Tissue-Mimetic Culture Enhances Mesenchymal Stem Cell Secretome Capacity to Improve Regenerative Activity of Keratinocytes and Fibroblasts in vitro.

Mesenchymal stem/stromal cells (MSCs) are a heterogenous population of multipotent and highly secretory cells currently being investigated in the field of wound healing for their ability to augment tissue responses. The adaptive response of MSC populations to the rigid substrate of current 2D culture systems has been considered to result in a deterioration of regenerative "stem-like" properties. In this study, we characterize how the improved culture of adipose-derived mesenchymal stem cells (ASCs) within a tissue-mimetic 3D hydrogel system, that is mechanically similar to native adipose tissue, enhances their regenerative capabilities. Notably, the hydrogel system contains a porous microarchitecture that permits mass transport, enabling efficient collection of secreted cellular compounds. By utilizing this 3D system, ASCs retained a significantly higher expression of ASC "stem-like" markers while demonstrating a significant reduction in senescent populations, relative to 2D. Additionally, culture of ASCs within the 3D system resulted in enhanced secretory activity with significant increases in the secretion of proteinaceous factors, antioxidants, and extracellular vesicles (EVs) within the conditioned media (CM) fraction. Lastly, treatment of wound healing cells, keratinocytes (KCs) and fibroblasts (FBs), with ASC-CM from the 2D and 3D systems resulted in augmented functional regenerative activity, with ASC-CM from the 3D system significantly increasing KC and FB metabolic, proliferative, and migratory activity. This study demonstrates the potential beneficial role of MSC culture within a tissue-mimetic 3D hydrogel system that more closely mimics native tissue mechanics, and subsequently how the improved phenotype augments secretory activity and potential wound healing capabilities of the MSC secretome.