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The central question addressed in this study is whether cells with different sizes have different responses to matrix stiffness. We used methacrylated hyaluronic acid (MeHA) hydrogels as the matrix to prepare an in vitro 3D microniche in which the single stem cell volume and matrix stiffness can be altered independently from each other. This simple approach enabled us to decouple the effects of matrix stiffness and cell volume in 3D microenvironments. Human mesenchymal stem cells (hMSCs) were cultured in individual 3D microniches with different volumes (2800, 3600, and 6000 μm 3 ) and stiffnesses (5, 12, and 23 kPa). We demonstrated that cell volume affected the cellular response to matrix stiffness. When cells had an optimal volume, cells could form clear stress fibers and focal adhesions on soft, intermediate, or stiff matrix. In small cells, stress fiber formation and YAP/TAZ localization were not affected by stiffness. This study highlights the importance of considering cellular volume and substrate stiffness as important cues governing cell-matrix interactions.

Cellular Volume and Matrix Stiffness Direct Stem Cell Behavior in a 3D Microniche