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Optimization of Murine Keratinocyte Culture for the Production of Graftable Epidermal Sheets
Author(s) -
Rouabhia Mahmoud,
Germain Lucie,
Bélanger François,
Guignard Rina,
Auger François A.
Publication year - 1992
Publication title -
the journal of dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.9
H-Index - 65
eISSN - 1346-8138
pISSN - 0385-2407
DOI - 10.1111/j.1346-8138.1992.tb03234.x
Subject(s) - laboratory flask , plating efficiency , epidermis (zoology) , cell culture , keratinocyte , fibronectin , tissue culture , petri dish , chemically defined medium , chemistry , biology , cell , andrology , in vitro , anatomy , biochemistry , medicine , microbiology and biotechnology , genetics
The aim of the present study was to optimize murine epidermal cell cultures in order to obtain graftable sheets. Newborn (1–3 days old) Balb/c mice skin were used to optimize culture media and plating cell concentration, then epidermal sheet production, and grafting. Epidermal cells were plated at various concentrations in different culture media containing low (0.1 mM) or high (>1 mM) Ca 2+ levels. After a 3 day culture at the 10 4 cells/cm 2 plating cell concentration, the percentage of differentiated cells was more than 80% in the high Ca 2+ culture medium and less than 50% with bulky cells in the low Ca 2+ culture medium. Under these conditions confluence was not obtained. At the 10 5 cells/cm 2 seeding inoculum, the percentage of confluence increased to 95–100% during the first 72 h of culture in both high and low Ca 2+ culture media. Three‐day‐old culture showed stratified multilayer epidermal sheets in the high calcium medium, and monolayer epidermal sheets were present in the low calcium medium after seeding keratinocytes in fibronectin precoated flasks. Seven days after plating, post confluent cultures were composed of a high percentage of differentiated cells (90%) with an increase in shedding cells in the medium. Considering the above morphological observations, sheets obtained with 10 5 cells/cm 2 in MCDB‐153 (A), DME‐HAM (B) or GMEM (C) media after 3 days in culture were grafted. Twenty days after grafting, histological analysis of biopsies showed an epidermal structure and organization comparable to normal murine epidermis without hair follicles. Epidermal transplants showed a complete basement membrane, hemidesmosomes, and tonofilament bundles. Sheets obtained after seven day culture in all media showed lower coverage of the wound bed. These studies point out the importance of the plating cell and Ca 2+ concentrations, and the culture time for murine keratinocyte confluence and differentiation to obtain graftable epidermal sheets.