Abstract
Summary: Cell-assisted lipotransfer has shown much promise as a technique to improve fat graft retention in both mouse and human studies. However, the literature varies as to whether fresh stromal vascular fraction or cultureexpanded adipose-derived stromal cells are used to augment volume retention. The authors' study sought to determine whether there was a significant advantage to using adipose-derived stromal cells over unpurified stromal vascular fraction cells in a mouse model of cell-assisted lipotransfer. (Plast. Reconstr. Surg. 139: 911, 2017.) 
C
ell-assisted lipotransfer, the addition of supplemental cells to autologous fat, has been promoted as a technique to enhance fat graft volume retention. [1] [2] [3] [4] A recent meta-analysis of 17 clinical cell-assisted lipotransfer studies concluded that it was more efficacious than autologous fat grafting alone, 5 although systematic reviews have highlighted the need for further studies (particularly given variability in methodologies reported) and more detailed investigations to define safety. 6, 7 As it stands, the current body of literature consists of a mixture of two different cell-assisted lipotransfer techniques: supplementation of fat with autologous stromal vascular fraction cells, and supplementation with in vitroexpanded adipose-derived stromal cells.
Although the use of fresh stromal vascular fraction is the most easily accomplished in the clinical setting, it means using a notoriously heterogenous cell population. That is, stromal vascular fraction consists of fibroblasts, endothelial cells, pericytes, preadipocytes, and various immune cells, in addition to adipose-derived stromal cells. 3, [8] [9] [10] Furthermore, the exact proportion/quality of adipose-derived stromal cells present in stromal vascular fraction varies between both individual patients and the body sites from which lipoaspirate is obtained. [11] [12] [13] This is an important consideration even in noncell-assisted lipotransfer fat grafting, as Philips et al. have shown a correlation between graft retention and the number of CD34 + cells present within a patient's adipose tissue.
14 Although adipose-derived stromal cells can be identified within stromal vascular fraction as the CD34 + / CD31 − /CD45 − proportion of cells by flow cytometry, they can also be defined as the adherent portion of stromal vascular fraction in vitro. 10 Thus, the rationale for the use of in vitroexpanded cells in cell-assisted lipotransfer is the ability to obtain a potentially larger number of more homogenous cells. However, use of an in vitro culture system represents a significant hurdle to be overcome before cell-assisted lipotransfer is approved for clinical use, and based on studies from our own laboratory, large numbers of supplemental cells may not be needed for enhancement of fat graft retention. 15 We therefore compared whether use of adiposederived stromal cells isolated through flow cytometry would better enhance fat graft volume with cell-assisted lipotransfer, or whether freshly obtained heterogeneous stromal vascular fraction would provide equivalent results, obviating the need for additional steps to isolate adipose-derived stromal cells.
Disclosure: The authors have no financial interest to declare in relation to the content of this article.

Plastic and Reconstructive Surgery • April 2017

MaterialS anD MethoDS

Cell harvest and Flow Cytometry
Fresh human lipoaspirate was obtained from three female patients, aged 45 to 55 years, with a body mass index ranging from 24 to 30 kg/m 2 , using suction-assisted lipectomy. Donors had no medical comorbidities, and informed consent was obtained in accordance with a Stanford Institutional Review Board-approved protocol (no. 2188). Lipoaspirate was processed for stromal vascular fraction as described previously. 16 Briefly, the tissue was washed with phosphate-buffered saline and digested with 0.075% type II collagenase (Sigma-Aldrich Co., St. Louis, Mo.). Red blood cell lysis buffer was used for removal of erythrocytes. Cells were counted and a portion set aside for combination with lipoaspirate for stromal vascular fraction-supplemented fat grafts.
The remaining cells were washed in staining buffer (1% fetal bovine serum, 1% P188, and 1% penicillin-streptomycin) before resuspension in a solution containing anti-CD45, anti-CD34, and anti-CD31 antibodies (eBioscience, Inc., San Diego, Calif.). A BD FACSAria II (BD Biosciences, San Jose, Calif.) was used for flow cytometry, with live CD34
− cells (adipose-derived stromal cells) isolated after gating to exclude debris.
Fat Processing and Grafting
Lipoaspirate was centrifuged for 3 minutes at 1200 g to separate the oil and aqueous components for removal. Either stromal vascular fraction cells or adipose-derived stromal cells were gently mixed with lipoaspirate to a concentration of 5 × 10 4 cells per milliliter of fat. This concentration was used based on our previous study determining the optimum concentration of cells to be used in cell-assisted lipotransfer for maximum fat graft retention. 15 Patient cells and fat were matched for all experiments (n = four animals per patient for stromal vascular fraction and adipose-derived stromal cells). Processed fat was then loaded into 1-cc syringes, and 200-μl grafts were placed beneath the scalps of immunocompromised mice, using a 14-gauge cannula.
imaging analysis
Micro-computed tomographic imaging was performed during week 0 after grafting for baseline volume measurements, and subsequently at 8 weeks. 17 A MicroCAT-II in vivo computed tomography scanner (ImTek, Inc./Siemens, Munich, Germany) was used, with fat distinguished from skin and bone by pixel intensity. As described previously, adipose tissue volume at each time point was calculated using Inveon Acquisition Workplace software (Siemens Medical Solutions, Malvern, Pa.) for interpolation of voxels based on pixel intensity. 18 Fat graft volume retentions were calculated as percentage change from baseline. + (includes endothelial cells) accounting for 21.9 ± 2.4 percent and 15.9 ± 7.4 percent of cells, respectively (Fig. 1) . Stromal vascular fraction-supplemented grafts had final volume retentions of 53.13 ± 24.59 percent, whereas the adipose-derived stromal cell-supplemented grafts retained 67.77 ± 7.007 percent of the original volume at 8 weeks. An F test to compare the variances of each group found them to be significantly different (p < 0.01). Thus, an unpaired t test with the Welch correction was used for analysis, determining that the differences in volume retention between the groups were statistically significant (p < 0.05) (Fig. 2) .
DiSCuSSion
Although stromal vascular fraction is undoubtedly efficacious in enhancing fat graft take, the large standard deviation of the volume retention speaks to the varied effects of using a heterogenous cell population. In contrast, use of purified adipose-derived stromal cells (a "standardized" cell population) allows for more predictable outcomes, and ultimately facilitates superior volume retention. As there is not yet a way to predetermine the quality of a patient's fat, the potential costs, both monetary and in terms of patient morbidity/dissatisfaction, of suboptimal outcomes must be weighed against the cost of adding a cell enrichment step to the cell-assisted lipotransfer procedure.
That said, with the abundance of stromal vascular fraction processing systems currently in clinical trials, the addition of a simplified procedure for immediate enrichment for CD34 + cells is conceivable. In fact, a U.S. Food and Drug Administration-approved device that positively selects for hematopoietic stem cells from peripheral blood samples, based on CD34 expression, is currently on the market. Although the CliniMACS Plastic and Reconstructive Surgery • April 2017 CD34 Reagent System (Miltenyi Biotec, Inc., San Diego, Calif.) is currently approved only for bone marrow transplants in cases of acute myeloid leukemia, the principles behind such a device could easily be extrapolated to human stromal vascular fraction samples.
Thus, with continued developments in automated cell processing systems, the benefits of improved and predictable volume retention seen with the use of adipose-derived stromal cells in cell-assisted lipotransfer could be realized without the need for additional time and inherent complications associated with in vitro culture. 

{'paper_id': '4519470', 'title': 'Purified Adipose-Derived Stromal Cells Provide Superior Fat Graft Retention Compared with Unenriched Stromal Vascular Fraction', 's2_url': 'https://api.semanticscholar.org/CorpusID:4519470', 'source': 'S2ORC'}