EXPERIMENTAL
Numerical Measurement of Viable andNonviable Adipocytes and Other Cellular
Components in Aspirated Fat Tissue
Hirotaka Suga, M.D.
Daisuke Matsumoto, M.D.
Keita Inoue, M.D.
Tomokuni Shigeura, M.S.
Hitomi Eto, M.D.
Noriyuki Aoi, M.D.
Harunosuke Kato, M.D.
Hiroaki Abe, M.D.
Kotaro Yoshimura, M.D.
Tokyo and Yokohama, Japan

Background: A reliable method with which to assay viability and number ofadipocytes and other cellular components in adipose tissue remains to beestablished.
Methods: The authors assessed cell viability and number obtained from 1 g ofsuctioned adipose tissue and respective layers (the top, middle, and bottom
layers) before and after digestion and centrifugation, using cell staining with
Hoechst 33342 and propidium iodide and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazoliumhydroxide (XTT) andglycerol-3-phosphate dehydrogenase assays (n ⫽ 10). The correlation between
the number of prepared cells (adipocytes, adipose stromal cells, and white blood
cells) and the resulting values from the XTT and glycerol-3-phosphate dehydrogenase assays was also examined (n ⫽ 5). The cell composition of the stromal
vascular fraction isolated from the same adipose tissue was determined bymulticolor flow cytometry (n ⫽ 5).
Results: Hoechst 33342 and propidium iodide staining allowed distinguishingof viable adipocytes from lipid droplets, dead adipocytes, and cells other thanadipocytes. The authors obtained 6.9 ⫻ 105 nonruptured adipocytes from 1 gof suctioned adipose tissue; 30 percent of the original adipocytes appeared tohave been ruptured. Both the XTT and glycerol-3-phosphate dehydrogenase
assays provided good correlations between the number of viable adipocytes andresulting values, but only the glycerol-3-phosphate dehydrogenase assay wasstrictly specific for adipocytes. The ratio of adipose stromal cells to adipocytes
was found to be much larger than previously described.
Conclusion: Single use or a combination of the viability assays used in thisstudy can appropriately determine the number of adipocytes and other cells,
although it remains difficult to assess original cells directly without tissue
dissociation. (Plast. Reconstr. Surg. 122: 103, 2008.)

A

dipose tissue has been widely studied in basic research, especially in relation to obesity
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 havebeen identified in adipose tissue, resulting in a
new field in adipose biology.2–7 Despite the intenseinterest in adipose tissue, however, its cellular composition— either excised or aspirated—remains un-

known. Although cell counting with cell staining,8 –13
colorimetric assays—namely, the 2,3-bis(2-methoxy4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]2H-tetrazoliumhydroxide (XTT) and/or 3-(4,5-2yl)-2,5-ditetrazolium bromide (MTT) assays9,14 –17—
and the glycerol-3-phosphate dehydrogenase assay9,10,18
have been used in previous studies to analyze cellnumber and viability in adipose tissue, a reliable
method has not yet been established. Specifically,
the number of mature adipocytes is difficult to

From the Department of Plastic Surgery, University of Tokyo
School of Medicine, and the Division of Research and Development, Biomaster, Inc.
Received for publication July 20, 2007; accepted December
14, 2007.
Copyright ©2008 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0b013e31817742ed

Disclosure: The authors do not have any commercial associations or financial disclosures that mightpose or create a conflict of interest with information
presented in this article.

www.PRSJournal.com

103

Plastic and Reconstructive Surgery • July 2008
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 thescant cytoplasm of mature adipocytes.12 Other
staining methods should be used to distinguishviable adipocytes, 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 ahighly colored formazan product.19 This assay provides a good correlation between the number of
viable cells and the resulting formazan absorbancevalues, 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/stromal cells,
these other cells could influence the total absorbance values.
In the glycerol-3-phosphate dehydrogenase assay, glycerol-3-phosphate dehydrogenase activity isdetermined by measuring the decrease in the reduced form of nicotinamide adenine dinucleotide
concentration.18 This assay has the advantage of adipocyte specificity, but extracellular glycerol-3-phosphate dehydrogenase from damaged adipocytes ismeasured together with intracellular glycerol-3phosphate dehydrogenase from viable adipocytes,
affecting the accuracy of viability analysis. Furthermore, a correlation between the number of viableadipocytes and the resulting values of glycerol-3phosphate dehydrogenase activity has not beenreported.
Thus, how many adipocytes and other cellularcomponents 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, andthe glycerol-3-phosphate dehydrogenase assay.

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 homoge-

104

neous samples possible, making an effort not toinclude debris or clumps. The sample was divided
by weighing 1 g for each specimen. One sample ofthe five was directly subjected to the XTT assay,
and another sample was directly subjected to theglycerol-3-phosphate dehydrogenase 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 and incubated at 37°C for 30
minutes. The digestion was terminated with 2 mlof phosphate-buffered saline containing 10% fetal
bovine serum, and the digested adipose tissue wascentrifuged at 430 g for 5 minutes. After centrifugation, the top, middle, and bottom layers wereassessed by a cell-staining morphometric assay, theXTT assay, and the glycerol-3-phosphate dehydrogenase assay, respectively (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 phosphate-bufferedsaline to a total of 5 ml. First, to distinguish adipocytes from floating lipid droplets released frombroken adipocytes, cells were stained with Nile
Red (AdipoRed) (Cambrex, Walkersville, Md.)
and Hoechst 33342 (Dojindo, Kumamoto, Japan).
Cells stained with both Nile Red and Hoechst
33342 were defined as adipocytes, and cells
stained only with Hoechst 33342 but negative for
AdipoRed were defined as nonadipocytes. Particles stained only with AdipoRed were defined as
floating lipid droplets.
Next, to distinguish viable cells from deadcells, cells were stained with Hoechst 33342 andpropidium iodide (Sigma-Aldrich, St. Louis, Mo.).
This staining distinguished four types of nucleatedcells: viable adipocytes (positive for Hoechst
33342, negative for propidium iodide, and including lipid); dead adipocytes (positive for propidium iodide and including lipid); viable cells
other than adipocytes (positive for Hoechst 33342,
negative for propidium iodide, and not including
lipid); and dead cells other than adipocytes (positive for propidium iodide and not including
lipid). Erythrocytes, which are not stained with thenuclear staining, were not counted in this study.
Samples were placed on a hematocytometer under a microscope. At least three fields (⫻100) wererandomly photographed and the number of cells

Volume 122, Number 1 • Measurement of Viable Adipocytes

Fig. 1. A schematic diagram of tissue sampling and processing. One sample was directly subjected to the XTT assay; another sample was directly subjected to the glycerol-3-phosphate
dehydrogenase assay. The other three samples were processed by digestion with collagenase
and centrifugation. After centrifugation, the top, middle, and bottom layers were assessed bya cell-staining morphometric assay, the XTT assay, and the glycerol-3-phosphate dehydrogenase assay, respectively.

in each group counted. Then, the total cell number obtained from 1 g of suctioned adipose tissue
was calculated.
XTT Assay
A Cell Proliferation Kit II (XTT) (Roche Diagnostics, Indianapolis, Ind.) was used according
to the manufacturer’s instructions. In brief, 1 g ofeach sample was suspended in phosphate-buffered saline containing 10% fetal bovine serum toa 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 six-wellplate for 6 hours at 37°C. After the incubation, 150
␮l 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, phosphate-buffered saline containing
10% fetal bovine serum and no cells, was subtracted from each result.
To examine any correlation between the number of viable cells and the resulting values from the
XTT assay for each cell type, several concentrations of adipocytes, white blood cells, and adiposederived stem cells were prepared for assessment by

the XTT assay. Adipocytes were separated from
adipose tissue by digestion and centrifugation as
described above, and viable adipocytes in the top
layer were used. White blood cells were obtained
from the peripheral blood of five healthy donors.
Adipose-derived stem/stromal cells were separated from adipose tissue as described previously6
and cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum. Adipose-derived stem/stromal cells at passage 1 or 2
were used. For each cell type, samples of 1, 2, 5,
and 10 ⫻ 104 viable cells/ml were prepared; thecell number was determined by cell staining with
Hoechst 33342 and propidium iodide for adipocytes, and it was determined by cell counter (NucleoCounter; Chemometec, Allerod, Denmark)for white blood cells and adipose-derived stem/
stromal cells.
Glycerol-3-Phosphate Dehydrogenase 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 thencentrifuged at 430 g for 5 minutes. A 1-ml aliquot

105Plastic and Reconstructive Surgery • July 2008of the aqueous layer was taken and again centrifuged at 17,700 g for 5 minutes. The supernatant
after the second centrifugation was diluted 10
times with an enzyme-extracting reagent, and theoptical absorption was measured at 340 nm for 10
minutes on a 96-well plate after addition of twice thevolume of a substrate reagent. Glycerol-3-phosphate
dehydrogenase activity was calculated based on thefollowing formula: Glycerol-3-phosphate dehydrogenase activity (U/ml) ⫽ ⌬OD ⫻ 0.482 ⫻ 10, where⌬OD is change in optical density per minute.
To examine correlation between the number of
viable cells and the resulting values of glycerol-3phosphate dehydrogenase activity in each cell type,
we assessed several concentrations of adipocytes,
white blood cells, and adipose-derived stem/stromalcells using the glycerol-3-phosphate dehydrogenase
assay, following the design used for the XTT assay.
Flow Cytometry
To determine the presence of nonadipocytes,
we used multicolor flow cytometric analysis to
examine cells in the bottom layer, the stromal
vascular fraction, for surface marker expression.
The following monoclonal antibodies conjugated to fluorochromes were used: anti-CD31PE, anti-CD34-PE-Cy7, and anti-CD45-FITC (BD
Biosciences, San Jose, Calif.). Cells were analyzed
with a LSR II (BD Biosciences), and cell composition percentages were determined according tosurface marker expression profiles.
Statistical Analysis
Data are expressed as mean ⫾ SEM. Comparisons of multiple groups were made using one-way
analysis of variance with Bonferroni’s multiple t test.
Values of p ⬍ 0.05 were considered statistically significant.

RESULTS
Cell Staining
After digestion and centrifugation, many purelipid droplets, not cells, were included together withcells 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 otherthan adipocytes; and 7.5 ⫾ 0.72 ⫻ 105 dead cells
other than adipocytes, which were distinguished bycell staining with Hoechst 33342 and propidium iodide (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 than adipocytes (Table 1).

106

XTT Assay
Adipose tissue before digestion resulted in thehighest value, which was similar to the sum of thethree layers after digestion. The top layer produced a much higher value than that of the bottom layer, although the number of viable cells
directly counted by cell staining was higher in thebottom layer (Fig. 5).
Good correlations between the number of prepared viable cells and the XTT values were respectively obtained in adipocytes, white blood
cells, and adipose-derived stem/stromal cells. Adipocytes showed higher absorbance than whiteblood cells and adipose-derived stem/stromal cells,
especially at a high concentration, differences thatwere statistically significant (Fig. 6). Thus, the results
suggest that the value derived from the XTT assay
would not be proportional to the total viable cellnumber when different cell types are mixed.
Glycerol-3-Phosphate Dehydrogenase Assay
Adipose tissue before digestion and centrifugation showed the highest value. After digestion andcentrifugation, the value of the top layer, which wasregarded as intracellular glycerol-3-phosphate dehydrogenase from floating nonruptured adipocytes,
was more than twofold that of the middle layer,
which was regarded as extracellular glycerol-3-phosphate dehydrogenase released from ruptured adipocytes. The bottom layer, which contained no adipocytes, showed a low value (Fig. 7).
The glycerol-3-phosphate dehydrogenase assay provided a good correlation between thenumber of prepared viable adipocytes and theresulting values, whereas white blood cells andadipose-derived stem/stromal cells showed noglycerol-3-phosphate dehydrogenase activity
even at a high concentration (Fig. 8).
Flow Cytometry
Flow cytometric analysis revealed that thestromal vascular fraction contained CD45-positive cells, corresponding to blood-derived cells
at 37 ⫾ 5.2 percent; adipose-derived stem/stromal cells (CD45⫺CD31⫺CD34⫹) at 37 ⫾ 4.0 percent; and endothelial cells (CD45⫺CD31⫹CD34⫹) at
15 ⫾ 4.9 percent (Fig. 9 and Table 2).

DISCUSSION
In the present study, suctioned fat samples
obtained from the abdomen or thighs were used.
Although regional (donor-site) differences in adipose cell components may exist, a previous study
reported that no statistical differences in adipose

Volume 122, Number 1 • Measurement of Viable Adipocytes

Fig. 2. Cell staining with AdipoRed and Hoechst 33342. Bright field (above, left), AdipoRed (above, right),
Hoechst 33342 (below, left), and merge (below, right). A white arrow indicates an adipocyte. A yellow arrow
indicates a pure lipid droplet. Scale bar ⫽ 100 ␮m.

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 inadipose stromal cell yield were observed between
the samples from the abdomen and the thigh(data not 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 through 4.
Lipid droplets the same size as adipocytes (50 to120 ␮m) cannot be distinguished from adipocytes
without nuclear staining. In previous studies,8,10,13
trypan blue staining was performed, but it cannot
distinguish pure lipid droplets from viable adipocytes because it does not stain either of them well.

The previous studies8,10,13 reported adipocyte numbers(2.5 to 90 million/ml) which were much larger
than those in our result (0.7 million/ml), and thedifference may arise from the confusion 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 deadcells. We combined propidium iodide, whichstained 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 otherthan adipocytes, and dead cells other than adipocytes. A combination of fluorescein diacetate andpropidium iodide may be another option.12
We found that many nonadipocyte cells were
present in the top layer after collagenase digestion

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Plastic and Reconstructive Surgery • July 2008

Fig. 3. Cell staining with Hoechst 33342 and propidium iodide. Bright field (above, left), propidium
iodide (above, right), Hoechst 33342 (below, left), and merge (below, right). The white arrow indicates a
viable adipocyte. The yellow arrow indicates a dead adipocyte. The white arrowhead indicates a viable
cell other than adipocytes. The yellow arrowhead indicates a dead cell other than adipocytes. Scale bar⫽ 100 ␮m.

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 bycentrifugation. A previous study using ceiling culture showed that cells tightly attached to matureadipocytes could generate fibroblastic cell popuTable 1. Number of Cells Obtained from 1 g of
Adipose Tissue (ⴛ105)*
Adipocytes

Other Cells

Layer

ViableDeadViableDead

Top
MiddleBottom

4.4 ⫾ 0.66
00

2.5 ⫾ 0.28
00

1.5 ⫾ 0.23
0
10.0 ⫾ 2.8

7.5 ⫾ 0.72
02.3 ⫾ 0.34

*Data are expressed as mean ⫾ SEM (n ⫽ 10).

108

lations with multiple differentiation potential,20
and the cells were regarded as adipose-derived
stem/stromal cells. Thus, the nonadipocyte cells
detected in the top layer may contain adiposederived stem/stromal cells and white blood cells,
although more than 80 percent of the nonadipocyte cells were dead in the current study.
Among colorimetric assays of cell viability, theXTT and MTT assays have been 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 not shown),
unlike the XTT assay. In the MTT assay, the resulting colored formazan product is insoluble andrequires a dissolving procedure,19 which may impair reproducibility, especially when using adipose tissue.

Volume 122, Number 1 • Measurement of Viable Adipocytes
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 fromthe XTT assay in the top layer compared with thebottom 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 with white blood cells andadipose-derived stem/stromal cells. This difference may be attributable to the increase in mitochondrial capacity (number of mitochondrion
profiles, concentration of numerous mitochondrial proteins, and oxygen consumption rate) during
adipogenesis.21 Some authors have reported in previ-

ous studies14,16 that the XTT assay is well suited toassessing adipose tissue viability; however, it is not
specific for adipocytes, and careful interpretationof results is needed when contamination by othercell types is present. Our results demonstrate thatthe 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.
Glycerol-3-phosphate dehydrogenase is normally intracellular, and extracellular glycerol-3phosphate dehydrogenase is considered proportional to the amount of adipocyte destruction.9,18

Fig. 4. Representative views of each layer stained with Hoechst 33342 and propidium iodide. In the top layer, four typesof cells (viable adipocytes, dead adipocytes, viable cells other than adipocytes, and dead cells other than adipocytes) andpure lipid droplets were observed. No cells were detected in the middle layer, whereas nonadipocytes, viable or dead,were seen in the bottom layer. Scale bar ⫽ 100 ␮m.

Fig. 5. Results of the XTT assay for 1 g of suctioned adipose tissue and respective layers after digestion and centrifugation (n ⫽ 10). Adipose tissue
before digestion resulted in the highest value, which was similar to the sumof the three layers after digestion. Statistical significance was detected between any combinations of groups. Values are mean ⫾ SEM.

109

Plastic and Reconstructive Surgery • July 2008
Therefore, examining total glycerol-3-phosphate
dehydrogenase in adipose tissue is not appropriate for viability assessment. To gain a full understanding of the results of previous studies, wewould have to confirm whether the reported glycerol-3-phosphate dehydrogenase value was intracellular or extracellular or a mixture of both. In
our study, the glycerol-3-phosphate dehydrogenase activity of the top layer was regarded as intracellular glycerol-3-phosphate dehydrogenasefrom nonruptured adipocytes, although this layer
appeared to contain both viable and dead (but not

ruptured) adipocytes. These latter adipocytes may
die because of the effects of collagenase, although
they did not rupture and the intracellular contents
were not released into the middle layer. The glycerol-3-phosphate dehydrogenase activity of the
middle layer indicated that approximately 30 percent of total adipocytes were ruptured. The rate ofthis extracellular glycerol-3-phosphate dehydrogenase can change, depending on adipose tissue
sampling methods22 and the digestion procedure.18
The glycerol-3-phosphate dehydrogenase assay issuperior to the XTT assay because of its strict

Fig. 6. Correlation between the number of prepared viable cells and theresulting values of the XTT assay (n ⫽ 5). Good correlations were obtained inadipocytes, white blood cells (WBCs), and adipose-derived stem/stromalcells (ASCs). Values are mean ⫾ SEM.

Fig. 7. Results of the glycerol-3-phosphate dehydrogenase assay for 1 g ofadipose tissue and respective layers after digestion and centrifugation (n ⫽10). Adipose tissue before digestion and centrifugation showed the highest
value. The bottom layer, which contained no adipocytes, showed a low
value. Statistical significance was detected between any combinations ofgroups. Values are mean ⫾ SEM.

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Volume 122, Number 1 • Measurement of Viable Adipocytes

Fig. 8. Correlation between the number of prepared viable cells and theresulting values of the glycerol-3-phosphate dehydrogenase assay (n ⫽ 5).
The glycerol-3-phosphate dehydrogenase assay provided a good correlation in adipocytes. White blood cells (WBCs) and adipose-derived stem/stromal cells (ASCs) showed no glycerol-3-phosphate dehydrogenase activity
even at a high concentration. Values are mean ⫾ SEM.

Fig. 9. 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 were regarded as adipose-derived stem/stromal cells(ASCs), whereas CD45–CD31⫹CD34⫹ cells were regarded as endothelial cells.

111Plastic and Reconstructive Surgery • July 2008
Table 2. Cell Composition in the Bottom Layer*
Adipose-Derived Cells

CD45
CD31
CD34
Percentage in all cells
Percentage in adipose-derived cells
Calculated percentage in all cells

Blood-Derived Cells

ASCs

Endothelial Cells

Other Cells

⫹

⫺
⫺
⫹

⫺
⫹
⫹
62.8 ⫾ 5.2
21.4 ⫾ 5.8
14.6 ⫾ 4.9⫺⫺⫺

36.8 ⫾ 5.2
36.8 ⫾ 5.2

60.7 ⫾ 8.5
37.4 ⫾ 4.0

17.9 ⫾ 3.8
11.2 ⫾ 3.1

ASCs, adipose-derived stem/stromal cells; ⫹, positive for the cell surface marker; ⫺, negative for the cell surface marker.
*Data are expressed as mean ⫾ SEM (n ⫽ 5).

specificity for adipocytes, which the current findings support. Although the previous studies9,10,18
appropriately used the glycerol-3-phosphate dehydrogenase assay, a control curve using premeasured adipocytes or a combination of direct cellcounting is needed to evaluate the absolute number of adipocytes.
From 1 g of suctioned adipose tissue, we obtained 6.9 ⫻ 105 adipocytes (viable-to-dead ratio,
4.4:2.5) and 2.1 ⫻ 106 nonadipocyte cells (viableto-dead ratio, 11.5:9.8). Based on the glycerol-3phosphate dehydrogenase data for the top andmiddle layers, we speculate that 1 g of 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 to 150 ␮m, with a mean size
between 90 and 100 ␮m regardless of the severityof obesity. Assuming that adipocytes are cubes (106␮m3) or spheres (5.23 ⫻ 105 ␮m3) with a side ordiameter of 100 ␮m, the number of adipocytes in
1 ml adipose tissue would correspond to 0.95 million or 1.81 million, respectively, under the condition that adipocytes occupy 95 percent of adipose
tissue volume; thus, the theoretical number of adipocytes in the 1 ml of adipose tissue is likely to be
between the two numbers. This suggestion is supported by the results of a previous study23 indicatingthat adipocyte number per 1 g of adipose tissue is1.51 ⫾ 0.47 million (mean ⫾ SD). Because theadipocyte number of noncentrifuged suctioned
adipose tissue is likely to be smaller than that of
intact adipose tissue,26 the estimated number inour study (1 million per 1 ml of suctioned adipose
tissue) seems to be reasonable.
Multicolor flow cytometry revealed that 37 percent of nonadipocyte cells in stromal vascular fraction (the bottom layer) are adipose-derived stem/
stromal cells, a ratio consistent with our previous

112

findings.5 Assuming that the nonadipocyte cellcomposition in the top layer is similar to that of thebottom layer, we can speculate that the number ofadipose-derived stem/stromal cells in 1 g of suctioned adipose tissue is approximately half that of
adipocytes. Considering also that the number ofadipose-derived stem/stromal cells in suctioned
adipose tissue is significantly smaller than in intact
adipose tissue,6 the adipose-derived stem/stromal
cell number in intact adipose tissue may be similar
to that of adipocytes. In addition, it may be muchlarger than that described in a previous histologic
study,27 which reported percentages of adipocytes
and stromal cells in the adipose tissue as 89 to 98
percent and 2 to 10 percent, respectively. In ahistologic assessment of the adipose tissue, a single
100-␮m adipocyte, for example, could appear in
12 serial sections (8 ␮m thick; 96 ␮m in all), buteach section contains different adipose-derived
stem/stromal cells; thus, a single adipocyte could
be repeatedly counted by mistake, and a careful
evaluation of the specimens is necessary for counting cell numbers. Therefore, thicker sections, as
thick as 200 ␮m, combined with an appropriate
calculation formula such as that used in a previous
study25 may contribute to avoiding mistakes in cellcounting.

CONCLUSIONS
Single use or a combination of the viability
assays used in this study can accurately determine the number of viable adipocytes and othercells, although other assays such as the adenosine triphosphate assay28 and glucose transport
experiment29 should be further investigated for
applications to adipose tissue. The major limitation of the current study is that the assays are
performed only after enzyme digestion and centrifugation, which partly destroys adipocytes andother cells. Thus, directly assessing the original
adipocytes and other cellular components without tissue dissociation is difficult. A three-di-Volume 122, Number 1 • Measurement of Viable Adipocytes
mensional measurement that can allow analysis
without tissue dissociation should be developed
for a more accurate anatomical evaluation of
adipose tissue.
Kotaro Yoshimura, M.D.
Department of Plastic Surgery
University of Tokyo School of Medicine
7-3-1, Hongo, Bunkyo-Ku
Tokyo 113-8655, Japan
yoshimura-pla@h.u-tokyo.ac.jp

ACKNOWLEDGMENT

This study was financially supported by Grants-inAid contract no. B2-19390452 from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
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