EXPERIMENTAL

Fate of Adipose-Derived Stromal Vascular

Fraction Cells after Co-implantation with
Fat Grafts: Evidence of Cell Survival and
Differentiation in Ischemic Adipose Tissue
Su Fu, M.D.
Jie Luan, M.D.
Minqiang Xin, M.D.
Qian Wang, M.D.
Ran Xiao, M.D., Ph.D.
Yunzhou Gao, M.D.
Beijing, People’s Republic of China
Background: Some studies have suggested that adipose-derived stromal vascu-
lar fraction is a potential cell source responsible for the improved quality and
long-term retention of fat grafts, but studies that have clearly demonstrated the
survival and differentiation potential of the implanted stromal vascular fraction
cells as being dynamic phenomena have not been widely reported.
Methods: The authors isolated stromal vascular fraction cells from C57BL/6J-
GFP mice. Green fluorescence protein–positive stromal vascular fraction cells
were mixed with minced inguinal adipose tissue harvested from C57BL/6J
mice and then co-implanted into BALB/c nude mice. The survival of im-
planted green fluorescence protein–positive stromal vascular fraction cells
was tracked by in vivo fluorescence imaging for 56 days. Immunofluorescence
staining was used to analyze the differentiation of green fluorescence protein–
positive stromal vascular fraction cells occurring in ischemic adipose tissue at
7, 14, 28, 35, 42, or 56 days.
Results: The fluorescence signal intensity fell drastically within the first 14 days
after co-implantation and continued to decrease thereafter, with 17.3 percent
signal intensity (relative to day 1) at 56 days. Immunofluorescence staining re-
vealed that some green fluorescence protein–positive cells can spontaneously
differentiate into adipocytes from day 7, and some of the implanted stromal
vascular fraction cells can incorporate into new blood vessels.
Conclusions: The authors show convincing evidence for dynamic changes of
stromal vascular fraction cells after co-implantation with fat grafts. The results
prove the principle that implanted stromal vascular fraction cells can survive
in the ischemic microenvironment of fat grafts and participate in the process
of adipogenesis and angiogenesis. (Plast. Reconstr. Surg. 132: 363, 2013.)

A
utologous fat grafting today has become
a well-established and frequently applied
procedure of soft-tissue augmentation for
both cosmetic and reconstructive purposes. How-
ever, the volume of lipoaspirated fat that can be
transferred in a single session has been limited
by a variable but unpredictable rate of fat absorp-
tion.1–4 Efforts at overcoming this have focused
on harvesting techniques, fat manipulation, and
From the Breast Plastic and Reconstructive Surgery Center,
Plastic Surgery Hospital, Plastic Surgery Institute, and the
Department of Pathology, Chinese Academy of Medical Sci-
ences, Peking Union Medical College.
Received for publication November 30, 2012; accepted Feb-
ruary 19, 2013.
Copyright © 2013 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0b013e31829588b3
related approaches.5–11 However, there is no con-
sensus regarding the best fat grafting technique
and the longevity of results. Recently, a signifi-
cant increase in interest in regenerative medicine
using adipose-derived stromal vascular fraction
cells has emerged as a novel therapeutic option
for fat grafting.12–17
As the largest endocrine organ in the body,
adipose appears to provide an ideal pool of mul-
tipotent, undifferentiated, and nucleated cell
populations that are involved in homeostasis and
regenerative efforts.18–20 This has led plastic sur-
geons to seek techniques that involve digestion,
Disclosure: The authors have no conflicts of inter-
est to disclose.

www.PRSJournal.com 363
 Plastic and Reconstructive Surgery • August 2013
isolation, and concentration of adipose-derived
stromal vascular fraction cells, to be added back
to the lipoaspirated tissues to restore native cel-
lular quantitative levels. This technique has been
reported by Yoshimura et al. and is termed cell-
assisted lipotransfer.21–23
In breast augmentation trials, Yoshimura and
colleagues have combined stromal vascular frac-
tion cells with lipoaspirates from equal volumes
of adipose tissue. The authors note improved fat
grafting in the presence of the stromal vascular
fraction cells, with retention of volume for more
than 1 year without evidence of fibrosis.21–23 Some
studies implicate the potential benefits of stro-
mal vascular fraction cells, possibly attributable to
their ability to promote angiogenesis and/or to
enhance adipocyte progenitor proliferation and
differentiation.24,25 However, the actual mecha-
nisms of stromal vascular fraction cells in the
repair and regeneration process are still poorly
understood.26 Of equal importance is to under-
stand the fate of the implanted stromal vascular
fraction cells. Can these cells survive for a long
time in the ischemic microenvironment of fat
grafts? To date, studies that have clearly demon-
strated the survival and differentiation of stromal
vascular fraction cells as being dynamic phenom-
ena have not been widely reported.
In this study, we introduced an animal model
of cell-assisted lipotransfer to investigate the fate
of the implanted stromal vascular fraction cells
and related cellular events occurring in the repair
process. We provide evidence that subpopulations
of stromal vascular fraction cells can survive long
term in hosts after co-implantation with fat grafts.
Moreover, we demonstrate that the spontaneous
adipogenic differentiation of implanted stromal
vascular fraction cells occurs over time in vivo. We
also show that implanted stromal vascular fraction
cells are able to participate in the formation of
new blood vessels following ischemia.
MATERIALS AND METHODS
Animals
C57BL/6J–green fluorescence protein mice
were purchased from the Institute of Labora-
tory Animal Sciences, Chinese Academy of
Medical Sciences. C57BL/6J mice (B6) and
BALB/c nude mice (CAnN.Cg-Foxn1nu/CrlVr)
were purchased from the Chinese Academy of
Military Medical Sciences. Female mice with an
average age of 8 weeks were used for this study.
Animal care and experimental procedures were
performed under approval from the Animal
364
Care and Ethics Committee of Peking Union
Medical College.
Flow Cytometry of Stromal Vascular Fraction
Cells Isolated from Mouse Adipose Tissue
For a flow cytometry analysis experiment, we
isolated the stromal vascular fraction cells from
B6 mice. Briefly, under general anesthesia with
pentobarbital sodium (Sigma Chemical Company,
St. Louis, Mo.) at 0.1 mg/100 g, the inguinal adi-
pose tissue of B6 mice (n = 20) was harvested and
washed extensively with sterile phosphate-buff-
ered saline (HyClone Laboratories, Inc., Logan,
Utah). Acquired fat was collected, minced finely,
and then digested with 0.075% collagenase type I
(Sigma) in a prewarmed orbital shaker at 37°C for
60 minutes. A rotation of 150 rpm was applied to
ensure a thorough mixing and thus digestion of
the cell preparation. The digested fat was centri-
fuged for 10 minutes at 600 g at room tempera-
ture. The resulting pellet (stromal vascular fraction
cells) was resuspended in high-glucose Dulbecco’s
Modified Eagle Medium containing 10% fetal
bovine serum (Gibco, Carlsbad, Calif.) and cen-
trifuged at 600 g for 10 minutes. After washing in
phosphate-buffered saline, the suspension was fil-
tered through a 35-μm mesh and incubated with
the following monoclonal antibodies conjugated
to fluorochromes at 4°C for 30 minutes: mouse
anti–CD31-fluorescein isothiocyanate (eBiosci-
ence, Inc., San Diego, Calif.), mouse anti–CD34-
fluorescein isothiocyanate (eBioscience), mouse
anti–CD45-fluorescein isothiocyanate (eBiosci-
ence), and mouse anti–Sca-1-fluorescein isothio-
cyanate (eBioscience). After the washing steps, the
labeled cells were analyzed by flow cytometry using
a FACSCalibur flow cytometer and CellQuest Pro
software (BD Biosciences, Franklin Lakes, N.J.).
Cell-Assisted Lipotransfer Animal Model
To distinguish implanted stromal vascular
fraction cells from grafted adipose tissue, we
introduced a new animal model of cell-assisted
lipotransfer. The green fluorescence protein
mice, B6 mice, and BALB/c nude mice were
enrolled into this study. We isolated the stromal
vascular fraction cells from green fluorescence
protein mice as described above. The stromal vas-
cular fraction pellet was washed and resuspended
in sterilized phosphate-buffered saline. Cell num-
bers of freshly isolated stromal vascular fraction
cells were determined by trypan blue staining.
The inguinal adipose tissue was obtained from B6
mice and minced finely. Then, 0.5 ml (0.360 g) of
the minced adipose tissue was mixed with either
Volume 132, Number 2 • Stromal Vascular Fraction in Fat Grafts

Fig. 1. A cell-assisted lipotransfer animal model was developed to track the fate of stromal vascu-
lar fraction (SVF) cells after co-implantation with fat grafts. Inguinal adipose tissue was obtained
from a B6 mouse. Stromal vascular fraction cells were isolated from a green fluorescence protein
(GFP) mouse. Then, 0.5 ml (0.360 g) of the minced adipose tissue harvested from the B6 mouse
was mixed with 5 × 105 green fluorescence protein–positive stromal vascular fraction cells and
then co-implanted onto the skulls of BALB/c nude mice.

phosphate-buffered saline (10 μl) or 5 × 105 stro-
mal vascular fraction cells (5 × 105 cells in 10 μl of
phosphate-buffered saline) harvested from green
fluorescence protein mice and injected subcu-
taneously onto the skulls of BALB/c nude mice
using a 16-gauge needle to minimize physical dam-
age on adipocytes during injection (Fig. 1). There
were 60 mice in the green fluorescence protein–
positive stromal vascular fraction cells and phos-
phate-buffered saline control groups, respectively.
Four nude mice from each group were subjected
to fluorescence imaging after surgery. Eight nude
mice from each group were killed at each time
point (days 1, 7, 14, 28, 35, 42, and 56). The fat
grafts were harvested from each mouse, weighed,
and subjected to histologic examination.
protein–positive stromal vascular fraction cells
was dissected from mice, fixed with 4% parafor-
maldehyde/phosphate-buffered saline solution,
embedded in paraffin, and sectioned at 3 μm for
immunostaining. Sections were then incubated
with the following primary antibodies: rabbit anti-
perilipin A (Abcam, Cambridge, Mass.) and rat
anti-CD31 (Abcam). Isotypic antibody was used
as a negative control for each staining. For visu-
alization, Alexa Fluor 594-conjugated secondary
antibodies (Invitrogen, Carlsbad, Calif.) were
used. Sections were also stained with 4′,6-diamid-
ino-2-phenylindole. All the sections were imaged
on a Zeiss fluorescence microscope (Carl Zeiss,
Oberkochen, Germany). Images were acquired
and analyzed by AxioVision software (Carl Zeiss).

In Vivo Fluorescence Imaging Statistical Analysis

To determine the survival of implanted green
fluorescence protein–positive stromal vascular
fraction cells in living animals, four nude mice
from each group were subjected to fluorescence
imaging on days 1, 7, 14, 28, 35, 42, and 56 after
cell-assisted lipotransfer. Fluorescence intensity
was quantified by drawing a region of interest over
the signals in images in units of photons per sec-
ond per square centimeter per steradian using the
IndiGo software provided with the in vivo imaging
system (NightOWL LB983; Berthold Technolo-
gies, Bad Wildbad, Germany).
Immunofluorescence Staining for Grafted
Adipose Tissue
After 7, 14, 28, 35, 42, or 56 days, the grafted
adipose tissue containing green fluorescence
Data were expressed as the mean ± SD. SPSS soft-
ware version 18.0 (SPSS, Inc., Chicago, Ill.) was used
to perform one-way analysis of variance for evalua-
tion of the differences of grafts weight between the
two groups at each time point. Differences were
­considered statistically significant at p < 0.05.
RESULTS
Flow Cytometric Analysis and Expression of Stem
Cell Markers in the Stromal Vascular Fraction
Cells from Mouse Adipose Tissue
To confirm that stromal vascular fraction cells
are composed of multiple cell types, including
adipocyte progenitors, vascular progenitor cells,
stromal cells, and yet undefined stem/progenitor
cells, we analyzed adipose tissue of B6 mice by flow

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cytometry. The stromal vascular fraction cells were
isolated and stained for CD31 (endothelial cells),
CD34 (capillary endothelial cells, bone marrow
stroma, and a small subpopulation of mouse bone
marrow cells), CD45 (all nucleated hematopoietic
cells), and Sca-1 (a major phenotypic marker for
mouse hematopoietic progenitor/stem cell sub-
set). The total population of CD31+ cells in the
mouse stromal vascular fraction cell sample was
as low as 5.54 percent. Only a small population of
cells expressed CD34 (8.69 percent) in the stro-
mal vascular fraction cells. The CD45+ population
constituted approximately more than half of the
cells present in the freshly isolated stromal vascu-
lar fraction cells (61.10 percent), and the stem cell
antigen 1 (Sca-1) was expressed on the surface of
36.67 percent of the cells in the stromal vascular
fraction cells (Fig. 2 and Table 1).
Fat Survival Assessment
To assess fat survival in our cell-assisted lipo-
transfer animal model, 0.5 ml (0.360 g) of fat
mixed with either phosphate-buffered saline or 5
× 105 green fluorescence protein–positive stromal
vascular fraction cells was initially injected subcuta-
neously onto the skulls of each nude mouse. All of
the animals survived. The fat grafts were dissected
out and weighed at scheduled time points. The fat
survival was evaluated by mass measurement. The
graft mass was calculated as follows (Table 2): at

Fig. 2. Flow cytometric histograms of the stromal vascular fraction cells freshly isolated from adipose tissue
of B6 mice, stained for CD31-FITC (marker for endothelial cells), CD34-FITC (marker for capillary endothelial
cells, bone marrow stroma, and a small subpopulation of mouse bone marrow cells), CD45-FITC (marker for all
nucleated hematopoietic cells), and Sca-1-FITC (marker for mouse hematopoietic progenitor/stem cell subset),
and appropriate isotype control antibody (data not shown). FITC, fluorescein isothiocyanate. See Table 1 for the
quantification of stem cell markers expressed in the stromal vascular fraction cells.

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Table 1.  Quantification of Stem Cell Markers demonstrate that implanted green fluorescence
Expressed in the Stromal Vascular Fraction Cells from protein–positive stromal vascular fraction cells sur-
Mouse Adipose Tissue* vived in the fat grafts during the follow-up of 56
CD31 (%) CD34 (%) CD45 (%) Sca-1 (%) days (Fig. 4, above). No fluorescence signals were
5.54 8.69 61.10 36.67
observed in the phosphate-buffered saline control
FITC, fluorescein isothiocyanate.
group (date not shown). The fluorescence signal
*Flow cytometric analysis was performed on the stromal vascu- intensity fell drastically within the first 14 days after
lar fraction cells of mouse adipose tissues using antibodies against co-implantation and continued to decrease there-
CD31-FITC, CD34-FITC, CD45-FITC, and Sca-1-FITC. Flow cytomet-
ric analysis demonstrates that cells expressing the stem cell markers after, with 17.3 percent signal intensity (relative to
CD31, CD34, CD45, and Sca-1 are present within the stromal vascular day 1) at 56 days (Fig. 4, below).
fraction cells of mouse adipose tissue.
Evidence of Adipogenic Differentiation of
56 days, the graft mass of the phosphate-buffered Implanted Green Fluorescence Protein–Positive
saline control group was 0.115 g, and the graft Stromal Vascular Fraction Cells as Being
mass of the green fluorescence protein–positive Dynamic Phenomena
stromal vascular fraction cell group was 0.137 g. To evaluate the in vivo adipogenic potential of
Data were compared between the green fluores- the implanted green fluorescence protein–positive
cence protein–positive stromal vascular fraction stromal vascular fraction cells, immunofluores-
cell group and the phosphate-buffered saline cence staining of sections from grafted adipose
control group at each time interval. The results tissues was performed using anti–perilipin anti-
indicated that there was a statistically significant body (a specific marker for viable adipocytes).
difference in fat survival between these two groups Implanted green fluorescence protein–positive
at day 56 (p < 0.001) (Table 2 and Fig. 3). stromal vascular fraction cells were visualized 7 days
after co-implantation. Green fluorescence protein–
Implanted Green Fluorescence Protein–Positive positive, perilipin-positive adipocytes were found in
Stromal Vascular Fraction Cells Survive in the Fat the grafted adipose tissues, and some dying adipo-
Grafts during the Follow-Up of 56 Days cytes completely lost perilipin expression (Fig. 5,
To track the survival of stromal vascular frac- above). At 14 days, an increased number of small,
tion cells after co-implantation with fat grafts in round, green fluorescence protein–positive, per-
vivo, the BALB/c nude mice were imaged by in ilipin-positive adipocytes (<50 μm in diameter)
vivo fluorescence imaging at the scheduled time were detected in the fat grafts (Fig. 5, second row).
points. The fluorescence signal of green fluores- Surprisingly, immunofluorescence staining also
cence protein was detected and quantified as the revealed an increase in the number of green fluo-
number of emitted photons per second in the rescence protein–negative, perilipin-positive small
region of fat grafting. The changes of fluorescence adipocytes (<50 μm in diameter) on day 14. These
intensity can reflect the changes in the number of cells were negative for green fluorescence protein,
indicating that they were newly generated adipo-
implanted green fluorescence protein–positive
cytes derived from the adipose tissue of B6 mice
stromal vascular fraction cells. Fluorescence images
rather than implanted green fluorescence protein–
positive stromal vascular fraction cells. The shapes
Table 2.  Comparison of Graft Mass between Fat
of green fluorescence protein–positive, perilipin-
plus Phosphate-Buffered Saline and Fat plus Green
Fluorescence Protein–Positive Stromal Vascular positive cells changed over time, as they finally
Fraction Cell Groups at Scheduled Time Points* became lipid droplet–containing unilocular adipo-
cytes at 56 days (Fig. 5, below). These results clearly
Time Fat plus Fat plus GFP+
Point (day) PBS (g) SVF Cells (g) p† demonstrate that implanted stromal vascular frac-
tion cells can differentiate into adipocytes under
1 0.350 ± 0.004 0.351 ± 0.005 0.796
7 0.340 ± 0.004 0.337 ± 0.005 0.384 the ischemic microenvironment of fat grafts in vivo
14 0.223 ± 0.008 0.237 ± 0.004 0.001 and contribute to adipose tissue regeneration.
28 0.164 ± 0.005 0.190 ± 0.006 <0.001
35 0.141 ± 0.006 0.165 ± 0.008 <0.001
42 0.129 ± 0.005 0.153 ± 0.008 <0.001 Implanted Green Fluorescence Protein–Positive
56 0.115 ± 0.006 0.137 ± 0.005 <0.001 Stromal Vascular Fraction Cells Participate in
PBS, phosphate-buffered saline; GFP+, green fluorescence protein– Vascular Regeneration In Vivo
positive; SVF, stromal vascular fraction.
*Data are expressed as the mean ± SD (n = 8 in each group). To further assess the therapeutic value of the
†A value of p < 0.05 is considered significant. implanted green fluorescence protein–positive

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Fig. 3. Mass of adipose tissue at each time point (n = 8). The mass decreased sharply on day 14, fol-
lowed by a further decrease over time. Statistical analysis was performed between the two groups
at each time point (*p < 0.05). The mass was higher in the green fluorescence protein–positive (GFP)
stromal vascular fraction (SVF) cell group on day 56. *p < 0.05 versus phosphate-buffered saline (PBS)
control group.

Fig. 4. Fluorescence imaging of implanted green fluorescence protein–positive stromal vascular fraction
cells. (Above) Fluorescent images of a representative mouse over time. The images demonstrate continuous
fluorescence signals within the region of fat grafting during the 56-day period and indicate the long-term
survival of implanted green fluorescence protein–positive stromal vascular fraction cells in ischemic adipose
tissue. (Below) Quantification of fluorescence intensity of the region of interest in mice. The fluorescence
intensity changed with time after co-implantation, as evidenced by a dramatic decrease within the first 14
days and a continued decrease thereafter. The fluorescence intensity of the region of interest at 56 days was
only 17.3 percent that of the day-1 signal intensity.

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Fig. 5. Immunofluorescence staining analysis of the adipogenic differentiation of green fluorescence protein (GFP)–positive stro-
mal vascular fraction cells after co-implantation with fat grafts. Stromal vascular fraction cells (green) overlap with 4′,6-diamidino-
2-phenylindole (DAPI) (blue) in perilipin-positive (red) adipose tissue in situ. Arrows indicate the area shown at high magnification
in the inset. (Above) Green fluorescence protein–positive, perilipin-positive adipocytes were detected on day 7 (white arrows).
Large lipid droplets resulting from adipocyte death were negative for perilipin. (Second row) At 14 days, an increased number
of small, round, perilipin-positive adipocytes were detected (merged image, yellow arrows). Note that some of them were green
fluorescence protein–positive cells (white arrows). (Third row, fourth row, and below) The shapes of green fluorescence protein–
positive, perilipin-positive cells changed over time, as they finally became lipid droplet–containing unilocular adipocytes at 56
days (white arrows) (original magnification, × 20). Scale bars = 200 μm.

stromal vascular fraction cells in neovasculariza-
tion, immunofluorescence staining of sections
from grafted adipose tissue was performed using
anti-CD31 antibody. We observed an increased
neovascularized capillary density in the fat grafts
from day 14 to day 56 after the co-implantation,
indicating the existence of a vascular regeneration
process in the fat grafts (Fig. 6). At 28 days, green
fluorescence protein–positive, CD31+ vascular
endothelial cells were detected next to the CD31+
vascular structures (Fig. 6, second row). At 56 days,
green fluorescence protein–positive, CD31+ vas-
cular endothelial cells completely incorporated
into the vascular structures (Fig. 6, below). Some
green fluorescence protein–positive, CD31+ vascu-
lar endothelial cells were also detected among the
new blood vessels (Fig. 6, third row). These results
show that implanted stromal vascular fraction

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 Plastic and Reconstructive Surgery • August 2013

Fig. 6. Immunofluorescence staining analysis of the angiogenic differentiation of green fluorescence protein (GFP)–positive stro-
mal vascular fraction cells after co-implantation with fat grafts. Stromal vascular fraction cells (green) overlap with CD31 (red) and
4′,6-diamidino-2-phenylindole (DAPI) (blue) in grafted adipose tissue in situ. Arrows indicate the area shown at high magnification
in the inset. (Above) At 14 days, neovascularized vessels were rarely observed. Yellow arrows indicate detected endothelial cells. A
few green fluorescence protein–positive stromal vascular fraction cells, accumulated as small clusters, were also detected (white
arrows). (Second row) At 28 days, the CD31+ vascular structures were observed (merged image, yellow arrow). Green fluorescence
protein–positive, CD31+ vascular endothelial cells were detected next to the vascular structure (white arrows). (Third row) Some
green fluorescence protein–positive, CD31+ vascular endothelial cells (white arrows) were detected among the new blood vessels
(merged image, yellow arrows). (Below) Green fluorescence protein–positive, CD31+ vascular endothelial cells (white arrows) com-
pletely incorporated into the vascular structures (merged image, yellow arrow) (original magnification, × 20). Scale bars = 200 μm.

cells participate in vascular regeneration of the
ischemic adipose tissue in vivo and therefore may
represent a promising tool for the angiogenesis
for fat grafting.
DISCUSSION
During the past decade, regenerative medi-
cine has recognized the potential value and
availability of a heterogeneous, undifferentiated,
nucleated cell population in adipose-derived
stromal vascular fraction.12,13,19,20 These findings
sparked extensive clinical and research efforts
to promote successful autologous fat grafting
using the multipotent nature of stromal vascular
fraction cells within fat. Theoretically, fat graft-
ing is a logical application for adipose-derived
cell therapies, because the isolated cells presum-
ably are involved in the repair process of injured
adipose tissue.26 However, previous understand-
ing of the nature of stromal vascular fraction cells
and their ultimate outcome after co-implantation
with fat grafts remains far from satisfactory. In this
study, the observations that we were able to make
were focused specifically on the survival and dif-
ferentiation of implanted stromal vascular frac-
tion cells.
In vivo fluorescence imaging has increasingly
become an important noninvasive technique for
evaluating the fate of implanted cells over time.

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With regard to the cell labeling method, many
published results support the green fluorescence
protein transgenic mouse, which ubiquitously
expresses green fluorescence protein in every cell,
as a generally useful tool for cell-tracking stud-
ies.27 Therefore, we established a new cell-assisted
lipotransfer animal model with C57BL/6J-green
fluorescence protein, C57BL/6J, and BALB/c
nude mice to distinguish the implanted stromal
vascular fraction cells from grafted adipose tissue.
Our experiment of using stromal vascular fraction
cells isolated from the green fluorescence protein
transgenic mouse allows us to continuously moni-
tor the viability of implanted stromal vascular frac-
tion cells in vivo.
Results from in vivo fluorescence imaging
indicated that implanted green fluorescence
protein–positive stromal vascular fraction cells
were retained in the fat grafts through 56 days
after implantation. Compared with day 1, serial
fluorescence imaging indicated that cell numbers
decrease by approximately 47.2 percent within
14 days and by approximately 17.3 percent at
56 days. These results demonstrate that the sub-
populations of stromal vascular fraction cells can
survive long term in hosts after co-implantation
with fat grafts; however, most of these cells tend
to die rapidly at early stages. This observed phe-
nomenon of cell loss is similar to that reported
by Suga et al. in their surgically induced fat pad
ischemia model.28–30 The reasons for the dramatic
decrease of cell number at early stages is obscure
but probably are related to the host local environ-
ment (e.g., lack of nutrition in the fat grafts). In
our study, we noted that complete vascularization
of the grafts could not be observed before day 14.
Therefore, increasing the nutrient supply to the
fat grafts at early stages (e.g., local administration
of platelet-rich plasma to the site of implantation)
would likely provide a much more favorable envi-
ronment to promote long-term survival and thera-
peutic effectiveness of implanted stromal vascular
fraction cells.
Some researchers believe that the therapeutic
effects of implanted stromal vascular fraction cells
may be attributable to their paracrine functions
rather than cellular contributions. For example,
hematopoietic stem cells within adipose stromal
vascular fraction have been reported to express
many growth factors, including vascular endo-
thelial growth factor, hepatocyte growth factor,
insulin-like growth factor 1, and fibroblast growth
factor 2.31 However, in the present study, we found
evidence that implanted green fluorescence
protein–positive stromal vascular fraction cells
spontaneously differentiated into adipocytes in
fat grafts. We observed the dynamic morphologic
changes of green fluorescence protein–positive,
perilipin-positive cells from day 7 to day 56. Our
experiment also provided evidence of intrinsic
regenerative changes in fat grafting. Small adipo-
cytes (<50 μm in diameter) strongly stained for
perilipin but negative for green fluorescence pro-
tein were observed on day 14, indicating that they
were newly generated adipocytes differentiated
from the adipose-derived stromal vascular frac-
tion of B6 mice. These results demonstrate the
cellular contribution of stromal vascular fraction
cells to adipose tissue regeneration.
We also evaluated the angiogenic differen-
tiation potential of implanted stromal vascular
fraction cells in this study. Previous studies have
highlighted the significant value of stromal vas-
cular fraction cells in angiogenesis. Koh et al.
suggested that freshly isolated stromal vascular
fraction cells can effectively induce new vessel for-
mation through the dynamic reassembly of blood
endothelial cells at the site of implantation.32 Our
findings strongly support previous research show-
ing that implanted stromal vascular fraction cells
can participate in vascular regeneration of the
ischemic adipose tissue. However, it remains to
be determined whether these green fluorescence
protein–positive, CD31+ cells were derived from
the differentiation of hematopoietic components
of stromal vascular fraction. Because our flow
cytometric analyses revealed that approximately
5.54 percent of stromal vascular fraction cells con-
sist of endothelial cells, these CD31+ cells might
directly incorporate into blood vessels without
further differentiation. Therefore, it would be
intriguing to isolate the pure endothelial cells or
the hematopoietic component from stromal vas-
cular fraction, respectively, and assess their effi-
cacy in vascular regeneration.
Although the fat survival assessment is not
the focus of this study, our results confirmed the
efficacy of implanted stromal vascular fraction
cells in a cell-assisted lipotransfer animal model.
However, analysis of graft mass on days 56 showed
that only 38 percent of the initial graft mass was
retained in the presence of stromal vascular frac-
tion cell supplementation. This result is not as
good as that reported by Moseley et al.33 This is
presumably because of a relatively higher volume
of fat (0.5 ml) injected over the skull of each
mouse (the more fat grafted, the lower its sur-
vival rate). In addition, we think the low number
of implanted green fluorescence protein–positive
stromal vascular fraction cells (5 × 105 cells per
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 Plastic and Reconstructive Surgery • August 2013
mouse) might be another reason for the lower
fat survival in our study. Further study is needed
to understand the dose-response relationship of
treatment with stromal vascular fraction cells.
CONCLUSIONS
In conclusion, the authors show convincing
evidence of dynamic changes of stromal vascular
fraction cells after co-implantation with fat grafts.
Although most implanted stromal vascular frac-
tion cells died shortly after co-implantation, some
of the stromal vascular fraction cells remained alive
in the ischemic microenvironment of fat grafts
and participated in the process of adipogenesis
and angiogenesis. This information is essential for
us to be able to optimize or promote specific stem
cell activity and lineage differentiation in future
soft-tissue regenerative clinical applications. For
therapeutic purposes, using freshly isolated stro-
mal vascular fraction cells is beneficial in terms
of convenience and safety. However, it remains
unclear how cross-talk between adipocytes and cel-
lular components of the stromal vascular fraction
affects their respective functions. Our next move
will be to focus on the isolation and purification
of the subpopulation of stromal vascular fraction
cells and to determine their respective therapeu-
tic values in fat grafting.
Jie Luan, M.D.
Breast Plastic and Reconstructive Surgery Center
Plastic Surgery Hospital
Chinese Academy of Medical Sciences
Peking Union Medical College
33 Badachu Road
Shijingshan District
Beijing 100144, People’s Republic of China
doctorluanjie@sina.com
ACKNOWLEDGMENT
This research was supported by funding from the
Plastic Surgery Hospital, Chinese Academy of Medical
Sciences.
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