EXPERIMENTAL Imaging the Stromal Vascular Fraction during Soft-Tissue Reconstruction Jacqueline M. Bliley, M.Sc. Latha Satish, M.Sc., Ph.D. Meghan M. McLaughlin, B.S. Russell E. Kling, M.D. James R. Day, B.S. Tara L. Grahovac, M.D. Lauren E. Kokai, Ph.D. Wensheng Zhang, Ph.D. Kacey G. Marra, Ph.D. J. Peter Rubin, M.D. Pittsburgh, Pa.
Background: Although fat grafting is an increasingly popular practice, suboptimal volume retention remains an obstacle. Graft enrichment with the stromal vascular fraction has gained attention as a method of increasing graft retention. However, few studies have assessed the fate and impact of transplanted stromal vascular fraction on fat grafts. In vivo imaging techniques can be used to help determine the influence stromal vascular fraction has on transplanted fat. Methods: Stromal vascular fraction was labeled with 1,1′-dioctadecyl-3,3,3′,3′tetramethylindotricarbocyanine iodide (DiR), a near-infrared dye, and tracked in vivo. Proliferation and differentiation of labeled cells were assessed to confirm that labeling did not adversely affect cellular function. Different doses of labeled stromal vascular fraction were tracked within fat grafts over time using the in vivo imaging system. Results: No significant differences in differentiation and proliferation were observed in labeled versus unlabeled cells (p > 0.05). A pilot study confirmed that stromal vascular fraction fluorescence was localized to fat grafts and different cell doses could be distinguished. A larger-scale in vivo study revealed that stromal vascular fraction fluorescence was statistically significant (p < 0.05) between different cell dose groups and this significance was maintained in higher doses (3 × 106 and 2 × 106 cells/ml of fat graft) for up to 41 days in vivo. Conclusions: DiR labeling allowed the authors to differentiate between cell doses and confirm localization. This article supports the use of DiR labeling in conjunction with in vivo imaging as a tool for imaging stromal vascular fraction within fat grafts. (Plast. Reconstr. Surg. 136: 1205, 2015.) CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.
A
utologous fat grafting is a technique used to reconstruct soft-tissue defects1 of the face2,3 and breast4; however, there are limits to its use. Problems with fat grafting include insufficient vasculature at the injection site, leading to unpredictable retention rates.5 Cellular therapies with adipose-derived stem cells and the stromal vascular fraction are clinically used as a means of preserving adipose.6,7 It is hypothesized that these cells mediate angiogenesis within transplanted adipose by secreting angiogenic growth factors.8 Stromal vascular fraction–enriched fat grafts exhibit decreased From the Departments of Plastic Surgery and Bioengineering, University of Pittsburgh; and the McGowan Institute for Regenerative Medicine. Received for publication September 19, 2014; accepted May 6, 2015. Copyright © 2015 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000001815
necrosis compared with unenriched grafts.9 Despite the positive effects associated with these therapies, few studies have assessed the fate, localization, and behavior of stromal vascular fraction within fat grafts over time. In vivo imaging combined with infrared labeling represents a novel technique of tracking cells within fat grafts. Infrared labeling allows the user to track specific ligand antagonists or cells because of the high-penetrance of infrared light into tissues and, subsequently, the high signal-to-noise ratio of infrared targets within tissues.10 In particular, 1,1′-dioctadecyl3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), an infrared fluorescing carbocyanine dye, can label cells, which can then be tracked using the in vivo imaging system. Disclosure: The authors have no financial interests to declare in relation to the content of this article.
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Plastic and Reconstructive Surgery • December 2015 Current research with in vivo infrared imaging monitors biological processes, including tumor, lymphatic, and vascular formation.11–13 DiR labeling has also detected the differences between normal and leukemic hematopoietic stem cell homing.14 DiR-labeled macrophages have also been used to assess granuloma formation.15 In this study, DiR labeling did not alter cellular activity (based on in vitro proliferation and differentiation assays). DiR-labeled stromal vascular fraction was combined with fat grafts and tracked using the in vivo imaging system. Cell dose and localization within the transplanted fat was confirmed. This method lends it applicability to many studies to assess the location, integration, and behavior of stromal vascular fraction with the ultimate goal of guiding clinical decisions regarding stromal vascular fraction dose.
PATIENTS AND METHODS The following materials were used: neuropaddies, phosphate-buffered saline (Gibco, Carlsbad, Calif.), Luer-lock syringes, type II collagenase (Worthington Biochemical Corp., Lakewood, N.J.), standard plating medium [Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F12 (Corning, Manassas, Va.), 10% fetal bovine serum (Atlas Biologicals, Fort Collins, Colo.), 1% penicillin/streptomycin, 1% Fungizone (Lonza, Walkersville, Md.), and 0.001% dexamethasone (Sigma-Aldrich Corp., St. Louis, Mo.)], Vybrant DiR (Invitrogen, Carlsbad, Calif.), MACS buffer (Miltenyi Biotec, San Diego, Calif.), Cyquant proliferation assay (Invitrogen), adipocyte differentiation media (Zen-Bio, Inc., Research Triangle Park, N.C.), AdipoRed assay (Lonza), 96-well microplate (Ibidi, Martinsried, Germany), ketamine (Butler Schein, North Dublin, Ohio), xylazine (Vedco, Inc., St. Joseph, Mo.), and ketoprofen (Fort Dodge Animal Health, Overland Park, Kan.). Human Subject, Stromal Vascular Fraction Isolation, and Cell Culture The University of Pittsburgh Institutional Review Board approved tissue collection under PRO13020131. Abdominal subcutaneous fat was harvested using a 17-gauge bucket handle cannula attached to 10-ml Luer-lock syringe. Handheld suction was created by gently pulling back on the syringe.16 For in vitro and in vivo experiments, stromal vascular fraction was isolated from donor-matched adipose tissue.17 Lipoaspirate was digested in type II collagenase at 37°C for 30 minutes. The tissue was centrifuged at 1000 rpm for 10 minutes. The pellet was then suspended
in erythrocyte lysis buffer and centrifuged at 1000 rpm for 10 minutes. Cells were labeled with DiR directly after isolation for all experiments (see later under DiR Labeling). Lipoaspirate Processing and Cell Enrichment Lipoaspirate was processed by means of the Coleman technique.3,18 Adipose tissue was aspirated and syringes were centrifuged for 3 minutes at 3000 rpm. Subsequently, the upper oil and lower aqueous layer were removed. Stromal vascular fraction–enriched fat grafts (6 × 106, 3 × 106, 2 × 106, 1.5 × 106, 1 × 106, and 0.5 × 106 cells/ml of graft) were prepared. Stromal vascular fraction was suspended in 100 µl of phosphate-buffered saline per milliliter of fat graft and gently mixed by means of Luer-to-Luer transfer. Homogenous mixing of cells was confirmed using the in vivo imaging system. Control grafts were mixed with unlabeled cells at the same concentration. Grafts were subsequently separated into 0.5ml aliquots for injection. DiR Labeling Vybrant DiR was prepared at 2.5 mg/ml in ethanol and stored at 4°C according to the manufacturer’s protocol. Cells were suspended at 1 × 106 cells/ml in serum-free medium. DiR dye (5 µl/1 ml of media) was added to the suspension and incubated for 20 minutes at 37°C in 5% carbon dioxide.19 After incubation, labeled cells were centrifuged at 1500 rpm for 5 minutes at 37°C. The cells were washed in serum containing medium four times to ensure that unbound dye was completely removed. Labeled cells were combined with fat grafts for in vivo experiments or used for in vitro flow cytometry, imaging, or functional assays. Labeling Efficiency Flow cytometry was used to determine DiR labeling efficiency. Unlabeled cells served as a negative control. Cells were suspended in magneticactivated cell sorting buffer at 1 × 106 cells/ml and then subjected to flow cytometry using the APC-Cy7-A laser (BD Biosciences, San Jose, Calif.). Labeling efficiency was determined relative to the total number of live cells within the sample. Proliferation, Differentiation, and Dye Maintenance Proliferation and differentiation studies were performed to confirm that labeling did not alter cell behavior. Dye maintenance studies were completed to determine whether cells could be tracked after adipocyte differentiation. Cells (passage
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction 0) were labeled and plated at 10,000 cells/well in a 96-well plate. The CyQuant kit was used to assess cell proliferation between labeled (n = 8) and unlabeled cells (n = 8) for 24, 48, and 72 hours. Proliferation was compared to a DNA standard curve, and fluorescence was measured using a Tecan SpectraFluor microplate reader (Tecan Systems, Inc., San Jose, Calif.).
For differentiation studies, labeled and unlabeled cells were incubated in adipocyte differentiation medium (n = 8) and standard plating medium (n = 8). After a 14-day incubation, lipid accumulation was quantified using the AdipoRed Assay. Fluorescence was quantified using a Tecan SpectraFluor microplate reader. To determine dye maintenance following differentiation, cells were differentiated
Fig. 1. Establishing a mixing technique for stromal vascular fraction within soft-tissue grafts. Unlabeled and DiR-labeled cells (1 × 106 cells/ml) were combined with the fat graft to confirm uniform distribution of cells within the grafts. Plates were imaged using the in vivo imaging system.
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Plastic and Reconstructive Surgery • December 2015 into adipocytes, and confocal microscopy was used to assess DiR-dye preservation. In Vitro Dye Longevity Stromal vascular fraction was labeled as described previously and seeded at 5000 cells/cm2 in multiple 175-cm2 culture flasks. Individual flasks were used at each time point to avoid passaging cells during the
study. At specified time points, subsets of cells (n = 6) were lifted and suspended at 100,000 cells/100 µl of phosphate-buffered saline. Unlabeled cells were used as negative controls. Cell suspension plates were imaged with the in vivo imaging system at 750nm excitation and 780-nm emission. Exposure time and imaging field of view were kept constant for all time points. Signal intensity was quantified as the
Fig. 2. DiR labeling efficiency. Stromal vascular fraction cells labeled with DiR were subjected to flow cytometry to determine labeling efficiency. Unlabeled cells were used as a negative control, and little to no background fluorescence was observed. Approximately 60 percent of the stromal vascular fraction population was labeled with DiR.
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction
Fig. 3. No significant differences were observed in proliferation of labeled (n = 8) versus unlabeled cells (n = 8) at 24, 48, or 72 hours. Paired t tests were used to analyze differences in proliferation between labeled and unlabeled cells at 24, 48, and 72 hours, with values of p < 0.05 being considered statistically significant.
sum of the number of photons detected within our region of interest using Xenogen Living Image software (Xenogen Corp., Alameda, Calif.). In Vivo Dye Longevity Xenograft Implantation Female athymic nude mice, 6 weeks old (Harlan Laboratories, Inc., Indianapolis, Ind.), were housed at the University of Pittsburgh Division of Laboratory Animal Resources. Mice were housed under controlled conditions (20° to 23°C, 40 to 60% humidity, and 12-hour light/dark cycles), fed
a standard imaging diet (alfalfa-free), and given sterile water ad libitum. The University of Pittsburgh Institutional Animal Care and Use Committee approved all experiments. Mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg ketamine) and xylazine (12 mg/kg). Half-milliliter injections of processed lipoaspirate (on the left flank) and DiRlabeled cell-enriched grafts (on the right flank) were injected subcutaneously onto the dorsum of each mouse. Processed lipoaspirate–alone animals
Fig. 4. Adipocyte differentiation of labeled and unlabeled adipose-derived stem cells. No significant difference in the amount of differentiation was observed between labeled and unlabeled cells at 14 days (indicated by NS on the graph). Both labeled (n = 8) and unlabeled (n = 8) cell populations differentiated following exposure to differentiation medium, with large lipid inclusions evident at the 14-day time point. Values are given as mean ± SD.
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Plastic and Reconstructive Surgery • December 2015 were used as a control for autofluorescence. A “fan technique” with a 16-gauge cannula was used. In Vivo Imaging At specified time points, mice were anesthetized with controlled isoflurane (3 to 4%) and DiRlabeled cells were imaged within the fat using the in vivo imaging system. Dorsolateral images were taken of both enriched and nonenriched grafts. Exposure time and imaging field of view were constant for all time points (1 second). Signal intensity was quantified as the sum of the number of photons detected in the region of interest. A small pilot study was performed with two animals per group to confirm that labeled stromal vascular fraction could be visualized within implanted fat. Another study was then performed with six animals per group to investigate statistical significance between groups.
Statistical Analysis All values are reported as mean ± SD. A oneway analysis of variance was performed to determine statistical significance between groups at the alpha = 0.05 level. The sample size for each group was determined by using the G*Power analysis program. Given an alpha level set at 0.05, a desired statistical power level of 0.8, the minimum number of sample subjects to achieve statistical significance is six per group.
RESULTS Lipoaspirate Processing and Cell Enrichment DiR-labeled stromal vascular fraction was admixed into prepared grafts by means of Luer-toLuer transfer. This technique ensured homogeneous
Fig. 5. Dye maintenance following differentiation. (Above, left) Unlabeled, undifferentiated cells showing DiR labeling (red) and nuclei (4′,6-diamidino-2-phenylindole, blue) (original magnification, × 40). (Above, right) DiR-labeled, differentiated cells showing lipid inclusions (green) and nuclei (4′,6-diamidino-2-phenylindole, blue) (original magnification, × 40). (Below, left) Unlabeled, differentiated cells (original magnification, × 40). (Below, right) High-magnification image (original magnification, × 60) of DiR-labeled differentiated cells. DiR labeling persists in cells after differentiating into adipocytes.
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction
Fig. 6. In vitro dye longevity experiment. (Above) Stromal vascular fraction was labeled with DiR and subsequently plated in multiple flasks. Individual flasks were used for each time point. On the day of imaging, cells were lifted and suspended in phosphate-buffered saline at a concentration of 100,000 cells/100 μl (column 1). Unlabeled cells at the same concentration were used as a negative control (column 2). Phosphate-buffered saline was also imaged to confirm the absence of autofluorescence (column 3). (Below) At specified time points, cells (n = 6) were lifted, suspended in 96-well plates, and imaged with the in vivo imaging system. Dotted line represents average background fluorescence of unlabeled cells suspended in phosphate-buffered saline. The values represent mean ± SD. Mean DiR fluorescence decreased gradually over time in culture, with a significant decrease in fluorescence seen at each imaging time point. DiR-labeled cells fluoresced for up to 63 days in vitro.
mixing of cells within the fat. Little to no background fluorescence was observed in processed lipoaspirate that contained unlabeled cells (Fig. 1). Labeling Efficiency Labeling efficiency was determined by flow cytometry. Unlabeled stromal vascular fraction displayed little autofluorescence. According to flow cytometry, approximately 60 percent of cells
were labeled with DiR after following our previously described protocol (Fig. 2). Proliferation and Differentiation No significant differences were observed in the proliferation of labeled (n = 8) and unlabeled cells (n = 8) at 24, 48, and 72 hours, suggesting that the dye did not alter normal cell division (Fig. 3).
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Plastic and Reconstructive Surgery • December 2015
Fig. 7. Fluorescence imaged at postoperative day (POD) 2 with the in vivo imaging system. Labeled cells could be tracked within fat grafts, despite a steady decrease in fluorescence over time. ROI, region of interest.
Both labeled (n = 8) and unlabeled (n = 8) cells showed large lipid inclusions after 14 days of differentiation. There was no significant difference in the amount of differentiation observed between labeled and unlabeled cells (p = 0.602), indicating that labeling did not interfere with the adipocyte differentiation (Fig. 4).
Dye Maintenance Confocal microscopy was used to verify that DiR labeling could be maintained following differentiation. Cells were labeled with DiR and subjected to a 14-day differentiation study. Labeled cells maintained DiR after differentiation, suggesting that cells could be tracked in vivo following adipocyte differentiation (Fig. 5).
Fig. 8. Large-scale in vivo imaging of DiR-labeled stromal vascular fraction within fat grafts over time. Statistically significant fluorescence was observed in all DiR-enriched groups compared with lipoaspirate-only controls (p < 0.05). In the higher cell dose groups (i.e., 3 ×106 and 2 ×106 cells/ml of fat graft), the stromal vascular fraction could be tracked within grafts for up to 41 days in vivo.
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction In Vitro Dye Longevity DiR-labeled cells fluoresced for up to 63 days in vitro, suggesting that the stromal vascular fraction could be tracked for up to 8 weeks in vivo (Fig. 6). Xenograft Implantation A small pilot study was performed with two animals per group. Animals received DiRlabeled, cell-enriched fat grafts with the following doses: 6 × 106, 3 × 106, and 1.5 × 106 cells/ ml of graft. All animals also received an internal lipoaspirate control to confirm cellular localization. Fluorescence intensity bounds were determined based on control background fluorescence. The pixel intensity represents the number of DiR molecules incorporated into cellular membranes, which has been shown to be proportional to cell number.19,20 Pilot data confirmed that stromal vascular fraction dose could be differentiated and followed over time (Fig. 7). Persistence of labeled cells within the grafts (data not shown) was also in accordance with in vitro dye longevity results. Graft and lipoaspirate-alone animals (six animals per dose) received DiR-labeled cell-enriched grafts with the following doses: 3 × 106, 2 × 106, 1 × 106, and 0.5 × 106 cells/ml. Within the clinical scenario, stromal vascular fraction dose is limited by fat yield and the success of the isolation. Thus, cell doses were decreased from the initial pilot study to represent a more clinically feasible number of cells (Fig. 7). Statistically significant fluorescence was observed in all DiR cell–enriched groups compared with lipoaspirate controls (p < 0.05). On postoperative day 1, a significant increase in fluorescence was observed in all groups with increasing DiR-labeled cell enrichment (p < 0.05), except between 1 × 106 and 0.5 × 106 cells/ml of graft. Significance persisted among these groups until postoperative day 21, when the fluorescence with 0.5 × 106 cells/ml was no longer significantly different from that observed with background fluorescence. However, all other groups maintained significant differences from each other and from lipoaspirate controls until postoperative day 41, when the dose of 1 × 106 cells/ml no longer possessed higher fluorescence compared with the lipoaspirate group (Fig. 8).
DISCUSSION With optimized cellular therapies, fat transfer may lead to more predictable retention. However, it is important to understand the mechanism by which stromal vascular fraction facilitates this retention. In our study, we labeled stromal vascular fraction with a nontoxic, infrared dye (i.e., DiR). Stromal vascular fraction, rather than adiposederived stromal cells, was chosen based on clinical relevance. Timely labeling of cells was important because we wanted to combine stromal vascular fraction with fat from the same patient, similar to the clinical scenario. By using a rapid dyeing technique, we were able to abrogate the use of more lengthy and tedious transfection protocols. Ultimately, the use of DiR was based on clinical relevance, its ability to rapidly and uniformly dye cells, and its low toxicity (based on cellular proliferation and differentiation experiments). Cell doses were based on previous research within our laboratory suggesting a range of 0.5 to 3 × 106 cells/ml to be effective in enhancing retention (data not shown). Similarly, Chang et al. enriched grafts with 1 × 106 stromal vascular fraction cells/ml of lipoaspirate to correct hemifacial lipoatrophy. Increased graft volume was observed in the stromal vascular fraction group at 6 months postoperatively.21 However, it should be noted that stromal vascular fraction dose is not standardized. In our study, a dose range was included because the optimal stromal vascular fraction dose has not been determined. Labeled cells were admixed into fat from the same patient and injected subcutaneously into the flanks of athymic mice. We were able to verify cell doses and track stromal vascular fraction within the fat until postoperative day 41 using the in vivo imaging system. Fluorescence intensity has previously been shown to be proportional to cell number.19,20 However, in our lower doses (0.5 to 1 × 106 cells/ml), we did not see a proportional increase in intensity with respect to cell number. One possible explanation for this is the decrease in the signal-to-noise ratio associated with lower doses. This means that fluorescence measurements of lower doses may be less accurate because of the decreased signal (i.e., lower cell numbers) compared with background fluorescence. Based on our data, it is possible that this threshold is below 1 × 106 cells/ml; however, further studies will be needed to confirm this observation. At the time of injection, fluorescence was homogeneous throughout the graft, implying
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Plastic and Reconstructive Surgery • December 2015 that stromal vascular fraction is distributed evenly (Fig. 1). After implantation, the fluorescence distribution changed, with more cells being located in the higher intensity/central region of the graft (Fig. 7). Furthermore, the bounds of fluorescence also differed significantly, with the larger doses displaying increased fluorescent area. In both of these cases, it is possible that the cells may be migrating; however, this observation will need to be confirmed in future studies. Our in vitro work confirmed dye longevity and maintenance of the dye following adipocyte differentiation. In dividing DiR-labeled cells, exponential signal decay was observed (2E + 08e−0.008x, R² = 0.99527). Thus, our in vitro work suggests that adipocyte differentiation may be protective against fluorescence loss compared with cell division. Because little research has been performed on the interaction of the stromal vascular fraction within transplanted grafts, it is difficult to discern whether fluorescence loss is associated with cell death, division, or replacement. Perhaps the most interesting data come from the differences observed in DiR fluorescence decay curves. A drastic decrease in fluorescence is observed in the group receiving 6 × 106 cells/ml in the immediate postoperative period (data not shown), which suggests that the graft may be “overloaded” with cells before the establishment of a vascular supply, resulting in cell death. Cell death is associated with a significant loss of fluorescence, as DiR exhibits strong fluorescence when incorporated into intact phospholipid bilayers.22,23 Prior research within our laboratory suggests that a dose that is too high may lead to unfavorable results (data not shown). Other authors have also reported problems with higher doses, including necrosis, cyst formation, and fibrogenesis.24,25 However, in the lower dose groups, stromal vascular fraction may be beneficial in promoting adipocyte viability and increasing vascularity within the graft (data not shown). Previous work by Kato et al. suggests that, following standard fat grafting, adipocytes in the central region of the graft die at 1 week postoperatively. After this period, cells adjacent to this region are replaced by adipocyte precursors in approximately 4 weeks.26 Although our research suggests that the stromal vascular fraction may be instrumental in maintaining adipocyte viability in the graft, we also believe that precursor remodeling will contribute to loss of fluorescence in the graft over time. Cell division may also result in decreased fluorescence, as more cells are needed to replace and remodel adipocytes in the regenerating zone of the graft. Thus, it is probable that
the fluorescence decay observed within the cellenriched grafts results from a combination of these events. Future work will focus on discerning these activities with DiR labeling to determine their contribution to the successful maintenance of transplanted fat. There is some concern that DiR may be transferred by cells within the graft. Lassailly et al. found that hematopoietic stem cells and cord blood mononuclear cells showed a high frequency of dye transfer.27 One suggested mechanism for this is trogocytosis, a membrane exchange mechanism common within these cell populations. Lassailly et al. posited that prolonged staining incubation and insufficient washing could have also led to this finding. For this reason, care was taken to ensure that stromal vascular fraction was thoroughly washed before mixing. In contrast, studies have also shown little to no dye transfer with DiR labeling. Lukas et al. investigated DiR labeling of axons derived from extraocular muscles into the trigeminal ganglia. Label specificity was confirmed using a dual label for calcitonin gene-related peptide immunoreactivity. No evidence of transfer to unspecific populations was noted.28 Eisenblätter et al. injected DiR-labeled monocytes to assess granuloma formation in lipopolysaccharide pellets. No dye transfer occurred between labeled and unlabeled cells based on flow cytometry of cells isolated from the pellets.15 Kalchenko et al. confirmed DiR selectivity to hematopoietic cells by their ability to “home” preferentially to tissues (i.e., bones and lymph nodes) and the inability of labeled fixed cells to do so.14 In our study, labeling was specific to nuclei and not diffuse throughout our graft (based on preliminary confocal microscopy data). Thus, we believe that little to no dye transfer is occurring. It is possible that dye transfer may be cell type specific. Future work is needed to determine the degree of dye transfer, if any, that may occur within this model.
CONCLUSIONS In this article, we describe a labeling technique to track stromal vascular fraction within fat grafts. However, this method can be applied to many in vivo cellular strategies with the goal of understanding how cell dose and behavior can promote regeneration and repair in tissues. Future work will include assessing dye transfer that may occur within this model and histology to determine prevalence and fate of injected cells.
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction J. Peter Rubin, M.D. Department of Plastic Surgery University of Pittsburgh Scaife Hall, Suite 6B 3550 Terrace Street Pittsburgh, Pa. 15261 [email protected]
references 1. Konczalik W, Siemionow M. Experimental and clinical methods used for fat volume maintenance after autologous fat grafting. Ann Plast Surg. 2014;72:475–483. 2. Arcuri F, Brucoli M, Baragiotta N, et al. The role of fat grafting in the treatment of posttraumatic maxillofacial deformities. Craniomaxillofac Trauma Reconstr. 2013;6:121–126. 3. Coleman SR. Structural fat grafting: More than a permanent filler. Plast Reconstr Surg. 2006;118(Suppl):108S–120S. 4. Spear SL, Pittman T. A prospective study on lipoaugmentation of the breast. Aesthet Surg J. 2014;34:400–408. 5. Philips BJ, Grahovac TL, Valentin JE, et al. Prevalence of endogenous CD34+ adipose stem cells predicts human fat graft retention in a xenograft model. Plast Reconstr Surg. 2013; 132: 845–858. 6. Moseley TA, Zhu M, Hedrick MH. Adipose-derived stem and progenitor cells as fillers in plastic and reconstructive surgery. Plast Reconstr Surg. 2006;118:121S–128S. 7. Zhu M, Zhou Z, Chen Y, et al. Supplementation of fat grafts with adipose-derived regenerative cells improves long-term graft retention. Ann Plast Surg. 2010;64:222–228. 8. Leblanc AJ, Nguyen QT, Touroo JS, et al. Adipose-derived cell construct stabilizes heart function and increases microvascular perfusion in an established infarct. Stem Cells Transl Med. 2013;2:896–905. 9. Matsumoto D, Sato K, Gonda K, et al. Cell-assisted lipotransfer: Supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006;12:3375–3382. 10. Frangioni JV. In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol. 2003;7:626–634. 11. Winkler AM, Rice PF, Weichsel J, et al. In vivo, dual-modality OCT/LIF imaging using a novel VEGF receptor-targeted NIR fluorescent probe in the AOM-treated mouse model. Mol Imaging Biol. 2011;13:1173–1182. 12. Proulx ST, Luciani P, Derzsi S, et al. Quantitative imaging of lymphatic function with liposomal indocyanine green. Cancer Res. 2010;70:7053–7062. 13. Hanyu A, Kojima K, Hatake K, et al. Functional in vivo optical imaging of tumor angiogenesis, growth, and metastasis prevented by administration of anti-human VEGF antibody in xenograft model of human fibrosarcoma HT1080 cells. Cancer Sci. 2009;100:2085–2092.
14. Kalchenko V, Shivtiel S, Malina V, et al. Use of lipophilic near-infrared dye in whole-body optical imaging of hematopoietic cell homing. J Biomed Opt. 2006;11:050507. 15. Eisenblätter M, Ehrchen J, Varga G, et al. In vivo optical imaging of cellular inflammatory response in granuloma formation using fluorescence-labeled macrophages. J Nucl Med. 2009;50:1676–1682. 16. Coleman SR. Hand rejuvenation with structural fat grafting. Plast Reconstr Surg. 2002;110:1731–1744; discussion 1745–1747. 17. Brayfield CA, Marra KG, Rubin JP. Adipose tissue regeneration. Curr Stem Cell Res Ther. 2010;5:116–121. 18. Coleman SR. Facial augmentation with structural fat grafting. Clin Plast Surg. 2006;33:567–577. 19. Shan L. Near-infrared fluorescence 1,1-dioctadecyl-3,3,3,3tetramethylindotricarbocyanine iodide (DiR)-labeled macrophages for cell imaging. In: Molecular Imaging and Contrast Agent Database (MICAD). Bethesda, Md: National Center for Biotechnology Information; 2004. 20. Ruan J, Song H, Li C, et al. DiR-labeled embryonic stem cells for targeted imaging of in vivo gastric cancer cells. Theranostics 2012;2:618–628. 21. Chang Q, Li J, Dong Z, Liu L, Lu F. Quantitative volumetric analysis of progressive hemifacial atrophy corrected using stromal vascular fraction-supplemented autologous fat grafts. Dermatol Surg. 2013;39:1465–1473. 22. Packard BS, Wolf DE. Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers. Biochemistry 1985;24:5176–5181. 23. Ozhalici-Unal H, Pow CL, Marks SA, et al. A rainbow of fluoromodules: A promiscuous scFv protein binds to and activates a diverse set of fluorogenic cyanine dyes. J Am Chem Soc. 2008;130:12620–12621. 24. Kakudo N, Tanaka Y, Morimoto N, et al. Adipose-derived regenerative cell (ADRC)-enriched fat grafting: Optimal cell concentration and effects on grafted fat characteristics. J Transl Med. 2013;11:254. 25. Yoshimura K, Aoi N, Suga H, et al. Ectopic fibrogenesis induced by transplantation of adipose-derived progenitor cell suspension immediately after lipoinjection. Transplantation 2008;85:1868–1869. 26. Kato H, Mineda K, Eto H, et al. Degeneration, regeneration, and cicatrization after fat grafting: Dynamic total tissue remodeling during the first 3 months. Plast Reconstr Surg. 2014;133:303e–313e. 27. Lassailly F, Griessinger E, Bonnet D. “Microenvironmental contaminations” induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking. Blood 2010;115:5347–5354. 28. Lukas JR, Aigner M, Denk M, Heinzl H, Burian M, Mayr R. Carbocyanine postmortem neuronal tracing: Influence of different parameters on tracing distance and combination with immunocytochemistry. J Histochem Biochem. 1998;46:901–910.
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Background: Although fat grafting is an increasingly popular practice, suboptimal volume retention remains an obstacle. Graft enrichment with the stromal vascular fraction has gained attention as a method of increasing graft retention. However, few studies have assessed the fate and impact of transplanted stromal vascular fraction on fat grafts. In vivo imaging techniques can be used to help determine the influence stromal vascular fraction has on transplanted fat. Methods: Stromal vascular fraction was labeled with 1,1′-dioctadecyl-3,3,3′,3′tetramethylindotricarbocyanine iodide (DiR), a near-infrared dye, and tracked in vivo. Proliferation and differentiation of labeled cells were assessed to confirm that labeling did not adversely affect cellular function. Different doses of labeled stromal vascular fraction were tracked within fat grafts over time using the in vivo imaging system. Results: No significant differences in differentiation and proliferation were observed in labeled versus unlabeled cells (p > 0.05). A pilot study confirmed that stromal vascular fraction fluorescence was localized to fat grafts and different cell doses could be distinguished. A larger-scale in vivo study revealed that stromal vascular fraction fluorescence was statistically significant (p < 0.05) between different cell dose groups and this significance was maintained in higher doses (3 × 106 and 2 × 106 cells/ml of fat graft) for up to 41 days in vivo. Conclusions: DiR labeling allowed the authors to differentiate between cell doses and confirm localization. This article supports the use of DiR labeling in conjunction with in vivo imaging as a tool for imaging stromal vascular fraction within fat grafts. (Plast. Reconstr. Surg. 136: 1205, 2015.) CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.
A
utologous fat grafting is a technique used to reconstruct soft-tissue defects1 of the face2,3 and breast4; however, there are limits to its use. Problems with fat grafting include insufficient vasculature at the injection site, leading to unpredictable retention rates.5 Cellular therapies with adipose-derived stem cells and the stromal vascular fraction are clinically used as a means of preserving adipose.6,7 It is hypothesized that these cells mediate angiogenesis within transplanted adipose by secreting angiogenic growth factors.8 Stromal vascular fraction–enriched fat grafts exhibit decreased From the Departments of Plastic Surgery and Bioengineering, University of Pittsburgh; and the McGowan Institute for Regenerative Medicine. Received for publication September 19, 2014; accepted May 6, 2015. Copyright © 2015 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000001815
necrosis compared with unenriched grafts.9 Despite the positive effects associated with these therapies, few studies have assessed the fate, localization, and behavior of stromal vascular fraction within fat grafts over time. In vivo imaging combined with infrared labeling represents a novel technique of tracking cells within fat grafts. Infrared labeling allows the user to track specific ligand antagonists or cells because of the high-penetrance of infrared light into tissues and, subsequently, the high signal-to-noise ratio of infrared targets within tissues.10 In particular, 1,1′-dioctadecyl3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), an infrared fluorescing carbocyanine dye, can label cells, which can then be tracked using the in vivo imaging system. Disclosure: The authors have no financial interests to declare in relation to the content of this article.
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Plastic and Reconstructive Surgery • December 2015 Current research with in vivo infrared imaging monitors biological processes, including tumor, lymphatic, and vascular formation.11–13 DiR labeling has also detected the differences between normal and leukemic hematopoietic stem cell homing.14 DiR-labeled macrophages have also been used to assess granuloma formation.15 In this study, DiR labeling did not alter cellular activity (based on in vitro proliferation and differentiation assays). DiR-labeled stromal vascular fraction was combined with fat grafts and tracked using the in vivo imaging system. Cell dose and localization within the transplanted fat was confirmed. This method lends it applicability to many studies to assess the location, integration, and behavior of stromal vascular fraction with the ultimate goal of guiding clinical decisions regarding stromal vascular fraction dose.
PATIENTS AND METHODS The following materials were used: neuropaddies, phosphate-buffered saline (Gibco, Carlsbad, Calif.), Luer-lock syringes, type II collagenase (Worthington Biochemical Corp., Lakewood, N.J.), standard plating medium [Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F12 (Corning, Manassas, Va.), 10% fetal bovine serum (Atlas Biologicals, Fort Collins, Colo.), 1% penicillin/streptomycin, 1% Fungizone (Lonza, Walkersville, Md.), and 0.001% dexamethasone (Sigma-Aldrich Corp., St. Louis, Mo.)], Vybrant DiR (Invitrogen, Carlsbad, Calif.), MACS buffer (Miltenyi Biotec, San Diego, Calif.), Cyquant proliferation assay (Invitrogen), adipocyte differentiation media (Zen-Bio, Inc., Research Triangle Park, N.C.), AdipoRed assay (Lonza), 96-well microplate (Ibidi, Martinsried, Germany), ketamine (Butler Schein, North Dublin, Ohio), xylazine (Vedco, Inc., St. Joseph, Mo.), and ketoprofen (Fort Dodge Animal Health, Overland Park, Kan.). Human Subject, Stromal Vascular Fraction Isolation, and Cell Culture The University of Pittsburgh Institutional Review Board approved tissue collection under PRO13020131. Abdominal subcutaneous fat was harvested using a 17-gauge bucket handle cannula attached to 10-ml Luer-lock syringe. Handheld suction was created by gently pulling back on the syringe.16 For in vitro and in vivo experiments, stromal vascular fraction was isolated from donor-matched adipose tissue.17 Lipoaspirate was digested in type II collagenase at 37°C for 30 minutes. The tissue was centrifuged at 1000 rpm for 10 minutes. The pellet was then suspended
in erythrocyte lysis buffer and centrifuged at 1000 rpm for 10 minutes. Cells were labeled with DiR directly after isolation for all experiments (see later under DiR Labeling). Lipoaspirate Processing and Cell Enrichment Lipoaspirate was processed by means of the Coleman technique.3,18 Adipose tissue was aspirated and syringes were centrifuged for 3 minutes at 3000 rpm. Subsequently, the upper oil and lower aqueous layer were removed. Stromal vascular fraction–enriched fat grafts (6 × 106, 3 × 106, 2 × 106, 1.5 × 106, 1 × 106, and 0.5 × 106 cells/ml of graft) were prepared. Stromal vascular fraction was suspended in 100 µl of phosphate-buffered saline per milliliter of fat graft and gently mixed by means of Luer-to-Luer transfer. Homogenous mixing of cells was confirmed using the in vivo imaging system. Control grafts were mixed with unlabeled cells at the same concentration. Grafts were subsequently separated into 0.5ml aliquots for injection. DiR Labeling Vybrant DiR was prepared at 2.5 mg/ml in ethanol and stored at 4°C according to the manufacturer’s protocol. Cells were suspended at 1 × 106 cells/ml in serum-free medium. DiR dye (5 µl/1 ml of media) was added to the suspension and incubated for 20 minutes at 37°C in 5% carbon dioxide.19 After incubation, labeled cells were centrifuged at 1500 rpm for 5 minutes at 37°C. The cells were washed in serum containing medium four times to ensure that unbound dye was completely removed. Labeled cells were combined with fat grafts for in vivo experiments or used for in vitro flow cytometry, imaging, or functional assays. Labeling Efficiency Flow cytometry was used to determine DiR labeling efficiency. Unlabeled cells served as a negative control. Cells were suspended in magneticactivated cell sorting buffer at 1 × 106 cells/ml and then subjected to flow cytometry using the APC-Cy7-A laser (BD Biosciences, San Jose, Calif.). Labeling efficiency was determined relative to the total number of live cells within the sample. Proliferation, Differentiation, and Dye Maintenance Proliferation and differentiation studies were performed to confirm that labeling did not alter cell behavior. Dye maintenance studies were completed to determine whether cells could be tracked after adipocyte differentiation. Cells (passage
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction 0) were labeled and plated at 10,000 cells/well in a 96-well plate. The CyQuant kit was used to assess cell proliferation between labeled (n = 8) and unlabeled cells (n = 8) for 24, 48, and 72 hours. Proliferation was compared to a DNA standard curve, and fluorescence was measured using a Tecan SpectraFluor microplate reader (Tecan Systems, Inc., San Jose, Calif.).
For differentiation studies, labeled and unlabeled cells were incubated in adipocyte differentiation medium (n = 8) and standard plating medium (n = 8). After a 14-day incubation, lipid accumulation was quantified using the AdipoRed Assay. Fluorescence was quantified using a Tecan SpectraFluor microplate reader. To determine dye maintenance following differentiation, cells were differentiated
Fig. 1. Establishing a mixing technique for stromal vascular fraction within soft-tissue grafts. Unlabeled and DiR-labeled cells (1 × 106 cells/ml) were combined with the fat graft to confirm uniform distribution of cells within the grafts. Plates were imaged using the in vivo imaging system.
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Plastic and Reconstructive Surgery • December 2015 into adipocytes, and confocal microscopy was used to assess DiR-dye preservation. In Vitro Dye Longevity Stromal vascular fraction was labeled as described previously and seeded at 5000 cells/cm2 in multiple 175-cm2 culture flasks. Individual flasks were used at each time point to avoid passaging cells during the
study. At specified time points, subsets of cells (n = 6) were lifted and suspended at 100,000 cells/100 µl of phosphate-buffered saline. Unlabeled cells were used as negative controls. Cell suspension plates were imaged with the in vivo imaging system at 750nm excitation and 780-nm emission. Exposure time and imaging field of view were kept constant for all time points. Signal intensity was quantified as the
Fig. 2. DiR labeling efficiency. Stromal vascular fraction cells labeled with DiR were subjected to flow cytometry to determine labeling efficiency. Unlabeled cells were used as a negative control, and little to no background fluorescence was observed. Approximately 60 percent of the stromal vascular fraction population was labeled with DiR.
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction
Fig. 3. No significant differences were observed in proliferation of labeled (n = 8) versus unlabeled cells (n = 8) at 24, 48, or 72 hours. Paired t tests were used to analyze differences in proliferation between labeled and unlabeled cells at 24, 48, and 72 hours, with values of p < 0.05 being considered statistically significant.
sum of the number of photons detected within our region of interest using Xenogen Living Image software (Xenogen Corp., Alameda, Calif.). In Vivo Dye Longevity Xenograft Implantation Female athymic nude mice, 6 weeks old (Harlan Laboratories, Inc., Indianapolis, Ind.), were housed at the University of Pittsburgh Division of Laboratory Animal Resources. Mice were housed under controlled conditions (20° to 23°C, 40 to 60% humidity, and 12-hour light/dark cycles), fed
a standard imaging diet (alfalfa-free), and given sterile water ad libitum. The University of Pittsburgh Institutional Animal Care and Use Committee approved all experiments. Mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg ketamine) and xylazine (12 mg/kg). Half-milliliter injections of processed lipoaspirate (on the left flank) and DiRlabeled cell-enriched grafts (on the right flank) were injected subcutaneously onto the dorsum of each mouse. Processed lipoaspirate–alone animals
Fig. 4. Adipocyte differentiation of labeled and unlabeled adipose-derived stem cells. No significant difference in the amount of differentiation was observed between labeled and unlabeled cells at 14 days (indicated by NS on the graph). Both labeled (n = 8) and unlabeled (n = 8) cell populations differentiated following exposure to differentiation medium, with large lipid inclusions evident at the 14-day time point. Values are given as mean ± SD.
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Plastic and Reconstructive Surgery • December 2015 were used as a control for autofluorescence. A “fan technique” with a 16-gauge cannula was used. In Vivo Imaging At specified time points, mice were anesthetized with controlled isoflurane (3 to 4%) and DiRlabeled cells were imaged within the fat using the in vivo imaging system. Dorsolateral images were taken of both enriched and nonenriched grafts. Exposure time and imaging field of view were constant for all time points (1 second). Signal intensity was quantified as the sum of the number of photons detected in the region of interest. A small pilot study was performed with two animals per group to confirm that labeled stromal vascular fraction could be visualized within implanted fat. Another study was then performed with six animals per group to investigate statistical significance between groups.
Statistical Analysis All values are reported as mean ± SD. A oneway analysis of variance was performed to determine statistical significance between groups at the alpha = 0.05 level. The sample size for each group was determined by using the G*Power analysis program. Given an alpha level set at 0.05, a desired statistical power level of 0.8, the minimum number of sample subjects to achieve statistical significance is six per group.
RESULTS Lipoaspirate Processing and Cell Enrichment DiR-labeled stromal vascular fraction was admixed into prepared grafts by means of Luer-toLuer transfer. This technique ensured homogeneous
Fig. 5. Dye maintenance following differentiation. (Above, left) Unlabeled, undifferentiated cells showing DiR labeling (red) and nuclei (4′,6-diamidino-2-phenylindole, blue) (original magnification, × 40). (Above, right) DiR-labeled, differentiated cells showing lipid inclusions (green) and nuclei (4′,6-diamidino-2-phenylindole, blue) (original magnification, × 40). (Below, left) Unlabeled, differentiated cells (original magnification, × 40). (Below, right) High-magnification image (original magnification, × 60) of DiR-labeled differentiated cells. DiR labeling persists in cells after differentiating into adipocytes.
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction
Fig. 6. In vitro dye longevity experiment. (Above) Stromal vascular fraction was labeled with DiR and subsequently plated in multiple flasks. Individual flasks were used for each time point. On the day of imaging, cells were lifted and suspended in phosphate-buffered saline at a concentration of 100,000 cells/100 μl (column 1). Unlabeled cells at the same concentration were used as a negative control (column 2). Phosphate-buffered saline was also imaged to confirm the absence of autofluorescence (column 3). (Below) At specified time points, cells (n = 6) were lifted, suspended in 96-well plates, and imaged with the in vivo imaging system. Dotted line represents average background fluorescence of unlabeled cells suspended in phosphate-buffered saline. The values represent mean ± SD. Mean DiR fluorescence decreased gradually over time in culture, with a significant decrease in fluorescence seen at each imaging time point. DiR-labeled cells fluoresced for up to 63 days in vitro.
mixing of cells within the fat. Little to no background fluorescence was observed in processed lipoaspirate that contained unlabeled cells (Fig. 1). Labeling Efficiency Labeling efficiency was determined by flow cytometry. Unlabeled stromal vascular fraction displayed little autofluorescence. According to flow cytometry, approximately 60 percent of cells
were labeled with DiR after following our previously described protocol (Fig. 2). Proliferation and Differentiation No significant differences were observed in the proliferation of labeled (n = 8) and unlabeled cells (n = 8) at 24, 48, and 72 hours, suggesting that the dye did not alter normal cell division (Fig. 3).
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Plastic and Reconstructive Surgery • December 2015
Fig. 7. Fluorescence imaged at postoperative day (POD) 2 with the in vivo imaging system. Labeled cells could be tracked within fat grafts, despite a steady decrease in fluorescence over time. ROI, region of interest.
Both labeled (n = 8) and unlabeled (n = 8) cells showed large lipid inclusions after 14 days of differentiation. There was no significant difference in the amount of differentiation observed between labeled and unlabeled cells (p = 0.602), indicating that labeling did not interfere with the adipocyte differentiation (Fig. 4).
Dye Maintenance Confocal microscopy was used to verify that DiR labeling could be maintained following differentiation. Cells were labeled with DiR and subjected to a 14-day differentiation study. Labeled cells maintained DiR after differentiation, suggesting that cells could be tracked in vivo following adipocyte differentiation (Fig. 5).
Fig. 8. Large-scale in vivo imaging of DiR-labeled stromal vascular fraction within fat grafts over time. Statistically significant fluorescence was observed in all DiR-enriched groups compared with lipoaspirate-only controls (p < 0.05). In the higher cell dose groups (i.e., 3 ×106 and 2 ×106 cells/ml of fat graft), the stromal vascular fraction could be tracked within grafts for up to 41 days in vivo.
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction In Vitro Dye Longevity DiR-labeled cells fluoresced for up to 63 days in vitro, suggesting that the stromal vascular fraction could be tracked for up to 8 weeks in vivo (Fig. 6). Xenograft Implantation A small pilot study was performed with two animals per group. Animals received DiRlabeled, cell-enriched fat grafts with the following doses: 6 × 106, 3 × 106, and 1.5 × 106 cells/ ml of graft. All animals also received an internal lipoaspirate control to confirm cellular localization. Fluorescence intensity bounds were determined based on control background fluorescence. The pixel intensity represents the number of DiR molecules incorporated into cellular membranes, which has been shown to be proportional to cell number.19,20 Pilot data confirmed that stromal vascular fraction dose could be differentiated and followed over time (Fig. 7). Persistence of labeled cells within the grafts (data not shown) was also in accordance with in vitro dye longevity results. Graft and lipoaspirate-alone animals (six animals per dose) received DiR-labeled cell-enriched grafts with the following doses: 3 × 106, 2 × 106, 1 × 106, and 0.5 × 106 cells/ml. Within the clinical scenario, stromal vascular fraction dose is limited by fat yield and the success of the isolation. Thus, cell doses were decreased from the initial pilot study to represent a more clinically feasible number of cells (Fig. 7). Statistically significant fluorescence was observed in all DiR cell–enriched groups compared with lipoaspirate controls (p < 0.05). On postoperative day 1, a significant increase in fluorescence was observed in all groups with increasing DiR-labeled cell enrichment (p < 0.05), except between 1 × 106 and 0.5 × 106 cells/ml of graft. Significance persisted among these groups until postoperative day 21, when the fluorescence with 0.5 × 106 cells/ml was no longer significantly different from that observed with background fluorescence. However, all other groups maintained significant differences from each other and from lipoaspirate controls until postoperative day 41, when the dose of 1 × 106 cells/ml no longer possessed higher fluorescence compared with the lipoaspirate group (Fig. 8).
DISCUSSION With optimized cellular therapies, fat transfer may lead to more predictable retention. However, it is important to understand the mechanism by which stromal vascular fraction facilitates this retention. In our study, we labeled stromal vascular fraction with a nontoxic, infrared dye (i.e., DiR). Stromal vascular fraction, rather than adiposederived stromal cells, was chosen based on clinical relevance. Timely labeling of cells was important because we wanted to combine stromal vascular fraction with fat from the same patient, similar to the clinical scenario. By using a rapid dyeing technique, we were able to abrogate the use of more lengthy and tedious transfection protocols. Ultimately, the use of DiR was based on clinical relevance, its ability to rapidly and uniformly dye cells, and its low toxicity (based on cellular proliferation and differentiation experiments). Cell doses were based on previous research within our laboratory suggesting a range of 0.5 to 3 × 106 cells/ml to be effective in enhancing retention (data not shown). Similarly, Chang et al. enriched grafts with 1 × 106 stromal vascular fraction cells/ml of lipoaspirate to correct hemifacial lipoatrophy. Increased graft volume was observed in the stromal vascular fraction group at 6 months postoperatively.21 However, it should be noted that stromal vascular fraction dose is not standardized. In our study, a dose range was included because the optimal stromal vascular fraction dose has not been determined. Labeled cells were admixed into fat from the same patient and injected subcutaneously into the flanks of athymic mice. We were able to verify cell doses and track stromal vascular fraction within the fat until postoperative day 41 using the in vivo imaging system. Fluorescence intensity has previously been shown to be proportional to cell number.19,20 However, in our lower doses (0.5 to 1 × 106 cells/ml), we did not see a proportional increase in intensity with respect to cell number. One possible explanation for this is the decrease in the signal-to-noise ratio associated with lower doses. This means that fluorescence measurements of lower doses may be less accurate because of the decreased signal (i.e., lower cell numbers) compared with background fluorescence. Based on our data, it is possible that this threshold is below 1 × 106 cells/ml; however, further studies will be needed to confirm this observation. At the time of injection, fluorescence was homogeneous throughout the graft, implying
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Plastic and Reconstructive Surgery • December 2015 that stromal vascular fraction is distributed evenly (Fig. 1). After implantation, the fluorescence distribution changed, with more cells being located in the higher intensity/central region of the graft (Fig. 7). Furthermore, the bounds of fluorescence also differed significantly, with the larger doses displaying increased fluorescent area. In both of these cases, it is possible that the cells may be migrating; however, this observation will need to be confirmed in future studies. Our in vitro work confirmed dye longevity and maintenance of the dye following adipocyte differentiation. In dividing DiR-labeled cells, exponential signal decay was observed (2E + 08e−0.008x, R² = 0.99527). Thus, our in vitro work suggests that adipocyte differentiation may be protective against fluorescence loss compared with cell division. Because little research has been performed on the interaction of the stromal vascular fraction within transplanted grafts, it is difficult to discern whether fluorescence loss is associated with cell death, division, or replacement. Perhaps the most interesting data come from the differences observed in DiR fluorescence decay curves. A drastic decrease in fluorescence is observed in the group receiving 6 × 106 cells/ml in the immediate postoperative period (data not shown), which suggests that the graft may be “overloaded” with cells before the establishment of a vascular supply, resulting in cell death. Cell death is associated with a significant loss of fluorescence, as DiR exhibits strong fluorescence when incorporated into intact phospholipid bilayers.22,23 Prior research within our laboratory suggests that a dose that is too high may lead to unfavorable results (data not shown). Other authors have also reported problems with higher doses, including necrosis, cyst formation, and fibrogenesis.24,25 However, in the lower dose groups, stromal vascular fraction may be beneficial in promoting adipocyte viability and increasing vascularity within the graft (data not shown). Previous work by Kato et al. suggests that, following standard fat grafting, adipocytes in the central region of the graft die at 1 week postoperatively. After this period, cells adjacent to this region are replaced by adipocyte precursors in approximately 4 weeks.26 Although our research suggests that the stromal vascular fraction may be instrumental in maintaining adipocyte viability in the graft, we also believe that precursor remodeling will contribute to loss of fluorescence in the graft over time. Cell division may also result in decreased fluorescence, as more cells are needed to replace and remodel adipocytes in the regenerating zone of the graft. Thus, it is probable that
the fluorescence decay observed within the cellenriched grafts results from a combination of these events. Future work will focus on discerning these activities with DiR labeling to determine their contribution to the successful maintenance of transplanted fat. There is some concern that DiR may be transferred by cells within the graft. Lassailly et al. found that hematopoietic stem cells and cord blood mononuclear cells showed a high frequency of dye transfer.27 One suggested mechanism for this is trogocytosis, a membrane exchange mechanism common within these cell populations. Lassailly et al. posited that prolonged staining incubation and insufficient washing could have also led to this finding. For this reason, care was taken to ensure that stromal vascular fraction was thoroughly washed before mixing. In contrast, studies have also shown little to no dye transfer with DiR labeling. Lukas et al. investigated DiR labeling of axons derived from extraocular muscles into the trigeminal ganglia. Label specificity was confirmed using a dual label for calcitonin gene-related peptide immunoreactivity. No evidence of transfer to unspecific populations was noted.28 Eisenblätter et al. injected DiR-labeled monocytes to assess granuloma formation in lipopolysaccharide pellets. No dye transfer occurred between labeled and unlabeled cells based on flow cytometry of cells isolated from the pellets.15 Kalchenko et al. confirmed DiR selectivity to hematopoietic cells by their ability to “home” preferentially to tissues (i.e., bones and lymph nodes) and the inability of labeled fixed cells to do so.14 In our study, labeling was specific to nuclei and not diffuse throughout our graft (based on preliminary confocal microscopy data). Thus, we believe that little to no dye transfer is occurring. It is possible that dye transfer may be cell type specific. Future work is needed to determine the degree of dye transfer, if any, that may occur within this model.
CONCLUSIONS In this article, we describe a labeling technique to track stromal vascular fraction within fat grafts. However, this method can be applied to many in vivo cellular strategies with the goal of understanding how cell dose and behavior can promote regeneration and repair in tissues. Future work will include assessing dye transfer that may occur within this model and histology to determine prevalence and fate of injected cells.
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Volume 136, Number 6 • Imaging the Stromal Vascular Fraction J. Peter Rubin, M.D. Department of Plastic Surgery University of Pittsburgh Scaife Hall, Suite 6B 3550 Terrace Street Pittsburgh, Pa. 15261 [email protected]
references 1. Konczalik W, Siemionow M. Experimental and clinical methods used for fat volume maintenance after autologous fat grafting. Ann Plast Surg. 2014;72:475–483. 2. Arcuri F, Brucoli M, Baragiotta N, et al. The role of fat grafting in the treatment of posttraumatic maxillofacial deformities. Craniomaxillofac Trauma Reconstr. 2013;6:121–126. 3. Coleman SR. Structural fat grafting: More than a permanent filler. Plast Reconstr Surg. 2006;118(Suppl):108S–120S. 4. Spear SL, Pittman T. A prospective study on lipoaugmentation of the breast. Aesthet Surg J. 2014;34:400–408. 5. Philips BJ, Grahovac TL, Valentin JE, et al. Prevalence of endogenous CD34+ adipose stem cells predicts human fat graft retention in a xenograft model. Plast Reconstr Surg. 2013; 132: 845–858. 6. Moseley TA, Zhu M, Hedrick MH. Adipose-derived stem and progenitor cells as fillers in plastic and reconstructive surgery. Plast Reconstr Surg. 2006;118:121S–128S. 7. Zhu M, Zhou Z, Chen Y, et al. Supplementation of fat grafts with adipose-derived regenerative cells improves long-term graft retention. Ann Plast Surg. 2010;64:222–228. 8. Leblanc AJ, Nguyen QT, Touroo JS, et al. Adipose-derived cell construct stabilizes heart function and increases microvascular perfusion in an established infarct. Stem Cells Transl Med. 2013;2:896–905. 9. Matsumoto D, Sato K, Gonda K, et al. Cell-assisted lipotransfer: Supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006;12:3375–3382. 10. Frangioni JV. In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol. 2003;7:626–634. 11. Winkler AM, Rice PF, Weichsel J, et al. In vivo, dual-modality OCT/LIF imaging using a novel VEGF receptor-targeted NIR fluorescent probe in the AOM-treated mouse model. Mol Imaging Biol. 2011;13:1173–1182. 12. Proulx ST, Luciani P, Derzsi S, et al. Quantitative imaging of lymphatic function with liposomal indocyanine green. Cancer Res. 2010;70:7053–7062. 13. Hanyu A, Kojima K, Hatake K, et al. Functional in vivo optical imaging of tumor angiogenesis, growth, and metastasis prevented by administration of anti-human VEGF antibody in xenograft model of human fibrosarcoma HT1080 cells. Cancer Sci. 2009;100:2085–2092.
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