Generation of human epidermal constructs on a collagen layer alone.
Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone. In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization of these layers was poor compared with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization. The lack of epithelial-mesenchymal interactions that occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.
Riva
Federica
F
Department of Experimental Medicine, Histology and Embryology Unit, University of Pavia, Pavia, Italy. federica.riva@unipv.it
Casasco
Andrea
A
Nespoli
Emanuele
E
Cornaglia
Antonia Icaro
AI
Casasco
Marco
M
Faga
Angela
A
Scevola
Silvia
S
Mazzini
Giuliano
G
Calligaro
Alberto
A
D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D001706:Biopsy; D006690:Bisbenzimidazole / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D003470:Culture Media; D004247:DNA / Q000378:metabolism; D062109:Dermatologic Surgical Procedures; D000078404:Epidermal Cells; D004817:Epidermis / Q000502:physiology / Q000648:ultrastructure; D000091344:Filaggrin Proteins; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure; D018613:Microscopy, Confocal; D008875:Middle Aged; D009924:Organ Culture Techniques; D011498:Protein Precursors / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D015415:Biomarkers; D003470:Culture Media; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D005456:Fluorescent Dyes; D007381:Intermediate Filament Proteins; D011498:Protein Precursors; C032253:involucrin; D003094:Collagen; D004247:DNA; D014357:Trypsin; D006690:Bisbenzimidazole