Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases.

The liver is the largest solid organ in the body and is responsible for hundreds of functions, including nutrient and xenobiotic/drug metabolism, serum protein production, nutrient storage, and immune system response. [1] Models of these processes in both healthy and diseased states, as well as their responses to therapeutic interventions, are of intense interest in drug discovery and development. [2][3][4] The increasing commercial availability and utility of human donor liver cells are driving the advancement of in vitro models of human liver, supplementing animal models that fail to capture the full complexity of human liver phenotypes. [3][4][5] An evergrowing number of in vitro liver model applications involve aspects of liver immune responses. [6][7][8][9] Such responses are mediated not just by cells classically associated with the immune system but also by hepatocytes and other nonparenchymal cells, which all produce immunemodulating signaling molecules. It is thus crucial to consider how the in vitro microenvironment regulates the potential for hepatocytes and other cell types to respond to perturbations, which may then, in turn, influence other metabolic, [6] toxicity, [10] and disease