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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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).
The authors declare no competing interests.
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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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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). https://doi.org/10.1038/s41596-022-00695-6