Tridimensional configurations of human mesenchymal stem/stromal cells to enhance cell paracrine potential towards wound healing processes

•Side-by-side comparison of three 3D configurations vs 2D monolayer MSC cultures.•3D MSC cultures include spheroids and alginate encapsulated spheroids or single cells.•MSC spheroids exhibit the highest oxidative stress resistance and paracrine potential.•Encapsulation supports low spheroids aggregation and high oxidative stress resistance.•Encapsulated MSC have higher angiogenic and chemotactic potential as spheroids. This study proposes to use alginate encapsulation as a strategy to assess the paracrine activity of 3D- and 2D-cultured human bone marrow mesenchymal stem/stromal cells (BM MSC) in the setting of wound repair and regeneration processes. A side-by-side comparison of MSC culture in three different 3D configurations (spheroids, encapsulated spheroids and encapsulated single cells) versus 2D monolayer cell culture is presented. The results reveal enhanced resistance to oxidative stress and paracrine potential of 3D spheroid-organized BM MSC. MSC spheroids (148±2μm diameter) encapsulated in alginate microbeads evidence increased angiogenic and chemotactic potential relatively to encapsulated single cells, as supported by higher secreted levels of angiogenic factors and by functional assays showing the capability of encapsulated MSC to promote formation of tubelike structures and migration of fibroblasts into a wounded area. In addition, a higher expression of the anti-inflammatory factor tumor necrosis factor alpha-induced protein 6 (TSG-6) was demonstrated by RT-PCR for encapsulated and non-encapsulated spheroids. Culture of spheroids within an alginate matrix maintains low aggregation levels below 5% and favors resistance to oxidative stress. These are important factors towards the establishment of more standardized and controlled systems, crucial to explore the paracrine effects of 3D-cultured MSC in therapeutic settings.