In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a+Langerin+HLA-DR+ MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO4 and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro . For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and CXCL8) was observed followed by complete reepithelialization. Seven days after wounding, tissue integrity, metabolic activity, and cytokine levels had returned to the prewounded state. In conclusion, immortalized human gingiva KC and fibroblasts can be used to make physiologically relevant GE, which resemble either the healthy gingiva or a neoplastic disease model. These organotypic models will provide valuable tools to investigate oral mucosa biology and can also be used as an animal alternative for drug targeting, vaccination studies, microbial biofilm studies, and testing new therapeutics.

After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a physiologically relevant full-thickness skin equivalent model (SE-LC). We describe differences and similarities in the mechanisms regulating LC migration and plasticity upon allergen or irritant exposure. The skin equivalent consisted of a reconstructed epidermis containing primary differentiated keratinocytes and CD1a(+) MUTZ-LC on a primary fibroblast-populated dermis. Skin equivalents were exposed to a panel of allergens and irritants. Topical exposure to sub-toxic concentrations of allergens (nickel sulfate, resorcinol, cinnamaldehyde) and irritants (Triton X-100, SDS, Tween 80) resulted in LC migration out of the epidermis and into the dermis. Neutralizing antibody to CXCL12 blocked allergen-induced migration, whereas anti-CCL5 blocked irritant-induced migration. In contrast to allergen exposure, irritant exposure resulted in cells within the dermis becoming CD1a(-)/CD14(+)/CD68(+) which is characteristic of a phenotypic switch of MUTZ-LC to a macrophage-like cell in the dermis. This phenotypic switch was blocked with anti-IL-10. Mechanisms previously identified as being involved in LC activation and migration in native human skin could thus be reproduced in the in vitro constructed skin equivalent model containing functional LC. This model therefore provides a unique and relevant research tool to study human LC biology in situ under controlled in vitro conditions, and will provide a powerful tool for hazard identification, testing novel therapeutics and identifying new drug targets.