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Patient-derived and mouse endo-ectocervical organoid generation, genetic manipulation and applications to model infection


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, 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.

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Fig. 1: Schematic representation of endo-ectocervical stem cell isolation, organoid generation, cultivation and characterization.
Fig. 2: Human and mouse endo-ectocervical organoids.
Fig. 3: Genetic manipulation of human ectocervical stem cells and organoid generation.
Fig. 4: Chlamydia trachomatis infections of endocervical and ectocervical organoids.

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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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 Würzburg 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 information

Authors and Affiliations



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.

Corresponding author

Correspondence to Cindrilla Chumduri.

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The authors declare no competing interests.

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Nature Protocols thanks Yoshitaka Hippo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Chumduri, C. et al. Nat. Cell Biol. 23, 184–197 (2021):

Koster, S. et al. Nat. Commun. 13, 1030 (2022):

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Gurumurthy, R.K., Koster, S., Kumar, N. et al. Patient-derived and mouse endo-ectocervical organoid generation, genetic manipulation and applications to model infection. Nat Protoc 17, 1658–1690 (2022).

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