Malassezia interaction with a reconstructed human epidermis: Keratinocyte immune response.

The immediate immune response developed by the keratinocytes against Malassezia yeasts has been addressed yielding conflicting results. This study aims the assessment of cytokines and antimicrobial peptides gene expression elicited by M. sympodialis and M. furfur once in contact with a reconstructed human epidermis. A yeast suspension was prepared in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with Tween 60 and oleic acid to obtain approximately 1 × 106 cells in a volume of 100 µL. Clinical isolates of M. sympodialis (from pityriasis versicolor) and M. furfur (from seborrhoeic dermatitis) were inoculated, separately, onto a reconstructed human epidermis. A distinct expression pattern was found between the two tested species, with a tendency for overexpression of pro-inflammatory cytokines very soon after infection, whereas no significant expression or gene downregulation was often noticed following 24 and 48 h of incubation. A possible Malassezia species-dependent immune response pattern is highlighted.

Malassezia colonisation on a reconstructed human epidermis: Imaging studies.

BACKGROUND: Biofilm formation represents a major microbial virulence attribute especially at epithelial surfaces such as the skin. Malassezia biofilm formation at the skin surface has not yet been addressed. OBJECTIVE: The present study aimed to evaluate Malassezia colonisation pattern on a reconstructed human epidermis (RhE) by imaging techniques. METHODS: Malassezia clinical isolates were previously isolated from volunteers with pityriasis versicolor and seborrhoeic dermatitis. Yeast of two strains of M furfur and M sympodialis were inoculated onto the SkinEthic™ RHE. The tissues were processed for light microscopy, wide-field fluorescence microscopy and scanning electron microscopy. RESULTS: Colonisation of the RhE surface with aggregates of Malassezia yeast entrapped in a multilayer sheet with variable amount of extracellular matrix was unveiled by imaging techniques following 24, 48, 72 and 96 hours of incubation. Whenever yeast were suspended in RPMI medium supplemented with lipids, the biofilm substantially increased with a dense extracellular matrix in which the yeast cells were embedded. Slight differences were found in the biofilm architectural structure between the two tested species with an apparently higher entrapment and viscosity in M furfur biofilm. CONCLUSION: Skin isolates of M furfur and M sympodialis were capable of forming biofilm in vitro at the epidermal surface simulating in vivo conditions. Following 24 hours of incubation, without added lipids, rudimental matrix was barely visible, conversely to the reported at plastic surfaces. The amount of biofilm apparently increased progressively from 48 to 96 hours. A structural heterogeneity of biofilm between species was found.

Characterization of a New Reconstructed Full Thickness Skin Model, T-Skin™, and its Application for Investigations of Anti-Aging Compounds.

BACKGROUND: We have characterized a new reconstructed full-thickness skin model, T-Skin™, compared to normal human skin (NHS) and evaluated its use in testing anti-aging compounds. METHODS: The structure and layer-specific markers were compared with NHS using histological and immunohistological staining. In anti-aging experiments, T-SkinTM was exposed to retinol (10 µM) or vitamin C (200 µM) for 5 days, followed by immunohistological staining evaluation. RESULTS: T-Skin™ exhibits a well stratified, differentiated and self-renewing epidermis with a dermal compartment of functional fibroblasts. Epidermal (cytokeratin 10, transglutaminase 1), dermo-epidermal junction (DEJ) (laminin 5, collagen-IV, collagen VII) and dermally-located (fibrillin 1, procollagen I) biomarkers were similar to those in NHS. Treatment of T-Skin™ with retinol decreased the expression of differentiation markers, cytokeratin 10 and transglutaminase 1 and increased the proliferation marker, Ki67, in epidermis basal-layer cells. Vitamin C increased the expression of DEJ components, collagen IV and VII and dermal procollagen 1. CONCLUSIONS: T-Skin™ exhibits structural and biomarker location characteristics similar to NHS. Responses of T-Skin™ to retinol and vitamin C treatment were consistent with those of their known anti-aging effects. T-Skin™ is a promising model to investigate responses of epidermal, DEJ and dermal regions to new skin anti-ageing compounds.

ISO 10993-23 In vitro irritation testing for medical devices: Substantiating applicability to mild irritants and non-extractables.

Irritation testing is an integral part of the biocompatibility assessment of medical devices and has historically been conducted on animals, either by direct contact or with polar and non-polar solvent extracts. In 2018 an ISO-sponsored interlaboratory validation study demonstrated that two reconstituted human epidermis (RhE) based assays, which were adapted from validated methods used for industrial chemicals, produced results essentially equivalent to those obtained with in vivo tests. This led to the publication of the ISO 10993-23:2021 standard on irritation testing, which states that RhE-based assays are now the preferred method. The 2018 validation study evaluated strong irritants, so we tested nine mild irritants (GHS Category 3), neat and spiked at different concentrations into medical device extracts, per ISO 10993-23:2021. The results substantiated the applicability of RhE-based assays for evaluating mild irritants in medical device extracts. Moreover, the 2018 validation study tested solid extractable medical device materials but did not consider non-extractable medical device materials (e.g., creams, gels, or sprays). By testing nine marketed non-extractable materials, either neat or spiked with irritants, we also confirmed that RhE-based assays are readily applicable to such medical device materials.

Reconstructed Human Epidermis: An Alternative Approach for In Vitro Bioequivalence Testing of Topical Products.

The use of in vitro human skin permeation tests is of value when addressing the quality and equivalence of topical drug products in Europe and the US. Human skin is the membrane of choice for these studies. The use of human skin as a membrane is hindered by limited access, high variability of results, and limited applicability for drugs with low skin permeability. Reconstructed human epidermis (RhE) models are validated as skin surrogates for safety tests and have been explored for percutaneous absorption testing. Clotrimazole poorly permeates human skin and is widely available for topical treatments. In this study, clotrimazole creams were used to test the ability of RhE to be used as biological membrane for bioequivalence testing, based on the Draft Guideline on Quality and Equivalence of Topical Products (CHMP/QWP/708282/2018) using a discriminative and modified in vitro permeation test (IVPT). To fulfill the validation of a discriminatory method, Canesten® 10 mg/g cream was compared with a test product with the same drug strength, along with two "negative controls" dosed at a 50% and 200% drug strength. Products were compared in finite dose conditions, regarding maximal flux (Jmax) and the total amount of drug permeated (Atotal). The results showed the discriminatory power of the method among the three drug strengths with no interference of the placebo formulation. The study design and validation complied with the requirements established in the guideline for a valid IVPT. This new test system allowed for the equivalence comparison between test and comparator product. Higher permeability of the RhE compared to human skin could be observed. This arose as a strength of the model for this modified IVPT bioequivalence testing, since comparing permeation profiles among products is envisaged instead of drawing absolute conclusions on skin permeation extent. These results may support the acceptance of RhE as biological membranes for modified IVPT in bioequivalence testing of topical products.

SkinEthic™ RHE for in vitro evaluation of skin irritation of medical device extracts.

According to ISO 10993 standards for biocompatibility of medical devices, skin irritation is one of the three toxicological endpoints to be always addressed in a biological risk assessment. This work presents a new protocol to assess this endpoint in vitro rather than in vivo. The protocol was adapted to medical devices extracts from the OECD TG 439 with the SkinEthic™ RHE model as test system. It was challenged with irritant chemicals, Sodium Dodecyl Sulfate, Lactic Acid and Heptanoic Acid spiked in polar solvents, sodium chloride solution or phosphate buffer saline and non-polar solvent, Sesame Oil. Cell viability measured by MTT reduction after 24 h exposure was used as readout. Quantification of IL-1α release as secondary readout did not increased performance. Samples of heat-pressed polyvinyl chloride (PVC) and silicone sheets infused with or without known irritant (4% Genapol-X80, 6% Genapol-X100 and 15% SDS) were tested after extraction in polar and non-polar solvents. Medical device extracts are classified irritant when the cell viability is inferior or equal to 50%, compared to the negative controls tissues, in at least one extraction solvent. The correct classification of all the samples confirmed the good performance of this new protocol for in vitro skin irritation of medical devices extracts with the SkinEthic™ RHE model. Seven naïve laboratories were trained in prevision of the Round Robin Study to evaluate Reconstructed Human Epidermis (RhE) models as in vitro skin irritation test for detection of irritant potential in medical device extracts.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.