Introduction
Although fat tissue has been used as a filler material for more than 100 years,1,2 there are several problems to be resolved, including unpredictability and fat necrosis resulting in infection and calcification.2,3 Autologous fat transfer, however, is almost the only method of soft tissue augmentation that can be performed without detectable scarring on either a donor or a recipient site and without complications associated with foreign materials. Because fat tissue is more easily damaged by ischemia compared to other tissues such as skin and bone, transferring fat tissue as quickly as possible after harvesting is recommended. Thus, it is of great interest to investigate how adipose tissue viability after aspiration changes over time depending on preservation methods. Clinically, aspirated fat tissue is usually preserved at room temperature in a suction bottle, but how aspirated fat in the suction bottle changes during the postoperative hours is unknown.
It was recently revealed that adipose tissue is a remarkable source of multipotent stem cells,4,5 which can differentiate into adipogenic, chondrogenic, osteogenic, myogenic, neurogenic, endothelial, and other lineages6. Adipose-derived stem cells (ASCs) have already been used in some clinical trials, including treatments for bone defects7 and rectovaginal fistula,8 and soft tissue augmentation such as breast enhancement, breast reconstruction, and facial rejuvenation.9 ASCs may be clinically used or banked also for other therapeutic purposes in the near future. In practice, there is some time lag between liposuction and cell isolation; it takes a few to several hours for liposuction surgery depending on the volume and sites to suction, and a few hours to even a day or two for transportation from an operation room to a cell processing unit. To maximize the potentiality of adipose aspirates as a stem cell source, it is very important to optimize protocols for their preservation.
Thus, aspirated fat is now valuable as an autologous filler material and as an abundant stem cell source. We sought to comprehensively evaluate the influences of preservation at differential temperatures on the viability of aspirated fat and ASCs.

Materials and Methods
Human Tissue Sampling
We obtained liposuction aspirates from 17 healthy female donors undergoing liposuction of the abdomen or thighs after informed consent using an IRB-approved protocol. The adipose portion of the liposuction aspirates was subjected to assays, as described below. Excised fat obtained from a tummy-tuck patient was also used for comparison.

Cell processing and culture
Stromal vascular fractions (SVF) were isolated from the fatty portion of liposuction aspirates as previously described.10 Briefly, the aspirated fat was washed with PBS and digested on a shaker at 37oC in PBS containing 0.075% collagenase for 30 min. Mature adipocytes and connective tissues were separated from pellets by centrifugation (800 ×g, 10 min). The pellets were resuspended and filtered with a 100-μm mesh (Millipore, MA, USA). Freshly isolated SVF was plated (30,000 cells/cm2) on gelatin-coated dishes and cultured at 37oC in an atmosphere of 5% CO2 in humid air. The culture medium was M-199 containing 10% FBS, 100 IU penicillin, 100 mg/mL streptomycin, 5 μg/mL heparin, and 2 ng/mL acidic FGF. Medium was replaced every third day. After 7 days, adherent cells were trypsinized and counted with a cell counter (NucleoCounterTM, ChemoMetec, Allerod, Denmark).

Flow cytometry analysis
Adherent ASCs were examined for surface marker expression using flow cytometry after 1 week of culture. The following monoclonal antibodies were used: CD29-PE, CD31-PE, CD34-PE, CD45-PE, CD90-PE, CD133-PE, CD144-PE, HLA-A,B,C-PE, Tie-2-PE (BD Biosciences, San Diego, CA, USA), CD105-PE (Serotec, Oxford, UK), and Flk-1-PE (Techne, NJ, USA). Cells were incubated with the directly conjugated monoclonal antibodies in PBS containing 0.5% bovine serum albumin (BSA) for 30 min at 4oC, then washed with PBS containing 0.2% BSA and diluted in PBS containing 0.1% BSA. Flow cytometric analyses were performed using an LSR2R (Becton Dickinson, San Jose, CA, USA).

Quantitative analysis of damaged adipocytes in aspirated fat
To assess damaged adipocytes in aspirated fat, we measured the ratios of oil and fat volumes after centrifugation, as follows. The fatty portion of liposuction aspirates was divided into 20 tubes (15-mL conical tubes) and preserved at room temperature. After preservation for 1, 2, 4, and 24 hours, five of the tubes were centrifuged at 2330 ×g for 5 min to separate the oil, fat, and fluid into distinct layers from top to bottom (Fig. 1). The oil ratio was calculated as follows: oil ratio = (oil volume)/[(oil volume) + (fat volume)]. Data were collected from lipoaspirates obtained from six patients.

Scanning electron microscope study
After preservation at room temperature or 4oC, aspirated fat was fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M cacodylate buffer for a week at room temperature, and then fixed in 1% osmium tetroxide. After dehydration, samples were dried with a super critical point CO2 dryer (HCP-2, Hitachi, Tokyo, Japan), sputter-coated with Pt-Pd, and examined with a scanning electron microscope (SEM) (S3500N, Hitachi).

Statistical analysis
Results were expressed as mean ± standard error (S.E.). Paired or unpaired t-tests were used to compare each parameter.

Results
Morphology of adipocytes in aspirated fat
Aspirated fat was preserved at 4oC and fixed for the SEM study at different time points. Adipocytes from aspirated fat almost retained their round shape and showed no significant morphological differences compared with those of excised fat (Fig. 2a). No remarkable change in adipocyte morphology was found in aspirated fat tissues on days 0, 1, and 3 (Fig. 2b-d).
Aspirated fat was preserved for 1, 2, 4, or 24 hours at room temperature and evaluated by SEM, as well (Fig. 2e-h). No remarkable difference in adipocyte morphology was identified.

Degeneration of adipocytes with preservation time
Because we clinically experience a gradual increase of oil volume in lipoaspirates, oil ratio (=oil volume/[oil volume + fat volume]) was used as an index of degeneration of adipocytes in aspirated fat. Oil ratio increased over time during preservation at room temperature (Fig. 3). The oil ratio at 4 hours was greater than that at 1 hour, and at 24 hours was significantly greater than ratios at 1 and 2 hours.

ASC yield from aspirated fat preserved at room or cool temperature
When preserved at room temperature, ASC yield was maintained up to 4 hours and remarkably decreased at 24 hours (Fig. 4). On the other hand, after preservation at 4oC, almost the same number of ASCs was isolated from aspirated adipose tissue on days 0 and 1 (Fig. 5) The number of isolated ASCs was extensively decreased in some cases on day 2 and in all cases on day 3. Statistical significance was seen between days 0 and 3 (P<0.001) and between days 1 and 3 (P<0.001).

Surface marker expression of ASCs isolated from aspirated fat preserved at a cool temperature
To examine changes in the biological properties of ASCs based on preservation time at a cool temperature, surface marker analysis was performed on ASCs isolated from aspirated fat tissues preserved for 0, 1, 2, and 3 days at 4oC (Table 1).

ASC yield from cryopreserved aspirated fat
We also evaluated the possibility of isolating ASCs from aspirated fat cryopreserved for 30 days (n=3). ASCs were harvested from the cryopreserved aspirated fat, but the ASC yield was significantly less than that obtained from fresh aspirated fat (Fig. 6).

Discussion
The SEM assay showed that no significant morphological change in adipocytes was found among aspirated fat tissues preserved either at 4oC for up to 3 days or at room temperature for up to 24 hours. However, quantitative analysis by measuring the oil ratio revealed that preserved adipocytes were partly degenerated and ruptured over preservation time when stored at room temperature. Thus, preservation at room temperature resulted in damage to some adipocytes that may have been located superficially; however, the remaining adipocytes retained almost-intact morphology.
In this study, damaged adipocytes were evaluated by measurement of the oil ratio after centrifugation. Boschert et al.11 reported that centrifugation at greater than 100 ×g caused adipose cell destruction; however, we recently found that centrifugation at 400 ×g increased the oil portion in lipoaspirates but further centrifugation did not significantly damage adipocytes or increase oil volume.12 In addition, our histological examinations of centrifuged aspirated fat with light and scanning electron microscopes showed that adipocytes appeared to be intact even after centrifugation 4300 ×g.12 Therefore, we considered that the increased oil volume after centrifugation (for 5 min at 2330 ×g) in the present study was attributable to adipocyte damage from preservation at room temperature. Thus, the present result indicated preservation at room temperature for 4 hours significantly damaged adipocytes in aspirated fat; thus, lipotransfer should be performed as quickly as possible after aspiration, especially when a large volume of aspirated fat is to be transplanted.
Since the reports showing that adipose tissue contains multipotent stem cells,4,5 aspirated adipose tissue has been regarded as not only a filler material but also as an abundant source of stem cells. ASCs reside in adipose tissue as progenitors of adipocytes, but it has been suggested that ASCs can differentiate into vascular endothelial cells,13,14 can release angiogenetic factors under hypoxic conditions,15 and can contribute to a higher graft take of transplanted fat.14,16 In the current study, ASC yield was maintained up to 4 hours at room temperature, and an ASC yield similar to that of fresh aspirated fat was obtained from that preserved at 4oC for 24 hours. This finding indicates that a one-day delay in isolation of ASCs from aspirated fat can be appropriate when the tissue is stored in a refrigerator. Therefore, overnight cooling transportation of aspirated fat to a specialized cell processing center for isolation and banking of ASCs can be regarded as practical, although ASC yield after 2 or 3 days would be uncertain, even with preservation at 4 oC.
Isolated ASCs can be frozen, thawed, and cultured again as well as almost any other cell type. However, whether aspirated fat tissue can be frozen as an effective filler material or a source of ASCs has not yet been established. In this study, we tried to isolate ASCs from aspirated fat cryopreserved for 30 days as well as from fresh aspirated fat. The ASC yield from cryopreserved fat was much lower than that of fresh aspirated fat. We tried several kinds of freezing conditions (rapid or slow freezing) and other freezing media (DMEM or M199 containing 10% DMSO with 1% methylcellulose or 1% trehalose or 1-20% gelatin, or their mixture), but the ASC yield was not improved (data not shown). Although there were a number of red blood cells contaminating cell fractions isolated from fresh aspirated fat, almost no red blood cells contaminated those from cryopreserved aspirated fat. Our result with ASC yield from cryopreserved fat contradicts a recent report17 showing that the ASC yield from cryopreserved lipoaspirates was about 90% of that from fresh lipoaspirates. The reported ASC yield from cryopreserved adipose is 3.7 ± 1.4 × 105 cells/mL after 2-week culture,17 which is comparable to our result (6.7 ± 4.7 × 104 cells/mL after a 1-week culture) because ASCs proliferate 10?100 times in a week depending on culture conditions. However, the reported ASC yield from fresh adipose (4.1 ± 1.4 × 105 cells/mL after 2-week culture17) was much less than that in this study (7.9 ± 1.5 × 105 cells/mL after 1-week culture). It is unknown why reported ASC yields from fresh adipose differ between the two studies, but it may the result of different methods of cell isolation.
In conclusion, we have demonstrated how ASC yield from aspirated fat changes depending on preservation conditions and time periods. Preservation for 4 hours at room temperature significantly damaged adipocytes but did not significantly alter ASC yield. ASC yield significantly decreased with preservation for 24 hours at room temperature but not with preservation at 4oC. Thus, aspirated fat can be transported to a cell processing center for cell isolation on the day following harvesting and for subsequent banking if it is kept at 4oC. ASC yield from cryopreserved aspirated fat was minimal, and a further optimization of methodology of freezing and preservation is needed for practical use of cryopreservation of aspirated fat intended as an ASC source.

Acknowledgment
We thank Dr. Satoru Fukuda for his assistance in the histological assay with SEM.

Figure Legends

Fig. 1
Analysis of adipocyte damage in lipoaspirates by centrifugation.
After centrifugation, aspirated fat tissue was separated into distinct layers from top to bottom: the oil, fat, and fluid layers. Adipocyte damage by preservation was quantified by calculating the oil ratio in the volume as follows: oil ratio = (oil volume)/[(oil volume) + (fat volume)]. Figure 3 shows the results of the oil ratio calculations.

Fig. 2
Comparison with a scanning electron microscope of human aspirated fat tissues after preservation at 4oC or room temperature.
(a) Excised adipose tissue was fixed immediately after the operation. (b-h) Aspirated fat tissues preserved at 4oC were fixed on Day 0 (b), Day 1 (c), or Day 3 (d), while those preserved at room temperature were fixed at 1 hour (e), 2 hours (f), 4 hours (g), or 24 hours (h) after the operation. Each sample was treated for evaluation with scanning electron microscopy (SEM), and representative photos are shown. No significant morphological changes over time were found by SEM in aspirated fat, even in samples stored at 4oC for 3 days or at room temperature for 24 hours. Scale bar: 250 μm.

Fig. 3
Oil ratios of aspirated fat preserved at room temperature.
Oil ratios in aspirated fat preserved at room temperature for 1, 2, 4, or 24 hours are shown. Statistical analysis was performed using paired t-tests between groups. The oil volume ratio gradually increased with storage time, likely because of breakdown of adipocytes. Values are mean + S.E. *P<0.05.

Fig. 4
ASC yield after preservation at room temperature.
We preserved aspirated adipose tissue at room temperature for 1, 2, 4, or 24 hours and processed for isolation of ASCs, which were then cultured for 1 week. Ratios of ASC yield to control (1 hour preservation) were calculated; data were obtained from three patients, and statistical analysis was performed using paired t-tests between groups. ASC yield seemed to be maintained for up to 4 hours of preservation and remarkably decreased when preserved for 24 hours at room temperature. Values are mean + S.E. *P<0.05.

Fig. 5
ASC yields after preservation at 4oC.
We preserved aspirated fat tissues at 4oC for 0, 1, 2, and 3 days and processed them for isolation of ASCs, which were then cultured for 1 week. Ratios of ASC yield to control (Day 0: no preservation) were calculated; data were obtained from 14 patients (data for Day 2 came from 4 of the 14 patients), and statistical analysis was performed using unpaired t-tests between groups. A statistical difference in ASC yield was not found between days 0 and 1, whereas ASC yield significantly decreased on days 2 and 3. Values are mean + S.E. *P<0.05.

Fig. 6
ASC yields from cryopreserved lipoaspirates.
Fresh aspirated adipose tissue was mixed with an equal amount of freezing medium, cooled to -80oC in a programmable freezing system, and stored at -80oC for 1 month. The cryopreserved adipose tissue was thawed and processed to isolate ASCs, which were then cultured for 1 week. The ratio of ASC yield to control (fresh lipoaspirates) was calculated. Data were obtained from three patients, and statistical analysis was performed using paired t-tests between groups. ASCs were isolated from cryopreserved aspirated fat (6.7 ± 4.7 × 104 cells/mL after 1 week culture), but the yield was much less than that of the fresh fat. Values are mean + S.E. *P<0.05.

Table 1
Surface marker expression of ASCs isolated from aspirated fat tissues preserved at 4oC.
ASCs were isolated from aspirated fat preserved at 4oC for 0, 1, 2, and 3 days, and flowcytometric analyses were performed on ASCs after culture for 1 week. Few differences in expression profile of principal surface markers were observed among groups, suggesting that biological properties of ASCs do not change by preservation at 4oC for up to 3 days. Values are mean ± S.E.

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