title: The effect of isolation and culture methods on epithelial stem cell populations and their progeny-toward an improved cell expansion protocol for clinical application.
abstract: OBJECTIVE
  The use of cultured epithelial keratinocytes in the treatment of burns and skin graft donor sites is well established in clinical practice. The most widely used culture method for clinical use was originally developed by Rheinwald and Green 40 years ago. This system uses irradiated mouse dermal fibroblasts as a feeder cell layer to promote keratinocyte growth, a process that is costly and labor-intensive for health care providers. The medium formulation contains several components of animal origin, which pose further safety risks for patients. Improvements and simplification in the culturing process would lead to clear advantages: improved safety through reduction of xenobiotic components and reduction in cost for health care providers by dispensing with feeder cells.
  
  
  METHODS
  We compared the Rheinwald and Green method to culture in three commercially available, feeder-free media systems with defined/absent components of animal origin.
  
  
  RESULTS
  During the isolation process, short incubation times in high-strength trypsin resulted in increased numbers of liberated keratinocyte stem cells compared with longer incubation times. All three commercially available media tested in this study could support the expansion of keratinocytes, with phenotypes comparable to cells expanded using the established Rheinwald and Green method. Growth rates varied, with two of the media displaying comparable growth rates, whereas the third was significantly slower.
  
  
  CONCLUSIONS
  Our study demonstrates the suitability of such feeder-free media systems in clinical use. It further outlines a range of techniques to evaluate keratinocyte phenotype when assessing the suitability of cells for clinical application.
authors.affiliation: Blond McIndoe Research Foundation, Queen Victoria Hospital, West Sussex, United Kingdom; Barts Cancer Institute, Barts and The London School of Medicine and Dentistry, London, United Kingdom.
authors.forename: Catherine
authors.initials: C
authors.lastname: Lenihan
authors.affiliation: Blond McIndoe Research Foundation, Queen Victoria Hospital, West Sussex, United Kingdom; Brighton and Sussex Medical School, University of Sussex, Brighton, United Kingdom.
authors.forename: Caroline
authors.initials: C
authors.lastname: Rogers
authors.affiliation: Blond McIndoe Research Foundation, Queen Victoria Hospital, West Sussex, United Kingdom; School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom.
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Metcalfe
authors.affiliation: Blond McIndoe Research Foundation, Queen Victoria Hospital, West Sussex, United Kingdom; School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom. Electronic address: yella.martin@blondmcindoe.org.
authors.forename: Yella H
authors.initials: YH
authors.lastname: Martin
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques / Q000379:methods*; D061252:Feeder Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051379:Mice; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
keywords: burns; cell therapy; keratinocyte; skin; sprayed autologous keratinocytes; stem cells; wound healing