title: Formation of keratinocyte multilayers on filters under airlifted or submerged culture conditions in medium containing calcium, ascorbic acid, and keratinocyte growth factor.
abstract: Three-dimensional (3D) cell culture is a powerful in vitro technique to study the stratification and differentiation of keratinocytes. However, culture conditions, including culture media, supplements, and scaffolds (e.g., collagen gels with or without fibroblasts), can vary considerably. Here, we evaluated the roles of calcium, L-ascorbic acid phosphate magnesium salt n-hydrate (APM), and keratinocyte growth factor (KGF) in a chemically defined medium, EpiLife, in 3D cultures of primary human epidermal keratinocytes directly plated on polycarbonate filter inserts under airlifted or submerged conditions. Eight culture media containing various combinations of these three supplements were examined. Calcium was necessary for the stratification and differentiation of keratinocytes based on the localization of keratins and involucrin. However, the localization patterns of keratins and integrin β4 were partially disrupted and Ki67-positive basal cells almost disappeared 3 weeks after airlift. The addition of KGF, but not APM, prevented these changes. Further addition of APM markedly improved the tissue architecture, including basal cell morphology and the appearance of keratohyalin granules and localized involucrin in the upper suprabasal cells, even after 1 week. Although the submerged culture also formed cornified epithelium-like multilayers, involucrin was localized in the cornified layer, where nuclei were often found. Based on these results, it is most effective to culture keratinocytes at the air-liquid interface in EpiLife medium supplemented with calcium, APM, and KGF to form well-organized and orthokeratinized multilayers as skin analogues.
authors.affiliation: Department of Oral Rehabilitation, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Seo
authors.affiliation: Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Kitagawa
authors.affiliation: Department of Oral Rehabilitation, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsuura
authors.affiliation: Department of Oral Rehabilitation, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Sato
authors.affiliation: Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan. tinaitj@college.fdcnet.ac.jp.
authors.forename: Tetsuichiro
authors.identifier: http://orcid.org/0000-0002-3684-960X
authors.initials: T
authors.lastname: Inai
mesh_terms: D000328:Adult; D001205:Ascorbic Acid / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry* / Q000494:pharmacology*; D051523:Fibroblast Growth Factor 7 / Q000494:pharmacology*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism
chemical_list: D003470:Culture Media; D051523:Fibroblast Growth Factor 7; D001205:Ascorbic Acid; D002118:Calcium
keywords: Cytokeratins; Integrin β4; Involucrin; Keratinocytes; Organotypic culture