Three-dimensional imaging of cell and extracellular matrix elasticity using quantitative micro-elastography.

Recent studies in mechanobiology have revealed the importance of cellular and extracellular mechanical properties in regulating cellular function in normal and disease states. Although it is established that cells should be investigated in a three-dimensional (3-D) environment, most techniques available to study mechanical properties on the microscopic scale are unable to do so. In this study, for the first time, we present volumetric images of cellular and extracellular elasticity in 3-D biomaterials using quantitative micro-elastography (QME). We achieve this by developing a novel strain estimation algorithm based on 3-D linear regression to improve QME system resolution. We show that QME can reveal elevated elasticity surrounding human adipose-derived stem cells (ASCs) embedded in soft hydrogels. We observe, for the first time in 3-D, further elevation of extracellular elasticity around ASCs with overexpressed TAZ; a mechanosensitive transcription factor which regulates cell volume. Our results demonstrate that QME has the potential to study the effects of extracellular mechanical properties on cellular functions in a 3-D micro-environment.

Stem Cell Mechanosensation on Gelatin Methacryloyl (GelMA) Stiffness Gradient Hydrogels.

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.

Volume Adaptation Controls Stem Cell Mechanotransduction.

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.

Hydrogels with an embossed surface: An all-in-one platform for mass production and culture of human adipose-derived stem cell spheroids.

Stem cell spheroids have been studied extensively in organoid culture and therapeutic transplantation. Herein, hydrogels with an embossed surface (HES) were developed as an all-in-one platform that can enable the rapid formation and culture of a large quantity of size-controllable stem cell spheroids. The embossed structure on the hydrogel was adjustable according to the grit designation of the sandpaper. Human adipose-derived stem cells (hADSCs) were rapidly assembled into spheroids on the hydrogel, with their size distribution precisely controlled from 95 ±â€¯6 µm to 181 ±â€¯15 µm depending on surface roughness. The hADSC spheroids prepared from the HES demonstrated expression of stemness markers and differentiation capacity. In addition, HES-based spheroids showed significantly greater VEGF secretion than spheroids grown on a commercially available low-attachment culture plate. Exploiting those advantages, the HES-based spheroids were used for 3D bioprinting, and the spheroids within the 3D-printed construct showed improved retention and VEGF secretion compared to the same 3D structure containing single cell suspension. Collectively, HES would offer a useful platform for mass fabrication and culture of stem cell spheroids with controlled sizes for a variety of biomedical applications.

Developing a high-throughput platform to direct adipogenic and osteogenic differentiation in adipose-derived stem cells.

The popular reductionist approach towards stem cell differentiation has identified an array of factors that can induce lineage-specific differentiation. Whether these variables direct differentiation in a multivariable setting in the same way as a single-variable setting is a question of interest. Polyacrylamide hydrogels of specific stiffness were microcontact printed with fibronectin in specific shapes and sizes to construct 54 different extracellular matrix types of defined stiffness, shape, and area. Three media types were also used to investigate the effect of both biomechanical and biochemical inducers of differentiation on adipose-derived stem cells. Stiffness was found to be a significant regulator of both adipogenic (3 kPa) and osteogenic (35 kPa) differentiation across all conditions. Biochemically induced osteogenic differentiation occurred as well as increased osteogenesis on larger extracellular matrix areas (above 5,000 µm2 ). The absence of clear trends for all variables demonstrates the atypical expression patterns that arise when variables that may work competitively or synergistically are considered together in a single, high-throughput system.