Castagnoli Carlotta, Alotto Daniela, Cambieri Irene, Casimiri Raffaella, Aluffi Matteo, Stella Maurizio, Alasia Simone Teich, Magliacani Gilberto
Department of Plastic Surgery and Burn Unit Skin Bank, CTO, Via Zuretti 29, 10126, Turin, Italy.
Burns. 2003 Dec;29(8):759-67. doi: 10.1016/j.burns.2003.01.001.
Cell viability assessment in allograft skin is an essential step to ensure a supply of good quality allograft skin for clinical repair of wounds. It is widely recognised that 'take' of allografts is strongly influenced grafted by tissue viability. The aim of this study was to set-up storage protocols that maintain high viability of the allograft after harvest, treatment and storage. In this study, the viability of post-mortem allografts (n=350) harvested from 35 different donors, was investigated using the MTT salt assay. The conditions of preparation and storage of the allograft included: 1. Fresh skin samples (about 12, 30, and 60h after harvesting). 2. The same specimens (stored at 4 and 37 degrees C) tested for at least 1 month. 3. Samples after cryopreservation and thawing. 4. Thawed specimens tested daily for at least 6 days. Parallel histomorphological analysis performed, under each of these conditions, showed a correlation between changes in structure and changes in viability as measured by the MTT quantitative assay. The viability index (VI) of skin is expressed as the ratio between the optical density (O.D.) produced in the MTT assay by the skin sample and its weight in grams. The percentage viability index is the ratio of the VI of the fresh sample (considered as 100% viability) and the value of specimens from the same harvest batch after storage or cryopreservation. The results indicated that samples tested within 12-30h from harvesting have an average viability index of about 75 with little variation. Samples tested within 60h have an average viability index of 40, showing a viability decrease of about 50%. A protocol to treat skin within a maximum of 30h was, therefore, set-up. The data suggested that skin stored at 37 degrees C, undergoes a viability increase during the first 2 days after harvesting. However, the viability under these conditions then decreased very quickly. After 6 days of preservation at this temperature the samples were no longer viable (PVI = 0). The tissue structure started to become damaged after 3 days. On the other hand, skin stored at 4 degrees C, showed a very slow viability decrease. After 15 days, viability was still almost 25% of the fresh sample. The tissue architecture showed no signs of damage under these conditions until day 7 from harvesting. MTT analysis was performed on the specimens cryopreserved with DMSO at 10%. These measurements were compared to viability assessment of the same fresh skin samples (considered as 100%) that were analysed within 30h from harvesting. The average PVI of thawed skin was 54% of the fresh sample. This result demonstrates that the viability of cryopreserved skin is comparable to the viability of fresh skin stored at 4 degrees C for 4 days. The PVI of thawed skin samples decreased dramatically within 24h, and had reached 0% within 6 days.
评估异体皮肤的细胞活力是确保为伤口临床修复提供高质量异体皮肤的关键步骤。人们普遍认识到，异体皮肤的“存活”受移植组织活力的强烈影响。本研究的目的是建立收获、处理和储存后能保持异体皮肤高活力的储存方案。在本研究中，使用MTT盐分析法研究了从35个不同供体获取的350个死后异体皮肤（尸源异体皮肤）的活力。异体皮肤的制备和储存条件包括：1. 新鲜皮肤样本（收获后约12、30和60小时）。2. 相同标本（分别储存在4℃和37℃）至少测试1个月。3. 冷冻保存和解冻后的样本。4. 解冻后的标本每天测试至少6天。在这些条件下进行的平行组织形态学分析表明，结构变化与MTT定量分析所测活力变化之间存在相关性。皮肤的活力指数（VI）表示为皮肤样本在MTT分析中产生的光密度（O.D.）与其克重的比值。活力指数百分比是新鲜样本（视为100%活力）的VI与同一收获批次储存或冷冻保存后标本值的比值。结果表明，收获后12 - 30小时内测试的样本平均活力指数约为75，变化很小。60小时内测试的样本平均活力指数为40，活力下降约50%。因此，制定了在最长30小时内处理皮肤的方案。数据表明，储存在37℃的皮肤在收获后的头2天活力增加。然而，在此条件下活力随后迅速下降。在此温度下保存6天后，样本不再有活力（PVI = 0）。3天后组织结构开始受损。另一方面，储存在4℃的皮肤活力下降非常缓慢。15天后，活力仍几乎是新鲜样本的25%。在收获后第7天之前，在此条件下组织结构未显示受损迹象。对用10% DMSO冷冻保存的标本进行MTT分析。将这些测量结果与收获后30小时内分析的相同新鲜皮肤样本（视为100%）的活力评估进行比较。解冻皮肤的平均PVI为新鲜样本的54%。这一结果表明，冷冻保存皮肤的活力与储存在4℃ 4天的新鲜皮肤活力相当。解冻皮肤样本的PVI在24小时内急剧下降，6天内降至0%。
