Mammalian proteins incorporate the LPXTG motif (247, 30). Here, we report how we initial defined a modular, synthetic, dissolvable ECM (“MSD-ECM”) composition appropriate for functional co-culture of epithelial and stromal cells, using the endometrium as a model epithelial-stromal interaction. We then investigated the kinetics of gel IGFBP-1 Proteins MedChemExpress dissolution as a function of enzyme and substrate concentrations also as gel crosslinking parameters, establishing a protocol that allowed fast dissolution of MSD-ECM gels made use of for co-cultures. The dissolution protocol was employed to study the effects of SrtAmediated dissolution on viability and signaling properties of endometrial cells and an more highly sensitive epithelial cell type, main hepatocytes. After evaluating the robustness in the dissolution process having a quantitative assay of 31 cytokines, growth aspects, and MMPs recovered from gels, we then compared the SrtA-mediated approach to typical degradation with proteolytic enzyme. We then investigated the relative concentrations of these molecules as detected within the culture supernate in comparison to the neighborhood microenvironment in the gel, applying quantitative recovery soon after dissolution. Lastly, we demonstrated how the temporal evolution from the cytokine network PDGF Proteins Biological Activity activated in response to stimulation of endometrial epithelial-stromal co-cultures with an inflammatory cue, interleukin 1 (IL-1), was revealed with greater depth and fidelity working with measurementsAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptBiomaterials. Author manuscript; readily available in PMC 2018 June 01.Valdez et al.Pagemade on proteins recovered in the dissolved MSD-ECM gel, compared to measurements on proteins from the standard culture supernate.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptResultsFunctionalized PEG hydrogels crosslinked with peptide substrates for SrtA support endometrial stromal-epithelial co-cultures Even though functionalized PEG hydrogels have already 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 demands 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 could be interrogated through SrtAmediated gel dissolution. We constructed on our earlier model from 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 best of encapsulated endometrial stromal cells (35). For this function, we extended the investigation of gel properties to include SrtA-mediated dissolution, and focused on recreating a glandular co-culture by coencapsulating epithelial and stromal cells within the functionalized PEG gels. Within this work, multi-arm PEG macromers activated with vinyl sulfone (PEG-VS) were partially functionalized together with the adhesion peptide PHSRN-K-RGD (36, 37) and crosslinked using a defined peptide containing substrates for both endogenous matrix metalloproteinases (MMPs) and exogenous SrtA (see Approaches for complete sequences). Hydrogel crosslinks are as a result topic to both cell-mediated remodeling at the same time as on-demand dissolution via addition of SrtA and GGG. PHSRN-K-RGD is actually a peptide mimic of integrin 51-binding domain in the 9th and 10th Type III repeats in fibronectin (F.