344 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS horse serum, a 1:100 dilution of a standard penicillin-streptomycin solution for tissue culture and a 100 unit/cc. suspension of Mycostatin. The medium for cultures that are to be maintained for two weeks or longer contains an additional 5 per cent supplement of beef embryo extract and a 10 per cent supplement of ascitic fluid (9). These constituents are all a[ailable from Microbiological Associates. Each graft receives 4 cc. of fresh medium three times a week. Care must be exercised to remove and renew the medium without wrinkling the skin, since optimum diffusion of nutrients through the tissue is desired. After ten days in culture, 0.1 cc. of the cortisone suspension is added directly to the fluid medium. In the experiments utilizing human skin a similar technique is employed. The donor site, however, depends on the available patients who volunteer. It is usually the thigh. The area is depilated and prepared with PHiso- hex* and 70 per cent ethanol. The skin is removed in the operating room by routine surgical procedure. Generally, strips of skin 2 cm. wide and 12 cm. in length are cut at 18 thousandths of an inch. Epidermis and some dermis is obtained although the grafts are not full thickness. The strips are then divided into 6 grafts, each 2 cm? in area. The grafts are transported to the laboratory in sterile containers and prepared for tissue culture as described for the mouse auricular skin. Large culture flasks roughly 200 cc. in volume are used. One cubic centimeter of the cortisone suspension is added to each vessel and flame dried. Twenty-five cubic centimeters of the described medium is inserted under each graft and renewed once a week. In every series some grafts were sacrificed for histologic examination. The criterion in our study for the maintenance of the structural integrity of the auricular skin is the subsequent successful grafting of the skin to the original donor. The grafting procedure is described in the literature (10). The human skin was homografted to voluntary recipients and the course of these grafts was compared to that of skin homografted directly, without prior residence in tissue culture. RESULTS Basic studies to determine the nutritional requirements of adult skin in vitro have been completed. In a series of preliminary studies, 66 grafts of full-thickness auricular skin of mice were maintained in different media containing various nutritional supplements without the addition of corti- sone. A complex medium containing serum proteins proved to be essential for prolonged maintenance of the grafts in vitro. Intact auricular grafts were successfully maintained in this medium for two weeks using the flota- tion technique developed in this laboratory. Of 30 auricular grafts cultured for three weeks in this medium 16 per cent survived. At the end of three * 3.0 per cent solution of hexachlorophene in PHisoderm.

MAINTENANCE OF SKIN IN VITRO AS AN ORGAN SYSTEM 345 weeks in culture a considerable growth of embryonic fibroblasts and epi- thelial cells was observed on the floor of the flasks. The abundance of mitotic figures indicated that these cells were in a state of active pro- liferation. The grafts, however, had a waxy appearance, were markedly translucent in areas, and were extremely friable. This was due to the partial separation of the dermal and epidermal layers, which was visible grossly and confirmed upon histologic examination. The failure of the majority of the grafts to "take" when transplanted to the donors seemed to be due to differentiation and not to inadequate nutrition. In the series of experiments which followed cortisone was incorporated in the cultures in the manner described and the differentiation of the fibroblasts was notably suppressed. Eight of the ten auricular grafts cultured by this method for three weeks were successfully grafted. Eight grafts were maintained for one month and two of these survived. Ten grafts of human skin were similarly treated for periods up to two weeks. All of these grafts were observed to be viable upon homotrans- plantation. They remained intact, became vascularized and were in good condition until the typical homograft rejection phenomenon occurred. D•set•ssxo• It is apparent from the results that although intact skin has been main- tained in tissue culture for several weeks, further refinements are indi- cated. If this system is to be applicable to many problems involving skin, e.g., homograft rejection, the period of time that the skin may be main- tained in culture must be extended. The addition of steroid compounds more active than cortisone may increase this period by more effectively suppressing dedifferentiation. Furthermore, corticosteroids primarily affect only the connective tissue elements, other compounds may be found which inhibit the aledifferentiation of the epithelium and if used in con- iunction with cortical steroids they may greatly increase the period of successful maintenance in vitro. Other purely mechanical problems remain to be solved. For example, at present, studies on human skin are limited to split-thickness grafts. A major factor preventing the maintenance of thicker skin in vitro is the shrinking, and curling which occurs when the skin is released from the tension it is normally under in the body. Possibly a device could be made which would hold the skin under variable tension in culture so that the nutrients would diffuse uniformly throughout the tissue. The advantages of using this technique, however, in physiologic research on the skin are numerous. The intact skin is isolated in tissue culture from the humoral mechanisms which regulate its metabolism. It is there- fore possible to study the direct effects of agents upon the chemical and cytological metabolism of the skin. The possible intervention of con-