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Isolation, cultivation and transfection of human keratinocytes
Author(s) -
Zare Sona,
Zarei Mohammad Ali,
Ghadimi Tayyeb,
Fathi Fardin,
Jalili Ali,
Hakhamaneshi Mohammad Saeed
Publication year - 2014
Publication title -
cell biology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 77
eISSN - 1095-8355
pISSN - 1065-6995
DOI - 10.1002/cbin.10218
Subject(s) - transfection , keratinocyte , involucrin , cytokeratin , human skin , foreskin , fibronectin , cell culture , microbiology and biotechnology , plasmid , laminin , biology , genetic enhancement , gene , immunology , biochemistry , extracellular matrix , genetics , immunohistochemistry
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.