Development of healthy endometrium and endometriosis models for high throughput phenotypic screening

Introduction:: Endometriosis affects 10-15% of menstruating people and is characterized by the presence of invasive endometrial-like tissue outside of the uterus, leading to chronic pelvic pain and infertility. Current diagnosis methods often miss microscopic endometriosis adhesions and there is currently no cure for endometriosis, highlighting the need for improved diagnostic and treatment options. The objective of this study was to develop 3D in vitro models of healthy endometrium and endometriosis that can interface with high throughput screening methods. These platforms can be used to evaluate thousands of compounds simultaneously, empowering large-scale mechanistic studies to investigate the onset and progression of endometriosis as well as enable drug discovery and personalized medicine.




Materials and Methods:: Multilayer multicellular constructs were fabricated in specialized 96-well plates using human microvascular endothelial cells (hMVECs) and either primary human endometrial epithelial cells (HEEC) or primary human endometriosis epithelial cells (12Z) (Fig 1A). The bottom composite hydrogel contained 8.7% w/v GelMa, 0.6 mg/mL fibrinogen, and 1.9 mg/mL collagen type I, while the top composite hydrogel contained 10% w/v PEGDA, 0.13 mg/mL collagen type IV, 0.17 mg/mL fibronectin, and 0.5 µg/mL laminin. We evaluated changes in endometrial lining thickness over time by taking 85 µm z-stack images of each well every three hours for 24 hours. Images were then assessed automatically using a custom image analysis software we developed to extract the z-position of the uppermost and lowermost cells.

Results, Conclusions, and Discussions:: Results: We observed an increase in endometrial lining thickness when endometrial epithelial cells were co-cultured with endothelial cells, but a decrease in endometrial lining when they were cultured alone (Figure 1B, C). These observations were consistent across cell types and biomaterials. Additionally, we observed greater endometrial lining thickness on the composite hydrogels compared to Matrigel for both healthy and diseased models, suggesting increased biological relevance. Lastly, we observed that the healthy endometrial cells build on top of each other over time, whereas the endometriosis cells invaded down into the surrounding matrix. This invasiveness was not seen to the same extent on Matrigel, indicating the composite gels better capture the phenotypic behaviors observed in endometriosis.

Conclusion: We developed physiomimetic models of healthy endometrium and endometriosis that closely resemble the native tissue in terms of layered architecture and cell interaction. These models can interface with high throughput screening methods, can be evaluated automatically, and are suitable for large-scale mechanistic studies aimed at understanding the onset and progression of endometriosis.


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