Role of mesenchymal stem cell conditioned medium on wound healing using a developed 3D skin model.

Alternative 3-dimensional (3D) skin models that replicate in vivo human skin are required to investigate important events during wound healing, such as collective cell migration, epidermal layer formation, dermal substrate formation, re-epithelialisation and collagen production. In this study, a matched human 3D skin equivalent model (3D-SEM) was developed from human skin cells (fibroblast and keratinocytes), characterised using haematoxylin and eosin, immunofluorescence staining and microRNA profiling. The 3D-SEM was then functionally tested for its use in wound healing studies. Mesenchymal stem cells (MSCs) were isolated and characterised according to the criteria stipulated by the International Society for Cell Therapy. Cytokine and growth factor secretions were analysed by enzyme-linked immunosorbent assay. MSC-conditioned medium (MSC-CM) was then tested for wound healing capacity using the developed 3D-SEM at different timepoints i.e., at one, two and four weeks. The constructed 3D-SEM showed consistent development of skin-like structures composed of dermal layers and epidermal layers, with the ability to express epidermal differentiation markers and full stratification. They also showed prolonged longevity in culture media, retaining full differentiation and stratification within the four weeks. MicroRNA profiling revealed a strong correlation in microRNA expression between the developed 3D-SEM and the original native skin (p<0.001; R=0.64). Additionally, MSC-CM significantly enhanced migration, proliferation and differentiation of epidermal cells in the wounded models compared to control models at the different timepoints. In conclusion, in this study, the developed 3D-SEM mimicked native skin at the cellular and molecular levels, and clearly showed the important stages of skin regeneration during the healing process. MSC secretome contains growth factors that play a pivotal role in the healing process and could be used as a therapeutic option to accelerate skin healing.

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.