Insulin-transferrin-selenium as an alternative to foetal serum for epidermal equivalents.

OBJECTIVE: Organotypic skin models are powerful tools for research in development, ageing and diseases. They have become more and more complex with the use of multiple cell types. This requires a culture medium adapted to optimize the development of such in vitro skin. Foetal bovine serum (FBS) is the most complete supplement in existence at the moment, providing at once growth factors, vitamins, hormones and other circulating compounds. However, this cocktail suffers from batch variability and its animal origin is ethically questionable. More importantly, its biological activities may interfere with the study of certain signalling pathways. Here, we present a strategy for constructing an epidermal equivalent using a defined culture medium without serum. METHODS: An epidermal equivalent was constructed with primary human keratinocytes cultured using an insulin-transferrin-selenium (ITS) medium. Determination of steady-state gene expression levels and the immunohistological characterization of keratinocyte markers were performed to compare the ITS medium condition with a reference model, where keratinocytes were co-cultured with fibroblasts in the presence of FBS. RESULTS: The data show that the ITS medium promoted the expression of keratinocyte proliferation and differentiation markers at the protein and transcript levels in a similar way to that of the reference model. CONCLUSION: We show that culture using the ITS medium appears as a viable replacement for FBS in the construction of epidermal equivalents, opening the way to signal transduction studies.

Development of an in vitro model of menopause using primary human dermal fibroblasts.

OBJECTIVE: To overcome the current lack of in vitro models to specifically reproduce hormonal skin ageing in women, and in search of active ingredients with innovative efficacy claim for cosmetic skin care, we developed a cell culture-based model by simulating menopause's hormonal decline and assessed several parameters of collagen metabolism. METHODS: Human dermal fibroblasts were incubated with media containing 17ß-oestradiol, progesterone, dehydroepiandrosterone, growth hormone and insulin-like growth factor-1 at concentrations corresponding to those of non-menopausal women's sera and then of menopausal women's sera. We measured cell proliferation [by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)], matrix metalloproteinase-1 and metalloproteinase-3 (MMPs) release (by enzyme-linked immunosorbent assay - ELISA), total collagen deposition (by Sirius red staining), types I and III collagen deposition (by ELISA), and types I and III procollagen gene expression (by real-time q-RT-PCR). RESULTS: Our results showed a significant decrease over time in cell proliferation, collagen deposition and type III/type I collagen ratio, together with an increase in MMP release, when cells were incubated in media containing sex hormones at menopausal levels. This is consistent with in vivo data from menopausal women available in the literature. Surprisingly, procollagen gene expression was only reduced within the first hours and increased afterwards when compared with non-menopausal culture conditions. CONCLUSION: Our results demonstrated that the increased procollagen synthesis with menopausal conditions was not sufficient to compensate for the MMPs' catabolic effects and/or the impaired procollagen protein maturation, resulting in a decrease in extracellular collagen content. These findings add to the overall understanding of hormone-dependent skin behaviour and highlight the suitability of this in vitro model for cosmetic actives testing aiming to underpin claims of anti-ageing efficacy, specifically for menopausal women, regarding collagen metabolism and balance of types, for maintenance of dermal mechanical properties.

Improved procedures for in vitro skin irritation testing of sticky and greasy natural botanicals.

Skin irritation evaluation is an important endpoint for the safety assessment of cosmetic ingredients required by various regulatory authorities for notification and/or import of test substances. The present study was undertaken to investigate possible protocol adaptations of the currently validated in vitro skin irritation test methods based on reconstructed human epidermis (RhE) for the testing of plant extracts and natural botanicals. Due to their specific physico-chemical properties, such as lipophilicity, sticky/buttery-like texture, waxy/creamy foam characteristics, normal washing procedures can lead to an incomplete removal of these materials and/or to mechanical damage to the tissues, resulting in an impaired prediction of the true skin irritation potential of the materials. For this reason different refined washing procedures were evaluated for their ability to ensure appropriate removal of greasy and sticky substances while not altering the normal responses of the validated RhE test method. Amongst the different procedures evaluated, the use of a SDS 0.1% PBS solution to remove the sticky and greasy test material prior to the normal washing procedures was found to be the most suitable adaptation to ensure efficient removal of greasy and sticky in-house controls without affecting the results of the negative control. The predictive capacity of the refined SDS 0.1% washing procedure, was investigated by using twelve oily and viscous compounds having known skin irritation effects supported by raw and/or peer reviewed in vivo data. The normal washing procedure resulted in 8 out of 10 correctly predicted compounds as compared to 9 out of 10 with the refined washing procedures, showing an increase in the predictive ability of the assay. The refined washing procedure allowed to correctly identify all in vivo skin irritant materials showing the same sensitivity as the normal washing procedures, and further increased the specificity of the assay from 5 to 6 correct predictions out of 7 non irritants as compared to the normal washing procedures. In addition, when exposed to non-irritant oily and viscous materials, tissues rinsed with 0.1% SDS generally showed increased viabilities accompanied by decreased variabilities as compared to the normal washing procedures. Similar results were obtained when testing typical in-house natural botanical ingredients. In conclusion, the use of a refined washing procedure making use of SDS 0.1% in PBS was found a suitable procedure to ensure efficient removal of greasy and sticky materials, leading to an increased predictive capacity and decreased variability of the tissue responses while maintaining its sensitivity and not affecting untreated tissues morphology and viability.

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

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

Thymic epithelial cells mediate a Bcl-2-independent protection of single-positive thymocytes from dexamethasone-induced apoptosis.

Intrathymic maturation of thymocytes is essential for the proper formation of T-cell repertoire. This process involves two major biochemical pathways, one initiated by the recognition of MHC/peptide by the T-cell receptor and the other mediated by glucocorticoids. These hormones seem to affect thymocyte maturation by increasing the threshold of TCR-mediated positive and negative selection, and by inducing apoptosis of nonselected thymocytes. We have previously reported that an SV40-immortalized murine thymic epithelial cell line, namely 2BH4, was able to protect thymocytes from dexamethasone-induced apoptosis. Here we show that this protection is independent of cell-to-cell contact and does not seem to involve a Bcl-2-mediated resistance, since incubation of thymocytes with 2BH4 cells or its supernatant does not interfere with the levels of this antiapoptotic molecule. The protection conferred by 2BH4 cells, or by a primary culture of thymic stromal cells, is specific for the CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes, whereas the broad-spectrum caspase inhibitor z-VAD-fmk blocks apoptosis induced by dexamethasone in all thymocyte subpopulations. Our results suggest that positively selected single-positive thymocytes are still susceptible to glucocorticoid-induced apoptosis but are protected from it through the action of a heat-stable protein(s) released by thymic stromal cells.