Three-dimensional cell culture (3DCC) improves secretion of signaling molecules of mesenchymal stem cells (MSCs)

OBJECTIVES: The secretome of mesenchymal stem cells (MSCs), also called MSC-conditioned media (MSC-CM), represents one of the promising strategies for cellular therapy and tissue repair and regeneration. MSC-CM contains growth factors and cytokines that control many cellular responses during development and regeneration. Traditional 2D cell culture (2DCC) has previously been used to generate MSC-CM while evidence has proved that the physiological and biological behaviors of cells in 2DCC are significantly different from those in 3D cell culture (3DCC). Therefore, the objective is to compare the content of MSC-CM generated from traditional 2DCC and 3DCC using a 3D scaffold. METHODS: Adipose tissue-derived MSCs (AT-MSCs) were isolated from four donors (N = 4) and characterized according to the criteria stipulated by the International Society for Cell Therapy (ISCT). MSCs at passage 3 were grown in traditional 2DCC until 70% confluence and MSC-CM were collected at 24, 48, and 94 h. On the other hand, MSCs at passage 3 were grown on a polystyrene scaffold for 10 days to generate a 3D model of MSCs, and then MSC-CM was collected at 24, 48, and 94 h. MSC-CM from both 2DCC and 3DCC were analyzed for protein content using ELISA. Haematoxylin eosin (HE) staining and immunofluorescence (IF) were used to characterize the 3DCC of MSCs. RESULTS: MSCs from 2DCC were fibroblast like cells, and flow cytometry showed they were positive for CD73 and CD105 while being negative for CD14, CD19, and HLA-DR. They were also able to differentiate into adipocytes, osteoblasts, and chondrocytes. HE and IF showed that MSCs formed 3D model structures on the polystyrene scaffold. MSC-CM collected from both 2DCC and 3DCC contained growth factors, e.g., platelet derived growth factor (PDGF-AB), transforming growth factor-1 (TGF-1), hepatocyte growth factor (HGF), stromal derived factor-1 (SDF-1), interleukin 1 (IL-1), and interleukin 6 (IL-6). Concentrations of biomolecules secreted by MSCs in 3DCC were significantly higher than in 2DCC. CONCLUSION: It could be concluded that 3DCC of MSCs using a polystyrene scaffold is a novel approach to generate MSC secretome for therapeutic applications.