A Human Fallopian Tube Model for Investigation of <em>C. trachomatis</em> Infections

Abstract

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2 . As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4 .Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell-culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-γ mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7 . We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9 . For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue. Video LinkThe 2. Incubate for one hour at 37 °C during constant shaking. 3. Add 20 ml DMEM with 10% FCS and 240 μl Cycloheximide. 4. Incubate in a humidified incubator for 48 h at 37 °C and 5% CO 2 . 5. Add 5 ml sterile glass beads, strong shake to detach and destroy C. trachomatis harboring HeLa-229 cells. 6. Collect supernatant and add another 5 ml of sterile glass beads. 7. Rock three times for 1 min to destroy all HeLa-229 cells and release C. trachomatis. 8. Centrifuge for 5 min at 1000 rpmi and 4 °C to get rid of cellular debris. 9. Collect the supernatant for infection of 8 to 10 175 cm 2 cell culture flasks confluent grown with HeLa 229 cells.10. Incubate every flask with 4 ml SPG buffer and 4 ml supernatant from previous infection for one hour at 37 °C with constant shaking. 11. After one hour add 15 ml DMEM with 10% FCS and 230 μl cycloheximide followed by 48 h incubation at 37 °C and 5% CO 2 . 12. Add 5 ml sterile glass beads, shake to detach and destroy C. trachomatis harboring HeLa-229 cells. 13. Collect the supernatant and add another 5 ml of sterile glass beads. 14. Rock three times for 1 min to destroy all HeLa-229 cells and release C. trachomatis. 15. Centrifuge for 5 min at 1000 rpmi at 4 °C to get rid of cellular debris. 16. Collect the supernatant. 17. Fill 40 ml of supernatant in 50 ml Falcon tubes, centrifuge for 99 min with 11000 rpmi. 18. After centrifugation discard the supernatant and wash the pellet with 1 ml SPG buffer.

Elaborations on Corallopyronin A as a Novel Treatment Strategy Against Genital Chlamydial Infections

“…We aimed to test the efficacy of CorA against C. trachomatis as close as possible to the human in vivo setting. Therefore, we applied an ex vivo fallopian tube model that has been previously established for the analysis of host-pathogen interactions (Jerchel et al, 2012). Initially, we checked whether hypoxia affects the infectious outcome.…”

“…Preparation of human fallopian tubes was performed as described (Jerchel et al, 2012). In brief, the tissue of human fallopian tubes was collected at peripartum sterilization on maternal request during cesarean sections or during hysterectomies due to symptomatic uterine fibroids in the luteal phase.…”

Organ‐on‐a‐Chip Systems for Women's Health Applications

“…Hence a major focus has been to develop 3D in vitro models to allow for the study of origin, transformation, and spread of malignant cells and the role of risk factors, including bacterial infection and gonadal hormones . Fallopian tube 3D in vitro models from human and animal primary cells or explanted tissues are also being used for the study of risk factors of infertility and tubal ectopic pregnancy, such as infection with Chlamydia trachomatis and smoking . In the future, microphysiological systems with iPSC‐derived human tissue and physiological tube architecture could provide additional disease models.…”

Murine Endometrial Organoids to Model Chlamydia Infection

“…Further, the use of three-dimensional (3D) and multi-cell type models have allowed for incorporating aspects of cell-cell interactions and tissue architecture (Nogueira et al, 2017 ; Edwards et al, 2019 ). Finally, whole tissue or ex vivo models are among the most relevant established systems, as they exhibit a more complete environment of infection (Hutchinson et al, 1979 ; Cooper et al, 1990 ; Roth et al, 2010 ; Jerchel et al, 2012 ; Kessler et al, 2012 ). However, they cannot be maintained long-term and are not easily accessible.…”