Skin Lightening

MatTek’s MelanoDerm™ System consists of normal, human-derived epidermal keratinocytes (NHEK) and melanocytes (NHM) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHM within co-cultures undergo spontaneous melanogenesis leading to tissues of varying levels of pigmentation. The tissues are produced using serum free medium without artificial stimulators of melanogenesis such as TPA and IBMX. The cultures are grown on cell culture inserts at the air-liquid interface, allowing for topical application of skin lighteners or self-tanning agents, providing a useful in vitro means to evaluate cosmetic and pharmaceutical agents designed to modulate skin pigmentation. For more information on MelanoDerm, click here.

Skin Lightening Application Note

METHODS

Cell Sources: Normal human epidermal keratinocytes (NHEK) were isolated from neonatal foreskins or obtained from commercial sources.  Normal human melanocytes were isolated from neonatal foreskins. Melanocytes were harvested from normal black (NHM-B), Caucasian (NHM-C), or Asian (NHM-A) skin tissue and are tested for infectious agents such as HIV-1, Hepatitis B, and Hepatitis C.  Additional information on the MelanoDerm and EpiDermTM models can be found in data and technical specification sheets available from MatTek Corporation.

Production of 3-D Organotypic Tissues: Primary NHEK and NHM were seeded onto microporous membrane inserts and cultured at the air-liquid interface for up to 21 days to produce the differentiated 3D MelanoDerm™ tissue model.

Melanin Assay: A quantitative assay to determine melanin content in the tissue was performed using the SOLVABLE™ melanin assay.  Briefly, MelanoDerm tissues were placed in SOLVABLE™ at 98°C overnight.  The following day, samples were cooled and centrifuged to pellet any insoluble material.  200µl of each sample were added to a 96-well plate and read at 490nM. Synthetic melanin (Sigma) was carried through the above procedure and a standard curve was constructed so that melanin content of unknown samples could be determined.

Color measurement: A Konica Minolta CM700d chromameter was used to evaluate L*(D65) of MelanoDerm tissues treated with various skin lightening actives and formulations.  Treatments occurred on days 0, 1, 2, 5 6 and 7 and chromametric measurements were taken on days 0-14 post-treatment.

 

RESULTS

A
A
B
B

Top views of MelanoDerm. Progressive lightening at 6, 10 and 14 day intervals, presenting lightening effects of topically applied test articles (Figure A), and a top view of MelanoDerm containing Asian, black, and caucasian melanocytes (Figure B).

MEL Quantitation

Melanin quantitation and Spectrophotometric measurement of MelanoDerm tissues (containing NHM-B) following treatment with cosmetic ingredients and formulations.  L* (D65) values were taken on days 1, 2, 6, 10 and 14 following treatment using a Konica Minolta CM700d spectrophotometer.  The SOLVABLE™ melanin assay was used to quantify melanin levels in MelanoDerm tissues after 6, 10 and 14 days of treatment.

For more information, view Technical References.