Table 1.
Cryopreservation with trehalose and dimethyl sulfoxide combined.
| Ref. | Level of Evidence OCEBM |
Methods, Slow Cooling and Rapid Rewarming |
CPA Concentration mol/L, Groups | Temperature, °C | Time | Results |
|---|---|---|---|---|---|---|
| Pu et al. [21] 2004 | Level 3 | Adipose aspirates from lipoplasty were randomised to each group and were evaluated by viable adipocyte counts, G3PDH assay and routine histology. | (1) Simple cryopreservation with liquid nitrogen only; (2) 0.5 mol/L DMSO + 0.2 mol/L trehalose; and (3) Control group with fresh adipose | −196 | 20 min | Group 2 showed significantly higher adipocyte viability and superior cellular function of adipocytes compared to group 1. (2.15 ± 0.68 vs. 1.04 ± 0.35 × 106/mL, P < 0.0001) |
| Pu et al. [25] 2006 | Level 3 | Cryopreserved adipocytes from cosmetic lipoplasty were administered into the posterior scalp of a nude mouse. Gross appearance of fat grafts was observed for up to 16 weeks. At the end of the study, final graft weight and volume and corresponding histology were determined. | (1) 0.5 mol/L DMSO + 0.2 mol/L trehalose solution; (2) Simple cryopreservation with liquid nitrogen only; and (3) Control group with fresh adipose aspirates | −196 | 20 min | Group 1 showed greater graft weight, volume and retained tissue architecture when compared to group 2 (P < 0.0001); the fresh control group showed a greater retained volume (47.7% ± 18.6%), and this was statistically significant relative to both groups 1 (35.3% ± 7.8%, P < 0.05) and 2 (6.5% ± 3.7%, P < 0.0001). |
| De Rosa et al. [26] 2009 | Level 3 | Different concentrations of CPA were evaluated to preserve ADSCs for future clinical applications. | (1) 1% DMSO (0.1 mol/L), 9% trehalose (0.26 mol/L), 90% FBS; (2) 4% DMSO (0.6 mol/L), 6% trehalose (0.18 mol/L), 90% FBS; (3) 8% DMSO (1.1 mol/L), 2% trehalose (0.06 mol/L), 90% FBS; and (4) 10% DMSO (1.4 mol/L), 90% FBS | −196 | 1, 6 and 12 months | The best freezing solution consisted of 90% FBS, 4% DMSO (0.6 mol/L) and 6% trehalose. The thawed cells in this group showed superior differentiation efficiency and higher levels of antigen expression, similar levels found in fresh isolates. |
| Cui et al. [27] 2010 | Level 3 | 0.5 mL of cryopreserved adipocyte grafts was thawed and injected into the posterior scalps of mice for 8 weeks. Graft volume, weight and histology were evaluated at the end of the study. | (1) 0.5 mol/L DMSO + 0.2 mol/L trehalose; (2) 0.35 mol/L trehalose; and (3) Control (fresh fat graft) | −196 | 20 min | Groups 1 and 2 showed no statistically significant difference in maintained volume (vs. 46.1% ± 14.4% vs. 38.2% ± 10.1%, NS) and weight (38.9% ± 14.7% vs. 34.1% ± 12.1%, NS). Both cryopreservation groups were found to be inferior to control (both P < 0.05). |
| Pu et al. [22] 2006 | Level 3 | In vitro study measuring the rate of growth and viable cell count (after 2 weeks) of fresh vs. cryopreserved (with fast rewarming) adipocyte aspirates. | (1) 0.5 mol/L DMSO + 0.2 mol/L trehalose and (2) Control fresh adipose aspirates | −196 | 20 min | The cryopreserved aspirates produced 90% of the cell count from fresh aspirates (3.7 ± 1.4 × 105 processed lipoaspirate cells per millilitre aspirates vs. 4.1 ± 1.4 × 105 cells/mL). |
| Cui et al. [23] 2007 | Level 3 | Different CPAs and their concentrations were tested in vitro. | (1) Fresh adipose aspirates; (2) Cryopreserved adipose aspirates without cryoprotectants; and (3) Cryopreserved adipose aspirates with cryoprotectants—0.2 mol/L DMSO + 0.1 mol/L trehalose, 0.5 mol/L DMSO + 0.2 mol/L trehalose, 0.25 mol/L trehalose, 0.5 mol/L trehalose, 1.0 mol/L DMSO, 1.5 mol/L DMSO | −196 | 20 min | The combination of 0.5 mol/L DMSO and 0.2 mol/L trehalose produced the greatest adipocyte count; group 3 produced a significantly higher adipocyte count than group 2 (2.06 ± 0.54 × 106/mL vs. 1.07 ± 0.41 × 106/mL, P < 0.0011); group 1 displayed only a marginally higher adipocyte count relative to group 3 (vs. 2.57 ± 0.56 × 106/mL vs. 2.06 ± 0.54 × 106/mL, P = 0.083); group 3 displayed less tissue shrinkage relative to group 2. |
| Cui et al. [23] 2007 | Level 3 | 1 mL fat graft was injected into nude mice and subsequently harvested 4 months later and analysed for volume, weight and histology. Maintenance of tissue architecture was rated as per the following scale: “5-pristine cellular architecture in all sections examined; 4-mild disruption of cellular architecture in <50% of sections; 3-mild disruption of cellular architecture in >50% of sections; 2-severe disruption of cellular architecture in <50% of sections; 1-severe disruption of cellular architecture in >50% of sections”. | (1) Fresh adipose aspirates; (2) Cryopreserved adipose aspirates without CPAs; and (3) Cryopreserved adipose aspirates with CPAs—0.5 mol/L DMSO + 0.2 mol/L trehalose | −196 | 1 week | Retained graft volume and weight were significantly higher in group 3 compared to group 2 (both P < 0.0001); histology showed extensive tissue fibrosis in group 2, in contrast to relatively preserved tissue architecture with very little fibrosis in group 3; the mean histological rating score in group 1 was significantly higher than that of group 2 (4.60 ± 0.22 vs. 1.50 ± 0.26, P < 0.0001). |
| Pu et al. [28] 2010 | Level 3 | The fat graft samples from both groups were evaluated with trypan blue vital staining, G3PDH assay and routine histology. | (1) 0.5 mol/L DMSO and 0.2 mol/L trehalose and (2) Fresh fat graft control | −196 | 20 min | Groups 1 and 2 showed similar adipocyte counts (3.46 ± 0.91 vs. 4.12 ± 1.11 × 106/mL, P = 0.22); activity of G3PDH was significantly higher in group 2 compared with group 1 (0.66 ± 0.09 vs. 0.47 ± 0.09 U/mL, P < 0.001); histological analysis showed mainly normal structure of fragmented fatty tissues in both groups. |
OCEBM: Oxford Centre for Evidence-Based Medicine; CPA: Cryoprotective agent; G3PDH: Glycerol-3-phosphate dehydrogenase; DMSO: Dimethyl sulfoxide; FBS: Foetal bovine serum; NS: Not significant; ADSC: Adipose-derived stem cell.