Local remodeling of synthetic extracellular matrix microenvironments by co-cultured endometrial epithelial and stromal cells enables long-term dynamic physiological function

Abstract

Mucosal barrier tissues, comprising a layer of tightly-bonded epithelial cells in intimate molecular communication with an underlying matrix-rich stroma containing fibroblasts and immune cells, are prominent targets for drugs against infection, chronic inflammation, and other disease processes. Although human in vitro models of such barriers are needed for mechanistic studies and drug development, differences in extracellular matrix (ECM) needs of epithelial and stromal cells hinder efforts to create such models. Here, using the endometrium as an example mucosal barrier, we describe a synthetic, modular ECM hydrogel suitable for 3D functional co-culture, featuring components that can be remodeled by cells and that respond dynamically to sequester local cell-secreted ECM characteristic of each cell type. The synthetic hydrogel combines peptides with off-the-shelf reagents and is thus accessible to cell biology labs. Specifically, we first identified a single peptide as suitable for initial attachment of both endometrial epithelial and stromal cells using a 2D semi-empirical screen. Then, using a co-culture system of epithelial cells cultured on top of gel-encapsulated stromal cells, we show that inclusion of ECM-binding peptides in the hydrogel, along with the integrin-binding peptide, leads to enhanced accumulation of basement membrane beneath the epithelial layer and more fibrillar collagen matrix assembly by stromal cells over two weeks in culture. Importantly, endometrial co-cultures composed of either cell lines or primary cells displayed hormone-mediated differentiation as assessed by morphological changes and secretory protein production. A multiplex analysis of apical cytokine and growth factor secretion comparing cell lines and primary cells revealed strikingly different patterns, underscoring the importance of using primary cell models in analysis of cell-cell communication networks. In summary, we define a “one-size-fits-all” synthetic ECM that enables long-term, physiologically responsive co-cultures of epithelial and stromal cells in a mucosal barrier format.

On-demand dissolution of modular, synthetic extracellular matrix reveals local epithelial-stromal communication networks

“…Therefore, the local in-gel measurements may be a more accurate method to reveal unknown interactions in complex 3D systems. These proof-of-principle studies with cell lines demonstrate the potential for this approach for detailed hypothesis-driven mechanistic studies with primary cells, which produce cytokines and growth factors more abundantly than cell lines (35). …”

“…We previously observed that the PHSRN-K-RGD peptide enhanced epithelial and stromal behaviors (attachment to gels, epithelial monolayer properties) compared to the minimal peptide RGD (35). In those studies, epithelial cells were cultured on functionalized gels.…”

“…Although functionalized PEG hydrogels have been used for epithelial (31), endothelial (32), connective tissue (33), and stromal cells (34), co-cultures of epithelial and stromal cells require tuning matrix properties to meet the needs of both cell types (35). Hence, we first established an endometrial stromal and epithelial co-culture in functionalized PEG gels as a model of a complex, multicellular, 3D system that can be interrogated through SrtA-mediated gel dissolution.…”

“…Hence, we first established an endometrial stromal and epithelial co-culture in functionalized PEG gels as a model of a complex, multicellular, 3D system that can be interrogated through SrtA-mediated gel dissolution. We built on our previous model of the endometrial mucosal barrier, in which we defined a functionalized PEG gel composition suitable for supporting functional viability of an endometrial epithelial monolayer cultured on top of encapsulated endometrial stromal cells (35). For this work, we extended the investigation of gel properties to include SrtA-mediated dissolution, and focused on recreating a glandular co-culture by co-encapsulating epithelial and stromal cells within the functionalized PEG gels.…”

A Gelatin Hydrogel to Study Endometrial Angiogenesis and Trophoblast Invasion

“…EECs were grown to confluence, passaged, and seeded at a density of 4 x 10 5 cells/mL on 5 wt% GelMA hydrogels cast in Ibidi angiogenesis μ slides [35]. Cells were allowed to attach for 6 hours and then hydrogels were washed once with cell growth medium to remove any unattached cells.…”