]. Soc. Cosmet. Chem. 21, 925-932 (Dec. 9, 1970) Effect of All-trans-Retinoic Acid on Human Skin Epithelial Cells in Vitro MARVIN A. KARASEK, Ph.D.* Presented May 26-27, 1970, New York City Synopsis--A method to study GROWTH and KERATINIZATION of postembryonic human SKIN epithelial cells in cell culture is presented and the effect of all-trans-RETINOIC ACID (vitamin A acid) on the behavior of human skin cells in vitro is described. In the complete absence of retinoic acid, epidermal cell growth is markedly retarded. At low concentrations of the acid (less than 10 /•g/ml), growth of skin cells is stimulated and keratin formation is decreased. At higher levels of retinoic acid (greater than 10 •g/ml), all growth is strongly inhibited. INTRODUCTION Changes in the levels of circulating vitamin A induced either by a diet deficient in vitamin A or by oral administration of excess vitamin A produce marked changes in the skin of both primates and non- primates. Sherman reported that animals on a diet deficient in vitamin A show an increase in keratinization and a reduced rate of growth of epidermal tissue (1). After oral administration of vitamin A, Pinkus and Hunter noted a decrease in keratinized cells on the forearm of adults, an increase in the number of nucleated cells, and a decrease in the mi- totic response of the epidermal cells (2). Effects of vitamin A on keratinization have also been reported in vitro. In 1953, Fell and Mellanby demonstrated an antikeratinizing effect of vitamin A on organ cultures of skin from chick embryos maintained in * Department of Dermatology, Stanford University School of Medicine, Palo Alto, Calif. 925

926 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS a plasma clot (3). This effect could be specifically described as a conver- sion from horn to mucin protein. Rothberg observed similar changes in embryonic chicken skin maintained in a chemically defined medium (4). An effect of vitamin A or one of its derivatives on the behavior of postembryonic mammalian skin in vitro has not been reported. Although it is clear that both vitamin A and, more recently, vitamin A acid (5-8) have an effect on the postembryonic epidermal cell, neither the primary target cell for vitamin A activity nor the mechanism of ac- tion in either embryonic or postembryonic skin is understood. In this study, evidence is presented that the postembryonic human epidermal cell in cell culture retains the capacity to synthesize proteins with characteristics of mature keratin, and that retinoic acid may either accelerate or depress epidermal cell •owth depending on concentration, EXPERIMENTAL Materials Skin Cultures Human foreskin tissue obtained 3 days postpartum, or abdominal skin from human autopsies, was used. Skin fragments of uniform di- mensions were obtained by a 3-mm stainless steel punch. Explants were maintained in a CO2 incubator by procedures previously described (9). Chemicals A stock suspension (1 mg/ml) of vitamin A acid (retinoic acid)* was prepared by producing a uniform suspension of the acid in Eagle's Mini- mal Essential Medium by sonication for 5 minutes in a Brownwill Soni- cator* at maximum energy. Appropriate dilutions of the stock solu- tion were made in complete Eagle's Minimal Essential Medium con- taining 10% ether-extracted calf serum. The calf serum was prepared by extraction of the serum with 2 volumes of ether with stirring for 24 hours at 0øC. The ether was removed by low pressure distillation, and the calf serum sterilized by filtration through a Millipore filter. Cell Growth New cell growth was determined by direct measurement with a microscope ocular fitted with a calibration scale. * Eastman Organic Chemicals, Eastman Kodak Co., Rochester, N. Y. * Brownwill Scientific, Division of Will Scientific Inc., Rochester, N.Y.