Hyaluronic Acid Microparticles Promote Uniform Differentiation and Survival of Stem Cells in Spheroid Cultures

Three-dimensional (3D) spheroid cultures offer a physiologically relevant environment for stem cell differentiation but face challenges with oxygen and nutrient delivery, leading to uneven differentiation and apoptosis in larger spheroids. This study addresses these limitations by incorporating hyaluronic acid (HA) microparticles into AdMSC spheroids. HA microparticles, synthesized via an inverse emulsion method, were confirmed to be cross-linked and porous, enhancing diffusion and microenvironmental support. Spheroids containing HA microparticles showed significantly improved cell viability and reduced apoptosis, evidenced by TUNEL staining and the upregulation of BCL2, alongside the downregulation of Caspase3 and Caspase7. Enhanced chondrogenic and adipogenic differentiation was confirmed through histological staining, immunohistochemistry, and gene expression analysis, with the 30% HA group demonstrating the most uniform and robust differentiation. These findings highlight HA microparticles as an effective tool for overcoming diffusion limitations in spheroid cultures, enabling uniform differentiation and improved cell survival. This approach holds promise for regenerative medicine applications such as cartilage repair, adipose tissue engineering, and advanced tissue modeling.