UVA-induced ROS Formation Study: Gentle Serum

1.0 Purpose

 

To use a MatTek EpiDerm tissue model to assess the ability of the Aeonia Gentle Serum to protect against UVA induced ROS formation.

 

2.0 Summary of Test

 

The testing model used for this study was the MatTek EpiDerm. This skin model consists of normal human - derived epidermal keratinocytes that have been cultured to form a multilayered, highly differentiated model of the human epidermis. Ultrastructural analysis has revealed the presence of keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Markers of mature epidermis specific differentiation such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase have been localized in this model. The MatTek EpiDerm is also mitotically and metabolically active.

In this study MatTek EpiDerm tissues were topically loaded with an ROS sensitive dye, CM-H2DCFDA. When ROS are formed, they can react with this dye to produce a fluorescent end product, with the intensity of the fluorescence proportional to the extent of ROS formation. After the tissues were loaded with the dye, they were topically treated with the test materials and then exposed to UVA irradiation. At various time points during the irradiation, the tissue fluorescence was measured and used as an index of ROS formation caused by UVA light exposure. Test materials were considered to protect against UVA light ROS formation if tissues treated with the material had a fluorescence intensity which was less than intensity measured in untreated tissues exposed to UVA light.

 

3.0 Methods 

 

3.1 Preparation of Tissue

Upon arrival, the MatTek EpiDerm Tissues were stored at 4°C until used. Prior to use, the tissues to be used were removed from the agarose-shipping tray and placed into a 6-well plate containing 0.9 ml of hydrocortisone free assay medium (37±2°C). The tissues were allowed to incubate for one hour at 37±2°C and 5±1% CO2. After this incubation period, 100 μl of 5 μM CM-H2DCFDA (ROS sensitive dye) prepared in PBS was applied topically to the tissues and the tissues were incubated for an additional one hour. At the end of the incubation period the tissues were rinsed three times with 300 μl of PBS and transferred to a 24 well plate containing 300 μl of PBS. 

 

3.2 Application of Test Material

Prior to exposing the cells to UVA light, 6 μl of test material was applied topically to the tissues and spread evenly across the tissue using a cell scraper. Non-UVA exposed tissues and untreated tissues were topically treated with PBS, while the positive control tissues were treated with PBS supplemented with 150 μg/ml Trolox. After the application of the test material a baseline fluorescence measurement was made of the surface of the tissues using an excitation wavelength of 485 nm and an emission wavelength of 518 nm.

 

3.3 UVA Exposure

The EpiDerm tissues were exposed to UVA light at room temperature using a XX-20BLB UV Bench Lamp emitting 365 nm light at an intensity of 2 mW/cm2. Non-UVA exposed tissues were also kept at room temperature protected from light. Changes in fluorescence were measured at 15, 30 and 45 minutes (UVA doses of 1.8, 3.6 and 5.4 J/cm2, respectively).

 

3.4 Calculations

Nine fluorescent measurements were made across the surface of the tissue for each measurement and the average of these nine measurements was used as the indicator of ROS formation for each tissue. The baseline fluorescence for each tissue was then subtracted from the 15 and 30-minute values to calculate the change in ROS formation with UVA exposure. Mean fluorescence values (RFU) were then determined for each treatment at each time point. Treatments within each time point were then compared using an ANOVA. 

 

4.0 Results 

 

The results for the ROS assay are presented in Table and Graph 1. In this study, ROS formation associated with one of the treatments was significantly greater than the other treatments, therefore its results are presented in Graph 1b. All values are expressed in relative fluorescence units (RFUs). Values are expressed as means ± standard deviation (n=3 per treatment). An (*) denotes values that are significantly different from the Untreated tissues (p<0.05).

 

5.0 Discussion

 

In this study, exposure of the MatTek EpiDerm tissues to UVA light resulted in a significant increase in the formation of ROS. This increase in ROS formation was prevented in tissues treated with the Aeonia Gentle Serum, with reduction in ROS observed at all three time points.

 

Table 1. UVA ROS Formation Results

 

Graph 1. UVA ROS Formation Results