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Recent studies have found discordant mechanosensitive outcomes when comparing 2D and 3D, highlighting the need for tools to study mechanotransduction in 3D across a wide spectrum of stiffness. A gelatin methacryloyl (GelMA) hydrogel with a continuous stiffness gradient ranging from 5 to 38 kPa was developed to recapitulate physiological stiffness conditions. Adipose-derived stem cells (ASCs) were encapsulated in this hydrogel, and their morphological characteristics and expression of both mechanosensitive proteins (Lamin A, YAP, and MRTFa) and differentiation markers (PPARγ and RUNX2) were analyzed. Low-stiffness regions (∼8 kPa) permitted increased cellular and nuclear volume and enhanced mechanosensitive protein localization in the nucleus. This trend was reversed in high stiffness regions (∼30 kPa), where decreased cellular and nuclear volumes and reduced mechanosensitive protein nuclear localization were observed. Interestingly, cells in soft regions exhibited enhanced osteogenic RUNX2 expression, while those in stiff regions upregulated the adipogenic regulator PPARγ, suggesting that volume, not substrate stiffness, is sufficient to drive 3D stem cell differentiation. Inhibition of myosin II (Blebbistatin) and ROCK (Y-27632), both key drivers of actomyosin contractility, resulted in reduced cell volume, especially in low-stiffness regions, causing a decorrelation between volume expansion and mechanosensitive protein localization. Constitutively active and inactive forms of the canonical downstream mechanotransduction effector TAZ were stably transfected into ASCs. Activated TAZ resulted in higher cellular volume despite increasing stiffness and a consistent, stiffness-independent translocation of YAP and MRTFa into the nucleus. Thus, volume adaptation as a function of 3D matrix stiffness can control stem cell mechanotransduction and differentiation.

Stiffness gradient hydrogels are a useful platform for studying mechanical interactions between cells and their surrounding environments. Here, we developed linear stiffness gradient hydrogels by controlling the polymerization of gelatin methacryloyl (GelMA) via differential UV penetration with a gradient photomask. Based on previous observations, a stiffness gradient GelMA hydrogel was created ranging from ~ 4 to 13 kPa over 15 mm (0.68 kPa/mm), covering the range of physiological tissue stiffness from fat to muscle, thereby allowing us to study stem cell mechanosensation and differentiation. Adipose-derived stem cells on these gradient hydrogels showed no durotaxis, which allowed for the screening of mechanomarker expression without confounding directed migration effects. In terms of morphological markers, the cell aspect ratio showed a clear positive correlation to the underlying substrate stiffness, while no significant correlation was found in cell size, nuclear size, or nuclear aspect ratio. Conversely, expression of mechanomarkers (i.e. Lamin A, YAP, and MRTFa) all showed a highly significant correlation to stiffness, which could be disrupted via inhibition of non-muscle myosin or Rho/ROCK signalling. Furthermore, we showed that cells plated on stiffer regions became stiffer themselves, and that stem cells showed stiffness-dependent differentiation to fat or muscle as has been previously reported in the literature.

We present the first quantitative measurements of hydrate formation probability, nucleation rate and growth on a water droplet suspended within a high pressure natural gas by acoustic levitation.

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