Human adipocyte viability testing: a new assay

Abstract

Background: Surgical experience and anecdotal data on the most effective method of harvesting, preparing, and injecting autologous fat grafts are inconsistent and conflicting. Because the limitation of fat grafting is its resorption, understanding how various handling techniques affect adipocyte survival is crucial to optimizing its long-term survival.

Objective: We sought to develop a method for assaying fat viability in its clinically used form and then to test several common techniques used in fat grafting for their effects on the viability of the fat.

Methods: We performed the well-established MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] cell survival and proliferation assay on fat, but the colored enzyme-breakdown product could not be released into the supernatant for spectrophotometric analysis. An entirely new protocol was developed that allowed the MTT assay to quantitate the viability of free fat grafts. The assay was able to distinguish between different quantities of live fat and to quantify the decrease in viability when the fat is stored. We subjected the fat to various treatments, including insulin and Triton-X 100 detergent, (Sigma Aldrich, St. Louis, MO) centrifugation, extrusion through different types and sizes of needles, and freezing.

Results: With the exception of detergent, which decreased viability, all other treatments had no statistically significant effect on adipocyte survival. Freezing did not result in decreased cell viability.

Conclusions: It is unlikely that variations in the clinical results of free fat grafting are the result of the handling techniques examined in this study.