Bioengineered Pigmented Skin Equivalent for UV and Cosmetic Testing

Drug Discovery Assay – reference number: B127

Overview

Testing Information

Assay Description

This platform can be used to assess either the detrimental impact of UV exposure on skin health, protective effect of a potential sunscreen or the ability of candidate actives to invoke pigmentary changes in the skin’s appearance. A comparison can be drawn against the anti-pigmentary effects of a known modulator of melanogenesis: kojic acid, an active commonly used in cosmetic formulations due to its skin tone lightening effects, that in this case may be used as a reference compound. Similarly for UV studies, a well-documented UV exposure of solar-simulated UV dose of 3.3 Jcm−2 (96.5% UVA, 3.5% UVB) representative of northern hemisphere solar exposure, may be used as a reference condition for induction of photodamage in UV studies.  The specific results that will be provided upon request are the effects of test articles on: Transepidermal water loss (TEWL), melanin index (MI), individual topography index (ITA), histology, cytokine release, melanin distribution, epidermal melanocyte density. 

Test Article Requirements

Test article(s) to be provided by the Sponsor in storable aliquots at required test concentrations with information on the diluent vehicle used. Stock solutions are prepared in distilled water unless otherwise requested. Sponsor to provide sufficient test article to run the entire study. 

Suggested Testing

A minimum of triplicate per condition with a maximum of 30 models per experiment. Several experiments can be run in a single study to increase model throughput.

Study Outline

Rationale and Experimental Design

To construct the bioengineered tissue representative of pigmented human skin, our researchers first generate a dermal foundation. The dermal foundation consists of fibroblasts seeded within Avletex® Scaffold, which populate the scaffold and neosynthesise endogenous extracellular matrix (ECM), along with providing paracrine support to the overlying epidermal tissue, in the form of soluble growth factors. Keratinocytes and melanocytes are then seeded upon the dermal foundation and following proliferation and differentiation phases, form a mature pigmented constructs at the air-liquid interface (ALI). Melanocytes representative of darkly, moderately or lightly pigmented skin are available depending on application and produce tissue representative of each skin tone. 

Schematic representation of culture process. Downstream applications include both pigmentary studies through addition of single active to the culture medium or topical formulation application, and UV challenge. 

Once skin constructs have matured and pigmentation has developed, characterized by melanosomes distributed throughout the epidermal layers, application of the test actives can commence. This may be in the form of topical application of a complete formulation, soluble addition to culture medium of a candidate active or UV protection/challenge as desired. End point analyses include inflammatory markers, pigmentary changes, melanocyte density measurements and skin damage marker expression. 

Addition of Test Articles

Test articles can take the form of solubilized compounds added directly to the culture medium, or topically applied formulations to the surface of the skin. It is recommended a minimum of triplicates should be used for each condition. An example of the conditions assessed for three test agents would be as follows: 

1. Untouched Control
2. Sham Control 
3. Test Agent 1
4. Test Agent 2
5. Test Agent 3 
6. Positive Control 

End Point Analysis

There are a range of endpoints that can be used to assess effectiveness in this system including:  

• Barrier Integrity: Histological assessment, immunofluorescent staining, transepidermal water loss (TEWL) measurements 

• Pigmentary Changes: Melanin index, individual topography angle (ITA), Fontana-Masson histological staining  

• Melanocyte Density: immunofluorescence detection and quantification of epidermal melanocytes 

• Dermal Changes: immunofluorescent detection of extracellular matrix (ECM) components 

• Cytotoxicity: Histological assessment, TUNEL assay to detect and quantify apoptotic cells 

Example data

Skin Tone Modulation: Kojic acid reduces pigmentation in vitro

Addition of known modulator of melanogenesis, kojic acid, to the culture medium, reduces skin equivalent gross pigmentation in a concentration dependent manner (A). Histological analysis reveals no cytotoxic activity, with all skin equivalents appearing viable and a reduction in melanin can be visualized through Fontana-Masson staining (B). Quantification of pigmentary changes depict a significant reduction in both ITA (C) and melanin index (D) at the highest concentration tested. Skin equivalents were treated over a 10 day period. The action of kojic acid is well characterized and can act as a positive control in pigmentation studies (data represent mean ± SEM, n = 6). Statistical significance described compared with untreated control *p ≤ 0.05, ****p < 0.0001. Scale bars: 50 μm. 

Goncalves K, De Los Santos Gomez P, Costello L, Smith L, Mead H, Simpson A, Przyborski S. Investigation into the effect of skin tone modulators and exogenous stress on skin pigmentation utilizing a novel bioengineered skin equivalent. Bioeng Transl Med. 2022 Sep 26;8(2):e10415. doi: 10.1002/btm2.10415. PMID: 36925688; PMCID: PMC10013773 

UV Protection in vitro: Endogenous melanin in pigmented skin constructs is protective against UV-induced damage 

Demonstration of the photoprotective effects of melanin. Changes in ITA (A) and melanin index (B) demonstrate a tanning effect in UV exposed pigmented skin equivalents. Location of melanosomes within keratinocytes, apical to the nucleus in vitro is reminiscent of supranuclear cap formation in vivo, and essential for melanin’s protective function (C). Melanocyte density (D/E) is significantly increased following UV exposure. the mean ± SEM from ×3 independent experiments with ×3 skin equivalents per condition, with an unpaired, two-tailed t-test used to determine statistical significance, **p < 0.01, ***p < 0.001. Scale bars: 50 μm (Ca–d), 10 μm (Ce–h), 100 μm (Da,b), 10 μm (Dc,d). 

Analysis of pro-inflammatory cytokine release following UV exposure in pigmented and non-pigmented skin equivalents. Cytokine array conduced on conditioned medium samples reveals in an increase in proinflammatory factors to a greater degree in non-pigmented skin equivalents irradiated with UVR, further evidencing the protective role of melanin. mean ± SEM from ×2 independent experiments with ×3 technical repeats per condition per time point. Ordinary two-way ANOVA with Tukey correction for multiple comparisons was used to determine statistical significance, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. AU, arbitrary units. 

De Los Santos Gomez P, Costello L, Goncalves K, Przyborski S. Comparison of photodamage in non-pigmented and pigmented human skin equivalents exposed to repeated ultraviolet radiation to investigate the role of melanocytes in skin photoprotection. Front Med (Lausanne). 2024 Apr 18;11:1355799. doi: 10.3389/fmed.2024.1355799. PMID: 38698778; PMCID: PMC11063240.