Ghosh M M, Boyce S G, Freedlander E, MacNeil S
Department of Plastic, Reconstructive, and Burns Surgery, Northern General Hospital, Sheffield, United Kingdom.
J Burn Care Rehabil. 1995 Jul-Aug;16(4):407-17. doi: 10.1097/00004630-199507000-00006.
This study investigated the effect of short-term storage on the viability and in vitro attachment efficiency of cultured epithelial autograft sheets. Four storage protocols were investigated: overnight at 37 degrees C in keratinocyte culture medium, overnight at 4 degrees C in phosphate-buffered saline solution, overnight at -80 degrees C in cryopreservation medium (containing 10% dimethyl sulphoxide), and 1 week at -80 degrees C in cryopreservation medium. Viability was assessed before and after storage by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. All the storage conditions significantly reduced viability compared with fresh sheets, and no significant decrease was seen when the sheets stored under the different protocols were compared with each other. The best viability obtained was 60% of that of the fresh sheets. The in vitro viability of these stored sheets was then compared with that of the fresh sheets by culturing them on deepidermized acellular allodermis and assessing the composites formed by light microscopy and the MTT assay. The fresh sheets attached and formed a histologically demonstrable composite with the dermal substrate, whereas none of the stored sheets formed demonstrable composites. The MTT assay demonstrated that composites formed with the stored sheets had less than 10% viability compared with composites formed with fresh sheets. It is concluded that under the conditions of storage examined, the viability of cultured epithelial autograft sheets was significantly reduced, but up to 60% of viability could be retained in some cases. However, the subsequent in vitro attachment and proliferation of such preserved sheets on allogeneic deepidermized dermis was poor compared with that of fresh cultured epithelial autograft sheets.
本研究调查了短期储存对培养的自体上皮移植片活力及体外附着效率的影响。研究了四种储存方案：在角质形成细胞培养基中于37℃过夜储存；在磷酸盐缓冲盐溶液中于4℃过夜储存；在含有10%二甲基亚砜的冷冻保存培养基中于-80℃过夜储存；以及在冷冻保存培养基中于-80℃储存1周。通过3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）法在储存前后评估活力。与新鲜移植片相比，所有储存条件均显著降低了活力，且不同方案储存的移植片相互比较时未观察到显著下降。获得的最佳活力为新鲜移植片的60%。然后，将这些储存移植片与新鲜移植片的体外活力进行比较，方法是将它们培养在去表皮的脱细胞同种异体真皮上，并通过光学显微镜和MTT法评估形成的复合物。新鲜移植片附着并与真皮基质形成了组织学上可证实的复合物，而储存的移植片中没有形成可证实的复合物。MTT法表明，与新鲜移植片形成的复合物相比，储存移植片形成的复合物活力不到10%。得出的结论是，在所研究的储存条件下，培养的自体上皮移植片的活力显著降低，但在某些情况下可保留高达60%的活力。然而，与新鲜培养的自体上皮移植片相比，这种保存的移植片随后在同种异体去表皮真皮上的体外附着和增殖情况较差。
