title: An evaluation of the choice of feeder cell growth arrest for the production of cultured epidermis.
abstract: Growth arrested 3T3 cells have been used as feeder cells in human epidermal keratinocyte cultures to produce cultured epidermal autografts for the treatment of burns. The feeder cells were ideally growth-arrested by gamma-irradiation. Alternatively, growth arrest by mitomycin C treatment is a cost effective option. We compared the functional efficacy of these two approaches in keratinocyte cultures by colony forming efficiency, the net growth area of colonies, BrdU labeling and histological features of cultured epidermal sheets. The growth area estimation involved a semi-automated digital technique using the Adobe Photoshop and comprised of isolation and enumeration of red pixels in Rhodamine B-stained keratinocyte colonies. A further refinement of the technique led to the identification of critical steps to increasing the degree of accuracy and enabling its application as an extension of colony formation assay. The results on feeder cell functionality revealed that the gamma irradiated feeders influenced significantly higher colony forming efficiency and larger growth area than the mitomycin C treated feeders. The BrdU labeling study indicated significant stimulation of the overall keratinocyte proliferation by the gamma irradiated feeders. The cultured epidermal sheets produced by gamma feeders were relatively thicker than those produced by mitomycin C feeders. We discussed the clinical utility of mitomycin C feeders from the viewpoint of cost-effective burn care in developing countries.
authors.affiliation: Cell Biology Laboratory, National Institute of Pathology (ICMR), New Delhi, India.
authors.forename: Rishi Man
authors.initials: RM
authors.lastname: Chugh
authors.affiliation: Cell Biology Laboratory, National Institute of Pathology (ICMR), New Delhi, India; Department of Medical Elementology and Toxicology, Jamia Hamdard, New Delhi, India.
authors.forename: Madhusudan
authors.initials: M
authors.lastname: Chaturvedi
authors.affiliation: Cell Biology Laboratory, National Institute of Pathology (ICMR), New Delhi, India. Electronic address: yernenilk@icmr.org.in.
authors.forename: Lakshmana Kumar
authors.initials: LK
authors.lastname: Yerneni
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D049109:Cell Proliferation; D018920:Coculture Techniques / Q000379:methods*; D003114:Colony-Forming Units Assay; D000078404:Epidermal Cells; D004817:Epidermis / Q000254:growth & development; D061252:Feeder Cells / Q000187:drug effects* / Q000528:radiation effects; D005720:Gamma Rays; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology*; D041701:Swiss 3T3 Cells; D023822:Tissue Engineering
chemical_list: D000477:Alkylating Agents; D016685:Mitomycin
keywords: BrdU labeling; Cultured epidermis; Gamma irradiation; Growth area quantification; Keratinocyte colony formation assay; Mitomycin C