Ex Vivo Skin Explants and Bioengineered 3D Human Skin In Vitro Models

Assay Description

REPROCELL has established ex vivo human skin models and in vitro three-dimensional (3D) autologous human skin equivalents (HSEs) as physiologically relevant platforms for high-throughput drug testing, cosmetic and skincare research, disease modelling, and precision medicine. Our 3D fabricated skin models consist of primary keratinocytes and fibroblasts derived from human skin punch biopsies in the clinical setting.

We can engineer both healthy and diseased in vitro HSEs as organotypic 3D skin cultures to address different aspects of cutaneous biology, as well as pathophysiology in common skin disorders such as atopic dermatitis, psoriasis and melanoma. In addition, we can provide innovative, tailored assays on the anti-inflammatory effect of test compounds in immunocompetent ex vivo human skin models including mRNA profiling of explants through quantitative PCR (qPCR).

Figure 1. Histochemical analysis of our in vitro 3D human skin model compared with native human skin

Study Outline

Deidentified full-thickness 3-mm punch skin biopsies from both healthy and diseased donors are provided through our clinical network, and then transferred to REPROCELL and processed in the laboratory in an efficient, timely manner. Skin biopsies are utilized to establish primary cell cultures of epidermal keratinocytes and dermal fibroblasts through multistep enzymatic dissociation of cutaneous tissue into single cells without affecting their cellular integrity. Primary human skin cells are expanded as the low-passage monolayers, cryopreserved in xeno-free freezing medium, and can be easily recovered for further 2D assays and molecular analyses or 3D in vitro human skin culture. Organotypic 3D skin models are developed through a coculture of primary keratinocytes and fibroblasts on Alvetex scaffolds in a serum-fortified medium at the air-liquid interface to optimize the self-assembly and shorten the feeding cycle.

REPROCELL’s 3D skin substitutes are maintained in the prolonged culture that is required for their cell differentiation and expansion, as well as expression of epidermal differentiation markers, generation of dermo-epidermal junction (DEJ), and secretion of dermal-specific extracellular matrix (ECM) components. Our 3D in vitro skin models can be used as a tool to address a wide range of biological questions in the presence of different test compounds or analytes within the recommended intervals that meet client requirements. On other hand, cell culture supernatants or conditioned media can be collected for various phenotypic analyses, such as multiplex ELISA (enzyme-linked immunosorbent assay) or liquid chromatography-mass spectrometry (LC/MS).

Experimental Design

An example of the experimental conditions for one test compound using ex vivo skin explants is presented below.

• Positive control (skin explant from diseased donor)
• Negative control (skin explant from healthy donor)
• Test compound (treated skin explant from diseased donor)

Testing information

Test compounds or articles need to be provided by the Sponsor in storable aliquots at the required test concentrations with information on a diluent used. Stock solutions are prepared in deionized water unless otherwise requested. The sponsor also needs to provide sufficient test compound(s) to run the entire study.

We strongly suggest that a minimum of 5 to 10 donors should be used per each condition.

Exclusion Criteria

No specific exclusion criteria are in place than to reject macroscopically diseased/necrotic tissue or skin substitutes with microbial contamination in culture.

However, a list of specific exclusion criteria can be requested by the Sponsor and will be thoughtfully considered and justified when used for a specific study with a specific test compound(s), rather than routinely incorporated into the study protocol.

Figure 2: Histochemical analysis revealing migration of melanocytes (indicated by arrow) in our 3D skin model.