The matrix: building more bioactive extracellular matrices via tuning of substrate stiffness

Abstract

Background & Aim Mesenchymal stromal cells (MSCs) have long been at the forefront of cellular therapy and regenerative medicine, and they are being explored in more than 700 NIH-funded trials for their therapeutic potential. The low frequency of MSCs in the human body, poor yield of MSCs from tissue biopsies, and high number of MSCs required for clinical applications demand that tissue-isolated MSCs undergo a lengthy expansion ex vivo, during which they exhibit senescence and become unsuitable for therapeutic applications. Decellularized extracellular matrices (dECM) present a novel solution to this issue, as MSCs grown on dECM maintain greater viability as compared to those grown on conventional tissue culture substrates. The effect of environmental parameters on extracellular matrix bioactivity requires further elucidation. Particularly, we will investigate the influence of substrate stiffness on the composition of MSC-derived ECM, to produce customized dECM which enhances cellular growth and functionality. This is achieved by culturing MSCs on hydrogels of varying stiffnesses, and assessing the bioactivity of the ECM produced during culture. Methods, Results & Conclusion Mechanical properties of tunable stiffness polyacrylamide gels were verified using an Instron Microtester. Cells used were isolated from the decidua basalis, and ECM was sourced from decidua basalis hTERT-transfected MSCs. ECM deposition was induced by addition of ascorbic acid. Instron measurements confirm the elastic moduli of fully-hydrated polyacrylamide gels to be 4 kPa, 10 kPa, and 40 kPa, tailored to mimic the stiffness of adipose tissue, skeletal muscle tissue, and collagenous bone tissue, respectively. MSCs exhibit a spindle-shaped morphology when cultured on the hydrogels. Confocal microscopy reveals microarchitectural differences between ECM deposited on softer vs. stiffer hydrogels. We have shown that MSCs can be grown on hydrogels of varying stiffnesses, and can be induced to laydown extracellular matrix. The functionalization of the gels with collagen I to aid cell adhesion has been confirmed with immunofluorescence. The deposition of ECM has also been confirmed with confocal microscopy, and the alignment of ECM fibrils has been shown to depend on substrate stiffness. Further work will involve proteomic characterization of the dECMs, and culturing MSCs on the dECMs to assess bioactivity. We expect that the substrate stiffness will modulate the ECM bioactivity, allowing customization of tailored dECM for MSC expansion.