Use of Human Skin Equivalent Cultures for Cosmetic Benefit Screening
Sara McPhail, B.S.; Eric Messersmith, B.S.; Rachelle Thomas, B.S.; Lisa Mullins, B.S.; Rosemarie Osborne, Ph.D.;
Procter and Gamble Miami Valley Innovation Center, Beauty Technology Division
Gary Grove, Ph.D.; CyberDERM
Transepidermal water loss (TEWL) is a clinical measure of skin barrier health widely used in the cosmetics industry, both for assessments of irritation and for improvements in skin barrier health. The availability of multi-layered, differentiated epithelial equivalents presents an opportunity to directly correlate in vitro barrier effects to clinical results.
Modification of DermaLab Probe:
A DermaLab probe (CyberDerm, Media, PA) was used to make transepidermal water loss measures. To isolate the tissue culture well from its surroundings an adapter was designed and constructed.
All tissues and media were supplied by MatTek Corporation, Ashland, MA. Three types of tissues with distinct barrier properties were used for the experiments. These were:
EpiOcular: human-derived epidermal keratinocytes cultured to form a stratified, squamous epithelium similar to that found in the cornea (lowest barrier)
EpiDerm 200: human-derived epidermal keratinocytes cultured to form a multilayered, highly differentiated model of the human epidermis. (highest barrier)
Epiderm 201: human-derived epidermal keratinocytes cultured in an identical manner to EpiDerm 200, except that the culture period is decreased by 3 days. This prevents full maturation of the stratum corneum.
For all cultures, each culture well was maintained in a 6-well plate in 1 mL of the supplied medium. Culture medium was replenished every 24 hours for the duration of the experiment. Test materials, where used, were added directly to the tissue culture wells to insure maximum absorption.
Modification of DermaLab Probe:
A DermaLab probe (CyberDerm, Media, PA) was used to make transepidermal water loss measures. To isolate the tissue culture well from its surroundings an adapter was designed and constructed. Tissue culture plates were allowed to pre-equilibrate inside a temperature and humidity controlled environment within a laminar flow hood. The DermaLab probe was placed directly over each culture well to begin the measure. Following achievement of steady-state water loss, TEWL was recorded for one minute for each sample.
Occlusion of EpiOcular cultures with petrolatum demonstrates:
- Modifications to the instrument probe have produced a good seal around the culture insert.
- The dynamic range of the system is quite large.
TEWL was used to show barrier increase in the underdeveloped EpiDerm 201 over time.
Histological results verify that stratum corneum development occurs over this time period.
Although less than 100 um thick, the EpiDerm 200 cultures provide a substantial and measurable barrier beyond that provided by the tissue culture insert membrane:
EWL can successfully be measured from human epithelial equivalents using existing clinical instrumentation, with modification to the instrument probe.
TEWL measurements parallel development of the stratum corneum in skin equivalent cultures, as observed histologically.
Skin barrier benefits may be measured using TEWL, although a large sample size may be required.
Measurement of skin injury or barrier disruption was not addressed in this study, but is a potential reapplication of this method.
Ms. Paula Hartwig, P&G Clinical