Human Cervix In Vitro Models of Healthy, Neoplastic and Cancerous Tissues Based on Organ-on-Chip Technology

Abstract

Women’s health has received little attention in biomedical research, leading to an insufficient understanding of gynecologic diseases and sex-specific differences in the efficacy and safety of drug treatments. The cervix uteri is part of the female reproductive system and numerous women worldwide suffer from infections and disorders of the cervix, including the precancerous lesion cervical intraepithelial neoplasia (CIN) and cervical cancer (CC), that may arise upon persistent infection with the human papillomavirus (HPV). Given the absence of adequate drugs for CIN and the frequent recurrence, side effects and complications after CC treatment, there is a pressing need to develop optimized therapies. To address this issue, this thesis aimed to develop complex human in vitro models of the cervix as a tool to advance basic research and to facilitate the exploration of novel treatment options in an academic context. In vitro tissue models were generated by employing Organ-on-Chip (OoC) technology, which integrates microfabrication techniques and tissue engineering. A microfluidic platform was developed iteratively, resulting in three generations of platforms, each of which benefited from insights gained from concurrent tissue engineering experiments. The third version comprises two fluidically independent systems within a microscope slide-sized platform, each featuring four open-top tissue wells for cervical tissues. The normal ectocervix consists of a multilayered squamous epithelium covering a stromal layer. Donor-derived keratinocytes, fibroblasts, and endothelial cells were isolated, characterized, and utilized as species- and tissue-specific cell sources for the engineering of normal and diseased ectocervical tissues. In the Cervix-on-Chip (CXoC), the combination of a hydrogel mimicking the extracellular matrix, and a mechanically robust scaffold proved to be the most promising method for generating a fibroblast-rich stromal layer. By generating a multilayered, differentiated ectocervical epithelium on top of the stroma using primary keratinocytes, a physiological tissue architecture of normal ectocervical tissue was closely emulated. This model can be applied for basic research as well as for infection studies. A diseased tissue model was generated in the CIN-on-Chip (CINoC), incorporating a stromal layer covered with an aberrant epithelium generated from the SCC cell line SiHa to emulate the pathophysiological tissue architecture. In a more complex model, the perfused microchannels of the CINoC were lined with cervical endothelial cells to more closely mimic the vasculature. In the CC-on-Chip (CCoC), SiHa spheroids were embedded in a fibroblast-spiked stromal environment to mimic infiltrating cancerous nests. Tissue engineering allowed for dissecting the effects of co-cultivation, demonstrating enhanced viability and proliferation of the cancerous nests in the presence of fibroblasts. Treatment with the chemotherapeutic agent cisplatin at clinically relevant routes of administration and dosing resulted in reduced tissue viability, highlighting the platform’s applicability for drug testing. Furthermore, the model allows for the integration and recruitment of donor-derived neutrophils from the microvasculature-like channel into the tissue, all while maintaining their ability to form neutrophil extracellular traps. In the future, these models can advance our understanding of disease mechanisms, including studies involving the microbiome and pathogens related to sexually transmitted infections, as well as the development of improved drugs and (immune)therapeutic options.