Scientific Forum. Human Adipocyte Viability Testing: A New Assay

Transcription

1 Human Adipocyte Viability Testing: A New Assay Julia W. MacRae, MD; Sunil S. Tholpady, PhD, MS; Adam J. Katz, MD; Thomas G. Gampper, MD; David B. Drake, MD; Roy C. Ogle, PhD; and Raymond F. Morgan, MD From the Department of Plastic and Reconstructive Surgery, University of Virginia Health System, Charlottesville, VA. Dr. Tholpady is also affiliated with the Department of Cell Biology; Dr. Ogle is also affiliated with the Departments of Cell Biology and Neurosurgery. 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. (Aesthetic Surg J 2003;23: ) The use of fat in plastic surgery is a vital part of the surgeon s armamentarium. The harvest, preparation, and reimplantation of fat grafts into a second surgical site are straightforward, minimally invasive, and cost-effective. These qualities have made fat grafting a common procedure in plastic surgery. Despite the obvious utility of fat grafting, strikingly little objective data have been accumulated with regard to the best method of performing this procedure. A practitioner reviewing the literature in an attempt to decide how to best perform the technique will find largely anecdotal experience and recommendations. 1 7 Furthermore, much of the existing literature sets forth confusing and contradictory information. For example, Coleman 8 advocates centrifuging the cells at 3000 rpm to consolidate them, whereas the work of Chajchir, 9 involving histologic analysis, suggests that centrifugation at 1000 or 5000 rpm destroys almost all living fat. Saline-solution washing of harvested fat is a standard technique for many practitioners; Chajchir 10 insists, however, that exposure of the cells to saline solution causes a loss of the fibrin which aids in the adhesion of the injected material to surrounding tissue. Ellenbogen 11 advocates bathing the cells in insulin for 45 to 60 minutes, as well as implanting 4- to 6-mm pearls of fat instead of performing needle or cannula harvesting. 2 Other variables that may bewilder the surgeon searching for the optimal technique include the diameter of the harvesting cannula, the diameter of the injection needle, and whether warming or chilling the cells is beneficial. Gatti 4 advises his patients to expect a minimum of 3 fat grafting sessions for an appreciable augmentation, whereas Coleman 8 has found, with few exceptions, that fat placed stably maintains its shape and consistency over time and has every indication of permanence. No matter the technical differences, however, most surgeons agree that injecting grafts with a high percentage live fat cells improves permanence in fat grafting. Testing the viability of free fat grafts in vitro is a challenging task that has not been satisfactorily achieved. Current methods of testing adipocyte viability (eg, glucose uptake, trypan-blue staining, and G6PDH uptake assay [reduced glucose-6-phosphate dehydrogenase]) rely on cell A ESTHETIC S URGERY J OURNAL ~ July/August

2 Figure 1. Time course of fat viability loss. isolation procedures. These procedures do not accurately reflect fat viability in the intact tissue because isolation of adipocytes eliminates the stromal component surrounding the fat cells. The stromal component provides the structural and biochemical support necessary for the maintenance of the adipocytes. Therefore any estimate viability in isolated fat cells does not reflect the viability of fat cells in the clinical environment. A model for testing fat grafts in the actual form in which they are used clinically would be useful in the evaluation of fat-handling techniques. One well-known and simple cell viability test is the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assay, a colorimetric assay in which active mitochondria cleave the pale-yellow substrate to yield a dark-purple formazan product (Chemicon International, Temecula, CA). Even newly dead cells will not cleave MTT, so the assay is very sensitive to the presence of living cells. However, no reports have been made of the MTT assay s being used to assay adipocyte viability. Because the major criticisms of fat grafting are the tendency for grafts to be resorbed and the inability to predict long-term results, knowing whether a particular handling technique tends to increase or decrease the likelihood of fat survival could predict a graft s long-term survival. The purpose of this study was to develop a method for assaying the viability of fat in its clinically used form and then to test several common techniques used in fat grafting for their effects on fat s viability. Methods Sample collection We obtained fresh tissue, collected during abdominoplasty, after it had undergone routine pathologic analysis in the University of Virginia Department of Surgical Pathology. Fat was then suctioned by hand with a 3.7- mm diameter Tulip cannula (Tulip Medical, San Diego, CA) on a 35-mL syringe. Time-course viability determination Samples (2 ml of whole-fat grafts) were placed in a 10-mL Falcon tube (Fischer Scientific, Pittsburgh, PA) and incubated at 37 C for 24 hours. Next, 200 μl of MTT solution AB (MTT plus phosphate-buffered saline solution) was added to the day 1 samples, and the tubes were gently inverted. A similar procedure was performed 24, 48, and 72 hours later for the day 2, day 3, and day 4 samples. After being incubated for 1 hour, the fat was divided into aliquots of 500, 250, and 125 mg (8 samples of each weight and incubation day) in 1-mL Eppendorf tubes. These aliquots were sonicated to disrupt the cell membranes and diluted with 200 μl of corn oil (Sigma Chemical Co, St. Louis, MO). The samples were then centrifuged at 16,000 g for 10 minutes in a microcentrifuge. At this point, each sample consisted of an upper lipid layer (with the purple formazan product) and a pellet containing cellular debris. The lipid layer was carefully pipetted from the top of the sample and read at 266 Aesthetic Surgery Journal ~ July/August 2003 Volume 23, Number 4

3 Figure 2. Differential viability in response to differing fat handling conditions. 470 nm in a spectrophotometer. We graphed the results using a Microsoft Excel spreadsheet (Microsoft Corp, Redmond, WA). Viability testing of handling techniques Fat was subjected to a variety of treatments, described below. Fat collected with the 3.7-mm cannula and not treated in any way was considered the control. Control tissue was compared with tissue collected with the use of a 2.1-mm cannula or by means of sharp dissection alone in 4- to 6-mm pearls. Some control tissue was subjected to the following treatments: addition of insulin (2 μl/ml fat), centrifugation at 1000 and 5000 rpm for 5 minutes, and extrusion through a Coleman needle, an 18-gauge needle, or a 23-gauge needle. Another sample was frozen (24 hours at 20 C), and yet another was treated with Triton-X 100 detergent (1:1 ml). The fat supplemented with Triton-X 100 served as a negative control because detergent causes lysis in fat cells. The samples, 2 ml of fat, were placed in Falcon tubes with 2 ml of Dulbecco s modified Eagle medium and gently inverted. This allowed the fat to be suspended in medium and optimized contact between the fat particles and the MTT solution. After 24 hours of incubation (or, in 1 sample, 20 freezing), 100 μl of MTT was added and allowed to react for 1 hour, with gentle mixing of the samples. The samples were then processed as described above for spectrophotometric analysis. We compared the data generated in this fashion using standard analysis of variance. Results Fat grafts exhibit decreased MTT signal as cells die We assayed fat samples for viability over the course of 4 days to establish the sensitivity of the MTT assay to cell death. As can be seen in Figure 1, the strength of the MTT signal is time- and concentration-dependent. As time in culture passes, fat cells die because of a lack of nutrients. This is reflected in Figure 1 as the progressive decrease in absorbance (and, proportionally, live fat cells) over the 4 days. In addition, a relatively linear relationship exists between absorbance and the quantity of fat analyzed. This is reflected in the slopes of the lines in Figure 1. The linearity displayed by the absorbance allows quantitation of live cells over a wide range of concentrations. Differential handling of fat grafts After concluding that the MTT assay was sensitive to cell death over a range of concentrations, we subjected fat to clinically applicable circumstances. Because of conflicting information in the literature on each of the methods, we tried all of them during the same trial so that interpatient variation would not confound statistical analysis. Enough fat was harvested from each abdominoplasty to suffice for each condition that was to be tested. Figure 2 shows the different variables tested and the viability of each group. We detected no statistically significant difference among any of the treatment groups (P <.05). The only difference observed was in the negative control that was exposed to Triton-X 100 detergent. In this sample, the vast majority of cells died, and the signal derived from the MTT assay was proportionally and significantly lower. Discussion Fat grafting has an extensive range of uses in plastic surgery. Given the great potential of this procedure, scientific studies should be conducted to optimize every Human Adipocyte Viability Testing: A New Assay A ESTHETIC S URGERY J OURNAL ~ July/August

4 parameter of the free fat grafting procedure. Optimization of the process to maximize cell viability may increase the reliability of the process over the short and long term if cell death plays any role in the outcome of fat grafts. We have conducted a methodologic study of the effects of different commonly used preimplantation treatments on cell viability. Measurement of cell viability is, in many systems, a simple matter. The most basic of these assays is the [ 3 H]thymidine-incorporation assay. In this assay, cells are labeled with tritiated thymidine. As these cells undertake basic activities, they incorporate tritium into their DNA. This assay is extremely sensitive; total DNA can be measured by proxy of tritium measurements. Total DNA is then correlated with the total number of cells in the system. However, the radioactivity associated with this assay and the impossibility of labeling tissue in vivo made this assay unusable for the purposes of this study. Other assays, such as G6PDH uptake assay, trypanblue exclusion, or glucose uptake, have been used to quantitate basic cellular activity as a measure of cell number. These assays have been optimized for cells in culture. They were of limited use because of the cell isolation steps required for the assay. The isolation process involves digestion of the cellular stroma with collagenase, as well as multiple washing, filtering, and purification steps. This process eliminates the soft-tissue stroma, present in whole-fat grafts, that protects the adipocytes from trauma. In the case of trypan-blue assay, the purification process leads to a falsely high estimate of viable cells because once cell membrane integrity is lost, adipocytes undergo lysis (which releases their lipid contents) and therefore cannot be visualized as nonviable cells. The MTT assay was finally chosen because of its sensitive nature and established use in other studies. This assay has been compared with lactate dehydrogenase release and trypan-blue exclusion in human epithelial cells and has been found to be the most sensitive assay of cell viability. 12 MTT, a tetrazolium compound, is cleaved by mitochondrial enzymes. The breakdown products form a purple crystal that can be solubilized and spectrophotometrically quantified. Increases in the chromogenic product correlate with increases in the number of living cells because the compound is not broken down by the mitochondrial enzymes of even recently dead cells. In testing the MTT assay for use on adipocytes, we found that fat cells responded vigorously, turning dark purple as a result of tetrazolium breakdown. Normally, the addition of hydrochloric acid/isopropanol at this point would solubilize the purple crystal into the supernatant. In the case of fat, the purple remained tightly bound in the tissue mass. Despite multiple attempts to release the compound using acids, bases, and detergents, we could not solubilize the product. At this point, we discovered that the breakdown products were actually lipophilic and that they were trapped within the lipid of the adipocytes. We developed an entirely new protocol that involved reacting the fat, sonicating it to disrupt the cell membranes, centrifuging the samples, and decanting the upper lipid layer, which was then diluted for spectrophotometric analysis. The results of this assay in whole fat grafts showed that the assay was successful in detecting different quantities of live fat and in determining the decrease in viability for each day in an incubator. Experimental knowledge of the kinetics of cell death over time is critical because these data can be correlated with the in vivo situation. Because free fat autografts show signs of revascularization 4 days after transplantation, 13 the remaining live cells in the fat samples incubated for 4 days may reflect the adipocytes destined to survive. Furthermore, testing of different treatment techniques showed that none of the commonly used techniques appears to adversely affect adipocyte viability or enhance viability. Likewise, freezing the fat did not have a negative effect on cell viability. This finding may reflect the fact that adipocytes filled with lipid undergo very little volume change when frozen compared with water-filled cells. Their cell membranes are therefore less likely to burst. Conclusion The variability seen clinically in the results of free fat grafting is probably not caused by the handling techniques tested above. Judging from the findings of this tudy, clinicians should use the techniques they find most effective in their practice. Centrifugation, which has been shown not to harm adipocyte viability, may be used to separate the fat from any water soluble solution associated with the fat (eg, saline solution washes or medium). It is possible that the harvest and implantation sites, as well as individual patient differences, account for the unpredictability of response to fat grafting. References 1. Coleman SR. Facial recontouring with lipostructure. Clin Plast Surg 1997;24: Ellenbogen R. Free autogenous pearl fat grafts in the face: a preliminary report of a rediscovered technique. Ann Plast Surg 1986;6: Ellenbogen R. Fat transfer: current use in practice. Clin Plast Surg 2000;27: Aesthetic Surgery Journal ~ July/August 2003 Volume 23, Number 4

5 4. Gatti JE. Permanent lip augmentation with serial fat grafting. Ann Plast Surg 1999;42: Har-Shai Y. An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects. Plast Reconstr Surg 1999;104: Shiffman MA, Mirrafati S. Fat transfer techniques: the effect of harvest and transfer methods on adipocyte viability and review of the literature. Dermatol Surg 2001;27: Ullmann Y. Enhancing the survival of aspirated human fat injected into nude mice. Plast Reconstr Surg 1998;101: Coleman SR. Structural fat grafts: the ideal filler? Clin Plast Surg 2001;28: Chajchir A, Benzaquen I, Moretti E. Comparative experimental study of autologous adipose tissue processed by different techniques. Aesthetic Plast Surg 1993;17: Chajchir A, Benzaquen I. Fat-grafting injection for soft-tissue augmentation. Plast Reconstr Surg 1989;84: Ellenbogen R. Fat transplantation. Plast Reconstr Surg 1987;79: da Costa A.O. Comparative analysis of three methods to assess viability of mammalian cells in culture. Biocell 1999;23: Billings E Jr. Historical review and present status of free fat graft autotransplantation in plastic and reconstructive surgery [see comments]. Plast Reconstr Surg 1989;83: Presented at The Aesthetic Meeting 2002 of The American Society for Aesthetic Plastic Surgery, Inc., Las Vegas, NV, April 29, 2002; and at the 45th Annual Scientific Meeting of the Southeastern Society of Plastic and Reconstructive Surgeons (SEPRS), Hilton Head, SC, June 21, Winner of the 2002 SEPRS Residents Competition. This study was funded in part by a grant from the Aesthetic Surgery Education and Research Foundation. Accepted for publication March 18, Reprint requests: Roy C. Ogle, PhD, Department of Plastic Surgery, Box , University of Virginia Health Sciences Center, Charlottesville, VA Copyright 2003 by The American Society for Aesthetic Plastic Surgery, Inc X/2003/$ doi: /maj Human Adipocyte Viability Testing: A New Assay A ESTHETIC S URGERY J OURNAL ~ July/August