Recently considerable progress has been made in the use of controlled freezing in conjunction with protective agents for the preservation of viability of cells and tissues for prolonged periods of time (1). In many instances it is advantageous to preserve oral tissues in a viable state for extended periods to permit additional assays of the cultured tissues at a future date. METHODS Human gingival tissues, as a test object, obtained at the time of periodontal surgery were placed immediately in Hanks-Eagles basal medium, washed thoroughly in the same medium to remove adherent blood and debris, and then cut into 2 to 3 mm3 fragments. The fragments were placed in 16 by 125 mm screw cap test tubes with 5 ml Hanks-Eagles basal medium containing 7.5% dimethylsulfoxide. In addition, half the tubes contained 1% calf serum. The tubes were placed in racks and frozen in a Linde BF 3-2 freezing apparatus at a rate of -1° C per minute to -30° C, and then at a rate of -10° C per minute from -30° C to -150° C. At the proper time, adjustments were made in the instrument to allow for the continuous addition of liquid nitrogen to compensate for the release of latent heat of fusion within the specimens. After freezing, the tissues were transfered and stored in the vapor phase of liquid nitrogen (-150° C) (2). Tissues were removed from the storage refrigerator after varying periods from one hour up to 14 months and were thawed rapidly by agitation in a 37° C water bath. Some of the tissue fragments were planted in small plastic petri dishes and cultured in Hanks-Eagles basal medium containing 10% calf serum, 50 units/ml penicillin, 50 jug/ ml streptomycin and 50 units/ml Mycostatin in a moist atmosphere of 5% C02 in air. In order to determine the suitability of these tissues for enzyme histochemical studies, other stored fragments were refrozen on dry ice, mounted on a microtome chuck and sectioned in a cryostat. Sections were incubated in the appropriate media for the demonstration of individual dehydrogenases, acid and alkaline phosphatases and nonspecific esterase. In general, for succinic dehydrogenase the medium was prepared according to Nachlas, et al. (3); for NAD-dependent malic, isocitric, glutamic, lactic, ot-glycerophosphate, /3-hydroxybutyric, and NADP-dependent glucose-6-phosphate dehydrogenases, the media were prepared according to the method of Hess, Scarpelli and Pearse (4) ; and for NADH2- and NADPH2-dehydrogenases the media were prepared according to Scarpelli, Hess and Pearse (5). Nitro-BT was the tetrazolium salt used, and coenzyme concentrations in the incubating media were 0.1 M. Menadione at a concentration of approximately 10* M was used in the incubation medium for the detection of glycerophosphate dehydrogenase. Medium for the demonstration of acid phosphatase was prepared according to the method of Barka (6), for alkaline phosphatase according to the method of Burstone (7), and for nonspecific esterase according to Holt and Withers (8). Fresh, non-stored gingival tissues were also cultured and stained as described above. Tissues incubated in the absence of substrates were negative. RESULTS After 14 months storage at - 150°C gingival cells were still capable of growing in tissue culture (Figs. 1 and 2). Cell migration was usually evident within seven days in culture and generally a monolayer of epithelial-like cells was clearly demonstrable by the tenth day in culture. The cultures continued to grow, and after 31/2 to 4 weeks nearly filled the bottoms of the petri dishes (3.5 cm diameter) (Fig. 1). After 414 to 5 weeks the cultures began to show signs of degeneration with areas of piling up of cells and other areas of cell breakdown. Fibroblast-like cells were seen in the area of the expiant. No differences were noted in growth characteristics in tissues frozen either with or without serum added to the freezing medium. Cell migration in fresh tissue cultures was usually evident within 5 to 6 days in culture and monolayers of epithelial-like cells were generally established by the 10th day in culture. Histochemically, enzyme activity was demonstrated for all enzymes tested. The localization of the reaction products was similar to t- 137 138 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY FIG. 1 Four week culture of human gingiva stained with toluidine blue. Tissue had been frozen and stored 14 months before culture. Expiant removed from center of 3.5 cm diameter petri dish. r -^v- *T ?•* - A ? * k “ : “ Y * ^ * , * '-V .'.v* ?# J-.* I.* #*. ''V FIG. 2 Photomicrograph of epithelial-like cells in culture stained with toluidine blue at pH 7. Tissue had been frozen and stored 14 months before culture. (Orig. mag X 250) that of fresh frozen tissue (Figs. 3 and 4). In surface (Fig. 3). Connective tissue cells and general, the oxidative enzymes showed greatest blood vessels showed varying degrees of ac-activity in the deeper layers of the epi- tivity for the different enzymes. The epithelium with decreasing activity toward the thelium of the gingiva stained moderately for FROZEN-STORED GINGIVA 139 ?'..*.? “ f. '?.-., - •..?>'...^ - FIG. 3 Human gingiva stained 15 minutes at 37°C for NADH2-dehydrogenase activity. Counterstained with Van Gieson's picrofuchsin. Note similarity of enzyme distribution between (A) fresh-frozen gingiva and (B) gingiva frozen and stored 14 months. Greater activity is evident in the deeper layers of the epithelium with decreasing activity toward the surface. (Orig. mag. X 125) Human gingiva stained for 10 minutes at 37°C for acid phosphatase activity. Note similarity of enzyme distribution between (A) fresh-frozen gingiva and (B) gingiva frozen and stored 14 months. Epithelium shows moderate activity with greater activity in the spinous and cornified layers. (Orig. mag. X 125) acid phosphatase with greater activity in the was also noted in the connective tissue ground spinous and cornified layers (Fig. 4). Alkaline substance in some areas. Nonspecific esterase phosphatase activity was limited principally to activity was demonstrated in gingival epi- the endothelium of capillaries. Minimal activity thelium and connective tissues. Within the 140 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY FIG. 5 Section of gingiva stained 25 minutes at 37°C for lactic dehydrogenase activity after 14 months storage. Counterstamed with Van Gieson's picrofuchsin. Note localized loss of enzyme activity in lower left portion of photomicrograph. (Orig. mag. X 125) epithelium the enzyme distribution was found to be rather patchy as with fresh frozen tissues. A few of the specimens studied showed localized areas with complete loss of enzyme activity suggesting that some of the cells in the tissue did not survive the freezing procedure (Fig. 5). CONCLUSIONS AND SUMMARY Despite the loss of some cells, these findings demonstrate the feasibility of using controlled freezing in conjunction with protective agents for the prolonged storage of viable tissues for future histochemical, biochemical or tissue culture purposes. Additional data will be necessary to estimate the degree of cell survival and to quantify the amount of enzyme preservation. However, 12 of 14 expiants retained sufficient viability to grow in culture after 14 months storage and enzyme activity was demonstrable histochemioally within incubation periods usual for fresh frozen gingiva. The histochemical demonstration of active enzymes in the tissues after storage would presuppose that they were present in the tissue before freezing and therefore cannot be construed to be a test for viability in and by itself. On the other hand, the complete loss of enzyme activity from areas of a tissue could indicate a loss of viability.
