Three-dimensional, soft neotissue arrays as high throughput platforms for the interrogation of engineered tissue environments

Local signals from tissue-specific extracellular matrix (ECM) microenvironments, including matrix adhesive ligand, mechanical elasticity and micro-scale geometry, are known to instruct a variety of stem cell differentiation processes. Likewise, these signals converge to provide multifaceted, mechanochemical cues for highly-specific tissue morphogenesis or regeneration. Despite accumulated knowledge about the individual and combined roles of various mechanochemical ECM signals in stem cell activities on 2-dimensional matrices, the understandings of morphogenetic or regenerative 3-dimenstional tissue microenvironments remain very limited. To that end, we established high-throughput platforms based on soft, fibrous matrices with various combinatorial ECM proteins meanwhile highly-tunable in elasticity and 3-dimensional geometry. To demonstrate the utility of our platform, we evaluated 64 unique combinations of 6 ECM proteins (collagen I, collagen III, collagen IV, laminin, fibronectin, and elastin) on the adhesion, spreading and fate commitment of mesenchymal stem cell (MSCs) under two substrate stiffness (4.6 kPa, 20 kPa). Using this technique, we identified several neotissue microenvironments supporting MSC adhesion, spreading and differentiation toward early vascular lineages. Manipulation of the matrix properties, such as elasticity and geometry, in concert with ECM proteins will permit the investigation of multiple and distinct MSC environments. This paper demonstrates the practical application of high through-put technology to facilitate the screening of a variety of engineered microenvironments with the aim to instruct stem cell differentiation.