Fluorescent Visualization of Mesenchymal Stromal Cells on the Surface of Full-Layer Excisional Skin Wounds in Mice

Application of mesenchymal stromal cells improves skin regeneration due to immunomodulatory action and facilitation of extracellular matrix remodeling. However, there is a need to deepen our knowledge about the main mechanisms involved in healing stimulation via cell therapeutics. The purpose of the work was to assess the survival of mesenchymal stromal cells on the surface of full-thickness skin wounds in mice. Material and methods. Adult male Balb / C mice (n = 27) at the age of 5-6 months were used for modeling the skin wounds. To mimic the healing process in humans and prevent contraction, the edges of the wounds were fixed with a polymer medical plaster and glue. Viable or lethally damaged green fluorescent protein labeled cells within blood plasma-based gel in concentration 200-250×103 were applied on the wound surface. Plasma-based gel was prepared by direct mixture of human platelet poor plasma with calcium chloride and serum. Lethal cell damage was achieved by rapid immersion of suspension into liquid nitrogen with following rapid thawing without addition of any cryoprotectants. Cell membrane disruption was tested by Trypan blue staining. The reparative activity of mesenchymal stromal cells was evaluated at day 3, 7, and 14 after wounding using a planimetric method. Cell fluorescence in vivo and in vitro was confirmed by confocal microscopy. Photographs were then analyzed using the ImageJ 1.50b software. Results and discussion. The obtained results showed that rapid freezing with following rapid thawing led to disruption of plasma membrane in 98-99% of cells in suspension. Cell damage was accompanied by complete loss of fluorescence. When applied on the wound surface without any additional manipulations, transfected green fluorescent protein labeled positive cells remained viable and maintained fluorescence up to 5 days. Plasma-based gel ensured optimal conditions for cell survival in wounding site acting as a supportive extracellular matrix. The application of mesenchymal stromal cells suspension with high viability accelerated the wound closure in mice by 12-14% compared to spontaneous healing (control group) at day 3 of examination. Positive healing effect persisted for at least 1 week, while lethally damaged cells had a short-term stimulating effect. Planimetric assay of wound closure fully confirmed our data achieved using a confocal microscopy. Conclusions. Reparative effect of mesenchymal stromal cells observed at the early stages of wound healing could be driven by the release of growth factors improving vascularization and proliferation of cells in surrounding tissues. Obtained results indicate that the rate of wound healing depends on mesenchymal stromal cells viability and correlates with terms of their survival in vivo.