342 (11) JOURNAL OF THE SOCIETY Ol: COSMETIC CHEMISTS a. Whitman, R., Proc. $ci. Sect., Toilet Goods ?lssoc., No. 18, 27 (1952). b. Reed, R. E., Den Beste, M., and Humoiler, F. L., •. $oc. Cosmetic Chemists, 1, 109 (1947). c. Stoves, J. l,., Mfg. Chemist, 22, 387 (1951). (12) Stein, H. H., and Guarnaccio, J., ?lnal. Chim. atcta, in press. (13) Jones, C. B., and Mecham, D. K., ?lrch. Biochem., 3, 193 (1943). (14) Brown, A. E., U.S. Patent No. 2,717,228 (1955). (15) Heilingotter, R., Parfumerie u. Kosmetik, 31, 190 (1950) ?lm. Perfumer and Essent. Oil Rev., 66, 17 (1955). (16) Sanford, D., and Humoiler, F. L., ?lnal. Chem., 19, 404 (1947). (17) Brown, A. E., U.S. Patent No. 2,688,972 (1954). 18) Eau de Colognefabriek, J. C. Boldoot N.V., Dutch Patent No. 75,320 (1954). THE MAINTENANCE OF SKIN IN VITRO AS AN ORGAN SYSTEM* By ANGELICA FINDLE¾ and RONALD GILLETTE, PH.D.t Presented February 3, 1960, New York Chapter IN ou}t STUDIES on the homotransplantation of tissues a technique was devised to maintain full-thickness skin in tissue culture as an intact organ. Recent investigations (1, 2, 3) have indicated that treatment of tissues in vitro prior to grafting procedures, may defer the typical course of homograft rejection. In most tissue culture problems the object is to implant small pieces of tissue in vitro and to study the reactions of indi- vidual living cells. In such systems, the central portion of the tissue ex- plant become necrotic, generally in three to five days, and studies are done on the proliferating cellular outgrowth. Many of these migrating cells differentiate into embryonic forms. However, in order to explore the possibilities of adapting skin in vitro, a technique was needed which would provide for the maintenance of pieces of adult skin of adequate size for subsequent grafting and for the preservation of its structural integrity for a period of time long enough to permit experimentation. The problems encountered in any long term maintenance of intact tissue in vitro are too numerous to detail here. According to Paul in his recent text "Cell and Tissue Culture" (4), the name organ culture is a misnomer since it usually refers to the maintenance of small pieces of organs in vitro or to the growth of intact embryonic organs. Perfusion techniques em- ployed to maintain adult organs, e.g., heart and kidney, for short term experiments depend largely upon direct anastomoses of the apparatus with the vascular system of the organs and are therefore not applicable to skin. * The work reported in this paper was supported in part by Grant No. 4554 from the U.S. Public Health Service. t The New York Hospital, Cornell Medical Center, The Dept. of Surgery (Plastic), New York 21, N.Y.

MAINTENANCE OF SKIN IN VITRO AS AN ORGAN SYSTEM 343 Our research indicated that there were two salient limiting factors present in maintaining organ cultures of adult skin. These were (1) the diffi- culty of supplying adequate nourishment to the multilayered tissue by dif- fusion alone and (2) the process of cellular dedifferentiation which occurs in vitro, with subsequent deterioration of the architecture of the skin. In a series of experiments using both animal and human skin a technique w/rs developed by which we are largely able to circumvent these problems. Adult skin has been successfully maintained in vitro for as long as four weeks. MATERIALS AND METHODS The initial technical problem was to obtain full-thickness skin which would be thin enough to permit adequate diffusion of nutrients to the epidermis. In the series of experiments in animals the ears of adult mice were chosen as donor sites. By using the auricular skin as our experimental tissue we are able to obtain full-thickness grafts, with the epidermal, derreal, and fatty layers intact, which are extremely thin and are large enough to be followed after grafting, i.e., around 20 ram. 2 in area. Also, when transplanted to the dorsum of a mouse the auricular skin retains its characteristic appearance and can always be easily grossly identified. The technique used to obtain the auricular skin is our adaptation (5) of a method originated by Edgerton (6). Immediately after excision, the graft is soaked for one-half hour in a Petri dish containing a suspension of 200 units/cc. of Mycostatin in Earle's Balanced Saline solution. The graft is positioned in the suspension with the epithelial surface downward on a piece of gauze. Next, the graft is placed for one hour on gauze saturated with a solution of 2000 unit/cc. crystalline penicillin potassium and 250 mg/cc. Streptomycin in Earle's Saline. Care is taken to spread the graft evenly over the gauze, with the epithelial surface upward. Before the grafts are placed in culture flasks, 0.1 cc. of a •25 mg/cc. aqueous suspension of Cortisone Acetate is introduced with a tuberculin syringe into each flask. The flask is held tilted so that the liquid streaks down one side of the vessel. The water is evaporated from the suspension by gently flaming the flask and an even film of cortisone remains adherent to the vessel wall. The cortisone is added to inhibit the differentiation and the subsequent migration of the fibroblasts from the dermis (7). Each graft is then transferred with long fine pointed forceps, to the sterile 4.5 culture flask and evenly spread out over the bottom surface. The addition of the culture medium detaches the graft from the glass surface so that it floats freely, with the epithelial surface upward and the dermal layer in contact with the medium. The culture medium determined to be adequate through experimenta- tion (8), consists of Eagle's Medium with a 10 per cent supplement of