An outcome analysis and long-term viability of cryopreserved cultured epidermal allografts: assessment of the conservation of transplantable human skin allografts

Abstract

PURPOSE:To assess the viability of cultured epithelium and preserved by freezing for periods varying from one month to one year. METHODS:Samples of cultured epithelium were incubated in cryoprotectant medium (Group A), packed in aluminum envelopes and packed in polystyrene boxes. The boxes were subjected to a temperature of-70 o C. After freezing for a period of time ranging from one to 12 months, cultured epithelial samples were assessed for their viability by vital staining (Trypan blue) and metabolic analysis based on glucose consumption and lactate production. Samples of not frozen cultured epithelium (Group B) were also tested for viability and the results obtained were used as comparison parameter for the variation of viability. RESULTS:Statistical analysis between the group A and B indicate that the mean age of the donors (p=0.51) and the culture time (p=1.18) showed no statistical difference. In 30 days we obtained 37% of the original viability of cultured epithelium, 25% at six months and one year, less than 15%. This trend was confirmed statistically with a reduction of approximately 1.8% of the original viability epithelium cultured every 30 days of storage. In the analysis by lactate production, similar results were observed. In the analysis by the glucose consumption results were not significant. The viability indices show statistically significant difference between the group A and B (p<0.0001). CONCLUSIONS:Although cryopreserved cultured epithelium showed significant reduction of viability, all samples remained viable. It was also found that the viability of cryopreserved cultured epithelial decreased as a function of storage time.

Current insights into skin banking: storage, preservation and clinical importance of skin allografts

“…48 In 2013, Schiozer et al estimated viability at 37%, 25% and 15% of the original values after 1, 6 and 12 months, respectively, at −70°C: these values were estimated by the trypan blue viability assay and metabolic analysis based on glucose consumption and lactate production. 46 In our experience with the MTT assay, the greatest loss in viability occurred in the first 15 days of storage. Indeed, loss of cell viability was 54.4%, 65% and 69% after 15 days, 6 and 12 months, respectively, compared with fresh skin (prefreezing).…”

“…36,[39][40][41][42][43] To maintain cell integrity and tissue structure, skin grafts should be incubated in cryoprotectants, cooled gradually and thawed rapidly when needed for clinical use. [44][45][46] Cryopreserved skin grafts are stored at −130°C in vapor-phase nitrogen or at −196°C in liquid nitrogen.…”

“…[43][44][45][46][47][48][49] In general, the decay of cell viability is inversely correlated with storage time and depends on storage temperature. Most authors agree that acceptable viability is obtained with a storage temperature of −80°C or below (−130°C).…”

“…15,18,47,49 Similar results were observed with storage at −70°C. 46,50 Most studies have investigated the viability of skin allografts cryopreserved for 2 and 4 weeks: very few have focused on long-term storage (≥2 months).…”

“…33,44 For allografts subject to long-term cryopreservation, many authors prefer storage below −130°C to avoid ice crystal formation and cell damage. 45,46 Typical temperatures are −135°C, 47 −150°C and −170°C.…”