International Journal of Cosmetic Science, 2016, 38, 60–67
doi: 10.1111/ics.12249
Establishment and characterization of a reconstructed Chinese human epidermis model J. Qiu*, L. Zhong*, M. Zhou*,†, D. Chen*,†, X. Huang*, J. Chen*, M. Chen*, H. Ni* and Z. Cai*,† *L’Oreal Research and Innovation, 550 Jin Yu Road, Pudong, Shanghai, and †Shanghai EPISKIN Biotechnology Co. Ltd, 1299 Zhang Heng Road, Pudong, Shanghai, China
Received 14 May 2015, Accepted 10 June 2015
Keywords: cell culture, Chinese, industrialization, keratinocyte, reconstructed human epidermis, skin barrier, skin structure
Synopsis OBJECTIVES: In vitro reconstructed human epidermis is a powerful tool for both basic research and industrial applications in dermatology, pharmacology and the cosmetic field. METHODS: By growing keratinocytes of Chinese origin on a collagen matrix after a submerged culture followed by an air–liquid interface culture, an in vitro reconstructed Chinese human epidermis model was obtained. This Chinese epidermis model was further characterized. RESULTS: The reconstructed human epidermis model (China EpiSkin model) exhibits morphological features similar to native skin and shows similar expression profile of proliferation (Ki67) and differentiation (K14 and K10 cytokeratins, filaggrin) markers. Corneodesmosomes, lamellar lipids, desmosomes, keratohyalin granules, keratin filaments and membrane-coating granules are also observed at the ultrastructure level. Moreover, China EpiSkin model contains most of the major lipid classes normally found in the native skin and potentially could present the properties of skin barrier. More importantly, the model production achieves high reproducibility and low intra- and inter-batch variations. CONCLUSION: This is the first reconstructed Chinese human epidermis model reported to meet the high quality standard with industrialized production criteria. This China EpiSkin model can be used for both skin research and safety assessment in vitro. sume Re OBJECTIFS: L’epiderme humain reconstitue in vitro est un outil puissant pour la recherche fondamentale et les applications industrielles dans la dermatologie, la pharmacologie et le domaine cosmetique. METHODES: En cultivant des keratinocytes d’origine chinoise sur une matrice de collagene apres une culture immergee suivie d’une culture d’interface air-liquide, un modele d’epiderme humain chinois reconstitue in vitro a ete obtenu. Ce modele Episkin Chinois a ete caracterise. RESULTATS: Le modele d’epiderme humain reconstruit (China model EpiSkin) presente des caracteristiques morphologiques simi la peau native et montre le m^eme profil d’expression du laires a marqueur de la proliferation (Ki67) et des marqueurs de la Correspondence: Zhenzi Cai, L’ Oreal Research and Innovation, 550 Jin Yu Road, Pudong, Shanghai, China and Shanghai EPISKIN Biotechnology Co. Ltd, 1299 Zhang Heng Road, Pudong, Shanghai, China. Tel.: +86 21 2061 1550; fax: +86 21 2061 1418; e-mail: [email protected]
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differenciation (cytokeratines K14 et K10, filaggrine). Les corneodesmosomes, les lipides lamellaires, les desmosomes, les grains de keratohyaline, les filaments de keratine et les granules couvrant la membrane sont egalement observes au niveau de l’ultrastructure. En outre, le modele EpiSkin Chinois contient la plupart des grandes classes de lipides se trouvant normalement dans la peau native et pourrait potentiellement presenter les proprietes de barriere de la peau. Plus important encore, la production du modele atteint une grande reproductibilite et des variations intra- et inter-lots bien faibles. CONCLUSION: Ceci est le premier modele d’epiderme reconstruit la norme de haute qualite avec des d’origine chinoise qui repond a criteres de production industrialisee. Ce modele China EpiSkin peut ^etre utilise a la fois pour la recherche sur la peau et de l’evaluation de la securite in vitro. Introduction Skin is an important human organ which is essential to the organism survival as a protective barrier. Skin prevents the loss of endogenous water and minerals and protects the body from harmful environmental exposure, such as xenobiotic, microbial invasion, sun UV rays and pollution. Additionally, skin fulfils a wide variety of functions, including thermoregulation, sensitivity, excretion, vitamin D generation, etc. Skin is a multilayered structure organ. Its outermost compartment is the epidermis which is composed of four layers, from the stratum basale (basal layer), the stratum spinosum (spinous layer), the stratum granulosum (granular layer) up to the stratum corneum (horny layer) [1]. Keeping the multilayered configuration of the epidermis is necessary to sustain the protective function of human skin. Keratinocytes are the most abundant epidermal cells (more than 90%). They undergo a differentiation process from the stratum basale (SB) to the stratum corneum (SC). Abnormal development of keratinocytes can result in various skin diseases. However, the fine underlying mechanism of epidermal differentiation is not fully understood. Cells or epidermal models are widely used and are required for fundamental, applied and clinical research. Technical progress especially in keratinocyte culture at air–liquid interface brought reconstructed human epidermis (RHE) to reality in laboratory conditions [2, 3]. As the technique of keratinocyte culture was improved by Howard Green, a great deal of research has been carried out to investigate the growth and differentiation of the epidermis following this technique [4–8].
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
Jie Qiu et al.
Chinese human epidermis model
Reconstructed human epidermis in vitro is also a useful tool for dermatology and cosmetic research. It can be used to screen active substances and to assess the safety and/or efficacy of cosmetic ingredients and products. Test methods using RHE have been validated for evaluating skin irritancy, corrosivity and phototoxicity [9–13]. In fact, the Organization for Economic Co-operation and Development (OECD) has accepted several RHEs including EpiSkin (EpiSkinTM, Lyon, France) model as a validated reference method (VRM) in the TG431 testing guideline for in vitro skin corrosion test [14]. The EpiSkin method was also validated by the European Center for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee as an alternative method to fully replace the Draize acute skin irritation test in 2007. It was adopted in both the European Commission (EU) test Method B46 and the OECD TG439 as a validated reference method for in vitro skin irritation assessment [15, 16]. Skins from different human populations can present distinct features. Some reports studied the in vivo difference in the structure of SC, barrier function and reaction to irritants among skins of various ethnic origins [17–19]. Currently, most commercially available skin models are based on Caucasian cells [13, 20, 21]. We developed a reconstructed epidermis model from Chinese human keratinocytes based on the technique used for the EpiSkinTM model [2, 20–22]. Analysis of morphological architecture and microstructure, location of differentiation markers, barrier function and batch reproducibility was performed. Our data show that this China EpiSkin model is comparable to native skin in structure, as well as expression and distribution of biological markers. More importantly, the production of China EpiSkin model reaches steady performance among different batches and can be scaled up. All these results ensure that this in vitro Chinese epidermis model can be used as a powerful tool to investigate and better understand normal skin features and conduct in vitro safety assessment of cosmetic ingredients and/or products to meet local development needs in China. Materials and methods Materials and antibodies Culture media (DMEM/F12) were purchased from Gibco (Grand Island, NY, U.S.A.). Lipid standards were from Sigma-Aldrich (St. Louis, MO, U.S.A.). Anti-K10 and anti-K14 antibodies were from Dakocytomation (Hamburg, Germany). Anti-filaggrin and anti-involucrin antibodies were purchased from Abcam (Cambridge, UK). Chemically modified bovine collagen-based membranes were from Symatese (Lyon, France). All other supplements were from Life Technologies (Madison, WI, U.S.A.).
Human epidermis was reconstructed based on the technology transferred from EPISKIN SA, France, as described by Roguet et al. and Tinois et al. [21, 22]. The chemically modified bovine collagenbased membrane as support was previously fixed to the bottom of a plastic insert by an O-ring, and one tissue kit is constituted by 12 inserts [22]. NHKs (0.3 9 106 cell cm 2) were seeded onto the collagen membrane. After an immersion culture for keratinocyte proliferation, cells were further exposed to air–liquid interface to sustain keratinocyte differentiation. Then, all the tissue samples were incubated at 37°C with 5% CO2 and saturated humidity. Before transportation, China EpiSkin models were put on gel medium (agarose gel and culture medium mixture) to fix the inserts in the 12-well plates. Then, the plates were wrapped by aluminium foil bags for shipment at room temperature. Histology China EpiSkin model was fixed in 10% formaldehyde and processed for embedding in paraffin. Five-micrometre vertical sections were stained with haematoxylin and eosin for light microscopic examination. HE results were scored by investigators to grade the quality of the reconstructed human epidermis model, on a zero-to-four scale in the following aspects, respectively: general organization, nucleation of basal layer, intercellular space of basal layer, stratification of epidermis, adherence to support, granular cells, horny layer, etc. [22]. Immunohistochemistry Five-micrometre paraffin sections were used to perform immunohistochemistry. For detecting K10, K14, Ki67 and filaggrin, antigen retrieval was performed by digesting sections with 0.25% trypsin at 37°C for 15 min. For involucrin, sections were immersed in 0.01 M sodium citrate buffer (pH6.0) at 100°C for 30 min and then cooled to room temperature in 30 min. The primary antibody incubation was carried out at room temperature for 1 h, followed by staining with the Dako strept ABC complex/HRP kit according to the manufacturer’s instructions. Nucleus was stained by incubating 5-lm sections with haematoxylin for 3 min at room temperature. The following antibody dilutions were used for immunodetection of specific targets:
Cell culture and China EpiSkin model reconstruction Samples of normal adult foreskin free of HIV-1, 2, hepatitis C and hepatitis B were collected from circumcision after obtaining informed consent. Normal human keratinocytes (NHKs) were isolated from these samples and cultivated as described by Rheinwald and Green on irradiated 3T3 feeder cells and used at early passages [23]. The culture medium was Dulbecco’s modified Eagle’s medium (DMEM) and HAM’s F12 (3 : 1) supplemented with 10% fetal calf serum and 1.8 9 10 4 M adenine, 5 lg mL 1 insulin, 2 9 10 9 M triiodothyronine, 5 lg mL 1 transferrin, 0.4 lg mL 1 hydrocortisone, 10 10 M cholera toxin and 10 ng mL 1 epidermal growth factor.
Antigen
Dilution
K10 K14 Filaggrin Ki67
1 1 1 1
: : : :
100 20 200 200
Cell viability and barrier function The viability of the cultures was assessed with MTT assay according to the protocol previously described by Katoh et al. [24]. The barrier function was evaluated by the ability of the China EpiSkin to resist fast penetration of a cytotoxic marker chemical, that is 50 lL gradient concentrations of sodium dodecyl sulphate (SDS, 0.5–1.5 mg mL 1) topically applied to the model samples and
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
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Results Morphology of reconstructed epidermis model in comparison with native human epidermis Haematoxylin and eosin staining was used to investigate the morphology of the skin model compared to that of the tissue of origin, namely the foreskin. The China EpiSkin model presented a wellstratified structure, including basal layer, spinous layer, granular layer and horny layer (Fig. 1). The chronological development of this model was also investigated (Fig. 2, upper panel). D6, D8 and D10 represent the specimens at 6, 8 and 10 days, respectively, after seeding keratinocytes onto the collagen matrix. Morphology status after transportation is shown in Fig. 2 (lower panel). D6+G1+M1 represent samples which were embedded in gel for 1day transportation and cultured in maintenance medium for 1 additional day before being used for assessment. In terms of culture duration, D6+G1+M1 is comparable to D8. In the same way, D6+G1+M3 represents samples measured after the 3-day culture following transportation which are comparable to D10 samples. All these epidermal equivalents are fully differentiated and form multilayered epidermis similar to normal human epidermis (Fig. 1). The basal layers adhere to the collagen matrix, and many cells in this layer show cubical shapes. There are 4–5 layers of flattened cells, which are spinous cells above the basal layer. Granular cells are very remarkable with significant keratohyalin granules. The uppermost layer is horny layer whose thickness increases with time in culture. In conclusion, China EpiSkin model shows morphological characteristics highly similar to that of native human epidermis. The quality of the skin model was well maintained following transportation and additional culture phase before application.
B
The expression and distribution of epidermis differentiation markers Besides optical morphology, biological markers were used to further characterize the differentiation of China EpiSkin model at Day 6. As shown in Fig. 3, basal layer marker keratin K14 was expressed only in basal layer just as the native human epidermis (Fig. 3A,E). The keratinocyte proliferation can be observed by Ki67 immunohistochemical staining. Proliferation was localized in the basal layer (Fig. 3B,F). The density of proliferative keratinocytes in China EpiSkin model was similar to that of native epidermis. Early differentiation marker keratin K10 was expressed and located in all suprabasal layers (Fig. 3C,G). Filaggrin, a major keratohyalin component, was localized in granular cell layer (Fig. 3D,H). Thus, all the differentiation markers were expressed and showed the same location as in native human epidermis.
C
The lipid composition in horny layer of China EpiSkin model The lipid composition of skin is a key determinant of epidermal barrier function, which is pivotal to keep the normal epidermal function. The lipid profile of China EpiSkin model is shown in Fig. 4. The lipid extracts derived from China EpiSkin model contained major epidermal lipid classes, including cholesterol esters (CE), triglycerides (TG), free fatty acids (FFA), ceramides (CER), glucosphingolipid (CEREB), cholesterol sulphate (CHOL SO4) and phospholipids (the dark lane under the cholesterol sulphate). Among these, we can observe from Fig. 4 that the level of CHOL is very high in both native skin and China EpiSkin model. The amount of FFA, CER4 and CERB in native skin and
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Figure 5 Ultrastructure of the reconstructed human epidermis model in transmission electron microscopy. A whole view of corneocytes and stratum granulosum (SG) together was shown in A, the horny layer with a dense keratin pattern in corneocytes was shown in B, and arrow indicates the corneodesmosome. In the stratum granulosum (C), the typical desmosomes (D), keratohyalin granules (KHG), keratin filaments (K) and membrane-coating granules (G) were recognized. Scale bars are indicated within the pictures. 109 9 254 mm (300 9 300 DPI).
in China EpiSkin model is also very similar, all at moderate level. CHOL SO4 is detected in both native foreskin and China EpiSkin model, but it shows slightly higher level in China
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
Jie Qiu et al.
Chinese human epidermis model
Figure 3 Immunohistochemical staining of reconstructed human epidermis and native human epidermis. Paraffin sections of China EpiSkin model (Day 6) and native skin samples with immunohistochemical staining by anti-K14, Ki67, K10, filaggrin antibodies (brown staining). 49 9 19 mm (300 9 300 DPI).
A
B
Figure 4 High-performance thin-layer chromatographic separation of lipids extracted from the reconstructed human epidermis model. Lipids of three batches of Day 6 samples (A) and 3 different days of culture (Day 6, Day 8, Day 10, B) were extracted and separated by HPTLC with ceramide development system. CE, cholesterol esters; TG, triglycerides; CHOL, cholesterol; FFA, free fatty acid; CER 2, ceramide 2; CER 3, ceramide 3; CER 4, ceramide 4; CEREB, galactocerebrosides; CHOL SO4, cholesteryl sulphate. 180 9 181 mm (300 9 300 DPI).
(pH6.8) in darkness for 1 h at room temperature. After rinsing twice with the buffer, specimens were dehydrated in a gradient ethanol series, acetone and embedded in an epoxy resin (#618). Ultrathin
sections (50–60 nm) were prepared using a Reichert ultramicrotome, contrasted with uranyl acetate and lead citrate and examined under a Philips CM120 electron microscope at 60 kV.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
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Results Morphology of reconstructed epidermis model in comparison with native human epidermis Haematoxylin and eosin staining was used to investigate the morphology of the skin model compared to that of the tissue of origin, namely the foreskin. The China EpiSkin model presented a wellstratified structure, including basal layer, spinous layer, granular layer and horny layer (Fig. 1). The chronological development of this model was also investigated (Fig. 2, upper panel). D6, D8 and D10 represent the specimens at 6, 8 and 10 days, respectively, after seeding keratinocytes onto the collagen matrix. Morphology status after transportation is shown in Fig. 2 (lower panel). D6+G1+M1 represent samples which were embedded in gel for 1day transportation and cultured in maintenance medium for 1 additional day before being used for assessment. In terms of culture duration, D6+G1+M1 is comparable to D8. In the same way, D6+G1+M3 represents samples measured after the 3-day culture following transportation which are comparable to D10 samples. All these epidermal equivalents are fully differentiated and form multilayered epidermis similar to normal human epidermis (Fig. 1). The basal layers adhere to the collagen matrix, and many cells in this layer show cubical shapes. There are 4–5 layers of flattened cells, which are spinous cells above the basal layer. Granular cells are very remarkable with significant keratohyalin granules. The uppermost layer is horny layer whose thickness increases with time in culture. In conclusion, China EpiSkin model shows morphological characteristics highly similar to that of native human epidermis. The quality of the skin model was well maintained following transportation and additional culture phase before application.
B
The expression and distribution of epidermis differentiation markers Besides optical morphology, biological markers were used to further characterize the differentiation of China EpiSkin model at Day 6. As shown in Fig. 3, basal layer marker keratin K14 was expressed only in basal layer just as the native human epidermis (Fig. 3A,E). The keratinocyte proliferation can be observed by Ki67 immunohistochemical staining. Proliferation was localized in the basal layer (Fig. 3B,F). The density of proliferative keratinocytes in China EpiSkin model was similar to that of native epidermis. Early differentiation marker keratin K10 was expressed and located in all suprabasal layers (Fig. 3C,G). Filaggrin, a major keratohyalin component, was localized in granular cell layer (Fig. 3D,H). Thus, all the differentiation markers were expressed and showed the same location as in native human epidermis.
C
The lipid composition in horny layer of China EpiSkin model The lipid composition of skin is a key determinant of epidermal barrier function, which is pivotal to keep the normal epidermal function. The lipid profile of China EpiSkin model is shown in Fig. 4. The lipid extracts derived from China EpiSkin model contained major epidermal lipid classes, including cholesterol esters (CE), triglycerides (TG), free fatty acids (FFA), ceramides (CER), glucosphingolipid (CEREB), cholesterol sulphate (CHOL SO4) and phospholipids (the dark lane under the cholesterol sulphate). Among these, we can observe from Fig. 4 that the level of CHOL is very high in both native skin and China EpiSkin model. The amount of FFA, CER4 and CERB in native skin and
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Figure 5 Ultrastructure of the reconstructed human epidermis model in transmission electron microscopy. A whole view of corneocytes and stratum granulosum (SG) together was shown in A, the horny layer with a dense keratin pattern in corneocytes was shown in B, and arrow indicates the corneodesmosome. In the stratum granulosum (C), the typical desmosomes (D), keratohyalin granules (KHG), keratin filaments (K) and membrane-coating granules (G) were recognized. Scale bars are indicated within the pictures. 109 9 254 mm (300 9 300 DPI).
in China EpiSkin model is also very similar, all at moderate level. CHOL SO4 is detected in both native foreskin and China EpiSkin model, but it shows slightly higher level in China
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
Jie Qiu et al.
Chinese human epidermis model
EpiSkin model. The amount of CE, TG and CER3 is all very low either in native foreskin or in China EpiSkin model though in latter the level is slightly higher. The amount of CER2 is at very low level in native skin, and it is almost undetected in China EpiSkin model.
batches, and the ODs of the extracted (solubilized) dye from the untreated tissues were 20-fold greater than the ODs of solvent control (Fig. 6D). All of these parameters fulfil the production release criteria according to the OECD TG439 [16]. Discussion
Ultrastructural features of the China EpiSkin model As shown in Fig. 5A, fully differentiating epidermis was developed in the China EpiSkin model. The transmission electron microscopy investigation of this model revealed the presence of corneodesmosomes (arrowed) abundantly in stratum corneum (SC) intercellular spaces (Fig. 5B), as visualized by transmission electron microscopy with ruthenium tetroxide staining. In stratum granulosum (SG) layer, most of the classic morphological criteria of terminal epidermal differentiation were recognized (Fig. 5C), such as complete desmosomes (D), homogeneous keratohyalin granules (KHG), keratin filaments (K) and membrane-coating granules (G). Reproducibility of the production of China EpiSkin model For a functional skin model, the low variability among production batches and along time is an essential and critical factor for its reliable use in research and safety assessment application. The reproducibility of 50 batches China EpiSkin models during 18 months was determined by HE scoring for morphology, cell viability and IC50 to SDS for barrier function. The samples were scored according to the morphology of HE staining. The scores of samples from the 50 batches were compared to show a good interbatch reproducibility (Fig. 6A). Furthermore, regarding the barrier function, all the SDS IC50 values were found between 1 and 3 mg mL 1 (Mean SD: 1.295 0.091 mg mL 1). These results did not only show that the stratum corneum of China EpiSkin model was maturely differentiated and robust enough to resist rapid SDS penetration and damage, but also indicated that the production quality of China EpiSkin model is stable with low interbatch variability (Fig. 6B). Cell viability measured by MTT assay was used to assess the qualitative difference among batches. As shown in Fig. 6C, all optical densities (ODs) of formazan produced in MTT assay were found between 0.6 and 1.5 among the 50
Figure 6 Reproducibility of reconstructed human epidermis model. Fifty batches of China EpiSkin model were assessed by scoring HE staining (A). The optical density (OD) of tissue batches was obtained by MTT measurement (C). The ratio of OD570 of samples to negative control (extraction buffer only) was expressed as fold increase of negative control OD (D). A series of SDS concentrations was topically applied to the tissues, and IC50 value was determined by MTT (B). 125 9 74 mm (300 9 300 DPI).
The production of 3D skin models represents a growing trend in the field of tissue engineering. As an alternative method to animal test [26], these in vitro models, mimicking the native skin, can allow to investigate key biochemical and physical mechanisms of skin homeostasis and functions. Nowadays, some reconstructed human epidermis models are commercially available. Among them, the most accepted models include EpiSkin, SkinEthic (Lyon, France), EpiDerm (Ashland, MA, U.S.A.) and most recently LabCyte EPI-MODEL (Aichi, Japan). The testing methods base on these four models have been referred to define the alternative test methods endorsed by OECD to assess skin corrosion and skin irritation [11, 12, 14, 16, 22, 27–30]. In addition, other culture skin models, such as the Leiden reconstructed human epidermal model, the KeraSkinTM model and epiCSTM model, have been reported in the evaluation and validation efforts for in vitro skin tolerance testing according to ECVAM performance standard [10, 31, 32]. China EpiSkin model was reconstructed with keratinocytes of Chinese origin, and this is the first Chinese reconstructed human epidermis model to meet the high quality standard with industrialized production criteria. Histological analysis indicated that keratinocyte differentiation occurred in a similar way as that in the human epidermis. China EpiSkin model presents a well-stratified structure with basal layer, several spinous layers, granular layers and horny layer. Keratohyalin granules are observed in the granular layer. Through transportation/shipment process in agarose gel, tissue samples show the same quality as those in culture conditions for the same period. Keratinocyte differentiation was also validated through differentiation markers’ expression pattern. The markers, representing different differentiation steps, showed an expression profile similar to that in the human epidermis. Filaggrin labelling was located in granular cells, keratin K14 labelling was observed in basal layer, whereas early differentiation marker keratin K10 was expressed by
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© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
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all suprabasal cells as observed in vivo. Proliferative keratinocytes were visualized in basal layer by Ki67 staining, with a density similar to that of human epidermis. Barrier function is the basic attribute of epidermis. It is affected by the lipid composition of epidermis [33–35]. Ponec et al. showed that reconstructed human epidermis models contained major epidermal lipid classes but deviated from native epidermis in some lipid components, including high ceramide 2, low ceramides 5 and 6, and ceramide 7 [36, 37]. The fact that China EpiSkin model contains most of the major lipid classes found in native skin indicates the presence of major factors influencing the skin barrier properties. However, when compared to the human epidermis, it also demonstrated higher amount of CHOL SO4, cholesterol ester, triglycerides and ceramide 3, and lower amount of ceramide 2. Furthermore, ultrastructural features, observed through transmission electron microscopy, indicated the presence of multilayered stratum corneum in the reconstructed epidermis, which mainly contributes to the barrier function [38]. In addition, according to the ECVAM performance standards, a skin model need to present a proper barrier function [31]. In other words, the skin models should contain stratum corneum and lipid composition to resist the rapid penetration of cytotoxic marker chemicals such as SDS and Triton X-100 [31]. The effect of SDS, a well-known reference compound for irritancy, on the epidermis viability, is considered as an indicator of both the barrier function of the epidermis and the physiological state of viable cells. Our results demonstrated China EpiSkin model also presents a potential of barrier functions and viable cell response. This property is estimated by determination of the IC50 of SDS after a fixed exposure time. In this study, IC50 of SDS application for 18 h was measured in 50 batches of China EpiSkin model and the mean value (with standard deviation) was shown to be
1.295 0.091 mg mL 1. The IC50 for the skin models validated by ECVAM in the same manner is 1.0–3.0 mg mL 1 for EpiSkin model and 1.0–4.0 mg mL 1 for LabCyte EPI-MODEL [31]. Our observation confirmed that the barrier function of China EpiSkin model is similar to that of EpiSkin model or Labcyte EPI-MODEL and therefore meets the requirement described in the ECVAM performance standards. Meanwhile, the results of HE scoring and cell viability test showed the high reproducibility of the model from 50 production batches. In conclusion, a reconstructed epidermis model based on Chinese keratinocytes with good reproducibility and stability was developed. This model can mimic native human epidermis with steady and persistent quality and meets the general and functional conditions referred in OECD TG439 regarding reconstructed human epidermis. This model could respond to the unmet need of the scientific community in China, where there is an absence of well-characterized and commercially available reconstructed epidermis model with industrialization capability. Thus, the China EpiSkin model could serve as a useful tool for cosmetic research in China. It could also be considered as a reliable and relevant 3-dimensional in vitro model for developing in vitro alternative and predictive toxicology test methods, making further validation process a possible reality in China, and thus pave the way for applying in vitro skin safety evaluation for cosmetic ingredients and products. Acknowledgements We thank Fabien GIRARD, Jose COTOVIO, Delphine GUILLON, Bruno BERNARD (L’OREAL Research and Innovation, Paris, France) and Marie-Helene TEISSIER, Estelle TINOIS-TESSONNEAUD, Charbel BOUEZ (EPISKIN SA, Lyon, France) for their great support in this study and in the preparation of this publication.
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Irritation: Reconstructed Human Epidermis Test Method (2013). Berardesca, E. and Maibach, H. Ethnic skin: overview of structure and function. J. Am. Acad. Dermatol. 48(6 Suppl), S139–S142 (2003). Rawlings, A.V. Ethnic skin types: are there differences in skin structure and function? Int. J. Cosmet. Sci. 28, 79–93 (2006). Richards, G.M., Oresajo, C.O. and Halder, R.M. Structure and function of ethnic skin and hair. Dermatol. Clin. 21, 595–600 (2003). Tinois, E., Faure, M., Chatelain, P., Vallier, P. and Schmitt, D. Growth and differentiation of human keratinocytes on extracellular matrix. Arch. Dermatol. Res. 279, 241–246 (1987). Tinois, E., Tiollier, J., Gaucherand, M., Dumas, H., Tardy, M. and Thivolet, J. In vitro and post-transplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute. Exp. Cell Res. 193, 310–319 (1991). Roguet, R., Cohen, C., Leclaire, J., Tessonneaud, E., Gagne, C. and Teissier, M.H. Use of a standardized reconstructed epidermis kit to assess in vitro the tolerance and the efficacy of cosmetics. Int. J. Cosmet. Sci. 22, 409–419 (2000). Rheinwald, J.G. and Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–343 (1975). Katoh, M., Hamajima, F., Ogasawara, T. and Hata, K. Assessment of human epidermal model LabCyte EPI-MODEL for in vitro
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skin irritation testing according to European Centre for the Validation of Alternative Methods (ECVAM)-validated protocol. J. Toxicol. Sci. 34, 327–334 (2009). Boelsma, E., Gibbs, S., Faller, C. and Ponec, M. Characterization and comparison of reconstructed skin models: morphological and immunohistochemical evaluation. Acta Derm. Venereol. 80, 82–88 (2000). MacNeil, S. Progress and opportunities for tissue-engineered skin. Nature 445, 874– 880 (2007). Alepee, N., Tornier, C., Robert, C. et al. A catch-up validation study on reconstructed human epidermis (SkinEthic RHE) for full replacement of the Draize skin irritation test. Toxicol. In Vitro 24, 257–266 (2010). Roguet, R., Cohen, C., Dossou, K.G. and Rougier, A. Episkin, a reconstituted human epidermis for assessing in vitro the irritancy of topically applied compounds. Toxicol. In Vitro 8, 283–291 (1994). Tornier, C., Rosdy, M. and Maibach, H.I. In vitro skin irritation testing on reconstituted human epidermis: reproducibility for 50 chemicals tested with two protocols. Toxicol. In Vitro 20, 401–416 (2006). Tornier, C., Amsellem, C., Fraissinette, A.B. and Alepee, N. Assessment of the optimized SkinEthic Reconstructed Human Epidermis (RHE) 42 bis skin irritation protocol over 39 test substances. Toxicol. In Vitro 24, 245– 256 (2010). ECVAM SIVS: Performance standards for applying human skin models to in vitro skin irritation testing (2007). Online: http:// ecvam.jrc.ec.europa.eu
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32. El, G.A., Siamari, R., Willemze, R. and Ponec, M. Leiden reconstructed human epidermal model as a tool for the evaluation of the skin corrosion and irritation potential according to the ECVAM guidelines. Toxicol. In Vitro 22, 1311–1320 (2008). 33. Joo, K.M., Hwang, J.H., Bae, S., Nahm, D.H., Park, H.S., Ye, Y.M. and Lim, K.M. Relationship of ceramide-, and free fatty acid-cholesterol ratios in the stratum corneum with skin barrier function of normal, atopic dermatitis lesional and non-lesional skins. J. Dermatol. Sci. 77, 71–74 (2015). 34. Matsui, T. and Amagai, M. Dissecting the formation, structure, and barrier function of the stratum corneum. Int. Immunol. 27, 269–280 (2015). 35. Yang, Q. and Guy, R.H. Characterisation of skin barrier function using bioengineering and biophysical techniques. Pharm. Res. 32, 445–457 (2015). 36. Ponec, M., Gibbs, S., Pilgram, G., Boelsma, E., Koerten, H., Bouwstra, J. and Mommaas, M. Barrier function in reconstructed epidermis and its resemblance to native human skin. Skin Pharmacol. Appl. Skin Physiol. 14 (Suppl 1), 63–71 (2001). 37. Ponec, M., Boelsma, E., Gibbs, S. and Mommaas, M. Characterization of reconstructed skin models. Skin Pharmacol. Appl. Skin Physiol. 15(Suppl 1), 4–17 (2002). 38. Ponec, M., Boelsma, E., Weerheim, A., Mulder, A., Bouwstra, J. and Mommaas, M. Lipid and ultrastructural characterization of reconstructed skin models. Int. J. Pharm. 203, 211–225 (2000).
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doi: 10.1111/ics.12249
Establishment and characterization of a reconstructed Chinese human epidermis model J. Qiu*, L. Zhong*, M. Zhou*,†, D. Chen*,†, X. Huang*, J. Chen*, M. Chen*, H. Ni* and Z. Cai*,† *L’Oreal Research and Innovation, 550 Jin Yu Road, Pudong, Shanghai, and †Shanghai EPISKIN Biotechnology Co. Ltd, 1299 Zhang Heng Road, Pudong, Shanghai, China
Received 14 May 2015, Accepted 10 June 2015
Keywords: cell culture, Chinese, industrialization, keratinocyte, reconstructed human epidermis, skin barrier, skin structure
Synopsis OBJECTIVES: In vitro reconstructed human epidermis is a powerful tool for both basic research and industrial applications in dermatology, pharmacology and the cosmetic field. METHODS: By growing keratinocytes of Chinese origin on a collagen matrix after a submerged culture followed by an air–liquid interface culture, an in vitro reconstructed Chinese human epidermis model was obtained. This Chinese epidermis model was further characterized. RESULTS: The reconstructed human epidermis model (China EpiSkin model) exhibits morphological features similar to native skin and shows similar expression profile of proliferation (Ki67) and differentiation (K14 and K10 cytokeratins, filaggrin) markers. Corneodesmosomes, lamellar lipids, desmosomes, keratohyalin granules, keratin filaments and membrane-coating granules are also observed at the ultrastructure level. Moreover, China EpiSkin model contains most of the major lipid classes normally found in the native skin and potentially could present the properties of skin barrier. More importantly, the model production achieves high reproducibility and low intra- and inter-batch variations. CONCLUSION: This is the first reconstructed Chinese human epidermis model reported to meet the high quality standard with industrialized production criteria. This China EpiSkin model can be used for both skin research and safety assessment in vitro. sume Re OBJECTIFS: L’epiderme humain reconstitue in vitro est un outil puissant pour la recherche fondamentale et les applications industrielles dans la dermatologie, la pharmacologie et le domaine cosmetique. METHODES: En cultivant des keratinocytes d’origine chinoise sur une matrice de collagene apres une culture immergee suivie d’une culture d’interface air-liquide, un modele d’epiderme humain chinois reconstitue in vitro a ete obtenu. Ce modele Episkin Chinois a ete caracterise. RESULTATS: Le modele d’epiderme humain reconstruit (China model EpiSkin) presente des caracteristiques morphologiques simi la peau native et montre le m^eme profil d’expression du laires a marqueur de la proliferation (Ki67) et des marqueurs de la Correspondence: Zhenzi Cai, L’ Oreal Research and Innovation, 550 Jin Yu Road, Pudong, Shanghai, China and Shanghai EPISKIN Biotechnology Co. Ltd, 1299 Zhang Heng Road, Pudong, Shanghai, China. Tel.: +86 21 2061 1550; fax: +86 21 2061 1418; e-mail: [email protected]
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differenciation (cytokeratines K14 et K10, filaggrine). Les corneodesmosomes, les lipides lamellaires, les desmosomes, les grains de keratohyaline, les filaments de keratine et les granules couvrant la membrane sont egalement observes au niveau de l’ultrastructure. En outre, le modele EpiSkin Chinois contient la plupart des grandes classes de lipides se trouvant normalement dans la peau native et pourrait potentiellement presenter les proprietes de barriere de la peau. Plus important encore, la production du modele atteint une grande reproductibilite et des variations intra- et inter-lots bien faibles. CONCLUSION: Ceci est le premier modele d’epiderme reconstruit la norme de haute qualite avec des d’origine chinoise qui repond a criteres de production industrialisee. Ce modele China EpiSkin peut ^etre utilise a la fois pour la recherche sur la peau et de l’evaluation de la securite in vitro. Introduction Skin is an important human organ which is essential to the organism survival as a protective barrier. Skin prevents the loss of endogenous water and minerals and protects the body from harmful environmental exposure, such as xenobiotic, microbial invasion, sun UV rays and pollution. Additionally, skin fulfils a wide variety of functions, including thermoregulation, sensitivity, excretion, vitamin D generation, etc. Skin is a multilayered structure organ. Its outermost compartment is the epidermis which is composed of four layers, from the stratum basale (basal layer), the stratum spinosum (spinous layer), the stratum granulosum (granular layer) up to the stratum corneum (horny layer) [1]. Keeping the multilayered configuration of the epidermis is necessary to sustain the protective function of human skin. Keratinocytes are the most abundant epidermal cells (more than 90%). They undergo a differentiation process from the stratum basale (SB) to the stratum corneum (SC). Abnormal development of keratinocytes can result in various skin diseases. However, the fine underlying mechanism of epidermal differentiation is not fully understood. Cells or epidermal models are widely used and are required for fundamental, applied and clinical research. Technical progress especially in keratinocyte culture at air–liquid interface brought reconstructed human epidermis (RHE) to reality in laboratory conditions [2, 3]. As the technique of keratinocyte culture was improved by Howard Green, a great deal of research has been carried out to investigate the growth and differentiation of the epidermis following this technique [4–8].
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
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Reconstructed human epidermis in vitro is also a useful tool for dermatology and cosmetic research. It can be used to screen active substances and to assess the safety and/or efficacy of cosmetic ingredients and products. Test methods using RHE have been validated for evaluating skin irritancy, corrosivity and phototoxicity [9–13]. In fact, the Organization for Economic Co-operation and Development (OECD) has accepted several RHEs including EpiSkin (EpiSkinTM, Lyon, France) model as a validated reference method (VRM) in the TG431 testing guideline for in vitro skin corrosion test [14]. The EpiSkin method was also validated by the European Center for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee as an alternative method to fully replace the Draize acute skin irritation test in 2007. It was adopted in both the European Commission (EU) test Method B46 and the OECD TG439 as a validated reference method for in vitro skin irritation assessment [15, 16]. Skins from different human populations can present distinct features. Some reports studied the in vivo difference in the structure of SC, barrier function and reaction to irritants among skins of various ethnic origins [17–19]. Currently, most commercially available skin models are based on Caucasian cells [13, 20, 21]. We developed a reconstructed epidermis model from Chinese human keratinocytes based on the technique used for the EpiSkinTM model [2, 20–22]. Analysis of morphological architecture and microstructure, location of differentiation markers, barrier function and batch reproducibility was performed. Our data show that this China EpiSkin model is comparable to native skin in structure, as well as expression and distribution of biological markers. More importantly, the production of China EpiSkin model reaches steady performance among different batches and can be scaled up. All these results ensure that this in vitro Chinese epidermis model can be used as a powerful tool to investigate and better understand normal skin features and conduct in vitro safety assessment of cosmetic ingredients and/or products to meet local development needs in China. Materials and methods Materials and antibodies Culture media (DMEM/F12) were purchased from Gibco (Grand Island, NY, U.S.A.). Lipid standards were from Sigma-Aldrich (St. Louis, MO, U.S.A.). Anti-K10 and anti-K14 antibodies were from Dakocytomation (Hamburg, Germany). Anti-filaggrin and anti-involucrin antibodies were purchased from Abcam (Cambridge, UK). Chemically modified bovine collagen-based membranes were from Symatese (Lyon, France). All other supplements were from Life Technologies (Madison, WI, U.S.A.).
Human epidermis was reconstructed based on the technology transferred from EPISKIN SA, France, as described by Roguet et al. and Tinois et al. [21, 22]. The chemically modified bovine collagenbased membrane as support was previously fixed to the bottom of a plastic insert by an O-ring, and one tissue kit is constituted by 12 inserts [22]. NHKs (0.3 9 106 cell cm 2) were seeded onto the collagen membrane. After an immersion culture for keratinocyte proliferation, cells were further exposed to air–liquid interface to sustain keratinocyte differentiation. Then, all the tissue samples were incubated at 37°C with 5% CO2 and saturated humidity. Before transportation, China EpiSkin models were put on gel medium (agarose gel and culture medium mixture) to fix the inserts in the 12-well plates. Then, the plates were wrapped by aluminium foil bags for shipment at room temperature. Histology China EpiSkin model was fixed in 10% formaldehyde and processed for embedding in paraffin. Five-micrometre vertical sections were stained with haematoxylin and eosin for light microscopic examination. HE results were scored by investigators to grade the quality of the reconstructed human epidermis model, on a zero-to-four scale in the following aspects, respectively: general organization, nucleation of basal layer, intercellular space of basal layer, stratification of epidermis, adherence to support, granular cells, horny layer, etc. [22]. Immunohistochemistry Five-micrometre paraffin sections were used to perform immunohistochemistry. For detecting K10, K14, Ki67 and filaggrin, antigen retrieval was performed by digesting sections with 0.25% trypsin at 37°C for 15 min. For involucrin, sections were immersed in 0.01 M sodium citrate buffer (pH6.0) at 100°C for 30 min and then cooled to room temperature in 30 min. The primary antibody incubation was carried out at room temperature for 1 h, followed by staining with the Dako strept ABC complex/HRP kit according to the manufacturer’s instructions. Nucleus was stained by incubating 5-lm sections with haematoxylin for 3 min at room temperature. The following antibody dilutions were used for immunodetection of specific targets:
Cell culture and China EpiSkin model reconstruction Samples of normal adult foreskin free of HIV-1, 2, hepatitis C and hepatitis B were collected from circumcision after obtaining informed consent. Normal human keratinocytes (NHKs) were isolated from these samples and cultivated as described by Rheinwald and Green on irradiated 3T3 feeder cells and used at early passages [23]. The culture medium was Dulbecco’s modified Eagle’s medium (DMEM) and HAM’s F12 (3 : 1) supplemented with 10% fetal calf serum and 1.8 9 10 4 M adenine, 5 lg mL 1 insulin, 2 9 10 9 M triiodothyronine, 5 lg mL 1 transferrin, 0.4 lg mL 1 hydrocortisone, 10 10 M cholera toxin and 10 ng mL 1 epidermal growth factor.
Antigen
Dilution
K10 K14 Filaggrin Ki67
1 1 1 1
: : : :
100 20 200 200
Cell viability and barrier function The viability of the cultures was assessed with MTT assay according to the protocol previously described by Katoh et al. [24]. The barrier function was evaluated by the ability of the China EpiSkin to resist fast penetration of a cytotoxic marker chemical, that is 50 lL gradient concentrations of sodium dodecyl sulphate (SDS, 0.5–1.5 mg mL 1) topically applied to the model samples and
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
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Results Morphology of reconstructed epidermis model in comparison with native human epidermis Haematoxylin and eosin staining was used to investigate the morphology of the skin model compared to that of the tissue of origin, namely the foreskin. The China EpiSkin model presented a wellstratified structure, including basal layer, spinous layer, granular layer and horny layer (Fig. 1). The chronological development of this model was also investigated (Fig. 2, upper panel). D6, D8 and D10 represent the specimens at 6, 8 and 10 days, respectively, after seeding keratinocytes onto the collagen matrix. Morphology status after transportation is shown in Fig. 2 (lower panel). D6+G1+M1 represent samples which were embedded in gel for 1day transportation and cultured in maintenance medium for 1 additional day before being used for assessment. In terms of culture duration, D6+G1+M1 is comparable to D8. In the same way, D6+G1+M3 represents samples measured after the 3-day culture following transportation which are comparable to D10 samples. All these epidermal equivalents are fully differentiated and form multilayered epidermis similar to normal human epidermis (Fig. 1). The basal layers adhere to the collagen matrix, and many cells in this layer show cubical shapes. There are 4–5 layers of flattened cells, which are spinous cells above the basal layer. Granular cells are very remarkable with significant keratohyalin granules. The uppermost layer is horny layer whose thickness increases with time in culture. In conclusion, China EpiSkin model shows morphological characteristics highly similar to that of native human epidermis. The quality of the skin model was well maintained following transportation and additional culture phase before application.
B
The expression and distribution of epidermis differentiation markers Besides optical morphology, biological markers were used to further characterize the differentiation of China EpiSkin model at Day 6. As shown in Fig. 3, basal layer marker keratin K14 was expressed only in basal layer just as the native human epidermis (Fig. 3A,E). The keratinocyte proliferation can be observed by Ki67 immunohistochemical staining. Proliferation was localized in the basal layer (Fig. 3B,F). The density of proliferative keratinocytes in China EpiSkin model was similar to that of native epidermis. Early differentiation marker keratin K10 was expressed and located in all suprabasal layers (Fig. 3C,G). Filaggrin, a major keratohyalin component, was localized in granular cell layer (Fig. 3D,H). Thus, all the differentiation markers were expressed and showed the same location as in native human epidermis.
C
The lipid composition in horny layer of China EpiSkin model The lipid composition of skin is a key determinant of epidermal barrier function, which is pivotal to keep the normal epidermal function. The lipid profile of China EpiSkin model is shown in Fig. 4. The lipid extracts derived from China EpiSkin model contained major epidermal lipid classes, including cholesterol esters (CE), triglycerides (TG), free fatty acids (FFA), ceramides (CER), glucosphingolipid (CEREB), cholesterol sulphate (CHOL SO4) and phospholipids (the dark lane under the cholesterol sulphate). Among these, we can observe from Fig. 4 that the level of CHOL is very high in both native skin and China EpiSkin model. The amount of FFA, CER4 and CERB in native skin and
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Figure 5 Ultrastructure of the reconstructed human epidermis model in transmission electron microscopy. A whole view of corneocytes and stratum granulosum (SG) together was shown in A, the horny layer with a dense keratin pattern in corneocytes was shown in B, and arrow indicates the corneodesmosome. In the stratum granulosum (C), the typical desmosomes (D), keratohyalin granules (KHG), keratin filaments (K) and membrane-coating granules (G) were recognized. Scale bars are indicated within the pictures. 109 9 254 mm (300 9 300 DPI).
in China EpiSkin model is also very similar, all at moderate level. CHOL SO4 is detected in both native foreskin and China EpiSkin model, but it shows slightly higher level in China
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
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Figure 3 Immunohistochemical staining of reconstructed human epidermis and native human epidermis. Paraffin sections of China EpiSkin model (Day 6) and native skin samples with immunohistochemical staining by anti-K14, Ki67, K10, filaggrin antibodies (brown staining). 49 9 19 mm (300 9 300 DPI).
A
B
Figure 4 High-performance thin-layer chromatographic separation of lipids extracted from the reconstructed human epidermis model. Lipids of three batches of Day 6 samples (A) and 3 different days of culture (Day 6, Day 8, Day 10, B) were extracted and separated by HPTLC with ceramide development system. CE, cholesterol esters; TG, triglycerides; CHOL, cholesterol; FFA, free fatty acid; CER 2, ceramide 2; CER 3, ceramide 3; CER 4, ceramide 4; CEREB, galactocerebrosides; CHOL SO4, cholesteryl sulphate. 180 9 181 mm (300 9 300 DPI).
(pH6.8) in darkness for 1 h at room temperature. After rinsing twice with the buffer, specimens were dehydrated in a gradient ethanol series, acetone and embedded in an epoxy resin (#618). Ultrathin
sections (50–60 nm) were prepared using a Reichert ultramicrotome, contrasted with uranyl acetate and lead citrate and examined under a Philips CM120 electron microscope at 60 kV.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
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Results Morphology of reconstructed epidermis model in comparison with native human epidermis Haematoxylin and eosin staining was used to investigate the morphology of the skin model compared to that of the tissue of origin, namely the foreskin. The China EpiSkin model presented a wellstratified structure, including basal layer, spinous layer, granular layer and horny layer (Fig. 1). The chronological development of this model was also investigated (Fig. 2, upper panel). D6, D8 and D10 represent the specimens at 6, 8 and 10 days, respectively, after seeding keratinocytes onto the collagen matrix. Morphology status after transportation is shown in Fig. 2 (lower panel). D6+G1+M1 represent samples which were embedded in gel for 1day transportation and cultured in maintenance medium for 1 additional day before being used for assessment. In terms of culture duration, D6+G1+M1 is comparable to D8. In the same way, D6+G1+M3 represents samples measured after the 3-day culture following transportation which are comparable to D10 samples. All these epidermal equivalents are fully differentiated and form multilayered epidermis similar to normal human epidermis (Fig. 1). The basal layers adhere to the collagen matrix, and many cells in this layer show cubical shapes. There are 4–5 layers of flattened cells, which are spinous cells above the basal layer. Granular cells are very remarkable with significant keratohyalin granules. The uppermost layer is horny layer whose thickness increases with time in culture. In conclusion, China EpiSkin model shows morphological characteristics highly similar to that of native human epidermis. The quality of the skin model was well maintained following transportation and additional culture phase before application.
B
The expression and distribution of epidermis differentiation markers Besides optical morphology, biological markers were used to further characterize the differentiation of China EpiSkin model at Day 6. As shown in Fig. 3, basal layer marker keratin K14 was expressed only in basal layer just as the native human epidermis (Fig. 3A,E). The keratinocyte proliferation can be observed by Ki67 immunohistochemical staining. Proliferation was localized in the basal layer (Fig. 3B,F). The density of proliferative keratinocytes in China EpiSkin model was similar to that of native epidermis. Early differentiation marker keratin K10 was expressed and located in all suprabasal layers (Fig. 3C,G). Filaggrin, a major keratohyalin component, was localized in granular cell layer (Fig. 3D,H). Thus, all the differentiation markers were expressed and showed the same location as in native human epidermis.
C
The lipid composition in horny layer of China EpiSkin model The lipid composition of skin is a key determinant of epidermal barrier function, which is pivotal to keep the normal epidermal function. The lipid profile of China EpiSkin model is shown in Fig. 4. The lipid extracts derived from China EpiSkin model contained major epidermal lipid classes, including cholesterol esters (CE), triglycerides (TG), free fatty acids (FFA), ceramides (CER), glucosphingolipid (CEREB), cholesterol sulphate (CHOL SO4) and phospholipids (the dark lane under the cholesterol sulphate). Among these, we can observe from Fig. 4 that the level of CHOL is very high in both native skin and China EpiSkin model. The amount of FFA, CER4 and CERB in native skin and
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Figure 5 Ultrastructure of the reconstructed human epidermis model in transmission electron microscopy. A whole view of corneocytes and stratum granulosum (SG) together was shown in A, the horny layer with a dense keratin pattern in corneocytes was shown in B, and arrow indicates the corneodesmosome. In the stratum granulosum (C), the typical desmosomes (D), keratohyalin granules (KHG), keratin filaments (K) and membrane-coating granules (G) were recognized. Scale bars are indicated within the pictures. 109 9 254 mm (300 9 300 DPI).
in China EpiSkin model is also very similar, all at moderate level. CHOL SO4 is detected in both native foreskin and China EpiSkin model, but it shows slightly higher level in China
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 60–67
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EpiSkin model. The amount of CE, TG and CER3 is all very low either in native foreskin or in China EpiSkin model though in latter the level is slightly higher. The amount of CER2 is at very low level in native skin, and it is almost undetected in China EpiSkin model.
batches, and the ODs of the extracted (solubilized) dye from the untreated tissues were 20-fold greater than the ODs of solvent control (Fig. 6D). All of these parameters fulfil the production release criteria according to the OECD TG439 [16]. Discussion
Ultrastructural features of the China EpiSkin model As shown in Fig. 5A, fully differentiating epidermis was developed in the China EpiSkin model. The transmission electron microscopy investigation of this model revealed the presence of corneodesmosomes (arrowed) abundantly in stratum corneum (SC) intercellular spaces (Fig. 5B), as visualized by transmission electron microscopy with ruthenium tetroxide staining. In stratum granulosum (SG) layer, most of the classic morphological criteria of terminal epidermal differentiation were recognized (Fig. 5C), such as complete desmosomes (D), homogeneous keratohyalin granules (KHG), keratin filaments (K) and membrane-coating granules (G). Reproducibility of the production of China EpiSkin model For a functional skin model, the low variability among production batches and along time is an essential and critical factor for its reliable use in research and safety assessment application. The reproducibility of 50 batches China EpiSkin models during 18 months was determined by HE scoring for morphology, cell viability and IC50 to SDS for barrier function. The samples were scored according to the morphology of HE staining. The scores of samples from the 50 batches were compared to show a good interbatch reproducibility (Fig. 6A). Furthermore, regarding the barrier function, all the SDS IC50 values were found between 1 and 3 mg mL 1 (Mean SD: 1.295 0.091 mg mL 1). These results did not only show that the stratum corneum of China EpiSkin model was maturely differentiated and robust enough to resist rapid SDS penetration and damage, but also indicated that the production quality of China EpiSkin model is stable with low interbatch variability (Fig. 6B). Cell viability measured by MTT assay was used to assess the qualitative difference among batches. As shown in Fig. 6C, all optical densities (ODs) of formazan produced in MTT assay were found between 0.6 and 1.5 among the 50
Figure 6 Reproducibility of reconstructed human epidermis model. Fifty batches of China EpiSkin model were assessed by scoring HE staining (A). The optical density (OD) of tissue batches was obtained by MTT measurement (C). The ratio of OD570 of samples to negative control (extraction buffer only) was expressed as fold increase of negative control OD (D). A series of SDS concentrations was topically applied to the tissues, and IC50 value was determined by MTT (B). 125 9 74 mm (300 9 300 DPI).
The production of 3D skin models represents a growing trend in the field of tissue engineering. As an alternative method to animal test [26], these in vitro models, mimicking the native skin, can allow to investigate key biochemical and physical mechanisms of skin homeostasis and functions. Nowadays, some reconstructed human epidermis models are commercially available. Among them, the most accepted models include EpiSkin, SkinEthic (Lyon, France), EpiDerm (Ashland, MA, U.S.A.) and most recently LabCyte EPI-MODEL (Aichi, Japan). The testing methods base on these four models have been referred to define the alternative test methods endorsed by OECD to assess skin corrosion and skin irritation [11, 12, 14, 16, 22, 27–30]. In addition, other culture skin models, such as the Leiden reconstructed human epidermal model, the KeraSkinTM model and epiCSTM model, have been reported in the evaluation and validation efforts for in vitro skin tolerance testing according to ECVAM performance standard [10, 31, 32]. China EpiSkin model was reconstructed with keratinocytes of Chinese origin, and this is the first Chinese reconstructed human epidermis model to meet the high quality standard with industrialized production criteria. Histological analysis indicated that keratinocyte differentiation occurred in a similar way as that in the human epidermis. China EpiSkin model presents a well-stratified structure with basal layer, several spinous layers, granular layers and horny layer. Keratohyalin granules are observed in the granular layer. Through transportation/shipment process in agarose gel, tissue samples show the same quality as those in culture conditions for the same period. Keratinocyte differentiation was also validated through differentiation markers’ expression pattern. The markers, representing different differentiation steps, showed an expression profile similar to that in the human epidermis. Filaggrin labelling was located in granular cells, keratin K14 labelling was observed in basal layer, whereas early differentiation marker keratin K10 was expressed by
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all suprabasal cells as observed in vivo. Proliferative keratinocytes were visualized in basal layer by Ki67 staining, with a density similar to that of human epidermis. Barrier function is the basic attribute of epidermis. It is affected by the lipid composition of epidermis [33–35]. Ponec et al. showed that reconstructed human epidermis models contained major epidermal lipid classes but deviated from native epidermis in some lipid components, including high ceramide 2, low ceramides 5 and 6, and ceramide 7 [36, 37]. The fact that China EpiSkin model contains most of the major lipid classes found in native skin indicates the presence of major factors influencing the skin barrier properties. However, when compared to the human epidermis, it also demonstrated higher amount of CHOL SO4, cholesterol ester, triglycerides and ceramide 3, and lower amount of ceramide 2. Furthermore, ultrastructural features, observed through transmission electron microscopy, indicated the presence of multilayered stratum corneum in the reconstructed epidermis, which mainly contributes to the barrier function [38]. In addition, according to the ECVAM performance standards, a skin model need to present a proper barrier function [31]. In other words, the skin models should contain stratum corneum and lipid composition to resist the rapid penetration of cytotoxic marker chemicals such as SDS and Triton X-100 [31]. The effect of SDS, a well-known reference compound for irritancy, on the epidermis viability, is considered as an indicator of both the barrier function of the epidermis and the physiological state of viable cells. Our results demonstrated China EpiSkin model also presents a potential of barrier functions and viable cell response. This property is estimated by determination of the IC50 of SDS after a fixed exposure time. In this study, IC50 of SDS application for 18 h was measured in 50 batches of China EpiSkin model and the mean value (with standard deviation) was shown to be
1.295 0.091 mg mL 1. The IC50 for the skin models validated by ECVAM in the same manner is 1.0–3.0 mg mL 1 for EpiSkin model and 1.0–4.0 mg mL 1 for LabCyte EPI-MODEL [31]. Our observation confirmed that the barrier function of China EpiSkin model is similar to that of EpiSkin model or Labcyte EPI-MODEL and therefore meets the requirement described in the ECVAM performance standards. Meanwhile, the results of HE scoring and cell viability test showed the high reproducibility of the model from 50 production batches. In conclusion, a reconstructed epidermis model based on Chinese keratinocytes with good reproducibility and stability was developed. This model can mimic native human epidermis with steady and persistent quality and meets the general and functional conditions referred in OECD TG439 regarding reconstructed human epidermis. This model could respond to the unmet need of the scientific community in China, where there is an absence of well-characterized and commercially available reconstructed epidermis model with industrialization capability. Thus, the China EpiSkin model could serve as a useful tool for cosmetic research in China. It could also be considered as a reliable and relevant 3-dimensional in vitro model for developing in vitro alternative and predictive toxicology test methods, making further validation process a possible reality in China, and thus pave the way for applying in vitro skin safety evaluation for cosmetic ingredients and products. Acknowledgements We thank Fabien GIRARD, Jose COTOVIO, Delphine GUILLON, Bruno BERNARD (L’OREAL Research and Innovation, Paris, France) and Marie-Helene TEISSIER, Estelle TINOIS-TESSONNEAUD, Charbel BOUEZ (EPISKIN SA, Lyon, France) for their great support in this study and in the preparation of this publication.
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