Investigation into the significant role of dermal-epidermal interactions in skin ageing utilising a bioengineered skin construct.

Increased prevalence of skin ageing is a growing concern due to an ageing global population and has both sociological and psychological implications. The use of more clinically predictive in vitro methods for dermatological research is becoming commonplace due to initiatives and the cost of clinical testing. In this study, we utilise a well-defined and characterised bioengineered skin construct as a tool to investigate the cellular and molecular dynamics involved in skin ageing from a dermal perspective. Through incorporation of ageing fibroblasts into the dermal compartment we demonstrate the significant impact of dermal-epidermal crosstalk on the overlying epidermal epithelium. We characterise the paracrine nature of dermal-epidermal communication and the impact this has during skin ageing. Soluble factors, such as inflammatory cytokines released as a consequence of senescence associated secretory phenotype (SASP) from ageing fibroblasts, are known to play a pivotal role in skin ageing. Here, we demonstrate their effect on epidermal morphology and thickness, but not keratinocyte differentiation or tissue structure. Through a novel in vitro strategy utilising bioengineered tissue constructs, this study offers a unique reductionist approach to study epidermal and dermal compartments in isolation and tandem.

3D Approaches to Culturing Bovine Skin: Explant Culture versus Organotypic Skin Model.

INTRODUCTION: Digital dermatitis (DD) in cattle appears with high prevalence; nevertheless, the knowledge on its pathogenesis is still limited. In this context, in vitro skin models represent a valuable tool to facilitate the study of DD. METHODS: Two in vitro skin models were established using bovine distal limb skin: a skin explant model and an organotypic skin model. For the skin explant model, skin samples were cultured with an air-liquid interface for up to 7 days. Besides routine histopathological examination, readout parameters were Ki-67 and cleaved Caspase-3 stainings. For the organotypic model, primary keratinocytes were layered on top of a dermal equivalent containing mainly mitotically inactive fibroblasts and maintained for up to 21 days. At regular intervals (days 7, 14, and 21), cultured skin samples were taken for (immuno)histological analysis. RESULTS: Both cultures could be maintained for the entire duration of the intended culture period. In the histopathological assessment, explant skin cultures showed ballooning degeneration of keratinocytes and segmental necrosis starting at day 5 of culturing. Initially, basal keratinocytes in the organotypic model differentiated as demonstrated by positive Keratin 14, Desmoglein-1, Loricrin, and Involucrin immunofluorescent stainings. Ki-67 was observed occasionally and suprabasally still after 21 days of culture. CONCLUSION: Both in vitro models proved dependable and constitute a viable option for replacing experiments on live animals, each with its own benefits. Whereas skin explants include all cell types available in vivo and can therefore reflect realistic cell-cell interactions and signaling pathways, the organotypic model offers a higher standardization and reproducibility. Depending on the focus of future studies, both models can be used for specific experimental purposes of bovine dermatological research in general or specialized questions concerning (infectious) claw diseases as, e.g., DD.

Human epidermis organotypic cultures, a reproducible system recapitulating the epidermis in vitro.

The translatability of research is highly dependent on models that recapitulate human tissues and organs. Here, we describe a procedure for the generation of human epidermis organotypic cultures (HEOCs) from primary keratinocytes isolated from foreskin and adult skin as well as from an immortalized keratinocyte cell line (KerTr). We tested several media conditions to develop a defined HEOC growing and expansion media. We characterized the HEOCs and show that in optimal culture conditions they express the proliferation marker Ki67, the basement membrane protein collagen 17 (col17) and the epidermal differentiation markers keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), transglutaminase 1 (TGM1), transglutaminase 3 (TGM3) and filaggrin (FLG). Thus, they recapitulate the human epidermis and are stratified from the basal layer to the stratum corneum. These HEOC can be generated reproducibly on a large scale, making it an invaluable model for screening therapeutic compounds and also for the study of pathologies affecting the epidermis.

Altered expression of S100 fused-type proteins in an atopic dermatitis skin model.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder with elevated interleukin (IL)-4 and IL-13 signatures and extensive barrier dysfunction, which is correlated with the downregulation of filaggrin (FLG). FLG is a member of the S100 fused-type protein family and this family also includes cornulin (CRNN), filaggrin-2 (FLG2), hornerin (HRNR) repetin (RPTN), trichohyalin (TCHH) and trichohyalin-like 1 (TCHHL1). The present study aimed to examine the effects of IL-4 and IL-13 and the downregulation of FLG on the expression of S100 fused-type proteins using a three-dimensional (3D) AD skin model by immunohistochemical study and quantitative polymerase chain reaction. In the 3D AD skin model, which was generated by a stimulation of recombinant IL-4 and IL-13, the expression of FLG, FLG2, HRNR and TCHH was decreased, while that of RPTN was increased in comparison to the 3D control skin. In the FLG knockdown (KD) 3D skin model, which was generated using FLG siRNA, the expression of HRNR was increased. The expression of the other proteins did not differ to a statistically significant extent. The expression of fused-S100 type protein family members may differ in AD skin. This suggests that these proteins play different roles in the pathogenesis of AD.

Requisite instruments for the establishment of three-dimensional epidermal human skin equivalents-A methods review.

Human skin equivalents (HSEs) are three-dimensional skin organ culture models raised in vitro. This review gives an overview of common techniques for setting up HSEs. The HSE consists of an artificial dermis and epidermis. 3T3-J2 murine fibroblasts, purchased human fibroblasts or freshly isolated and cultured fibroblasts, together with other components, for example, collagen type I, are used to build the scaffold. Freshly isolated and cultured keratinocytes are seeded on top. It is possible to add other cell types, for example, melanocytes, to the HSE-depending on the research question. After several days and further steps, the 3D skin can be harvested. Additionally, we show possible markers and techniques for evaluation of artificial skin. Furthermore, we provide a comparison of HSEs to human skin organ culture, a model which employs human donor skin. We outline advantages and limitations of both models and discuss future perspectives in using HSEs.

CC chemokine ligand 20 (CCL20) positively regulates collagen type I production in 3D skin equivalent tissues.

Chemokines are a group of small proteins that induce chemoattraction and inflammation and contribute to the differentiation and homeostasis of various cell types. Here we explored the role of chemokines, extracellular matrix production, and myofibroblast differentiation in self-assembled skin equivalents (SASE), a three-dimensional (3D) skin-equivalent tissue model. We found that the expression of three chemokines, C-C motif chemokine ligand (CCL) 20, C-X-C motif chemokine ligand (CXCL) 5, and CXCL8, increased with differentiation to myofibroblasts. Addition of recombinant CCL20 to human skin fibroblast induced collagen Type I alpha 2 gene expression, but did not affect the expression of alpha smooth muscle actin expression. Conversely, siRNA gene knockdown of CCL20 effectively inhibited the expression of collagen Type I gene and protein. Furthermore, when the CCL20 gene in fibroblasts was knocked down in SASE, collagen Type I synthesis and stromal thickness were decreased. Taken together, these results have indicated the utility of SASE in understanding how cytokines such as CCL20 positively regulate extracellular matrix proteins such as collagen Type I production during myofibroblast differentiation in 3D tissues that mimic human skin.

An Interleukin-4 and Interleukin-13 Induced Atopic Dermatitis Human Skin Equivalent Model by a Skin-On-A-Chip.

Currently, the mechanism of progression of atopic dermatitis (AD) is not well understood because there is no physiologically appropriate disease model in terms of disease complexity and multifactoriality. Type 2 inflammation, mediated by interleukin (IL)-4 and IL-13, plays an important role in AD. In this study, full-thickness human skin equivalents consisting of human-derived cells were fabricated from pumpless microfluidic chips and stimulated with IL-4 and IL-13. The morphological properties, gene expression, cytokine secretion and protein expression of the stimulated human skin equivalent (HSE) epidermis were investigated. The results showed epidermal and spongy formations similar to those observed in lesions in AD, and decreased expression of barrier-related filaggrin, loricrin and involucrin genes and proteins induced by IL-4Rα signaling. In addition, we induced the expression of carbonic anhydrase II (CAII), a gene specifically expressed in the epidermis of patients with AD. Thus, AD human skin equivalents can be used to mimic the key pathological features of atopic dermatitis, overcoming the limitations of existing studies that rely solely on mouse models and have been unable to translate their effects to humans. Our results will be useful for future research on the development of therapeutic agents for atopic dermatitis.

Effects of Indole-3-Lactic Acid, a Metabolite of Tryptophan, on IL-4 and IL-13-Induced Human Skin-Equivalent Atopic Dermatitis Models.

Indole-3-lactic acid (I3LA) is a well-known metabolite involved in tryptophan metabolism. Indole derivatives are involved in the differentiation of immune cells and the synthesis of cytokines via the aryl hydrocarbon receptors for modulating immunity, and the indole derivatives may be involved in allergic responses. I3LA was selected as a candidate substance for the treatment of atopic dermatitis (AD), and its inhibitory effect on AD progression was investigated. Full-thickness human skin equivalents (HSEs) consisting of human-derived cells were generated on microfluidic chips and stimulated with major AD-inducing factors. The induced AD-HSEs were treated with I3LA for 7 days, and this affected the AD-associated genetic biomarkers and increased the expression of the major constituent proteins of the skin barrier. After the treatment for 14 days, the surface became rough and sloughed off, and there was no significant difference between the increased AD-related mRNA expression and the skin barrier protein expression. Therefore, the short-term use of I3LA for approximately one week is considered to be effective in suppressing AD.

Enhanced Nrf2 up-regulation by extracellular basic pH in a human skin equivalent system.

Extracellular basic pH regulates cellular processes in wounds, and consequently influenced wound healing. Oxidative defence system modulation in the skin helps heal wounds, inhibits skin ageing and improves the skin condition. Moreover, the role of keratinocyte growth factor (KGF) and nuclear factor erythroid 2-related factor 2 (Nrf2) in antioxidant systems has been reported in various skin models. However, the effects of extracellular basic pH on wound- or skin ageing-related skin damage have not been examined. Thus, we investigated the antioxidant systems affected by extracellular basic pH in a 3D human skin equivalent system (3HSE). Extracellular basic pH decreased KGF expression and enhanced the oxidative defence system, and thus activated Nrf2 in the 3HSE. Additionally, extracellular basic pH and KGF treatment up-regulated Nrf2 activation and its regulation of the oxidative defence system in the 3HSE. This indicates that Nrf2 up-regulation is enhanced by reactive oxygen species production, rather than KGF, and by extracellular basic pH of the skin. The inhibition of skin damage through pH imbalance and KGF regulation suggests that the development of pH-regulating or pH-maintaining materials may provide effective therapeutic strategies for maintaining a healthy skin.

3D skin models in domestic animals.

The skin is a passive and active barrier which protects the body from the environment. Its health is essential for the accomplishment of this role. Since several decades, the skin has aroused a strong interest in various fields (for e.g. cell biology, medicine, toxicology, cosmetology, and pharmacology). In contrast to other organs, 3D models were mostly and directly elaborated in humans due to its architectural simplicity and easy accessibility. The development of these models benefited from the societal pressure to reduce animal experiments. In this review, we first describe human and mouse skin structure and the major differences with other mammals and birds. Next, we describe the different 3D human skin models and their main applications. Finally, we review the available models for domestic animals and discuss the current and potential applications.

Skin Disease Models In Vitro and Inflammatory Mechanisms: Predictability for Drug Development.

Investigative skin biology, analysis of human skin diseases, and numerous clinical and pharmaceutical applications rely on skin models characterized by reproducibility and predictability. Traditionally, such models include animal models, mainly rodents, and cellular models. While animal models are highly useful in many studies, they are being replaced by human cellular models in more and more approaches amid recent technological development due to ethical considerations. The culture of keratinocytes and fibroblasts has been used in cell biology for many years. However, only the development of co-culture and three-dimensional epidermis and full-skin models have fundamentally contributed to our understanding of cell-cell interaction and cell signalling in the skin, keratinocyte adhesion and differentiation, and mechanisms of skin barrier function. The modelling of skin diseases has highlighted properties of the skin important for its integrity and cutaneous development. Examples of monogenic as well as complex diseases including atopic dermatitis and psoriasis have demonstrated the role of skin models to identify pathomechanisms and drug targets. Recent investigations have indicated that 3D skin models are well suitable for drug testing and preclinical studies of topical therapies. The analysis of skin diseases has recognized the importance of inflammatory mechanisms and immune responses and thus other cell types such as dendritic cells and T cells in the skin. Current developments include the production of more complete skin models comprising a range of different cell types. Organ models and even multi-organ systems are being developed for the analysis of higher levels of cellular interaction and drug responses and are among the most recent innovations in skin modelling. They promise improved robustness and flexibility and aim at a body-on-a-chip solution for comprehensive pharmaceutical in vitro studies.

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased ß-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.

A comparison of the in vitro permeation of 3-O-ethyl-l-ascorbic acid in human skin and in a living skin equivalent (LabSkin™).

OBJECTIVES: The safety assessment of personal care products often entails determining dermal absorption of their ingredients. Such experiments are typically performed in human or animal skin in vitro; however, ethical and safety considerations are associated with obtaining these tissues. Several human skin equivalent models (HSEs) have been developed as alternatives to human tissue. The barrier function of such models however, is normally less developed than human skin. Here, we examine the permeability of the HSE LabSkinTM to a model compound, 3-O-ethyl-l-ascorbic acid (EA) compared with human skin. METHODS: Skin uptake and permeation of EA was investigated in vitro using heat-separated human epidermis and LabSkinTM . Finite dose (5 µL cm-2 ) Franz-diffusion studies were conducted using 2 % (w/w) EA in a ternary solvent mixture comprising propylene glycol (PG), propylene glycol monolaurate (PGML), and isopropyl myristate (IPM). These excipients are commonly used in cosmetic products and they have been reported to promote permeation of EA in a different model, namely porcine skin. RESULTS: Permeation of EA through LabSkinTM was evident from 2 h; however, EA permeation in human skin was not detected until 5 h. Similar amounts of EA permeated through the two membranes at time points 8, 10, 12 and 24 h (p > 0.05). The cumulative amounts of EA delivered through LabSkinTM at 24 h were 41.3 ± 2.0 µg cm-2 , corresponding to 55.1 ± 1.8 % of the applied dose. Similar amounts permeated across human skin, 49.4 ± 4.1 µg cm-2 , accounting for 58.0 ± 4.2 % of the dose applied (p > 0.05). CONCLUSION: The permeation of EA in LabSkinTM compared well with results for human epidermis in terms of the permeation profiles and the cumulative amounts of EA that permeated. The data suggest that the skin barrier of the two models was similar with regard to their overall permeability to the hydrophilic active EA. The findings are promising for the use of LabSkinTM as a surrogate for human skin in permeability testing. Future studies will focus on exploring the reproducibility and robustness of LabSkinTM for delivery of other actives that span a range of physicochemical properties.

OBJECTIFS: L'évaluation de la sécurité des produits de soins personnels implique souvent de déterminer l'absorption cutanée de leurs ingrédients. Ces expérimentations sont généralement réalisées in vitro sur la peau humaine ou animale ; cependant, des considérations éthiques et de sécurité sont associées à l'obtention de ces tissus. Plusieurs modèles équivalents de peau humaine (Human Skin Equivalent, HSE) ont été développés comme alternatives au tissu humain. La fonction barrière de ces modèles est cependant normalement moins développée que la peau humaine. Ici, nous examinons la perméabilité du HSE LabSkin™ à un composé modèle, l'acide 3-O-éthyl-l-ascorbique (EA) en le comparant à la peau humaine. MÉTHODES: L'absorption cutanée et la perméation de l'EA ont été étudiées in vitro à l'aide d'épiderme humain séparé par la chaleur et de LabSkin™. Des études de diffusion de Franz à dose limitée (5 µL cm-2 ) ont été réalisées en utilisant 2 % (p/p) d'EA dans un mélange de solvant ternaire contenant du propylène glycol (PG), du propylène glycol monolaurate (PGML) et du myristate d'isopropyle (IPM). Ces excipients sont fréquemment utilisés dans les produits cosmétiques et il a été rapporté qu'ils favorisent la perméation de l'EA dans un modèle différent, à savoir la peau porcine. RÉSULTATS: La perméation de l'EA par LabSkin™ était évidente dès 2 h ; cependant, la perméation de l'EA dans la peau humaine n'a pas été détectée avant 5 h. Des quantités similaires d'EA ont pénétré les deux membranes aux points temporels 8, 10, 12 et 24 h (p > 0,05). Les quantités cumulées d'EA délivrées par LabSkin™ à 24 h étaient de 41,3 ± 2,0 µg cm-2 , correspondant à 55,1 ± 1,8 % de la dose appliquée. Des quantités similaires ont pénétré la peau humaine, 49,4 ± 4,1 µg cm-2 , représentant 58,0 ± 4,2 % de la dose appliquée (p > 0,05). CONCLUSION: La perméation de l'EA dans LabSkin™ a bien soutenu la comparaison quant aux résultats concernant l'épiderme humain en termes de profils de perméation et de quantités cumulées d'EA qui ont pénétré. Les données suggèrent que la barrière cutanée des deux modèles était similaire en ce qui concerne leur perméabilité globale à l'EA hydrophile actif. Les résultats sont prometteurs pour l'utilisation de LabSkin™ en tant que substitut de la peau humaine dans les tests de perméabilité. Les études futures se concentreront sur l'exploration de la reproductibilité et de la robustesse de LabSkin™ pour la délivrance d'autres principes actifs qui couvrent un éventail de propriétés physicochimiques.

Immortalization of Human Keratinocytes Using the Catalytic Subunit of Telomerase.

A new stable line of human keratinocytes was obtained. The cells have altered morphology, both abnormal chromosomal composition and expression of keratinocyte markers, do not show contact inhibition, could be cultured in various media and have limited stratification ability in vitro. Upon transplantation into nude mice the cells have tumorigenic properties.

Skin microbiota analysis in human 3D skin models-"Free your mice".

In the May issue of Experimental Dermatology 2018, we published a review article focusing on human 3D skin models in the context of microbiota research. The principal intention was to provide an overview of present and future concepts to use skin models in microbiota analyses. With the present viewpoint, we would like to draw the reader's attention again to the use of human skin models in microbiota research with the aim to highlight the benefits and necessity of human skin models to analyse the human skin-microbiota interaction. This is accompanied by a critical view on mice models that often are not suitable to analyse the functional impact of the human skin microbiota. In addition, we present novel and future concepts highlighting the benefits of human 3D skin models in microbiota research.

Subacute dermal toxicity of perfluoroalkyl carboxylic acids: comparison with different carbon-chain lengths in human skin equivalents and systemic effects of perfluoroheptanoic acid in Sprague Dawley rats.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are used in various fields but raise concerns regarding human health and environmental consequences. Among PFASs, perfluorooctanoic acid (PFOA) and short-chain perfluoroalkyl carboxylic acids (SC PFCAs) are detectable in skin-contact consumer products and have dermal absorption potential. Here, we investigated the effects of dermal exposure to PFOA and SC PFCAs using in vitro and in vivo models. Human skin equivalents were topically treated with 0.25 mM and 2.5 mM PFOA and SC PFCAs (perfluoropentanoic acid, PFPeA; perfluorohexanoic acid, PFHxA; and perfluoroheptanoic acid, PFHpA) for 6 days, and cell viability, interleukin (IL)-1α, oxidative stress markers (malondialdehyde, MDA; and 8-hydroxydeoxyguanosine, 8-OHdG), and histopathology were examined. MDA levels were significantly higher in the PFASs groups than in controls. Compared with SC PFCAs, 2.5 mM PFOA caused more IL-1α (p < 0.001) release, decreased skin thickness and microscopic abnormalities. To evaluate systemic effects, Sprague Dawley (SD) rats were dermally treated with 250 and 1000 mg/kg PFHpA for 2 weeks and clinical and anatomic pathology were assessed. At 1000 mg/kg, 83% of the rats died, with severe ulcerative dermatitis at the application site. Adverse PFHpA-treated systemic changes were observed in the kidney, liver and testes, and histopathologic lesions such as renal tubular necrosis, hepatocellular necrosis, and germ cell degeneration were seen at 250 and 1000 mg/kg. Our study suggests that SC PFCAs have fewer effects on the skin than PFOA, but SC PFCAs can have adverse effects on major organs with systemic exposure at high concentrations.

Electric field-induced changes in biomechanical properties in human dermal fibroblasts and a human skin equivalent.

PURPOSE: An electric field (EF) can be used to change the mechanical properties of cells and skin tissues. We demonstrate EF-induced elasticity changes in human dermal fibroblasts (HDFs) and a human skin equivalent and identify the underlying principles related to the changes. METHODS: HDFs and human skin equivalent were stimulated with electric fields of 1.0 V/cm. Change in cellular elasticity was determined by using atomic force microscopy. Effects of EF on the biomechanical and chemical properties of a human skin equivalent were analyzed. In cells and tissues, the effects of EF on biomarkers of cellular elasticity were investigated at the gene and protein levels. RESULTS: In HDFs, the cellular elasticity was increased and the expression of biomarkers of cellular elasticity was regulated by the EF. Expression of the collagen protein in the human skin equivalent was changed by EF stimulation; however, changes in density and microstructure of the collagen fibrils were not significant. The viscoelasticity of the human skin equivalent increased in response to EF stimulation, but molecular changes were not observed in collagen. CONCLUSIONS: Elasticity of cells and human skin equivalent can be regulated by electrical stimulation. Especially, the change in cellular elasticity was dependent on cell age.

Glucose Exerts an Anti-Melanogenic Effect by Indirect Inactivation of Tyrosinase in Melanocytes and a Human Skin Equivalent.

Sugars are ubiquitous in organisms and well-known cosmetic ingredients for moisturizing skin with minimal side-effects. Glucose, a simple sugar used as an energy source by living cells, is often used in skin care products. Several reports have demonstrated that sugar and sugar-related compounds have anti-melanogenic effects on melanocytes. However, the underlying molecular mechanism by which glucose inhibits melanin synthesis is unknown, even though glucose is used as a whitening as well as moisturizing ingredient in cosmetics. Herein, we found that glucose significantly reduced the melanin content of α-melanocyte-stimulating hormone (MSH)-stimulated B16 cells and darkly pigmented normal human melanocytes with no signs of cytotoxicity. Furthermore, topical treatment of glucose clearly demonstrated its whitening efficacy through photography, Fontana-Masson (F&M) staining, and multi-photon microscopy in a pigmented 3D human skin model, MelanoDerm. However, glucose did not alter the gene expression or protein levels of major melanogenic proteins in melanocytes. While glucose potently decreased intracellular tyrosinase activity in melanocytes, it did not reduce mushroom tyrosinase activity in a cell-free experimental system. However, glucose was metabolized into lactic acid, which can powerfully suppress tyrosinase activity. Thus, we concluded that glucose indirectly inhibits tyrosinase activity through conversion into lactic acid, explaining its anti-melanogenic effects in melanocytes.

A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture.

An important problem for researchers working in the field of dermatology is the preparation of the human skin equivalent (HSE). Here, we describe a simple and reliable protocol for preparing a skin model from the commercially available cell lines: keratinocytes, fibroblasts, and melanocytes. Importantly, in our 3D model, the keratinocytes are diverse that brings this model closer to the natural skin. For the production of HSE, we used available primary PCS-200-010, PCS-201-010, PCS-200-013, and immortalized CRL-4048 and CRL-4001 cell lines. We used genipin, which is necessary for collagen cross-linking and studied its cytotoxicity for keratinocytes and fibroblasts. The addition of 20 µM genipin reduced the shrinkage of the collagen in the constructs from 59% to 24% on day 12 of the culture of the construct. A higher concentration (80-200 µM) of genipin reduced shrinkage by 14% on average. Genipin in concentration 10 µM and below was not cytotoxic to the keratinocytes, and 150 µM and below to the fibroblasts. Hematoxylin and eosin staining showed that the morphology of HSEs was identical to that of native human skin. The immunohistochemical staining of the constructs showed the presence of vimentin-positive fibroblasts in the skin layer, while the melanocytes were in the epidermis and in the basal layer. We observed that the longer differentiation of constructs led to the higher secretion of GM-CSF, IL-10, IL-15, IL-1α, IL-6, IL-7, IL-8, and MCP-1. We also observed that the longer time of differentiation led to a more stable secretion of all analytes, which was reflected in the coefficient of variation. We described here a simple, reliable, and cost-effective production of the full-thickness human skin equivalents that can be used in the research and industry. With the global trend to decrease animal use for the research and testing, our HSE could be a useful testing tool and an alternative research model.

Tyrosinase-Targeting Gallacetophenone Inhibits Melanogenesis in Melanocytes and Human Skin-Equivalents.

Demands for safe depigmentation compounds are constantly increasing in the pharmaceutical and cosmetic industry, since the numerous relevant compounds reported to date have shown undesirable side effects or low anti-melanogenic effects. In this study, we reported three novel inhibitors of tyrosinase, which is the key enzyme in melanogenesis, identified using docking-based high throughput virtual screening of an in-house natural compound library followed by mushroom tyrosinase inhibition assay. Of the three compounds, gallacetophenone showed high anti-melanogenic effect in both human epidermal melanocytes and a 3D human skin model, MelanoDerm. The inhibitory effect of gallacetophenone on tyrosinase was elucidated by computational molecular modeling at the atomic level. Binding of gallacetophenone to the active site of tyrosinase was found to be stabilized by hydrophobic interactions with His367, Ile368, and Val377; hydrogen bonding with Ser380 and a water molecule bridging the copper ions. Thus, our results strongly suggested gallacetophenone as an anti-melanogenic ingredient that inhibits tyrosinase.