THE SKIN AS A COMMUNITY OF STRUCTURES the tissue cultivated in the presence of excess vitamin A utilised sulphate like a normal mucous membrane. Thus, the development of the tissue was not uniquely determined and could be influenced in the absence of the intact animal. No experiments on uptake of labelled cystine or methionine in such cultures have been reported, but would be of interest in making clear-cut the difference in metabolism of two types of epithelium. The effect of vitamin A and the relation to the action of oestrogens have been investigated using older tissues. Hardy, Biggers and Claringbold (1953) cultivated portions of the vagina of a prepuberal mouse and showed that this tissue retained its non-keratinising epithelium. However, when oestrogens such as oestradiol, oestrone and diethylstilboestrol were added to the medium then a typical squamous, keratinising epithelium was produced. This work was extended by the cultivation of similar tissue from prepuberal rats (Kahn, 1954), and in this case it was noted that the epithelium did change to a keratinising type if cultivation in the standard medium was continued for a sufficient length of time. Such a change was accelerated by the addition of oestrone to the medium, but was prevented if excess vitamin A was present. Tissue cultivated in the presence of excess vitamin A and then transferred to the standard medium underwent normal conversion to a keratinising condition. ADULT MAMMALIAN SKIN The ability of adult rabbit skin to survive in a stirred fluid mhdium at body temperature was investigated by Medawar (1947, 1948), using a grafting technique to determine whether survival had occurred. Small squares of skin consisting of epidermis and part of the dermis were removed from an animal, cultured under desired conditions and then transplanted to the animal from which they had been removed. In the presence of air or an air-oxygen mixture, cultivation for as long as eight days was possible during which time migration of epithelial cells from the epidermis and the hair follicles occurred to provide a layer enclosing the tissue specimen completely. The cells of this layer underwent normal division and gave rise to a true keratinised epidermis. The continued formation of this tissue was taken as evidence that the characteristic functional activity of the skin epidermis had not been impaired. When the enclosing epithelium was removed from the dermal tissue then successful grafting could be carried out. The skin was able to survive for a week at body temperature in the medium in the complete absence of atmospheric or dissolved oxygen, but under these conditions no movement nor division of the epithelial cells took place. In the presence of air, tissues cultured in media containing more than 10 -• M iodoacetate did not survive. Iodoacetate is a powerful inhibitor of certain enzymes involved in the production of energy from sugars, i.e., involved in glycolysis, and 121

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS was presumed to have had such an effect upon such enzymes in the skin culture. Tissue-culture studies on adult human skin, consisting of epidermis and a thin layer of dermis removed from the flexor surface of the forearm, have been made in relation to allergens and the toxic effects of antibiotics (Pomerat et alia, 1952 and 1953). Outgrowth of the epithelium, which appeared 'in two to three days after culture and reached a maximum in about six days, was used as a measure of continued activity of the tissue. A moderately close correlation was observed between the in vitro effects of antibiotics in restrict- ing outgrowth and their in vivo effects when applied to intact skin and mucous membranes. A comparison of the behaviour of cultures of adult human forearm skin with the skin of healing wounds in rats has been carried out by Washburn (1954) using histochemical procedures to reveal glycogen distribution and the distribution of ribo- and deoxyribonucleic acids. Glycogen is the form in which an animal normally stores carbohydrate, and can provide a source of energy for the activities of the tissues. In many tissues, the amount of ribonucleic acid increases w/th increase in protein synthesis. Deoxy- ribonucleic acid is specifically associated with the cell nucleus. In the normal human skin there is only a small amount of detectable glycogen in the epidermis, but when the cells from the surrounding epidermis and from hair follicle residues migrate over the surface of a healing skin wound, all the layers of the migrating epithelium, except the basal layer, in which the cells are actively dividing, contain glycogen. The glycogen disappears when keratinisation of the cells forming the epithelium of the wound takes place. In culture, the cells of the human skin which contained large amounts of glycogen were those which migrated and proliferated. Thus, a similarity between the metabolic processes involving carbohydrate in wound healing and in culture was suggested. In both wound-healing and skin-culture it was found that the content of ribonucleic acid remained unchanged during initial migration, but when proliferation occurred an increase in the content of this nucleic acid took place. There was no diminution of the deoxyribonucleic acid content of the culture during its activity. The interpretation of the results obtained in tissue culture studies in re- lation to the behaviour of that tissue in vivo must be treated with caution due to the need of materials such. as chick embryo extract and fowl plasma extract, for example, in the media. However, it should be noted that the constituents of the culture media are frequently obtained from animals of an entirely different class from those whose tissues are studied, yet normal development proceeded in the tissue with no indication of reaction against the foreign plasma proteins. Medawar was successful in maintaining his skin slices for four days without serum in the medium and survival was demonstrated by grafting. The extension of the use of tissue culture methods for toxicity 122