Introduction
Human adipose-derived stem (stromal) cells (ASCs)1,2 are promising as a tool of regenerative therapies for tissue defects of mesenchymal lineage, including fat,3 bone,4-8 and cartilage,9-11 as well as blood vessels.12-14 As emphasized for other types of stem/progenitor cells,15 harvested autologous ASCs will most probably be preserved for multiple clinical applications in the future. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies, including transportability of stem cells, pooling of cells to reach a therapeutic dose, and time for the completion of safety and quality control testing.15 Therefore, cryopreservation of ASCs 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 ASCs before and after long-term (6 months) cryopreservation under our defined protocol.

Materials and Methods
Human tissue samples
ASCs were harvested at surgery from lipoaspirates of 14 female patients, ages 20 to 51 years (mean, 29.6 years). Donor sites were abdomen and/or thighs. Doubling times were determined for ASCs from five patients before cryopreservation and from another four patients after 6 months of cryopreservation; therefore, the cell lines for the assays before and after cryopreservation were not identical. Similarly, four lines of ASCs were assayed for determination of chondrogenic potential with the micromass culture system (see below) before cryopreservation, while another five lines were examined after cryopreservation. For osteogenic and adipogenic differentiation assays, five lines of ASCs were assayed before cryopreservation, while a distinct four lines were examined after cryopreservation. Of these five and four lines, one in the before and after groups was from the same patient, and the others were all from separate patients. As for flow cytometry, we analyzed the expression of a set of surface markers (see below) of four lines of ASCs before cryopreservation and five lines after cryopreservation; one patient in both before and after; four other patients in the before, and three other patients in the after.

Cell isolation and culture
All protocols used for these procedures have been described previously.16

Cryopreservation of cells
ASCs were counted and resuspended in cryoprotective agent (Cell Banker IR, Wako Chemicals Co., Ltd., Osaka, Japan) at a density of 106 cells/mL. 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 min, then decreasing ?1°C/min until temperature reached ?50°C, followed by a decrease of ?5°C/min until ?80°C. 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, MI) for long-term storage at ?196°C.. Cells before cryopreservation were subjected to each assay at passage 1 or 2, while 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 in a 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 time
Fresh or cryopreserved ASCs were seeded onto six-well plates at a density of 1 × 105 cells/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?6 days. The cells were then sequentially trypsinized at intervals of 48 hours and counted with a cell counter (NucleoCounter, Chemometec, Denmark). Doubling time was calculated according to the following formula: doubling time = 48 hours/log2(N2/N1), where N1 is the first cell count and N2 is the cell count 48 hours later.

Induced differentiation of cultured ASCs
The protocol for chondrogenic differentiation (micromass culture system) has been described previously.17 DMEM/10% FBS was used for the culture medium as negative control. Quantification was done by measuring the micromass sizes on microphotographs with a scale using an image analysis software (Scion v.3.53, Scion Corporation, MA). Safranin O staining was performed for qualitative analysis.
To test osteogenic differentiation, calcium deposition was evaluated based on the ortho-cresolphthalein complexone (OCPC) method18 with the Calcium C-Test Wako Kit (Wako Chemicals) according to the manufacturer’s instructions. After 5 × 104 cells/60-mm dish were cultured in M199 containing 10% FBS for 6 days, the medium was replaced with osteogenic differentiation medium (DMEM containing 10% FBS, 50 μM ascorbate-2-phosphate, 0.1 μM dexamethasone, and 10 mM β-glycerophosphate) or control medium (DMEM with 10% FBS). Cells were then assayed after 1, 2, 3, and 4 weeks of differentiation culture.
For assessment of adipogenic differentiation, cells were seeded onto 96-well plates at a density of 1 × 104 cells/well. After 4 days of culture, medium was replaced with adipogenic differentiation medium (DMEM with 10% FBS, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 10 μM insulin, and 200 μM indomethacin) or control medium (DMEM with 10% FBS). Cells were further cultured for 1, 2, 3, or 4 weeks and stained with 5 μL/well of AdipoRed-Lipid Assay ReagentR (Cambrex, NJ). After 10 minutes of incubation, the fluorescence with excitation at 485 nm and emission at 535 nm was measured with a microplate spectrophotometer reader Model 680 (BioRad, CA).

Flow cytometry
Flow cytometric analyses were performed as described elsewhere.16 The following monoclonal antibodies conjugated to fluorochromes were used: anti-CD29(β1-integrin)-PE, CD34-PE, CD44-PE, CD49d-PE, CD90(Thy-1)-PE, CD117-PE, and Tie-2-PE (BD Biosciences, San Diego, CA); CD71-PE and CD105-PE (Serotec, Oxford, UK); and CD144-PE (Beckman Coulter, CA, USA).

Statistical analysis
Measured values were expressed as mean ± S.D. Student’s t-tests were used to compare the population means between groups when their population variances were 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.

Results
Proliferative capacity of fresh and cryopreserved ASCs
Doubling times were comparable between fresh and cryopreserved ASCs (Fig. 1), indicating that cryopreservation did not affect the proliferative capacity of the ASCs.

Chondrogenic potential of fresh and cryopreserved ASCs
The chondrogenic potential of nine lines of ASCs, consisting of four fresh cell lines and five cryopreserved lines, was assayed with the micromass culture protocol, followed by morphometry for quantification (Fig. 2A) and safranin O staining for qualitative analysis (Fig. 2B). Sizes of micromass, determined by measuring the areas 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 fresh and cryopreserved ASCs were not significantly different. The standard deviation of micromass sizes of cryopreserved ASCs cultured in chondrogenic medium was much larger than that of fresh cells, suggesting variable but definite effects of long-term cryopreservation on their chondrogenic potential. Moreover, although significant differences were detected between chondroinductive and control culture conditions in each group, the p value of this difference was lower in the cryopreserved cell group than in the fresh cell group. Thus, cryopreserved cells are more strongly influenced by culture medium than are fresh cells, suggesting that cryopreservation could exert some effects on ASC susceptibility to chondrogenic differentiation.
Microphotographs of safranin O-stained sections of micromass showed higher production of proteoglycan matrix in cells with chondrogenic induction than in controls in both fresh and cryopreserved ASCs (Fig. 2B). As with the observation from the quantification assay above, micromasses showed comparable degrees of chondrogenic matrix production in both groups, but cryopreserved ASCs compared to fresh ASCs appeared to exhibit a greater sensitivity to the chondroinductive culture environment.

Osteogenic and adipogenic potential of ASCs before and after cryopreservation
Fresh and cryopreserved ASCs were assayed for osteogenic (Fig. 3) and adipogenic (Fig. 4) potential. Osteogenic potential, determined as calcium deposition in intra- and extra-cellular spaces, was comparable between fresh and cryopreserved ASCs; in fact, cryopreserved cells surpassed fresh cells. Adipogenic potential, determined based on spectrophotometric absorbance using AdipoRed staining, showed no apparent difference between fresh and cryopreserved ASCs. These results indicate that long-term cryopreservation up to 6 months does not affect the osteogenic and adipogenic potential of ASCs.

Flow cytometric analysis of cell surface markers on ASCs
Cell surface marker expression is closely related to cell lineage and biological properties, including multipotentiality. Expression profiles of cell surface markers, as well as their change over passages, was analyzed with flow cytometry. ASCs before and after 6 months of cryopreservation 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 in the expression patterns were quite similar between the two groups of ASCs. Only CD34 declined with increased passage number; the other markers generally remained constant in both groups (Fig. 5 and Table 1). Therefore, as far as those analyzed marker subsets are concerned, the expression profile of cell surface markers of ASCs underwent little change through cryopreservation.

Discussion
In this study, we demonstrated that human ASCs preserve their proliferative capacity, mesenchymal multipotency (remaining chondrogenic, osteogenic, and adipogenic), and surface marker expression profiles after 6 months of cryopreservation. The only difference we detected between fresh and cryopreserved ASCs in this study was the larger variability in chondrogenic differentiation potentials of cryopreserved ASCs. Although the results do not guarantee the validity of preservation longer than 6 months for ASCs, 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 human ASCs vital for potential cell therapies.
Human fat is readily obtained from liposuction and frequently used as a filler material for soft tissue augmentation.19,20 Because multiple lipoinjection is frequently necessary for maximizing cosmetic results, some cosmetic surgeons are strongly inclined to store and repeatedly use the harvested fat. Thus, cryopreserved human fat tissue has been intensively investigated,21,22 but there have been few reports about the effects of cryopreservation on ASCs.23-26 The three reports by Thirumalas et al.23-25 studying human ASCs as isolated cells mainly focused on the physical effect of freezing on cell membrane integrity; another report26 focused on ASC yield from cryopreserved fat. To our knowledge, prior to the present study there have been no reports investigating the potential of cryopreserved human ASCs as a tool for cell therapy.
With advances in tissue engineering and regenerative medicine, use of adult stem cells may be a solid therapeutic option in the near future, and our data can contribute to developing protocols for regenerative therapies with cryopreserved ASCs. 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.27,28 Based on the findings regarding these antecedent stem cells, it is likely that we can reasonably extend the period of cryopreservation of ASCs, though the safety of doing so should be further examined.
In this study, the influences of different cryoprotective agents and cryopreservation methods were not examined. The cryoprotective agent used in this study contains FBS as a supplement, which contributes to the maintenance of viability of ASCs during freezing and thawing. Given the point-of-view that animal-derived biological ingredients should be completely eliminated from the process for cells to be used clinically in cell therapy, the cryoprotective agent may have to be further optimized for ASC storage.

FIGURE LEGENDS

Figure 1.
Doubling time of fresh and cryopreserved human ASCs.
Five lines of human ASCs before the freezing and thawing process (Fresh) and four lines harvested after 6 months of cryopreservation (Cryopreserved) were assayed for cell proliferation rate, and their doubling times were determined. NS: no significant difference. Values are mean + S.D.

Figure 2.
Chondrogenic potentials of fresh and cryopreserved human ASCs.
A. Four lines of human ASCs prior to the freezing and thawing process (Fresh) and five lines harvested after being cryopreserved for 6 months (Cryopreserved) were subjected to micromass culture in the defined chondrogenic differentiation medium (blue columns) or control medium (grey columns) to assess chondrogenic potential. Micromass sizes were determined using image analysis software on microphotographs with scale as an area [mm2]. NS: no significant difference.
B. Microphotographs of representative sections of micromass. Paraffin-embedded sections were stained with safranin-O and counterstained with hematoxylin: fresh ASCs in chondrogenic medium (above left), fresh ASCs in control medium (above right), cryopreserved ASCs in chondrogenic medium (below left), and cryopreserved ASCs in control medium (below right).

Figure 3.
Osteogenic differentiation potentials of human adipose-derived stem cells (ASCs) before and after long-term cryopreservation.
A. Fresh (red lines) and cryopreserved (blue lines) ASCs derived from a single patient were assayed for calcium deposition after 1, 2, 3, and 4 weeks of cell culture with osteogenic differentiation medium (closed circles) or control medium (closed rectangles). Fresh ASCs with (Fresh/induced) or without (Fresh/uninduced) osteogenic induction are indicated with red circles and red rectangles, respectively; cryopreserved ASCs with (Cryopreserved/induced) or without (Cryopreserved/induced) osteogenic induction are indicated with blue circles and blue rectangles, respectively.
B. Five lines of human ASCs prior to the freezing and thawing process (Fresh) and four lines after cryopreservation for 6 months (Cryopreserved) were assayed for calcium deposition to assess osteogenic potential with (blue columns) or without (grey columns) osteogenic induction. NS: no significant difference.

Figure 4.
Adipogenic differentiation potentials of human ASCs before and after long-term cryopreservation.
A. Fresh and cryopreserved ASCs derived from the same patient were assayed for oil deposition as a quantification of adipogenic differentiation at the indicated time point. Fresh ASCs with (Fresh/induced) or without (Fresh/uninduced) osteogenic induction are indicated with red circles and red rectangles, respectively; cryopreserved ASCs with (Cryopreserved/induced) or without (Cryopreserved/induced) osteogenic induction are indicated with blue circles and blue rectangles, respectively.
B. Five lines of human ASCs prior to the freezing and thawing process (Fresh) and four lines after cryopreservation for 6 months (Cryopreserved) were assayed for lipid deposition to assess adipogenic potential with (blue columns) or without (grey columns) adipogenic induction. NS: no significant difference.

Fig. 5.
Representative data of cell surface marker expression in fresh and cryopreserved ASCs (passages 0 and 7) obtained from a single patient.
ASCs before (Fresh) and after 6 months of cryopreservation (Cryopreserved) were analyzed at passages 0 and 7 with flow cytometry for expression of a selected set of cell surface markers. The representative data from fresh and cryopreserved ASCs (passages 0 and 7) obtained from a single patient are shown. IgG1 indicates the negative control using a non-specific mouse immunoglobulin G1 species as an antibody to determine background fluorescence.

Table 1
Expression of cell surface markers in fresh and cryopreserved human ASCs.
Percentages of positive cells for each surface marker are shown. Data were collected from four fresh lines and five 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 ± S.D.

REFERENCES
1. 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.
2. Zuk, P. A., Zhu, M., Mizuno, H., et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 7: 211, 2001.
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. Tissue Eng., in press, 2006.
4. Halvorsen, Y. C., Wilkison, W. O. and Gimble, J. M. Adipose-derived stromal cells--their utility and potential in bone formation. Int. J. Obes. Relat. Metab. Disord. 24 Suppl 4: S41, 2000.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. Awad, H. A., Halvorsen, Y. D., Gimble, J. M., et al. Effects of transforming growth factor beta1 and dexamethasone on the growth and chondrogenic differentiation of adipose-derived stromal cells. Tissue Eng. 9: 1301, 2003.
11. 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.
12. 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.
13. Miranville, A., Heeschen, C., Sengenes, C., et al. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation 110: 349, 2004.
14. Cao, Y., Sun, Z., Liao, L., et al. Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem. Biophys. Res. Commun. 332: 370, 2005.
15. Hubel, A. Parameters of cell freezing: implications for the cryopreservation of stem cells. Transfus. Med. Rev. 11: 224, 1997.
16. Yoshimura, K., Shigeura, T., Matsumoto, D., et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J. Cell. Physiol. 208: 64, 2006.
17. 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.
18. Connerty, H. V. and Briggs, A. R. Determination of serum calcium by means of orthocresolphthalein complexone. Am. J. Clin. Pathol. 45: 290, 1966.
19. Fulton, J. E., Suarez, M., Silverton, K., et al. Small volume fat transfer. Dermatol. Surg. 24: 857, 1998.
20. 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.
21. Ullmann, Y., Shoshani, O., Fodor, L., et al. Long-term fat preservation. J. Drugs Dermatol. 3: 266, 2004.
22. Shoshani, O., Ullmann, Y., Shupak, A., et al. The role of frozen storage in preserving adipose tissue obtained by suction-assisted lipectomy for repeated fat injection procedures. Dermatol. Surg. 27: 645, 2001.
23. Thirumala, S., Gimble, J. M. and Devireddy, R. V. Transport phenomena during freezing of adipose tissue derived adult stem cells. Biotechnol. Bioeng. 92: 372, 2005.
24. 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.
25. Devireddy, R. V., Thirumala, S. and Gimble, J. M. Cellular response of adipose derived passage-4 adult stem cells to freezing stress. J. Biomech. Eng. 127: 1081, 2005.
26. Pu, L. L., Cui, X., Fink, B. F., et al. Adipose aspirates as a source for human processed lipoaspirate cells after optimal cryopreservation. Plast. Reconstr. Surg. 117: 1845, 2006.
27. Rowley, S. D. Hematopoietic stem cell cryopreservation: a review of current techniques. J. Hematother. 1: 233, 1992.
28. Kobylka, P., Ivanyi, P. and Breur-Vriesendorp, B. S. Preservation of immunological and colony-forming capacities of long-term (15 years) cryopreserved cord blood cells. Transplantation 65: 1275, 1998.