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Arch Aesthetic Plast Surg > Volume 20(2); 2014 > Article
Lee and Kim: Effects of Long-Term Cryopreservation on Fat Grafts: An Experimental Study

Abstract

Background

Although microfat grafting is now used to augment soft tissue, resorption of some amount of fat is inevitable. There are no consistent guidelines for the duration of fat storage. This study evaluated absolute fat mass and pathological changes according to storage duration.

Methods

Nude mice were injected with fresh fat or fat that had been stored for 3 weeks, 5 months, 9 months, 15 months, or 22 months. After 15 weeks, fat graft weight and pathology (viable cells, structural integrity, microvessel formation, cystic degeneration, fibrosis, and cellular infiltration) were assessed.

Results

After 15 weeks, the average weight of the remaining fat was 486 mg in the control group and 298, 160, 180, 106, 88, and 80 mg in the 3-week and 5-, 9-, 15-, 22-, and 36-month storage groups, respectively. The average weight of fat tissue significantly decreased to less than 20% in the 5-month group. Also, there was a significant decrease in structural integrity and an increase in cystic degeneration in the 5-month group. Tissue vascularization tended to decrease according to the duration of cryopreservation.

Conclusions

The mean weight of the fat grafts preserved in a general freezer was reduced by 61.3% compared with that of the fresh fat group, which was not statistically significant. The mean fat graft weight was, however, significantly reduced following storage in a general freezer for longer than 5 months. In addition, there were decreases in viable adipocytes and increases in fibrocystic degeneration and inflammatory changes when long-term preserved fat was grafted.

INTRODUCTION

The main drawback of free fat injection is the unpredictable resorption of the grafted fat, the rate of which has been reported to be between 30% and 70%. The reasons for this variability include different donor site preparation techniques, harvesting methods, instruments, fat grafting and injection techniques, grafting intervals, and methods of analysis. These variations have made it difficult to compare studies [1-5].
The high and diverse absorption rates result in uncertain clinical outcomes, and absorption requires additional grafts, which mean repeated harvesting procedures. Repeated harvesting procedures can increase cost and surgical risks, as well as patient pain and discomfort. Thus, overcorrection and repeated injections are generally accepted solutions, although cryopreservation of the harvested fat has been attempted in a number of studies. However, the problems of resorption and unpredictable results are even greater with cryopreserved fat [5-7]. There is no universal agreement on the fate of the cryopreserved fat, and the survival of adipocytes preserved in a general freezer without a cryoprotective agent is still controversial.
The mitochondrial activity of adipocytes has been found to be properly maintained when cells are preserved at –20°C for 7 days, with no deterioration or change in the adipocytes compared with fresh fat [8]. Given that fat grafts are usually reinjected after a 6-month period, most prior studies focused on analyzing the viability of adipocytes at a single time point, and the effects of the duration of storage on engraftment of adipose tissue remains to be established.
Therefore, the objective of this study was to test the viability of fat grafts by the duration of storage in an in vivo model.

METHODS

Fat harvesting and processing

Fat was obtained from the midabdomen of a healthy female donor (38 years old) undergoing elective fat graft surgery after informed consent using an institutional review board-approved protocol. The harvested areas were first injected with a tumescent solution (1,000 mL Hartmann, 25 mL of 2% lidocaine, and 1 mL of 1:1,000 epinephrine). While applying negative pressure equivalent of 2 cc backward in 10 cc syringe (Coleman System, Santa Barbara, Canada) using an 18-ga cannula (Coleman System, Santa Barbara, Canada), approximately 42 cc of fat tissue was aspirated from the lower abdomen.
The harvested fat was centrifuged at 3,000 rpm for 3 minutes, and the middle layer was transferred to 1-mL Luer-Lok syringes (Becton, Dickinson and Company, New Jersey, USA) and sealed. The syringes were insulated with a cotton pad to allow slow cooling and stored at –20°C in a general freezer that cooled the syringes at a rate of 1°C/min. The fat tissue was cryopreserved here for 3 weeks and 5, 9, 15, 22, and 36 months. A cryoprotective agent was not used. Fat aliquots that were to be transplanted as “fresh fat” were kept at 4°C for 3 hours before transplantation. Cryopreserved fat aliquots were quickly thawed in a water bath at 20°C, which reduces recrystallization and is considered the preferred method to reduce cell damage. The conditions of the process of slow freezing and rapid thawing were similar to that used clinically.

Animal model

Six-week-old CD-1 male nude mice (weighing between 20 g and 25 g) were used in this study. The animals were kept under a constant laminar flow of air and allowed to feed ad libitum on standard laboratory chow and water. One milliliter of fat was weighed and injected into the subdermal scalp using a 16-ga sharp needle (Fig. 1). CD-1 male nude mice were divided into six experimental groups (3 weeks and 5, 9, 15, 22, and 36 months) and one control group, each containing five mice (Table 1).

Data collection

Mice were euthanized after 15 weeks. The grafted fat was dissected and weighed (Fig. 2). The weight of the surviving fat was expressed as the percent weight (% weight) that remained from the initial weight. In the case of macrocyst formation containing necrotic material, the cyst portion, including its capsule, was excluded from the measurement. For histological examination, the central part of the dissected fat tissue was fixed in 4% formaldehyde, embedded in paraffin, and stained with standard hematoxylin and eosin. Evidence of inflammation, such as neutrophil adhesion and perivascular infiltration, was examined, and microvessel formation was estimated to assess angiogenesis at high power (100×).
Five randomly selected fields from each slide were viewed to assess viable cells (the presence of nucleated fat cells), structural integrity, microvessel formation, cystic degeneration, fibrosis, and infiltration of inflammatory cells. Each of these parameters was graded on a semiquantitative scale of 0–5 by relative evaluation: 0, absence; 1, minimal; 2, minimal to moderate; 3, moderate; 4, moderate to extensive; 5, extensive presence.

Statistical analysis

Statistical analysis was performed using SAS 9.3 software (SAS Institute Inc., North Carolina, USA). One-way analysis of variance (ANOVA) was used for comparing the % weight. Kruskal–Wallis nonparametric ANOVA was employed for comparing histological parameters. A P value of less than 0.05 was considered to be statistically significant.

RESULTS

After 15 weeks, the average weight of the remaining fat graft was 486 mg for the fresh fat-injected group (control group) and 298, 160, 180, 106, 88, and 80 mg for the 3-week and 5-, 9-, 15-, 22-, and 36-month cryopreserved fat-injected groups (experimental groups), respectively. About 55% and 34% of the original weight was preserved after 15 weeks in the control and experimental groups, respectively (P >0.05). The average weight of fat tissue, however, significantly decreased to less than 20% in the 5-month group (P<0.05) and decreased to less than 10% in both the 22- and 36-month groups (Table 2, Fig. 3).
Various degrees of fibrosis and cystic degeneration, and numbers of cells with a lymphocyte, macrophage, and nucleated adipocyte appearance were seen in all groups. There was a significant decrease in structural integrity and an increase in cystic degeneration in the 5-month group (Fig. 4). Microvessel formation tended to decrease according to the duration of cryopreservation, but no statistically significant differences were seen (Table 3).

DISCUSSION

The optimal procedure for fat grafting has been subjected to much scrutiny and speculation. Scientific studies such as the present one have attempted to test various protocols for free fat grafting. Storage of fat grafts would allow for multiple grafting sessions with only a single harvesting event, reducing patient discomfort, as well as the cost and time spent by both the patient and physician [9-11]. Freezing fat did not have a significantly negative effect on cell viability. This may be because adipocytes, filled with lipids, undergo very few volume changes with freezing compared with water-filled cells. Therefore, their cell membranes are less likely to burst [12]. Many studies have shown that storing fat below –20°C is an effective way to maintain cell viability [12-15].
Our current nude mouse study was conducted to determine the optimal storage duration for fat grafts by determining the effect of storage duration on cell viability and comparing the results with those for fresh fat injection. In this study, free fat with a volume of 1 cc was injected into the scalp of each mouse to inject an even amount of free fat. In the process of harvesting specimens 15 weeks after the injection, macrocysts were removed, and grossly surviving fat was harvested. When grafting, stored fat in a 1-cc syringe was directly grafted without measuring its mass. This may be a limitation of the study such that the volume was measured when grafting and the mass was measured when harvesting. The volume of the fat could be accurately measured when grafting, but the fat present after successful grafting becomes a bulk of fat tissue with an irregular shape. It is clear that measuring the mass rather than measuring the volume of the lump is more precise. Under the hypothesis that grossly healthy tissue, excluding scar tissue after debridement of necrotic portions, shows an uniform density in general, the mass after harvesting is regarded as proportional to the volume of the surviving fat.
In this study, 34% of adipose tissue that was stored in a general freezer with no cryoprotective agent was still present 3 weeks after being injected, but after 5 months, less than 20% remained and resorption was increased. These results suggest that reinjection of frozen adipose tissue more than 5 months after harvest is not advisable, considering the high resorption rate. However, there appears to be no differences between adipose tissue kept for 3 years and that kept for 5 months, with only a 10% difference in the transplanted mass and the presence of viable cells. Based on this study, experiments to determine the optimal and clinically appropriate time for injection between 3 weeks and 5 months is necessary.
On histological analysis, vascularization of tissue decreased as preservation time increased but was insignificant (P>0.05). Reduced vascularization in response to reduced fat graft survival is expected, because angiogenesis is important for fat graft survival. However, not all histological findings of neovascularization indicate increased fat graft survival. As the cryopreservation period increases, there are more inflammatory reactions that have a negative influence on fat graft survival, causing neovascularization. The histological findings observed in this study should be regarded as interrelated phenomena, not certain factors or causes regarding fat survival.
In conclusion, both the weight and volume of frozen adipose tissue are reduced after 5 months. Thus, we believe that a maximal volume-expanding effect can be obtained using adipose tissue that has been frozen for less than 5 months. However, there is no difference between adipose tissue maintained at –20°C for 5 months and that maintained for 3 years, with both sets of tissue containing about 10% of the original transplanted mass after 15 weeks. Thus, it appears that, if necessary, the storage period can be prolonged. One limitation of this study is that the number of animals in the experimental group was low. Prolongation of the storage period should be studied with a larger experimental group.

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

Fig. 1.
Fat injected into scalp of nude mice. One milliliter of fat was weighed and injected into the subdermal scalp by using a 16 G sharp needle.
aaps-2014-20-2-70f1.jpg
Fig. 2.
Fat specimen from nude mice’s scalp. The grafted fat was dissected from group 1 mice.
aaps-2014-20-2-70f2.jpg
Fig. 3.
Percent weight curve by group. Percent weight of fat tissue was significantly decreased to less than 20% in the 5-month group.
a)Significant difference (P<0.05).
aaps-2014-20-2-70f3.jpg
Fig. 4.
Histologic finding of group 2b mice. There was significant decrease in structural integrity and an increase in cystic degeneration in the 5-month group (H&E, original magnification ×100).
aaps-2014-20-2-70f4.jpg
Table 1.
Experimental groups
Group Treatment Fat transplant subgroups n
1 Fresh fat injection 1 Fresh 5
2 Cryopreserved fat injection 2a 3 weeks 5
2b 5 months 5
2c 9 months 5
2d 15 months 5
2e 22 months 5
2f 36 months 5

Fat samples for each subgroup were harvested from the same donor.

Table 2.
Percent weight by group
Group % Weight (weight) Standard deviation (SD)
Group 1 (fresh) 55 (486) 19.5
Group 2a (3 weeks) 34 (298) 12.3
Group 2b (5 months) 18 (160)a) 10.6
Group 2c (9 months) 21 (180)a) 24.7
Group 2d (15 months) 12 (106)a) 11.1
Group 2e (22 months) 10 (88)a) 11.1
Group 2f (36 months) 9 (80)a) 12.3

Group 1: Fresh fat injection; Group 2: Cryopreserved fat injection.

a) Significant difference (P<0.05); when compared with group 1.

Table 3.
Histological parameters by group
Parameter Fat sample preservation period
Group 1 (Fresh) Group 2a (3 weeks) Group 2b (5 months) Group 2c (9 months) Group 2d (15 months) Group 2e (22 months) Group 2f (36 months)
Viable cells 3.2 2.75 2.0 1.2 0.8 1.12 0.7
Structural integrity 3.9 2.80 1.7a) 1.7 0.9 1.12 0.8
Microvessel formation 2.5 2.38 2.0 1.5 1.4 1.50 1.0
Cystic degeneration 0.9 1.50 2.5a) 1.6 1.5 1.50 1.3
Fibrosis 1.3 2.30 2.5 3.2 4.0 3.50 3.8
Cellular infiltration 1.3 2.13 2.7 2.8 3.1 2.40 3.3

Group 1: Fresh fat injection; Group 2: Cryopreserved fat injection.

a) Significant difference (P<0.05).

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