Reconstituted Human Epidermis Research Articles

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.

Cytokine induced 3-D organotypic psoriasis skin model demonstrates distinct roles for NF-κB and JAK pathways in disease pathophysiology.

Psoriasis vulgaris is an inflammatory skin disease that affects 2%-3% of the population worldwide. One of the major challenges in discovering novel therapies is the poor translatability of animal models to human disease. Therefore, it is imperative to develop human preclinical models of psoriasis that are amenable to pharmacological intervention. Here, we report a 3-D reconstituted human epidermis (RHE) culture system treated with cytokines commonly associated with psoriasis (TNFα, IL-17A and IL-22) that reproduced some key features of the human disease. The effects on epidermal morphology, gene transcription and cytokine production, which are dysregulated in psoriasis were assessed. Certain morphological features of psoriatic epidermis were evident in cytokine-stimulated RHEs, including hypogranulosis and parakeratosis. In addition, RHEs responded to a cytokine mix in a dose-dependent manner by expressing genes and proteins associated with impaired keratinocyte differentiation (keratin 10/K10, loricrin), innate immune responses (S100A7, DEFB4, elafin) and inflammation (IL-1α, IL-6, IL-8, IL-10, IL-12/23p40, IL-36γ, GM-CSF and IFNγ) typical of psoriasis. These disease-relevant changes in morphology, gene transcription and cytokine production were robustly attenuated by pharmacologically blocking TNFα/IL-17A-induced NF-κB activation with IKK-2 inhibitor IV. Conversely, inhibition of IL-22-induced JAK1 signalling with ABT-317 strongly attenuated morphological features of the disease but had no effect on NFκB-dependent cytokine production, suggesting distinct mechanisms of action by the cytokines driving psoriasis. These data support the use of cytokine-induced RHE models for identifying and targeting keratinocyte signalling pathways important for disease progression and may provide translational insights into novel keratinocyte mechanisms for novel psoriasis therapies.

Ethosomes for Coenzyme Q10 Cutaneous Administration: From Design to 3D Skin Tissue Evaluation.

Ethosome represents a smart transdermal vehicle suitable for solubilization and cutaneous application of drugs. Coenzyme Q10 is an endogenous antioxidant whose supplementation can counteract many cutaneous disorders and pathologies. In this respect, the present study describes the production, characterization, and cutaneous protection of phosphatidylcholine based ethosomes as percutaneous delivery systems for coenzyme Q10. CoQ10 entrapment capacity in ethosomes was almost 100%, vesicles showed the typical ‘fingerprint’ structure, while mean diameters were around 270 nm, undergoing an 8% increase after 3 months from production. An ex-vivo study, conducted by transmission electron microscopy, could detect the uptake of ethosomes in human skin fibroblasts and the passage of the vesicles through 3D reconstituted human epidermis. Immunofluorescence analyses were carried on both on fibroblasts and 3D reconstituted human epidermis treated with ethosomes in the presence of H2O2 as oxidative stress challenger, evaluating 4-hydroxynonenal protein adducts which is as a reliable biomarker for oxidative damage. Notably, the pretreatment with CoQ10 loaded in ethosomes exerted a consistent protective effect against oxidative stress, in both models, fibroblasts and in reconstituted human epidermis respectively.

Formulating O/W Emulsions with Plant-Based Actives: A Stability Challenge for an Effective Product

Quality, safety, and efficacy concerns added to instability, poor absorption, and the dispersion of actives are common problems while formulating plant-based cosmetics. Furthermore, a correct balance between the stability of the emulsion, the sensory profile, and the high efficacy has to be considered to formulate an effective product. In this paper, we demonstrate that rheology is a methodological tool that can be used while designing a new product. In particular, we developed an O/W emulsion which is easy to spread on irritated skin, and that can soothe the redness and discomfort caused by the exposure to both physical and chemical irritating agents. The green active mixture consists of three natural raw materials: Bosexil®, Zanthalene®, and Xilogel®. Each ingredient has a well-demonstrated efficacy in terms of soothing, anti-itching, and moisturizing properties respectively. Starting from the selection of a new green emulsifying system, through the analysis of the rheological properties, we obtained a stable and easy-to-apply o/w emulsion. The efficacy of the optimized product was assessed in vitro on intact and injured skin using the SkinEthic™ Reconstituted Human Epidermis (RHE) as a biological model.

Development of an In Vitro Method to Estimate the Sensitization Induction Level of Contact Allergens.

Recently, an in vitro procedure, which combines the epidermal equivalent potency assay with assessment of IL-18 to provide a single test for identification and classification of skin sensitizers, was developed and validated. This unit will describe a simple in vitro method for estimation of the expected sensitization induction level interpolating in vitro EC50 and IL-18 SI2 values to predict LLNA EC3 and/or human NOEL from standards curves generated using reference contact allergens, based on the use of Reconstituted human Epidermis (RhE).© 2018 by John Wiley & Sons, Inc.

Keratinocytes oxidative damage mechanisms related to airbone particle matter exposure

Epidemiological evidences have correlated airbone particulate matter (PM) to adverse health effects, mainly linking to pulmonary and cardiovascular disease. Nevertheless, only recently, some studies reported detrimental effects of PM on other organs such as skin. In a recent work, we have reported increased oxidative and inflammatory responses in Reconstituted Human Epidermis (RHE) exposed to ambient particles (CAPs) and we also demonstrated the ability of CAPs to penetrate the skin tissue.The present study was aimed to better understand the cellular mechanisms beyond the oxidative changes induced by CAPs (5–10–25μg/mL) in human immortalized keratinocytes (HaCaT).After 24h of treatment, CAPs were able to enter the cells leading to a decrease in viability, increased levels of 4-hydroxinonenal products (4-HNE) and IL-1α release. Overall these data, suggest lipid and protein oxidative damage, as well as an increase of inflammatory response after being challenged with CAPs. In addition, 3h after CAPs exposure we found a significant increase in NF-kB and Nrf2 translocation into the nucleus. In contrast, no differences in gene expression and enzymatic activity of Nrf2 target genes were detected. This last finding could be explained by the ability of CAPs to possibly alter the binding of Nrf2 to the ARE DNA sequence.

In Vitro Skin Models and Their Predictability in Defining Normal and Disease Biology, Pharmacology, and Toxicity.

In vitro skin model systems are increasingly being used both in the early evaluation of therapeutic drug candidates and in confirmatory mechanistic studies. The most commonly used of these in vitro model systems are reconstituted human epidermis (RHE) models. These RHE models consist solely of epidermal keratinocytes, which comes with some limitations but also with the advantage of focusing toxicologic and pharmacologic evaluation on keratinocytes alone. RHE models can generally be implemented more quickly, easily, and reproducibly than in vivo models and can thus be used for high throughput compound screening while potentially reducing the need for animal studies. Histologic evaluation of RHE sections can be done quite easily, and the sections are very amenable to quantification via image analysis, including automated analysis. RHE model systems can provide very valuable early indications of therapeutic candidate biology, pharmacology, and toxicity; and early results have demonstrated that RHE models have been quite predictive for in vivo pharmacologic and toxicologic effects on the skin, including clinical skin toxicity.

A new hair follicle-derived human epidermal model for the evaluation of sunscreen genoprotection

Induction of skin cancer is the most deleterious effect of excessive exposure to sunlight. Accurate evaluation of sunscreens to protect the genome is thus of major importance. In particular, the ability of suncare products to prevent the formation of DNA damage should be evaluated more directly since the Sun Protection Factor is only related to erythema induction. For this purpose, we developed an in vitro approach using a recently characterized reconstituted human epidermis (RHE) model engineered from hair follicle. The relevance of this skin substitute in terms of UV-induced genotoxicity was compared to ex vivo explants exposed to solar-simulated radiation (SSR). The yield of bipyrimidine photoproducts, their rate of repair, and the induction of apoptosis were very similar in both types of skin samples. In order to evaluate the protection afforded by sunscreen against DNA damage, bipyrimidine photoproducts were quantified in tissue models following SSR exposure in the presence or absence of a SPF50+ formula. A rather high DNA protection factor of approximately 20 was found in RHE, very similar to that determined for explants. Thus, RHE is a good surrogate to human skin, and also a convenient and useful tool for investigation of the genoprotection of sunscreens.

Sonicated and stirred copper oxide nanoparticles induce similar toxicity and pro-inflammatory response in N-hTERT keratinocytes and SZ95 sebocytes

The potential toxic and pro-inflammatory effects of rod-shaped copper oxide (CuO) nanoparticles (NPs; 10 ± 3 nm in thickness and 74 ± 17 nm in length) were studied on N-hTERT keratinocytes and SZ95 sebocytes and on reconstructed human epidermis. Non-sonicated and sonicated CuO NPs induced similar cellular toxicity. The toxic effect of CuO NPs (non-sonicated and sonicated) was more pronounced in keratinocytes than in sebocytes. Pro-oxidant effects of CuO NPs were demonstrated by showing increase in the production of reactive oxygen species and decrease of cellular glutathione. In addition, DNA-binding activities suggested that redox-sensitive transcription factors Nrf2 and NF-κB were implicated in the response of keratinocytes to CuO NPs. Transcriptomic analysis showed an increase in the abundance of transcript species coding for pro-inflammatory interleukins (e.g. IL-8 and IL-1α) and chemokines. In reconstituted human epidermis exposed topically to raw CuO NPs, no effect on the integrity, viability and inflammatory response was noticed.

KeraSkin™-VM: A novel reconstructed human epidermis model for skin irritation tests

Several alternative in vitro methods to evaluate skin irritants have been developed recently. In July 2010, OECD officially endorsed the validated reference method (VRM) that uses reconstituted human epidermis (RhE) models as replacements for the in vivo skin irritation test. This study evaluated the KeraSkin™-VM model, a novel human epidermis model that was reconstructed with Asian skin tissue using 20 reference chemicals according to the OECD TG 439 performance standard. The test chemicals were applied to the epidermal surface side for 45min and then rinsed, and then incubated for 42h post-treatment. An overall accuracy of 80%, sensitivity of 90% and specificity of 70% were obtained when the results from KeraSkin™-VM were compared with UN GHS categories, which was comparable to the EpiDerm™ Skin irritation test (SIT) rates. Furthermore, KeraSkin™-VM demonstrated good performance in terms of within-laboratory reproducibility and predictive capacity to screen skin irritants.

Expression of microRNA‐184 in keratinocytes represses argonaute 2

Interleukin-22 (IL-22) is a proinflammatory cytokine that has been associated with the pathogenesis of inflammatory skin disorders. However, the impact of IL-22 on microRNA (miRNA) expression in epidermal keratinocytes is unknown. Here we show that IL-22 induces miR-184 in reconstituted human epidermis (RHE) and in the HaCaT keratinocyte cell line. Exposure to IL-22 increased miR-184 expression 8- and 15-fold in RHE and HaCaT cells, respectively. Oncostatin M, an unrelated proinflammatory cytokine, also raised miR-184 expression in RHE and HaCaT keratinocytes. Pharmacologic and genetic inhibition demonstrated that cytokine-induced expression of miR-184 was mediated by signal transducer and activation of transcription 3 (STAT3). Argonaute 2 (AGO2), a member of the RNA-induced silencing complex (RISC), is a predicted miR-184 target. Using protein, messenger RNA and reporter analyses, we found that miR-184 regulates the expression of AGO2. We conclude that cytokine-induced miR-184 attenuates AGO2 expression in keratinocytes.

Successful micronucleus testing with the EPI/001 3D reconstructed epidermis model: Preliminary findings

Currently, the cosmetics industry relies on the results of in vitro genotoxicity tests to assess the safety of chemicals. Although the cytokinesis-block micronucleus (CBMN) test for the detection of cells that have divided once is routinely used and currently accepted by regulatory agencies, it has some limitations. Reconstituted human epidermis (RHE) is widely used in safety assessments because its physiological properties resemble those of the skin, and because it allows testing of substances such as hydrophobic compounds. Thus, the micronucleus test is being adapted for application in RHE-reconstructed tissues.Here we investigated whether two different reconstructed epidermis models (EPI/001 from Straticell, and RHE/S/17 from Skinethic) are suitable for application of the micronucleus test. We found that acetone does not modify micronucleus frequency, cell viability, and model structure, compared with non-treated RHE. Treatment of the EPI/001 model with mitomycin C and vinblastine resulted in a dose-dependent increase of micronucleus frequency as well as a decrease of tissue viability and of binucleated cell rate, while no changes of the epidermal structure were observed. The number of binucleated cells obtained with the RHE/S/17 model was too small to permit micronucleus testing. These results indicate that the proliferative rate of the tissue used is a critical parameter in performing the micronucleus test on a 3D model.

Comparison of Metabolism of Donepezil in Rat, Mini-Pig and Human, Following Oral and Transdermal Administration, and in an In Vitro Model of Human Epidermis

Donepezil hydrochloride is a centrally acting, reversible acetyl cholinesterase inhibitor approved for Alzheimer’s disease therapy. Donepezil is marketed as an oral tablet but a transdermal patch may have advantages as an alternate therapy. To determine whether the transdermal route alters known donepezil metabolism, this study compared plasma metabolites of donepezil hydrochloride with two routes of drug delivery, transdermal and oral, in three species; rat, mini-pig and human. The utility of a reconstituted human epidermis (RHE) in vitro model for transdermal drug biotransformation was also evaluated. In addition to donepezil, structures from three metabolic pathways were detectable in each matrix: 1) O demethylation (M1/M2; 2) N-dealkylation (M4); 3) N-oxidation (M6) utilizing LC/quadrupole–Ion trap MS/MS. In rats and humans, including RHE, the mean concentration levels of the donepezil and metabolites for both routes were found in the following order: donepezil>M4>M6>isomeric M1/M2 whilst in the pig, the order was the following: donepezil>M6>isomeric M1/M2>M4. Overall, there were no substantial differences in the plasma profiles between the rat and human. Donepezil metabolites were detectable in mini-pigs after both oral and transdermal administration, however, the concentrations varied from both rat and human. RHE showed quantitative and qualitative similarities between the metabolites produced in vitro with those found in human plasma after transdermal application. Thus, the data from the present study demonstrate that similar metabolites are found between the three species with rat most closely resembling humans and that the RHE model is a potentially valuable model to predict dermal metabolism.

The Effects of IL-20 Subfamily Cytokines on Reconstituted Human Epidermis Suggest Potential Roles in Cutaneous Innate Defense and Pathogenic Adaptive Immunity in Psoriasis

IL-19, IL-20, IL-22, IL-24, and IL-26 are members of the IL-10 family of cytokines that have been shown to be up-regulated in psoriatic skin. Contrary to IL-10, these cytokines signal using receptor complex R1 subunits that are preferentially expressed on cells of epithelial origin; thus, we henceforth refer to them as the IL-20 subfamily cytokines. In this study, we show that primary human keratinocytes (KCs) express receptors for these cytokines and that IL-19, IL-20, IL-22, and IL-24 induce acanthosis in reconstituted human epidermis (RHE) in a dose-dependent manner. These cytokines also induce expression of the psoriasis-associated protein S100A7 and keratin 16 in RHE and cause persistent activation of Stat3 with nuclear localization. IL-22 had the most pronounced effects on KC proliferation and on the differentiation of KCs in RHE, inducing a decrease in the granular cell layer (hypogranulosis). Furthermore, gene expression analysis performed on cultured RHE treated with these cytokines showed that IL-19, IL-20, IL-22, and IL-24 regulate many of these same genes to variable degrees, inducing a gene expression profile consistent with inflammatory responses, wound healing re-epithelialization, and altered differentiation. Many of these genes have also been found to be up-regulated in psoriatic skin, including several chemokines, beta-defensins, S100 family proteins, and kallikreins. These results confirm that IL-20 subfamily cytokines are important regulators of epidermal KC biology with potentially pivotal roles in the immunopathology of psoriasis.

Control of androgen receptor expression in human keratinocytes and in a reconstituted human epidermis model with selective antisense oligonucleotides

Association of locally increased androgen activity and skin disorders is obvious in acne and androgenetic alopecia in males. In addition, testosterone was unexpectedly found to perturb the epidermal barrier. Blockade of androgen action via androgen receptor (AR) antagonism accelerates wound healing in aged individuals. Androgen activity on skin can classically be inhibited by systemic administration of compounds, which have strong affinity for AR and antagonize androgen binding to AR molecules. In this study we applied a new technology to realize the same purpose: We tested the activity of antisense oligonucleotides against the AR in primary human foreskin keratinocytes, human non‐foreskin keratinocytes from young (30 y) and older (60 y) female donors, and reconstituted human epidermis (SkinEthic model). Reconstituted human epidermis is similar to in vivo human epidermis and features a functional permeability barrier. To transfer the antisense oligonucleotides into human keratinocytes an optimum liposome‐mediated transfection system with Poly‐L‐ornithine (12 μg/ml) over 4 h was used. The transfection efficiency was assessed using FITC‐labeled (ACTG)5 random oligonucleotides, which were localized in cytoplasmic structures of the keratinocytes. AR expression on the protein level was investigated by Western blotting. Transient transfection of foreskin keratinocytes with phosphorothioate antisense oligonucleotides (PTO) revealed a reduction of AR expression (≈25%) compared to native keratinocytes after 14 h recovery time. The AR knock down in epidermal keratinocytes of the compared women was stronger in the older, more differentiated keratinocytes. After 24 h, AR expression level have returned back to the level of non‐transfected cells. The effect could be reestablished by repetition of transfection. PTO and 2?O‐methylribosyl (MRO) antisense oligonucleotides decreased AR expression at levels varying between 46% and 70% in the air‐lifted reconstituted human epidermis after 18 h recovery time. The successful inhibition of AR expression in human keratinocytes and reconstituted human epidermis is the first step to develop topically efficient compounds with oligonucleotides.

Dynamic monitoring of glutathione redox status in UV-B irradiated reconstituted epidermis: effect of antioxidant activity on skin homeostasis

An “oxidative stress” should be considered as the consequence of a disbalance within the cutaneous tissue, between pro-oxidant processes and effective antioxidant defence systems and finally the result of a detrimental disturbance of the “cellular redox homeostasis”. Instead of monitoring some particular impairment or a specific biological target of oxidative stress, it is preferable to examine the global effect of a stress, which is revealed by a loss of cellular homeostasis. The measurement of glutathione (relative amount of reduced (GSH), an oxidized form (GSSG)), offers a suitable approximation of the cellular redox status,since glutathione plays a pivotal role in the biological antioxidant systems. It is involved in the “first line “ defence systems but also in repairing and recycling systems. This work aimed to develop an in vitro model that enables the dynamic monitoring of the glutathione redox status after UV-B irradiation. The method was performed in a commercially available Reconstituted Human Epidermis (RHE) model characterized by an high histo-morphological correspondance with normal human skin and by a very good inter batch reproducibility. With this experimental design it was possible to assess both the “physiological “ behaviour of the epidermis after irradiation and also the ability of a tested ingredient to preserve or to restore the redox cutaneous homeostasis. The epidermis were irradiated and allowed to different recovery time (1,5,8,24 h) After each recovery time the quantification of GSH and GSSG by HPLC has been performed on RHE homogenates prepared in carefully controlled conditions. A series of non treated epidermis has been used as reference control. The redox homeostasis loss (e.g. GSH depletion) has been observed immediately after irradiation and remained between 1 and 5 h, although partial recovery takes place. In the presence of antioxidant actives the GSH depletion was minimised, and a faster restoration of homeostasis has been observed. The GSH/GSSG ratio was differentially modified by the actives tested, providing important insights into their specific mechanism of action. The glutathione redox status appears a very sensitive sensor, and kinetic studies have correlated protection against oxidative stress with redox homeostasis preservation.

159 Strong reproducibility for 50 chemicals tested for their in vitro skin irritation potential on skinethic reconstituted human epidermis using multiple end point analysis

159 Strong reproducibility for 50 chemicals tested for their in vitro skin irritation potential on skinethic reconstituted human epidermis using multiple end point analysis

Characterization of the barrier function in a reconstituted human epidermis cultivated in chemically defined medium

The aim of the study was to characterize and assess the maturation process of the barrier function in a reconstituted human epidermis (REp) cultivated in a chemically defined medium. For this purpose histo-morphological analysis, percutaneous absorption studies and non-invasive measurements were performed. In order to understand the time course of the barrier development, REp cultivated from the same pool of normal human keratinocytes were harvested and measured after several increasing periods of exposure at the air-liquid interface. From these results we concluded that a maturation period of at least 16 days at the air-liquid interface was required for an optimal barrier function development of REp. At this time point, the permeability of the skin cultures for caffeine (CAF) was 20-25-fold higher than that of normal human skin (NHS) biopsies. Non-invasive measurements of skin water content, transepidermal water loss (TEWL) and pH were achieved after the same period of time. Results showed that the stratum corneum (SC) of REp was slightly more hydrated than that of ex vivo and in vivo, human skin. TEWL was slightly higher through REp than through NHS, and the pH of the REp models was very close to that of in vivo, normal human skin. In the latter part, assuming that this type of model could be routinely used, we quantified over a 1-year period the barrier function variability of this skin culture model between batches. The satisfactory results obtained with a 20% coefficient of variation indicated that this REp model has a consistent and reproducible barrier function. This leads us to suggest that the skin model might be considered an alternative membrane to normal human skin for permeation screening tests.

Comparison of tissue sources for the skin integrity function test (SIFT)

One of the in vitro models involved in an ECVAM-sponsored prevalidation study for acute skin irritation is the skin integrity function test (SIFT), which utilises full-thickness mouse skin. We have evaluated nine different skin types in order to identify the most useful model for assessing skin barrier function using transepidermal water loss (TEWL), electrical resistance (ER) and tritiated water flux (TWF) and sodium lauryl sulphate (SLS) as a standard skin irritant. Tissues were: human skin (epidermis and whole), reconstituted human epidermis (RHE), pig (dermatomed and whole), rabbit (whole), rat (epidermis and whole) and mouse (whole). Barrier function was measured following sodium lauryl sulphate (SLS) exposure and expressed as a damage ratio. Human epidermis gave good responses at high doses of SLS only. RHE had abnormally high permeability to water and therefore had little or no response to SLS. Pig skin gave low TEWL ratios and rabbit skin was a poor responder to SLS. Mouse whole skin performed best in this study, giving consistent high damage ratios to TEWL, ER and TWF following SLS treatment. Rat whole skin also performed well but was generally less responsive. We conclude that mouse skin is the best and most practical in vitro model for the SIFT approach for skin irritation prediction.