Combinatorial Extracellular Matrix Tissue Chips for Optimizing Mesenchymal Stromal Cell Microenvironment and Manufacturing

Despite the therapeutic potential of mesenchymal stromal cells (MSC), there is limited understanding of optimal extracellular matrix (ECM) environments to manufacture these cells. We developed tissue chips to study the effects of multi-factorial ECM environments under manufacturable stiffness ranges and multi-component ECM compositions. Manufacturing qualities of cell expansion potential, immunomodulation, and differentiation capacity were examined. The results show stiffness effects, with 900 kPa substrates supporting higher proliferation and osteogenic differentiation, along with anti-inflammatory IL10 expression, whereas 150 kPa substrates promoted adipogenic differentiation at 150 kPa, suggesting that optimal ECM environments may differ based on manufacturing goals. ECM biochemistries containing fibronectin and laminin further modulated MSC manufacturing qualities across various stiffnesses. Proteomic and transcriptomic analyses revealed unique ECM combinations that induced higher levels of angiogenic and immunomodulatory cytokines, compared to single factor ECMs. These findings demonstrate that optimized ECM environments enhance MSC manufacturing quality. Overall design: Short term gene changes were assessed in MSCs seeded on different substrate stiffnesses on various ECM combinations. After 48 hours in growth media, cells were lysed and RNA was extracted using GeneJET RNA isolation kit (Fisher Scientific) followed by RNA sequencing by Novogene Corporation (n=4).