Nishikawa A, Shimizu-Nishikawa K, Miller L
Department of Biological Sciences, University of Illinois, Chicago 60680.
In Vitro Cell Dev Biol. 1990 Dec;26(12):1128-34. doi: 10.1007/BF02623689.
Methods for the isolation and in vitro culture of larval and adult Xenopus laevis epidermal cells have been developed. Epidermal cells of stage 52-54 tadpoles and adult epidermal cells were enzymatically dissociated and purified (98%) by Percoll-density centrifugation and unit-gravity sedimentation. Both cell types attached on fibronectin-coated dishes and proliferated for 1 wk when the proper medium was used. There were four significant differences between larval and adult cells: a) Adult cells had a greater buoyant density than larval cells. b) Keratin synthesis patterns were markedly different. c) A combination of medium F12 and Eagle's minimum essential medium was optimal for growth of larval cells whereas MCDB151 medium was optimal for adult cells. d) Adult cells needed fetal bovine serum (greater than 5%) whereas larval cells grew without fetal bovine serum. In contrast to these differences, larval and adult cells had two similar properties: a) Insulin had a potent effect on the growth of both cells, and b) The optimal Ca++ concentration for cell growth was quite low for both cell types; 0.1 mM for larval cells and below 0.05 mM for adult cells. These results suggest that low Ca++ levels are essential for both cornifying (adult) and uncornifying (larval) amphibian keratinocytes. The culture techniques described herein for larval and adult epidermal cells provide a new in vitro model for analyzing development of the epidermis during amphibian metamorphosis.
已经开发出非洲爪蟾幼体和成体表皮细胞的分离及体外培养方法。52 - 54期蝌蚪的表皮细胞和成体表皮细胞经酶解后，通过Percoll密度离心和单位重力沉降进行纯化（纯度达98%）。当使用合适的培养基时，两种细胞类型均可附着于纤连蛋白包被的培养皿上并增殖1周。幼体和成体细胞之间存在四个显著差异：a）成体细胞的浮力密度大于幼体细胞。b）角蛋白合成模式明显不同。c）F12培养基和伊格尔最低必需培养基的组合对幼体细胞生长最为适宜，而MCDB151培养基对成体细胞最为适宜。d）成体细胞需要胎牛血清（大于5%），而幼体细胞在无胎牛血清的情况下也能生长。与这些差异相反，幼体和成体细胞有两个相似的特性：a）胰岛素对两种细胞的生长均有显著作用，b）两种细胞类型生长的最佳钙离子浓度都相当低；幼体细胞为0.1 mM，成体细胞低于0.05 mM。这些结果表明，低钙离子水平对两栖类角质形成细胞（成体的角质化细胞和幼体的非角质化细胞）都是必不可少的。本文所述的幼体和成体表皮细胞培养技术为分析两栖类变态过程中表皮的发育提供了一种新的体外模型。
