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. Author manuscript; available in PMC: 2022 Apr 8.
Published in final edited form as: Plast Reconstr Surg. 2016 Dec;138(6):983e–996e. doi: 10.1097/PRS.0000000000002790

A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield

Ruth Tevlin 1, Adrian McArdle 1, Elizabeth Brett 1, Michael T Chung 1, Kevin Paik 1, Eun Young Seo 1, Graham G Walmsley 1, Christopher R Duldulao 1, David Atashroo 1, Elizabeth Zielin 1, Stephanie Vistnes 1, Charles K F Chan 1, Derrick C Wan 1, Michael T Longaker 1
PMCID: PMC8992773  NIHMSID: NIHMS1791277  PMID: 27537222

Abstract

Background:

The authors have developed a novel protocol for isolating adipose-derived stem cells from human lipoaspirate. In this study, they compare their new method to a previously published standard protocol.

Methods:

Human adipose-derived stem cell isolation was performed using two methods to compare cell yield, cell viability, cell proliferation, and regenerative potential. The new and conventional isolation methods differ in two key areas: die collagenase digestion buffer constituents and the use of an orbital shaker. The osteogenic and adipogenic potential of adipose-derived stem cells isolated using both protocols was assessed in vitro, and gene expression analysis was performed. To assess the ability of the isolated cells to generate bone in vivo, die authors created critical-size calvarial defects in mice, which were treated with adipose-derived stem cells loaded onto hydroxyapatite-coated poly(lactic-co-glycolic acid) scaffolds. To test die ability of the isolated cells to enhance adipogenesis, die cells were added to lipoaspirate and placed beneath the scalp of immunocompromised mice. Fat graft volume retention was subsequently assessed by serial computed tomographic volumetric scanning.

Results:

The new method resulted in a 10-fold increased yield of adipose-derived stem cells compared with die conventional method. Cells harvested using die new method demonstrated significantly increased cell viability and proliferation in vitro (p < 0.05). New method cells also demonstrated significantly enhanced osteogenic and adipogenic differentiation capacity in vitro (p < 0.05) in comparison with die conventional method cells. Both cell groups demonstrated equivalent osteogenic and adipogenic regenerative potential in mice.

Conclusions:

The authors have developed a protocol that maximizes the yield of adipose-derived stem cells derived from lipoaspirate. The new method cells have increased osteogenic and adipogenic potential in vitro and are not inferior to conventional method cells in terms of their ability to generate bone and fat in vivo.

CLINICAL QUESTION/LEVEL OF EVIDENCE:

Therapeutic, V.

Large bone defects resulting from trauma, tumor resection, and nonunion of fractures are common reconstructive challenges. Over the past decade, reconstructive bone tissue engineering modalities, which couple stem-cell based therapy with biomaterials, have been studied extensively, becoming one of the most promising methods in both animal and human studies.1 The concept of critical-size defect reconstruction using mesenchymal stem cells harvested from bone marrow has been validated in numerous animal models.2–5

Adipose tissue represents an abundant, practical, and appealing alternate source of donor tissue for autologous cell replacement. In 2001, Zuk et al. described the adipose-derived stem cell.6,7 The cells possess properties similar to those of bone marrow–derived mesenchymal stem cells, including similar cell surface marker profiles and differentiation potential.8 However, compared with 100 ml of bone marrow aspirate, the yield of stem cells from 100 g of adipose tissue can be 300-fold more.9,10 Adipose-derived stem cells are more accessible than bone marrow–derived mesenchymal stem cells and represent a readily available, expandable building block for osseous regeneration.3,11–13 In addition, they are not restricted by the technical and ethical considerations with which embryonic or induced pluripotent stem cell use is fraught. Recently, our laboratory has shown that it is possible to isolate a subpopulation of adipose-derived stem cells with enhanced osteogenic potential.3,14

Taking example from lessons in fat grafting, where it is hypothesized that the degree of fat graft survival correlates with the number of viable adipocytes following transfer,15,16 we aim to preserve the viability of adipose-derived stem cells during harvest, isolation, and subsequent transplantation. Although improvement is required in each of these key events, in this article we present an enhanced method for isolation of adipose-derived stem cells from lipoaspirate. This isolation process yields an abundant population of the cells, which maintain multipotential differentiation capacity both in vitro and in vivo.

MATERIALS AND METHODS

Fat Processing and Harvesting

Lipoaspirate was obtained from healthy female patients undergoing elective procedures in accordance with the Stanford University Institutional Review Board. Gravity separation was performed and, importantly, the lipoaspirate of each patient was divided into two samples—one processed using the new method and the other processed using the conventional method—thus allowing direct comparison and elimination of differences resulting from the harvest method or clinical preferences of the attending surgeon.

Lipoaspirate Harvest Methods

The new method and the conventional method differ in two key areas: the constituents of the collagenase digestion buffer and the use of an orbital shaker.

New Method

Lipoaspirate was placed on ice for 1 hour to allow the fat to congeal and to separate out the fat and blood. Fresh collagenase digestion buffer was prepared using M199 medium, 2.2 mg/ml type I collagenase (Sigma Aldrich, St Louis, Mo.), 1000 U/ml DNAse, 1000× 1 mM calcium chloride, 10% bovine serum albumin, 100× poloxamer-188 (Sigma Aldrich), and 50× hydroxyethyl piperazineethanesulfonic acid (Life Technologies, Grand Island, N.Y.), and filtered using a 0.22-μm filter system. The congealed fat was transferred to a 500-ml sterile polyethylene terephthalate copolyester media bottle and an equal volume of collagenase digestion buffer was added to the fat. The lid was closed and sealed with Parafilm (Bemis NA, Neenah, Wis.). The fat/collagenase mixture was incubated at 37°C in a water bath for 10 minutes to activate the collagenase. The fat/collagenase mixture was then transferred to the orbital shaker for 20 minutes. Then, 125 ml of the digested fat was subsequently transferred to a 250-ml conical-bottom centrifuge bottle. Collagenase activity was then neutralized by addition of an equal volume of fluorescence-activated cell sorting buffer (1× phosphate-buffered saline, 2% fetal bovine serum, 1% poloxamer-188, and 1% penicillin/streptomycin). The solution was then centrifuged at 1500 rpm for 10 minutes at room temperature. Supernatant was aspirated and the stromal vascular fraction pellet resuspended in 15 ml of room temperature fluorescence-activated cell sorting buffer. The solution was strained through a 100-μm cell strainer. Fifteen milliliters of Histopaque (Sigma Aldrich), a commercially available density gradient separation medium, was added to a new 50-ml conical-bottom centrifuge bottle, and the strained cell solution was overlaid on top of the Histopaque in a 1:1 ratio. Solution was centrifuged at 1450 rpm for 15 minutes at room temperature with acceleration set to low and deceleration settings inactivated. The cloudy interface was transferred to a new conical-bottom centrifuge bottle and the tube was filled with fluorescence-activated cell sorting buffer. The solution was centrifuged at 1300 rpm at 4°C for 5 minutes. The supernatant was then aspirated and the pellet resuspended in 500 μl of fluorescence-activated cell sorting buffer in preparation for fluorescence-activated cell sorting.

Conventional Method

In the conventional method, stromal vascular fraction was isolated as described previously by Zuk et al.6 Briefly, raw lipoaspirates were washed and treated with 0.075% collagenase type I (Sigma Aldrich) in Hank’s Balanced Salt Solution (Cellgro, Manassas, Va.) for 1 hour at 37°C with gentle agitation. The cellular pellet was treated with Histopaque density gradient separation medium as described above and then resuspended in 500 μl of fluorescence-activated cell sorting buffer in preparation for fluorescence-activated cell sorting.

Fluorescence-Activated Cell Sorting

The stromal vascular fraction contains myriad other cells in addition to adipose-derived stem cells, including pericytes, smooth muscle cells, fibroblasts, and blood-derived cells.17 The phenotypic characterization of adipose-derived stem cells is still in its infancy18; however, it is accepted that freshly isolated adipose-derived stem cells can be identified as CD34+CD31−CD45− cells in the erythrocyte-depleted stromal vascular fraction, as depicted by the International Society for Cellular Therapy.18–20 For instance, CD45 (leukocyte common antigen) is the classic marker for identifying cells of hematopoietic origin except for red blood cells and should be excluded. In combination with CD45, CD31 allows the exclusion of CD45−CD31+ endothelial populations. CD34 is also cited by the International Society for Cellular Therapy as a potential marker that should be used to identify the stromal cell–containing population.

Fluorescence-activated cell sorting was performed on a BD FACSAria II (BD Biosciences, San Jose, Calif.), using a 100-μm nozzle. Cells were incubated for 30 minutes in a fluorescence-activated cell sorting buffer containing antibodies to CD34, CD31, and CD45 (BD Biosciences). In addition, recent studies in our laboratory have demonstrated that subpopulations of adipose-derived stem cells exist with varying osteogenic3,21,22 and adipogenic23 potential; thus, we subsequently examined whether the method of their harvest significantly affected the immunophenotype of the adipose-derived stem cell milieu. Thus, cells were probed with antibodies to CD90, CD105, bone morphogenetic protein receptor type 1a, and bone morphogenetic protein receptor type 1b to assess differences in cell surface phenotype between harvest methods.3,21–23

Bromodeoxyuridine Assay

Proliferation of new method versus conventional method adipose-derived stem cells was assessed by bromodeoxyuridine incorporation, as described previously.24

2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) Assay

A trypan blue exclusion assay was implemented to assess the viability of conventional versus new method adipose-derived stem cells. Viable, large cells were counted using light microscopy. After the cells were harvested, they were seeded onto 96-well plates. Cell proliferation was measured with a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-based assay to compare conventional versus new method adipose-derived stem cells (Cell Proliferation Kit II XTT; Roche Applied Science, Indianapolis, Ind.).18

In Vitro Osteogenic Differentiation Assay

For osteogenic differentiation, cells from the two experimental groups were seeded at equal densities (100,000 cells per well) onto six-well culture plates. After attachment, cells were grown to at least 80% confluence before being cultured in osteogenic differentiation medium (10% fetal bovine serum, 1% penicillin/streptomycin, 250 μM ascorbate-2-phosphate, and 10 mM glycerophosphate) (R&D Systems, Minneapolis, Minn.).25,26 Alkaline phosphatase and alizarin red staining and quantification were performed at 7 and 14 days respectively, as described previously.25,26 Finally, gene expression was analyzed after 0, 7, and 14 days of differentiation by quantitative real-time polymerase chain reaction.

In Vitro Adipogenesis

Freshly sorted cells were seeded onto six-well plates (100,000 cells per well). Oil red O staining was performed 7 days after exposure to adipogenic differentiation medium, as described previously.18 In brief, adipogenic differentiation medium, consisting of Dulbecco’s Modified Eagle Medium, 10% fetal bovine serum, 1% penicillin/streptomycin, 10 μg/ml insulin, 1 μM dexamethasone, 0.5 mM methylxanthine, and 200 μM indomethacin, was added after cell attachment. Oil red O staining was performed at 7 days of differentiation.18 Finally, specific gene expression was examined after 7 days by quantitative real-time polymerase chain reaction.

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction

RNA was harvested at 0, 7, and 14 after commencement of osteogenic/adipogenic differentiation medium to determine specific gene relative expression.27 Quantitative real-time polymerase chain reaction was performed with the Applied Biosystems Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, Calif.). The amount of polymerase chain reaction product was calculated using an external glyceraldehyde 3-phosphate dehydrogenase standard curve and LightCycler software (Roche). All values were normalized against glyceraldehyde 3-phosphate dehydrogenase expression in the corresponding samples. Specific gene primer sequences were obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank/).28

In Vivo Bone Formation: Mouse Calvarial Defect Model

All research was performed in accordance with the Stanford University Administrative Panel on Laboratory Animal Care. To examine in vivo osteogenesis, nonhealing, critical-size (4-mm) calvarial defects were created in the right parietal bone of 8-week-old male mice, as reported previously.29 Athymic nude mice [Crl:NU(NCr)-Foxn1nu; Charles River Laboratories, Wilmington, Mass.] were used for experiments in this study to minimize the effects of an immune reaction to retention of human adipose-derived stem cells.30 Hydroxyapatite-coated poly(lactic-co-glycolic acid) was fabricated by 85:15 poly(lactic-co-glycolic acid by solvent casting and a particulate leaching process, as illustrated previously.31 In preparation for cell engraftment, scaffolds were seeded with 150,000 adipose-derived stem cells in 125 μl of traditional medium in 96-well culture plates 24 hours before implantation, as reported previously.3 Animals were treated with the hydroxyapatite-coated poly(lactic-co-glycolic acid) scaffold seeded with either new or conventional method adipose-derived stem cells. Small-animal micro–computed tomographic scanning was used to follow calvarial defect healing.3 The area of the calvarial defect was evaluated by quantifying pixels in the defect. Percentage healing was then calculated by dividing the defect area by the size of the defect, as recorded on the immediate postoperative micro–computed tomographic scan.3

In Vivo Fat Grafting Mouse Model

Whole lipoaspirate was minimally processed for grafting by centrifuging at 1300 rpm for 6 minutes. The liquid blood/debris and oil layers were aspirated, and the tube was overturned on an absorbent pad for 5 minutes to remove any remaining oil. Freshly harvested new or conventional method adipose-derived stem cells were then added to the fat stromal layer at a ratio of 10,000 adipose-derived stem cells per 200 μl of fat. Fat and adipose-derived stem cell–supplemented fat were divided into two groups (new method/conventional method) and transferred to 1-cc syringes for injection into mouse scalps. A minimal incision was created in the scalp at the base of the skull, and a volume of 200 μl of fat was injected into the subcutaneous plane beneath the scalp using a 14-gauge blunt-tip cannula. Incisions were closed with 6-0 Vicryl suture (Ethicon, Inc., Somerville, NJ.). Small-animal micro-computed tomographic scanning was used to follow calvarial defect healing.3 Following a baseline volume measurement, serial imaging was performed every 2 weeks over a total of 8 weeks. Images were reconstructed as a three-dimensional surface with cubic-spline interpolation using the Inveon Acquisition Workplace (n = 3) (Siemens, Berlin, Germany).

In Vivo Cell Viability

After fluorescence-activated cell sorting, new and conventional method adipose-derived stem cells were then labeled separately with Qtracker 655 quantum dots (Thermo Fisher Scientific, Waltham, Mass.) for the purpose of identifying the cells ex vivo 48 hours later (to assess the viability of the implanted adipose-derived stem cells 48 hours after implantation). Qtracker 655 is a particle that is endocytosed and can be excited and analyzed using fluorescence-activated cell sorting. Forty-eight hours after implantation, the cells were digested from the fat grafts using a previously described fat harvest protocol.32 Using 4′,6-diamidino-2-phenylindole as the viability stain (dead cells, 4′,6-diamidino-2-phenylindole–positive; live cells, 4′,6-diamidino-2-phenylindole–negative), these harvested cells were then subjected to fluorescence-activated cell sorting analysis for cells positive for Qtracker 655 and negative for 4′,6-diamidino-2-phenylindole (i.e., live new method or conventional method adipose-derived stem cells).

Statistical Analysis

Means and standard error means were calculated from numerical data. Bar graphs represent mean ± SEM. Unless otherwise stated, statistical analysis was performed with a two-tail t test to compare the two experimental groups. A value of p < 0.05 was considered significant.

RESULTS

Fluorescence-Activated Cell Sorting

To accurately measure the adipose-derived stem cell yield isolated by the new method and the conventional method, we implemented fluorescence-activated cell sorting to count the yield of each method. We sorted adipose-derived stem cells using the following profile: CD45−CD31−CD34+.18,19 Fluorescence-activated cell sorting analysis of adipose-derived stem cells immediately after harvest revealed a higher yield of total cells isolated from the lipoaspirate in those samples that were processed using the new method in comparison with the conventional method. Furthermore, fluorescence-activated cell sorting revealed that the new method resulted in a significantly higher population (p = 0.0017, t test) of adipose-derived stem cells as confirmed by the cell surface marker profile, suggesting a more “fat-friendly” method of isolation compared with the conventional method (Fig. 1, left and above, right). Meanwhile, fluorescence-activated cell sorting analysis showed equivocal expression of CD90 and CD105 among adipose-derived stem cells from both harvest methods, but also showed higher expression of both bone morphogenetic protein receptor type 1a and type 1b among the new method cells harvested compared with conventional method cells (p = 0.0089) (Fig. 1, below, right).

Fig. 1.

Fig. 1.

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(Left) Fluorescence-activated cell sorting plots demonstrating the frequency of CD45−CD31−CD34+ cells from the stromal vascular fraction. (Above, left) Cells isolated using the new method. (Below, left) Cells isolated using the conventional method. (Above, right) Graph representing the cell yield following fluorescence-activated cell sorting following the conventional method (blue) versus the new method (red) (**p = 0.0017, t test). (Below, right) Graph showing percentage surface expression of (left to right) CD90, CD105, bone morphogenetic protein receptor type 1a, and bone morphogenetic protein receptor type 1b in new method (red) and conventional method (blue) adipose-derived stem cells (ASC). No significant differences exist with respect to CD90 and CD105 surface expression between new method and conventional method cells. Expression of bone morphogenetic protein receptor type 1a (BMPR1a) and bone morphogenetic protein receptor type 1b (BMPR1b) is shown to be statistically significantly higher in new method cells compared with conventional method cells (*p < 0.05).

Cell Viability and Cell Proliferation

We implemented an XTT assay to evaluate the inherent effect of each processing technique on the viability of equal numbers of adipose-derived stem cells isolated using either the new method or the conventional method. We demonstrated that new method cells were more viable than those harvested using conventional method cells. The increased cellular viability associated with the new method was significantly greater than that of the conventional method at multiple time points (4 hours, p = 0.0030; 8 hours, p = 0.0089; 12 hours, p < 0.0001; 16 hours, p = 0.0094; 20 hours, p = 0.0032; 24 hours, p = 0.003; 26 hours, p = 0.0138, and 48 hours, p = 0.0049; t test) after harvest (Fig. 2, above). In addition, on examination of cellular proliferation, we noted a significantly higher rate of cellular proliferation at all examined time points from 0 to 72 hours in the cells harvested with the new relative to the conventional method (0 hours, p = 0.0487; 24 hours, p = 0.0086; 48 hours, p = 0.0003; and 72 hours, p = 0.0001) (Fig. 2, below).

Fig. 2.

Fig. 2.

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(Above) Graphic representation of XTT assay results demonstrates greater viability of the adipose-derived stem cells isolated using the new method (red) versus conventional method (blue) (4 hours, **p = 0.0030; 8 hours, **p = 0.0089; 12 hours, ****p < 0.0001; 16 hours, **p = 0.0094; 20 hours, ***p = 0.0032; 24 hours, **p = 0.003; 36 hours, *p = 0.0138; and 48 hours, **p = 0.0049; t test). (Below) Graphic representation of bromodeoxyuridine (BrdU) assay results demonstrate a significantly higher rate of cellular proliferation at all examined time points from 0 to 72 hours in the cells harvested with the new method (red) relative to the conventional method (blue) (0 hours, *p = 0.0487; 24 hours, **p = 0.0086; 48 hours, ***p = 0.0003; and 72 hours, ***p = 0.0001).

In Vitro Osteogenic Differentiation Assay

To determine the in vitro osteogenic differentiation capacity of the cell populations, equal numbers of cells were cultured in osteogenic differentiation medium for 14 days. Alkaline phosphatase staining was performed at day 7, as this is an early marker of bone formation. Here, we found that new method adipose-derived stem cells had a significantly greater percentage alkaline phosphatase staining than conventional method cells (p < 0.05, t test) (Fig. 3).

Fig. 3.

Fig. 3.

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(Left) Representative bright-field micrographs illustrating alkaline phosphatase staining of adipose-derived stem cells isolated using the conventional method (above, left) versus the new method (below, left). (Right) Graphic representation of the alkaline phosphatase quantification after osteogenic differentiation in vitro demonstrates significantly greater alkaline phosphatase staining of the adipose-derived stem cells isolated using the new method (red) compared with the conventional method (blue) (*p < 0.05, t test).

Osteogenic Gene Expression

We queried whether this increase in osteogenic differentiation in vitro correlated with an increase in relative expression of osteogenic genes—Runt-related transcription factor-2 (Runx2) and osteopontin. Thus, we analyzed transcription levels for markers of osteogenic differentiation at baseline and 7 and 14 days after commencement of osteogenic differentiation. Here, in fact, we saw a significantly lower transcript level of Runx2 in the new method adipose-derived stem cells relative to the conventional method at day 0 (p = 0.0058, t test), suggesting perhaps that the conventional method may potentially isolate more terminally differentiated cells rather than a multipotent cell population (Fig. 4). This hypothesis is further supported by the finding that the osteopontin relative gene expression increases with time in those cells isolated by the new method, with the relative gene expression becoming significantly greater than the conventional method at day 14 after commencement of osteogenic differentiation (p = 0.0003, t test).

Fig. 4.

Fig. 4.

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Relative transcriptional expression of (above, left) alkaline phosphatase, (below, left) Runx2, (above, right) osteopontin, and (below, right) osteocalcin as determined by quantitative real-time polymerase chain reaction (new method, red; conventional method, blue).

In Vivo Osteogenic Regeneration

Furthermore, we questioned whether equal numbers of adipose-derived stem cells isolated by the new method would be noninferior to those isolated by the conventional method, with respect to in vivo osteogenesis. A critical-size calvarial defect model was implemented in mice to examine the in vivo osteogenic capacity of both experimental groups of adipose-derived stem cells. The cells, as confirmed by fluorescence-activated cell sorting using their surface marker profile, CD45−CD31−CD34+, were then seeded onto hydroxyapatite-coated poly(lactic-co-glycolic acid) scaffolds. Calvarial defect healing was followed with live micro–computed tomography at 0 and 8 weeks postoperatively, as described above. We found that there was equivalent in vivo bone regeneration seen in the defects treated with adipose-derived stem cells isolated by the new method and the conventional method (Fig. 5). On examination of calvarial defect percentage healing using high-resolution micro–computed tomography and analysis of the three-dimensional reconstructions, we saw equivalent bone formation in defects treated with both populations of cells.

Fig. 5.

Fig. 5.

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(Left) Micro–computed tomographic reconstructed images demonstrating the critical calvarial bone defects at week 0 and week 8 (above, left, conventional method adipose-derived stem cells; center, left, new method adipose-derived stem cells; below, left, control scaffold, no cells added). (Right) Graphic representation of the percentage healing at 8 weeks of calvarial defects treated with conventional method cells (blue) versus new method cells (red) demonstrates equivocal results. These results are significantly different from the acellular control scaffold (black), which exhibits a nonhealing critical-size defect (***p = 0.001).

In Vitro Adipogenic Differentiation Assay

To examine the adipogenic potential of the adipose-derived stem cells harvested by both isolation methods, we cultured equal numbers of cells, freshly isolated by fluorescence-activated cell sorting, in adipogenic differentiation medium for 14 days. Oil red O staining was performed at day 7 after starting adipogenic differentiation medium. Interestingly, we noted that the cells isolated using the new method had a significantly greater percentage oil red O staining relative to the control, conventional method (p = 0.0249, t test) (Fig. 6).

Fig. 6.

Fig. 6.

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(Left) Bright-field microscopy illustrating oil red O staining of adipose-derived stem cells isolated using the conventional method (above, left) versus the new method (below, left). (Right) Graphic representation of the oil red O staining quantification after adipogenic differentiation in vitro assay demonstrates significantly greater oil red O staining of the adipose-derived stem cells isolated using the new method (red) versus the conventional method (blue) (*p < 0.05, t test).

Adipogenic Gene Expression

We further questioned whether increased in vitro adipogenesis was attributable to increased relative expression of genes related to adipogenesis—peroxisome proliferator activated receptor-γ (PPARγ), lipoprotein lipase (LPL), and fatty acid binding protein-4 (FABP4). Therefore, we analyzed transcription levels for markers of adipogenic differentiation at baseline and 7 and 14 days after commencement of adipogenic differentiation medium. Interestingly, we found significantly increased expression of each of the three genes examined (PAPARγ, LPL, and FABP4) in adipose-derived stem cells isolated by the new method in comparison with the conventional method (Fig. 7) (p < 0.05, t test).

Fig. 7.

Fig. 7.

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Relative transcriptional expression of (left) FABP4, (center) LPL, and (right) PPARγ as determined by quantitative real-time polymerase chain reaction.

In Vivo Fat Graft Retention

To further assess the functionality of adipose-derived stem cells in vivo, we used a soft-tissue model of fat grafting combined with cell-assisted lipotransfer. The cells harvested using both the conventional method and the new method were added to 200-μl fat grafts and injected subcutaneously under the mouse scalp. Micro–computed tomographic scans at weeks 0, 2, 4, 6, and 8 showed that grafts that received cells harvested from either method had higher volume retention than grafts that did not receive cells (p = 0.0058, t test). However, grafts supplied with new method cells did not show significantly different retention compared with conventional method cells (Fig. 8, left).

Fig. 8.

Fig. 8.

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(Left) Graphic representation of fat graft retention showing an unsupplemented fat graft (black, fat alone) compared with grafts supplemented with cells obtained using either the conventional method (blue) or the new method (red). Although significant differences exist between the fat-alone control group and groups supplied with adipose-derived stem cells, the graft retention shown with either population of harvested cells is equivocal. Data are normalized to week 0 (**p > 0.01). (Right) Fluorescence-activated cell sorting plots show viability of implanted cells ex vivo. Labeled cells are identified by the presence of the quantum dot label detected using alkaline phosphatase, and analyzed for viability with 4′,6-diamidino-2-phenylindole, detected using Pacific blue. Implanted cell viability was greater in new method compared with conventional method adipose-derived stem cells (conventional method, 41 percent viability; new method, 54.6 percent viability).

In Vivo Cell Viability

Fluorescence-activated cell sorting analysis of labeled cells 48 hours after implantation in fat grafts showed higher cell viability in new method compared with conventional method adipose-derived stem cells. Viability was confirmed in fluorescence-activated cell sorting plots by analyzing cells positive for Qtracker 655 and negative for 4′,6-diamidino-2-phenylindole, which was seen at 54.6 percent viability in the new method cells and 41 percent viability in the conventional method cells (Fig. 8, right).

DISCUSSION

Although it is normally discarded, adipose tissue also contains an easily accessible source of adipose-derived stem cells that may be used for a multitude of tissue-engineering purposes.33 At present, the ideal method of adipose-derived stem cell harvest from adipose tissue remains elusive. The technique first described by Zuk et al. has undergone little change in the past decade.6 Here, we present a novel method of isolating these cells that results in a significantly higher yield compared with the conventional method. Furthermore, new method adipose-derived stem cells had a higher rate of proliferation and viability relative to conventional method cells in vitro. In addition, new method cells were noninferior to the conventional method cells when tested for in vivo osteogenic and adipogenic capacity. Thus, the new method allows isolation of a greater number of viable adipose-derived stem cells with no inhibitory effect on the adipose-derived stem cell population on their regenerative capacity.

The new and conventional methods of adipose-derived stem cell isolation differ in two key areas: the collagenase digestion buffer constituents and the use of an orbital shaker. The collagenase digestion buffer includes calcium chloride, DNAse, bovine serum albumin, poloxamer-188, and hydroxyethyl piperazineethanesulfonic acid. These additional constituents result in a more hospitable environment for the cells released from adipose tissue during mechanochemical dissociation, in comparison with the conventional collagenase digestion buffer, which offers little support to the stromal vascular fraction cells. Notably, poloxamer-188, a copolymer surfactant, is known to stabilize cell membranes and is reported to be cyto-protective in a number of instances, including the management of sickle cell disease, experimental cardioprotection, and acute cartilage trauma.34–36 Hydroxyethyl piperazineethanesulfonic acid buffer, first described by Good et al. in 1966,37 is an organic chemical buffering agent, buffering the solution to maintain a pH 7.2 to 7.6, allowing for cellular protection during extended periods of manipulation outside of a carbon dioxide incubator, as in the case of adipose-derived stem cell isolation. We hypothesize that bovine serum albumin provides additional buffer to the cells, in addition to a minor contribution to nutrition of the targeted cells during adipose-derived stem cell isolation. Deoxyribonuclease, DNase, performs a house-keeping role during cell harvest by degrading DNA strands, released from cells lysed during mechanochemical digestion, which can increase viscosity and reduce cell recovery because of the toxicity to the neighboring cells.

In addition, the concentration of collagenase is higher in digestion buffer used in the new method relative to the conventional method (conventional method, 0.075% collagenase type I; new method, 0.22% collagenase type I). Following a measured enzyme titration, we have found that a higher collagenase concentration does not prove harmful to the isolated cell populations, as shown in the cell viability and proliferative indices, or to the multipotency of the isolated adipose-derived stem cells.

Furthermore, we suspect that use of an orbital shaker, instead of gentle agitation in a water bath, for the incubation stage allows for more efficient adipose tissue mechanochemical dissociation in the new method of adipose-derived stem cell harvest, because of increased exposure of the collagenase digestion buffer to the tissue surface area. One could argue that this exposes the cells to additional shear stress38; however, the new method cells were not only more plentiful but also more viable and proliferative relative to conventional method cells.

Strategies to prospectively enrich stromal vascular fraction of human adipose tissue to isolate homogenous stem subpopulations with enhanced osteogenic enrichment have been recently reported. Indeed, James et al. have shown that a subpopulation of cells in the stromal vascular fraction with a specific immunophenotype (i.e., CD146+CD34−CD45−), which the authors term “perivascular stem cells,” has increased osteogenic potential.39,40 In addition, our laboratory has identified that adipose-derived stem cells with high surface marker expression of CD90 and low expression of CD105 surface marker can be used to isolate a highly osteogenic subpopulation for bone tissue engineering.3,21 Furthermore, we have previously reported that positive selection for bone morphogenetic protein receptor type 1b promotes differentiation and specification of adipose-derived stem cells toward an osteogenic lineage,22 whereas positive selection for bone morphogenetic protein receptor type 1a enhances de novo adipogenesis of adipose-derived stem cells.23 When we probed the cells isolated using either the new method or the conventional method by fluorescence-activated cell sorting for specific adipose-derived stem cell subpopulations, we did not perceive a significant difference in CD90 or CD105 expression; interestingly, however, bone morphogenetic protein receptor type 1a and 1b expression did differ significantly between the two populations.

It is well described that adipose-derived stem cells exhibit a shift in phenotypic expression under in vitro conditions, which may alter the biology of stem cells3,41–43; thus, a method that ensures the maximal yield of adipose-derived stem cells harvested before further enrichment is optimal for tissue regeneration. There is a physiologic demand for multipotent cells for tissue regeneration. Thus far, the majority of clinical studies refer to the necessity to first expand adipose-derived stem cells ex vivo, before use in reconstructive procedures.44–47 The elusive goal of tissue regeneration is to define a practice of cell isolation and subsequent incorporation into reconstructive procedures without the need for culture expansion ex vivo, thus avoiding potential phenotypic cell shift and the risk of contamination.48,49 For instance, there is increasing emphasis on atraumatic adipose tissue procurement and subsequent placement of fat grafts.48,49

CONCLUSIONS

It is now well characterized that liposuction, first developed as a cosmetic surgical technique to remove excess fat, proffers a population of multipotent cells that can be exploited for regenerative medicine. To fully exploit the potential of adipose-derived stem cells in tissue regeneration, we need to develop techniques for cell procurement, isolation, and subsequent placement that respect the viability and regenerative potential of these multipotent cells. The new and conventional methods of adipose-derived stem cell isolation described above differ in two key areas: the constituents of the collagenase digestion buffer and the use of an orbital shaker. Here, we describe a technique of isolating a higher yield of more viable and proliferative adipose-derived stem cells, which can be implemented in tissue regeneration.

ACKNOWLEDGMENTS

The authors would like to acknowledge the following ongoing support for this work: R01DE02183, R21DE02423001, U01HL099776 (to M.T.L.), the Oak Foundation, the Hagey Laboratory for Pediatric Regenerative Medicine, the American College of Surgeons Franklin Martin Faculty Research Fellowship (to D.C.W.), the Stanford University Child Health Research Institute Faculty Scholar Award (to D.C.W), the Plastic Surgery Foundation/Plastic Surgery Research Council Pilot Grant (to R.T.), and the Stanford University Transplant and Tissue Engineering Center of Excellence Fellowship (to R.T. and A.M.).

Footnotes

Disclosure: The authors have no inancial interest in any of the products or devices mentioned in this article.

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