28 TESTING a RHE-MEL b RHE-SL RHE-SL+KA
160 140 120 100 80 60 40 20 0
100 40 58 * 56 * 20 0
RHE-MEL RHE-SL RHE- SL+KA
RHE-MEL RHE-SL RHE- SL+KA
c 60 * *
60 50 40 30 20 10 0
d RHE-SL spots * * RHE-SL spots + KA
RHE-MEL RHE-SL RHE- SL+KA
Figure 2: StratiCELL’s in vitromodel of hyperpigmented reconstructed human epidermis, with or without treatment with kojic acid. (a) Fontana-Masson staining (left) and high-resolution images taken by dermoscopy (right). (b) Quantification of melanin by image analysis after Fontana-Masson staining. (c) Measurement of pigmentation parameters by dermoscopy image analysis. (d) High-resolution images of hyperpigmented spots, taken by dermoscopy. RHE-MEL: reconstructed human epidermis with melanocytes; RHE-SL: hyperpigmented reconstructed human epidermis; KA: kojic acid.
techniques, opening up new perspectives towards the complexification of models in particular for applications in toxicology, pharmacology or regenerative medicine.5 While these basal state models are
perfectly adapted to evaluate the effects of compounds, plant or marine extracts, or even finished products on skin physiology and epidermal homeostasis, they can also be subjected beforehand to stressful conditions to which we are exposed daily, such as UV, infrared or blue light, urban pollution, organic solvents, temperature, or microbial challenges. The disturbances caused by these environmental aggressions and the skin defence mechanisms are increasingly well characterised and can easily be monitored by cellular and molecular biology assays. In addition, many publications report the
integration of other cell types for specific applications, such as primary melanocytes of different phototypes, immune T cells, iPSC- derived human sensory neurons, or components of the skin microbiota (commensal or opportunistic bacteria, Malassezia yeasts, dermatophytes, etc.). In a concern for quality and standardisation,
a discussion that followed the keynote session New Developments in Skin and Epidermal Equivalent Models at the 2019 Barrier Function of Mammalian Skin Gordon Research Conference, pointed out that there is a clear need for a consensus on the quality standard and validation of which organotypic 3D skin models are suitable for skin barrier research and a recommendation paper has been published.6
Reconstructed epidermis for the modelling of skin pathologies and pigmentation disorders Multiple skin pathologies or disorders originate from a deregulation of the immune system, leading to the secretion of pro-inflammatory cytokines by effector cells such as CD4 or CD8 lymphocytes, to a cascade of inflammatory responses from keratinocytes, and to a loss of efficiency of the epidermal barrier.7 Additionally, deregulation of paracrine
communication between fibroblasts, keratinocytes and melanocytes, may also lead to the development of pigmentary disorders such
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as actinic or solar lentigo. Prevalence of those pigmentary spots, increases significantly with age, in areas exposed daily to solar radiation and atmospheric pollutants. Even though little is known about the origin of hyper-pigmented spots, it seems to involve fibroblastic growth factors with melanogenic properties.8 RHE is a simple yet relevant approach for
modelling certain inflammatory pathologies such as psoriasis or atopic eczema, or pigmentary disorders such as lentigo actinic or vitiligo. The role played by lymphocytes or fibroblasts can in fact be substituted by the addition of a cocktail of cytokines or growth factors in the culture medium, leading to a response of the epidermis close to the physiological response (Fig 1).
Modelling of inflammatory pathologies (atopic eczema, psoriasis) and disturbance of the epidermal barrier Epidermal disturbances in the context of atopic eczema can be induced in RHE by the application in the culture medium of a cocktail of Th-2 cytokines (interleukins IL-4 and IL-13), produced in a physiological context by Th-2 polarised CD4 lymphocytes.9
Stimulation of RHE
by these two cytokines disturbs tissue morphology (observation of spongiosis by histological analysis), reduces the efficiency of the cutaneous barrier, and induces deregulation of the synthesis and transport of lipids and components of the cornified envelope. An increase in cytokines produced by keratinocytes and possessing chemotactic properties is also observed. These perturbations can be attenuated by different classes of molecules, such as Liver X receptor (LXR) agonists,10
Janus
kinase inhibitors (JAK), or antibodies directed against the IL-4 receptor, blocking the stimulation of the pathway. The same approach can be applied to For example, the addition of Th-17
psoriasis.11
cytokines has the effect of inducing the pro- inflammatory cascade dependent on IκB-ζ and NF- κB, leading to overexpression of psoriasis genes IL-19, IL-23, β-defensin-2 and psoriasin (S100A7). Here too, inhibitors of the p38 protein kinase or the NF-κB pathway can be used as reference therapy. A Th-22-type stimulation can reproduce the phenomenon of hyperplasia characteristic of
psoriasis lesions, as well as a loss of barrier components such as filaggrin and loricrin, and a disruption of the involucrin expression profile.12 Hyperplasia can be limited, for example, by a treatment in parallel with cyclosporine. Furthermore, in both atopic eczema and
psoriasis, a disturbance in the ceramide profile is associated with excessive permeability of the stratum corneum. In addition, a correlation between the average length of the fatty acid chains from ceramides and the effectiveness of the barrier function has been demonstrated.13,14 Deregulation of Th-1 cytokine release, in particular interferon(IFN)-γ, leads to a decrease in the expression of elongases (ELOVLs) and ceramide synthases (CerS), with an impact on the number of carbons in the fatty acid chains of ceramides. This has been demonstrated upon a 7-days IFN-γ challenge of RHE. Downregulation of ELOVLs and CerS by IFN-γ is mediated through a STAT1-independent pathway, and is prevented by pyridone-6, a pan-JAK inhibitor. 15,16
Modelling of skin pigmentary disorders (lentigo, vitiligo) Solar lentigines, commonly known as ‘age spots’, appear on areas exposed to the sun (back of hands, shoulders, face, forearms). They are very common in elderly people of Caucasian or Asian origin. A link with excessive exposure to air pollutants has recently been established on the basis of epidemiological studies, in particular on populations living in extremely polluted areas in Beijing province in China.17 Several studies suggest an involvement of
dermal fibroblasts in the appearance of pigmentary spots, through an increased secretion of cytokines having an influence on the growth of melanocytes and the intensity of pigmentation, such as growth factors like HGF, KGF/FGF-7 or SCF.8,18 Based on the publication of Chen et al.,19
StratiCELL has developed a model of hyper- pigmented RHE or individualised spots by applying a cocktail of melanogenic factors (Fig 2). In addition to overproduction of melanin, which can be demonstrated by dermoscopy, spectrophotometry or histology (Fontana- Masson staining), the epidermis also presents a hyperplasia characteristic of pigment spots in
November 2020
Relative % of melanin content, compared to RHE-SL
Individual Typological Angle mean (ITA)
Pigmentation Index mean (PI)
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