title: Isolation, cultivation and transfection of human keratinocytes.
abstract: 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.
authors: Department of Biological Sciences and Biotechnology, Faculty of Science, University of Kurdistan, Sanandaj, Iran.
authors: Sona
authors: S
authors: Zare
authors: Mohammad Ali
authors: MA
authors: Zarei
authors: Tayyeb
authors: T
authors: Ghadimi
authors: Fardin
authors: F
authors: Fathi
authors: Ali
authors: A
authors: Jalili
authors: Mohammad Saeed
authors: MS
authors: Hakhamaneshi
mesh_terms: D000954:Antigens, Surface / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000737:chemistry / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D005353:Fibronectins / Q000737:chemistry; D052816:Foreskin / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053547:Keratin-14 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D008055:Lipids / Q000737:chemistry; D008297:Male; D014162:Transfection
chemical_list: D000954:Antigens, Surface; D024042:Collagen Type I; D003470:Culture Media; D005353:Fibronectins; D053547:Keratin-14; D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins
keywords: effectene; human keratinocyte; lipofectamine 2000; serum free medium; transfection