrigel for 5 d to form organoids of an intermediate
size. They are then removed from the Matrigel and co-incubated with Chlamydia trachomatis for 2 h

a b
Endocervical columnar Ectocervical stratified
organoid organoid
Matrigel Matrigel
C. trachomatis
C. trachomatis
Organoid
fragment
Infected columnar Infected stratified

organoid organoid
Epithelial cell
infected with Epithelial cell
C. trachomatis infected with
C. trachomatis
Uninfected
epithelial cell Uninfected
epithelial cell
c Human endocervical organoids d Human ectocervical organoids e Human ectocervical organoids

Uninfected
100 µm 50 µm 50 µm
C. trachomatis
100 µm 100 µm 50 µm
Fig. 4 | Chlamydia trachomatis infections of endocervical and ectocervical organoids. a,b, Schematic representation for Chlamydia trachomatis
(C. trachomatis) infection of endocervical (a) and ectocervical (b) organoids. c,d, Representative wide-field microscopic fluorescence images of human
endocervical (c) and ectocervical (d) organoids uninfected or 5 d post-infection with GFP-Chlamydia trachomatis (green). e, Human ectocervical
organoids uninfected or 5 d post-infection with mCherry-Chlamydia trachomatis K strain (red).
to achieve an efficient infection before plating back into Matrigel. When planning infection experi-
ments, care should be taken to include appropriate uninfected controls cultured in the same medium
as the infected sample.
Comparison with other methods

Another study22 also described a protocol for generating and propagating adult stem cell-derived
organoids from human endo- and ectocervix. There are some differences between the Lohmussaar
et al. protocol and our protocol4,22 in terms of tissue digestion, the extracellular matrix used for
organoid generation and the medium composition used for endo- and ectocervical organoids.
Lohmussaar et al. use the Wnt agonist (RSPO1) in the ectocervical medium, in contrast to our
protocol4,22, where we have found Wnt signaling agonists WNT3A and RSPO1 to be inhibitory to the
growth of human ectocervical organoids. Further, their ectocervical organoid medium composition

includes FGF7 and p38 inhibitor and does not include EGF, in contrast to our ectocervical medium.
Similar to our endocervical organoid culture4,22, Lohmussaar et al. found that the addition of Wnt
agonists is critical for their generation and long-term culture. However, in contrast to WNT3A used
in our endocervical medium, their endocervical medium consists of WNT surrogates (U-Protein
Express, cat. no. N001) and CHIR (Stemgent, cat. no. 04-0004-10) for WNT pathway activation in
addition to β-estradiol. While our organoids were generated by plating cells in Matrigel, Lohmussaar
et al. used Cultrex BME RGF type 2 from Trevigen (cat. no. 3533-005-02).
Other strategies also have been used for genetic manipulation of organoids, including short
hairpin RNA (shRNA)-mediated stable knockdown23 and targeted genome editing by CRISPR/
Cas924. Stable gene knockdowns were achieved in fallopian tube epithelial cells by transduction of
cells in 2D culture with a replication-deficient virus containing specific shRNAs. Once selected, the
shRNA expressing 2D cells were seeded in Matrigel for the generation of organoid lines. For genetic
manipulation using CRISPR/Cas9, fallopian tube organoids were fragmented and electroporated with
a vector containing the guide RNA (gRNA) directed against the gene of interest; then, cells were
plated for organoid recovery. These methods can be applied for sequence-specific genome editing of
endocervical organoids since endocervical and fallopian tube organoids are monolayered columnar
epithelium and can be propagated using fragmentation. However, for stratified ectocervical
epithelial organoids, 2D stem cell cultures can be used for efficient genetic manipulation followed by
generation of 3D organoids similar to the protocol described for organids expressing HPV16 E6E7 in
this protocol.
C. trachomatis infection models have also been established for murine endometrial organoids and
human fallopian tube organoids that recapitulate the columnar epithelium of the native tissue. Two
methods have been developed to infect these columnar organoids with C. trachomatis efficiently. The
bacterium can be microinjected into the lumen of murine endometrial organoids25. This method has
the advantage that the integrity of the organoid is minimally disturbed and the multiplicity of
infection (MOI) can be defined more precisely, resulting in minimal differences between replicates.
However, microinjection is time consuming, and the handling is laborious.
Alternatively, as we described here in our protocol for the infection of endocervical organoids,
fallopian tube organoids are broken into fragments before infection, which are then incubated in
suspension with the pathogen for a specific time. The fragments are then re-embedded in Matrigel
and overlaid with medium26. Compared with infection by microinjection, here the actual MOI may
differ from the calculated MOI. Further, the integrity of the organoids is disrupted, but owing to the
organoid properties of self-renewal, the fragments re-form into complete organoids after a short
time27,28. Thus, infection by fragmentation is efficient for C. trachomatis infection of columnar
organoids of the FRT. While both microinjection- and fragmentation-based infection protocols were
not ideal for stratified ectocervical organoids, no alternate protocols have been described so far for
Chlamydia infections of ectocervical organoids.
Future applications of endocervical and ectocervical organoids

The endocervical and ectocervical organoids are ideal 3D near-physiological epithelial tissues to study
and model cervix biology, host–pathogen interactions and disease development. The microbiota plays
a vital role in the physiology and function of the cervix. Endocervical and ectocervical organoids allow
in vitro recapitulation of the in vivo interactions between epithelial tissue and microbes, including
commensal, symbiotic, pathogenic bacteria, viruses and fungi. Owing to the complex 3D structures of
epithelial organoids comprising multiple cellular subtypes, they facilitate understanding of coloni-
zation dynamics, invasion of infections and cellular tropism of various pathogens.
In addition to single infections, co-infections are often discovered in clinical practices and can have
effects ranging from antagonistic to synergistic in disease development. Thus, modeling co-infection
of Treponema pallidum (syphilis), Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas
vaginalis, HPV, HSV, HIV, etc. will provide insights into cervical disease development and enable
formulation of treatments or preventive strategies. Further, these organoids are ideal for investiga-
tions into host-targeted mechanisms against antibiotic-resistant pathogens. Cervical mucus con-
stitutes a defined chemical composition that prevents pathogen ascension yet supports spermatozoa’s
entrance into the uterus29. Hormonal changes are known to influence mucus production and
composition, resulting in altered susceptibility to infections29. These organoids therefore will allow
researchers to investigate cervix epithelial response to hormonal changes and their impact on stem
cell regeneration, mucus production and innate defense against pathogens.

The long-term culturability of organoids also offers a unique opportunity to study chronic or
repeated infections and their influence on host cells. Previously, the tissue explant culture systems
have been used to study the molecular changes with epithelial cells and pathogen interactions with
cervix tissue30,31. Even though this method provides a natural infection model, the limited availability
of tissue for explant culture and their limited lifespan makes it challenging to study the effects of
chronic infections on epithelial cells. This problem can be overcome by the 3D organoid models, as
organoids can be generated and expanded from small tissue biopsies; the infected organoids could
be cultured for the long term, thus enabling investigations of molecular changes associated with
chronic infections.
Further, the organoid models offer an excellent opportunity to study different stages of histo-
pathological and molecular changes of the epithelium during cervical cancer development, including
metaplasia, cervical intraepithelial neoplasia and cancers. The epithelial, immune or stromal inter-
actions can be modeled by adapting these organoids for co-cultures with immune or stromal cells or
mimetics recapitulating tissue microenvironmental signals in healthy homeostasis and disease
development. Further, generating organoids from all subtypes of cervical cancers would be invaluable
for drug testing and developing immunotherapy approaches, as well as for personalized medicine. We
have demonstrated that cervical organoids are amenable for genetic manipulation. Hence, they can be
used for genome editing with lentiviruses or CRISPR/Cas9 systems to study the effects of gene
knockouts, knockins or point mutations on tissue biology and disease development. Integration of
cancer-specific mutation signatures would allow the role of cancer driver mutations in epithelial cell
homeostasis changes to be studied and to perform drug screens.
Potential limitations of endocervical and ectocervical organoids

One of our organoid model’s limitations, similar to other organoid models, is that it is devoid of
microenvironmental signals providing a niche compared with in vivo epithelium. Since adult stem
cell-derived organoids are composed of only epithelial cells, the microenvironment provided by the
stromal cells, immune cells and blood vessels is absent in this system. However, different techniques
have been used to mimic tissue microenvironmental conditions to recapitulate in vivo conditions,
including co-culturing organoids with immune cells, stromal cells or secreted components of niche
cells32. Our models can use these approaches to study the effect of tissue microenvironmental signals
in healthy homeostasis, infections and disease development.
Certain pathogens depend on the apical surface of the epithelium to establish infections. For
ectocervical organoid infections, only the basal stem cell layer and basal surface of these cells are
accessible to pathogens and might limit certain infection experiments that strictly depend on the
apical surface. To overcome this in our previous publication33, we adapted the human ectocervical
stem cells to grow on transwell-based air–liquid interface cultures, where the luminal apical surface of
the stratified epithelium is accessible to infections. However, the infection protocol we provide here
for endocervix supports the infections of the epithelium’s apical and basal surfaces. Overall, the
cervical organoid model and adaptation of our protocols would offer great potential for further
advancing the study of FRT biology.
Materials
! CAUTION Wear proper personal protective equipment and refer to material safety data sheets of
chemical reagents before use ! CAUTION Follow biosafety precautions whenever experimenting with
biological material and infectious or potentially infectious agents. Decontaminate all working places
before and after work, including all liquid or solid waste that came in contact with the infectious agent,
and immediately decontaminate spills. Use a biological safety cabinet, appropriate protective laboratory
coats and disposable gloves. If gloves become contaminated, change them immediately. Wash hands
after handling infectious agents and removal of gloves, and before leaving the laboratory. Minimize the
creation of aerosols, droplets or accidental release, and limit the working area. When the biohazardous
material needs to be transported outside the laboratory, use a secondary container that can be closed and
follow national or international rules and regulations.
Biological materials
! CAUTION All disposal material and liquid waste must be autoclaved when working with biological
materials. Tissue residue and suspensions that may contain cells or cell debris must be decontaminated
with Korsolex before autoclaving. For this purpose, waste bottles can be prepared with the appropriate

volume of Korsolex, and all liquids produced when working with biological material can be transferred
to these waste bottles, which can be easily autoclaved.
Primary tissue
● Samples of healthy human and mouse cervical tissue, removed by surgery ! CAUTION Ethical approval
from the local authority must be obtained ! CAUTION Human body fluids and tissue samples may
contain viruses and other agents. Hence, they pose a risk of infection if not handled with care.
Therefore, appropriate personal protective equipment, access to containment level 2 facilities and
cooperation between scientists and the clinical team are essential CRITICAL Our human cervix
c
samples were obtained from the Department of Gynecology, Charité University Hospital, Campus
Virchow Clinic, and August-Viktoria Klinikum Berlin, Department of Gynecology and Obstetrics. The
scientific usage of human tissue samples was approved by the Charité University Hospital’s ethics
committee, Berlin (EA1/059/15), and informed consent to use their tissue samples was obtained from
all participants CRITICAL The scientific usage of mouse tissue samples was approved by the national
c
legal, institutional and local authorities at Max Planck Institute for Infection Biology. All procedures
involving animals were performed in accordance with the ethical regulations for research on animals
CRITICAL Stem cells were isolated within 2–3 h of tissue removal.
c
Cell lines
● 3T3-J2 cells (kind gift from Craig Meyers; Howard Green laboratory, Harvard University, RRID:
CVCL_W667) are required for maintaining ectocervical stem cells in 2D
●
293T cells (ATCC CRL-11268; RRID: CVCL_1926) are used for WNT3a/R-spondin-1 testing and to
produce lentiviral particles for the genetic manipulation of primary cells
● L-Wnt3a cells (ATCC CRL-2647; RRID: CVCL_0635) and 293T-HA-Rspo1-Fc cells (kind gift from
Clevers Lab, Netherlands) are required to produce WNT3a- and R-spondin-1-conditioned medium,
respectively34,35
● End1/E6E7 cells (ATCC CRL-2615; RRID: CVCL_3684) were used as a positive control for validation
of oncogene integration ! CAUTION The cell lines should be tested regularly for mycoplasma
contamination. Furthermore, it is advisable to use cell lines in early passages.
Bacteria strains
●
For infection experiments, we use Chlamydia trachomatis LGV L2 from lymphatic isolate 434 Bu
(ATCC VR-902B) that expresses GPF and Chlamydia trachomatis K strain that expresses mCherry26
! CAUTION Handling of infectious agents such as Chlamydia trachomatis requires appropriate
personal protective equipment, and work must be done at containment level 2 as Chlamydia
trachomatis is a human pathogen ! CAUTION Ethical and gene technological approval from the local
authority must be obtained CRITICAL All Chlamydia trachomatis infection experiments with
c
human organoids were approved by the ethics committee of the Charité University Hospital, Berlin
(EA1/059/15) and the GenTech committee of the LAGeSo, Berlin (269/00-14; 269/00-00)
CRITICAL For prolonged storage, Chlamydia trachomatis infectious progeny should be aliquoted
c
and stored at −80 °C36.
Plasmids
● For genetic manipulation of ectocervical stem cells, we transfected 293T cells with pMD2.G (Addgene,
RRID: Addgene_12259) and packaging plasmid psPAX2 (Addgene, RRID: Addgene_12260) together
with pLXSN HPV16 e6e7 (Addgene, RRID: Addgene_52394) that encodes HPV16 E6E7. The plasmids
pMD2.G and psPAX2 were deposited in Addgene by Didier Trono, and pLXSN16E6E7 by Denise
Galloway.
Reagents
Culture reagents
● Dulbecco’s phosphate-buffered saline, without Ca2+ and Mg2+ (Gibco, cat. no.14190-169)
●
Collagenase type II (Calbiochem, cat. no. 234155)
CRITICAL Since collagenases have different enzymatic profiles and the protocol described here is
c
established for type II, we do not recommend using an alternative type.

● TrypLE Express (Gibco, cat. no. 12605-028)

●
Advanced DMEM/F12 (ADF; Gibco, cat. no. 12634)
● Glutamax, 100× (Gibco, cat. no. 35050-038)
● HEPES (Gibco, cat. no. 15630-056)
● Collagen type I, from rat tail (Sigma, cat. no. C3867)
CRITICAL Since the existing collagen types have different functions, using a different type than type
c
I could influence primary cell growth behavior.
● R-spondin-1-conditioned growth medium (see ‘Reagent setup’)
● WNT3a conditioned growth medium (see ‘Reagent setup’)
● Human EGF (Gibco, cat. no. PHG0311)
● Mouse EGF (Invitrogen, cat. no. PMG8041)
● Human Noggin (Peprotech, cat. no. 120-10C)
●
Mouse Noggin (Peprotech, cat. no. 250-38-100)
● Human FGF-10 (Peprotech, cat. no.100-26-B)
● B27 supplement, 50× (Gibco, cat. no.17504044)
● N2 supplement, 100× (Gibco, cat. no.17502048)
● Nicotinamide (Sigma, cat. no. N0636)
● N-acetyl-L-cysteine (Sigma, cat. no. A9165-5G)
● TGF-β inhibitor (A83-01) (Calbiochem, cat. no.616454)
● ROCK inhibitor (Y-27632 dihydrochloride monohydrate) (Sigma, cat. no. Y0503)
● FSK (Sigma, cat. no. F6886)
● Hydrocortisone (Sigma, cat. no. H0888-1G)
● Penicillin–streptomycin, 10,000 U/mL (Gibco, cat. no.15140-12)
! CAUTION Penicillin–streptomycin solution is suspected of damaging fertility or the unborn child;

avoid skin contact by wearing protective gloves/protective clothing/eye protection/face protection.
● DMEM (Gibco, cat. no. 10938-025)
● Fetal calf serum (FCS; Biochrome, cat. no. S0115)
● Sodium pyruvate (Sigma, cat. no. S8636)
● 0.05 % (wt/vol) Trypsin–EDTA (Gibco, cat. no.25300-096)
● Mitomycin C (Fisher Scientific, cat. no.10182953)
! CAUTION Mitomycin C is toxic if swallowed and suspected of causing cancer; avoid skin contact and
inhalation by wearing protective gloves/protective clothing/eye protection/face protection.
● Matrigel (basement membrane matrix, growth factor reduced) (Corning, cat. no. 356231)
CRITICAL The protocols listed here were established using Matrigel. The use of another substitute
c
may affect organoid growth.

●
Cryo-SFM (Promo cell, cat. no. C-29910)
● Zeocin (100 mg/ml), (Invitrogen cat. R250-01)
● L-Glutamine (Gibco, cat. no. 25030-024)
● Na-pyruvate (Sigma, cat. no. S8636)
● Hank’s balanced salt solution (Gibco, cat. no. 14025-092)
● Poly-L-lysine (Merck, cat. no. A-005-C)
● Korsolex (Carl Roth, cat. no. AK41.1)
! CAUTION Korsolex is harmful if swallowed or inhaled. It causes severe skin burns and eye damage,
and it may cause an allergic skin reaction, asthma symptoms or breathing difficulties if inhaled. It is
suspected of causing genetic defects and may cause cancer. Further, it is toxic to aquatic life with long-
lasting effects. Thus, avoid breathing gas/mist/vapours/spray and wear protective gloves/protective
clothing/eye protection/face protection.
Chemical reagents for genetic manipulation

●
FuGENE 6 (Promega, cat. no. E2691)
! CAUTION FuGENE is highly flammable; avoid skin contact by wearing protective gloves/protective
clothing.
● Opti-MEM (Thermo Scientific, cat. no. 31985070)
● Polybrene (Sigma, cat. no. H9268)
● Blasticidin (Gibco, cat. no. A11139-03)
! CAUTION Blasticidin is harmful if swallowed; avoid skin contact by wearing protective gloves/
protective clothing, and wash hands thoroughly after handling.

Chemical reagents for PCR and agarose gel electrophoresis
● DNA gel loading dye, 6× (Fermentas, cat. no. R0611)
● dNTPs (Thermo Scientific, cat. no. R0182)
● Taq reaction buffer (New England Biolabs, cat. no. B9014SVIAL)
● Taq DNA polymerase 5,000 U/mL (New England Biolabs, cat. no. M0273AVIAL)
●
GeneRuler 1 kb DNA ladder (Thermo Scientific, cat. no. SM0312)
● GeneRuler 50bp DNA ladder (Thermo Scientific, cat. no. SM0371)
● LE agarose (Biozym, cat. no. 840004)
● MgCl
2 (Promega, cat. no. A351H)
● DNAse/RNAse-free water (Invitrogen, cat. no. 10977049)
● Tris base (AppliChem, cat. no. A1086.5000)
● Boric acid (Roth, cat. no. 6943.3)
! CAUTION Boric acid is suspected of damaging fertility or the unborn child; avoid skin contact by
wearing protective gloves/protective clothing/eye protection/face protection.
● EDTA-Na
2 (Roth, cat. no. 8043.2)
● Ethidium bromide solution (Roth, cat. no. 2218.2)
! CAUTION Avoid exposure to ethidium bromide solution as it is suspected of causing genetic defects;
use a fume hood, and wear protective gloves/protective clothing/eye protection/face protection.
Primers for PCR

● Primer sequences to test for HPV status were used as previously published19. The following primers
were used in this protocol:
○ GAPDH forward: 5′-GGTATCGTGGAAGGACTCATGAC-3′
reverse: 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′
○ HPV16 forward: 5′-AGCTGTCATTTAATTGCTCATAACAGTA-3′
reverse: 5′-TGTGTCCTGAAGAAAAGCAAAGAC-3′
Chemical reagents for IHC

● Mowiol 4-88 (Sigma, cat. no. 81381)
● PFA (Sigma, cat. no.4441244)
! CAUTION PFA is highly toxic; avoid contact with skin, eyes or mucous membranes. It is harmful if
swallowed or inhaled, causes severe skin burns and eye damage, may cause an allergic skin reaction and
breathing difficulties if inhaled, is suspected of causing genetic defects and may cause cancer. Thus,
avoid breathing gas/mist/vapours/spray and skin contact, and wear protective gloves/protective
clothing/eye protection/face protection and a mask (P2 level).
●
Ethanol (Merck, cat. no. 1.00983.2511)
● 2-Propanol (Merck, cat. no. 1.09634.2511)
● Xylene (Roth, cat. no. 9713.2)
● Acetone (Roth, cat. no. 5025.6)
● Target retrieval solution, 10× (Dako, cat. no. S1699)
CRITICAL Other target retrieval solutions were not tested.

c
Chemical reagents for western blot sample preparation

●
Sodium dodecyl sulfate (SDS; Serva, cat. no. 20765,03)
●
Glycerol (Roth, cat. no. 4043.1)
● β-Mercaptoethanol (Carl Roth, cat. no. 4227.1)
! CAUTION Avoid exposure to β-mercaptoethanol, as it is harmful if swallowed or inhaled, causes

severe skin burns and eye damage, is a danger to life in case of skin contact, may cause an allergic skin
reaction, may cause damage to internal organs (heart, liver) in case of prolonged exposure and is toxic
to aquatic life with long-lasting effects. Thus, wear protective gloves/protective clothing/eye protection/
face protection, use a fume hood, prevent release into the environment and thoroughly clean
contaminated surfaces.
● Bromophenol blue (AppliChem, cat. no. A2331,0005)
Antibodies and stains for IHC and western blot

● Rabbit-anti-cytokeratin 5-Alexa488 (1:300, Abcam, cat. no ab193894)
● Rabbit-anti-cytokeratin 8 (1:200, Abcam, cat. no ab59400)

Table 1 | Table of antibodies used for IHC including dilutions and RRIDs
Antibody target Manufacturer Cat. no. Dilution RRID
Mouse-anti-p63 Abcam ab735 1:200 AB_305870

Rabbit-anti-p63 Abcam ab53039 1:200 AB_881860
Mouse-anti-E-cadherin BD Biosciences 610181 1:300 AB_397580
Rabbit-anti-Ki67 Abcam ab16667 1:200 AB_302459
Rat-anti-Ki67-FITC eBioscience 11-5698 1:200 AB_11151689
Rabbit-anti-KRT5 Abcam ab52635 1:250 AB_869890
Rabbit-anti-cytokeratin 5-Alexa488 Abcam ab193894 1:200 AB_2893023
Mouse-anti-KRT7 Santa Cruz sc-23876 1:100 AB_2265604
Rabbit-anti-cytokeratin 7 Abcam ab181598 1:8,000 AB_2783822
Rabbit-anti-cytokeratin 7-Alexa555 Abcam ab209601 1:300 AB_2728790
Rabbit-anti-CSTA Sigma HPA001031 1:200 AB_1078610
Rabbit-anti-AGR2 Proteintech 12275-1-AP 1:200 AB_2225096
Mouse-anti-MUC5B Abcam ab77995 1:250 AB_2146987
Rabbit-anti-GDA Sigma HPA019352 1:250 AB_1855593
Rabbit-anti-cytokeratin 17 Abcam ab109725 1:200 AB_10889888
Mouse-anti-cytokeratin 19 Abcam ab7754 1:250 AB_306048
Mouse-anti-cytokeratin 18 Abcam ab668 1:200 AB_305647
Rabbit-anti-Loricrin Abcam ab85679 1:50 AB_2134912
Goat-anti-Chlamydia MOMP AbD Serotec 1990-0804 1:500 AB_620522
Mouse-anti-phospho γH2AX (Ser139) Millipore 5636 1:500 AB_309864
Rabbit-anti-cytokeratin 8 Abcam ab59400 1:200 AB_942041
Rabbit-anti-MSH6 (EPR3945) Abcam ab92471 1:200 AB_2144959
Alexa Fluor 488 AffiniPure F(ab′)2 Jackson Immuno Research 711-546-152 1:150 AB_2340619
fragment donkey anti-rabbit IgG (H+L)
Cy3 AffiniPure F(ab′)2 fragment Jackson Immuno Research 711-166-152 1:100 AB_2313568
donkey anti-rabbit IgG (H+L)
Alexa Fluor 647 AffiniPure Jackson Immuno Research 711-605-152 1:150 AB_2492288
donkey anti-rabbit IgG (H+L)
Cy5 AffiniPure donkey anti-mouse IgG (H+L) Jackson Immuno Research 715-175-151 1:150 AB_2340820
Alexa Fluor 488 AffiniPure Donkey anti-goat Jackson Immuno Research 705-545-147 1:150 AB_2336933
IgG (H+L)
Alexa Fluor 647 AffiniPure donkey anti- Jackson Immuno Research 715-605-150 1:150 AB_2340862
mouse IgG (H+L)
Cy3 AffiniPure F(ab′)2 fragment Jackson Immuno Research 115-165-006 1:150 AB_2338681
goat anti-mouse IgG
Alexa Fluor 647 AffiniPure donkey anti-goat Jackson Immuno Research 705-605-003 1:150 AB_2340436
IgG (H+L)
Alexa Fluor 488 AffiniPure donkey anti- Jackson Immuno Research 715-545-151 1:150 AB_2341099
mouse IgG (H+L)
●
Donkey-anti-rabbit-Cy3 (1:150, Jackson ImmunoResearch, cat. no 711-166-152)
● Hoechst (1:2,000, Sigma, cat. no B2261)
● See Tables 1 and 2 for details of other antibodies used for IHC and western blot analysis of organoids
in refs. 4,13
Equipment
Culture equipment
● Fine scissors (e.g., B Braun, cat. no. BC050R)
●
Fine forceps (e.g., Bochem, cat. no. 1130)
● Biological safety cabinet (e.g., Heraeus Hera Safe from Thermo Fisher Scientific, cat. no. 10110910)
● Cell culture incubator with 5% CO , 37 °C and 35 °C (e.g., Heracell 150i CO incubator from Thermo
2 2
Fisher Scientific, cat. no. 16406639)

Table 2 | Table of antibodies used for western blot analysis, including dilutions and RRIDs
Antibody target Manufacturer Cat. no. Dilution RRID
Rabbit-anti-MSH6 (EPR3945) Abcam ab92471 1:1,000 AB_2144959

Mouse-anti-MSH2 (3A2B8C) Abcam ab52266 1:1,000 AB_2144800
Rabbit-anti-MLH1 (EPR3894) Abcam ab92312 1:10,000 AB_2049968
Mouse-anti-Chlamydia HSP60 (A57-E4) Enzo Life Sciences ALX-804-071-R100 1:500 AB_2051632
Mouse-anti-Chlamydia HSP60 (A57-E4) GeneTex GTX25486 1:1,000 AB_380556
Mouse-anti-β-actin Sigma A5441 1:10,000 AB_476744
Mouse-anti-Rb (4H1) Cell Signaling 9309 1:2,000 AB_823629
Rabbit-anti-pRb (Ser807/811) Cell Signaling 9308 1:1,000 AB_331472
Mouse-anti-E2F1 (8G9) Abcam ab135251 1:250 AB_2631246
Mouse-anti-Golgin-84 BD Biosciences 611382 1:500 AB_398904
Mouse-anti-p53 (DO-1) Santa Cruz sc-126 1:500 AB_628082
ECL mouse IgG, HRP-linked whole Ab Amersham NA931 1:2,000 AB_772210
ECL rabbit IgG, HRP-linked whole Ab Amersham NA934 1:2,000 AB_772206
● High-speed centrifuge (e.g., Eppendorf, cat. no. 5810 R)

● Water bath 37 °C (e.g., GFL Gesellschaft für Labortechnik, cat. no. 1002)
●
Bright-field microscope (e.g., OLYMPUS IX50)
●
Gamma irradiation system (e.g., MDS Nordion, Gammacell 40 Exactor Low Dose Rate Research
Irradiator)
● Calibrated pipettes (0.5–10 µL Eppendorf, cat. no. 3120000020; 10–100 µL Eppendorf, cat. no.
3120000046; 100–1,000 µL Eppendorf, cat. no. 3120000062; 20–200 µL Eppendorf, cat. no.
3120000054; 2–20 µL Eppendorf, cat. no. 3120000038)
● Filter tips (10 μL filter tips, Fisher Scientific, cat. no. 10394121/2139; 20 μL filter tips, Fisher Scientific,
cat. no. 10282761; 200 μL filter tips, Fisher Scientific, cat. no. 10029040; 1,000 μL filter tips, Fisher
Scientific, cat. no. 10045460)
● Conical centrifuge tubes (15 mL, Sarstedt, cat. no. 62.554.502; 50 ml, Sarstedt, cat. no. 62.547.254)
● Microcentrifuge tubes (1.5 mL, Sarstedt, cat. no. 72.690.001)
●
0.22 µm filter (Millipore, cat. no. SLGP033RS)
●
40 μm cell strainer (BD Falcon, cat. no. 352340)
● 10 cm Petri dish (Sarstedt, cat. no. 430167)
● 48-Well adherent culture plate (TPP, cat. no. P92448)
● 24-Well adherent culture plate (TPP, cat. no. P92424)
● Six-well adherent culture plate (TPP, cat. no. P92406)
● Cell culture flasks (T25, TPP, cat. no. P90026; T75, TPP, cat. no. P90076; T150, TPP, cat. no. P90151)
● Cell freezing container Mr. Frosty (Thermo Scientific, cat. no. 5100-0001)
● 2 mL cryogenic vials (Nunc, cat. no. 375418)
● Ice bucket (e.g., FisherScientific, cat. no. 15572891)
● Coated stainless steel Dewar carrying flask (e.g., Fisher Scientific, cat. no. 10613321)
●
Sorvall centrifuge and rotor (e.g., Sorvall RC 6 Plus,Thermo Scientific, cat. nos. 46910 and 28020)
●
Neubauer counting chamber (Marienfeld, cat. no. 0640210)
Equipment additionally required for genetic manipulation

● PCR system thermal cycler (e.g., Gene Amp 9700 Thermo Fisher)
● Gel documentation system (e.g., GeneGenius Syngene)
● DNA isolation kit (e.g., QIAmp DNA Mini Kit, QIAGEN, cat. no. 51306)
Equipment additionally required for Chlamydia trachomatis infection

● Fluorescence microscope (e.g., Olympus CKX41)
● Incubated tabletop orbital shaker set to 35 °C (e.g., Thermo Scientific, cat. no. SHKE420HP)
● PaVrafilm (Bemis, cat. no. PM996)

Equipment for IHC

● Confocal microscope (e.g., Leica TCS SP8, Leica Microsystems GmbH)
● Tissue processor (e.g., Leicabiosystems, Leica TP1020)
● Paraffin embedding station (e.g., Microm AP250)
● Paraffin rotation microtome (e.g., Microm HM315)
●
Tissue cassette (e.g., Simport Scientific, cat. no. M490-11)
● Glass slides (e.g., Thermo Scientific, cat. no. J3800AMNZ)
● Coverslips (e.g., Marienfeld Superior, cat. no. 0101172)
● Pap pen (Biozol, cat. no. CED-MU12)
Reagent setup
Growth medium for preparation of WNT3a- and R-spondin-1-conditioned medium
Supplement DMEM with 2 mM glutamine, 1 mM Na-pyruvate, 10% (vol/vol) FCS and 1.25 µL/mL
Zeocin. The medium can be stored at 4 °C for 1–2 weeks.
Harvest medium for preparation of WNT3a- and R-spondin-1-conditioned medium

Supplement DMEM with 2 mM glutamine, 1 mM Na-pyruvate and 5% (vol/vol) FCS.
CRITICAL Do not use any antibiotics in the harvesting medium. The medium can be stored at 4 °C
c
for 1–2 weeks.
ADF++ for human cervical organoid cultivation and Chlamydia trachomatis infection
Prepare ADF, 10 mM HEPES and 1% (vol/vol) Glutamax. Store at 4 °C for up to 1 month.
ADF+++ for human cervical organoid cultivation and Chlamydia trachomatis infection
Prepare ADF, 10 mM HEPES, 1% (vol/vol) Glutamax and 5% (vol/vol) FCS (heat-inactivated). Store
at 4°C for up to 1 month.
Collagenase type II solution for human cervical organoid cultivation and Chlamydia
trachomatis infection
Dissolve 100 mg collagenase II in 200 mL of Hank’s balanced salt solution to get a 0.5 mg/mL
concentration. Filter sterilize using 0.22 µm filter, make aliquots of 5 mL in 15 mL tubes and store at
−20 °C for up to 2 months. Thaw the aliquots at 4 °C overnight and warm at 37 °C for 5 min
before use.
Collagen type I working solution for human cervical organoid cultivation and Chlamydia
trachomatis infection
Prewarm collagen type I solution (4 mg/mL) and 1× PBS by incubating for 5 min at 37 °C in a water
bath. Prepare a working solution of collagen type I solution in warm PBS by diluting at a 1:100 ratio.
Reagent must be made up fresh each time.
Matrigel for human cervical organoid cultivation and Chlamydia trachomatis infection
Thaw Matrigel on ice overnight at 4 °C. Mix well, and make 1 mL aliquots in prechilled 1.5 mL
Eppendorf tubes on ice. Matrigel aliquots can be stored at −20 °C until the expiry date specified by
the company.
CRITICAL Always keep original stock and aliquots on ice. The 1 mL aliquots can be thawed on ice
c
in 2 h.
Mouse ectocervical organoid medium

Prepare ADF, 12 mM HEPES, 1% (vol/vol) GlutaMax, 1% (vol/vol) B27, 1% (vol/vol) N2, 10 ng/mL
mouse EGF, 100 ng/mL mouse noggin, 100 ng/mL human FGF-10, 1.25 mM N-acetyl-L-cysteine,
2 μM TGF-β R kinase inhibitor IV, 10 μM ROCK inhibitor (Y-27632), 10 mM nicotinamide and
10 μM FSK. This medium can be stored at 4 °C for up to 2 weeks.
Mouse endocervical organoid medium

mouse EGF, 100 ng/mL mouse noggin, 100 ng/mL human FGF-10, 1.25 mM N-acetyl-L-cysteine,

10 mM nicotinamide, 2 μM TGF-β R kinase inhibitor IV and 10 μM ROCK inhibitor (Y-27632) with
25% (vol/vol) WNT3A- and 25% (vol/vol) R-spondin-1-conditioned medium. This medium can be
stored at 4 °C for up to 2 weeks.
Human ectocervical organoid medium

Prepare ADF, 12 mM HEPES, 1% (vol/vol) GlutaMax, 1% (vol/vol) B27, 1% (vol/vol) N2, 0.5 μg/mL
hydrocortisone, 10 ng/mL human EGF, 100 ng/mL human noggin, 100 ng/mL human FGF-10,
1.25 mM N-acetyl-L-cysteine, 2 μM TGF-β R kinase inhibitor IV, 10 μM ROCK inhibitor (Y-27632),
10 mM nicotinamide and 10 μM FSK. This medium can be stored at 4 °C for up to 2 weeks.
Human endocervical organoid medium

human EGF, 100 ng/mL human noggin, 100 ng/mL human FGF-10, 1.25 mM N-acetyl-L-cysteine,
10 mM nicotinamide, 2 μM TGF-β R kinase inhibitor IV and 10 μM ROCK inhibitor (Y-27632) with
25% (vol/vol) WNT3A- and 25% (vol/vol) R-spondin-1-conditioned medium. This medium can be
stored at 4 °C for up to 2 weeks.
Complete DMEM to culture 3T3-J2 cell line

Prepare Dulbecco’s Modified Eagle Medium (DMEM), heat-inactivated 10% (vol/vol) FCS, 2 mM
L-glutamine, 1 mM sodium pyruvate and 1% (vol/vol) penicillin–streptomycin solution. This medium
can be stored at 4 °C for up to 2 weeks.
! CAUTION Penicillin–streptomycin solution is harmful; avoid skin contact.
Mitomycin C stock preparation

Dissolve 2.0 mg of mitomycin C in 5 mL of sterile water to get a 0.4 mg/mL stock concentration.
Filter sterilize the solution using 0.22 µm filter, make aliquots and store at 4 °C in the dark, effective
for 2–3 weeks until precipitate forms. This reagent can be stored at 4 °C for up to 3 weeks.
! CAUTION Do not use the precipitated solution as it induces toxicity in cells.
! CAUTION Mitomycin C is harmful; avoid skin contact and inhalation.
10% (wt/vol) SDS for western blot sample preparation

Dissolve 10 g of SDS in 100 mL of distilled water in a glass beaker inside a fume hood. This reagent
can be stored at 25 °C for up to 6 months.
! CAUTION Weigh SDS under a fume hood. Wear a mask to avoid inhalation of SDS powder.
1 M Tris–HCL pH 6.8 for western blot sample preparation

Dissolve 30.2 g of Tris base in 250 mL of distilled water, and adjust to pH 6.8. This reagent can be
stored at 4 °C for up to 1 year.
6× Laemmli buffer for western blot sample preparation

Dissolve 3.75 mL 1 M Tris–HCl pH 6.8 (375 mM), 3 mL glycerol (60%), 1 g SDS (10%), 1.5 mL
β-mercaptoethanol and a pinch of bromophenol blue, and fill the volume up to 10 mL with distilled
water, aliquot and freeze at −20 °C. This reagent can be stored at −20 °C for up to 1 year. Avoid
freeze–thaw cycles.
! CAUTION Avoid exposure to β-mercaptoethanol, as it is harmful; use a fume hood. Thoroughly clean
contaminated surfaces.
0.5% (vol/vol) TBE buffer for PCR

Prepare Tris–HCl (89.15 mM), boric acid (88.95 mM) and EDTA-Na2 (2 mM). This reagent can be
stored at 25 °C for up to 3 months.
! CAUTION Boric acid is harmful; avoid exposure.
1.5% (wt/vol) agarose gel for PCR

Dissolve 1.5 g LE agarose in 100 mL 0.5% (vol/vol) TBE buffer. Boil up to dissolve the powder. Add
5 µL ethidium bromide solution. The gel must be made up fresh each time.
! CAUTION Avoid exposure to ethidium bromide solution, as it is harmful; use a fume hood.

Immunofluorescence blocking solution for IHC

Dissolve 1 g BSA in 100 mL (1%) of 1× PBS. Add 2 mL FCS (2%), filter through 0.2 µm membrane
filter and store at −20 °C as 10 mL aliquots. This reagent can be stored at −20 °C for up to
1 month.
4% PFA solution for IHC

Heat 900 mL water to ~60 °C. Next, add stepwise 1 mL 0.5 M NaOH, 40 g PFA and 100 mL 10× PBS.
Allow the solution to come to 25 °C, and adjust the pH to 7.4. Prepare aliquots, and store them at
−20 °C until use for up to 1 year. Before use, prewarm PFA solution to 37 °C.
! CAUTION PFA is highly toxic; avoid contact with skin, eyes or mucous membranes. Use suitable
protective gloves and a mask (P2 level).
Mowiol preparation for IHC

Dissolve 35 g glycerol and 12 g Mowiol in 30 mL distilled water, slowly stirring without forming any
air bubbles for 2 h at 25 °C. Add 60 mL of 0.2 M Tris–HCl (pH 8.5). Incubate for 1 h at 50 °C on a
magnetic stirrer overnight. Once Mowiol dissolves, clarify the solution by centrifugation for 15 min at
5,000g in 50 mL Falcon tubes at 25 °C. Aliquot 10 mL Mowiol solution in 15 mL Falcon tube. The
aliquots can be stored at −20 °C for up to 1 year.
Production of WNT3a- or R-spondin-1-conditioned medium

As described in refs. 34,35, the production of WNT3a- and R-spondin-1-conditioned media follows the
same protocol. To produce WNT3a-conditioned medium, use L-Wnt3a cells, and for R-spondin-1-
conditioned medium, use 293T-HA-Rspo1-Fc cells.
CRITICAL 293T-HA-Rspo1-Fc cells are loosely adherent. Take care that cells do not detach while
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adding medium.
1 On day 1, seed 1 million L-Wnt3a or 293T-HA-Rspo1-Fc cells in a T75 flask in 12 mL of growth
medium, and incubate in a CO2 incubator for 2 d.
2 On day 3, split cells at a 1:4 ratio into two T150 flasks. For this, remove the medium and wash once
with warm PBS. Overlay the cells with 3 mL of TrypLE, and incubate for 3–4 min at 37 °C. Transfer
the suspension into a Falcon tube. Then add a complete growth medium to dilute TrypLE, and
centrifuge at 300g for 5 min at 25 °C.
3 Remove the supernatant, and resuspend the pellet in 10 mL of the growth medium. To obtain a
homogeneous cell suspension, carefully pipette up and down with a 10 mL pipette. Distribute the
cells equally into two T150 flasks, add 20 mL of growth medium per flask and incubate in a CO2
incubator for 3 d.
4 On day 6, split cells at a 1:4 ratio into eight T150 flasks. For this, repeat steps 2–3 above to harvest
cells. Distribute the cells equally into eight T150 flasks. Add 20 mL of growth medium per flask, and
incubate in a CO2 incubator for 3 d.
5 On day 9, split cells at 1:6 ratio into 48 T150 flasks. For this, repeat steps 2–3 above to harvest cells.
Distribute the cells equally into 48 T150 flasks. Add 20 mL of growth medium per flask, and
incubate in a CO2 incubator for 2 d.
6 On day 11, discard the growth medium, add 20 mL of harvest medium to each flask and incubate
the cells for 4–5 d. Change the medium from six flasks at a time.
CRITICAL Do not add medium directly on top of cells, which causes detachment of adherent
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cells. Always make sure flasks are free from microbial contamination.
7 On days 15–16, undertake harvest 1. Collect the cell supernatant in 100 mL Sorvall centrifuge tubes,
and spin at 1,500g for 10 min at 4 °C. Collect the supernatant, and filter sterilize by passing through
0.2 µm filter units. Store the filtered supernatant at 4 °C. To produce higher quantities of WNT3a-
or R-spondin-1-conditioned medium, add 20 mL of the harvest medium to each flask and incubate
for an additional 2–3 d.
CRITICAL Before harvesting, make sure the color of the medium changes to orange/yellow,
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indicative of cells growing at a higher density.

8 Repeat the step from day 15 (step 7 above) up to three more times, and pool the filtered
supernatants. Test the activity of WNT3a or R-spondin-1 from each harvest by using a Wnt
reporter cell line (293T WntR)37. Prepare a 10 mL aliquot in 15 mL Falcon tubes, and store the
WNT3a- and R-spondin-1-conditioned medium at −20 °C.

Testing the activity of WNT3a or R-spondin-1
To test the activity of WNT3a or R-spondin-1, use 293T WntR cells that are transfected with a
7 TCF/LEF promoter-binding site that drives GFP expression upon Wnt pathway activation37.
1 On day 1, add 100 µL per well of 0.01% poly-L-lysine solution to 48-well plates to coat the surface,
and incubate at 25 °C for 30 min.
2 Air dry the plate, and wash with sterile ddH2O. Store the coated plate with ddH2O at 4 °C
until use.
3 Seed 293T WntR cells (20,000 cells per 250 µL medium per well) in a coated 48-well plate.
4 Incubate the plate for 24 h at 37 °C and 5% CO2.
5 On day 2, mix the WNT3a or R-spondin-1 supernatant with DMEM medium at different dilutions
(e.g., 60%, 30%, 20% 15%, 10%, 5%, 2.5%, 0.5%).
CRITICAL Perform measurements as triplicates, and test together with a negative control
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(DMEM medium only) and WNT3a or R-spondin-1 known concentration as positive control.
6 Remove the medium from the 293T WNT reporter cells.
CRITICAL Use a 5 mL pipette, and do not empty the whole plate at once; cells might get dry.
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7 Pipette supernatant dilutions onto the cells, 250 µL per well.

CRITICAL Be careful; cells may detach by the force of the solution.
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8 Incubate plates for 24 h at 37 °C and 5% CO2.

9 On day 3, remove the medium by flipping the plate on an absorbing paper, then fix the cells with
250 µL per well 3.7% PFA, and stain nuclei with Hoechst 1:1,000 (10 mg/mL stock) for 30 min in
the dark at 25 °C or 4 °C.
CRITICAL Be careful; cells may detach by the force of the solution.
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10 Remove PFA by flipping the plate on an absorbing paper.

11 Fill the plate with PBS, and remove PBS by flipping the plate on an absorbing paper.
12 Fill the plate with 250 µL per well PBS, and store the plate in PBS at 4 °C until fluorescence is
measured with an automated microscope. If active WNT3a or R-spondin-1 is present, cells will
express GFP.
Preparation of collagen-type-I-coated plate or flask

CRITICAL Collagen-coated plates/flasks should be prepared before initiation of cell isolation.
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Alternatively, collagen-coated plates/flasks can be prepared the previous day and stored at 4 °C.
1 Prewarm collagen type I and PBS by incubating for 5 min at 37 °C in a water bath.
2 Prepare a working collagen type I solution in warm PBS by diluting at a 1:100 ratio.
CRITICAL Prepare the solution fresh each time.
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3 Add 1 mL per well of collagen solution to six-well plate/2 mL per T25 flask/4 mL per T75 flask to
coat the surface of the culture dish or flask.
4 Incubate culture dish or flask at 37 °C for 1–3 h at 25 °C or overnight at 4 °C.
5 Remove the collagen solution completely by using a 1 mL pipette.
6 Air dry for 10 min before using it for the culture.
CRITICAL Collagen-coated plates can be stored at 4 °C after completely air drying for 1 h.
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Preparation of 3T3-J2 cell line (feeder cells for ectocervical epithelial stem cells)
CRITICAL Cell lines need to be prepared and kept ready when splitting ectocervix primary stem cells.
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Cell line culture needs to be planned 1–2 weeks before splitting 2D passage 0 to 2D passage 1 of
ectocervix stem cells.
1 Seed 1 × 105 cells per T25 flask in 5 mL complete DMEM medium, and incubate at 37 °C and 5%
CO2 in a humidified incubator.
CRITICAL Do not allow cells to grow to full confluence because this will induce cell stress.
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2 When cells reach 90% confluence, remove the medium and wash the monolayer briefly with warm
1× PBS.
3 Add 1 mL warm fresh trypsin–EDTA solution on top of the cell monolayer, spreading it evenly all
over the surface.
4 Incubate at 37 °C for 3–4 min until cells detach from the plate.
CRITICAL Cells may require tapping the flask to aid in detachment.
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5 Dilute with 10 mL of complete DMEM, and count the number of cells per mL using a Neubauer
counting chamber.
CRITICAL 3T3-J2 cells can be split 1:2 to use for co-culture and up to 1:10 for passaging.
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6 When cells are 80% confluent, growth arrest cells. Do one of the following:
● Irradiate cells with a total dose of 30 Gy. Afterward, incubate the flask at 37 °C and 5% CO until
2
needed, but use within 1 d.
! CAUTION Follow the instructions for operating the machine from an authorized person.
CRITICAL Calculate the required time from the irradiation machine’s current dose per minute
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(Gy/min) to reach a 30 Gy dose.
● Add 0.1 mL of mitomycin C stock to a T25 flask containing 3T3-J2 cells with 5 mL of complete
DMEM medium (8 μg/mL final concentration of mitomycin C), and incubate at 37 °C with 5%
CO2 for 3 h. Then aspirate off mitomycin-C-containing medium, and wash four times with 5 mL
warm sterile PBS. Washed cells are ready to use for co-culture. Alternatively, add 8 mL complete
DMEM to the monolayer and store the cells in a 37 °C incubator with 5% CO2 for up to 2 d
until needed.
Procedures
Procedure 1: mouse ectocervix and endocervix organoid culture
Tissue preparation for mouse ectocervix and endocervix isolation ● Timing 1 h
1 Euthanize mice at age 5–20 weeks using the appropriate ethically approved technique. We
euthanize mice using the cervical dislocation method.
2 Place euthanized mice on a dissection platform with the ventral side facing up. Stretch the legs, and
firmly pin them to the dissection platform with wall needles.
3 Sterilize the abdominal and the genital surface with 70% (vol/vol) ethanol.
4 Using sterile scissors, cut open skin from the abdomen up to the genital part and transversely
toward the hind limb. Firmly pin the opened skin to the dissection platform.
5 Carefully lift out the entire genital tissue using bent forceps by holding at the middle part of the
cervix region and cutting out the tissue, then place in a 10 cm dish.
CRITICAL STEP This must be done without causing any damage using aseptic technique.
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6 Remove any connective and adipose tissues, wash tissue once with PBS and transfer to a sterile
10 cm Petri dish.
7 Separate the ectocervix and endocervix as indicated in Fig. 1, and place them in a separate 10
cm dish.
CRITICAL STEP Use separate scissors to cut the ectocervix and endocervix region from the cervix
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junction to avoid contamination of ectocervical cells into the endocervical sample.
Cell isolation and 3D organoid culture ● Timing 8–12 d

CRITICAL The same procedure is followed for generating both endocervical and ectocervical
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organoids for the respective dissected tissues.

8 Using sterile scissors and forceps, mince the tissue (with 100 µL PBS) into small pieces by holding
tissue with forceps and cutting tissue into small fragments using scissors (<0.5 mm3 thickness).
9 Transfer the minced tissue into a 15 mL Falcon tube, add 3 mL of prewarmed collagenase II
(0.5 mg/mL) per tube and incubate for 45 min at 37 °C, 180 rpm in a horizontal position in a shaker.
CRITICAL STEP Cover the Falcon tube cap with Parafilm to avoid leakage of solution from the tube
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during the incubation period.

10 Centrifuge at 1,000g for 5 min at 4 °C, discard the supernatant, add 3 mL warm TrypLE and
resuspend the tissue pieces and cells using a 5 mL pipette by pipetting up and down five times.
11 Incubate for 15 min at 37 °C, 180 rpm, in an orbital shaker, keeping the tube in the horizontal
position. Again, cover the Falcon tube cap with Parafilm to avoid leakage of solution from the tube
during the incubation period.
12 Pipette up and down 20 times with a 1 mL pipette to dissociate cell aggregates. Centrifuge at 1,000g
for 5 min at 4 °C, and discard the supernatant. Resuspend the pellet in 10 mL of ADF++, and mix
by inverting the tube several times.
? TROUBLESHOOTING
13 Pass the solution through a 40 µm cell strainer into a 50 mL Falcon tube to filter tissue debris.
Centrifuge at 1,000g for 5 min at 4 °C, and discard the supernatant.
14 Resuspend the cells in 3 mL ADF++, and count the number of cells using a Neubauer counting
chamber. Take the required number of cells (10,000 cells per well) into an Eppendorf tube, and
centrifuge at 1,000g for 5 min at 4 °C. Remove the supernatant, leaving 5–10 µL for resuspending
the cells before adding Matrigel medium.

Box 1 | Cryopreservation and thawing of organoids
Procedure
Freezing organoids ● Timing 30 min
CRITICAL Always freeze organoids on the second or third day of culture to increase organoid growth efficiency after thawing. One 50 µL drop
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Matrigel full of organoids is sufficient for one vial cryopreservation.

1 Remove the medium, and break up the Matrigel drop with 1 mL ice-cold ADF++.
2 Transfer the organoids with the surrounding Matrigel into an Eppendorf tube, spin at 500g for 5 min at 4 °C, and remove the supernatant and
Matrigel with a 1 mL pipette tip.
3 Resuspend the organoids in 0.5 mL of Cryo-SFM, transfer them to a labeled cryovial.
4 Transfer the cryovial to an isopropanol-containing freezing container (Mr. Frosty precooled at 4 °C), and place it at −80 °C overnight.
CRITICAL STEP Cryovials can be stored for up to 3 months at −80 °C before transfer into liquid nitrogen.
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5 On the next day, transfer the cryovials to liquid nitrogen.

! CAUTION Wear protective clothes, including gloves and a face mask, while handling liquid nitrogen.
PAUSE POINT Organoids can be frozen in liquid nitrogen indefinitely.
j
Thawing organoids ● Timing 30 min

CRITICAL Keep 37 °C water bath, thawed Matrigel, prewarmed 24-well plates and 3D organoid medium ready for the culture.
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1 Thaw cryovials quickly by shaking the vial in a 37 °C water bath till the vial contains a small ice clump.
2 Transfer the organoids with the freezing mixture into a 15 mL Falcon tube.
3 Add 5 mL ADF++ dropwise, and mix while adding; centrifuge at 500g for 5 min at 4 °C.
4 Remove the supernatant, and resuspend the pellet in 50 µL of Matrigel.
5 Dispense 50 µL drops of the Matrigel and cell suspension carefully onto the middle of a well of a prewarmed 24-well cell culture plate.
6 Incubate for 15 min at 37 °C and 5% CO2 to allow Matrigel polymerization.
7 Add 500 µL warm respective organoid medium to each well, and place it back inside the 37 °C and 5% CO2 incubator for an appropriate time.
CRITICAL STEP Do not retain more than 10 µL medium for a well of cells as more medium will
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dilute the Matrigel and affect its polymerization and formation of organoids.
? TROUBLESHOOTING
15 Resuspend the pellet in Matrigel (50 µL per well) (volume of Matrigel needs to be adjusted to the
number of wells that need to be seeded), and place Matrigel in the middle of a well of a prewarmed
24-well plate.
CRITICAL STEP It is important to avoid formation of air bubbles because they can cause uneven
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distribution of organoids and interfere with imaging.

16 Place the plate for 15 min in the incubator at 37 °C for Matrigel to polymerize.
CRITICAL STEP If the incubation time is too short, the Matrigel drop may dissolve when the
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medium is added.
17 Add 500 µL of warm mouse endo-/ectocervical organoid medium, and place the plate back inside
the cell incubator (37 °C and 5% CO2) for 8–10 d.
18 Replace medium every 3–4 d with fresh mouse endocervical/ectocervical organoid medium.
Biobank the organoids on the second or third day of culture (Box 1). Once organoids have grown to
an optimal size, they can be further expanded (Box 2).
? TROUBLESHOOTING
19 (Optional) Characterize organoids by performing IHC or prepare cell extracts for western blot
analysis (Box 3).
Procedure 2: human endocervix organoid culture

Isolation of epithelial cells from human endocervix ● Timing 5–6 h
1 Obtain biological material from the clinic in sterile plastic tubes containing PBS.
2 Transfer the tissue sample into a sterile 10 cm Petri dish filled with the appropriate amount of PBS.
Wash the tissue to remove mucus or blood content by agitating tissue with sterile forceps in PBS, and
discard the PBS. Add fresh PBS, and repeat the process until mucus and blood is removed from tissue.
3 Using sterile scissors and forceps, mince the tissue (without PBS) into small pieces (<0.5 mm3
thickness) by holding tissue with the forceps.
? TROUBLESHOOTING
4 Transfer the minced tissue into a 15 mL Falcon tube, add 5 mL of prewarmed collagenase II (0.5 mg/mL)
solution per tube and incubate for 2.5 h at 37 °C, 180 rpm in a horizontal position in the orbital shaker.
CRITICAL STEP To avoid leakage of the solution from the tube during the incubation period, seal the
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Falcon tube cap with Parafilm.

5 Centrifuge the suspension at 1,000g for 5 min at 4 °C, discard the supernatant and resuspend the
tissue pieces in 5 mL warm TrypLE using a 5 mL pipette.

Box 2 | Organoid splitting

Procedure
Splitting ectocervical organoids ● Timing 2 h
CRITICAL Ectocervix organoids are ready for passaging when they reach a diameter of ~150 µm, which usually occurs on days 7–9 for mouse
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organoids and days 12–14 for human organoids, and should be split in a 1:5 ratio.
1 Remove the old medium, and add ice-cold ADF++ (500 µL per well) on the Matrigel drop.
? TROUBLESHOOTING
2 Mix Matrigel up and down three to five times with a 1 mL pipette tip along with ADF++, and transfer the suspension into a 15 mL Falcon tube.
Additionally, add 4 mL cold ADF++.
3 Centrifuge at 300g for 5 min at 4 °C (keep centrifuge precooled at 4 °C).
CRITICAL STEP To avoid disrupting the pellet, first remove only the upper supernatant with 5 mL pipette followed by the Matrigel layer
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carefully with 1 mL pipette tip.

? TROUBLESHOOTING
4 Add 1 mL warm TrypLE to the pellet, and resuspend gently with a 1 mL tip by aspirating only 200 µL up and down three to four times. Place the
tube at an inclined position in a 37 °C shaking water bath/shaking incubator for 30 min at 100 rpm.
5 Rinse a narrowed Pasteur pipette with ADF++, and break the organoids by pipetting suspension six times up and down. Alternatively, pass
through a 26 G needle and break the organoids.
CRITICAL STEP It is essential to rinse glass Pasteur pipette with ADF++ to avoid organoids sticking to the glass wall.
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6 Add 3 mL of ADF++, count the number of cells using a Neubauer counting chamber and take an aliquot of 10,000 cells to seed in one well
(take a proportionate number of cells to seed more than one well).
7 Centrifuge at 1,000g for 5 min at 4 °C. Remove the supernatant, leave behind 500 µL, resuspend the pellet in the remaining medium and
transfer to a 1.5 mL Eppendorf tube.
8 Centrifuge at 1,000g for 5 min at 4 °C, remove the supernatant and resuspend the pellet in Matrigel (50 µL per well) (volume of Matrigel needs
to be adjusted according to the number of wells to be seeded). Place the Matrigel and cell suspension in the middle of a well of a prewarmed
24-well plate.
CRITICAL STEP Remove as much ADF++ as possible, which will otherwise dilute the Matrigel, affecting the proper polymerization of the
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Matrigel and the organoid formation, and avoid forming air bubbles.
CRITICAL STEP If the incubation time is too short, the Matrigel drop may dissolve when the medium is added.
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10 Add 500 µL of warm ectocervical organoid medium, and place the plate back inside the 37 °C and 5% CO2 incubator for 10–14 d.
11 Replace medium every 3–4 d with fresh ectocervical organoid medium. Once organoids have grown to an optimal size, they can be further
expanded by repeating this section or can be infected at day 5 of culture with Chlamydia trachomatis as described in Procedure 6.
Splitting endocervical organoids ● Timing 1.5 h

CRITICAL Endocervical organoids are ready for passaging when they reach an average diameter of ~500 µm, which usually occurs on days 10–12
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for mouse organoids and days 18–20 for human organoids, and should be split in a 1:2 ratio.
1 Remove the old medium, and add 500 µL per well ice-cold ADF++ on the Matrigel.
? TROUBLESHOOTING
2 Mix Matrigel up and down three to five times with a 1 mL pipette tip along with ADF++ medium, and transfer the suspension into a 15 mL
Falcon tube. Add 4 mL cold ADF++. Centrifuge at 300g for 5 min at 4 °C.
3 Remove only the upper supernatant with 5 mL pipette, followed by the Matrigel layer carefully without disturbing the pellet with 1 mL
pipette tip.
? TROUBLESHOOTING
4 Add 1 mL warm TrypLE to the pellet, and resuspend gently with a 1 mL tip by aspirating only 200 µL up and down three to four times. Place the
tube in a 37 °C water bath for 5 min.
5 Rinse a narrowed Pasteur pipette with ADF++, and break the organoids by pipetting suspension six times up and down. Alternatively, pass
through a 26 G needle to break the organoids.
CRITICAL STEP It is essential to rinse glass Pasteur pipette with ADF++ to avoid organoids sticking to the glass wall.
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6 Add 3 mL of ice-cold ADF++, and centrifuge at 300g for 5 min at 4 °C.

7 Remove the supernatant, leave behind 500 µL, resuspend the pellet in the remaining medium and transfer to a 1.5 mL Eppendorf tube.
8 Centrifuge at 300g for 5 min at 4 °C, and remove the supernatant.
CRITICAL STEP Remove as much supernatant as possible to avoid dilution of the Matrigel.
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9 Resuspend the pellet in Matrigel (50 µL per well) (volume of Matrigel needs to be adjusted according to the number of wells to be seeded), and
place Matrigel in the middle of a well of a prewarmed 24-well plate.
CRITICAL STEP It is important to avoid formation of air bubbles because they can cause uneven distribution of organoids and interfere with
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imaging.
CRITICAL STEP If the incubation time is too short, the Matrigel drop may dissolve when the medium is added.
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11 Add 500 µL warm endocervical organoid medium to each well, and place it back inside the 37 °C and 5% CO2 incubator for 3 weeks.
12 Replace medium every 3–4 d with the fresh endocervical organoid medium. Once organoids have grown to an optimal size, they can be further
expanded by repeating this section or can be infected with Chlamydia trachomatis as described in Procedure 5.
? TROUBLESHOOTING
6 Incubate the resuspended tissue pieces in TrypLE for 15 min at 37 °C, 180 rpm in a horizontal
position in the orbital shaker. Pipette up and down five times with a 5 mL pipette to dissociate cell
aggregates. Next, add 5 mL ADF++, and pipette up and down five times with a 5 mL pipette.
CRITICAL STEP To avoid leakage of the solution from the tube during the incubation period, seal
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the Falcon tube cap with Parafilm.

Box 3 | Characterization of ectocervical and endocervical organoids
Procedure
IHC of organoids ● Timing 2 d
CRITICAL This method can be applied to both mouse and human samples. Antibodies successfully used to characterize the organoids by IHC
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are listed in Table 1.

! CAUTION Since PFA is highly toxic, dispose the PFA solution in accordance with local regulations separately from other waste.
Harvesting and fixation of organoids ● Timing 1.5 h

1 Remove the medium from the 24-well plate, and add ice-cold 1× PBS with 0.1% BSA, mix up and down three to four times gently with 1 mL
pipette tip and transfer the solution into glass tubes.
2 Add 4 mL of ice-cold 1× PBS with 0.1% BSA to the glass tube, and allow the organoids to settle down by gravity while keeping them on ice for
15 min.
3 Remove the supernatant using a 5 mL pipette, leaving behind 1 mL of solution. Again add 5 mL of ice-cold 1× PBS with 0.1% BSA to the glass
tube, and allow the organoids to settle down while keeping them on ice for 15 min. Repeat this process three times to remove all the Matrigel
from the organoids. Do final wash with 5 mL of ice-cold 1× PBS without 0.1% BSA.
4 Add 5 mL of prewarmed 4% PFA, and incubate for 1 h at 25 °C.
5 Exchange the PFA with 1× PBS, and proceed with the dehydration and paraffinization process.
Dehydration ● Timing 3 h
6 Dehydrate the organoids by an ascending alcohol series. Add 5 mL of 70%, 80%, 90%, 2× 100% ethanol for 20 min, each at 25 °C. Next, add
100% isopropanol, 100% acetone twice for 20 min, each at 25 °C.
PAUSE POINT Organoids in acetone can be stored for 1 week at 4 °C.
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CRITICAL STEP Tightly seal the glass cap with parafilm to avoid evaporation of acetone.
Embedding organoids with paraffin ● Timing 2.5 h

CRITICAL Keep the heating plate at 66 °C, to obtain molten paraffin, and the cooling plate ready.
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7 Place paraffin embedding metal jar on top of the heating plate for 2–3 min to heat up to 66 °C.
8 Carefully remove the acetone leaving 1 mL in the tube, transfer organoids in acetone into a paraffin embedding metal jar using a 3 mL Pasteur
pipette and allow acetone to evaporate.
9 As soon as acetone evaporates, carefully drip 500 µL of molten paraffin (66 °C) on top of organoids, let it stand for at least 10 min at 66 °C to
ensure penetration of paraffin into the organoid cells. Make sure that all the organoids are at the bottom.
10 Transfer the embedding metal jar to a cooling plate (−5 °C), place a labeled plastic embedding cassette on the metal jar, make up the cassette
with molten paraffin and incubate for 1 h to solidify.
11 Make 5-µm-thick sections of paraffin-embedded organoids using a microtome, and transfer them onto adhesion glass slides.
CRITICAL STEP To avoid losing the section during rehydration, let the slides dry at 37 °C until water evaporates. Slides can be stored at 25 °C
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until rehydration and immunofluorescence labeling.

? TROUBLESHOOTING
Immunofluorescence staining ● Timing 1.5 d

12 Perform rehydration of the sections by immersing slides in series, twice with 100% xylene for 10 min, 100% ethanol for 2 min, and once with
90% ethanol, 70% ethanol, 50% ethanol and water for 2 min each.
13 Perform antigen retrieval by transferring the slides into a slide jar containing 1× target retrieval reagent preheated to 95 °C, incubate for 30 min
in a water bath, cool down for 20 min at 25 °C and wash ten times with running tap water.
14 Mark a circle around the organoid section with a Pap pen, and proceed to immunofluorescence analysis.
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15 Add 50 µL of blocking solution to the organoid section, and incubate for 1 h at 25 °C inside a moistened chamber.
CRITICAL STEP For co-staining with the prelabeled primary antibodies (e.g., prelabeled anti-KRT5-488), the procedure is slightly modified
c
between steps 15 and 19. After this step, incubate the sections overnight with diluted primary antibody solution excluding anti-KRT5-488. After
washing with 1× PBS, incubate the sections in the secondary antibody solution without DNA dye. After incubation with secondary antibodies,
wash the sections with PBS and incubate for a second time in the blocking buffer for 1 h at 25 °C. Then incubate the sections with anti-KRT5-
488 antibody diluted in the blocking buffer and Hoechst for 2 h at 25 °C in a moistened chamber before continuing with step 19 below.
16 Discard the blocking solution, add 50 µL of diluted primary antibody solution and incubate overnight at 4 °C in a dark moistened chamber.
17 The next day, discard the primary antibody and wash it five times with 1× PBS for 2–5 min.
18 Add 50 µL of the diluted fluorochrome-conjugated secondary antibody solution along with 1:2,000 Hoechst dye for DNA staining, and incubate
for 1 h at 25 °C in a moistened chamber. Dilute the secondary antibodies 1:150 in the blocking buffer.
19 Discard secondary antibody, wash five times with 1× PBS for 2–5 min and rinse one time with distilled water.
20 Mount the section with a drop of Mowiol, place a coverslip above and let the slide dry at 25 °C in the dark. Stained slides can be stored at 4 °C
in the dark until fluorescence microscopy.
CRITICAL STEP Avoid air bubbles while mounting; otherwise, they may lower the viewing resolution under the microscope.
c
Preparation of cell extracts for western blot analysis ● Timing 15 min

CRITICAL This method can be applied to both mouse and human samples.
c
! CAUTION For all analyses, we followed the wet blot method as described in ref. 36. Antibodies successfully used for western blot analysis are
listed in Table 2.
1 Carefully aspirate the medium from the well, add ice-cold PBS, mix up and down three to four times with 1 mL pipette to detach the Matrigel
drop, and transfer the complete solution into a 1.5 mL Eppendorf tube.
2 Centrifuge at 1,000g for 10 min at 4 °C, and discard the supernatant. If any residual Matrigel remains, add 1 mL of ice-cold PBS, centrifuge again
at 1,000g for 10 min at 4 °C and discard the supernatant. Repeat the step until Matrigel has been removed completely, then resuspend the
pellet in 2× Laemmli buffer (~50 µL per well of 24-well plate) and heat the sample at 95 °C for 5 min, vortex and store at −20 °C until use.

7 Allow the tissue to settle down in the tube by gravity for 2 min. Transfer the supernatant containing
dissociated cells into a new 50 mL Falcon tube through the 40 µm cell strainer.
8 Resuspend the settled tissue in 5 mL ADF++, and repeat Step 7 another two times.
9 Centrifuge the Falcon tube at 1,000g for 5 min at 4 °C to pellet the dissociated cells. Discard the
supernatant.
Culturing human endocervical cells to 3D organoids ● Timing 3 weeks

10 Remove the supernatant and resuspend the cells (30,000 cells per well) carefully in ice-cold Matrigel
(50 µL per well). Take 50 µL of the Matrigel containing cell suspension, and place it in the middle of
a well of a prewarmed 24-well plate.
c CRITICAL STEP It is important to avoid formation of air bubbles because they can cause uneven
? TROUBLESHOOTING
c
medium is added.
12 Add 500 µL warm human endocervical organoid medium to each well, and place it back inside the
37 °C and 5% CO2 incubator for 3 weeks.
13 Replace medium every 3–4 d with a fresh human endocervical organoid medium. Biobank the
organoids on the second or third day of culture (Box 1). Once organoids reach an average
diameter of ~500 µm, they can be passaged following the endocervical organoid splitting procedure
(Box 2).
? TROUBLESHOOTING
analysis (Box 3).
Procedure 3: human ectocervix 2D stem cell and organoid culture

Isolation of epithelial cells from human ectocervix ● Timing 5–6 h
1 Isolate and dissociate human ectocervix epithelial cells as for endocervical cells in Procedure 2,
Steps 1–9. At this point, do one of the following:
● Directly generate organoids from cells isolated from tissue, by following Procedure 3, Steps 2–3
● Culture stem cells in 2D for expansion and subsequent 3D organoid generation, our preferred
method for generating ectocervical organoids, by following Procedure 3, Steps 4–29.

2 Remove the supernatant and resuspend the cells (10,000 cells per well) carefully in ice-cold Matrigel
(50 µL per well). Take 50 µL of the Matrigel-containing cell suspension, and place it in the middle
of a well of a prewarmed 24-well plate.
3 From here on, follow Procedure 3, Steps 26–28.
2D human ectocervix stem cell culture in collagen-coated plates/flasks ● Timing 2–3 weeks
4 Resuspend the cell pellet gently with a 5 mL pipette in an appropriate amount of human
ectocervical organoid medium, and transfer the cell suspension into a collagen-coated plate/flask
(see ‘Preparation of collagen-type-I-coated plate or flask’ in ‘Reagent setup’). Use 2 mL for one well
of a six-well plate or 5 mL for one T25 flask.
5 Incubate the cells at 37 °C and 5% CO2 in a humidified incubator for 2–3 weeks. Change the
medium every 3–4 d.
CRITICAL STEP Once cells reach 80% confluency, proceed to splitting and culturing in the
c
presence of feeder cells for long-term maintenance of stem cells.
Co-culture of human ectocervical stem cells with 3T3-J2 cell line ● Timing 2 weeks
6 When primary cells reach 80–90% confluence, remove the medium and wash twice with 5 mL of
warm PBS.
CRITICAL STEP Do not allow cells to grow to full confluence because this will induce cell stress.
c
7 Add 4 mL of TrypLE to the flask, and incubate for 8–15 min at 37 °C in a CO2 incubator. Check
under a microscope if all cells are detached. Cells may require tapping the flask to aid in detachment.
8 Transfer cell suspension into a 15 mL Falcon tube.
9 Separate the cells by pipetting the suspension six times up and down with a prepared glass Pasteur
pipette or using a 26 G needle.

10 Dilute TrypLE solution by adding 5 mL of ADF++ medium, and centrifuge at 1,000g for 5 min
at 4 °C.
11 Remove the supernatant, and resuspend the pellet in 4 mL of human ectocervical organoid
medium.
12 Add resuspended cells to the T25 flask containing feeder cells (3T3-J2) treated with mitomycin C or
irradiation (see ‘Preparation of 3T3-J2 cell line’ in ‘Reagent setup’). Use 0.1 million primary cells in
human ectocervical organoid medium (4 mL) per one T25 flask.
13 Incubate the cells at 37 °C in 5% CO2 until epithelial stem cells reach 70–80% confluency. Usually,
this takes 6–8 d.
CRITICAL STEP Replace medium every 3–4 d with a fresh human ectocervical organoid medium,
c
and check the confluence.
14 Once cells reach 70–80% confluence, remove the medium and incubate with 2 mL of warm TrypLE
for 1–2 min at 37 °C in a CO2 incubator to remove 3T3-J2 cells. Check under a microscope to make
sure all feeder cells are detached.
? TROUBLESHOOTING
15 Keep the flask in an upright position, and immediately add 5 mL of warm PBS to the bottom of the
flask, tilt the flask to a horizontal position, rinse the monolayer by tilting flask left and right side,
and discard the supernatant.
! CAUTION Do not add the PBS directly on the surface of the monolayer, which may detach
epithelial cells by the force of the solution.
16 Add 4 mL of TrypLE to the flask, and incubate for 8–15 min at 37 °C in a CO2 incubator. Check
under a microscope whether all cells are detached. Cells may require tapping the flask to aid in
detachment.
17 Transfer cell suspension into a 15 mL Falcon tube.
18 Separate the cells by pipetting the suspension six times up and down with a prepared glass Pasteur
pipette or using a 26 G needle.
19 Dilute TrypLE solution by adding 5 mL of ADF++ medium, and centrifuge at 1,000g for 5 min
at 4 °C.
PAUSE POINT Cells can be cryopreserved by freezing as described for organoids in Box 1,
j
steps 3–5 (‘Cryopreservation and thawing of organoids’).

20 (Optional) If cells are not going to be immediately used to generate 3D organoids, remove the
supernatant and resuspend the pellet in the appropriate amount of human ectocervical organoid
medium. At this point, do one of the following:
● Maintain the cells in 2D culture by splitting in a 1:4–1:5 ratio and repeating Steps 12–20 of
Procedure 3
? TROUBLESHOOTING
● Perform genetic manipulation with cells as described in Procedure 4
Culturing of human ectocervix 3D organoids from 2D stem cells ● Timing 2 weeks

21 Thaw 1 mL aliquot of Matrigel on ice for at least 2 h before starting the experiment. Matrigel should
be completely thawed and mixed two to three times before use.
CRITICAL STEP It is important to thaw the Matrigel completely and avoid the formation of air
c
bubbles, as bubbles and inappropriate thawing can lead to wrong Matrigel volumes being pipetted.
22 Prewarm 24-well plates by placing them at 37 °C for at least 1 h or overnight.
23 Resuspend the cells from Step 17 in 5 mL of ADF++, and mix up and down using a 5 mL pipette.
Count the number of cells using a Neubauer counting chamber, and take an aliquot of 10,000 cells
to seed in one well. Take the proportionate number of cells to seed more than one well.
? TROUBLESHOOTING
24 Centrifuge at 1,000g for 5 min at 4 °C, and remove the supernatant.
CRITICAL STEP Remove as much ADF++ as possible, which will otherwise dilute the Matrigel
c
and affect the proper polymerization of Matrigel and organoid formation.

25 Resuspend the cell pellet carefully in ice-cold Matrigel (50 µL per well). Dispense 50 µL drops of the
suspension carefully onto the middle of a well of a prewarmed 24-well cell culture plate.
CRITICAL STEP It is important to avoid formation of air bubbles because they can cause uneven
c

26 Incubate the cell culture plate for 15 min at 37 °C and 5% CO2 to allow the Matrigel to polymerize.
c
medium is added.

27 Overlay the Matrigel drop with 500 µL of warm human ectocervical organoid medium, and
incubate at 37 °C and 5% CO2.
28 Change medium every 3–4 d with a human ectocervical organoid medium. Biobank the organoids
on the second or third day of culture (Box 1). After 14 d of cultivation, the organoids mature and
reach an average diameter of ~150 µm. They can be passaged following the ectocervical organoid
splitting procedure (Box 2).
? TROUBLESHOOTING
analysis (Box 3).
Procedure 4: genetic manipulation of human ectocervical epithelial stem cells ● Timing

1–2 months
! CAUTION Gene technological approval from the local authority must be obtained.
! CAUTION Since HPV16 E6E7 is cancerogenic, access to containment level 2 facilities is an essential
prerequisite.
CRITICAL All genetic manipulation experiments with human epithelial cells were approved by the
c
GenTech committee of the LAGeSo, Berlin (269/00-08; 269/00-13).

! CAUTION We recommend distinguishing working places from regular primary cell culture and virus
work. Further, it is recommended to include appropriate controls for transfection, transduction and
selection efficiency.
! CAUTION All liquid and disposal materials that have come in contact with lentivirus must be
disinfected before autoclaving.
Lentivirus generation ● Timing 5 d

1 Day 1: seed ~2 × 106 293T cells into 10 cm cell culture dishes with 13 mL DMEM supplemented
with 2 mM L-glutamine, 1 mM pyruvate and 10% (vol/vol) FCS.
! CAUTION Prepare at least one cell culture dish for each DNA construct and one dish for empty
vector control.
2 Day 2: 293T cells should be ~50% confluent.
3 Create a Master Mix of FuGENE6 reagent and Opti-MEM for a 10 cm cell culture dish (per dish
mix 15.6 µL of FuGENE6 + 192.4 µL of Opti-MEM = 208 µL Master Mix). First, add Opti-MEM to
a polypropylene tube, then add FuGENE6 reagent directly into the Opti-MEM. Mix by gently
swirling the tube.
CRITICAL STEP Ensure that FuGENE6 reagent does not come in contact with the walls of the
c
tube before it has been diluted.

! CAUTION FuGENE is harmful; avoid skin contact.
! CAUTION For the reaction steps, do NOT use polystyrene tubes; instead, use polypropylene tubes.
CRITICAL STEP Start with the transfection in the late afternoon as incubation time should not
c
exceed 15 h.
4 Incubate for 5 min at room temperature (RT, ~25 °C). During incubation, prepare the DNA mix for the
construct of interest and empty vector control. We used 2.6 µg of DNA construct pLXSN HPV16 E6E7,
1.95 µg psPAX2 and 0.65 µg pMD2.G (VSVG) for transfection. Make up the DNA mix to 52 µL with
Opti-MEM. As a control, we prepared one additional DNA mix with psPAX2 and pMD2.G (VSVG) only.
5 Add the FuGENE6 reagent Master Mix to the tube with the DNA mix (208 µL Master Mix + 52 µL
DNA Mix = 260 µL). Mix by gently swirling the tube.
CRITICAL STEP Add the Master Mix directly into the liquid, not onto the tube’s walls, for
c
efficient transfection complex formation with the DNA.

6 Incubate for 20–30 min at RT. Gently add the DNA-FuGENE6 reagent Mix dropwise to the cells
without touching the sides of the dish. Swirl to disperse the mixture evenly.
7 Incubate the cells for 12–15 h at 37 °C and 5% CO2.
8 Day 3: after 15 h of incubation, replace the medium with fresh 13 mL DMEM supplemented with
2 mM L-glutamine and 1 mM pyruvate, 10% (vol/vol) FCS and 1% (vol/vol) penicillin–streptomycin.
9 Incubate the cells at 37 °C and 5% CO2 for 48 h until required in Step 13.
Transduction and selection of ectocervical cells ● Timing 3 weeks

10 Day 4: Coat six-well plates with collagen (1:100 in PBS) for 1 h at 37 °C and 5% CO2, as described
in ‘Preparation of collagen-type-I-coated plate or flask’ in ‘Reagent setup’.
CRITICAL STEP Ensure that the surface is covered entirely with the solution.
c

11 Remove the collagen solution, and let the surface of the well dry for a few minutes under sterile
conditions.
12 Seed ~2 × 106 ectocervical epithelial stem cells per well into the collagen-coated six-well plates.
Use human ectocervical organoid culture medium and cells from Procedure 3, Step 18.
CRITICAL STEP Besides the well you plan to transduce, seed three wells, one well for empty
c
vector control, one for only polybrene treatment and one with untreated cells, to compare
phenotype.
13 Day 5: collect the medium from the 293T cells from Step 9 in a 50 mL Falcon tube, and filter
(0.45 µm) the supernatant to remove any cell debris.
14 Concentrate the lentiviral particles by following the manufacturer’s protocol of the Lenti-XTM
Concentrator (Clontech), and resuspend the pellet in 3–5 mL ADF++ according to the
pellet size.
15 Add 50 µL virus solution per six-well plate containing ectocervical epithelial stem cells (confluence
should be ~50%), and add 10 µL polybrene (1 mg/mL). Swirl to disperse mixture evenly, and
incubate overnight at 37 °C and 5% CO2. As controls, add 10 µL polybrene (1 mg/mL) to one
six-well plate containing primary cells, add 50 µL of the virus solution containing empty vector
control or envelope and packaging plasmid only to one additional well with primary cells
and leave one well untreated. Store the viral particles in ADF++ at −80 °C in cryotubes for
later use.
CRITICAL STEP Note that the transduction efficiency of these viruses can be reduced compared
c
to a fresh virus; hence, we recommend preparing a fresh virus.

16 Day 6: start selecting by adding blasticidin (0.5 µg/mL) to the human ectocervical organoid
medium.
! CAUTION Blasticidin is harmful; avoid skin contact.
CRITICAL STEP All wells containing human ectocervical primary cells should be treated equally,
c
and control cells (polybrene-treated well; well transduced with virus-containing only packaging and
envelope plasmid; well that was left untreated) should die within 1 week.
CRITICAL STEP If cells have already reached 80% confluence, passage them and start the
c
selection earliest 2 d after passaging on collagen-coated plates. Otherwise, there is a risk that
transduced cells will die from excessive stress.
17 For splitting, remove the medium, follow Procedure 3, Steps 4–9, and add the resuspended cells
to a collagen-coated plate (see ‘Preparation of collagen-type-I-coated plates or flask’ in ‘Reagent
setup’). Incubate the cells at 37 °C and 5% CO2 in a humidified incubator. Change the medium
every 2–3 d.
CRITICAL STEP Use the human ectocervical organoid medium supplemented with 1% (vol/vol)
c
penicillin–streptomycin and blasticidin (0.5 µg/mL) for optimal growth conditions of the
primary cells.
18 Second/third week: when the selection is successful, and cell confluence reaches 80%, do one of the
following:
● Passage the cells on cell culture flasks containing irradiated 3T3-J2 cells, following instructions
from Procedure 3, Steps 4–18

● Culture cells as 3D organoids as described in Procedure 3, Steps 19–26, then proceed to Procedure
4, Step 19, for validation of oncogene integration

CRITICAL Use the human ectocervical organoid medium without antibiotics when planning
c
infection experiments.
? TROUBLESHOOTING
Validation of oncogene integration ● Timing 6 h

CRITICAL Genetic manipulation of human ectocervical stem cells should be validated by PCR.
c
19 Carefully aspirate the medium from the well, add ice-cold PBS, mix up and down three to four
times with 1 mL pipette to detach the Matrigel drop, and transfer the complete solution into a
1.5 mL Eppendorf tube.
20 To isolate DNA for PCR and RNA for further characterization, centrifuge at 300g for 10 min
at 4 °C, discard the supernatant, resuspend the pellet in an appropriate amount of lysis
buffer (from kit manufacturer), pass the solution through a 26 G needle and follow the
manufacturers’ isolation protocol. Store the isolated RNA at −80 °C and DNA at 4 °C until used for
PCR analysis.

CRITICAL STEP We isolate DNA and RNA with the AllPrep DNA/RNA Mini Kit from Qiagen
c
(cat. no. 80204).
CRITICAL STEP Use water as a negative control and DNA from cells having integrated HPV16
c
E6E7 like End1 cell line (ATCC CRL-2615) as a positive control.
? TROUBLESHOOTING
21 For the PCR mix, use 100 ng DNA template, 0.5 µL of each forward and reverse primer (10 µM)
(we used primers to amplify HPV16 and GAPDH as a control; see ‘Materials’), 0.5 µL dNTPs
(10 mM), 2.5 µL 10× NEB buffer, 1 µL NEB Taq polymerase (5,000 U/mL) and 1 µL MgCl2
(25 mM), and make up to 25 µL with DNAse/RNAse-free water.
22 Use following PCR cycling conditions: initial denature of 5 min at 95 °C followed by 35 cycles at
95 °C for 30 s, at 55 °C for 30 s and 72 °C for 1 min. The final extension is at 72 °C for 5 min and a
minimum of 1 h at 4 °C.
23 Process the PCR products (15 µL) using agarose gel electrophoresis with a 1.5% (wt/vol) agarose gel
and 0.5% (vol/vol) TBE buffer supplemented with ethidium bromide solution at 120 V for 60 min.
As a size reference, use 5 μL of a 50 bp DNA ladder.
Procedure 5: Chlamydia trachomatis infection of human endocervical organoids ● Timing

1.5 h
! CAUTION Culture organoids before infection at least one passage without adding antibiotics to the
organoid medium; otherwise, infectivity will be reduced.
! CAUTION We recommend distinguishing working places between regular primary cell culture and
infection work.
CRITICAL Always include uninfected controls that are cultured and handled in the same way as the
c
infected sample.
! CAUTION Liquid waste generated by Chlamydia trachomatis infection of organoids and further
cultivation must be decontaminated with Korsolex before autoclaving. All disposal material that has
come in contact with primary cells or the pathogen must be autoclaved.
1 Precool the ADF++ and ADF+++ according to the number of wells with organoids you want to
use for the infection experiment, place Matrigel on ice and prewarm the 24-well cell culture plate.
CRITICAL STEP For infection experiments, use endocervical organoids, ~14 d post-splitting, and
c
work under sterile conditions.

2 Carefully remove the old cell culture medium without disrupting the Matrigel drop containing
endocervical organoids.
3 Add 500 µL per well ice-cold ADF++ on the Matrigel. Detach the Matrigel drop with a 1 mL
pipette tip by moving backwards and forward on the well’s surface, and mix the Matrigel with the
ice-cold ADF++ by pipetting up and down.
4 Transfer the suspension from all wells into a single 15 mL Falcon tube.
5 Add 4 mL ice-cold ADF++, and centrifuge at 300g for 5 min at 4 °C. Remove the supernatant with
a 5 mL pipette, and resuspend the pellet in 1 mL ADF++.
6 Pass the organoids six times through a 26 G needle and 1 mL syringe to get organoid fragments.
7 Divide the organoid suspension into two equal aliquots for infection and uninfected control into
15 mL Falcon tubes. For comparable uninfected control, handle both infected and uninfected
control aliquots identically.
8 Centrifuge at 300g for 5 min at 4 °C. Remove the supernatant, and resuspend the pellet carefully in
250 µL of ADF+++.
9 Add the appropriate amount of Chlamydia trachomatis infective progenies according to the MOI of
interest to one aliquot with organoids. We used MOI = 10.
CRITICAL STEP From this step, work under the infection bench to avoid contamination of other
c
cell cultures.
10 Incubate the organoid fragments for 15 min at 35 °C and 5% CO2, followed by 5 min on ice.
11 Centrifuge at 300g for 5 min at RT. Discard the supernatant, and resuspend the pellet gently in
50 μL per well ice-cold Matrigel. After centrifugation, handle one sample at a time and keep
Matrigel on ice while pipetting. Resuspend the pellet carefully to distribute the organoids.
CRITICAL STEP Thaw the Matrigel completely on ice before use, and always keep on ice to avoid
c
solidification. Remove as much as possible of the supernatant to minimize dilution of the Matrigel.
Further, avoid the formation of air bubbles because they can result in pipetting an incorrect
Matrigel volume.

12 Dispense 50 µL drops of the suspension carefully onto the middle of a well of a prewarmed 24-well
cell culture plate.
CRITICAL STEP Avoid creating air bubbles and the Matrigel droplets touching the wall of
c
the well as this might cause uneven distribution and formation of organoids and interfere with
imaging.
14 Add 500 µL warm endocervical organoid medium without antibiotics to each well, and place it back
inside the 35 °C and 5% CO2 incubator for an appropriate time. Change the medium every 2–3 d
until sample harvest.
? TROUBLESHOOTING
analysis (Box 3).
Procedure 6: Chlamydia trachomatis infection of human ectocervical organoids ● Timing

3–4 h
! CAUTION Culture organoids before infection at least one passage without adding antibiotics to the
organoid medium; otherwise, infectivity will be reduced.
! CAUTION We recommend distinguishing working places between regular primary cell culture and
infection work.
CRITICAL Always include uninfected controls that are cultured and handled in the same way as the
c
infected sample.
! CAUTION Liquid waste generated by Chlamydia trachomatis infection of organoids and further
cultivation must be decontaminated with Korsolex before autoclaving. All disposal material that has
come in contact with primary cells or the pathogen must be autoclaved.
1 Precool the ADF++ and ADF+++ according to the number of wells with organoids you want
to use for the infection experiment, place Matrigel on ice and prewarm the 24-well cell culture
plate.
CRITICAL STEP For infection experiments, use nondifferentiated ectocervical organoids, 5–6 d
c
post-seeding as single cells.

2 Carefully remove the old cell culture medium without disrupting the Matrigel drop containing 5- to
6-day-old ectocervical organoids.
3 Add 500 µL per well ice-cold ADF++ medium on the Matrigel. Detach the Matrigel drop with a
1 mL pipette tip by carefully moving backward and forward on the surface of the well, and mix the
Matrigel gently with the ice-cold ADF++ by pipetting up and down.
4 Transfer the suspension from all wells into a single 15 mL Falcon tube.
5 Add 4 mL ice-cold ADF++, and centrifuge at 300g for 5 min at 4 °C. Remove the supernatant with
a 5 mL pipette, and resuspend the pellet in 2 mL ADF++.
6 Divide the organoid suspension into two aliquots into 15 mL Falcon tubes, one for infection and one
for uninfected control.
7 Centrifuge at 300g for 5 min at 4 °C. Remove the supernatant, and resuspend the pellet carefully in
250 µL of ADF+++.
8 Add the appropriate amount of Chlamydia trachomatis infective progenies according to the MOI of
interest to one aliquot with organoids. We used MOI = 10.
CRITICAL STEP From this step, work under the infection bench to avoid contamination of other
c
cell cultures.
9 Incubate the organoids with the inoculum for 2 h at 35 °C with gentle shaking at ~100 rpm. Handle
the control identical to the infection sample.
CRITICAL STEP Avoid organoid clumping as this might disrupt organoid growth.
c
10 From here, follow instructions from Procedure 4, Steps 11–14.

CRITICAL STEP As ectocervical epithelial cells require WNT-deficient medium in Step 14, add
c
500 µL warm ectocervical organoid medium without antibiotics to each well and place it back inside
the 35 °C and 5% CO2 incubator for an appropriate time.
? TROUBLESHOOTING
analysis (Box 3).

Troubleshooting
Troubleshooting advice can be found in Table 3.
Table 3 | Troubleshooting table
Step Problem Possible reason Solution
Procedure 1, Step 12; Few epithelial cells Insufficient digestion of tissue Cut the tissue into a smaller size;
Procedure 2, Step 3 pipette up and down more times after
TrypLE treatment
Procedure 1, Step 14; Organoids are overcrowded Seeded excess amount of epithelial cells Always count and seed an appropriate
Procedure 2, Step 13; and medium turns yellow in per Matrigel number of cells per Matrigel. Higher
Procedure 3, Step 23 color (Fig. 2i,k) density of organoids in the Matrigel
may induce stress to epithelial cells and
sudden death of organoids due to
inadequate availability of growth
components from the medium
Procedure 1, Step 18; Collapsed Matrigel and Matrigel was not adequately mixed Completely thaw the Matrigel stock
Procedure 2, Step 10; adherent cells (Fig. 2h,j) during aliquoting overnight at 4 °C in ice. Mix properly
Procedure 3, Step 28 before aliquoting
Too much medium present during mixing Remove all the medium, leaving behind
with Matrigel only 10–30 µL (depending on the
volume of Matrigel that needs to be
added). Make sure additional medium
did not accumulate from the wall of the
tube while prechilling on ice
Matrigel touches wall Place the drop of Matrigel in the middle
of the well
Procedure 1, Step 18 Growth of ectocervical Ectocervical epithelial cells contaminated Cut the tissue at the correct region in
organoids in endocervical with endocervical cells during tissue the ectocervix and endocervix junction.
organoid culture harvest or quiescent ectocervical lineage- Use a smaller and separate scissor to
specific stem cells in the endocervical cut ectocervix and endocervix regions
tissue reactivate and proliferate in the Ensure that WNT3a- or R-spondin-1-
presence of the WNT3a- or R-spondin-1- conditioned or deficient medium is used
deficient medium for the appropriate tissue region
Procedure 2, Step 10; No organoid growth Not enough epithelial cells Seed a higher density of epithelial cells
Procedure 3, Step 26; in the initial culture
Box 2, step 12 Growth factor missing in the 3D medium Ensure that the growth factors were
reconstituted at recommended
concentration, used within the expiry
date, and double-check each growth
factor addition during 3D medium
preparation
Procedure 3, Step 14 Colonies are small, and Cells were diluted at too high a ratio Seed a higher density of epithelial cells
feeders detach in the initial culture
High passage of feeder cells To support the growth of the small
colonies, prepare feeder cells as
described in ‘Preparation of 3T3-J2 cell
line’ in ‘Reagent setup’, but in a Falcon
tube. Next, add growth-arrested feeders
to the cell culture medium. Change
medium every 3–4 d
Procedure 3, Step 20 Cells grow slowly Isolated cells are few, or cells were split During the medium change, leave 1/3 of
at a high ratio the old medium and add only 2/3
fresh medium
Procedure 3, Step 28; Fewer organoids after Organoids lost during aspiration, Do not use a vacuum aspirator to
Box 2, step 1 harvest organoids stuck to the tube or pipette tip remove the Matrigel. Use a 1 mL pipette
to remove Matrigel after centrifugation.
Use low-protein-binding tubes and low-
retention pipette tips
Procedure 3, Step 28; Organoids suspended in Matrigel was not cold during harvest Use ice-cold ADF++ to harvest
Box 2, step 3 the Matrigel after organoids, and mix Matrigel three to
centrifugation four times with ADF++
Table continued

Table 3 (continued)
Step Problem Possible reason Solution
Procedure 4, Step 18 Transduced cells die during Blasticidin concentration was too high, or Repeat transduction with a higher virus
the selection transduction did not work concentration, but start selecting with a
low Blasticidin concentration and
increase stepwise every 3–4 d
Procedure 4, Step 20 DNA/RNA concentration is Organoids lost during aspiration, Use a 1 mL pipette to remove Matrigel
extremely low organoids stuck to the tube or pipette tip after centrifugation. Use low-protein-
binding tubes and low-retention
pipette tips
Lysis was unsuccessful Freeze the sample in lysis buffer
overnight at −80 °C before starting the
isolation protocol
Procedure 5, Step 14; Organoids show Chlamydia trachomatis MOI was too high Always determine the inclusion forming
Procedure 6, Step 10 disintegration units(IFU) of your Chlamydia trachomatis
stock, and estimate how many cells are
in one well of organoids, e.g., by
trypsinizing one well and counting
Reduce the amount of Chlamydia
trachomatis per well
No inclusions can be MOI was too low, or Matrigel was not Increase the MOI. Make sure that the
detected completely removed before incubation Matrigel is removed before incubation
with Chlamydia trachomatis with the bacteria
Box 3, step 11 Instead of organoids, only Paraffin did not penetrate the organoids. Increase incubation time with paraffin at
holes are visible in the Holes are from air bubbles 60 °C
sections Avoid air bubbles
Box 3, step 14 Organoids are lost after Organoids did not stick enough to Incubate overnight at 25 °C or for 1 h at
dehydration the slide 37 °C before staining
Timing
Reagent setup
Production of WNT3a- or R-spondin-1-conditioned medium: 16 d
Testing the activity of WNT3a or R-spondin-1: 3 d
Preparation of collagen-type-I-coated plate or flask: 2–3 h
Preparation of 3T3-J2 cell line (feeder cells for ectocervical epithelial stem cells): 1 week
Procedure 1
Tissue preparation for mouse ectocervix and endocervix isolation: 1 h
Cell isolation and 3D organoid culture: 8–12 d
Procedure 2
Isolation of epithelial cells from human endocervix: 5–6 h
Culturing human endocervical cells to 3D organoids: 3 weeks
Procedure 3
Isolation of epithelial cells from human ectocervix: 5–6 h
2D human ectocervix stem cell culture in collagen-coated plates/flasks: 2–3 weeks
Co-culture of human ectocervical stem cells with 3T3-J2 cell line: 2 weeks
Culturing of human ectocervix 3D organoids from 2D stem cells: 2 weeks
Procedure 4
Genetic manipulation of human ectocervical epithelial stem cells: 1–2 months
Validation of oncogene integration: 6 h
Procedure 5
Infection of endocervical organoids: 1.5 h

Procedure 6
Infection of ectocervical organoids: 3–4 h
Box 1
Freezing organoids: 30 min
Thawing organoids: 30 min
Box 2
Organoid splitting: 2–3 h
Splitting ectocervical organoids: 2 h
Splitting endocervical organoids: 1.5 h
Box 3
IHC of organoids: 2 d
Harvesting and fixation of organoids: 1.5 h
Dehydration: 3 h
Embedding organoids with paraffin: 2.5 h
Immunofluorescence staining: 1.5 d
Preparation of cell extracts for western blot analysis: 15 min
Anticipated results
We have successfully generated human ectocervical and endocervical organoids from more than 80%
of tissue biopsies and 90% of mouse tissues using this protocol. The possible reasons behind the lack
of organoid generation from the remaining tissues include the suboptimal sampling of the human
tissue biopsy, drying of the tissue biopsy, loss of cells during centrifugation steps, incorrect cell
seeding density and accidental omitting of medium components. Therefore, we recommend estab-
lishing efficient sampling, storage and transportation protocols together with clinical partners. Handle
samples carefully during each centrifugation step, ensure the cell pellet is not lost, count the cells and
plate appropriate numbers and monitor the cultures daily at the initial stages.
Once established, the endocervix and ectocervix organoids can be identified by light microscopic
observation (Fig. 2a–e), H&E staining or immunofluorescence staining for specific markers (Fig. 2f,g).
In some instances, owing to excessive dilution of Matrigel, while resuspending the cells, the Matrigel
loses its integrity after a couple of days, and the organoids come in contact with the surface of the well
and start to grow as 2D monolayers (Fig. 2h,j). In such instances, we recommend collecting the cells
from the culture plate by TrypLE treatment followed by centrifugation and plating again with fresh
Matrigel to solve the problem. While performing infection experiments, the MOI should be adjusted
by counting cells; too high MOI would disintegrate the organoids. The establishment of infections
can be monitored using fluorescent-protein-expressing bacteria or viruses or IHC staining with
specific antibodies.
Data availability
The main data discussed in this protocol were generated as part of the studies published in the
supporting primary research papers by Chumduri et al.4 and Koster et al.13. Source data are provided
with this paper.
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Acknowledgements
We are thankful to the patients for donating tissue for research. We thank M. Mangler and staff at Vivantes Klinikum Berlin, Charité
University Medicine, Berlin, Germany. We thank the late J. Angermann for the technical help with lentivirus production. This work was
initiated at the Max Planck Institute for Infection Biology, Berlin. C.C. is funded by the University of Wü rzburg and DFG (GRK 2157).
T.F.M. acknowledges funding from BMBF via the Infect-ERA program CINOCA; N.K. is funded by DFG-DAAD and DFG (GRK2157).

Author contributions
C.C. and R.K.G. developed human and mouse endocervical and ectocervical organoid culture systems in ref. 4, and T.F.M. provided the
infrastructure and guidance. S.K. and N.K. carried out experiments adopting the organoid culture system. S.K., C.C. and R.K.G. developed
the genetic manipulation and infection protocols for the organoid system. C.C., R.K.G., S.K. and N.K. wrote the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to Cindrilla Chumduri.
Peer review information Nature Protocols thanks Yoshitaka Hippo and the other, anonymous, reviewer(s) for their contribution to the
peer review of this work.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Received: 4 June 2021; Accepted: 10 March 2022;

Published online: 11 May 2022
Related links
Key references using this protocol
Chumduri, C. et al. Nat. Cell Biol. 23, 184–197 (2021): https://doi.org/10.1038/s41556-020-00619-0
Koster, S. et al. Nat. Commun. 13, 1030 (2022): https://doi.org/10.1038/s41467-022-28569-1

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PROTOCOL

Patient-derived and mouse endo-ectocervical

1658 NATURE PROTOCOLS | VOL 17 | JULY 2022 | 1658–1690 | www.nature.com/nprot

NATURE PROTOCOLS | VOL 17 | JULY 2022 | 1658–1690 | www.nature.com/nprot 1659

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Endocervical cells Columnar Transcriptome/genomic

Epithelial Stratified Genetical manipulation

Maintenance of organoid cultures

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200 µm 200 µm 600 µm 150 µm 150 µm

f Endocervical organoids g Ectocervical organoids h i

Genetic manipulation of organoids

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hCEcto hCEctoE6E7 End1 Water

Chlamydia trachomatis infection of organoids

NATURE PROTOCOLS | VOL 17 | JULY 2022 | 1658–1690 | www.nature.com/nprot 1663

Infected columnar Infected stratified

c Human endocervical organoids d Human ectocervical organoids e Human ectocervical organoids

Comparison with other methods

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Future applications of endocervical and ectocervical organoids

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Potential limitations of endocervical and ectocervical organoids

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and stored at −80 °C36.

established for type II, we do not recommend using an alternative type.

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● Collagen type I, from rat tail (Sigma, cat. no. C3867)

● WNT3a conditioned growth medium (see ‘Reagent setup’)

● Human EGF (Gibco, cat. no. PHG0311)

● Mouse EGF (Invitrogen, cat. no. PMG8041)

● Human Noggin (Peprotech, cat. no. 120-10C)

● B27 supplement, 50× (Gibco, cat. no.17504044)

● N2 supplement, 100× (Gibco, cat. no.17502048)

● Nicotinamide (Sigma, cat. no. N0636)

● N-acetyl-L-cysteine (Sigma, cat. no. A9165-5G)

● TGF-β inhibitor (A83-01) (Calbiochem, cat. no.616454)

● ROCK inhibitor (Y-27632 dihydrochloride monohydrate) (Sigma, cat. no. Y0503)

● FSK (Sigma, cat. no. F6886)

● Hydrocortisone (Sigma, cat. no. H0888-1G)

● Penicillin–streptomycin, 10,000 U/mL (Gibco, cat. no.15140-12)

! CAUTION Penicillin–streptomycin solution is suspected of damaging fertility or the unborn child;

● Fetal calf serum (FCS; Biochrome, cat. no. S0115)

● Sodium pyruvate (Sigma, cat. no. S8636)

● 0.05 % (wt/vol) Trypsin–EDTA (Gibco, cat. no.25300-096)

● Mitomycin C (Fisher Scientiﬁc, cat. no.10182953)

may affect organoid growth.

● L-Glutamine (Gibco, cat. no. 25030-024)

● Na-pyruvate (Sigma, cat. no. S8636)

● Hank’s balanced salt solution (Gibco, cat. no. 14025-092)

● Poly-L-lysine (Merck, cat. no. A-005-C)

● Korsolex (Carl Roth, cat. no. AK41.1)

Chemical reagents for genetic manipulation

● Polybrene (Sigma, cat. no. H9268)

● Blasticidin (Gibco, cat. no. A11139-03)

1668 NATURE PROTOCOLS | VOL 17 | JULY 2022 | 1658–1690 | www.nature.com/nprot

● LE agarose (Biozym, cat. no. 840004)

● Tris base (AppliChem, cat. no. A1086.5000)

● Boric acid (Roth, cat. no. 6943.3)