Event Abstract Back to Event On-demand dissolution of 3D synthetic extracellular matrix for systems biology assays Jorge Valdez1 and Linda Griffith1 1 Massachusetts Institute of Technology, Biological Engineering, United States Introduction: Three-dimensional cell culture formats are desirable in applications ranging from mechanistic analysis of tissue behavior to modeling human responses to new drugs. There is a growing interest in analyzing paracrine interactions between stroma, epithelia, and immune cells in 3D physiological cultures, however the commonly-employed proteolytic methods used to recover cells from 3D gels are often inefficient and can impair fidelity of cytokine measurements and cell surface protein expression, and are not well-suited to allow recovery of locally-released cytokines. Here, we present a method for rapidly (<5 mins) dissolving synthetic polyethylene glycol (PEG)-based matrices using a bacterial transpeptidase with almost no known substrates in native mammalian proteins, in a manner that preserves cell viability and phenotype upon recovery from the gel, and greatly preserves cytokine levels for multiplex measurements compared common proteases. In brief, the approach relies on inclusion of a substrate for the enzyme in the gel crosslinks, allowing the gel to be dissolved by addition of the enzyme and a second simple substrate for the transpeptidase; hence it is translatable to any synthetic gel system. The approach is robust to a range of crosslinking densities, PEG macromer properties, and gel functionalities. Materials and Methods: Endometrial stromal and epithelial cells were encapsulated in PEG gels formed by either norbornene or vinyl sulfone crosslinking with the peptide sequences containing motifs recognized by metalloproteinases in addition to the LPRTG Sortase A (sortA) substrate. PEG macromers were partially functionalized with fluorescein-labelled RGD for dissolution characterization. Epithelial cells were cultured for 5 days before gel dissolution. Gels were dissolved by addition of recombinantly-produced sortA[1],[2] and the sortase substrate GGG. Cell viability upon recovery was measured with trypan blue and viability within gels was evaluated with a live/dead assay using confocal microscopy. Cytokine levels after exposure of sortA, trypsin, or liberase were measured by Luminex according to vendor’s specifications. Results and Discussion: We used sortase chemistry to recover cells from a 3D PEG hydrogel in <5 minutes without decreasing their viability (Fig. 1). We characterized the dissolution method across a wide range of relevant hydrogel mechanical conditions. We observed that sortase mediated-gel degradation preserved gland structures formed by epithelial cells. We compared the effect of sortase/GGG, trypsin, and liberase, on cytokine level measurements by Luminex and saw 16 out of 27 cytokine signals were annihilated by trypsin and/or liberase, while only IL-15 was affected by SrtA (Fig. 2). Conclusions: We developed a robust, easily transferable system for recovering cells from 3D matrices in a bio-orthogonal matter. This method does not affect cell viability, preserves cytokine levels compared to trypsin and collagenase, and could be extremely useful in cell signaling studies and morphogenesis assays. Funding from NSF EBICS center