COSMETIC

Influences of Centrifugation on Cells andTissues in Liposuction Aspirates: OptimizedCentrifugation for Lipotransfer and Cell Isolation

Masakazu Kurita, M.D.Daisuke Matsumoto, M.D.Tomokuni Shigeura, M.S.

Katsujiro Sato, M.D.Koichi Gonda, M.D.Kiyonori Harii, M.D.

Kotaro Yoshimura, M.D.

Tokyo and Yokohama, Japan

Background: Although injective autologous fat transplantation is one of themost attractive options for soft-tissue augmentation, problems such as unpre-dictability and fibrosis resulting from fat necrosis limit its universal acceptance.Centrifugation is one of most common methods for overcoming these difficul-ties. This study was performed to investigate quantitatively the effects of cen-trifugation on liposuction aspirates to optimize centrifugal conditions for fattransplantation and isolation of adipose-derived stem cells.Methods: Liposuction aspirates, obtained from eight healthy female donors,were either not centrifuged or centrifuged at 400, 700, 1200, 3000, or 4200 g for3 minutes. The volumes of the oil, adipose, and fluid portions and numbers ofblood cells and adipose-derived cells in each portion were examined. Theprocessed adipose tissues (1 ml) were injected into athymic mice, and grafts wereharvested and weighed at 4 weeks. Morphologic alterations were observed usinglight and scanning electron microscopy.Results: Centrifugation concentrated adipose tissues and adipose-derived stemcells in the adipose portion and partly removed red blood cells from the adiposeportion. Centrifugation at more than 3000 g significantly damaged adipose-derived stem cells. Centrifugation enhanced graft take per 1 ml centrifugedadipose but reduced calculated graft take per 1 ml adipose before centrif-ugation.Conclusions: Excessive centrifugation can destroy adipocytes and adipose-derived stem cells, but appropriate centrifugation concentrates them, re-sulting in enhanced graft take. The authors tentatively recommend 1200 gas an optimized centrifugal force for obtaining good short- and long-termresults in adipose transplantation. (Plast. Reconstr. Surg. 121: 1033, 2008.)

Soft-tissue augmentation by autologous fattransplantation is an attractive therapy, butsome problems remain, including the low

survival rate of transplanted adipose tissue andformation of calcifications. To overcome thesedifficulties, a number of technical improve-ments in harvesting, processing, and injectionwere reported.1–9 Among these, we focused oncentrifugation of liposuction aspirates. Centrifuga-tion is also an important process for isolating adi-pose-derived stem cells from liposuction aspirates.

Centrifugation, which has been used in pro-cessing lipoaspirates since the 1980s,10 is nowwidely used to concentrate aspirated fat and toremove oil, fluid, and blood products. Variousviews and ideas regarding centrifugation of li-poaspirates have been reported,1,3,5,7–9,11–13 and thecurrent consensus is as follows. Centrifugationconcentrates adipocytes3,5,7,8 and separates themfrom substances that may degrade adipocytes,such as blood cells, lipids, proteases, andlipases,3,5,8 but does not enhance immediate fat

From the Department of Plastic Surgery, University of TokyoSchool of Medicine; Department of Plastic Surgery, KyorinUniversity School of Medicine; and Division of Research andDevelopment, Biomaster, Inc.Received for publication May 24, 2006; accepted August 25,2006.Copyright ©2008 by the American Society of Plastic Surgeons

DOI: 10.1097/01.prs.0000299384.53131.87

Disclosures: A centrifugation device (Lipokit;Medikan Corp., Seoul, Korea) and syringes madespecific for the device (Medikan Corp.) were pro-vided to authors for research purposes by the com-pany.

www.PRSJournal.com 1033

tissue viability.9 Centrifugation may enhance thetotal amount of transplanted fat,11 although ex-cessive centrifugal force may damage intactadipocytes.7,9,13,14

Undifferentiated stromal cells present in adi-pose tissue recently became a focus. Stromal vas-cular fractions isolated from liposuction aspiratescontain multiple cell populations,15,16 one ofwhich has been identified as adipose-derived stemcells.17 Adipose-derived stem cells can differenti-ate into various lineages forming adipose, bone,cartilage, neuronal, and vasculature tissues. Ourrecent research18 showed that both the fatty andfluid portions of liposuction aspirates contain a sig-nificant amount of adipose-derived stem cells, andwe characterized freshly isolated adipose-de-rived stem cells as CD31–CD34�CD45–CD90�

CD105–CD146– cells. The presence of adipose-de-rived stem cells has clinical implications for au-tologous fat transfer because adipose-derived stemcells may contribute to neoangiogenesis in theacute phase by acting as endothelial progenitorcells19 or angiogenic-factor-releasing cells.20 Ad-ipose-derived stem cells are known to secreteangiogenic factors such as vascular endothelialgrowth factor and hepatocyte growth factor un-der hypoxic conditions20 and to contribute toincreased capillary density and blood flow.19 Inaddition, they can affect long-term survival oftransplanted adipose by acting as preadipocytes.

We have recognized positive effects of centrif-ugation on fat transfer in our clinical experience,but the force-dependent influences of centrifuga-tion on liposuction aspirates have not been wellstudied thus far. The purposes of this study were(1) to determine the anatomical and physiologicalterations of cell and tissue components in lipo-suction aspirates during the centrifugation pro-cess, and (2) to optimize centrifugation parame-ters for autologous lipotransfer and isolation ofadipose-derived stem cells.

MATERIALS AND METHODS

Centrifugation of Liposuction AspiratesLiposuction aspirates were obtained from sur-

gery performed on the abdomen or thigh regionsof eight healthy female donors aged 21 to 38 yearswith informed consent approved by our institu-tional review board. Infiltration of saline (tumes-cent solution), liposuction, and subsequent cen-trifugation of syringes were conducted using asingle combined machine (Lipokit; MedikanCorp., Seoul, Korea) (Fig. 1, above). Liposuctionaspirates were divided and poured into disposable

sterilized syringes (50 ml) with a filter piston(Medikan). The filter piston was specifically de-signed for separation of the oil from the otherportions by centrifugation (Fig. 1, below). Afterbeing placed upright for 10 minutes, specimenswere divided into two portions: a floating adiposeportion and a denser fluid portion. Syringes wereallotted to six groups (two syringes each) and cen-trifuged at 0, 400, 800, 1200, 3000, or 4200 g bymeans of the Lipokit for 3 minutes. After centrif-ugation, specimens were divided into three por-tions; oil (top), adipose (middle), and fluid (bot-tom) (Fig. 2). The volume and weight of eachportion were measured before and after centrif-ugation, and the specific gravity [weight (ingrams)/volume (in milliliters)] of the adiposeportion was calculated. The volume of the oilportion was measured by aspirating the oil onthe filter piston with a 10-ml syringe, and those

Fig. 1. Centrifugation of liposuction aspirates. (Above) A ma-chine for centrifugation used in this study. (Below) A disposablesterilized syringe (50 ml) with a filter piston. By centrifugation, oilwas shifted onto the piston (arrow).

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of the adipose and fluid portions were measuredby reading a scale on the centrifugation syringe(50 ml).

Counting of Blood Cells and Adipose-DerivedStem Cells

Although blood cells are thought to disturbengraftment of injected adipose and to be partlyextracted by centrifugation, there are no reliabledata on the extent of blood cell clearance by cen-trifugation. Adipose portion tissues before andafter centrifugation were digested at 37°C for 30minutes on a shaker with an equal volume of0.075% collagenase. Mature adipocytes and con-nective tissues were separated from pellets by cen-trifugation (400 g for 10 minutes). The pellet wasresuspended in phosphate-buffered saline andpassed through a 100-�m mesh filter (Millipore,Billerica, Mass.). Fluid portions (3 ml each) beforeand after centrifugation were centrifuged (400 gfor 10 minutes), and the pellets were resus-pended and passed through a 100-�m mesh fil-ter. Numbers of red blood cells and nucleatedcells in adipose and fluid portions were countedseparately with CellTec (Nihon Koden, Tokyo,Japan). Nucleated cells counted correspondedto the total of white blood cells, adipose-derivedstem cells, and other adipose-derived cells suchas endothelial cells.

Isolation, Culture, and Counting ofAdipose-Derived Stem Cells from the Adiposeand Fluid Portions

Adipose-derived stem cells were isolated sep-arately from the adipose and fluid portions ofliposuction aspirates as described before.18 Thecells were cultured on 100-mm gelatin-coateddishes with M-199 medium containing 10% fetalbovine serum, 100 IU penicillin, 100 mg/ml strep-tomycin, 5 ng/ml heparin, and 2 �g/ml acidicfibroblast growth factor, at 37°C, in a humid at-mosphere of air and 5% carbon dioxide. Afterbeing cultured for 7 days, cell counts were per-formed using a NucleoCounter (Chemometec,Denmark).

Fat Transplantation to Nude MiceTo examine the influences of centrifugation

on engraftment of adipose tissue, lipoaspirates ob-tained from a single donor were divided into sixgraft materials using centrifugations at 0, 400, 700,1200, 3000, and 4200 g. Six milliliters of eachmaterial was injected into the backs of six 5-week-old nude mice using an 18-gauge needle, with 1 mlinjected per mouse. The same experiment wasrepeated twice using lipoaspirates from anotherdonor. Consequently, a total of 72 mice were usedas recipients of human fat transplantation. Ani-mals were killed 4 weeks after fat transplantation,and transplanted grafts were measured for weight.Harvested samples were fixed and processed for his-tology (see below). To estimate the net efficacy oftransplantation per volume of adipose portion be-fore centrifugation, a calculation was performedwith the following hypothetical equation: Putativegraft take of 1 ml uncentrifuged adipose � (grafttake of 1 ml centrifuged adipose) � (volume ofadipose portion after centrifugation)/(volume ofadipose portion before centrifugation).

Scanning Electron Microscopic StudyAdipose samples were fixed with paraformal-

dehyde and 2.5% glutaraldehyde in 0.2 M caco-dylate buffer for 1 week at room temperature, andfixed in 1% osmium tetroxide. After dehydration,they were dried with a supercritical point carbondioxide dryer (HCP-2; Hitachi, Tokyo, Japan),sputter-coated with platinum/palladium, and ex-amined with a scanning electron microscope(S3500N; Hitachi). Light microscopic examina-tion of hematoxylin and eosin–stained slides wasalso performed.

Fig. 2. The adipose and fluid portions of liposuction aspirateswere clearly separated by 3 minutes of centrifugation and stayedbelow the piston after that.

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Statistical AnalysesResults were expressed as mean � SE. Paired

t tests were performed to evaluate the differencesbetween centrifugal conditions, and no correctionwas made for multiple comparisons. Statistical sig-nificance was defined as p � 0.05.

RESULTSGross Effects of Centrifugation on the AdiposePortion of Liposuction Aspirates

A total of 78 syringes containing liposuctionaspirates were centrifuged with six different cen-trifugal forces. With increased centrifugal force,the volumes of the oil and fluid portions in-creased, and the volume of the adipose portiondecreased. Although there was no significant dif-ference between 3000 g and 4200 g, volumes of oil,adipose, and fluids altered significantly with in-creased centrifugal forces (Fig. 3, above). For 48samples, the specific gravity of the adipose portionwas measured; it did not change significantly ex-cept for a decrease between control and 400 g (Fig.3, below). On scanning electron microscopic ob-servation, the adipose portion was observed asclusters of spherically shaped adipose cells. Adi-pocyte size was not remarkably altered with in-creased centrifugal forces. In all samples, includ-ing uncentrifuged controls, clusters of adipocyteswere partially ruptured. The degree of rupturedadipocyte clusters seemed to vary among donorsubjects, but no remarkable difference was seenbetween the samples processed with different cen-trifugal forces from the same subject (Fig. 4).

Effects of Centrifugation on Numbers of RedBlood Cells, Adipose-Derived Stem Cells, andNucleated Cells

Numbers of red blood cells, adipose-derivedstem cells, and nucleated cells in the adipose andfluid portions were counted separately using 48samples. Although the total number of red bloodcells in the adipose and fluid portions did notchange significantly based on centrifugal forces(Fig. 5, above), red blood cells shifted significantlyfrom the adipose portion to the fluid portion at alldifferent centrifugal forces. In addition, a signif-icant difference was seen between 400 g and 700g but not between 700 g and more than 1200 g (Fig.6, above). Total numbers of nucleated cells, whichincluded white blood cells, adipose-derived stemcells, and other adipose-derived cells, did notchange significantly by centrifugation; nor werethere statistically significant shifts in numbers ofnucleated cells detected for any centrifugal

Fig. 3. (Above) Columns on the left, middle, and right demon-strate the volumetric proportions of the oil, adipose, and fluidportions, respectively, under each centrifugal condition. Statis-tical analysis was performed with paired t tests between groups.Green, orange, and red bars indicate statistical significances in theoil, adipose, and fluid portions, respectively. The significance lev-els are indicated with the number of asterisks (*p � 0.05; **p �0.01; ***p � 0.001). A significant volume reduction in the adiposeportion was observed not only between uncentrifuged and cen-trifuged samples but also between different centrifugal forces.The fluid and oil portions significantly increased in volume ina centrifugal force– dependent manner. Data representmeans � SE. (Below) Specific gravity of the adipose portiontends to decrease with increased centrifugal force, but thedifferences were not statistically significant. Data representmeans � SE.

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force (data not shown). The total number ofadipose-derived stem cells remained consistentup to 1200 g, whereas the number of adipose-derived stem cells decreased significantly atmore than 3000 g (Fig. 5, below). Unlike redblood cells, adipose-derived stem cells did notshift significantly between the adipose and fluidportions (Fig. 6, below).

Adipose Graft Survival of In Vivo ExperimentalModels

The results of the transplantation of uncen-trifuged or centrifuged adipose using 72 nudemice revealed that centrifugation significantly en-hanced the proportion of graft survival (Fig. 7,above). Significance was detected not only betweencontrol and all centrifugal forces, but also betweenincreased centrifugal forces, although resultsfrom centrifugation at 3000 g were superior tothose from 4200 g (Fig. 7, above).

Centrifugation made the original volume offat compact, and thus 1 ml of uncentrifuged fatand 1 ml of centrifuged fat differed originally infat volume. If we have a sufficient volume of as-pirated fat, we conclude that centrifugation canenhance the graft take. In contrast, if we tried toobtain the largest adipose graft by using 1 ml of

uncentrifuged adipose alone, it could be con-cluded by virtual calculation that the uncentri-fuged graft would be better than any centrifugedgrafts (Fig. 7, below).

Under microscopic observation, no remark-able difference was observed in cell integrity orstructure among samples centrifuged at differentforces. Even in samples centrifuged with a maxi-mum force of 4200 g, adipocytes survived well 4weeks after transplantation.

DISCUSSIONConcentration of the Graft

Although various authors have recommendedperforming precentrifugation of fat grafts, manyreports described centrifugal force using rpm (rev-olutions per minute) units.1,3,5,8,21–24 Working cen-trifugal forces with the same rpm value can differin terms of radius of centrifugation. For example,centrifugation at 3000 rpm with either a 12- or18-cm radius of gyration is equivalent to 1207 or1811 g, respectively. Thus, it is hard to compareour results with those from other previous reportsthat used rpm.

Our study showed that 3 minutes of centrifu-gation compacts aspirated fat and partly excludesoil, water, and blood cells, but not adipose-derived

Fig. 4. Scanning electron microscopic photographs of representative uncentrifuged and centrifuged samples derived from a singledonor. Clusters of spherically shaped adipocytes and intermittently dispersed ruptured cells were observed, regardless of centrifugalforces [left, control (uncentrifuged); center, 1200 g; right, 4200 g). Magnified photographs show that there are adipocytes that werenot morphologically altered even in samples centrifuged at 4200 g (below, right). Scale bar � 200 �m (above) and 50 �m (below).

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stem cells. Consequently, adipose tissue, extracel-lular matrix, and adipose-derived stem cells areconcentrated by centrifugation, likely contribut-ing to a boost in the graft take.25,26 The degrees ofconcentration and exclusion tended to be ele-vated with increased centrifugal force.

Damage to AdipocytesBoschert et al.7 reported that the quantity of

oil increased because of adipocyte destructionwhen specimens were centrifuged at more than100 g and thus concluded that centrifugation atgreater than 100 g was not appropriate for autol-

Fig. 5. Total numbers of red blood cells and adipose-derivedstem cells in the adipose and fluid portions of liposuction aspi-rates before and after centrifugation. Proportions of total (adi-pose and fluid portions) cell count at each centrifugal conditioncompared to control are presented. Significance was analyzedusing paired t tests for groups. Data represent means � SE (*p �

0.05; **p � 0.01; ***p � 0.001). (Above) Total numbers of redblood cells did not change significantly by centrifugation. (Below)Total number of adipose-derived stem cells showed no remark-able alteration after centrifugation up to 1200 g, but a significantdecrease was observed between control and samples centri-fuged at 3000 and 4200 g.

Fig. 6. Shift of red blood cells and adipose-derived stem cells

between the adipose and fluid portions by centrifugation. Pro-

portions of cell numbers contained in adipose and fluid por-

tions before and after centrifugation are presented. Signifi-

cance was analyzed using paired t tests between groups. Data

represent means � SE (*p � 0.05; **p � 0.01; ***p � 0.001).

(Above) Percentages of red blood cell count contained in the

adipose and fluid portions. Red blood cells shifted signifi-

cantly from the adipose portion to the fluid portion at all dif-

ferent centrifugal forces compared with control. In addition,

significance was seen between 400 g and 700 g, but not be-

tween 700 g and more than 1200 g, suggesting that centrifu-

gation at 700 g is enough and that more than 700 g may not be

necessary for red blood cell extraction from aspirated adipose.

(Below) Percentage of adipose-derived stem cell count con-

tained in the adipose and fluid portions. Adipose-derived

stem cells did not shift significantly between the adipose and

fluid portions at any level of centrifugal force.

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ogous fat transplantation. However, the results ofour fat graft experiments indicate that centrifu-gation with more than 100 g centrifugal force cansurely be used in aspirated fat transplantation.

Our results showed that the oil volume in-creased with increased centrifugal forces, but his-tologic findings did not clearly demonstrate de-struction of adipocytes. Based on our microscopicobservations, the degree of adipocyte destructiondiffered among patients but showed only minordifferences between different centrifugal forces.Scanning electron microscopic observationshowed that remnant oil was seen in the adiposeportion even after 3 minutes of centrifugation.Thus, we suggest that the increase in the oil por-tion does not necessarily mean an increase of adi-pocyte destruction by centrifugation but mayrather mean an increase in separation of oil fromthe adipose portion.

Damage and Distribution of Blood Cells andAdipose-Derived Stem Cells

Our results are mostly in accordance with theview previously reported that centrifugation sep-arates fat cells from lipid, blood cells, water, andwater-soluble ingredients such as proteases andlipases.3,5,7,8 To our knowledge, there have been noreports examining quantitatively the effects ofcentrifugation on blood cells and adipose-derivedstem cells in liposuction aspirates. Although cen-trifugation induced a slight shift of red blood cellsfrom the adipose portion to the fluid portion with-out change in total numbers of red blood cells, thevolume of the adipose portion was compacted toa greater extent than the shift of blood cells, andthus these blood cells were slightly concentratedin the adipose portion. Although a previous au-thor indicated that the presence of blood in theinjected fat stimulates macrophage activity to re-move the fat cells,5 the actual effect of the bloodin the graft has not clearly been elucidated. Thusfar, we cannot determine whether a decrease ofnumber and increase of concentration of redblood cells and white blood cells is advantageousor disadvantageous in fat grafting.

In contrast, the results indicated that adipose-derived stem cell yield after 1 week of culture wasalmost consistent up to 3000 g and decreased atmore than 3000 g. In addition, it was shown thatadipose-derived stem cells did not shift betweenthe adipose and fluid portions by centrifugation,likely because adipose-derived stem cells con-tained in the adipose portion are resident in orstrongly adhered to the adipose tissues. Accord-

Fig. 7. Transplantation of uncentrifuged and centrifuged adi-pose tissue lipoaspirates obtained from a single donor were di-vided into six graft materials using centrifugation at 0, 400, 700,1200, 3000, and 4200 g. Six milliliters of each material was in-jected into the backs of six 5-week-old nude mice using an 18-gauge needle, with 1 ml injected per mouse. The same experi-ment was repeated twice using lipoaspirates from anotherdonor. The surviving adipose tissues were harvested 4 weekslater. Significance was analyzed with paired t tests betweengroups. Data represent means � SE (*p � 0.05; **p � 0.01; ***p �

0.001). (Above) Weights of transplanted adipose tissues. Withcentrifugation at 1200 g or more, transplanted adipose tissue wassignificantly greater in weight than uncentrifuged control. Cen-trifugation significantly contributed to obtaining a better grafttake at least in short-term observations, although centrifugationat 4200 g might be excessive compared with 3000 g. (Below) Calcu-lated putative graft take per volume of uncentrifuged adipose. Val-ues were calculated as follows. Putative graft take of 1 ml uncentri-fuged adipose� (graft take of 1 ml centrifuged adipose)� (volumeof adipose portion after centrifugation)/(volume of adipose portionbefore centrifugation). Based on these putative calculations, if therewas a limited volume of aspirated adipose, graft take would be bestwhen it was not centrifuged before transplantation.

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ingly, centrifugation simply enhanced the densityof adipose-derived stem cells as a result of com-paction of the adipose portion. Condensation ofadipose-derived stem cells in fat graft may be ben-eficial for enhancing the fat graft survival rate forthe reasons discussed below.

Graft SurvivalWe propose that the aspirated fat graft takes

were grossly influenced by the balance of the neg-ative effects of destruction and the positive effectsof condensation by centrifugation. Our results re-vealed that the short-term survival rate of aspiratedadipose graft per volumetric unit after centrifu-gation increased with centrifugal forces up to 3000g. Condensation of the graft material and adipose-derived stem cells is thought to dominantly con-tribute to this enhancement.

However, it was also shown by a virtual calcu-lation that surviving fat graft per volumetric unitbefore centrifugation decreased by interventionwith centrifugation. Surviving fat graft decreasedat 400 g but did not decrease further with in-creased centrifugal forces; this outcome may implythat the decrease at 400 g could have resulted fromdestruction of adipocytes located especially in thesuperficial layers of adipose fragments and thatadipocytes in the inner layers may be protectedfrom mechanical injuries by the destroyed super-ficially located adipocytes.

Histologic findings of transplanted fat wereconsistent with previous reports,5,27 which foundthat centrifuged graft samples were similar to un-centrifuged ones. Even samples centrifuged at4200 g showed no remarkable differences in his-tology from controls after transplantation. It isthus suggested that once adipocytes succeed inavoiding critical damage during centrifugation,there will be no difference in the structural qualityof adipocytes between centrifuged and uncentri-fuged samples.

Our results of graft take with or without cen-trifugation indicate a clinical application for se-lective use of centrifugation. If there has only beena restricted amount of adipose (though it wouldbe very rare), centrifugation might not have beenthe best approach from the standpoint of the mosteffective use of restricted graft material. For ex-ample, by using 10 ml of uncentrifuged adiposetissue, we would have a 6.4-ml augmentation (64percent survival) if we transplanted the adiposewithout centrifugation. However, if we centri-fuged the 10 ml of adipose at 1200 g, we wouldobtain centrifuged and condensed adipose, with a

volume of 7.2 ml. After transplanting the 7.2 ml ofcentrifuged fat, we would have a 5.9-ml augmen-tation (82 percent survival). However, in most clin-ical cases, we can harvest a sufficient volume ofaspirated fat, and in such cases, we should centri-fuge aspirated fat before grafting to obtain thebest augmentation effects. For example, by har-vesting 13.8 ml of adipose tissue, we could trans-plant 10 ml of adipose centrifuged at 1200 g andachieve an 8.2-ml augmentation.

We suggest that adipose-derived stem cells andother adipose-derived cells are crucial for graftsurvivability in both the short and long term. Ourrecent report26 revealed that aspirated fat is rela-tively adipose-derived stem cell deficient com-pared with excised whole fat, which containslarge vessels and nerves, unlike aspirated fat,and that adipose-derived stem cells can surviveand reside between adipocytes or in the con-nective tissues of surviving adipose tissue aftertransplantation. The relative deficiency of adi-pose-derived stem cells may be the reason forthe lessened survival rate of aspirated fat aftertransplantation compared with excised fat re-ported by two previous experimental studies.28,29

Condensation of adipose-derived stem cells bycentrifugation may mean conversion of rela-tively stem-cell– deficient adipose to relativelystem-cell–rich adipose. This adipose-derivedstem cell condensation may enhance the fatgraft take26 and prevent long-term atrophy oftransplanted adipose by working as tissue-spe-cific progenitors.25,26

Excessive centrifugation can destroy adipo-cytes and adipose-derived stem cells. Centrifuga-tion, however, plays a beneficial role in concen-trating adipocytes, extracellular matrix, andadipose-derived stem cells, and in partially exclud-ing red blood cells. Extracellular matrix shouldmaintain its volume after transplantation, at leastin the short term, and exclusion of red blood cellsfrom graft materials may contribute to a bettersurvival rate of transplanted adipose.

The oil portion increased only after centrifu-gation at 400 g. Red blood cells were shifted at400 g. The adipose-derived stem cell-to-adipocyteratio, which may influence long-term atrophy ofgrafted adipose, was suggested to be relatively im-proved by centrifugation, but adipose-derivedstem cells were damaged at 3000 g. Graft takes ofcentrifuged adipose were best at 3000 g. Consid-ered together, these data lead us tentatively torecommend 1200 g as an optimized centrifugalforce among the tested centrifugal forces for ob-taining good short- and long-term results in adi-

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pose transplantation. Although our conclusionthat selective centrifugation with relatively highcentrifugal force may be beneficial for graft sur-vival contradicts the views or recommendations ofrecent authors,7,9,12 it is similar to the 1286 g rec-ommended by Coleman based on abundant clinicalexperience.13 Further investigation is necessary toelucidate the net efficacy of centrifugation on graftsurvival in clinical settings because the gross takeof transplanted tissue will be clinically influencedby various factors associated with harvesting, pro-cessing, and transplanting adipose tissues.

Kotaro Yoshimura, M.D.Department of Plastic Surgery

University of Tokyo School of Medicine7-3-1 Hongo, Bunkyo-Ku

Tokyo 113-8655, Japanyoshimura-pla@h.u-tokyo.ac.jp

ACKNOWLEDGMENTThis study was supported by grants-in-aid (con-

tract no. B2-16390507) from the Japanese Ministry ofEducation, Culture, Sports, Science, and TechnologyContract.

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24. Ramon, Y., Shoshani, O., Peled, I. J., et al. Enhancing the takeof injected adipose tissue by a simple method for concen-trating fat cells. Plast. Reconstr. Surg. 115: 197, 2005.

25. Masuda, T., Furue, M., and Matsuda, T. Novel strategy for softtissue augmentation based on transplantation of fragmentedomentum and preadipocytes. Tissue Eng. 10: 1672, 2004.

26. Matsumoto, D., Sato, K., Gonda, K., et al. Cell-assisted lipo-transfer (CAL): Supportive use of human adipose-derivedcells for soft tissue augmentation with lipoinjection. TissueEng. 12: 3375, 2006.

27. Carpaneda, C. A., and Ribeiro, M. T. Study of the histologicalterations and viability of the adipose graft in humans. Aes-thetic Plast. Surg. 17: 43, 1993.

28. Kononas, T. C., Bucky, L. P., Hurley, C., and May, J. W., Jr.The fate of suctioned and surgically removed fat after reim-plantation for soft-tissue augmentation: A volumetric andhistologic study in the rabbit. Plast. Reconstr. Surg. 91: 763,1993.

29. Fagrell, D., Enestrom, S., Berggren, A., and Kniola, B. Fatcylinder transplantation: An experimental comparative studyof three different kinds of fat transplants. Plast. Reconstr. Surg.98: 90, 1996.

Volume 121, Number 3 • Influence of Centrifugation on Cells

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DISCUSSION

Influences of Centrifugation on Cells and Tissues inLiposuction Aspirates: Optimized Centrifugation forLipotransfer and Cell Isolation

Charles L. Puckett, M.D.Steven L. Henry, M.D.

Columbia, Mo.

The authors present a sophisticated and some-what complex analysis of the role of centrifuga-

tion in fat grafting. This study addresses a numberof aspects regarding the value of centrifugationand probably needs to be read more than oncefor one to extract and evaluate all of the infor-mation. The methods used are sound and thediscussion is particularly germane to the read-er’s interpretation.

As with any good research, for each answeredquestion several additional ones are raised, butthis study does provide several interesting answers.Optimizing fat transfer has been an ongoing in-terest of ours, and clearly this article sheds addi-tional light on the subject. The authors have an-alyzed cell viability after centrifugation at a varietyof g forces. These data have been compared withours and with those of other contributors.1–3 Re-cent interest in the role of adipose-derived stemcells and their potentially positive effects on fatgraft take are explored and some important pointsare made. Since our work was published and pre-sented, the interest in adipose-derived stem cellshas clearly increased. This has been fostered by thetheoretical potential of adipose-derived stem cellsto enhance neoangiogenesis and, by inference,also enhance adipocyte viability and graft take.

The authors used fat from female liposuctionpatients and indicated that the sources were ab-dominal and thigh fatty deposits. No subsequentanalysis as to any differences in these sources wasmade and therefore no information can begleaned in terms of site designation and its po-tential role in fat graft survival.

Beyond the identification of viable cells, theauthors have pursued short-term cell culture sur-vival and proliferation and longer term survival intransplantation to the athymic nude mousemodel, assessed at 4 weeks. The authors concludethat although excessive centrifugation can destroy

adipocytes and adipose-derived stem cells, appro-priate centrifugation (lower g forces) increasedthe population of not only viable adipocytes butalso adipose-derived stem cells, and resulted inenhanced early graft take. They have settled on anoptimum centrifugation of 1200 g.

The authors also paid attention to the pres-ence of red blood cells in the concentrated adi-pocyte portion of their specimens. They make thetheoretical point that the presence of red bloodcells may negatively affect cell take and viability, astheir presence may stimulate macrophage activitythat could destroy fat cells. They report the inter-esting finding that centrifugation did not decreasethe number of adipose-derived stem cells but didresult in the removal of some red blood cells,presumably into the fluid layer. They specificallynote that the short-term survival rate of aspiratedadipose graft (per volumetric unit after centrifu-gation) increased with centrifugation. They con-clude that selective use of centrifugation has prob-able value in enhancing the viable adipocytepopulation, with no diminution in the number ofadipose-derived stem cells. They mention, refer-encing one of their earlier studies, that the pres-ence of reduced numbers of adipose-derived stemcells in aspirated fat grafts compared with wholefat transplants may be the explanation for therelatively lower survival rate of aspirated fat aftertransplantation.

Our personal conclusions from this studywould be in concert. Appropriate centrifugation(50 to 1200 g) can provide a more generous con-centration of viable fat cells without sacrifice ofadipose-derived stem cells while reducing the con-centration of potentially detrimental red bloodcells. The authors made no mention of the sam-pling area of the adipose layer, but our work

From the Division of Plastic Surgery, University of Missouri.Received for publication September 29, 2006.Copyright ©2008 by the American Society of Plastic Surgeons

DOI: 10.1097/01.prs.0000299385.55282.6f

Disclosure: Neither author has any financial in-terests in any of the products, drugs, or devicesmentioned in this article.

www.PRSJournal.com1042

clearly demonstrated a higher concentration ofviable adipocytes in the lowest segment of the fatlayer.2 Of parallel interest, Rohrich et al.4 showedno increase in adipocyte viability after centrifuga-tion at 500 g when compared with uncentrifugedspecimens. This would be countered by their (Roh-rich et al.) lack of indication of the layer fromwhere the sample was taken—that is, top, middle,or lower layer—and the use of higher g force (500g) in contrast to our recommendation of 50 g.

Another point made by the authors might ben-efit from additional interpretation. They observedgood cell viability even with higher g centrifuga-tion (4200 g). Our interpretation of this observa-tion would be that although centrifugation mayresult in more lysis of cells, it does not appear tocompromise the viability of the remaining intactcells in the aliquot of the centrifuged specimen.

The authors make the subtle point that cen-trifugation may not be appropriate in patients withimpoverished sources of transplantable fat cells.However, this is a problem rarely encountered inAmericans.

We believe that the authors have made theirpoints well and have analyzed these data accu-rately and in detail. We also believe that the ap-preciation of the valuable role of adipose-derivedstem cells in graft survival is at play here. It con-tinues to be apparent to us that appropriate cen-

trifugation, which we interpret to be lower g forcesand for short periods of time, can contribute toimproved fat graft transfer success and ultimatelyto its reliable use as the best (potentially perma-nent) method of soft-tissue augmentation. We ap-plaud the authors’ efforts to further define therole of centrifugation in aspirated fat grafts andfeel that there are several valuable “take-home”messages in this article.

Charles L. Puckett, M.D.Division of Plastic Surgery

University of MissouriOne Hospital Drive, M349

Columbia, Mo. 65212puckettc@health.missouri.edu

REFERENCES1. Boschert, M. T., Beckert, B. W., Puckett, C. L., and Concan-

non, M. J. Analysis of lipocyte viability after liposuction. Plast.Reconstr. Surg. 109: 761, 2002.

2. Puckett, C. L., and Beckert, B. W. In search of improved fattransfer viability: A quantitative analysis of the role of centrif-ugation and harvest site (Discussion). Plast. Reconstr. Surg. 113:396, 2004.

3. Butterwick, K. J. Lipoaugmentation for aging hands: A com-parison of the longevity and aesthetic results of centrifugedversus noncentrifuged fat. Dermatol. Surg. 28: 987, 2002.

4. Rohrich, R. J., Sorokin, E. S., and Brown, S. A. In search ofimproved fat transfer viability: A quantitative analysis of therole of centrifugation and harvest site. Plast. Reconstr. Surg.113: 391, 2004.

PRS Mission StatementThe goal of Plastic and Reconstructive Surgery� is to inform readers about significant developments in all areasrelated to reconstructive and cosmetic surgery. Significant papers on any aspect of plastic surgery—originalclinical or laboratory research, operative procedures, comprehensive reviews, cosmetic surgery—as well asselected ideas and innovations, letters, case reports, and announcements of educational courses, meetings,and symposia are invited for publication.

Volume 121, Number 3 • Discussion

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