The contributions of inflammatory cocktail and stromal cell origin on a scaffold-free 3D biofabricated SSuPer tissue of endometriosis and normal endometrium

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Endometriosis is a debilitating disease that affects up to 10% of U.S. women. While treatments exist, more than 50% of women with endometriosis will have disease recurrence. The lack of useful models continues to impede the discovery of aspects of pathophysiology that are amenable to novel interventions. Therefore, there is an urgent need to develop novel models of endometriosis to advance the field significantly. This application proposes to create 3D biofabricated SSuPer (Self-Supporting Perfused) tissues that recapitulate either endometriosis or normal endometrium using the Kenzan method. The Kenzan method uses the Regenova Bio 3D Printer to place multicellular spheroids onto a microneedle array, called a Kenzan. On Kenzan, the spheroids fuse through cell-cell interaction and secretion of their native extracellular matrix, allowing more intrinsic intercellular communication. Intercellular communication between epithelial and stromal cells is paramount in endometrial function. Preliminary data shows that the Kenzan method effectively biofabricates the first scaffold-free 3D bioprinted constructs comprised of both endometriotic epithelial and endometrial stromal cells. Further, SSuPer tissues contain microchannels that can be perfused in a biologically inert FABRICA bioreactor. Because human endometrial stromal fibroblasts are critical to uterine function, stromal fibroblasts from women with endometriosis can be used to model the abnormal cellular function, aberrant molecular signaling, and pro-inflammatory transcriptomic profiles that occur in endometriosis. Additionally, pro- inflammatory mediators are elevated in the peritoneal fluid of women with endometriosis and likely play a role in progesterone resistance. This application's objective is to biofabricate 3D models of both endometriosis and normal endometrium, which recapitulate the inflammatory, transcriptomic, and steroid hormone response faithfully. The central hypothesis is that both inflammatory cytokines and stromal cell origin (i.e., endometriosis or not) independently affect the endometriotic or endometrial phenotype of each SSuPer tissue. SSuPer tissues, comprised of epithelial and stromal cells from either endometriosis or normal endometrium, will be biofabricated and perfused with either media or media with an inflammatory cocktail. SSuPer tissues will be determined to be either endometriosis or normal endometrium by inflammatory marker secretion, expression of endometriosis biomarkers, and global transcriptomic changes. Secondary validation studies will focus on steroid hormone response. The research proposed in this application is innovative because it will generate novel scaffold-free 3D biofabricated SSuPer tissues that maintain high biological fidelity to endometriosis or normal endometrium. Rigorously validated in vitro models will provide useful models for investigating the pathophysiology of endometriosis and other uterine dysfunction diseases such as infertility and recurrent pregnancy loss. Highly reproducible models are necessary for testing novel therapies.