Skin Organoids As a Novel Tool for Analysis of the Skin Microenvironment In Vitro

Abstract

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Introduction. Skin has a complex layered structure designed to provide the first physical and immunologic barrier from the aggressive influence of environment. In vitro models containing native skin architecture include cell sheets-derived skin and bioprinted skin. However, these models show low viability in long-term in vitro studies (more than 14 days), require complex bioreactors for culture, and do not allow for non-invasive, real-time microscopy due to sample thickness. To overcome these limitations, we have generated a novel skin organoid containing the key cell types present in skin as a tool for disease modeling and drug testing in vitro. Materials and Methods. Multiple human skin cell types were induced to form spheroids at varying cellular concentrations. Cell viability in the organoids was determined using Live/Dead assay and skin structures were assessed by histology, immunohistochemistry and scanning electron microscopy (SEM) at 7, 14 and 21 days of culture in vitro. Cell-specific markers have been used to specify a location of different cells: Pan-Cytokeratin, G-Mel, Cytokeratin 71, CD146, Vimentin, and Adiponectin. Results. The skin organoids formed compact structures with high cell viability maintained up to 21 days. The skin organoids' layered organization was confirmed using SEM, Hematoxylin and Eosin (H&E) staining and Masson's Trichrome staining. Immunohistochemical staining showed that the surface of the epidermis was formed by keratinocytes and melanocytes and the center representing the dermal core was formed by fibroblasts, follicle dermal papillae cells, microvascular endothelial cells and adipocytes. The epidermal zone was stratified, organoids were uniformly pigmented, and the dermal core increased collagen deposition by 21 days. Conclusions. By utilizing cellular self-organization, we have created skin organoids containing the major skin cell types which localized with layer specificity to recreate the normal skin microenvironment. Thus, our novel skin organoids recapitulate normal human skin microanatomy and functional characteristics, and provide an efficient and cost effective in vitro model of human skin for a wide range of investigations.