Background: This study investigated fluorescent DAPI staining through antibody binding by measuring the fluorescent signal to confirm the presence of endothelial/vascular cells. The isolation of the Stromal Vascular Fraction (SVF) from adipose tissue yields material rich in multipotent cells. These multipotent cells have been used in numerous research studies to repair defects in bone, cartilage, and soft tissues. A key point in moving this work from research bench to the bedside for clinical studies is the ability consistently isolate, store and retrieve these SVF cells in a viable state.
Methods: Samples of human SVF were taken out of cryogenic storage and the cell viability was assessed using a hemocytometer with trypan blue and 6-diamidino-2-phenylindole (DAPI) for viability assays by light and fluorescent microscopy. The cells then underwent staining by antibodies to help identify specific cells types verifying cells of the SVF were isolated and retrieved. The antibody binding of seven cell type markers: CD31, CD34, CD44, CD45, Siglec, ALCAM and Collagen IV were assessed for staining of adipose tissue samples. These antibodies stain endothelial/vascular cells, mark for binding to stromal cells; extracellular matrix respectively was used to verify that SVF cells were successfully recovered from cryopreservation storage.
Results: The cell viability of the SVF was calculated to be 54 percent from the recovered cells. The antibody-treated cells were visualized for fluorescence and our results showed twenty-seven percent of the SVF fluoresced for CD31, forty-four percent for CD34, and twenty-five percent for CD44 cells.
Conclusion: The percent of multipotent stem cells recovered enabled our testing to proceed to antibody binding assays. It was concluded that 44 percent of CD34, a site specific maker for endothelial cells fluoresced, and no samples showed fluorescence by ALCAM a site specific marker for epithelial cells, verifying the presence of SVF cells.