Epidermal Skin Model Research Articles

Transdermal delivery of elastin peptide assisted by betaine-based deep eutectic solvent to ameliorate skin photoaging

The unique amino acid composition of elastin peptide (EP) makes it an excellent resource to obtain antioxidant peptides. It exhibits high elastase inhibitory activity with the potential to resist skin aging and is currently used in a many cosmetic products. However, the inherent low permeability of the skin limits its ability to penetrate the skin. To address this issue, a deep eutectic solvent (SAB) with excellent bioactivity was synthesized from betaine and succinic acid and used as a permeation enhancer to improve the absorption and utilization of EP in this paper. The results showed that low SAB concentrations significantly increased the transdermal delivery of EP. The 3D epidermal skin model (EpiKutis®) demonstrated that SAB/EP induced the synthesis of hyaluronic acid (HA) and filaggrin (FLG), accelerated skin barrier repair, and reduced water loss. Additionally, the zebrafish embryonic model showed that SAB/EP could reduce melanin secretion, decrease melanin deposition, and have an ameliorative effect on skin photoaging. Cellular experiments proved that SAB/EP can stimulate human skin fibroblasts to secrete procollagen I and elastin, improving skin elasticity and anti-wrinkle. The combination of EP and DES is a new attempt that is expected to be used as a safe and effective anti-wrinkle cosmetic material.

Flexible nano-liposomes-encapsulated recombinant UL8-siRNA (r/si-UL8) based on bioengineering strategy inhibits herpes simplex virus-1 infection

Herpes simplex virus-1 (HSV-1) infection can cause various diseases and the current therapeutics have limited efficacy. Small interfering RNA (siRNA) therapeutics are a promising approach against infectious diseases by targeting the viral mRNAs directly. Recently, we employed a novel tRNA scaffold to produce recombinant siRNA agents with few natural posttranscriptional modifications. In this study, we aimed to develop a specific prodrug against HSV-1 infection based on siRNA therapeutics by bioengineering technology. We screened and found that UL8 of the HSV-1 genome was an ideal antiviral target based on RNAi. Next, we used a novel bio-engineering approach to manufacture recombinant UL8-siRNA (r/si-UL8) in Escherichia coli with high purity and activity. The r/si-UL8 was selectively processed to mature si-UL8 and significantly reduced the number of infectious virions in human cells. r/si-UL8 delivered by flexible nano-liposomes significantly decreased the viral load in the skin and improved the survival rate in the preventive mouse zosteriform model. Furthermore, r/si-UL8 also effectively inhibited HSV-1 infection in a 3D human epidermal skin model. Taken together, our results highlight that the novel siRNA bioengineering technology is a unique addition to the conventional approach for siRNA therapeutics and r/si-UL8 may be a promising prodrug for curing HSV-1 infection.

Based on In Vitro and 3D Epidermal Skin Model: The Efficacy of Bletilla Striata Extract for Skin Care

Introduction: Bletilla Striata (Thunb. ex A. Murray) Rchb. f.) Is a native herb of the orchidaceae family. Modern pharmacological activity studies have shown that Bletilla striata mainly has pharmacological activities such as promoting blood circulation and hemostasis, promoting wound healing anticancer, anti-tumor disease-resistant pathogenic microorganisms, protecting mucosa, anti-ulcer, whitening and anti-oxygen, anti-aging, and immune regulation, which have been widely recognized and applied. Research on the efficacy of plant extracts mainly adopts in vitro testing methods, such as cell experiment and 3D skin model experiment. Objective: To evaluate the soothing effect of Bletilla striata extracton UVB-stimulated keratinocytes (HaCaT) and its barrier repair efficacy in Skinovo-Epi skin model. Methods: The soothing and barrier repairing effects of Bletilla striata extract were studied in vitro. The cytotoxicity of the sample was measured by MTT assay, and cytotoxicity to keratinocytes was evaluated by cell survival rate. The soothing effect was evaluated by detecting changes in inflammatory cytokines (IL-6, IL-1β, IL-8, TNF-α) secreted by UVB-stimulated keratinocytes with the sample treatment. Barrier repair efficacy was assessed in a Skinovo-Epi skin model by measuring changes in FLG (Filaggrin) and LOR (Loricrin) protein levels after topical application of the sample. Results: The sample showed low cytotoxicity on keratinocytes. The sample significantly reduced UVB-stimulated secretion of inflammatory cytokines in keratinocytes. Topical application of the sample increased FLG and LOR protein levels in the Skinovo-Epi skin model. Conclusions:Bletilla striata extract exhibits soothing and barrier repair activities in vitro. Bletilla striata extract reduces UVB-stimulated inflammation in keratinocytes and improves skin barrier function. The data provide novel experimental evidence supporting the innovative application of Bletilla striata for skin disorders. Bletilla striata has potential as a therapeutic herb for sensitive and damaged skin conditions.

A novel recombinant ORF7-siRNA delivered by flexible nano-liposomes inhibits varicella zoster virus infection

BackgroundVaricella zoster virus (VZV), which is a human restricted alpha-herpesvirus, causes varicella (chickenpox) and zoster (shingles). The subsequent post-herpetic neuralgia (PHN) due to VZV infection is excruciating for most patients. Thus, developing specific therapeutics against VZV infection is imperative. RNA interference (RNAi) represents an effective approach for alternative antiviral therapy. This study aimed to develop a novel anti-VZV therapeutics based on RNAi.ResultsIn this study, we screened and found the open reading frame 7 (ORF7) of the VZV genome was an ideal antiviral target based on RNAi. Therefore, a novel siRNA targeting ORF7 (si-ORF7) was designed to explore the potential of RNAi antiviral treatment strategy toward VZV. We used a bio-engineering approach to manufacture recombinant siRNA agents with high yield in E. coli. Then, the efficacy of recombinant ORF7-siRNA (r/si-ORF7) in inhibiting VZV infection both in cellular level and 3D human epidermal skin model was evaluated. The r/si-ORF7 was proved to inhibit the VZV replication and reduce the virus copy numbers significantly in vitro. Furthermore, flexible nano-liposomes were established to deliver r/si-ORF7 to 3D human epidermal skin model and found r/si-ORF7 also could inhibit the VZV infection, thus maintaining normal skin morphology.ConclusionsTaken together, our results highlighted that transdermal administration of antiviral r/si-ORF7 was a promising therapeutic strategy for functional cure of VZV infection.

Skin penetration of active folate salts and their effects on wound healing in vitro: new approaches for dermal folate application

# Objectives The skin has a high demand for folates because of its constant renewal and growth. Folates play an important role in the metabolism of healthy skin and in the regeneration of irritated or injured skin. The high proliferation rate supports wound healing in chronic and degenerative skin diseases. In contrast to systemic folate administration, there is little known on skin penetration of folates after topical application. Therefore, skin penetration with L-formyltetrahydrofolate di-arginine salt (L-FTHF-di-arginine), L-formyltetrahydrofolate calcium gluconate mixture (L-FTHF-Ca) and L-methyltetrahydrofolate dicholine salt (L-MTHF-dicholine) was studied. # Methods We investigated the epidermal penetration of 2.5% aqueous solution of L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine using *in vitro* reconstructed epidermal skin models. These multi-layered epidermis models closely emulate the barrier of human skin. Aqueous solutions of - L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine were applied topically to the keratinized layer. The folate concentration below the multi-layer was analyzed after 24 hours by high-performance liquid chromatography (HPLC)-analysis to detect penetration. In order to assess an effect on wound healing *in vitro*, the selected folates were tested in a keratinocyte scratch assay. # Results Analysis of the examined folates in the cell culture medium located opposite of the application site showed their penetration through the reconstructed epidermal model. In epidermal models to which L-FTHF di-arginine was added, L-MTHF dicholine was detected on the opposite site. L-FTHF salts were partially converted to L-MTHF. Improved wound healing was shown in keratinocyte scratch assay when the selected folates were added to the medium. # Conclusion The *in vitro* experiments on an epidermal skin model have revealed the following new findings: a) the different reduced folates investigated (L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine) penetrate well into and through the skin, in contrast to folic acid, which is commonly used; b) they neither disturb the viability of skin cells nor their barrier function, but even improve it; c) the proliferation and migration of epidermal cells, which is important for wound healing, is promoted; d) epidermal cells are able to convert L-FTHF locally into L-MTHF, i.e., to the dominant biologically active form in the circulation. This creates important prerequisites for a future topical application of folates for the treatment of certain skin diseases and the prophylaxis of skin aging processes.

Royal Jelly Protects against Epidermal Stress through Upregulation of the NQO1 Expression.

Royal jelly (RJ) is secreted by honeybees and has been used as an apitherapy to obtain healthy skin since ancient times. However, the mechanism of the protective effects of RJ against skin aging and skin diseases caused by skin stress and its components have not been clarified. In this study, we attempted to understand the effect of RJ on epidermal function and observed that NAD(P)H quinone dehydrogenase 1 (NQO1) is significantly induced by RJ in keratinocytes. The expression of NQO1 was also increased in the 3D epidermal skin model. NQO1 is involved in antioxidation and detoxification metabolism, and we found that RJ protects against the epidermal stress caused by UVB and menadione through the upregulation of NQO1. We identified 10-hydroxy-2-decenoic acid (10H2DA), a major fatty acid in RJ, as an active compound in this reaction as it induced the expression of NQO1 and protected the skin against oxidative stress. We demonstrated that the protective effect of RJ against epidermal stress is mediated through the upregulation of NQO1 by 10H2DA.

Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models

Organotypic skin tissue models have decades of use for basic research applications, the treatment of burns, and for efficacy/safety evaluation studies. The complex and heterogeneous nature of native human skin however creates difficulties for the construction of physiologically comparable organotypic models. Within the present study, we utilized bioprinting technology for the controlled deposition of separate keratinocyte subpopulations to create a reconstructed epidermis with two distinct halves in a single insert, each comprised of a different keratinocyte sub-population, in order to better model heterogonous skin and reduce inter-sample variability. As an initial proof-of-concept, we created a patterned epidermal skin model using GPF positive and negative keratinocyte subpopulations, both printed into 2 halves of a reconstructed skin insert, demonstrating the feasibility of this approach. We then demonstrated the physiological relevance of this bioprinting technique by generating a heterogeneous model comprised of dual keratinocyte population with either normal or low filaggrin expression. The resultant model exhibited a well-organized epidermal structure with each half possessing the phenotypic characteristics of its constituent cells, indicative of a successful and stable tissue reconstruction. This patterned skin model aims to mimic the edge of lesions as seen in atopic dermatitis or ichthyosis vulgaris, while the use of two populations within a single insert allows for paired statistics in evaluation studies, likely increasing study statistical power and reducing the number of models required per study. This is the first report of human patterned epidermal model using a predefined bioprinted designs, and demonstrates the relevance of bioprinting to faithfully reproduce human skin microanatomy.

18672 Evaluation of skin barrier and hydration effects of a new rebalancing moisturizing treatment cream in a human epidermal skin model

Background and Objective: Three critical elements of skin moisturization and optimal skin barrier health include enhancing skin’s natural lipid bilayer, natural moisturizing factors (NMF) and hyaluronic acid (HA). Herein we evaluated the skin barrier and hydration effects of a new rebalancing moisture treatment (TRMT).

Red Carrot Cells Cultured in vitro Are Effective, Stable, and Safe Ingredients for Skin Care, Nutraceutical, and Food Applications.

Plant biomasses growing in bioreactor could be developed as production systems for cosmetic ingredients, nutraceuticals and food additives. We previously reported that the red carrot cell line R4G accumulates high levels of anthocyanins, which are potent antioxidants with multiple health-promoting properties. To investigate the industrial potential of this cell line in detail, we tested extract for antioxidant and anti-inflammatory activity in the mouse monocyte/macrophage cell-line J774A.1 and in reconstructed skin tissue models. We also compared the R4G extract to commercial carrot extracts in terms of stability and metabolomic profiles. We found that the R4G extract have potent antioxidant and anti-inflammatory activities, protecting mammalian cells from the oxidative stress triggered by exposure to bacterial lipopolysaccharides and H2O2. The extract also inhibited the nuclear translocation of NF-κB in an epidermal skin model, and induced the expression of VEGF-A to promote the microcirculation in a dermal microtissue model. The anthocyanins extracted from R4G cells were significantly more stable than those found in natural red carrot extracts. Finally, we showed that R4G extract has similar metabolomic profile of natural extracts by using a combination of targeted and untargeted metabolomics analysis, demonstrating the safety of R4G carrot cells for applications in the nutraceutical and food/feed industries.

694 An epidermal model containing melanocytes for skin pigmentation and lightening studies

Considerable interest exists in evaluating raw materials and/or skin care formulations which cause lightening of the skin. These products are utilized to modulate one’s natural skin color or to combat skin pigmentation disorders such as melasma, dark spots, solar lentigo, and other hyperpigmentation lesions. To aid in the development and testing of such products, we have developed a skin whitening protocol using the epidermal skin model, MelanoDerm™, to evaluate both raw materials and skin lightening formulations. MelanoDerm is a highly differentiated, three-dimensional tissue culture model of human epidermis that contains normal human melanocytes (NHM) and keratinocytes (NHK). Epidermal tissues have been produced containing NHM of varying skin phototypes which follow the pigmentation level of the donor tissue, i.e. black > Asian > Caucasian. For lightening studies, tissues were treated topically three times a week over a two to three week period to mimic consumer application. Several over-the-counter skin lightening products were evaluated in cultures containing NHM from black and Asian donors. Over the treatment period, negative control cultures became increasingly pigmented with retention of normal epithelial morphology. In contrast, tissues treated topically with cosmetic skin lightening agents containing tyrosinase inhibitors such as kojic acid and magnesium ascorbyl phosphate remained lighter than the control cultures. The skin lightening effect on treated tissues was quantitatively evaluated for melanin content using a Solvable melanin assay and for skin brightness (L* value) using a hand-held spectrometer. Treated tissues showed significant changes in overall melanin content and brightness compared to control tissues. These results suggest that this model can provide valuable in vitro data for screening raw materials prior to the commencement of costly clinical trials and that it will be useful to study melanogenesis, skin lightening, and other pigmentation phenomena of the skin.

Protective effects of a comprehensive topical antioxidant against ozone-induced damage in a reconstructed human skin model

Tropospheric ozone (O3) is a source of oxidative stress. This study examined the ability of a topical antioxidant (WEL-DS) to inhibit O3-mediated damage in a human epidermal skin model. Four groups of tissues (N = 24) were compared: Group 1 (control) were untreated and unexposed; Group 2 were untreated and exposed to O3 (0.4 ppm, 4 h); Group 3 were pretreated with WEL-DS and unexposed; Group 4 were pretreated with WEL-DS and exposed to O3 (0.4 ppm, 4 h). Pretreated tissues were topically treated with 20 uL of WEL-DS and incubated for up to 20 h at 37 °C [humidified, 5% carbon dioxide (CO2)]. After 24 h, tissues were re-treated with WEL-DS and exposed to O3. Tissues were evaluated for Reactive Oxygen Species (ROS), hydrogen peroxide (H2O2), 4-hydroxynonenal (4-HNE) protein adducts, NF-κB p65 response and histology. In O3-exposed groups, WEL-DS significantly inhibited ROS formation vs. untreated tissues (p < 0.05). Pretreatment with WEL-DS inhibited H2O2 production vs. untreated tissues (p < 0.05), and decreased NF-κB p65 transcription factor signal. Oxidative stress induction in O3-exposed tissues was confirmed by increased levels of 4-HNE protein adducts (marker of lipid peroxidation); WEL-DS application reduced this effect. WEL-DS inhibited damage in tissues exposed to O3 with no significant changes in epidermal structure. A comprehensive topical antioxidant significantly diminished O3-induced oxidative damage in a human epidermal skin model.

Acremonidin E produced by Penicillium sp. SNF123, a fungal endophyte of Panax ginseng, has antimelanogenic activities

BackgroundGinseng extracts and ginseng-fermented products are widely used as functional cosmetic ingredients for their whitening and antiwrinkle effects. Recently, increasing attention has been given to bioactive metabolites isolated from endophytic fungi. However, little is known about the bioactive metabolites of the fungi associated with Panax ginseng Meyer. MethodsAn endophytic fungus, Penicillium sp. SNF123 was isolated from the root of P. ginseng, from which acremonidin E was purified. Acremonidin E was tested on melanin synthesis in the murine melanoma cell line B16F10, in the human melanoma cell line MNT-1, and in a pigmented 3D-human skin model, Melanoderm. ResultsAcremonidin E reduced melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 cells with minimal cytotoxicity. qRT–PCR analysis demonstrated that acremonidin E downregulated melanogenic genes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), while their enzymatic activities were unaffected. The antimelanogenic effects of acremonidin E were further confirmed in MNT-1 and a pigmented 3D human epidermal skin model, Melanoderm. Immunohistological examination of the Melanoderm further confirmed the regression of both melanin synthesis and melanocyte activation in the treated tissue. ConclusionThis study demonstrates that acremonidin E, a bioactive metabolite derived from a fungal endophyte of P. ginseng, can inhibit melanin synthesis by downregulating tyrosinase, illuminating the potential utility of microorganisms associated with P. ginseng for cosmetic ingredients.

2'-Fucosyllactose Attenuates Particulate Matter-Induced Inflammation via Inhibition of Hypoxia-Inducible Factor in Keratinocytes.

2'-Fucosyllactose (2FL) is the most abundant component of the oligosaccharide content in human milk. It has been reported that 2FL has the ability to protect against infectious disease caused by bacterial pathogens. In this study, we investigated the protective effects of 2FL on particulate matter (PM)10-induced pro-inflammatory cytokines in HaCaT keratinocytes. 2FL reduced PM10-induced excess expression of interleukin (IL)-6, IL-8, IL-1α and IL-1β in HaCaT keratinocytes. In addition, PM10 also increased hypoxia-inducible factor (HIF)-1α protein levels; however, 2FL inhibited the accumulation of HIF-1α protein and the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt stimulated by PM10. Furthermore, 2FL improved PM10-induced the decrease in epidermal thickness and integrity of the cornified layer in the reconstructed human epidermal skin model (RHE). In our results, 2FL inhibited PM10-induced pro-inflammatory mediators by regulating the HIF-1α/PI3K/Akt pathway and protected the skin epidermis against PM10 irritation. Taken together, these results suggest that 2FL can be used as a primary ingredient in cosmeceutical products to alleviate skin irritation and inflammation caused by urban air pollution.

Novel polylactic acid (PLA)-organoclay nanocomposite bio-packaging for the cosmetic industry; migration studies and in vitro assessment of the dermal toxicity of migration extracts

The exploitation of polylactide (PLA) nanocomposites (which integrate organically modified clays (organoclays) into polymers) in packaging for the cosmetics industry could provide a biodegradable alternative to the use of conventional plastics. In this study nanocomposites with a PLA polymer matrix and clay fillers organically modified with the quaternary ammonium salts, hexadecyl trimethyl ammonium bromide (HDTA) or octadecyl trimethyl ammonium chloride (TMSA), were produced and tested for their safe use in cosmetic packaging. To address concerns over the potential release of constituents from such nanocomposites, levels of total overall migration in a range of simulants (e.g. vegetable oil, aqueous media, and cosmetic formulations) was assessed 10 days post incubation at 40 °C following EU-Plastics Regulation 10/2011 concerning materials and articles in contact with foodstuffs. Total overall migration levels calculated for all PLA nanocomposites tested (maximum of 0.88 ± 0.44 mg/dm2) were well below the total established legislative migration limit (10 mg/dm2). Toxicity of the nanocomposite migration extracts to the skin was assessed in vitro. Exposure of skin cells (HaCaT immortalized human keratinocytes) and a full thickness epidermal skin model (EpiDerm™) to migration extracts did not result in any significant loss in cell viability or skin irritation (OECD TG 439). The results therefore indicate that the levels of migration from the nanocomposite measured was low, and that the nanocomposite migration extracts stimulated minimal toxicity to the skin. Up until now, the hazard of migration extracts from polymer-organoclay nanocomposites following dermal exposure has not been investigated and thus our study addresses a gap in knowledge. These findings can inform the safe design of bio-based biodegradable nanocomposite packaging (used by the cosmetics, and other industries) in the future to promote a more sustainable and greener economy for the plastics industry.

3D Cell Printing of Perfusable Vascularized Human Skin Equivalent Composed of Epidermis, Dermis, and Hypodermis for Better Structural Recapitulation of Native Skin.

Although skin cell-printing has exhibited promises for fabrication of functional skin equivalents, existing skin models through 3D cell printing are still composed of dermal and epidermal layers. However, a key hope for printing skin is to improve structural complexity of human skin over conventional construction, enabling the precise localization of multiple cell types and biomaterials. Here, the complexity of skin anatomy is increased using 3D cell printing. A novel printing platform is suggested for engineering a matured perfusable vascularized 3D human skin equivalent composed of epidermis, dermis, and hypodermis. The skin model is evaluated using functional markers representing each region of epidermis, dermis, and hypodermis to confirm tissue maturation. It is hypothesized that the vascularized dermal and hypodermal compartments that provide a more realistic microenvironment can promote cross-talks with the epidermal compartment, producing better recapitulation of epidermal morphogenesis. Skin stemness in epithelial tissue is investigated. These findings reveal that the full-thickness skin has more similarities to the native human skin compared with the dermal and epidermal skin model, indicating that it better reflects the actual complexity of native human skin. It is envisioned that it offers better predictive and reliable in vitro platform for investigation of mechanisms of pathological research and skin disease modeling.

Anti-Pigmentary Effect of (-)-4-Hydroxysattabacin from the Marine-Derived Bacterium Bacillus sp.

Bioactivity-guided isolation of a crude extract from a culture broth of Bacillus sp. has led to the isolation of (-)-4-hydroxysattabacin (1). The inhibitory effect of (-)-4-hydroxysattabacin (1) was investigated on melanogenesis in the murine melanoma cell line, B16F10, and human melanoma cell line, MNT-1, as well as a pigmented 3D-human skin model. (-)-4-Hydroxysattabacin treatment decreased melanin contents in a dose-dependent manner in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. Quantitative real time PCR (qRT–PCR) demonstrated that treatment with (-)-4-hydroxysattabacin down-regulated several melanogenic genes, including tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) while their enzymatic activities were unaffected. The anti-melanogenic effects of (-)-4-hydroxysattabacin were further demonstrated in a pigmented 3D human epidermal skin model, MelanodermTM, and manifested as whitening and regression of melanocyte activation in the tissue.

Inhibitory Effect of 4‐hydroxysattacin, Extract of Soil Bacteria, on Melanogenesis Using B16F10 Melanoma Cells And 3D Human Skin Model

Epidermal or dermal pigmentation is a skin disorder produced from accumulated melanins in hyperactivated melanocyte. Various chemicals or natural compounds have been reported as a therapeutic agent of hyperpigmentary disorders that manifests, post‐inflammatory, melanoderma, solar lentigo, or freckles. Here, we investigated melisma inhibitory effect of 4‐hydroxysattabacin, crude extract of soil bacteria, Bacillus sp, on melanogenesis in murine melanoma cell, B16F10, and UVB‐induced hyperpigmentation on 3D‐human skin model. Using alpha‐melanocyte stimulating hormone (a‐MSH) as triggering factor of pigmentation, 4‐hydroxysattabacin treatment decreased melanin contents in dose dependent manner. The Real‐Time Polymerase Chain Reaction result demonstrated 48 hour treatment of 4‐hydroxysattabacin produced down‐regulation of several melanogenic genes that encompass Tyrosinase, Tyrosinase‐related protein 1 (TRP‐1), and Tyrosinase‐related protein 2 (TRP‐2) while melanogenic enzyme activities were slightly affected. Moreover, diminution of Tyrosinase protein expression was confirmed by immunoblotting assay as well as anti‐melanogenic effects of 4‐hydroxysattabacin was proved on 3D human epidermal skin model, which supplant animal test for cosmetic testing. Finally, inhibitory effect of 4‐hydroxysattabacin was clarified both in vitro and 3D human skin model that is to say 4‐hydroxysattabacin would be a potential therapeutic agent for cutaneous hyperpigmentation.

The highly differentiated 3D epidermal skin model (epiCS®) to characterize skin sensitizers in mixtures

The highly differentiated 3D epidermal skin model (epiCS®) to characterize skin sensitizers in mixtures

Evaluation of effect during cell isolation process in alkaline comet assay using epidermal skin cells

The aim of the present study was to evaluate the effect of the cell isolation process in the alkaline comet assay using epidermal skin cells. When we explored the cell isolation method for the alkaline comet assay using the 3-dimensional (3D) human epidermal skin model, we found that DNA damage and cytotoxicity were induced during the cell isolation process. In particular, trypsin 5 min treatment with ethylene diamine tetraacetic acid (EDTA) showed about 5 times %DNA in the tail value compared to without EDTA treatment. In general, EDTA is commonly used for cell isolation, but it is known to induce genotoxicity due to secondary effects. We therefore evaluated the effect of EDTA and pH in the alkaline comet assay on a monolayer culture of rat keratinocytes. As a result, there was a significant increase of %DNA in tail values by treatment with 0.1 w/v% EDTA for 60 min; however, there was no difference in the %DNA in tail values between 0.1 w/v% EDTA/PBS(-) (pH 6.8) and 0.1 w/v% EDTA/PBS(-) (pH 7.4). These data imply that there is a need to control the EDTA conditions for cell isolation in the epidermal skin cells.

Leiden reconstructed human epidermal model as a tool for the evaluation of the skin corrosion and irritation potential according to the ECVAM guidelines

In the ECVAM validation studies two common skin protocols have been developed, the skin corrosion and skin irritation protocol. Both protocols include next to general and functional conditions that the skin model must meet, also the correct prediction of the activity of certain reference chemicals. For the skin corrosion protocol, the OECD TG 431 defined 12 reference chemicals that should be correctly predicted by the epidermal skin model. For skin irritation 20 test substances should meet the defined criteria. In this study we aimed to subject our Leiden human epidermal (LHE) model to both common protocols according to the ECVAM guidelines. The LHE model generated in this study has been fully characterized and shows very high similarities with the native skin. After minor technical changes in both protocols, corrosion classifications were obtained in concordance with those reported for the validated human skin models EpiSkin and EpiDerm. The results obtained with the common skin irritation protocol were very similar to that of earlier studies with the SkinEthic, EpiSkin and EpiDerm models. This means that the protocols and prediction models developed during the validation studies with a specific skin model can be used with other similar skin models. This study demonstrates that reconstructed human skin equivalents have been proven to be efficient and reliable alternatives to animal testing.