title: A simple human dermal model for assessment of in vitro attachment efficiency of stored cultured epithelial autografts.
abstract: This study investigated the effect of short-term storage on the viability and in vitro attachment efficiency of cultured epithelial autograft sheets. Four storage protocols were investigated: overnight at 37 degrees C in keratinocyte culture medium, overnight at 4 degrees C in phosphate-buffered saline solution, overnight at -80 degrees C in cryopreservation medium (containing 10% dimethyl sulphoxide), and 1 week at -80 degrees C in cryopreservation medium. Viability was assessed before and after storage by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. All the storage conditions significantly reduced viability compared with fresh sheets, and no significant decrease was seen when the sheets stored under the different protocols were compared with each other. The best viability obtained was 60% of that of the fresh sheets. The in vitro viability of these stored sheets was then compared with that of the fresh sheets by culturing them on deepidermized acellular allodermis and assessing the composites formed by light microscopy and the MTT assay. The fresh sheets attached and formed a histologically demonstrable composite with the dermal substrate, whereas none of the stored sheets formed demonstrable composites. The MTT assay demonstrated that composites formed with the stored sheets had less than 10% viability compared with composites formed with fresh sheets. It is concluded that under the conditions of storage examined, the viability of cultured epithelial autograft sheets was significantly reduced, but up to 60% of viability could be retained in some cases. However, the subsequent in vitro attachment and proliferation of such preserved sheets on allogeneic deepidermized dermis was poor compared with that of fresh cultured epithelial autograft sheets.
authors.affiliation: Department of Plastic, Reconstructive, and Burns Surgery, Northern General Hospital, Sheffield, United Kingdom.
authors.forename: M M
authors.initials: MM
authors.lastname: Ghosh
authors.forename: S G
authors.initials: SG
authors.lastname: Boyce
authors.forename: E
authors.initials: E
authors.lastname: Freedlander
authors.forename: S
authors.initials: S
authors.lastname: MacNeil
mesh_terms: D002478:Cells, Cultured; D015925:Cryopreservation; D003470:Culture Media; D004847:Epithelial Cells; D004848:Epithelium / Q000637:transplantation; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D008954:Models, Biological; D012038:Regeneration; D012867:Skin / Q000166:cytology; D012879:Skin Physiological Phenomena; D016038:Skin Transplantation / Q000379:methods; D014021:Tissue Preservation
chemical_list: D003470:Culture Media