Cell-Assisted Lipotransfer: Supportive Use of Human
Adipose-Derived Cells for Soft Tissue Augmentation
DAISUKE MATSUMOTO, M.D.,1KATSUJIRO SATO, M.D.,1KOICHI GONDA, M.D.,1
YASUYUKI TAKAKI, M.D.,2TOMOKUNI SHIGEURA, M.S.,2TAKAHIRO SATO, M.S.,2
EMIKO AIBA-KOJIMA, M.D.,1FUMIKO IIZUKA, M.D.,1KEITA INOUE, M.D.,1
HIROTAKA SUGA, M.D.,1and KOTARO YOSHIMURA, M.D.1
Injective 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 this
study, 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, and
ASC yield was lower in aspirated fat. Aspirated fat was transplanted subcutaneously into severe com-
bined immunodeficiency mice with (cell-assisted lipotransfer; CAL) or without (non-CAL) vascular
stromal 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 Willebrand
factor, suggesting differentiation into vascular endothelial cells. Another experiment that used vascular
stromal 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. These
findings may partly explain why transplanted aspirated fat does not survive well and suggest clinical
potential of the CAL method for soft tissue augmentation.
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,3–7chon-
genic15–17lineages. Adipose tissue is known to be rich in
characteristics and to differentiate into vascular endothelial
DIPOSE-DERIVED STROMAL (STEM) CELLS (ASCs) can be
obtained from liposuction aspirates1,2and differentiate
mesenchymal progenitors in surface marker expression pro-
file; 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 those
investigating bone defect23(fresh ASCs) and rectovaginal
fistula24(cultured ASCs) treatments and soft tissue aug-
mentation by cell-assisted lipotransfer25(our unpublished
data; fresh ASCs). If ASCs are harvested from a large
1Department of Plastic Surgery, University of Tokyo School of Medicine, Tokyo, Japan.
2Department of Research and Development, Biomaster Inc., Kanagawa, Japan.
Volume 12, Number 12, 2006
# Mary Ann Liebert, Inc.
clinically without cell expansion because a sufficient num-
ber can be obtained. Furthermore, the use of minimally
manipulated fresh cells may lead to greater safety and effi-
cacy 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 enhance-
problems to be resolved, such as unpredictability and a low
survival rate due to partial necrosis,26,27it is almost the only
method of soft tissue augmentation that can be performed
without detectable scarring on a donor or a recipient site and
without complications associated with foreign materials.
Thus, if the clinical efficacy and safety of the procedure can
be improved, it could be a strong cosmetic and reconstruc-
tive 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-rich
aspirated fat). In CAL, ASCs were supportively used to
boost the efficacy of autologous lipoinjection (resulting in a
higher survival rate and persistency of transplanted fat) and
to decrease known adverse effects of lipoinjection, such as
formation of fibrosis, pseudocyst, and calcification. In this
study, aspirated fat was compared with excised whole fat
(nonaspirated fat) in microscopic and electromicroscopic
histology and adherent cell yields. In addition, we evaluated
the effectiveness of CAL in animal models compared with
non–cell-assisted lipotransfer (non-CAL), and examined the
fate of ASCs in fat transplanted with the CAL method.
MATERIALS AND METHODS
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 (20–30g) was also taken. Aspirated fat was
used 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.
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 phosphate-
buffered saline (PBS) and digested on a shaker at 378C in
PBS containing 0.075% collagenase for 30min. Mature
adipocytes and connective tissues were separated from
pellets by centrifugation (800g, 10min). The pellets were
resuspended and filtered with a 100-mm mesh (Millipore,
MA). Freshly isolated SVFs were plated (30,000cells/cm2)
on gelatin-coated dishes and cultured at 378C in an atmo-
sphere of 5% carbon dioxide (CO2) in humid air. The culture
medium was M-199 containing10% fetal bovine serum, 100
IU penicillin, 100mg/mL streptomycin, 5mg/mL heparin,
and 2ng/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 then
processed in the same manner as aspirated fat.
Mouse models for transplantation
of human aspirated fat
Seven-week-old male severe combined immunodefi-
ciency (SCID) mice housed with free access to water and
standard chow diet were anesthetized by intraperitoneal
injection of 5mg/mL pentobarbital. For preparation of graft
material, human aspirated fat was washed with saline and
poured into 10-mL syringes, which were then placed upright
at room temperature for 10min. The infranatant fluid was
then discarded. The human aspirated fat (1mL¼900mg)
was subcutaneously injected into the back of the SCID mice
with or without freshly isolated SVF cells. For CAL, SVFs
taken from 4mL of aspirated fat was mixed with 1mL of
aspirated fat. Four weeks later, transplanted fat was har-
vested, weighed, and fixed with 4% paraformaldehyde, and
4mm sections were stained with hematoxylin-eosin.
Mouse models for tracing human SVF cells in CAL
For tracing the SVFs, SVF cells freshly isolated from
human aspirated fat were labeledby incubated with 5mg/mL
CM-DiI (Molecular Probes, Eugene, Oregon) for 1h before
transplantation. DiI-labeled SVFs taken from 4mL of aspi-
rated fat were mixed with 1mL of aspirated fat and then
injected under the back skin of a SCID mouse. In the same
manner, human aspirated fat was injected without DiI-
labeled SVFs as control. Four weeks later, transplanted fat
was harvested, fixed with 4% paraformaldehyde, and em-
bedded in optimal cutting temperature (OCT) compound by
mm-thick frozen sections were examined with a confocal
microscope system (Leica TCS SP2, Leica Microsystems
GmbH, Wetzlar, Germany).
Rat models for tracing rat green fluorescent
protein–labeled SVF in CAL by using
rat minced fat
Inguinal adipose tissue excised from green fluorescent
protein (GFP) rats [SD TgN(act-EGFP)OsbCZ-004; gener-
MATSUMOTO ET AL.
ated from Sprague-Dawley (SD) rats] was processed in the
same way as human excised fat. GFP-SVF taken from 1mL
of fat of the GFP rat was mixed with minced inguinal adi-
pose tissue (1mL) harvested from SD rats and then injected
under the back skin of an SD rat. Four weeks later, trans-
planted fat was harvested and forwarded to immunohistol-
ogy as well.
Frozen sections were prepared from transplanted fat em-
bedded in OCT compound. After fixation in 100% acetone
0.5% goat serum in PBS. Paraffin-embedded sections
ASC yeield from suctioned
fat / ASC yield from whole fat
#1#2 #3 Ave.
aspirated fatexcised fat
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 micro-
graph [SEM] photos; red scale bar¼200mm, white scale bar¼
40mm). The basic structure of adipose tissue was preserved in the
aspirated fat, while vascular vessels, especially large one, were
significantly 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 as-
pirated fat was significantly less (48%?13%) than that from
Comparison of human aspirated fat and excised whole
fat mass [mg]
non-CAL fatCAL fat
man aspirated fat and vascular stromal fractions. (A) Photographs
of CAL fat (right) and non-CAL fat (left) in severe combined
immunodeficiency mice. Human aspirated fat was transplanted
with (CAL fat) or without (non-CAL fat) human vascular stromal
fractions freshly isolated from the same aspirated fat. Trans-
planted fat was harvested at 4 weeks. (B) Weight of harvested
CAL fat and non-CAL fat. CAL fat weighed significantly more
than non-CAL fat (p<.05). (C) Histologic features of CAL fat
and non-CAL fat samples (red scale bar¼250mm, black scale
bar¼100mm). Central necrosis (*) was almost always seen in non-
CAL fat, and the surviving layer of CAL fat was thicker than that
of non-CAL fat. At higher magnification, microvasculature was
more frequently seen in CAL fat, especially in the outer layers,
than in non-CAL fat.
Cell-assisted lipotransfer (CAL) and non-CAL using hu-
were dewaxed, washed in PBS, and treated with protei-
nase K (DakoCytomation, Carpinteria, CA) for 6min and
blocked with 0.5% goat serum in PBS. Primary and sec-
room temperature. 40,6-diamidino-2-phenylindole (DAPI)
staining was performed with DAPI containing mounting
medium (Vector Laboratories, Burlingame, CA). The fol-
lowing antibodies and dilutions were used: anti-green
fluorescent protein (mouse, 1:500; Molecular Probes), von
Willebrand factor (rabbit, 1:500; DakoCytomation), and
antirabbit antibodies (goat, 1:200; Molecular Probes). Sec-
tions were imaged on a confocal microscope system (Leica
Scanning electron microscopic study
Aspirated and excised fat were fixed with 2% paraf-
ormaldehyde and 2.5% glutaraldehyde in 0.2M cacodylate
buffer for 1 week at room temperature, and then fixed in 1%
osmium tetroxide. After dehydration, they were dried with a
super-critical-point CO2dryer (HCP-2, Hitachi, Tokyo, Ja-
with a scanning electron microscope (S3500N, Hitachi).
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.
Comparison of histologic features and ASC yield
between aspirated fat and excised fat
Both aspirated fat and excised fat were harvested from
the 3 patients who underwent the tummy-tuck operation.
Histologic examinations with a light microscope and scan-
ning electron microscope showed that aspirated fat pre-
serves basic structures of adipose tissue and that mature
adipocytes keep normal adhesions with each others. The
only difference detected was that aspirated fat contained
fewer vascular structures, especially large ones, compared
with excised fat.
The same weights of aspirated fat and excised fat were
processed for isolation of SVFs. The cell isolation process
was performed within 2h after harvest. The isolated SVFs
were culturedfor1week,and thenumbers ofadherent ASCs
from the aspirated fat and the excised fat were counted and
compared. In all 3 patients, the number of adherent ASCs at
The ratio of normalized ASC number from aspirated fat to
that from excised fat was 0.48?0.13 (n¼3) (Fig. 1B).
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?45.3mg (n¼10) or
520.6?40.8mg (n¼11), respectively, while fresh adipose
tissue (1mL) before transplantation was approximately
900mg (Fig. 2A and B). Histology of transplanted fat
samples showed that the central region was necrotic in non-
CAL 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
Fate of human ASCs transplanted with
human aspirated fat
To trace human ASCs, SVFs freshly isolated from aspi-
rated fat were labeled with DiI and then transplanted with
human aspirated fat. In CAL fat, DiI-labeled ASCs were
occasionally detected between mature adipocytes and in the
connective tissue of the transplanted fat (Fig. 3). ASCs po-
sitive for both DiI and von Willebrand factor were detected
in CAL fat, suggesting that some transplanted ASCs were
differentiated into vascular endothelial cells (Fig. 4).
Fate of ASCs derived from GFP rats
in rat CAL models
To traceASCs transplanted with fat, minced fat ofSD 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 sup-
posed to be transplanted GFP-ASCs, were detected within
connective 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 von
Willebrand factor and GFP were occasionally detected. The
GFP-positive cells partly or entirely covered the inner sur-
face of vessels (Fig. 5B). It was suggested that some ASCs
differentiated into endothelial cells and contributed to an-
giogenesis during the surviving process of the adipose trans-
Adipose tissue is predominantly composed of extracellular
matrix; mature adipocytes; ASCs; vascular cells, such as
endothelial cells; and mural cells, such as pericytes and
vascular smooth muscle cells. We recently reported the cell
MATSUMOTO ET AL.
composition of SVFs freshly isolated from liposuction aspi-
rates, which is important information for the clinical use of
SVFs.22The SVFs are composed of heterogeneous popula-
tions, including blood-derived cells, which are composed of
50%–70% SVF cells. Adipose-derived cells (CD45?cells) in
SVFs isolated from aspirated fat were of the following types:
ASCs (70%–90%; CD31?CD34þCD45?CD90þCD105?
CD146?), vascular endothelial (progenitor) cells (3%–9%;
cytes (2%–5%; CD31?CD34?CD45?CD90þCD105?
CD146þ), and other cells. The cell composition of the SVFs
digestion for 30min, but not all adipose-derived cells can be
isolated from the SVFs in this process; thus, the cell com-
position may not accurately correspond to the actual cell
composition of the adipose tissue. The location of ASCs in
the adipose tissue is not clearly understood. Some ASCs are
supposed to be located in the connective tissues in adipose,
and others are located between adipocytes or around micro-
vasculature or macrovasculature.
Liposuction aspirates consist of 2 parts: a floating fatty
portion and an infranatant fluid portion. We have in-
vestigated cells derived from these portions of liposuction
aspirates and found that a substantial number of adipose-
derived cells, including ASCs and endothelial cells, can
be isolated from the fluid portion, although the number
from the fluid portion is smaller than that from the fatty
portion.22This finding supports a result of this study
showing that aspirated fat is relatively stem cell–deficient
compared with excised whole fat. The reason was not elu-
cidated here, but mechanical injury during the liposuc-
tion procedure and digestion by endogenous proteases
during the surgery or subsequent storage periods proba-
bly 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 the
aspirated fat, but vascular vessels, especially larger ones,
are significantly less detected in aspirated fat than in ex-
cised fat. It is well known that the honeycomb structures of
vascular and neural perforator networks are left intact
in aspirated sites after liposuction operation. (Click here
for supplementary material.) Thus, it is reasonable that
fewer ASCs, which also reside around capillaries and ves-
sels,were isolated fromaspirated fat,whichdoesnotcontain
larger vessels, than excised fat. Unlike our results, a recent
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-2-
phenylindole (DAPI). In CAL fat, DiI-labeled cells, which are
supposed to be co-transplanted adipose-derived stromal (stem)
cells, are located between mature adipocytes and in interstitial
connective tissue. The frequency of DiI-positive cells was 10%–
30%, although it varied among samples and at locations in a
sample. Scale bar¼50mm. The right column shows merges of DiI
and DAPI, and differential interference contrast (DIC) images.
Histologic features of cell-assisted lipotransfer (CAL)
using human aspirated fat and DiI-labeled vascular stromal frac-
tions. Normal excised fat (control fat) and non-CAL fat were also
assessed for comparison. Endothelial cells were immunostained
with von Willebrand factor (vWF). Nuclei were stained with 40,6-
diamidino-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 adipose-
derived stromal (stem) cells, were observed in connective tissue or
between adipocytes. Scale bar¼10mm. The right column shows
merges of immunostaining of vWF, DiI, DAPI, and differential
interference contrast images.
Immunohistology of cell-assisted lipotransfer (CAL) fat
study28that compared viable cell yield from fresh aspirated
versus fresh excised fat did not detect a significant differ-
ence. However, this finding was probably occurred because
the investigators centrifuged the aspirated fat and removed
fibrous structures and visible vessels from excised fat during
the cell isolation process.
The results of the present study suggest that addition of
ASCs to aspirated fat improves the efficacy of adipose
transfer, although the exact mechanisms remain to be elu-
cidated. By addition of ASCs to relatively ASC-poor fat
(aspirated fat), the aspirated fat is theoretically converted to
relatively ASC-rich fat. A recent study using fragmented
omentum tissue suggested the effects of co-transplantation
with preadipocytes, but the fragmented adipose tissue in that
study 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 in
this study as well as in previous studies,19–21ASCs can dif-
ferentiate into vascular endothelial cells that may contribute
to neoangiogenesis during the healing process after trans-
plantation. 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 were
located 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 asadipose progenitorcells
for future turnover of adipocytes. The relative deficiency of
ASCs in aspirated fat compared with excised fat may con-
tribute to the low long-term survival rate in non-CAL fat,
which is a well-known clinical phenomenon.26,27This hy-
pothesis is also supported by a recent study29showing that
fragmented omentum (mainly composed of adipose) with or
without 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,30the relative deficiency of
tissue-specific progenitor cells in non-CAL fat may affect
the coming turnover of the tissue and lead to its long-term
atrophy.Turnoverofadipocytesmay occurinthe earlystage
after transplantation in transplanted adipose tissue because
its vascularity was temporarily damaged; this outcome may
be the reason that atrophy of transplanted adipose is clini-
cally seen during the first 3 months.
Third, some ASCs may differentiate into mature adipo-
cytes and partly constitute transplanted fat. Although adi-
pogenic differentiation of labeled ASCs was not detected in
this 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 knowl-
edge, no previous report has clearly demonstrated labeled
cytoplasm of mature adipocytes.
Fourth, transplanted ASCs were kept in a hypoxic con-
dition in the acute phase after transplantation and may re-
lease angiogenic soluble factors such as vascular endothelial
growth factor and hepatocyte growth factor, accelerat-
ing neoangiogenesis from the surrounding host tissue in a
paracrine manner. Itwas reported that cultured human ASCs
produce and release these growth factors in a hypoxic con-
(fragmented fat of Sprague-Dawley [SD] rat and green fluorescent
protein – 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, all
adherent cells were GFP-positive. (B) Immunohistology of cell-
assisted lipoprotein fat in rat models. GFP was detected by im-
munostaining with anti-GFP antibody. Adipose tissue of SD rats
and GFP rats were also demonstrated for comparison. Vascular
endothelial 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, suggest-
ing that they were differentiated from co-transplanted GFP-ASCs.
The other endothelial cells were only positive for vWF, suggesting
that they were derived from the host SD rat. White scale bar¼30
mm, yellow scale bar¼10mm. The right column shows merges of
immunostaining of vWF, DiI, 40,6-diamidino-2-phenylindole, and
differential interference contrast images.
Cell-assisted lipotransfer (CAL) fat in rat models
MATSUMOTO ET AL.
dition.31This effect may also contribute to the decreased
volume of central necrosis and more prominent microvas-
culature in the outer layers of the CAL fat seen in this study.
Surgical injury accompanying the transplantation and the
subsequent hypoxic condition and wound healing process,
includinginflammatoryreactions, appeartotriggerASC dif-
ferentiation into specific lineages, such as adipocytes, vas-
cular endothelial cells, and mural cells. Because ASCs
are known to undergo adipogenic differentiation when co-
cultured with mature adipocytes,32aspirated adipose tissue
transplanted together with ASCs may contribute to acute
adipogenic differentiation of ASCs.
In conclusion, aspirated fat contains less vasculature and
fewer ASCs than excised fat. Transplanted aspirated fat sur-
vived better when transplanted with ASCs than without
ASCs. These findings may partly explain why transplanted
aspirated fat does not survive very well and suggest the
clinical potential of the CAL method. Soft tissue augmen-
tation with autologous fat, which leaves no incisional scar
and lacks the complications associated with foreign mate-
rials, can be a cosmetically ideal tool when its effectiveness
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 (con-
tract 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
MATSUMOTO ET AL.
FIG. S1. Download full-text
muscle 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.
Honeycomb structures in subcutaneous layers after liposuction. Vascular and neural perforators arising from the fascia or