BACKGROUND: Fat grafting bench research is difficult because many traditional endpoints cannot be used reliably with adipocytes. Manual cell counting with trypan blue is a common method of measuring cell viability. There are, however, multiple known limitations, including human error, inability to analyze cell size, overestimation of adipocyte viability, and labor intensity. In this study, the authors demonstrate the effectiveness of an improved method of accurate adipocyte analysis using an automated cell counter. METHODS: Human lipoaspirate was obtained, centrifuged, and digested. Samples were analyzed using a hemocytometer and an automated cell counter with two viability dyes. Results were then optimized by novel methods of preparation using carboxymethyl cellulose and formalin. RESULTS: Manual trypan blue cell counts ranged from 2,750,000 to 19,200,000 live cells/ml. Automated cell counts significantly reduced variability (3,230,000 to 4,290,000 cells/ml). Counting cells between 40 and 150 μm, which is more specific to adipocytes, yielded 1,040,000 to 1,420,000 viable cells/ml. Using a second viability dye, CellTiter Blue, cell counts ranged between 993,000 and 1,340,000 live cells/ml. Adding carboxymethyl cellulose substantially decreased sampling variability by 80 percent, and the use of formalin prevented the decrease in cell counts over 4 hours from 432,000 to 7,000 cells/ml. CONCLUSIONS: This novel method utilizing automated cell counters can more accurately identify the viable adipocyte population without the limitations of traditional cell counting. In addition, the use of carboxymethyl cellulose and formalin in the preparation process can decrease variability and stabilize cell counts over time. This is an efficient, specific, and reliable method of adipocyte analysis.
BACKGROUND: Fat grafting bench research is difficult because many traditional endpoints cannot be used reliably with adipocytes. Manual cell counting with trypan blue is a common method of measuring cell viability. There are, however, multiple known limitations, including human error, inability to analyze cell size, overestimation of adipocyte viability, and labor intensity. In this study, the authors demonstrate the effectiveness of an improved method of accurate adipocyte analysis using an automated cell counter. METHODS:Human lipoaspirate was obtained, centrifuged, and digested. Samples were analyzed using a hemocytometer and an automated cell counter with two viability dyes. Results were then optimized by novel methods of preparation using carboxymethyl cellulose and formalin. RESULTS: Manual trypan blue cell counts ranged from 2,750,000 to 19,200,000 live cells/ml. Automated cell counts significantly reduced variability (3,230,000 to 4,290,000 cells/ml). Counting cells between 40 and 150 μm, which is more specific to adipocytes, yielded 1,040,000 to 1,420,000 viable cells/ml. Using a second viability dye, CellTiter Blue, cell counts ranged between 993,000 and 1,340,000 live cells/ml. Adding carboxymethyl cellulose substantially decreased sampling variability by 80 percent, and the use of formalin prevented the decrease in cell counts over 4 hours from 432,000 to 7,000 cells/ml. CONCLUSIONS: This novel method utilizing automated cell counters can more accurately identify the viable adipocyte population without the limitations of traditional cell counting. In addition, the use of carboxymethyl cellulose and formalin in the preparation process can decrease variability and stabilize cell counts over time. This is an efficient, specific, and reliable method of adipocyte analysis.
Authors: Raymond M Esper; Michael Dame; Shannon McClintock; Peter R Holt; Andrew J Dannenberg; Max S Wicha; Dean E Brenner Journal: Cancer Prev Res (Phila) Date: 2015-10-20