Numerical measurement of viable and nonviable adipocytes and other cellular components in aspirated fat tissue

Background: A reliable method with which to assay viability and number of adipocytes and other cellular components in adipose tissue remains to be established. Methods: The authors assessed cell viability and number obtained from 1 g of suctioned adipose tissue and respective layers (the top, middle, and bottom layers) before and after digestion and centrifugation, using cell staining with Hoechst 33342 and propidium iodide and the 2,3-bis(2-methoxy-4-vitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazoliumhydroxide (XTT) and glycerol-3-phosphate dehydrogenase assays (n = 10). The correlation between the number of prepared cells (adipocytes, adipose stromal cells, and white blood cells) and the resulting values from the XTT and glycerol-3-phosphate dehydrogenase assays was also examined (n = 5). The cell composition of the stromal vascular fraction isolated from the same adipose tissue was determined by multicolor flow cytometry (n = 5). Results: Hoechst 33342 and propidium iodide staining allowed distinguishing of viable adipocytes from lipid droplets, dead adipocytes, and cells other than adipocytes. The authors obtained 6.9 X 10(5) nonruptured adipocytes from 1 g of suctioned adipose tissue; 30 percent of the original adipocytes appeared to have been ruptured. Both the XTT and glycerol-3-phosphate dehydrogenase assays provided good correlations between the number of viable adipocytes and resulting values, but only the glycerol-3-phosphate dehydrogenase assay was strictly specific for adipocytes. The ratio of adipose stromal cells to adipocytes was found to be much larger than previously described. Conclusion: Single use or a combination of the viability assays used in this study can appropriately determine the number of adipocytes and other cells, although it remains difficult to assess original cells directly without tissue dissociation.