A Method for the Cultivation of Dispersed Epithelial Cells from Mouse Palatal Mucosa

A cell dispersion method is described for establishing cultures of mouse palatal epithelial cells which are viable in primary cultures and subcultures for extended periods of time. The cells in vitro mainly displayed the normal morphologic characteristics of palatal epithelial cells. As demonstrated by labelling the cells with tritiated thymidine, the monolayers of epithelial cells possess a high rate of DNA synthesis. Karyotypes of the cultured cells show a high percentage of normal displays of chromosomal numbers typical of the mouse strain employed. The number of spontaneous aberrations observed is in agreement with previous investigations using other methods. OHIO J. SCI. 79(2): 73, 1979 Cultivation of adult oral epithelium in vitro for limited periods, mostly as organ or explant cultures, has been reported. Virtually without exception, either fibroblastic overgrowth of cultures (Rose et al 1967, Bracho et al 1970) or keratinization of cultured epithelial cells was observed (Porter 1960, Neiders and Weiss 1970). Milnek and Buchner (1975) recently described a simple and reliable method for the cultivation of human gingiva for epithelial outgrowth in which the fibroblastic emigration over a 3 week period was insignificant in comparison to the large epithelial outgrowth. Numerous attempts to cultivate disaggregated epithelial cells have been made in addition to the studies of epithelium grown in organ or explant cultures (Smulow and Glickman 1966, Karasek 1975, Rheinwald and Green 1975). In general, such efforts resulted in monolayers with greater growth but were usually maintained for very limited periods in primary culture. The present study is concerned with a method for the establishment of cultures of adult mouse palatal epithelium Manuscript received 25 May 1978 and in revised form 6 November 1978 (#78-29). Present address: Dept. of Microbiology and Physiology, University of Southern California, Los Angeles, CA. Present address: Dept. of Biology, Aldephi University, Garden City, NY. which are viable in primary cultures and in subcultures for extended periods of time, for studies of the morphology and karyotypes of the cells. MATERIALS AND METHODS Cell Culture Technique. Swiss albino male mice (5-6 weeks old) were sacrificed by cervical dislocation. Incisions were made at the posterior aspect of the hard palate and at the palatal margins of the teeth. The palatal mucosa was stripped from the hard palate and washed several times in Hanks' Balanced Salt Solution (HBSS). Samples consisted of pooled amounts of palatal epithelium from several mice cut into small fragments (~lmm) and washed twice with HBSS. The minced epithelial fragments were treated with a 0.1% solution of Pronase in HBSS for 30 to 60 minutes at 37°C in a trypsinizing flask until a slightly feathered appearance was noted to indicate intercellular separation. The sample was then centrifuged for 10 minutes at 131 x g and the resulting pellet was washed twice with PIBSS. Mechanical dispersion of the pellet was effected with a magnetic stirring bar for 30 minutes. The cell suspension was then decanted into centrifuge tubes and centrifuged at 131 x g for 15 minutes, then resuspended in sufficient growth medium to provide a concentration of approximately 250,000 cells/ml. The viability of the final cell suspension was determined by the trypan blue dye exclusion test (Merchant et al 1964). The cells were grown in Eagle's Basal Medium (BME) supplemented with 10% fetal calf serum (previously decomplemented by heating to 56°C for 40 minutes), 1% ^-glutamine and a 1% antibiotic-antimycotic mixture containing 10,000 units/ml of penicillin, 35fig/£ fungizone, and 10,000 Mg/l streptomycin. The cell sus-