Numerical measurement of viable and non-viable adipocytes and other cellularcomponents in aspirated fat tissue
Authors:
Hirotaka Suga,1 Daisuke Matsumoto,1 Keita Inoue,1 Tomokuni Shigeura,2Hitomi Eto,1 Noriyuki Aoi,1 Harunosuke Kato,1 Hiroaki Abe,1 and Kotaro
Yoshimura1
Affiliations:1

Department of Plastic Surgery, University of Tokyo School of Medicine,

7-3-1, Hongo, Bunkyo-Ku, Tokyo 113-8655, Japan.
2

Division of Research and Development, Biomaster Inc.

Yokohama Excellent III Bldg. 8F, 3-35, Minami-nakadori,Naka-ku, Yokohama, Kanagawa 231-0006, Japan.
Short title: Numerical measurement of viable adipocytes1Correspondence:
Kotaro Yoshimura, M.D.
Department of Plastic Surgery, University of Tokyo School of Medicine,
7-3-1, Hongo, Bunkyo-Ku, Tokyo 113-8655, Japan.
TEL: +81-3-5800-8948
FAX: +81-3-5800-9186
e-mail: yoshimura-pla@h.u-tokyo.ac.jp

2

Financial Disclosure and Products
The authors do not have any commercial associations or financial disclosures thatmight pose or create a conflict of interest with information presented in thismanuscript.
This study was financially supported by the following grant.
Grant: Contract grant sponsor: Grants-in-Aid by Japanese Ministry of Education,
Culture, Sports, Science, and Technology (MEXT); contract grant number: B219390452.

3

Abstract
Background: A reliable method to assay viability and number of adipocytes andother cellular components in adipose tissue remains to be established.
Methods: We assessed cell viability and number obtained from 1 g suctioned adipose
tissue and respective layers (the top, middle, and bottom layers) before and afterdigestion and centrifugation, using cell staining with Hoechst 33342 and propidium
iodide (PI) and the XTT and glycerol-3-phosphate dehydrogenase (GPDH) assays(n=10). The correlation between the number of prepared cells (adipocytes, adipose
stromal cells [ASCs], and white blood cells [WBCs]) and the resulting values fromthe XTT and GPDH assays was also examined (n=5). The cell composition of thestromal vascular fraction isolated from the same adipose tissue was determined bymulticolor flow cytometry (n=5).
Results: Hoechst 33342 and PI staining allowed distinguishing of viable adipocytes
from lipid droplets, dead adipocytes, and cells other than adipocytes. We obtained 6.9
× 105 non-ruptured adipocytes from 1 g suctioned adipose tissue; 30% of the original
adipocytes appeared to have been ruptured. Both the XTT and GPDH assays provided
good correlations between the number of viable adipocytes and resulting values, but
only the GPDH assay was strictly specific for adipocytes. The ratio of ASCs toadipocytes was found to be much larger than previously described.
Conclusions: Single use or a combination of the viability assays used in this study
can appropriately determine the number of adipocytes and other cells, although it
remains difficult to assess original cells directly without tissue dissociation.

4

Introduction
Adipose tissue has been widely studied in basic research, especially in relation toobesity and metabolic diseases such as diabetes.1 In the clinical setting, autologous fat
grafting has long been an effective technique for soft tissue augmentation.
Furthermore, multipotent stem cells have been identified in adipose tissue, resulting in
a new field in adipose biology.2-7 In spite of the intense interest in adipose tissue,
however, its cellular composition—either excised or aspirated—remains unknown.
Although cell counting with cell staining,8-13 colorimetric assays (the XTT and/or
MTT assays),9,14-17 and the glycerol-3-phosphate dehydrogenase (GPDH) assay9,10,18
have been used in previous studies to analyze cell number and viability in adipose
tissue, a reliable method has not yet been established. Specifically, the number ofmature adipocytes is difficult to measure mainly because of their fragility and thepresence of adjacent progenitors.
Trypan blue staining is a well established procedure for detecting viable cells.
In adipocytes, however, this technique is difficult because of the scant cytoplasm of
mature adipocytes.12 Other staining methods should be used to distinguish viableadipocytes, and differentiating adipocytes and pure lipid droplets is also necessary.
A colorimetric assay uses tetrazolium salt (XTT and/or MTT), which can be
reduced by active mitochondrial dehydrogenase in viable cells, yielding a highlycolored formazan product.19 This assay provides a good correlation between thenumber of viable cells and the resulting formazan absorbance values, but it is not
specific for adipocytes. In addition, because adipose tissue contains not only
adipocytes but also blood-derived cells, endothelial cells, and adipose-derived stem
cells (ASCs), these other cells could influence the total absorbance values.

5In the GPDH assay, GPDH activity is determined by measuring the decreasein NADH concentration.18 This assay has the advantage of adipocyte specificity, but
extracellular GPDH from damaged adipocytes is measured together with intracellular
GPDH from viable adipocytes, affecting the accuracy of viability analysis.
Furthermore, a correlation between the number of viable adipocytes and the resulting
values of GPDH activity has not been reported.
Thus, how many adipocytes and other cellular components exist in the adipose
tissue remains unclear.8,10,13 In this study, to seek a reliable method for evaluating
adipose tissue, we assessed the viability and number of cellular components obtained
before and after collagenase digestion of suctioned adipose tissue using cell staining,
the XTT assay, and the GPDH assay.

6Materials and methods
Tissue sampling and processing
After obtaining informed consent using an institutional review board-approved
protocol, we acquired adipose tissue from 10 healthy female donors undergoing
liposuction of the abdomen or thighs. We used a cannula with a 2.5-mm innerdiameter and a conventional liposuction machine. From each donor, five sample tubes
were prepared. We carefully collected the most homogeneous samples possible,
making an effort not to include debris or clumps. The sample was divided byweighing 1 g for each specimen. One sample of the five was directly subjected to the
XTT assay, and another sample to the GPDH assay. The other three samples were
processed by digestion with collagenase and centrifugation; each sample was mixed
with 2 ml of 0.075% collagenase (Wako Pure Chemical Industries, Osaka, Japan) in
phosphate-buffered saline (PBS), and incubated at 37 °C for 30 minutes. The
digestion was terminated with 2 ml of PBS containing 10% fetal bovine serum (FBS),
and the digested adipose tissue was centrifuged at 430 ×g for 5 minutes. Aftercentrifugation, the top, middle, and bottom layers were respectively assessed by acell-staining morphometric assay, the XTT assay, and the GPDH assay (Fig. 1). To
avoid waiting time, we performed these assays simultaneously. We also performed
each assay as immediately as possible.

Cell-staining morphometric assay
Respective layers after digestion and centrifugation were resuspended in PBS
to a total of 5 ml. First, to distinguish adipocytes from floating lipid droplets released
from broken adipocytes, cells were stained with Nile Red (AdipoRedTM) (Cambrex,
Walkersville, MD) and Hoechst 33342 (Dojindo, Kumamoto, Japan). Cells stained7

with both Nile Red and Hoechst 33342 were defined as adipocytes, while cells stained
only with Hoechst 33342 but negative for Nile Red were defined as non-adipocytes.
Particles stained only with Nile Red were defined as floating lipid droplets.
Next, to distinguish viable cells from dead cells, cells were stained with
Hoechst 33342 and propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO). This
staining distinguished four types of nucleated cells: viable adipocytes (positive for
Hoechst 33342, negative for PI, and including lipid); dead adipocytes (positive for PI
and including lipid); viable cells other than adipocytes (positive for Hoechst 33342,
negative for PI, and not including lipid); and dead cells other than adipocytes(positive for PI and not including lipid). Erythrocytes, which are not stained with thenuclear staining, were not counted in this study. Samples were placed on ahematocytometer under a microscope. At least three fields (×100) were randomly
photographed and the number of cells in each group counted. Then, the total cellnumber obtained from 1 g suctioned adipose tissue was calculated.

The XTT assay
A Cell Proliferation Kit II (XTT) (Roche Diagnostics, Indianapolis, IN) wasused according to the manufacturer’s instructions. In brief, 1 g of each sample wassuspended in PBS containing 10% FBS to a total of 5 ml. An XTT-labeling reagent
and electron-coupling reagent were mixed at a ratio of 50:1, and 2.5 ml of this reagent
mixture was added to the sample, followed by incubation in a 6-well plate for 6 hours
at 37 °C. After the incubation, 150 µl of the solution was transferred to a 96-well plate,
and the absorbance was measured at a test wavelength of 450 nm and a reference
wavelength of 650 nm. The baseline absorbance of the negative control, PBS
containing 10% FBS and no cells, was subtracted from each result.
8

To examine any correlation between the number of viable cells and theresulting values from the XTT assay for each cell type, several concentrations ofadipocytes, white blood cells (WBCs), and ASCs were prepared for assessment by the
XTT assay. Adipocytes were separated from adipose tissue by digestion andcentrifugation as described above, and viable adipocytes in the top layer were used.
WBCs were obtained from the peripheral blood of five healthy donors. ASCs wereseparated from adipose tissue as described previously6 and cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) containing 10% FBS. ASCs at passage 1 or 2
were used. For each cell type, samples of 1, 2, 5, and 10 × 104 viable cells/ml wereprepared; the cell number was determined by cell staining with Hoechst 33342 and PI
for adipocytes, and it was determined by cell counter (NucleoCounter, Chemometec,
Allerod, Denmark) for WBCs and ASCs.

The GPDH assay
A GPDH Assay Kit (Cell Garage, Tokyo, Japan) was used according to the
manufacturer’s instructions. In brief, 1 g sample (adipose tissue or respective layers
after digestion and centrifugation) was mixed with 0.25 M sucrose solution to a totalof 5 ml and homogenized. The mixture was then centrifuged at 430 ×g for 5 minutes.
A 1-ml aliquot of the aqueous layer was taken and again centrifuged at 17700 ×g for 5
minutes. The supernatant after the second centrifugation was diluted 10 times with an
enzyme-extracting reagent, and the optical absorption was measured at 340 nm for 10
minutes on a 96-well plate after addition of twice the volume of a substrate reagent.
GPDH activity was calculated based on the following formula: GPDH activity (U/ml)= ΔOD × 0.482 × 10 (ΔOD: change in optical density per minute).

9To examine correlation between the number of viable cells and the resulting
values of GPDH activity in each cell type, we assessed several concentrations ofadipocytes, WBCs, and ASCs using the GPDH assay, following the design used for
the XTT assay.

Flow cytometry
To determine the presence of non-adipocytes, we used multicolor flow cytometricanalysis to examine cells in the bottom layer, the stromal vascular fraction (SVF), forsurface marker expression. The following monoclonal antibodies conjugated tofluorochromes were used: anti-CD31-PE, anti-CD34-PE-Cy7, and anti-CD45-FITC(BD Biosciences, San Jose, CA). Cells were analyzed with a LSR II (BD Biosciences),and cell composition percentages were determined according to surface marker
expression profiles.

Statistical analysis
Data are expressed as mean ± SEM. Comparisons of multiple groups were madeusing one-way ANOVA with Bonferroni’s multiple t-test. Values of P<0.05 wereconsidered statistically significant.

10

Results
Cell staining
After digestion and centrifugation, many pure lipid droplets, not cells, were
included together with cells in the top layer (Fig. 2). The top layer contained 4.4 (±0.66) × 105 viable adipocytes; 2.5 (± 0.28) × 105 dead adipocytes; 1.5 (± 0.23) × 105
viable cells other than adipocytes; and 7.5 (± 0.72) × 105 dead cells other thanadipocytes, which were distinguished by cell staining with Hoechst 33342 and PI (Fig.
3). The middle layer contained no cells. The bottom layer contained 10.0 (± 2.8)× 105
viable cells other than adipocytes and 2.3 (± 0.34) × 105 dead cells other thanadipocytes (Table 1).

The XTT assay
Adipose tissue before digestion resulted in the highest and similar to the sumof three layers after digestion. The top layer produced a much higher value than thatof the bottom layer, although the number of viable cells directly counted by cellstaining was higher in the bottom layer (Fig. 4).
Good correlations between the number of prepared viable cells and the XTTvalues were respectively obtained in adipocytes, WBCs, and ASCs. Adipocytes
showed higher absorbance than WBCs and ASCs, especially at a high concentration,differences that were statistically significant (Fig. 5). Thus, the results suggest that thevalue derived from the XTT assay would not be proportional to the total viable cellnumber when different cell types are mixed.

The GPDH assay

11Adipose tissue before digestion and centrifugation showed the highest value.
After digestion and centrifugation, the value of the top layer, which was regarded as
intracellular GPDH from floating non-ruptured adipocytes, was more than twofoldthat of the middle layer, which was regarded as extracellular GPDH released fromruptured adipocytes. The bottom layer, which contained no adipocytes, showed a low
value (Fig. 6).
The GPDH assay provided a good correlation between the number of preparedviable adipocytes and the resulting values, while WBCs and ASCs showed no GPDH
activity even at a high concentration (Fig. 7).

Flow cytometry
Flow cytometric analysis revealed that the SVF contained CD45-positive cells,corresponding to blood-derived cells at 37 ± 5.2%; ASCs (CD45–, CD31–, CD34+) at
37 ± 4.0%; and endothelial cells (CD45–, CD31+, CD34+) at 15 ± 4.9% (Fig. 8; Table
2).

12

Discussion
In the present study, suctioned fat samples obtained from the abdomen or
thighs were used. Although regional (donor site) difference in adipose cellcomponents may exist, a previous study reported that no statistical differences inadipose tissue viability were demonstrated among abdominal fat, thigh fat, flank fat,or knee fat, based on the XTT assay.14 In our study, no differences in adipose stromal
cell yield were observed between the samples from the abdomen and the thigh (datanot shown).
In staining and counting of adipocytes, distinguishing them from pure lipid
droplets is necessary. In our study, many lipid droplets derived from ruptured
adipocytes were observed together with adipocytes, as shown in Figures 2 and 3.
Lipid droplets the same size as adipocytes (50 to 120 µm) cannot be distinguishedfrom adipocytes without nuclear staining. In previous studies,8,10,13 trypan blue
staining was performed, but it cannot distinguish pure lipid droplets from viableadipocytes because because it does not stain either of them well. The previous
studies8,10,13 reported adipocyte numbers (2.5–90 million/ml) which were much larger
than those in our result (0.7 million/ml), and the difference may arise from theconfusion of lipid droplets with adipocytes. Nuclear staining with Hoechst 33342 only
is not appropriate for a viability assay, because it also stains the nuclei of dead cells.
We combined PI, which stained only the nuclei of nonviable cells, with Hoechst
33342 staining, and with this combination, we distinguished four types of cells: viableadipocytes, dead adipocytes, viable cells other than adipocytes, and dead cells otherthan adipocytes. A combination of fluorescein diacetate and PI may be anotheroption.12

13

We found that many non-adipocyte cells were present in the top layer after
collagenase digestion and centrifugation, although the viable cell ratio was much
lower than that of the bottom layer. Cells, especially adherent cells, may have been
trapped in the floating layer and not released by centrifugation. A previous study
using ceiling culture showed that cells tightly attached to mature adipocytes could
generate fibroblastic cell populations with multiple differentiation potential,20 and thecells were regarded as ASCs. Thus, the viable non-adipocyte cells detected in the top
layer may contain ASCs as well as WBCs, although more than 80% of the viable nonadipocyte cells were dead in the current study.
Among colorimetric assays of cell viability, the XTT and MTT assays havebeen widely used. We first assessed the viability of adipose tissue using both the XTT
and MTT assays, but the MTT assay did not give reproducible results (data notshown) unlike the XTT assay. In the MTT assay, the resulting colored formazan
product is insoluble and requires a dissolving procedure,19 which may impair
reproducibility, especially when using adipose tissue.
We also found that the resulting values of the XTT assay differed among cell
types. This difference is likely the reason for the higher value from the XTT assay inthe top layer compared to the bottom layer; the results from the cell-staining assays
made it clear that the bottom layer contained a much larger number of viable cells.
Adipocytes exhibited much higher absorbance in the XTT assay compared to WBCsand ASCs. This difference may arise because of the increase in mitochondrial
capacity (number of mitochondrion profiles, concentration of numerous mitochondrial
proteins, and oxygen consumption rate) during adipogenesis.21 Some authors havereported in previous studies14,16 that the XTT assay is well suited to assessing adipose
tissue viability; however, it is not specific for adipocytes, and careful interpretation of14

results is needed when contamination by other cell types is present. Our results
demonstrate that the bottom layer, which contains no adipocytes, had a substantial
XTT value. Given this finding, it is possible that in previous studies, vascular stromal
fraction cells such as white blood cells, adipose stromal cells, and vascular endothelial
cells were mistakenly counted as adipocytes.
GPDH is normally intracellular, and extracellular GPDH is considered
proportional to the amount of adipocyte destruction.9,18 Therefore, examining total
GPDH in adipose tissue is not appropriate for viability assessment. To gain a fullunderstanding of the results of previous studies, we would have to confirm whether
the reported GPDH value was intracellular or extracellular or a mixture of both. In ourstudy, the GPDH activity of the top layer was regarded as intracellular GPDH from
non-ruptured adipocytes, although this layer appeared to contain both viable and dead(but not ruptured) adipocytes. These latter adipocytes may die because of the effectsof collagenase, although they did not ruptured and the intracellular contents were notreleased into the middle layer. The GPDH activity of the middle layer indicated thatapproximately 30% of total adipocytes were ruptured. The rate of this extracellular
GPDH can change, depending on adipose tissue sampling methods22 and the digestion
procedure.18 The GPDH assay is superior to the XTT assay because of its strict
specificity for adipocytes, which the current findings support. Although the previous
studies9,10,18 appropriately used the GPDH assay, a control curve using premeasured
adipocytes or a combination of direct cell counting is needed to evaluate the absolute
number of adipocytes.
From 1 g suctioned adipose tissue, we obtained 6.9 × 105 adipocytes(viable:dead = 4.4:2.5) and 2.1 × 106 non-adipocyte cells (viable:dead = 11.5:9.8).
Based on the GPDH data for the top and middle layers, we speculate that 1 g
15

suctioned adipose tissue originally contains approximately 1 million adipocytes (6.9 ×
105 adipocytes in the top layer and approximately 3 × 105 adipocytes in the middle
layer). Although the size of adipocytes may differ according to nutritional status,23
previous studies24,25 reported relatively similar adipocyte sizes, ranging from 50–150µm, with a mean size between 90 and 100 µm regardless of severity of obesity.
Assuming that adipocytes are cubes (106 µm3) or spheres (5.23 × 105 µm3) with aside or diameter of 100 µm, the number of adipocytes in 1 ml adipose tissue would
correspond to 0.95 million or 1.81 million under the condition that adipocytes occupy
95% of adipose tissue volume; thus, the theoretical number of adipocytes in the 1mlof adipose tissue is likely to be between the two numbers. This suggestion is
supported by the results of a previous study23 indicating that adipocyte number per 1 g
adipose tissue is 1.51 ± 0.47 million (mean ± SD). Because the adipocyte number of
non-centrifuged suctioned adipose tissue is likely to be smaller than that of intact
adipose tissue,26 the estimated number in our study (1 million per 1 ml suctioned
adipose tissue) seems to be reasonable.
Multicolor flow cytometry revealed that 37% of non-adipocyte cells in SVF(the bottom layer) are ASCs, a ratio compatible with our previous findings.5
Assuming that the non-adipocyte cell composition in the top layer is similar to that of
the bottom layer, we can speculate that the number of ASCs in 1 g suctioned adipose
tissue is about half that of adipocytes. Considering also that the number of ASCs insuctioned adipose tissue is significantly smaller than in intact adipose tissue,6 the ASC
number in intact adipose tissue may be similar to that of adipocytes. In addition, it
may be much larger than that described in a previous histological study,27 which
reported percentages of adipocytes and stromal cells in the adipose tissue as 89–98%
and 2–10%, respectively. In a histological assessment of the adipose tissue, a single
16

adipocyte 100 µm in size, for example, could appear in 12 serial sections (8 µm thick;
96 µm in all), but each section contains different ASCs; thus, a single adipocyte could
be repeatedly counted by mistake, and a careful evaluation of the specimens isnecessary for counting cell numbers. Therefore, thicker sections, as thick as 200 µm,
combined with an appropriate calculation formula such as that used in a previous
study25 may contribute to avoiding mistakes in cell counting.
In conclusion, single use or a combination of the viability assays employed inthis study can accurately determine the number of viable adipocytes and other cells,
although other assays like the ATP assay28 and glucose transport experiment29 should
be further investigated for applications to adipose tissue. The major limitation of thecurrent study is that the assays are performed only after enzyme digestion andcentrifugation, which partly destroys adipocytes and other cells. Thus, directly
assessing the original adipocytes and other cellular components without tissue
dissociation is difficult. A three-dimensional measurement that can allow analysis
without tissue dissociation should be developed for a more accurate anatomicalevaluation of adipose tissue.

17

References
1. Rosen, E. D., and Spiegelman, B. M. Adipocytes as regulators of energy balanceand glucose homeostasis. Nature 444: 847, 2006.
2. Katz, A.J., Llull, R., Hedrick, M.H., and Futrell, J.W. Emerging approaches to thetissue engineering of fat. Clin. Plast. Surg. 26: 587, 1999.
3. Zuk, P.A., Zhu, M., Mizuno, H., et al. Multilineage cells from human adipose
tissue: implications for cell-based therapies. Tissue Eng. 7: 211, 2001.
4. Casteilla, L., and Dani, C. Adipose tissue-derived cells: from physiology toregenerative medicine. Diabetes Metab. 32: 393, 2006.
5. Yoshimura, K., Shigeura, T., Matsumoto, D., et al. Characterization of freshly
isolated and cultured cells derived from the fatty and fluid portions of liposuction
aspirates. J. Cell. Physiol. 208: 64, 2006.
6. Matsumoto, D., Sato, K., Gonda, K., et al. Cell-assisted lipotransfer: Supportive useof human adipose-derived cells for soft tissue augmentation with lipoinjection. TissueEng. 12: 3375, 2006.
7. Gimble, J. M., Katz, A. J., and Bunnell, B. A. Adipose-derived stem cells forregenerative medicine. Circ. Res. 100: 1249, 2007.
8. Piasecki, J. H., Gutowski, K. A., Lahvis, G. P., and Moreno, K. I. An experimental
model for improving fat graft viability and purity. Plast. Reconstr. Surg. 119: 1571,
2007.
9. Wolter, T. P., von Heimburg, D., Stoffels, I., et al. Cyopreservation of mature
human adipocytes: In vitro measurement of viability. Ann. Plast. Surg. 55: 408, 2005.
10. Pu, L. L. Q., Cui, X., Fink, B. F., et al. The viability of fatty tissues within adipose
aspirates after conventional liposuction: A comprehensive study. Ann. Plast. Surg. 54:
288, 2005.
18

11. Tholpady, S. S., Aojanepong, C., Llull, R., et al. The cellular plasticity of humanadipocytes. Ann. Plast. Surg. 54: 651, 2005.
12. Moscatello, D. K., Dougherty, M., Narins, R. S., and Lawrence, N.
Cryopreservation of human fat for soft tissue augmentation: Viability requires use of
cryopreservation and controlled freezing and storage. Dermatol. Surg. 31: 1506, 2005.
13. Boschert, M. T., Beckert, B. W., Puckett, C. L., and Concannon, M. J. Analysis oflipocyte viability after liposuction. Plast. Reconstr. Surg. 109: 761, 2002.
14. Rohrich, R. J., Sorokin, E. S., and Brown, S. A. In search of improved fat transfer
viability: A quantitative analysis of the role of centrifugation and harvest site. Plast.
Reconstr. Surg. 113: 391, 2004.
15. Atik, B., Ozturk, G., Erdogan, E., and Tan, O. Comparison of techniques for longterm storage of fat grafts: An experimental study. Plast. Reconstr. Surg. 118: 1533,
2006.
16. Smith, P., Adams, W. P., Lipschitz, A. H., et al. Autologous human fat grafting:
Effect of harvesting and preparation techniques on adipocyte graft survival. Plast.
Reconstr. Surg. 117: 1836, 2006.
17. MacRae, J. W., Tholpady, S. S., Ogle, R. C., and Morgan, R. F. Ex vivo fat graft
preservation: Effects and implications of cryopreservation. Ann. Plast. Surg. 52: 281,
2004.
18. Lalikos, J. F., Li, Y., Roth, T. P., et al. Biochemical assessment of cellular damage
after adipocyte harvest. J. Surg. Res. 70: 95, 1997.
19. Roehm, N. W., Rodgers, G. H., Hatfield, S. M., and Glasebrook, A. L. An
improved colorimetric assay for cell proliferation and viability utilizing thetetrazolium salt XTT. J. Immunol. Methods 142: 257, 1991.

19

20. Miyazaki, T., Kitagawa, Y., Toriyama, K., et al. Isolation of two humanfibroblastic cell populations with multiple but distinct potential of mesenchymal
differentiation by ceiling culture of mature fat cells from subcutaneous adipose tissue.
Differentiation 73: 69, 2005.
21. Wilson-Fritch, L., Burkart, A., Bell, G., et al. Mitochondrial biogenesis andremodeling during adipogenesis and in response to the insulin sensitizer Rosiglitazone.
Mol. Cell. Biol. 23: 1085, 2003.
22. Shiffman, M. A., and Mirrafati, S. Fat transfer techniques: The effect of harvest
and transfer methods on adipocyte viability and review of the literature. Dermatol.
Surg. 27: 819, 2001.
23. van Harmelen, V., Skurk, T., Röhrig, K., et al. Effect of BMI and age on adipose
tissue cellularity and differentiation capacity in women. Int. J. Obes. 27: 889, 2003.
24. Garaulet, M., Hernandez-Morante, J.J., Lujan, J., et al. Relationship between fat
cell size and number and fatty acid composition in adipose tissue from different fat
depots in overweight/obese humans. Int. J. Obes. (Lond) 30: 899, 2006.
25. Sjöström, L., Björntorp, P., and Vrána, J. Microscopic fat cell size measurements
on frozen-cut adipose tissue in comparison with automatic determination of osmiumfixed fat cells. J. Lipid. Res. 12: 521, 1971.
26. Kurita, M., Matsumoto, D., Shigeura, T., et al. Influences of centrifugation oncells and tissues in liposuction aspirates: optimized centrifugation for lipotransfer andcell isolation. Plast. Reconstr. Surg., in press.
27. Rink, J. D., Simpson, E. R., Barnard, J. J., and Bulun, S. E. Cellularcharacterization of adipose tissue from various body sites of women. J. Clin.
Endocrinol. Metab. 81: 2443, 1996.

20

28. Pohjala, L., Tammela, P., Samanta, S. K., et al. Assessing the data quality inpredictive toxicology using a panel of cell lines and cytotoxicity assays. Anal.
Biochem. 362: 221, 2007.
29. Moore, J. H., Kolaczynski, J. W., Morales, L. M., et al. Viability of fat obtainedby syringe suction lipectomy: Effects of local anesthesia with lidocaine. Aesth. Plast.
Surg. 19: 335, 1995.

21

Figure legends
Fig. 1. A schematic diagram of tissue sampling and processing. One sample was
directly subjected to the XTT assay, and another sample to the GPDH assay. The
other three samples were processed by digestion with collagenase and centrifugation.
After centrifugation, the top, middle, and bottom layers were respectively assessed bya cell-staining morphometric assay, the XTT assay, and the GPDH assay.

Fig. 2. Cell staining with Nile Red and Hoechst 33342. Bright field (above, left), Nile
Red (above, right), Hoechst 33342 (below, left), and merge (below, right). A whitearrow indicates an adipocyte. A yellow arrow indicates a pure lipid droplet. Scale bar= 100 µm.

Fig. 3. Cell staining with Hoechst 33342 and PI.
(A) Bright field (above, left), PI (above, right), Hoechst 33342 (below, left), andmerge (below, right). A white arrow indicates a viable adipocyte. A yellow arrow
indicates a dead adipocyte. A white arrowhead indicates a viable cell other thanadipocytes. A yellow arrowhead indicates a dead cell other than adipocytes. Scale bar= 100 µm.
(B) Representative views of each layer stained with Hoechst 33342 and PI. In the top
layer, four types of cells (viable adipocytes, dead adipocytes, viable cells other thanadipocytes, and dead cells other than adipocytes) as well as pure lipid droplets wereobserved. No cells were detected in the middle layer, while non-adipocytes, viable ordead, were seen in the bottom layer. Scale bar = 100 µm.

22Fig. 4. Results of the XTT assay for 1 g suctioned adipose tissue and respective layers
after digestion and centrifugation (n = 10). Adipose tissue before digestion resulted in
the highest value, which is similar to the sum of three layers after digestion. Statisticalsignificance was detected between any combinations of groups. Values are mean ±SEM.

Fig. 5. Correlation between the number of prepared viable cells and the resulting
values of the XTT assay (n = 5). Good correlations were respectively obtained inadipocytes, WBCs, and ASCs. Values are mean ± SEM.

Fig. 6. Results of the GPDH assay for 1 g adipose tissue and respective layers afterdigestion and centrifugation (n = 10). Adipose tissue before digestion andcentrifugation showed the highest value. The bottom layer, which contained no
adipocytes, showed a low value. Statistical significance was detected between any
combinations of groups. Values are mean ± SEM.

Fig. 7. Correlation between the number of prepared viable cells and the resulting
values of the GPDH assay (n = 5). The GPDH assay provided a good correlation in
adipocytes. WBCs and ASCs showed no GPDH activity even at a high concentration.
Values are mean ± SEM.

Fig. 8. Multicolor flow cytometric analysis of cells in the bottom layer. CD45+ cells
were regarded as blood-derived cells. CD45– cells were regarded as adipose-derived
cells and were processed to the next analysis. CD45–CD31–CD34+ cells wereregarded as ASCs, while CD45–CD31+CD34+ cells were regarded as endothelial cells.
23

Table 1.

Number of cells obtained from 1 g adipose tissue (× 105)
Adipocytes,

Adipocytes,

Other cells,

Other cells,

viabledeadviabledead

Top layer

4.4 ± 0.66

2.5 ± 0.28

1.5 ± 0.23

7.5 ± 0.72

Middle layer

0

0

0

0

Bottom layer00

10.0 ± 2.8

2.3 ± 0.34

Data are expressed as mean ± SEM (n = 10).

24

Table 2.
Cell composition in the bottom layer

Adipose-derived cells

Blood-derived
cells

ASCs

Endothelial cells

Other cells

+

–

–

–

CD31

–

+

–

CD34

+

+

–

CD45

Percentage in all cells

36.8 ± 5.2

Percentage in adipose-

62.8 ± 5.2
60.7 ± 8.5

21.4 ± 5.8

17.9 ± 3.8

37.4 ± 4.0

14.6 ± 4.9

11.2 ± 3.1

derived cells
Calculated percentage in

36.8 ± 5.2

all cells+: positive for the cellsurface marker.
–: negative for the cell surface marker.
Data are expressed as mean ± SEM (n = 5).

25

Figure 1
Click here to download high resolution image

Figure 2
Click here to download high resolution image

Figure 3
Click here to download high resolution image

Figure 4
Click here to download high resolution image

Figure 5
Click here to download high resolution image

Figure 6
Click here to download high resolution image

Figure 7
Click here to download high resolution image

Figure 8
Click here to download high resolution image