EXPERIMENTAL
Preserved Proliferative Capacity andMultipotency of Human Adipose-Derived StemCells after Long-Term Cryopreservation
Koichi Gonda, M.D.
Tomokuni Shigeura, M.S.
Takahiro Sato, M.S.
Daisuke Matsumoto, M.D.
Hirotaka Suga, M.D.
Keita Inoue, M.D.
Noriyuki Aoi, M.D.
Harunosuke Kato, M.D.
Katsujiro Sato, M.D.
Syoko Murase, Ph.D.
Isao Koshima, M.D.
Kotaro Yoshimura, M.D.
Tokyo and Kanagawa, Japan

Background: Human adipose-derived stem (stromal) cells are promising as aregenerative therapy tool for defective tissues of mesenchymal lineage, including fat, bone, and cartilage, and blood vessels. In potential future clinicalapplications, adipose-derived stem cell cryopreservation could be an indispensable fundamental technology, as has occurred in other fields involving cell-based
therapies using hematopoietic stem cells and umbilical cord blood cells.
Methods: The authors examined the proliferative capacity and multipotency ofhuman adipose-derived stem cells isolated from lipoaspirates of 18 patients intotal before and after a 6-month cryopreservation following their defined protocol. Proliferative capacity was quantified by measuring doubling time in cell
culture, and multipotency was examined with differentiation assays for chondrogenic, osteogenic, and adipogenic lineages. In addition, expression profilesof cell surface markers were determined by flow cytometry and compared
between fresh and cryopreserved adipose-derived stem cells.
Results: Cryopreserved adipose-derived stem cells fully retained the potential
for differentiation into adipocytes, osteoblasts, and chondrocytes and for proliferative capacity. Flow cytometric analyses revealed that surface marker expression profiles remained constant before and after storage.
Conclusions: Adipose-derived stem cells can be cryopreserved at least for up to
6 months under the present protocol without any loss of proliferative or differentiation potential. These results ensure the availability of autologous banked
adipose-derived stem cells for clinical applications in the future. (Plast. Reconstr. Surg. 121: 401, 2008.)

Human adipose-derived stem (stromal) cells1,2
are promising as a tool of regenerative therapies for tissue defects of mesenchymal lineage, including fat,3 skeletal and cardiac muscles,4,5
bone,6 –10 cartilage,11–13 and blood vessels.14 –16 They
have already been clinically used for enhancing regeneration of fat,17,18 vasculature,19 and bone.20 In
addition, they are reported to be differentiated into
other cell types deriving from different germ layers
such as neural cells21 and hepatocytes.22 Several studies have demonstrated that human adipose-derived
stem cells can differentiate into vascular endothelial
cells14 –16 and augment tissue neovascularization in aparacrine manner in an ischemic environment.23
From the Department of Plastic Surgery, University of Tokyo
School of Medicine, and the Division of Research and Development, Biomaster, Inc.
Received for publication August 28, 2006; accepted February
8, 2007.
Copyright ©2008 by the American Society of Plastic Surgeons
DOI: 10.1097/01.prs.0000298322.70032.bc

Thus, it is currently thought that adipose-derived
stem cells contribute to bioengineering and transplantation of tissues not only by acting as tissuespecific precursor cells but also through reinforcing
vascularization of regenerating tissue, which underlines the importance of adipose-derived stem cells ingeneral applications of tissue engineering.
As emphasized for other types of stem/progenitor cells,24 harvested autologous adipose-derived stem cells will most probably be preserved for
multiple clinical applications in the future. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies, includingtransportability of stem cells, pooling of cells toreach a therapeutic dose, and time for the completion of safety and quality control testing.24

Disclosure: The authors declare that they have nocompeting financial interests.

www.PRSJournal.com

401

Plastic and Reconstructive Surgery • February 2008
Therefore, cryopreservation of adipose-derived
stem cells should be intensively investigated for
optimization as an indispensable fundamental
technology. As the first step to achieving this aim,
we examined the proliferative capacity and multipotency of human adipose-derived stem cells before and after long-term (6 months) cryopreservation under our defined protocol.

MATERIALS AND METHODS
Human Tissue Samples
Adipose-derived stem cells were harvested at surgery from lipoaspirates of 18 female patients, aged
20 to 51 years (mean, 32.3 years). Donor sites were
the abdomen and/or thighs. Before liposuction, patients received external ultrasound pretreatment at
the areas to be suctioned with External Ultrasonic
Assist (Silberg, Santa Rosa, Calif.) after epidural anesthesia. After saline solution supplemented with
0.1% lidocaine and 0.001% epinephrine was infiltrated into the adipose compartment, liposuction
was performed manually with a hollow blunt-tipped
cannula (diameter, 2 to 4 mm).
Doubling times were determined for adiposederived stem cells from six patients before cryopreservation and from another five patients after 6 months of cryopreservation; therefore, thecell lines for the assays before and after cryopreservation were not identical. Similarly, fivelines of adipose-derived stem cells were assayedfor determination of chondrogenic potential
with the micromass culture system (see below)
before cryopreservation, and another six lines
were examined after cryopreservation. For osteogenic and adipogenic differentiation assays,
six lines of adipose-derived stem cells were assayed before cryopreservation, whereas a distinct five lines were examined after cryopreservation. Of these six and five lines, one in the before
and after groups was from the same patient, and theothers were all from separate patients. As for flow
cytometry, we analyzed the expression of a set of
surface markers (see below) of five lines of adiposederived stem cells before cryopreservation and sixlines after cryopreservation; one patient both before
and after cryopreservation; four other patients before cryopreservation, and five other patients after
cryopreservation.
Ideally, cell lines subjected to each assay before and after cryopreservation should be identical, derived from the same donor patient. In determining the experimental design, however, wefound that adipose-derived stem cell yield from asingle patient was not large enough to perform all

402

the intended assays using both fresh and frozen
cells, unless a long-term cell culture was conducted before carrying out the assays. Therefore,
in some assays in this study, different cell lines
were used.
Cell Isolation and Culture
All protocols used for these procedures havebeen described previously.25 Briefly, the suctioned
fat was digested with 0.075% collagenase in phosphate-buffered saline for 30 minutes on a shaker
at 37°C. Mature adipocytes and connective tissues
were separated from pellets by centrifugation (800g for 10 minutes). Pellets were resuspended inerythrocyte lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM
ethylenediaminetetraacetic aced) and incubatedfor 5 minutes at room temperature. The pellets
were resuspended and passed through a 100-␮mmesh filter (Millipore, Billerica, Mass.), yielding
stromal vascular fraction containing adipose-derived
stem cells. Freshly isolated cells were plated in medium at a density of 5 ⫻ 106 nucleated cells per
100-mm gelatin-coated dish, and cultured at 37°C,
5% carbon dioxide, in humid air. The culture medium was M-199 containing 10% fetal bovine serum,
100 IU of penicillin, 100 mg/ml streptomycin, 5␮g/ml heparin, and 2 mg/ml acidic fibroblast
growth factor. Primary cells were cultured for 7 days
and were defined as passage 0. The medium was
replaced every 3 days, and cells were subcultured
every week. After primary culture for 7 days, attachedcells were subcultured by trypsinization and plated
in the same medium at a density of 2000 cells/cm2.
Cryopreservation of Cells
Adipose-derived stem cells were counted andresuspended in cryoprotective medium, Cell
Banker 1 (Wako Chemicals Co., Ltd., Osaka,
Japan) at a density of 106 cells/ml. The manufacturer does not disclose its ingredients but
states that it contains fetal bovine serum. They
were then frozen under temperature control
using a programmed freezer (PROFREEZE
TNP-87S2Q; Nihon Freezer Co., Ltd., Tokyo,
Japan) under a protocol of 4°C for 5 minutes,
then decreasing ⫺1°C per minute until the temperature reached ⫺50°C, followed by a decreaseof ⫺5°C per minute until ⫺80°C was reached.
Samples were then transferred to a liquid nitrogen tank (Isothermal Vapor Storage System
V-1500 with Series 2300 Auto-fill/Monitor System; Custom Biogenic Systems, Shelby Township, Mich.) for long-term storage at ⫺196°C.

Volume 121, Number 2 • Human Adipose-Derived Stem Cells
Cells before cryopreservation were subjected toeach assay at passage 1 or 2, whereas those after
6 months of cryopreservation underwent the assays at passage 2 or 3. Cells that had been cryopreserved for 6 months were rapidly thawed ina warm water bath set at 37°C, spun to remove
the cryoprotective agent, and seeded onto appropriate culture plates for each experiment.
Measurement of Doubling TimeFresh or cryopreserved adipose-derived stem
cells were seeded onto six-well plates at a densityof 1 ⫻ 105 cells per well and allowed to grow until
they reached logarithmic growth phase. It usually
took 3 days for cells to start proliferating logarithmically, but sometimes (especially at passage 0) it
took 5 to 6 days. The cells were then sequentiallytrypsinized at intervals of 48 hours and countedwith a cell counter (NucleoCounter; Chemo Metec,
Allerød, Denmark). Doubling time was calculated
according to the following formula: doubling time ⫽48 hours/log2(N2/N1), where N1 is the first cellcount and N2 is the cell count 48 hours later.

Medium and 10% fetal bovine serum was used forthe culture medium as negative control. Quantification was performed by measuring the micromass sizes on microphotographs with a scale using
image analysis software (Scion v.3.53; Scion Corp.,
Frederick, Md.). Safranin O staining was performed for qualitative analysis.
To test osteogenic differentiation, calcium
deposition was evaluated based on the orthocresolphthalein complexone method with theCalcium C-Test Wako Kit (Wako Chemicals) according to the manufacturer’s instructions. After 5 ⫻ 104 cells/60-mm dish were cultured in
M-199 containing 10% fetal bovine serum for 6days, the medium was replaced with osteogenic
differentiation medium (Dulbecco’s Modified
Eagle Medium containing 10% fetal bovine serum, 50 ␮M ascorbate-2-phosphate, 0.1 ␮M
dexamethasone, and 10 mM ␤-glycerophosphate) or control medium (Dulbecco’s Modi-

Induced Differentiation of Cultured
Adipose-Derived Stem Cells
The protocol for chondrogenic differentiation (micromass culture system) has been described previously.26 Dulbecco’s Modified Eagle

Fig. 1. Doubling time of fresh and cryopreserved human adipose-derived stem cells. Six lines of human adipose-derived stem
cells before the freezing and thawing process (Fresh) and fivelines harvested after 6 months of cryopreservation (Cryopreserved) were assayed for cell proliferation rate, and their doublingtimes were determined. NS, no significant difference. Values aremean ⫾ SD.

Fig. 2. Chondrogenic potentials of fresh and cryopreserved human adipose-derived stem cells. Five lines of human adiposederived stem cells before the freezing and thawing process(Fresh) and six lines harvested after being cryopreserved for 6
months (Cryopreserved) were subjected to micromass culture in
the defined chondrogenic differentiation medium (purple columns) or control medium (gray columns) to assess chondrogenic
potential. Micromass sizes were determined using image analysis software on microphotographs with scale as an area (in millimeters squared). NS, no significant difference.

403

Plastic and Reconstructive Surgery • February 2008
fied Eagle Medium with 10% fetal bovine serum). Cells were then assayed after 1, 2, 3, and4 weeks of differentiation culture.
For assessment of adipogenic differentiation,
cells were seeded onto 96-well plates at a densityof 1 ⫻ 104 cells/well. After 4 days of culture, medium was replaced with adipogenic differentiation
medium (Dulbecco’s Modified Eagle Medium
with 10% fetal bovine serum, 0.5 mM isobutyl
methylxanthine, 1 ␮M dexamethasone, 10 ␮M insulin, and 200 ␮M indomethacin) or control medium (Dulbecco’s Modified Eagle Medium with
10% fetal bovine serum). Cells were further cultured for 1, 2, 3, or 4 weeks and stained with 5
␮l/well of AdipoRed-Lipid Assay Reagent (Cambrex Corp., East Rutherford, N.J.). After 10 minutes of incubation, the fluorescence with excitation at 485 nm and emission at 535 nm wasmeasured with a microplate spectrophotometer
reader Model 680 (BioRad, Hercules, Calif.).

Flow Cytometry
Flow cytometric analyses were performed as
described elsewhere.25 The following monoclonal
antibodies conjugated to fluorochromes were used:anti-CD29(␤1-integrin)-PE, CD34-PE, CD44-PE,
CD49d-PE, CD90(Thy-1)-PE, CD117-PE, and Tie2-PE (BD Biosciences, San Diego, Calif.); CD71-PE
and CD105-PE (Serotec, Oxford, United Kingdom); and CD144-PE (Beckman Coulter, Fullerton, Calif.).
Statistical Analysis
Measured values were expressed as means ⫾ SD;t tests were used to compare the population means
between groups when their population varianceswere assumed to be equal, whereas Welch’s t test
was used when variances were unequal. F tests
were used to assess variances of the two groups
being compared.

Fig. 3. Chondrogenic potentials of fresh and cryopreserved human adipose-derived stem cells. Photomicrographs of representative sections of micromass. Paraffin-embedded sections were stained with safranin-O and counterstained with hematoxylin: fresh adipose-derived stem cells in chondrogenic medium(above, left), fresh adipose-derived stem cells in control medium (above, right), cryopreserved adiposederived stem cells in chondrogenic medium (below, left), and cryopreserved adipose-derived stem cells incontrol medium (below, right). Bar ⫽ 250 ␮m.

404Volume 121, Number 2 • Human Adipose-Derived Stem Cells
RESULTS
Proliferative Capacity of Fresh andCryopreserved Adipose-Derived Stem Cells
Doubling times were comparable between
fresh and cryopreserved adipose-derived stem
cells (Fig. 1), indicating that cryopreservation did
not affect the proliferative capacity of the adiposederived stem cells.
Chondrogenic Potential of Fresh andCryopreserved Adipose-Derived Stem Cells
The chondrogenic potential of nine lines ofadipose-derived stem cells, consisting of four freshcell lines and five cryopreserved lines, was assayed

Fig. 4. Osteogenic differentiation potentials of human adiposederived stem cells before and after long-term cryopreservation.
Fresh (red lines) and cryopreserved (blue lines) adipose-derived
stem cells derived from a single patient were assayed for calcium
deposition after 1, 2, 3, and 4 weeks of cell culture with osteogenic
differentiation medium (filled circles) or control medium (filled
rectangles). Fresh adipose-derived stem cells with (Fresh/induced) or without (Fresh/uninduced) osteogenic induction are indicated with red circles and red rectangles, respectively; cryopreserved adipose-derived stem cells with (Cryopreserved/induced)
or without (Cryopreserved/induced) osteogenic induction are indicated with blue circles and blue rectangles, respectively.

with the micromass culture protocol, followed bymorphometry for quantification (Fig. 2) and safranin O staining for qualitative analysis (Fig. 3).
Sizes of micromass, determined by measuring theareas of the masses using image analysis software,
could be assumed to reflect the capability of chondrogenic matrix production. Judged based on the
micromass sizes, chondrogenic potentials of freshand cryopreserved adipose-derived stem cells were
not significantly different. The SD of micromass
sizes of cryopreserved adipose-derived stem cells
cultured in chondrogenic medium was larger thanthat of fresh cells, suggesting variable but definite
effects of long-term cryopreservation on theirchondrogenic potential. Microphotographs of safranin O–stained sections of micromass showed higher
production of proteoglycan matrix in cells withchondrogenic induction than in controls in both
fresh and cryopreserved adipose-derived stem cells(Fig. 3). As with the observation from the quantification assay above, micromasses showed comparable
degrees of chondrogenic matrix production in bothgroups.

Fig. 5. Osteogenic differentiation potentials of human adiposederived stem cells before and after long-term cryopreservation. Sixlines of human adipose-derived stem cells before the freezing andthawing process (Fresh) and five lines after cryopreservation for 6
months (Cryopreserved) were assayed for calcium deposition to assess osteogenic potential with (purple columns) or without (gray columns) osteogenic induction. NS, no significant difference.

405

Plastic and Reconstructive Surgery • February 2008
Osteogenic and Adipogenic Potential of
Adipose-Derived Stem Cells before and after
Cryopreservation
Fresh and cryopreserved adipose-derived stem
cells were assayed for osteogenic (Figs. 4 and 5) andadipogenic (Figs. 6 and 7) potential. Osteogenic
potential, determined as calcium deposition in intracellular and extracellular spaces, was comparable
between fresh and cryopreserved adipose-derived
stem cells; in fact, cryopreserved cells surpassed freshcells. Adipogenic potential, determined based on
spectrophotometric absorbance using AdipoRed
staining, showed no apparent difference between
fresh and cryopreserved adipose-derived stem cells.
These results indicate that long-term cryopreservation up to 6 months does not affect the osteogenic
and adipogenic potential of adipose-derived stem
cells.
Fig. 7. Adipogenic differentiation potentials of human adiposederived stem cells before and after long-term cryopreservation.
Six lines of human adipose-derived stem cells before the freezing
and thawing process (Fresh) and five lines after cryopreservationfor 6 months (Cryopreserved) were assayed for lipid deposition toassess adipogenic potential with (purple columns) or without (graycolumns) adipogenic induction. NS, no significant difference.

Fig. 6. Adipogenic differentiation potentials of human adiposederived stem cells before and after long-term cryopreservation.
Fresh and cryopreserved adipose-derived stem cells derived
from the same patient were assayed for oil deposition as a quantification of adipogenic differentiation at the indicated time
point. Fresh adipose-derived stem cells with (Fresh/induced) orwithout (Fresh/uninduced) osteogenic induction are indicatedwith red circles and red rectangles, respectively; cryopreserved adipose-derived stem cells with (Cryopreserved/induced) or without(Cryopreserved/induced) osteogenic induction are indicated withblue circles and blue rectangles, respectively.

406

Flow Cytometric Analysis of Cell Surface
Markers on Adipose-Derived Stem Cells
Cell surface marker expression is closely related to cell lineage and biological properties, including multipotentiality. Expression profiles ofcell surface markers, and their change over passages, were analyzed with flow cytometry. Adiposederived stem cells before and after 6 months ofcryopreservation showed similar expression patterns of the cell surface markers selected for evaluation at all passage numbers examined (i.e., passages 0, 1, 2, 3, and 7). Also, sequential changes inthe expression patterns were quite similar between the two groups of adipose-derived stem
cells. Only CD34 declined with increased passage
number; the other markers generally remained
constant in both groups (Fig. 8 and Table 1).
Therefore, as far as those analyzed marker subsets
are concerned, the expression profile of cell surface markers of adipose-derived stem cells underwent little change through cryopreservation.

DISCUSSION
In this study, we demonstrated that humanadipose-derived stem cells preserve their proliferative capacity, mesenchymal multipotency (chon-

Volume 121, Number 2 • Human Adipose-Derived Stem Cells

Fig. 8. Representative data of cell surface marker expression in
freshandcryopreservedadipose-derivedstemcells(passages0and
7) obtained from a single patient. Adipose-derived stem cells before(Fresh) and after 6 months of cryopreservation (Cryopreserved) wereanalyzed at passages 0 and 7 with flow cytometry for expression of
a selected set of cell surface markers. The representative data from
freshandcryopreservedadipose-derivedstemcells(passages0and
7) obtained from a single patient are shown. IgG1 indicates thenegative control using a nonspecific mouse immunoglobulin G1
species as an antibody to determine background fluorescence.

drogenic, osteogenic, and adipogenic lineages),and surface marker expression profiles after 6
months of cryopreservation. The only difference
we detected between fresh and cryopreserved adipose-derived stem cells in this study was the larger
variability in chondrogenic differentiation potentials of cryopreserved adipose-derived stem cells.
Although the results do not guarantee the validityof preservation longer than 6 months for adiposederived stem cells, it is clinically of great importance that at least a single cycle of freezing, thawing, and storage at ⫺196°C up to 6 months does
not affect the biological characteristics of humanadipose-derived stem cells. Overall, the results ofthe present study would encourage practicing application of cryopreserved adipose-derived stem
cells for potential cell-based therapies.
Human fat is readily obtained from liposuction and frequently used as a filler material forsoft-tissue augmentation.18,27,28 Because multiple
lipoinjection is frequently necessary for maximizing cosmetic results, some cosmetic surgeons arestrongly inclined to store and repeatedly use theharvested fat. Thus, cryopreserved human fat tissue has been investigated,29 –31 but there have been
few reports about the effects of cryopreservation
on adipose-derived stem cells.32–35 The three reports by Thirumala et al.32–34 studying human adipose-derived stem cells as isolated cells focused
mainly on the physical effect of freezing on cell
membrane integrity; another report35 focused on
adipose-derived stem cell yield from cryopreserved fat. To our knowledge, before the present
study, there have been no reports investigating thepotential of cryopreserved human adipose-derived stem cells as a tool for cell therapy. One of
the advantages of adipose-derived stem cells compared with other adult stem cells is a large cell
yield; a large volume of adipose tissue can be obtained easily, with minimal morbidity. Accordingly, adipose-derived stem cells can be theoretically clinically used without cell expansion, but if
adipose-derived stem cells can be cultured andstored for clinical use, the value of adipose-derived
stem cells in cell-based therapy would be greatly
enhanced. The potential of clinical application of
adipose-derived stem cells in plastic surgery has
been reviewed elsewhere.18,36
The effects of cryopreservation have been investigated using other types of stem cells. Cryopreserved cord blood stem cells retain clonogenic
capacity and immunophenotypic composition
comparable to fresh cells,37 whereas cryopreservation does not markedly affect the rate of proliferation and multipotency of murine neural pre-407

Plastic and Reconstructive Surgery • February 2008
Table 1. Expression of Cell Surface Markers in Fresh and Cryopreserved Human Adipose-Derived Stem Cells*
CD29
␤1-Integrin
Fresh
Cryo.
CD34
Fresh
Cryo.
CD44
Pgp-1
Fresh
Cryo.
CD49d
␣4-Integrin
Fresh
Cryo.
CD71
Transferrin receptor
Fresh
Cryo.
CD90
Thy-1
Fresh
Cryo.
CD105
Endogrin
Fresh
Cryo.
CD117
c-kit
Fresh
Cryo.
CD144
VE-cadherin
Fresh
Cryo.
Flk-1
VEGFR-2 (KDR)
Fresh
Cryo.
Tie-2
Fresh
Cryo.

P0

P1

P2

P3

P7

Mean

98.6 ⫾ 1.0
91.1 ⫾ 11.3

99.1 ⫾ 0.3
84.6 ⫾ 35.2

99.3 ⫾ 0.2
98.3 ⫾ 1.7

99.4 ⫾ 0.3
99.2 ⫾ 0.5

99.5 ⫾ 0.4
90.7 ⫾ 13.8

99.2 ⫾ 0.6
92.8 ⫾ 17.3

52.6 ⫾ 30.3
45.1 ⫾ 32.8

26.7 ⫾ 26.5
31.8 ⫾ 18.7

8.9 ⫾ 14.9
10.5 ⫾ 7.6

25.7 ⫾ 20.5
15.3 ⫾ 11.8

9.9 ⫾ 4.2
10.5 ⫾ 14.2

25.4 ⫾ 25.4
22.6 ⫾ 22.6

98.2 ⫾ 0.9
96.5 ⫾ 4.9

99.2 ⫾ 0.3
99.2 ⫾ 0.8

99.6 ⫾ 0.2
98.6 ⫾ 1.8

99.6 ⫾ 0.2
99.7 ⫾ 0.2

99.7 ⫾ 0.2
97.1 ⫾ 3.8

99.3 ⫾ 0.7
98.2 ⫾ 3.0

86.1 ⫾ 2.8
81.0 ⫾ 14.6

92.4 ⫾ 5.5
95.5 ⫾ 5.1

97.9 ⫾ 1.8
96.0 ⫾ 3.0

98.3 ⫾ 1.0
98.2 ⫾ 1.2

98.9 ⫾ 0.8
86.8 ⫾ 17.6

94.6 ⫾ 5.8
91.5 ⫾ 11.8

2.7 ⫾ 3.2
1.8 ⫾ 2.6

11.7 ⫾ 23.6
6.1 ⫾ 9.6

2.3 ⫾ 2.1
3.5 ⫾ 4.8

4.7 ⫾ 7.6
5.6 ⫾ 5.4

0.9 ⫾ 1.0
4.4 ⫾ 5.8

4.6 ⫾ 11.2
4.3 ⫾ 5.8

98.8 ⫾ 1.3
99.1 ⫾ 0.7

99.7 ⫾ 0.2
99.7 ⫾ 0.3

99.8 ⫾ 0.2
99.8 ⫾ 0.2

99.8 ⫾ 0.2
99.8 ⫾ 0.2

99.9 ⫾ 0.0
99.3 ⫾ 0.7

99.6 ⫾ 0.7
99.5 ⫾ 0.5

89.4 ⫾ 11.1
88.3 ⫾ 20.7

90.7 ⫾ 9.6
97.8 ⫾ 1.9

97.6 ⫾ 2.1
93.1 ⫾ 13.6

92.4 ⫾ 14.2
96.9 ⫾ 3.2

97.6 ⫾ 1.0
91.4 ⫾ 9.9

93.4 ⫾ 9.2
93.5 ⫾ 11.7

13.6 ⫾ 17.2
4.6 ⫾ 6.2

19.6 ⫾ 18.7
10.1 ⫾ 9.2

13.4 ⫾ 10.1
16.2 ⫾ 12.8

11.4 ⫾ 15.5
16.0 ⫾ 16.3

24.3 ⫾ 26.0
14.4 ⫾ 20.5

16.6 ⫾ 17.6
12.3 ⫾ 13.7

0.2 ⫾ 0.2
0.2 ⫾ 0.2

0.2 ⫾ 0.2
0.2 ⫾ 0.2

0.2 ⫾ 0.1
0.1 ⫾ 0.2

0.5 ⫾ 1.0
0.7 ⫾ 1.5

0.5 ⫾ 0.9
0.2 ⫾ 0.3

0.3 ⫾ 0.6
0.3 ⫾ 0.7

5.4 ⫾ 2.4
1.8 ⫾ 1.2

7.2 ⫾ 5.3
4.0 ⫾ 3.6

5.7 ⫾ 3.3
4.3 ⫾ 2.8

5.6 ⫾ 6.2
6.4 ⫾ 6.5

10.9 ⫾ 11.5
7.6 ⫾ 7.5

7.0 ⫾ 6.4
4.8 ⫾ 5.0

12.9 ⫾ 27.5
8.1 ⫾ 19.1

6.0 ⫾ 12.3
7.2 ⫾ 14.6

6.7 ⫾ 11.9
6.5 ⫾ 8.9

13.7 ⫾ 26.4
18.1 ⫾ 29.2

21.3 ⫾ 40.3
16.0 ⫾ 33.2

12.0 ⫾ 23.7
11.2 ⫾ 21.8

*Adipose-derived stem cells before (Fresh) and after 6 months of cryopreservation (Cryo.) were analyzed at passages 0, 1, 2, 3, and 7 (P0, P1,
P2, P3, and P7) using flow cytometry for expression of a selected set of cell surface markers. Percentages of positive cells for each surface marker
are shown. Data were collected from five fresh lines and six cryopreserved lines; one line from the fresh and one from the cryopreserved were
derived from the same patient; the others were derived from different patients. Values are mean ⫾ SD.

cursor cells.38 These results and our present results
indicate that the freeze-and-thaw process andlong-term storage in liquid nitrogen do not critically alter the phenotype of stem/precursor
cells. A recently emerging concept of gene activity is that gene expression status is regulated
by epigenetic modifications to the genomic DNA
and its related histone proteins, in the form of
DNA methylation and covalent modifications ofhistone tails.39,40 These epigenetic modifications
are covalent and therefore are presumably unaffected by a cryopreservation process, resulting in
no essential changes in cell gene expression profileand phenotype. Although cryopreservation could
cause some damage to the structural and functional408

proteins of the cells and reduce their viability, immunophenotypic changes in the cells might scarcely
occur.
The only positive finding in this study is that
the SD of the chondrogenic potential of cryopreserved adipose-derived stem cells cultured inchondrogenic medium, based on micromass sizes,
was much larger than that of fresh cells. One explanation for this finding is that individual differences were simply greater in the cryopreserved
adipose-derived stem cell group than in the fresh
adipose-derived stem cell group. (Note that noneof the cell lines was identical between the twogroups.) Another explanation is that cryopreservation affected the chondrogenic differentiation

Volume 121, Number 2 • Human Adipose-Derived Stem Cells
potentials of some of the adipose-derived stem celllines both positively and negatively.
With advances in tissue engineering and regenerative medicine, use of adult stem cells may be
a solid therapeutic option in the near future, and ourdata can contribute to developing protocols for regenerative therapies with cryopreserved adipose-derived stem cells. As for other types of adult stem cells(e.g., hematopoietic stem cells and umbilical cord
blood cells), standard cryopreservation protocols
have been established, and the safety of long-term
storage has been demonstrated.41,42 Based on thefindings regarding these antecedent stem cells, it islikely that we can reasonably extend the period of
cryopreservation of adipose-derived stem cells, although the safety of doing so should be examinedfurther.
Before the present study, we had not fully optimized the protocol for cryopreservation of adipose-derived stem cells. Among the various parameters relating to cryopreservation of cells,
cryoprotective agents could significantly affect thesurvival rate and proliferation potential of the frozen cells after thawing.43– 46 We used the cryoprotective medium Cellbanker 1 because it is widelyused in Japan and our previous pilot study suggested that adipose-derived stem cell viability after
1 month of cryopreservation was better whenstored with Cellbanker 1 compared with otherwell-known cryopreservation agents such as dimethyl sulfoxide, trehalose, dextran, methylcellulose, and their combinations. The cryoprotective
medium used in this study contains fetal bovine serum as a supplement, which contributes to prevention of viability loss of adipose-derived stem cells
during freezing and thawing. Given the point of viewthat animal-derived biological ingredients should becompletely eliminated from the process for cells to
be used clinically in cell therapy, and to pursue conditions for better survival of the cryopreserved cells,
the cryoprotective agent may have to be further optimized for adipose-derived stem cell storage. The
effect of cooling rate on cell freezing is anothersignificant factor affecting cell viability after cryopreservation and should be determined for eachcell species respectively, depending on physiochemical properties, especially membrane permeability as a major parameter.24 We have adopted
the standard protocol of cooling rate for many
cultured cells, but the specific protocol for adipose-derived stem cells should again be worked
out for better survival rate, proliferation rate, anddifferentiation potentials after cryopreservation.

CONCLUSIONSA primary purpose of the present study was not
to define the best protocol for the cryopreservation of adipose-derived stem cells by comparing
various protocols or to assess the significance ofeach parameter relating to the cryopreservation of
adipose-derived stem cells but to determine theeffects of cryopreservation on the phenotypes,
proliferative potentials, and differentiation potentials of adipose-derived stem cells, which indicate
their potentials for cell-based therapy. Thus, ourresults might well be regarded as having beenobtained under specific conditions of cryopreservation that still remain to be optimized. Even under such storage conditions, however, we showedthat adipose-derived stem cells still retain their
proliferative and differentiation potentials after
long-term cryopreservation.
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

ACKNOWLEDGMENTS

This work was supported by Japanese Ministry of
Education, Culture, Sports, Science, and Technology
grant B2-16390507. The authors are grateful to A.
Kurata, A. Matsuura, and S. Kawarasaki for technicalsupport.
REFERENCES
1. Zuk, P. A., Zhu, M., Mizuno, H., et al. Multilineage cells fromhuman adipose tissue: Implications for cell-based therapies.
Tissue Eng. 7: 211, 2001.
2. Zuk, P. A., Zhu, M., Ashjian, P., et al. Human adipose tissue
is a source of multipotent stem cells. Mol. Biol. Cell 13: 4279,
2002.
3. Matsumoto, D., Sato, K., Gonda, K., et al. Cell-assisted lipotransfer (CAL): Supportive use of human adipose-derived
cells for soft tissue augmentation with lipoinjection. TissueEng. 12: 3375, 2006.
4. Rodriguez, A. M., Pisani, D., Dechesne, C. A., et al. Transplantation of a multipotent cell population from humanadipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J. Exp. Med. 201: 1397, 2005.
5. Miyahara, Y., Nagaya, N., Kataoka, M., et al. Monolayered
mesenchymal stem cells repair scarred myocardium after
myocardial infarction. Nat. Med. 12: 459, 2006.
6. Halvorsen, Y. C., Wilkison, W. O., and Gimble, J. M. Adiposederived stromal cells: Their utility and potential in boneformation. Int. J. Obes. Relat. Metab. Disord. 24(Suppl. 4): S41,
2000.
7. Dragoo, J. L., Samimi, B., Zhu, M., et al. Tissue-engineered
cartilage and bone using stem cells from human infrapatellar
fat pads. J. Bone Joint Surg. (Br.) 85: 740, 2003.
8. Cowan, C. M., Shi, Y. Y., Aalami, O. O., et al. Adipose-derived
adult stromal cells heal critical-size mouse calvarial defects.
Nat. Biotechnol. 22: 560, 2004.

409

Plastic and Reconstructive Surgery • February 2008
9. Hicok, K. C., Du Laney, T. V., Zhou, Y. S., et al. Human
adipose-derived adult stem cells produce osteoid in vivo.
Tissue Eng. 10: 371, 2004.
10. Peterson, B., Zhang, J., Iglesias, R., et al. Healing of critically
sized femoral defects, using genetically modified mesenchymal stem cells from human adipose tissue. Tissue Eng. 11: 120,2005.
11. Erickson, G. R., Gimble, J. M., Franklin, D. M., et al. Chondrogenic potential of adipose tissue-derived stromal cells in
vitro and in vivo. Biochem. Biophys. Res. Commun. 290: 763,
2002.
12. Awad, H. A., Halvorsen, Y. D., Gimble, J. M., et al. Effects oftransforming growth factor beta1 and dexamethasone on thegrowth and chondrogenic differentiation of adipose-derived
stromal cells. Tissue Eng. 9: 1301, 2003.
13. Huang, J. I., Zuk, P. A., Jones, N. F., et al. Chondrogenic
potential of multipotential cells from human adipose tissue.
Plast. Reconstr. Surg. 113: 585, 2004.
14. Planat-Benard, V., Silvestre, J. S., Cousin, B., et al. Plasticity
of human adipose lineage cells toward endothelial cells:
Physiological and therapeutic perspectives. Circulation 109:
656, 2004.
15. Miranville, A., Heeschen, C., Sengenes, C., et al. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation 110: 349, 2004.
16. Cao, Y., Sun, Z., Liao, L., et al. Human adipose tissue-derived
stem cells differentiate into endothelial cells in vitro andimprove postnatal neovascularization in vivo. Biochem. Biophys. Res. Commun. 332: 370, 2005.
17. Yoshimura, K., Matsumoto, D., and Gonda, K. A clinical trialof soft tissue augmentation by lipoinjection with adiposederived stromal cells (ASCs). In Proceedings of the 8th Annual
Meeting of Tissue Engineering Society International (TESI), Shanghai, China, October 22–25, 2005. Pp. 206 –207.
18. Moseley, T. A., Zhu, M., and Hedrick, M. H. Adipose-derived
stem and progenitor cells as fillers in plastic and reconstructive surgery. Plast. Reconstr. Surg. 118(3 Suppl.): 121S, 2006.
19. Garcia-Olmo, D., Garcia-Arranz, M., Herreros, D., et al. A
phase I clinical trial of the treatment of Crohn’s fistula byadipose mesenchymal stem cell transplantation. Dis. Colon
Rectum 48: 1416, 2005.
20. Lendeckel, S., Jodicke, A., Christophis, P., et al. Autologous
stem cells (adipose) and fibrin glue used to treat widespreadtraumatic calvarial defects: Case report. J. Craniomaxillofac.
Surg. 32: 370, 2004.
21. Safford, K. M., Hicok, K. C., Safford, S. D., et al. Neurogenic
differentiation of murine and human adipose-derived stromal cells. Biochem. Biophys. Res. Commun. 294: 371, 2002.
22. Talens-Visconti, R., Bonora, A., Jover, R., et al. Hepatogenic
differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal
stem cells. World J. Gastroenterol. 12: 5834, 2006.
23. Rehman, J., Traktuev, D., Li, J., et al. Secretion of angiogenic
and antiapoptotic factors by human adipose stromal cells.
Circulation 109: 1292, 2004.
24. Hubel, A. Parameters of cell freezing: Implications for the
cryopreservation of stem cells. Transfus. Med. Rev. 11: 224,
1997.
25. Yoshimura, K., Shigeura, T., Matsumoto, D., et al. Characterization of freshly isolated and cultured cells derived fromthe fatty and fluid portions of liposuction aspirates. J. Cell.
Physiol. 208: 64, 2006.

410

26. Johnstone, B., Hering, T. M., Caplan, A. I., et al. In vitro
chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238: 265, 1998.
27. Fulton, J. E., Suarez, M., Silverton, K., et al. Small volume fat
transfer. Dermatol. Surg. 24: 857, 1998.
28. Coleman, S. R. Structural fat grafting: More than a permanent filler. Plast. Reconstr. Surg. 118: 108S, 2006.
29. Ullmann, Y., Shoshani, O., Fodor, L., et al. Long-term fat
preservation. J. Drugs Dermatol. 3: 266, 2004.
30. Butterwick, K. J., Bevin, A. A., and Iyer, S. Fat transplantation
using fresh versus frozen fat: A side-by-side two-hand comparison pilot study. Dermatol. Surg. 32: 640, 2006.
31. Moscatello, D. K., Dougherty, M., Narins, R. S., et al. Cryopreservation of human fat for soft tissue augmentation: Viability requires use of cryoprotectant and controlled freezing
and storage. Dermatol. Surg. 31: 1506, 2005.
32. Thirumala, S., Gimble, J. M., and Devireddy, R. V. Transportphenomena during freezing of adipose tissue derived adultstem cells. Biotechnol. Bioeng. 92: 372, 2005.
33. Thirumala, S., Zvonic, S., Floyd, E., et al. Effect of various
freezing parameters on the immediate post-thaw membrane
integrity of adipose tissue derived adult stem cells. Biotechnol.
Prog. 21: 1511, 2005.
34. Devireddy, R. V., Thirumala, S., and Gimble, J. M. Cellularresponse of adipose derived passage-4 adult stem cells tofreezing stress. J. Biomech. Eng. 127: 1081, 2005.
35. Pu, L. L., Cui, X., Fink, B. F., et al. Adipose aspirates as asource for human processed lipoaspirate cells after optimal
cryopreservation. Plast. Reconstr. Surg. 117: 1845, 2006.
36. Tholpady, S. S., Llull, R., Ogle, R. C., Rubin, J. P., Futrell,
J. W., and Katz, A. J. Adipose tissue: Stem cells and beyond.
Clin. Plast. Surg. 33: 55, 2006.
37. Almici, C., Carlo-Stella, C., Wagner, J. E., et al. Clonogenic
capacity and ex vivo expansion potential of umbilical cord
blood progenitor cells are not impaired by cryopreservation.
Bone Marrow Transplant. 19: 1079, 1997.
38. Milosevic, J., Storch, A., and Schwarz, J. Cryopreservation
does not affect proliferation and multipotency of murine
neural precursor cells. Stem Cells 23: 681, 2005.
39. Cheung, P., and Lau, P. Epigenetic regulation by histone
methylation and histone variants. Mol. Endocrinol. 19: 563,
2005.
40. Weissmann, F., and Lyko, F. Cooperative interactions between epigenetic modifications and their function in theregulation of chromosome architecture. Bioessays 25: 792,
2003.
41. Rowley, S. D. Hematopoietic stem cell cryopreservation: A
review of current techniques. J. Hematother. 1: 233, 1992.
42. Quillen, K., and Berkman, E. M. Methods of isolation andcryopreservation of stem cells from cord blood. J. Hematother.
5: 153, 1996.
43. Karlsson, J. O., and Toner, M. Long-term storage of tissues
by cryopreservation: Critical issues. Biomaterials 17: 243, 1996.
44. Pegg, D. E. The history and principles of cryopreservation.
Semin. Reprod. Med. 20: 5, 2002.
45. Fuller, B. J. Cryoprotectants: The essential antifreezes toprotect life in the frozen state. Cryo Letters 25: 375, 2004.
46. Li, Y., Lu, R. H., Luo, G. F., et al. Effects of different cryoprotectants on the viability and biological characteristics ofporcine preadipocyte. Cryobiology 53: 240, 2006.