The Reconstructed Human Epidermis in vitro — a Model for Basic and Applied Research of Human Skin

Abstract

Background. The reconstructed human epidermis (RE) is an in vitro tissue-engineering construct similar to the native epidermis.
 Objective. 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.
 Materials and methods. 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.
 Results. 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.
 Conclusions. A full-layer human RE was obtained in vitro. 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.

Signatures of Dermal Fibroblasts from RDEB Pediatric Patients

“…Primary fibroblasts and keratinocytes were isolated from skin using a technique described earlier [ 68 ]. Briefly, skin was washed in Hank’s Balanced Salt Solution HBSS (PanEco, Moscow, Russia) with gentamicin (0.35 mg/mL, Dalhimpharm, Khabarovsk, Russia).…”

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination

“…A variety of strategies have been developed so far in the area of skin bioengineering. Here, we established skin equivalents from immKEB and immKHC by the method we used for primary keratinocytes [31]. The results obtained indicate that our immortalized keratinocytes fully retained the ability to undergo successive stages of differentiation and stratification with onset of appropriate marker expression.…”

Immortalization of Human Keratinocytes Using the Catalytic Subunit of Telomerase

“…Isolation and cultivation of primary keratinocytes was described previously [ 6 ]. Immortalization was performed by transduction of a lentiviral vector carrying the hTERT cDNA.…”

“…The creation of skin equivalents and the preparation of cryosections were carried out as described previously [ 6 ]. In one of the groups, the CnT-Prime Airlift culture medium (Ztn-Bio, United States) was replaced with DMEM/F12 with 10% serum, which was supplemented with ITS (Insulin–Transferrin–Selenium additive) and EGF (recombinant human epidermal growth factor) (Gibco, United Statets).…”