title: Production of epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium.
abstract: Rhenwald and Green's technique is currently the standard method for growing stratifying epidermal cell cultures. The serum free system developed in Ham's laboratory (MCDB 153) was designed to grow keratinocyte monolayers in clonogenic conditions. Our aim was to optimize conditions in serum-free MCDB 153 for culturing epidermal sheets from adult normal skin, and to assess the effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes. Sixteen strains derived from plastic surgery specimens (mean age of donors 37 years; range 5-89) were used. Primary cultures were seeded at an optimal density of 8 x 10(4) cells/cm2 in primary cultures and 10(4) cells/cm2 in secondary cultures in complete medium including EGF, insulin, hydrocortisone and bovine pituitary extract, supplemented with isoleucine, tyrosine, methionine, phenylalanine, tryptophane and histidine. Amino acid (AA) supplementation allows a 5.8-fold increase in cell counts at confluency and monolayers with densely packed cells are obtained. In AA supplemented cultures, confluency is obtained in 16 +/- 3 days in primary cultures and in 13 +/- 0.5 days at first passages. Switches to 1.1 mM calcium at first or second passages resulted in a significant increase in cell counts (P less than 0.001), when compared with AA supplemented low calcium cultures. Low temperature/low calcium cultures resulted in a 50% decrease in cell counts. Low temperature/high calcium cultures gave similar cell counts as the 37 degrees C controls. AA and calcium supplemented cultures were evaluated for differentiation markers: involucrin expression was increased, keratins 5, 6, 14, 17 were expressed, and the sheets were 6-10 layers thick by electron microscopy, with keratohyalin granules and cornified envelopes appearing at layers 3-6 (from basal layer). Dispase treatment allowed an easy detachment of these sheets. These results show that the culture medium MCDB 153 can be adapted without serum supplementation to batch culture of human adult keratinocytes to produce epidermal sheets suitable for grafting. They also indicate that extracellular calcium in physiological range of concentration is not a sufficient signal for growth arrest when other growth conditions are optimized.
authors.affiliation: Unité de Dermatologie, INSERM U 306, Université de Bordeaux II, France.
authors.forename: A M
authors.initials: AM
authors.lastname: Boisseau
authors.forename: P
authors.initials: P
authors.lastname: Donatien
authors.forename: J E
authors.initials: JE
authors.lastname: Surlève-Bazeille
authors.forename: J
authors.initials: J
authors.lastname: Amédée
authors.forename: M F
authors.initials: MF
authors.lastname: Harmand
authors.forename: J H
authors.initials: JH
authors.lastname: Bézian
authors.forename: J
authors.initials: J
authors.lastname: Maleville
authors.forename: A
authors.initials: A
authors.lastname: Taieb
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000596:Amino Acids / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002675:Child, Preschool; D003470:Culture Media; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects; D008297:Male; D008875:Middle Aged; D011498:Protein Precursors / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D016038:Skin Transplantation; D013696:Temperature
chemical_list: D000596:Amino Acids; D003470:Culture Media; D011498:Protein Precursors; C032253:involucrin; D002118:Calcium