Biomaterial Tools for Modeling the Interface of Endometriotic Lesions & Healthy Ovarian Tissue

Introduction:: Introduction: Endometriosis, marked by the presence of endometrial tissues outside of the uterus, impacts 1 in 10 menstruators of child-bearing age. This disease is marked by chronic pelvic pain, heavy menstrual bleeding, infertility, and in the case of ovarian endometriosis, an increased risk of ovarian cancer. Endometriosis is typically diagnosed through laparoscopic surgery, and on average, patients do not receive a diagnosis until a decade after the initial onset of symptoms. Endometriosis is treated through surgical excision of the lesions or hormone therapy. The likelihood of lesion recurrence post-resection is high, and many are not interested in hormonal birth control. With the goal of developing more robust treatment methods, we must fill gaps related to the etiology of endometriosis. Work pertaining to lesion etiology has been stunted by the lack of proper model systems. Rodents lack the spontaneous occurrence of endometriosis, and non-human primate models, although more physiologically relevant, require special facilities and are cost prohibitive. Thus, there is an urgent need to develop biomaterial tools to model the initiation of these lesions and their early interactions with their microenvironment. We propose one such method of characterizing these initial interactions utilizing a high-throughput microarray system of combinatorial, tissue-specific ECM proteins printed onto polyacrylamide (PA) islands. This discovery system lends itself to building more robust 3D in vitro model systems to further elucidate aspects of lesion etiology.


Materials and Methods:: Materials & Methods: Methacrylamide-functionalized gelatin (GelMA) was synthesized utilizing a previously established method. GelMA was characterized through NMR (Agilent 600MHz NMR), and compressive testing (Instron 5943). 3D hydrogels were prepared with 5wt% or 7wt% GelMA, PBS, and 0.1% w/v lithium phenyl phosphinate photoinitiator. GelMA was photopolymerized under a UV light (λ = 365 nm, 7.14 mW cm–2, AccuCure Spot System ULM-3-365) for 30s or 45s. Polyacrylamide (PA) hydrogels laden with single and pairwise combinations of ECM proteins were printed onto glass slide microarrays by the Underhill Lab at UIUC. These combinations were based on the ovaries and liver (tissue control). Endometriotic cells used for these arrays include human endometrial-endometriotic stromal cells (iEC-ESCs; Dr. Fazleabas, Michigan State University) and human endometriotic epithelial cells (12z; ABM). Healthy endometrial cells acting as cellular controls used for these arrays include human endometrial stromal cells (hESC, ABM) and human uterine epithelial cells (hEEC, Lifeline Cell Technology). Cells were seeded, incubated for 30 minutes, washed, incubated for 24 hours, washed, fixed-stained with DAPI-fluoromount, and imaged using a using a Zeiss Axioscan Z1. Cellular adhesion is quantified using an established pipeline in R. Endometriotic spheroids were formed utilizing ratios of cohorts of 12z cells and iEC-ESCs (100:0, 75:25, 50:50, 25:75, & 0:100 respectively) in 3k, 5k, and 50k cells/spheroid. Cells were seeded in low-adhesion, 96-well plates (CLS4520, Corning) for 24 hours at 37˚C, followed by 24 hours on a shaker at 60 rpm at 37˚C. These spheroids were added to GelMA precursor and UV polymerized.


Results, Conclusions, and Discussions:: Results: Using GelMA, we aimed to match the stiffness of natural ovarian tissue 3.3 ± 2.5 kPa. 5wt% and 7wt% GelMA hydrogels have significantly different (p< 0.05) elastic moduli of 2.14 kPa and 6.27 kPa respectively (Fig 1). Increasing UV exposure time, from 30s to 45s, significantly increases (p< 0.05) elastic moduli to 2.83 kPa and 8.11 kPa, respectively (Fig 1). Hence, we have identified a GelMA system which mimics ovarian stiffness. ECM combinations that demonstrated increased cellular adhesion in our microarray experiments will be included in our GelMA system to better recapitulate the complex ovarian microenvironment. We observed differential cellular adhesion (cell number) in our microarrays as a function of cell type (stromal or epithelial), cell health (endometriotic or healthy), substrate stiffness (1kPa or 6kPa), and ECM combinations (Fig 2). To create a more relevant model for endometriotic precursors, we sought to determine optimal conditions for spheroid culture. Endometriotic spheroids saw increased outgrowth when seeded with higher ratios of iEC-ESCs to 12z cells. (Fig 3).

Conclusions: Our findings are informing future work looking at coculture of healthy or diseased cells seeded onto the microarrays. As we downselect the most successful ECM combinations for attachment, we plan to implement these ECM proteins in our engineered hydrogel system to recapitulate the ovarian tissue in which lesions initiate. We will look at cohorts of endometriotic spheroids, comprised of endometriotic epithelial and stromal cells, in our engineered system alongside the inclusion of relevant ovarian epithelium (hOSECs, ScienCell). Further characterization of our system will include functional staining for vimentin for stromal populations, pan-cytokeratin for epithelial cells, and actin visualization. To best model the lesion microenvironment, we must harness relevant cellular systems, ECM, and mechanical properties. These complex models aim to provide insight that allows for the discovery of novel treatment targets.


Acknowledgements (Optional): : Acknowledgments: National Institutes of Health (R01 DK099528, R21 EB01949).

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