Episkin

Description

Cutaneous penetration assay




Models

Protocol principle (SkinEthic RHE model)


The purpose of this study is to determine the penetration of the test product after topical application on the reconstructed human epidermis models.


Detailed assay procedure

Test method



Duplicate in vitro reconstructed human epidermal tissues (size 0.5 cm²) at day 17 are dosed topically with the test product (at a concentration to be determined by the study sponsor) Untreated vehicule control are run in parallel The medium underneath the tissues is sampled (and replaced) at 1, 2, 3, 6, and 24 hours for further analysis. At the end of the experiment (24 hours), one of the tissues is assessed for histology (H&E staining) and one is used for viability assessment.


Evaluation of cell viability (MTT test)


For each condition, one culture is placed on 300 µl of 0,5 mg/ml MTT and incubated 3 hours at 37°C, 5% CO². Extraction is performed in 1.5 ml of isopropanol at room temperature, for a minimum of 2 hours. Optical density is measured at 570 nm. Results will be expressed as percentage of viability compared to negative control: % Viability = [OD (570nm) test compound / OD (570nm ) negative control)] x 100.


Histology


For each test condition, one tissue is fixed in a balanced 10% formalin solution and embedded in paraffin. Four micron vertical sections are stained with hematoxylin/eosin and photographed under a microscope.


References


Characterization of the barrier function in a reconstructed human epidermis cultivated in chemically defined medium.
N. Garcia, O. Doucet, M. Bayer, D. Fouchard, L. Zastrow and JP. Marty. Intl. J. of  Cosmetic Sciences, 24, p.25-34, 2002.
 
Skin penetration and metabolism of topical glucocorticoids in reconstructed epidermis and excised human skin. Gysler A., Kleuser B., Sippl W., Lange K., Korting H. C., Höltje H.-D., and Schäfer-Korting M. Pharmaceutical. Research. Vol. 16, 9; p.1386-1391, 1999.