A cell bank system for cultured epithelial sheet transplantation

1)组织活检皮肤和培养上皮片的保存和运输方法的发展。为了将培养上皮片技术纳入日本医疗体系，有必要建立提供培养上皮片的培养中心。我们检查了在冷藏条件下保存活检皮肤4天的最佳培养基。我们发现L-15培养基适合于此目的。接下来，我们研究了上皮片在脱离培养板后能在良好条件下保存多长时间。培养的上皮细胞片在Hanks液冷藏条件下可保存12小时，但不能保存24小时。这一结果促使我们开发了一种在冷冻条件下保存培养上皮片的新方法。我们发现含有聚乙二醇600的L-15培养基是最佳的培养基。体外培养的上皮细胞片可在-150℃的超深冷冻室中冷冻保存3个月以上。此外，在干冰中保存72小时对培养的表皮片没有任何不利的损害。我们也证实了培养的上皮细胞理论上可以在液氮中保存20年以上。2)上皮细胞培养片的临床应用评估。首先，我们对冷冻和非冷冻培养上皮片的有效性进行了比较分析。两者之间无显著性差异。基于这一结果，我们一般采用冷冻自体移植物培养的上皮片。我们尝试了培养上皮片的临床应用，主要用于复发性腿部溃疡、大疱性表皮松解症和严重烧伤。在大疱性表皮松解症中观察到明显的改善，其中溃疡上皮化的持续时间明显缩短，移植病变中由于机械力导致的水疱形成减少。在腿部溃疡中，使用冷冻保存的表皮角质形成细胞对培养的上皮片进行重复移植。提示在培养的上皮片移植中建立细胞库系统的可能性。少

1)Development of preservation and transportation method for biopsied skin and cultured epithelial sheet. For incorporating cultured epithelial sheet technique into Japanese medical care system, it is necessary to establish culture center for supplying cultured epithelial sheet. We examined the best medium to keep biopsied skins for 4 days under refrigerated condition. We found that L-15 medium is suitable for this purpose. Next, we studied how long cultured epithelial sheet could be stored in favorable condition after detachment from culture plate. Cultured epithelial sheet could be preserved in good condition under refrigerated condition with Hanks solution for 12 hours, but not foe 24 hours. This result led us to develop a new method to preserve cultured epithelial sheet under frozen condition. We found that L-15 medium containing polyethylene glycol 600 is the best medium for this purpose. Cultured epithelial sheet could be frozen and stored in ultra-deep freezer (-150ﾟC) for up to … More 3 months. Furthermore, storage in dry ice for 72 hours did not cause any unfavorable damage to cultured epiderm sheet. We also confirmed that cultured epithelial cells could be stored in liquid nitrogen for over 20 years theoretically. 2)Assessment of clinical application of cultured epithelial sheet. First, we performed the compared analysis for the effectiveness of frozen and non-frozen cultured epithelial sheet. There were no significant difference between both of them. Based on this result, we used frozen autograft cultured epithelial sheet in general. We tried clinical application of cultured epithelial sheet mainly for recurrent leg ulcer, epidermolysis bullosa and sever burn. The marked improvement was observed in epidermolysis bullosa, in which durati on of ulcer epihtelization was markedly shortened and blister formation due to mechanical force was decreased in transplanted lesions. In leg ulcer, repetitive transplantation of cultured epithelial sheet was performed using frozen-stored epidermal keratinocytes. Thus, the possibility of cell bank system in cultured epithelial sheet transplantation was suggested. Less