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Fine Tuning Lipoaspirate Viability forFat Grafting J. Lauren Crawford, M.D.
Background: The efficient harvest of abundant viable adipocytes for grafting is
Bradley A. Hubbard, M.D.
of considerable interest. Hand aspiration, low-g-force, short-duration centrifu- Stephen H. Colbert, M.D.
gation, and harvest of the lower sublayer of fat centrifugate maximize viable Charles L. Puckett, M.D.
adiopocytes, but this process is cumbersome with conventional equipment. The Columbia, Mo. Lipose Corporation (Greenwich, Conn.) has produced special syringes, filters,
and a low-g-force centrifuge (Viafill system) to facilitate this process. The adi-
pocyte viability using this system is presented.
Methods: Six women underwent fat graft harvest using the Viafill system from
the lower hips (n ⫽ 6) and/or upper hips (n ⫽ 3). After centrifugation for 2
minutes at 50 g, the lower, middle, and top sublayers of the adipose layer were
analyzed for viable adipocyte counts using trypan blue vital staining. Additional
samples from standard power-assisted liposuction were obtained and analyzed
similarly.
Results: The mean difference in square-root transformation of cell counts
between the bottom sublayer of centrifuged fat and the middle sublayer was 0.95
(95 percent CI, 0.61 to 1.3), and the difference between the middle and top
sublayers was 0.67 (CI, 0.50 to 0.84). Thus, the bottom sublayer had approxi-
mately 2.5 to 3 times more cells than the top sublayer. The difference between
the hand aspirate samples and the power-assisted liposuction samples was sig-
nificant (1.62; CI, 1.35 to 1.90).
Conclusions: This study reconfirms the authors' early findings that atraumatic
harvest of lipoaspirate yields high cell counts and that adipocyte density is
greatest at the lowest sublayer of centrifuged fat. The Viafill system provides a
more efficient and user-friendly system for fat grafting while maintaining cell
counts similar to the authors' technique using conventional equipment.
Reconstr. Surg. 126: 1342, 2010.) Fathasthepotentialtobetheidealsoft-tissue centrations exceeding those of bone marrow.8,9
filler because it is abundant, easily accessible, The regenerative qualities of lipoaspirate stem inexpensive, and host compatible, and be- cells have already found clinical applications by cause it can be harvested repeatedly. Successful reversing the damage of radiation injury6,7 and as and long-term results from free fat grafting used a source of stem cells in tissue engineering.10 The in soft-tissue augmentation have long been a goal applications of adipose progenitor cells will un- for plastic surgeons. A large array of applications doubtedly continue to grow in number.
has been reported previously: cosmetic enhance- The historical drawback to fat grafting has ment and rejuvenation, body contour improvement, been the unpredictable nature of its outcomes.
and reconstruction of scarred and radiation-dam- The most probable argument for this phenome- aged sites, among others.1–7 The realization thatfacial aging is partially a result of soft-tissue atro-phy has spurred renewed interest.
An exciting development has been the dem- Disclosures: None of the authors holds a financial
onstration that lipoaspirate contains stem cell con- position with the Lipose Corporation. The LiposeCorporation supplied funding for the facility fees From the Division of Plastic Surgery, University of Missouri. and equipment used in the liposuction procedures Received for publication August 17, 2009; accepted April 2, and the use and modification of the equipment discussed. No professional fees were obtained from Copyright 2010 by the American Society of Plastic Surgeons the patients or the Lipose Corporation.

Volume 126, Number 4 • Fine Tuning Lipoaspirate Viability non is Peer's cell survival theory.11 Simply stated, hydrophilic coated disposable cannula (Tulip the number of viable cells grafted correlates with Biomed, San Diego, Calif.) and the 20-ml Viafill sy- the ultimate volume of graft survival. Antiquated ringe. Samples (15 to 20 ml) were harvested from techniques have used traumatic approaches and, each area, and these syringes were then placed in a in retrospect, the poor or inconsistent results are 37°C warm water bath. After the hand-suctioned not surprising. Using the Coleman technique or specimens were taken, standard power-assisted lipo- similar atraumatic approaches, acceptable and re- suction (Accelerator-III; Byron Medical, Inc., Tuc- liable resorption rates have been obtained.12–15 son, Ariz.; MicroAire Surgical Instruments, Char- However, there is a large variability in the methods lottesville, Va.) using 4-mm cannulas was performed used in terms of harvest, processing, grafting at the same site and fat was collected in 1.5-liter method, and recipient location. In addition, there lipoaspirate canisters using atmosphere negative may be differences in the intrinsic quality of the pressure. Lipoaspirate volumes ranged between 600 adipose tissue from person to person or regionally and 1000 ml. The lipoaspirate canisters were main- within the same person. Thus, the published tained within the water bath with the other samples resorption rates vary wildly from 20 to 90 for less than 60 minutes.
The power-assisted liposuction lipoaspirate Because of such variability in techniques and canisters were gently stirred into a homogeneous inconsistencies in resorption rates, the ability to mixture, and one 15- to 20-ml sample was drawn efficiently harvest large quantities of viable fat cells into a Viafill syringe to serve as a control. Each continues to be of great interest. In 2001, we re- Viafill syringe was then placed into a filtered Viafill ported a method for obtaining the maximum centrifuge tube (Figs. 1 through 3) and placed number of viable fat cells to be used for grafting.19 within the specially fitted 50-g centrifuge (Lipose).
Since that study, our approach has been that of All tubes were spun at 50 g for 2 minutes. The hand aspiration; low-g-force, short-duration cen- filtered Viafill centrifuge tube allows the aqueous trifugation; and use of the lower centrifuged sub- layer and erythrocytes to flow out of the syringe tip layer of fat cells for our fat grafting cases. However, during the centrifugation process, leaving the ad- the process has been cumbersome and time con- ipose layer at the bottom, near the tip, and the oil suming using commercially available equipment.
layer at the top (Fig. 4). One-milliliter samples of Since that time, the Lipose Corporation (Green- adipose were taken from the bottom, middle, and wich, Conn.) has expressed an interest in produc- top sublayers of the centrifuged fat layer.
ing a line of equipment to facilitate this process.
Each of these samples was then processed The purpose of the present study is to investigate with 1 mg/ml of collagenase (collagenase type the technology of the Viafill (Lipose) fat graftharvest system and to assess lipocyte viability ascompared with our previous results.
PATIENTS AND METHODS
Six female subjects were enrolled in the study.
University of Missouri Health Sciences Institu-tional Review Board approval was obtained beforepatient enrollment. The average age of the sub-jects was 36 years (range, 24 to 47 years). None ofthe patients had significant medical histories andnone had undergone previous liposuction or bodycontouring procedures. Half of the subjects hadliposuction on bilateral upper and lower hips, andhalf had liposuction on bilateral lower hips alone,for a total of 18 body areas. Hand lipoaspirate andpower-assisted lipoaspirate specimens were ob-tained from each body area.
Fig. 1. The syringe and filter of the Viafill (Lipose) system pro-
For each patient, our standard tumescent fluid totype are shown. The same syringe used for harvest is placed (1 ml of 1:1000 epinephrine and 50 ml of 1% lido- within the filtered centrifuge tube and also can be used for caine per liter of lactated Ringer's solution) was in- distribution of the fat to smaller syringes for injection. After jected into the harvest site. Fat was aspirated by hand hand suctioning, the syringe is placed into the filter for using the Viafill system with a 2.10-mm ⫻ 12-cm

Plastic and Reconstructive Surgery • October 2010 Fig. 2. A syringe from the Viafill system with a plunger handle in
Fig. 4. Viafill syringe and centrifuge tube after centrifugation.
place and a syringe with the plunger unlocked and removed.
Note that the heavier aqueous layer and erythrocytes (arrow)have passed out of the syringe through the filter and the adiposeand oil layers remain.
Statistical analysis was completed with assis- tance from the Division of Biostatistics, Universityof Missouri. All statistical analysis was performedusing SAS v9 (SAS Institute, Inc., Cary, N.C.). Sub-group analysis was performed based on body area(upper or lower hips), laterality (right or left), andthe sublayer from within the centrifuged adiposelayer from which the specimen was taken (bottom,middle, or top).
The cell counts were skewed to the high end (i.e., few values were close to 0). We thereforefound it useful to analyze the data using the Fig. 3. The centrifuge prototype of the Viafill (Lipose) system
square-root transformation of the cell counts. Dif- prototype is shown with two filters and syringes in place.
ferences between square-root transformationswere then averaged. Because measurements weretaken on the same patient, there are dependencies 1, filtered; Worthington, Lakewood, N.J.) in a in the data attributable to multiple observations warm water bath. After 1 hour of collagenase on the same subject. Consequently, the patient was digestion, the specimens were diluted 1:1 with included in the analysis as a random effect and a trypan blue vital stain 0.4% solution (product mixed model analysis of variance was carried out.
T8154; Sigma, St. Louis, Mo.). A 100-␮l samplewas taken from each digested and stained prep-aration and the numbers of viable fat cells were counted with a hemocytometer under 400⫻ mag- Table 1 lists the mean and median cell counts nification. Five different squares from the hemo- for each centrifugate adipose sublayer from each cytometer grid were recorded three separate times body area and by harvest method. One body area for each 100-␮l sample. After the trypan blue di- in one patient had abnormally low cell counts; lution was factored in, the number of viable fat otherwise, the counts were distributed normally.
cells from each sample was determined. This fat The outlier was not found to adjust conclusions cell quantification method has been used previ- when left out or when kept in and was considered ously by our laboratory and has been described by insignificant. Figure 5 shows a box plot of the Moore et al.19,20 square root transformation of the cell counts from

Volume 126, Number 4 • Fine Tuning Lipoaspirate Viability Table 1. Lipocyte Cell Counts*
PAL, power-assisted liposuction.
*Lipocyte cell counts (in millions of cells per milliliter) separated by centrifuge sublayer (bottom, middle, and top), location (upper or lowerhip), and method of harvest (PAL or hand aspirate).
each centrifugate sublayer of the hand and power- assisted liposuction aspirates.
Debate continues in the literature as to the In all body areas, the hand aspirate samples best method of fat graft harvest. Despite the ab- had significantly higher mean cell counts than sence of an undisputed best method for long-term power-assisted liposuction samples (p ⬍ 0.0001).
volume restoration, a consensus seems to have As expected, the laterality, right compared with developed. A recent survey has shown that a total left, did not have a significant effect (p ⫽ 0.90).
of 70 percent of plastic surgeons performing fat Consistent with our previous work, the cell counts grafts use either the Coleman technique or an- at the bottom of the centrifugate fat sublayer were other atraumatic syringe aspiration technique, 54 greater than those of the middle sublayer, which percent and 16 percent, respectively.21 In addi- were greater than those of the top sublayer (p ⬍ tion, nearly half of these surgeons (49 percent) 0.0001). Somewhat surprisingly, the upper hip use centrifugation as opposed to other techniques area had significantly greater cell counts com- of fat preparation such as washing or gravity.21 As pared with the lower hip (p ⫽ 0.0003).
mentioned, there are many conflicting publica- Considering all sublayers and all body areas by tions comparing the different techniques for fat square-root analysis, the hand aspirate samples graft harvest. It is beyond the scope of this article had greater numbers of viable cells than power- to revisit all of these studies. However, our results assisted liposuction samples (mean difference in do reinforce the current consensus view, using square roots, 1.62; 95 percent CI, 1.35 to 1.90).
centrifugation and atraumatic harvest.
Similarly, the mean of the square roots from the Disagreement continues to surround the bottom sublayer was greater than that of the mid- centrifugation of the lipoaspirate before graft- dle sublayer (0.95; 95 percent CI, 0.61 to 1.3), and ing. In 2008, Kurita et al. found a decrease in the mean of the middle sublayer was greater than potentially detrimental erythrocytes, but found that of the top sublayer (0.67; 95 percent CI, 0.50 no difference in viable adipose cells after cen- to 0.84). By syllogism, the bottom sublayer square- trifugation at 1200 g.22 However, the authors did root transformation was also greater than that of not make a distinction between the sublayers of the top sublayer (mean difference, 1.62; 95 percent centrifuged fat. The filter system in the Lipose CI, 1.45 to 1.79). The mean difference in square-root centrifugation tube allows erythrocytes to be re- transformations from the upper hip area compared moved with the aqueous layer, thus addressing the with the lower hip area was statistically significant at potential erythrocyte problem. This makes avail- 1.63 (95 percent CI, 1.47 to 1.80).
able the direct transfer of the highest quantity of

Plastic and Reconstructive Surgery • October 2010 Fig. 5. Box plot of the square-root transformation of adipose cell counts
for hand aspirate and power-assisted liposuction (PAL). The area of har-
vest was ignored; therefore, there are 18 samples (12 lower and six upper
hip) represented by each box plot. Both groups were separated into the
bottom, middle, and top centrifuged samples. Box demarcates the first
and third quartiles; ⫹, mean; –, median; asterisks, outliers.
viable fat cells from the capture syringe to an in- head comparison would be flawed because the jection syringe for grafting. Our current study re- power-assisted liposuction samples represent the confirms our previous work (Boschert et al.) and average cell counts from a larger volume of aspi- that of Butterwick that low-force, short-duration rate. Regardless, it was not our intent to compare centrifugation does increase viable adipose cell power-assisted liposuction to Lipose syringes per counts.19,23 In addition, we have reconfirmed our se, but merely to show whether the Lipose system finding that viable cell density is greater at lower is efficient and user-friendly while maintaining sublayers of centrifuged fat.
cell counts similar to our previous work. Despite The purpose of the study was not to perform the introduction of potential confounding vari- a head-to-head comparison of harvest techniques; ables, order of harvest and volume of harvest, the thus, no "true" control group was created. We are present study found that atraumatic hand aspira- convinced from our previous work and that of tion provides 150 percent greater viable fat cells others that atraumatic hand aspiration is superior relative to conventional liposuction.
to other methods (e.g., power-assisted liposuction It was not the intention of our protocol to or conventional liposuction).19,24 Nevertheless, in investigate the superiority of donor sites for fat this study, atraumatic hand aspiration provided a grafting. Recently, such a study has been per- greater number of viable fat cells relative to con- formed. Rohrich et al. compared thigh, knee, ventional liposuction. This finding is limited by flank, and abdominal donor sites.25 They found no our harvesting of the hand-aspirated samples first difference in viable adipocytes obtained from in all body areas and the larger volume of power- these areas. Surprisingly, our results seem to con- assisted liposuction aspirate. Although this has not tradict this finding, and other studies would sug- been studied previously, the order of harvest could gest a potential difference among donor sites.
potentially have an effect on cell numbers. The Padoin et al. examined the effect of donor site on first pass of liposuction might obtain higher fat cell quantity of mesenchymal stem cells in fat aspirates counts because of less trauma or could obtain and found significant differences between some lower numbers because of the dilutional effect of regions.26 In our patients, the upper hip area adi- the tumescent fluid. This would be an interesting pocyte cell counts were significantly higher than concept for future study. In addition, a head-to- those of the lower hip. It is important to note our

Volume 126, Number 4 • Fine Tuning Lipoaspirate Viability small patient sample size and that both areas grafting procedures, these clinical answers are on were harvested in only half of the patients. Al- the horizon.
though statistical significance was reached, withthis small sample we feel that the potential dif-ferences in donor sites should be the focus of This study reconfirms our early findings that As mentioned previously, we have been dissatis- atraumatic harvest of fat grafts continues to yield fied with the commercially available equipment for high viable cell counts and that the adipose cell fat graft harvest, centrifugation, and grafting. The density is greatest at the lowest level of centrifuged Viafill system relieves many of these complaints. A fat. The Viafill system provides an efficient and single 20-ml syringe is connected by means of a Luer user-friendly system for fat graft harvest while lock to a harvesting cannula of choice. After the maintaining cell counts similar to those of our sample is acquired, the syringe is then secured into technique using conventional equipment.
a filtered centrifuge holder and the plunger handle Charles L. Puckett, M.D.
is removed. During centrifugation, the aqueous Division of Plastic Surgery layer and erythrocytes are filtered from the syringe University of Missouri into the centrifuge holder. With the plunger handle One Hospital Drive reattached, the same syringe can be connected to Columbia, Mo. 65212 smaller syringes (connectors provided) that canthen be used for grafting.
When comparing the results of this work to our previous studies, the Viafill system did yield 1. Coleman SR. Facial recontouring with lipostructure. Clin greater cell counts, 20 compared with 8 million Plast Surg. 1997;24:347–367.
cells per milliliter. Obviously, such a comparison 2. Coleman SR. Hand rejuvenation with structural fat grafting.
is scientifically flawed and therefore statistical Plast Reconstr Surg. 2002;110:1731–1744.
3. Gatti JE. Permanent lip augmentation with serial fat grafting.
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tom fat sublayer to contain more viable cells than 5. Roberts TL III, Weinfeld AB, Bruner TW, Nguyen K. "Uni- versal" and ethnic ideals of beautiful buttocks are best ob- the middle, and the middle to contain more than tained by autologous micro fat grafting and liposuction. Clin the top fat sublayer by approximately 150 percent Plast Surg. 2006;33:371–394.
each. Both studies also found that the bottom fat 6. Rigotti G, Marchi A, Galie M, et al. Clinical treatment of sublayer has 2.5 to 3 times more viable cells relative radiotherapy tissue damage by lipoaspirate transplant: A to the top fat sublayer. Thus, a gradient exists, with healing process mediated by adipose-derived adult stem cells.
Plast Reconstr Surg. 2007;119:1409–1422.
the density of viable cells increasing from top to 7. Phulpin B, Gangloff P, Tran N, Bravetti P, Merlin JL, Dolivet bottom of the centrifugate fat layer.
G. Rehabilitation of irradiated head and neck tissues by This study is limited by the lack of confirma- autologous fat transplantation. Plast Reconstr Surg. 2009;123: tion of viable cell counts obtained with trypan blue 8. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a staining with other methods. For example, 3-(4,5- source of multipotent stem cells. Mol Biol Cell. 2002;13:4279– dimethylthiazol-2-yl)-2,5-diphenyl tetra sodium bromide or glycerol-3-phophatase dehydrogenase 9. Strem BM, Hicok KC, Zhu M, et al. Multipotential differen- assays would have to be performed for the absolute tiation of adipose tissue-derived stem cells. Keio J Med. 2005; viable cell counts to be comparable to other works.
10. Mischen BT, Follmar KE, Moyer KE, et al. Metabolic and A viable cell count is not the only factor pre- functional characterization of human adipose-derived stem dicting the success of fat grafting techniques.
cells in tissue engineering. Plast Reconstr Surg. 2008;122:725– Peer's cell survival theory has scientific merit but certainly does not account for all the variables 11. Peer LA. Cell survival theory versus replacement theory. Plast Reconstr Surg. 1955;16:161–168.
involved.11 The popularity of cell counts as the 12. Billings E Jr, May JW. Historical review and present status of endpoint for fat graft research is certainly related free fat graft autotransplantation in plastic and reconstruc- to the ease by which they can be obtained. Adipose tive surgery. Plast Reconstr Surg. 1989;83:368–381.
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16. Nguyen A, Pasyk KA, Bouvier TN, Hassett CA, Argenta LC.
22. Kurita M, Matsumoto D, Shigeura T, et al. Influences of Comparative study of survival of autologous adipose tissue centrifugation on cells and tissues in liposuction aspirates: taken and transplanted by different techniques. Plast Reconstr Optimized centrifugation for lipotransfer and cell isolation.
Plast Reconstr Surg. 2008;121:1033–1041.
17. Boyce RG, Nuss DW, Kluka EA. The use of autogenous fat, 23. Butterwick KJ. Lipoaugmentation for aging hands: A com- fascia, and nonvascularized muscle grafts in the head and parison of the longevity and aesthetic results of centrifuged neck. Otolaryngol Clin North Am. 1994;27:39–68.
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18. Mattsudo PK, Toledo LS. Experience of injected fat grafting.
24. Pu LL, Coleman SR, Cui X, Ferguson RE Jr, Vasconez HC.
Aesthetic Plast Surg. 1988;12:35–38.
Autologous fat grafts harvested and refined by the Coleman 19. Boschert MT, Beckert BW, Puckett CL, Concannon MJ. Anal- technique: A comparative study. Plast Reconstr Surg. 2008; ysis of lipocyte viability after liposuction. Plast Reconstr Surg. 25. Rohrich RJ, Sorokin ES, Brown SA. In search of improved fat 20. Moore JH Jr, Kolaczynski JW, Morales LM, et al. Viability of fat transfer viability: A quantitative analysis of the role of cen- obtained by syringe suction lipectomy: Effects of local anesthe- trifugation and harvest site. Plast Reconstr Surg. 2004;113:391– sia with lidocaine. Aesthetic Plast Surg. 1995;19:335–339.
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