Three-Dimensional High-Throughput Cell Encapsulation Platform to Study Changes in Cell-Matrix Interactions

In their native extracellular microenvironment, cells respond to a complex array of biochemical and mechanical cues that can vary in both time and space. High throughput methods that allow characterization of cell-laden matrices are valuable tools to screen through many combinations of variables, ultimately helping to evolve and test hypotheses related to cell-ECM signaling. Here, we developed a platform for high-throughput encapsulation of cells in peptide-functionalized poly(ethylene glycol) hydrogels. Hydrogels were synthesized using a thiol-ene, photoclick reaction, which allowed the cell matrix environment to be modified in real time. Matrix signals were dynamically altered by in situ tethering of RGDS (0-1.5 mM), a fibronectin-derived adhesive peptide that induced more elongation than RLD or IKVAV, and/or by increasing the matrix modulus (1 to 6 kPa). This method was demonstrated with aortic valvular interstitial cells (VICs), a population of cells responsible for the pathological fibrosis and matrix remodeling that leads to aortic stenosis. VIC response to cell matrix interactions was characterized by quantifying cell morphology and the fraction of cells exhibiting alpha-smooth muscle actin (alpha SMA) stress fibers, a hallmark of the myofibroblast phenotype. VICs elongated in response to RGDS addition, with a dramatic change in morphology within 24 h. Myofibroblast activation was also dependent on RGDS addition, with VICs exhibiting high activation (16-24%) in 1 kPa gels with RGDS. Response to RGDS was path-dependent, with the amount of time exposed to the adhesive ligand important in determining VIC morphology and activation. Although VIC aspect ratios were dependent on the amount of time spent in a stiff vs soft gel, low levels of VIC activation (<= 4%) were observed in any gels cultured in higher modulus (6 kPa vs 1 kPa) microenvironments.