Department of Biological Sciences and Biotechnology, Faculty of Science, University of Kurdistan, Sanandaj, Iran.
Cell Biol Int. 2014 Apr;38(4):444-51. doi: 10.1002/cbin.10218. Epub 2014 Jan 6.
Human keratinocytes could be used in the repair of damaged skin, in tissue engineering applications, gene therapy and recently, the generation of iPS cells. We isolated human keratinocytes from foreskin and subsequently cultured them on fibronectin, collagen type I, gelatin and laminin-coated dishes that contained three different types of serum-free medium (epilife, KSM or CnT). We developed improved conditions for efficient transfection of these human keratinocytes by testing three common transfection methods and a GFP plasmid vector. The isolated cells showed typical keratinocyte morphology and expressed the epithelial cell specific antigen, cytokeratin 14. Collagen type 1, epilife medium and lipofectamin 2000 gave the best results for isolation and transfection of human keratinocytes. Our protocol can be used as a reproducible, simple and efficient method for isolation, cultivation and genetic manipulation of human keratinocytes, which may be useful in cell and gene therapy applications.
人角质形成细胞可用于修复受损皮肤，组织工程应用，基因治疗，最近还用于诱导多能干细胞生成。我们从包皮中分离出人角质形成细胞，随后在铺有纤维连接蛋白、Ⅰ型胶原、明胶和层粘连蛋白的培养皿中培养，这些培养皿中含有三种不同的无血清培养基（epilife、KSM 或 CnT）。我们通过测试三种常见的转染方法和 GFP 质粒载体，开发了提高这些人角质形成细胞转染效率的改良条件。分离出的细胞呈现典型的角质形成细胞形态，并表达上皮细胞特异性抗原细胞角蛋白 14。Ⅰ型胶原、epilife 培养基和 lipofectamin 2000 对人角质形成细胞的分离和转染效果最佳。我们的方案可作为一种可重复、简单有效的方法，用于人角质形成细胞的分离、培养和遗传操作，这可能对细胞和基因治疗应用有用。
