TISSUE ENGINEERING
Volume 12, Number 12, 2006
# Mary Ann Liebert, Inc.

Cell-Assisted Lipotransfer: Supportive Use of Human
Adipose-Derived Cells for Soft Tissue Augmentation
with Lipoinjection
DAISUKE MATSUMOTO, M.D.,1 KATSUJIRO SATO, M.D.,1 KOICHI GONDA, M.D.,1
YASUYUKI TAKAKI, M.D.,2 TOMOKUNI SHIGEURA, M.S.,2 TAKAHIRO SATO, M.S.,2
EMIKO AIBA-KOJIMA, M.D.,1 FUMIKO IIZUKA, M.D.,1 KEITA INOUE, M.D.,1
HIROTAKA SUGA, M.D.,1 and KOTARO YOSHIMURA, M.D.1

ABSTRACTInjective transfer of autologous aspirated fat is a popular option for soft tissue augmentation, but several
issues require attention, including unpredictability and a low survival rate due to partial necrosis. In thisstudy, histologic features and yield of adipose-derived stromal (stem) cells (ASCs) were compared between
human aspirated fat and excised whole fat. Aspirated fat contained fewer large vascular structures, andASC yield was lower in aspirated fat. Aspirated fat was transplanted subcutaneously into severe combined immunodeficiency mice with (cell-assisted lipotransfer; CAL) or without (non-CAL) vascularstromal fractions containing ASCs isolated from adipose tissue. The CAL fat survived better (35% larger
on average) than non-CAL fat, and microvasculature was detected more prominently in CAL fat,
especially in the outer layers. DiI-labeled vascular stromal fraction cells were found between adipocytes
and in the connective tissue in CAL fat, and some of these cells were immunopositive for von Willebrandfactor, suggesting differentiation into vascular endothelial cells. Another experiment that used vascularstromal fractions taken from green fluorescent protein rats also suggested that ASCs differentiated into
vascular endothelial cells and contributed to neoangiogenesis in the acute phase of transplantation. Thesefindings may partly explain why transplanted aspirated fat does not survive well and suggest clinicalpotential of the CAL method for soft tissue augmentation.

INTRODUCTIONADIPOSE-DERIVED STROMAL (STEM) CELLS (ASCs) can beobtained from liposuction aspirates1,2 and differentiate
into multiple lineages of mesodermal or ectodermal origins.
Human ASCs were shown by several in vitro and in vivo
studies to differentiate into adipogenic, osteogenic,3–7 chondrogenic,8–10 myogenic,11,12 cardiomyogenic,13,14 and neurogenic15–17 lineages. Adipose tissue is known to be rich in
microvasculature,18 and ASCs were shown to have angiogenic
characteristics and to differentiate into vascular endothelial
12cells experimentally.19–21 Human ASCs are distinct from othermesenchymal progenitors in surface marker expression profile; notably, only ASCs express stem-cell–associated marker
CD34 in higher percentages compared with bone marrow–derived mesenchymal stem cells and dermal fibroblasts.22
ASCs are being studied in clinical trials, including thoseinvestigating bone defect23 (fresh ASCs) and rectovaginal
fistula24 (cultured ASCs) treatments and soft tissue augmentation by cell-assisted lipotransfer25 (our unpublisheddata; fresh ASCs). If ASCs are harvested from a large
volume (e.g., 1 L) of liposuction aspirates, ASCs can be used

Department of Plastic Surgery, University of Tokyo School of Medicine, Tokyo, Japan.
Department of Research and Development, Biomaster Inc., Kanagawa, Japan.
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3376

clinically without cell expansion because a sufficient number can be obtained. Furthermore, the use of minimallymanipulated fresh cells may lead to greater safety and efficacy in actual treatments.
Aspirated fat is also used as injection material for soft
tissue augmentation to reconstruct inborn or acquired tissue
defects, or for such cosmetic treatments as breast enhancement or facial rejuvenation. Although lipoinjection has someproblems to be resolved, such as unpredictability and a low
survival rate due to partial necrosis,26,27 it is almost the only
method of soft tissue augmentation that can be performed
without detectable scarring on a donor or a recipient site andwithout complications associated with foreign materials.
Thus, if the clinical efficacy and safety of the procedure canbe improved, it could be a strong cosmetic and reconstructive tool for soft tissue augmentation.
Here, we provide evidence to support a novel method of
autologous tissue transfer, which we named cell-assisted
lipotransfer (CAL). CAL is a concurrent transplantation of
aspirated fat and ASCs (i.e., transplantation of ASC-richaspirated fat). In CAL, ASCs were supportively used toboost the efficacy of autologous lipoinjection (resulting in ahigher survival rate and persistency of transplanted fat) andto decrease known adverse effects of lipoinjection, such asformation of fibrosis, pseudocyst, and calcification. In thisstudy, aspirated fat was compared with excised whole fat(nonaspirated fat) in microscopic and electromicroscopic
histology and adherent cell yields. In addition, we evaluatedthe effectiveness of CAL in animal models compared with
non–cell-assisted lipotransfer (non-CAL), and examined thefate of ASCs in fat transplanted with the CAL method.

adipocytes and connective tissues were separated frompellets by centrifugation (800 g, 10 min). The pellets were
resuspended and filtered with a 100-mm mesh (Millipore,
MA). Freshly isolated SVFs were plated (30,000 cells/cm2)
on gelatin-coated dishes and cultured at 378C in an atmosphere of 5% carbon dioxide (CO2) in humid air. The culture
medium was M-199 containing 10% fetal bovine serum, 100
IU penicillin, 100 mg/mL streptomycin, 5 mg/mL heparin,and 2 ng/mL acidic fibroblast growth factor. After 7 days,
attached cells were passaged by trypsinization and cultured
in the same medium. Medium was replaced every third day.
The excised fat from patients who underwent a tummy tuck
was first minced with scissors into 3-mm pieces and thenprocessed in the same manner as aspirated fat.

MATERIALS AND METHODS

Mouse models for tracing human SVF cells in CAL

Human tissue sampling
We obtained liposuction aspirates from healthy female
donors undergoing liposuction of the abdomen or thighs.
Participants provided informed consent, and an institutional
review board—approved protocol was used. From 3 patients
who underwent both liposuction and tummy tuck, excised
adipose tissue (20–30 g) was also taken. Aspirated fat wasused as the cell source of vascular stromal fraction (SVFs)
containing ASCs. The excised fat obtained from patients
who underwent a tummy tuck was also used for isolation of
SVFs. Both the aspirated fat and the excised fat were also
used for histologic examination.

Mouse models for transplantationof human aspirated fat
Seven-week-old male severe combined immunodeficiency (SCID) mice housed with free access to water andstandard chow diet were anesthetized by intraperitoneal
injection of 5 mg/mL pentobarbital. For preparation of graft
material, human aspirated fat was washed with saline andpoured into 10-mL syringes, which were then placed uprightat room temperature for 10 min. The infranatant fluid wasthen discarded. The human aspirated fat (1 mL ¼ 900 mg)
was subcutaneously injected into the back of the SCID micewith or without freshly isolated SVF cells. For CAL, SVFs
taken from 4 mL of aspirated fat was mixed with 1 mL ofaspirated fat. Four weeks later, transplanted fat was harvested, weighed, and fixed with 4% paraformaldehyde, and
4 mm sections were stained with hematoxylin-eosin.

For tracing the SVFs, SVF cells freshly isolated fromhuman aspirated fat were labeled by incubated with 5 mg/mL
CM-DiI (Molecular Probes, Eugene, Oregon) for 1 h before
transplantation. DiI-labeled SVFs taken from 4 mL of aspirated fat were mixed with 1 mL of aspirated fat and theninjected under the back skin of a SCID mouse. In the samemanner, human aspirated fat was injected without DiIlabeled SVFs as control. Four weeks later, transplanted fat
was harvested, fixed with 4% paraformaldehyde, and embedded in optimal cutting temperature (OCT) compound by
freezing in isopentene cooled with liquid nitrogen. Then, 20mm-thick frozen sections were examined with a confocal
microscope system (Leica TCS SP2, Leica Microsystems
GmbH, Wetzlar, Germany).

Cell isolation and culture
SVFs were isolated from the fatty portion of liposuction
aspirates by using a procedure modified from Zuk et al.1
Briefly, the aspirated fat was washed with phosphatebuffered saline (PBS) and digested on a shaker at 378C in
PBS containing 0.075% collagenase for 30 min. Mature

Rat models for tracing rat green fluorescentprotein–labeled SVF in CAL by using
rat minced fat
Inguinal adipose tissue excised from green fluorescentprotein (GFP) rats [SD TgN(act-EGFP)OsbCZ-004; gener-

CELL-ASSISTED LIPOTRANSFER

A

aspirated fat

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excised fat

A
B

HE

fat mass [mg]

1000
800
600
n=10

400
n=11
200
0
non-CALSEM

B

ASC yeield from suctioned
fat / ASC yield from whole fat

C

non-CAL fat

CAL

CAL fat

1.0
0.8

x 40

0.6
0.4
0.2
0.0
#1

#2

#3

Ave.

x 100
FIG. 1. Comparison of human aspirated fat and excised whole
fat obtained from a single site of a single patient. (A) Histologic
features of aspirated fat and excised fat. (Hematoxylin-eosin[HE]–stained microphotographs and scanning electron micrograph [SEM] photos; red scale bar ¼ 200 mm, white scale bar ¼
40 mm). The basic structure of adipose tissue was preserved in theaspirated fat, while vascular vessels, especially large one, weresignificantly less detected in aspirated fat than in excised fat. (B)Adipose-derived stromal (stem) cell (ASC) yield from aspirated
fat and excised fat. Both tissues were processed for isolation of )vascular stromal fractions, which were then cultured for 1 week.
Ratios of ASC yield from aspirated fat to that from excised fat of
the same volume were calculated; data from 3 patients (#1–#3)
and their average value were demonstrated. ASC yield from aspirated fat was significantly less (48%  13%) than that fromexcised fat.

ated from Sprague-Dawley (SD) rats] was processed in thesame way as human excised fat. GFP-SVF taken from 1 mLof fat of the GFP rat was mixed with minced inguinal adipose tissue (1 mL) harvested from SD rats and then injectedunder the back skin of an SD rat. Four weeks later, transplanted fat was harvested and forwarded to immunohistology as well.

FIG. 2. Cell-assisted lipotransfer (CAL) and non-CAL using human aspirated fat and vascular stromal fractions. (A) Photographsof CAL fat (right) and non-CAL fat (left) in severe combined
immunodeficiency mice. Human aspirated fat was transplantedwith (CAL fat) or without (non-CAL fat) human vascular stromal
fractions freshly isolated from the same aspirated fat. Transplanted fat was harvested at 4 weeks. (B) Weight of harvested
CAL fat and non-CAL fat. CAL fat weighed significantly morethan non-CAL fat (p < .05). (C) Histologic features of CAL fat
and non-CAL fat samples (red scale bar ¼ 250 mm, black scalebar ¼ 100 mm). Central necrosis (*) was almost always seen in nonCAL fat, and the surviving layer of CAL fat was thicker than thatof non-CAL fat. At higher magnification, microvasculature wasmore frequently seen in CAL fat, especially in the outer layers,
than in non-CAL fat.

Immunostaining
Frozen sections were prepared from transplanted fat embedded in OCT compound. After fixation in 100% acetone
for 10 min at 48C, sections were rehydrated and blocked with
0.5% goat serum in PBS. Paraffin-embedded sections

MATSUMOTO ET AL.

3378

were dewaxed, washed in PBS, and treated with proteinase K (DakoCytomation, Carpinteria, CA) for 6 min andblocked with 0.5% goat serum in PBS. Primary and secondary antibody stainings were performed for 60 min each atroom temperature. 40 ,6-diamidino-2-phenylindole (DAPI)
staining was performed with DAPI containing mounting
medium (Vector Laboratories, Burlingame, CA). The following antibodies and dilutions were used: anti-green
fluorescent protein (mouse, 1:500; Molecular Probes), vonWillebrand factor (rabbit, 1:500; DakoCytomation), andAlexa Fluor 488 or Alexa Fluor 546 conjugated antimouse or
antirabbit antibodies (goat, 1:200; Molecular Probes). Sections were imaged on a confocal microscope system (LeicaTCS SP2).

Scanning electron microscopic study
Aspirated and excised fat were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M cacodylate
buffer for 1 week at room temperature, and then fixed in 1%
osmium tetroxide. After dehydration, they were dried with asuper-critical-point CO2 dryer (HCP-2, Hitachi, Tokyo, Japan), sputter-coated with platinum-palladium, and examinedwith a scanning electron microscope (S3500N, Hitachi).

Statistical analyses
Results were expressed as the mean  standard error. The
data were statistically analyzed using an unpaired Student
t-test. A p value less than .05 was considered to represent a
statistically significant difference.

Human aspirated fat transplantation
with or without SVF
Human aspirated fat was transplanted with or without
freshly isolated SVFs containing ASCs taken from the same
patient. The experiments were done 3 times using aspirated
fat from 3 patients.
Transplanted adipose tissue with or without ASCs (CAL
fat and non-CAL fat) was 712.3  45.3 mg (n ¼ 10) or
520.6  40.8 mg (n ¼ 11), respectively, while fresh adipose
tissue (1 mL) before transplantation was approximately
900 mg (Fig. 2A and B). Histology of transplanted fat
samples showed that the central region was necrotic in nonCAL fat samples and that the survived layer was thicker in
CAL fat than in non-CAL fat (Fig. 2C). Microvasculature
microscopically detected appeared to be prominent in CAL
fat, especially in the outer layers, but not in non-CAL fat(Fig. 2C).

Fate of human ASCs transplanted withhuman aspirated fat
To trace human ASCs, SVFs freshly isolated from aspirated fat were labeled with DiI and then transplanted withhuman aspirated fat. In CAL fat, DiI-labeled ASCs wereoccasionally detected between mature adipocytes and in theconnective tissue of the transplanted fat (Fig. 3). ASCs positive for both DiI and von Willebrand factor were detected
in CAL fat, suggesting that some transplanted ASCs weredifferentiated into vascular endothelial cells (Fig. 4).

Fate of ASCs derived from GFP rats
in rat CAL models
RESULTS
Comparison of histologic features and ASC yield
between aspirated fat and excised fat
Both aspirated fat and excised fat were harvested fromthe 3 patients who underwent the tummy-tuck operation.
Histologic examinations with a light microscope and scanning electron microscope showed that aspirated fat preserves basic structures of adipose tissue and that matureadipocytes keep normal adhesions with each others. The
only difference detected was that aspirated fat contained
fewer vascular structures, especially large ones, comparedwith excised fat.
The same weights of aspirated fat and excised fat were
processed for isolation of SVFs. The cell isolation process
was performed within 2 h after harvest. The isolated SVFs
were cultured for 1 week, and the numbers of adherent ASCs
from the aspirated fat and the excised fat were counted andcompared. In all 3 patients, the number of adherent ASCs at
1 week was greater in the excised fat than in the aspirated fat.
The ratio of normalized ASC number from aspirated fat tothat from excised fat was 0.48  0.13 (n ¼ 3) (Fig. 1B).

To trace ASCs transplanted with fat, minced fat of SD rats
was transplanted with (CAL) or without (non-CAL) SVFs
isolated from the inguinal adipose of GFP rats (GFP-SVF)(Fig. 5A). In CAL fat, GFP-positive cells, which are supposed to be transplanted GFP-ASCs, were detected withinconnective tissue and in some vessels (Fig. 5B). Most of
the vessels were derived from host (von Willebrand factor–
positive, GFP-negative), but vessels positive for both vonWillebrand factor and GFP were occasionally detected. The
GFP-positive cells partly or entirely covered the inner surface of vessels (Fig. 5B). It was suggested that some ASCs
differentiated into endothelial cells and contributed to angiogenesis during the surviving process of the adipose transplantation.

DISCUSSION
Adipose tissue is predominantly composed of extracellularmatrix; mature adipocytes; ASCs; vascular cells, such as
endothelial cells; and mural cells, such as pericytes andvascular smooth muscle cells. We recently reported the cell

CELL-ASSISTED LIPOTRANSFER

FIG. 3. Histologic features of cell-assisted lipotransfer (CAL)
fat using human aspirated fat and DiI-labeled vascular stromal
fractions. Normal excised fat and non-CAL fat were also used
for comparison. Nuclei were stained with 40 ,6-diamidino-2phenylindole (DAPI). In CAL fat, DiI-labeled cells, which aresupposed to be co-transplanted adipose-derived stromal (stem)
cells, are located between mature adipocytes and in interstitialconnective tissue. The frequency of DiI-positive cells was 10%–30%, although it varied among samples and at locations in asample. Scale bar ¼ 50 mm. The right column shows merges of DiI
and DAPI, and differential interference contrast (DIC) images.

composition of SVFs freshly isolated from liposuction aspirates, which is important information for the clinical use of
SVFs.22 The SVFs are composed of heterogeneous populations, including blood-derived cells, which are composed of
50%–70% SVF cells. Adipose-derived cells (CD45cells) in
SVFs isolated from aspirated fat were of the following types:
ASCs (70%–90%; CD31 CD34þ CD45 CD90þ CD105
CD146), vascular endothelial (progenitor) cells (3%–9%;CD31þ CD34þ CD45 CD90þ CD105low CD146þ), pericytes (2%–5%; CD31 CD34 CD45 CD90þ CD105
CD146þ), and other cells. The cell composition of the SVFs
was measured after the cell isolation process with collagenase
digestion for 30 min, but not all adipose-derived cells can beisolated from the SVFs in this process; thus, the cell composition may not accurately correspond to the actual cellcomposition of the adipose tissue. The location of ASCs in
the adipose tissue is not clearly understood. Some ASCs aresupposed to be located in the connective tissues in adipose,and others are located between adipocytes or around microvasculature or macrovasculature.

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FIG. 4. Immunohistology of cell-assisted lipotransfer (CAL) fat
using human aspirated fat and DiI-labeled vascular stromal fractions. Normal excised fat (control fat) and non-CAL fat were also
assessed for comparison. Endothelial cells were immunostainedwith von Willebrand factor (vWF). Nuclei were stained with 40 ,6diamidino-2-phenylindole (DAPI). Cells double-positive for vWF
and DiI (arrows), which were suggested to be vascular endothelial
cells differentiated from co-transplanted DiI-labeled adiposederived stromal (stem) cells, were observed in connective tissue or
between adipocytes. Scale bar ¼ 10 mm. The right column shows
merges of immunostaining of vWF, DiI, DAPI, and differential
interference contrast images.

Liposuction aspirates consist of 2 parts: a floating fatty
portion and an infranatant fluid portion. We have investigated cells derived from these portions of liposuction
aspirates and found that a substantial number of adiposederived cells, including ASCs and endothelial cells, canbe isolated from the fluid portion, although the number
from the fluid portion is smaller than that from the fatty
portion.22 This finding supports a result of this study
showing that aspirated fat is relatively stem cell–deficientcompared with excised whole fat. The reason was not elucidated here, but mechanical injury during the liposuction procedure and digestion by endogenous proteases
during the surgery or subsequent storage periods probably induces the release of ASCs from harvested aspirated
fat into the fluid portion. In addition, as shown in this study,
the basic structure of adipose tissue was preserved in theaspirated fat, but vascular vessels, especially larger ones,
are significantly less detected in aspirated fat than in excised fat. It is well known that the honeycomb structures ofvascular and neural perforator networks are left intact
in aspirated sites after liposuction operation. (Click here
for supplementary material.) Thus, it is reasonable thatfewer ASCs, which also reside around capillaries and vessels, were isolated from aspirated fat, which does not containlarger vessels, than excised fat. Unlike our results, a recent

3380

FIG. 5. Cell-assisted lipotransfer (CAL) fat in rat models(fragmented fat of Sprague-Dawley [SD] rat and green fluorescentprotein – vascular stromal fractions [GFP-SVF rat]). (A) Cultured
GFP–adipose-derived stromal (stem) cells (ASCs) (phase-contrast
and fluorescence images). When GFP-SVF cells were cultured, alladherent cells were GFP-positive. (B) Immunohistology of cellassisted lipoprotein fat in rat models. GFP was detected by immunostaining with anti-GFP antibody. Adipose tissue of SD ratsand GFP rats were also demonstrated for comparison. Vascularendothelial cells are only von Willebrand factor (vWF) positive in
SD fat but were all double positive for vWF and GFP in GFP fat.
In CAL fat, capillaries with endothelial cells partly or entirely
double positive for GFP and vWF (arrows) were detected, suggesting that they were differentiated from co-transplanted GFP-ASCs.
The other endothelial cells were only positive for vWF, suggestingthat they were derived from the host SD rat. White scale bar ¼ 30
mm, yellow scale bar ¼ 10 mm. The right column shows merges of
immunostaining of vWF, DiI, 40 ,6-diamidino-2-phenylindole, anddifferential interference contrast images.

study28 that compared viable cell yield from fresh aspirated
versus fresh excised fat did not detect a significant difference. However, this finding was probably occurred because
the investigators centrifuged the aspirated fat and removed

MATSUMOTO ET AL.
fibrous structures and visible vessels from excised fat during
the cell isolation process.
The results of the present study suggest that addition ofASCs to aspirated fat improves the efficacy of adipose
transfer, although the exact mechanisms remain to be elucidated. By addition of ASCs to relatively ASC-poor fat(aspirated fat), the aspirated fat is theoretically converted torelatively ASC-rich fat. A recent study using fragmented
omentum tissue suggested the effects of co-transplantation
with preadipocytes, but the fragmented adipose tissue in thatstudy was not ASC-poor.29
Partly on the basis of the present data, we can speculate
on the fate and roles of ASCs as follows. First, as shown inthis study as well as in previous studies,19–21 ASCs can differentiate into vascular endothelial cells that may contributeto neoangiogenesis during the healing process after transplantation. This effect may contribute to the decreased
amount of central necrosis and marked microvasculature in
the outer layers of CAL fat seen in this study.
Second, as suggested in this study, some ASCs werelocated between mature adipocytes and in the connective
tissue of CAL fat, as they had been in normal fat before
liposuction. They may play a role as adipose progenitor cells
for future turnover of adipocytes. The relative deficiency ofASCs in aspirated fat compared with excised fat may contribute to the low long-term survival rate in non-CAL fat,which is a well-known clinical phenomenon.26,27 This hypothesis is also supported by a recent study29 showing thatfragmented omentum (mainly composed of adipose) with orwithout preadipocytes (ASCs) was transplanted and the
postoperative atrophy of transplanted tissue was suppressed
when transplanted with preadipocytes. As it was reported
that adipocytes are replaced with the next generation every
1–2 years in normal adipose,30 the relative deficiency oftissue-specific progenitor cells in non-CAL fat may affectthe coming turnover of the tissue and lead to its long-term
atrophy. Turnover of adipocytes may occur in the early stage
after transplantation in transplanted adipose tissue because
its vascularity was temporarily damaged; this outcome may
be the reason that atrophy of transplanted adipose is clinically seen during the first 3 months.
Third, some ASCs may differentiate into mature adipocytes and partly constitute transplanted fat. Although adipogenic differentiation of labeled ASCs was not detected inthis study, the failure of detection may result from difficulty
in detecting GFP-labeled cytoplasm of mature adipocytes,
which are filled with lipid material; indeed, to our knowledge, no previous report has clearly demonstrated labeledcytoplasm of mature adipocytes.
Fourth, transplanted ASCs were kept in a hypoxic condition in the acute phase after transplantation and may release angiogenic soluble factors such as vascular endothelial
growth factor and hepatocyte growth factor, accelerating neoangiogenesis from the surrounding host tissue in aparacrine manner. It was reported that cultured human ASCs
produce and release these growth factors in a hypoxic con-

CELL-ASSISTED LIPOTRANSFER
dition.31 This effect may also contribute to the decreased
volume of central necrosis and more prominent microvasculature in the outer layers of the CAL fat seen in this study.
Surgical injury accompanying the transplantation and thesubsequent hypoxic condition and wound healing process,including inflammatory reactions, appear to trigger ASC differentiation into specific lineages, such as adipocytes, vascular endothelial cells, and mural cells. Because ASCs
are known to undergo adipogenic differentiation when cocultured with mature adipocytes,32 aspirated adipose tissuetransplanted together with ASCs may contribute to acuteadipogenic differentiation of ASCs.
In conclusion, aspirated fat contains less vasculature andfewer ASCs than excised fat. Transplanted aspirated fat survived better when transplanted with ASCs than without
ASCs. These findings may partly explain why transplanted
aspirated fat does not survive very well and suggest theclinical potential of the CAL method. Soft tissue augmentation with autologous fat, which leaves no incisional scarand lacks the complications associated with foreign materials, can be a cosmetically ideal tool when its effectivenessis improved.

ACKNOWLEDGMENTS
GFP rats [SD TgN(act-EGFP)OsbCZ-004] were kindly
provided by Professor Masaru Okabe (Osaka University,
Japan). Supported by grants-in-aid by Japanese Ministry of
Education, Culture, Sports, Science, and Technology (contract grant B2-16390507).

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Address reprint requests to:
Kotaro Yoshimura, M.D.
Department of Plastic Surgery
University of Tokyo School of Medicine
Tokyo
Japan
E-mail: yoshimura-pla@h.u-tokyo.ac.jp

FIG. S1. Honeycomb structures in subcutaneous layers after liposuction. Vascular and neural perforators arising from the fascia ormuscle are left intact after suctioning. Liposuction is usually performed with a metal cannula after infiltration of saline solution containing
lidocaine and adrenaline. This fact clearly supports that aspirated fat contains fewer vascular structures compared to excised fat.