Poster BB10 - Co-Culture of Epithelial and Stromal Cells on High Throughput Cellular Microarrays to Determine Role of Extracellular Matrix Composition on Endometriotic Cell Adhesion

Introduction: Alterations in extracellular matrix (ECM) composition and cell adhesion biomolecules are believed to play a role in endometriosis. However, the specific role of the cells’ interactions with these proteins in endometriosis is not fully understood. In this study, we presented ECM proteins and adhesion biomolecules commonly found in the ovaries (a primary site of ectopic endometrial cysts) and the liver (a less common site for control) on high throughput contact-printed cellular microarrays to cells derived from healthy and diseased endometrial tissues. These arrays were used to study the differences in the cellular adhesion responses between disease states and between epithelial and stromal cells (two cell types commonly found in these endometrial lesions) to the arrayed proteins and hydrogel stiffness. For the first time, this high throughput microarray platform was utilized for the co-culture of two cell types on the protein islands, allowing for the study of more complex cell interactions and a more physiologically relevant recapitulation of the tissue microenvironment.

Materials and

Methods: Protein microarrays were printed on polyacrylamide (PA) hydrogels of elastic modulus 1 and 6 kPa with controlled spotting of 42 physiologically relevant extracellular matrix protein combinations. Human endometrial stromal cells (ESCs), primary human endometrial epithelial cells (EECs), 12z endometriotic epithelial-like cells, and ectopic endometrial stromal cells (iEC-ESCs) were cultured in the presence of CellTracker dyes that stained the ESCs green and the EECs orange, to distinguish between cell types. The four cell types represent two disease states, endometriotic and healthy, each with a stromal and epithelial cell associated with it (iEC-ESC + 12z & EEC + ESC). For each disease state, the corresponding two cell types were seeded on the arrays at a physiologically relevant ratio of 3:1 stromal to epithelial cells and cultured for 24 hours. The fixed cells were stained with phalloidin and DAPI and imaged using a fluorescent light microscope. The images were processed using ImageJ and CellProfiler, then analyzed in R.

Results, Conclusions, and Discussions: Excitingly, we found that the two cell types could be co-cultured on the arrays simultaneously for both disease states (Figure 1A). Also, these cells seemed to have different patterns of attachment between cell types and disease states. Separating the cell populations on the islands presented a challenge, as thresholding by mean fluorescent intensity for the two channels was not enough to reliably separate these cells. To address this, we designed a decision tree that utilized the observed significant differences in cell area from prior monoculture experiments of the cells on the microarrays (Figure 1B) to help determine the distinction between stromal and epithelial cells. This decision tree assigned a likelihood measurement based on the z-score normalized cell area, which was used to help distinguish the cell types when neither fluorescent channel surpassed the threshold. When the cells were adequately separated, we observed that the proportions of stromal and epithelial cells present on an island were dependent on the microenvironment composition and disease state (Figure 1C). We also found the cellular adhesion profile on the ECM microenvironments differed between the monoculture of these cells and the co-culture of the stromal and epithelial cells together (Figure 1D). This new application of cellular microarrays for co-culture presents endless opportunities for the controlled and high throughput study of how the microenvironment modulates the interactions between cell types. Future work consists of going beyond cellular adhesion and looking into co-culture's effects on cell morphology and actin dynamics, proliferative potential, and spatial patterning within the island. With these insights, we hope to better elucidate the role of extracellular matrix composition in endometriosis through the study of cells alone and in co-culture.

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