Vestnik dermatologii i venerologiiVestnik dermatologii i venerologii0042-46092313-6294Rossijskoe Obschestvo Dermatovenerologov i Kosmetologov110710.25208/vdv1107Research ArticleThe Reconstructed Human Epidermis in vitro — a Model for Basic and Applied Research of Human SkinBeilinArkadii K.<p>Ph.D-student of Laboratory of Cell Biologyy; Junior scientific researcher of the Institute of Translational Medicine</p>arkadii.beilin@gmail.comhttps://orcid.org/0000-0001-9868-5623RippaAlexandra L.<p>Ph.D (Biol.), scientific researcher of Laboratory of Cell Biology</p>rippa86@yandex.ruhttps://orcid.org/0000-0001-7300-8466SharobaroValentin I.<p>Dr. Sci. (Med.), Prof., Professor of the Russian Academy of Sciences, Professor, Department of Plastic&Reconstructive Surgery, Cosmetology and Cell Technologies</p>sharobarovi@mail.ruGurskayaNadejda G.<p>Ph.D (Biol.), senior scientific researcher of the Institute of Translational Medicine; scientific researcher of Laboratory of Cell Biology</p>ngurskaya@mail.ruVorotelyakEkaterina A.<p>Dr. Sci. (Biol.), Prof., Corresponding Member of the Russian Academy of Sciences, Head of Laboratory of Cell Biology</p>vorotelyak@yandex.ruKoltzov Institute of Development Biology of Russian Academy of SciencesPirogov Medical University0208202096224342003202012052020Copyright © 2020, Beilin A.K., Rippa A.L., Sharobaro V.I., Gurskaya N.G., Vorotelyak E.A.2020<p style="text-align: justify;"><strong>Background.</strong> The reconstructed human epidermis (RE) is an <em>in vitro</em> tissue-engineering construct similar to the native epidermis.</p>
<p style="text-align: justify;"><strong>Objective.</strong> To develop a full-layer RE. Describe its structure: determine the presence of all layers of the epidermal component, including basal, spinous and granular layers and stratum corneum of the epidermis; detect the basement membrane, the border between the epidermal and mesenchymal component.</p>
<p style="text-align: justify;"><strong>Materials and methods.</strong> Isolation of keratinocytes and fibroblasts from human donor skin. Cultivation of keratinocytes and fibroblasts in vitro under 2D conditions, cell subculturing and 3D modeling of RE, obtaining cryosections, histological staining, immunohistochemical (IHC) study with antibodies to cytokeratins 14 and 10, Ki67 protein, loricrin, laminin 5 and plectin.</p>
<p style="text-align: justify;"><strong>Results.</strong> A technique was developed for the formation of RE. Histological examination showed that the stratification of keratinocyte layers occurs during the formation of RE. Layers are formed including basal, spinous and granular layers and stratum corneum. The IHC study has shown the proliferative activity of keratinocytes of the basal layer and has detected the presence of marker proteins of keratinocytes at different stages of differentiation. RE basal keratinocytes, like native ones, form hemidesmosomes and synthesize basement membrane proteins.</p>
<p style="text-align: justify;"><strong>Conclusions.</strong> A full-layer human RE was obtained <em>in vitro</em>. RE meets all the characteristics of the native epidermis and it is suitable for basic and practical research in the field of skin biology, dermatology, and cosmetology.</p>human epidermiskeratinocytesbasal layerspinous layergranular layerstratum corneumbasement membraneimmunohistochemistryреконструированный эпидермис человекакератиноцитыбазальный слойшиповатый слойгранулярный слойроговой слойбазальная мембранаиммуногистохимическое исследование[Vorotelyak E.A., Tsitrin E.B., Vasiliev A.V., Terskikh V.V. Cultured human keratinocytes retaining label for a long time. Dokl Biol Sci. 2005; 402:221-223. doi:10.1007/s10630-005-0094-x.][Chermnykh E.S., Vorotelyak E.A., Tkachenko S.B., Vasiliev A.V., Terskikh V.V. Proliferation of K19+ human epidermal keratinocytes in vitro. Dokl Biol Sci. 2007; 416: 406—408. doi:10.1134/s0012496607050250.][Blanpain C., Fuchs E. 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