3D Culture in Functionalized FN‐Silk Networks Facilitate Proliferation, Differentiation and Phenotypic Stability of Mature Human Primary Cells and Stem Cells

The recombinant functionalized silk protein FN-silk, including a cell adhesion motif from fibronectin, can form networks suitable for 3D culture of adherent cells. Such FN-silk networks have previously been shown to support the growth and differentiation of a wide array of cell types. Herein, we have developed a user-friendly methodology for the creation of free-floating FN-silk networks in 96-well plates with both mature human primary cells and stem cells. We show that human mesenchymal stem cells (hMSC) cultured in FN-silk networks form both cell-cell and cell-matrix contacts, resulting in tissue-mimicking 3D cultures. Viability and expression analysis revealed that hMSC in FN-silk networks have an initial proliferative phase with high cell viability and significantly lower hypoxia and apoptosis, compared to when cultured as scaffold-free spheroids. The FN-silk networks were shown to support differentiation of hMSC into adipocyte-like cells with well-maintained viability during the 3-week-long differentiation period, in contrast to the very poor long-term viability of scaffold-free 3D cultures. Improved adipogenesis was confirmed by lipid droplet staining, quantification of intracellular triglycerides, and secreted adiponectin levels, as well as expression analysis of multiple bona fide adipose markers. Lastly, we show that primary human hepatocytes maintain important functions and phenotypic markers when cultured in FN-silk networks, features that are lost rapidly during conventional 2D culture. We therefore propose FN-silk networks as a valuable scaffold for 3D human cell cultures, providing support for cell proliferation, differentiation, and the maintenance of critical tissue-specific functionality.