Cell types in addition to those previously described (Kruse et al. 1963 . J. Nat. Cancer Inst. 31 :109 ; Kruse and Miedema . 1965 . J. Cell Biol. 27 :273) were found to form multiple-layered cultures by perfusion-culture technique . Dense populations containing 43 X 10 6 embryonic rat muscle (NF-ER) cells, 23 X 10 6 diploid human tonsillar (NF JAM) cells, 77 X 10 6 human pleural effusion isolate (RPMI 2650) cells, 35 X 10 6 embryonic diploid human lung (Flow 2000) cells, 21 X 10 6 bovine lung (FB4BM) cells, 108 X 10 6 bat lung (TblLu) cells, and 81 X 10 6 SV-40 virus-transformed embryonic diploid human lung (WI-38VA13A) cells were obtained in 6-14 days from dilute inocula in T-60 or T-75 flasks ; these were equivalent to about 4, 3, 3, 4, 2, 4, and eight monolayers, respectively . Perfusion of an NF-ER culture for 6 wk with medium plus 10 % whole calf serum yielded a cell density equivalent to 12 monolayers (140 X 10 6 cells per T-75 flask) . This culture exhibited random labeling of nuclei from bottom to top after pulsing for 90 min with thymidine-3H . Medium plus 0 .1 %Jo serum maintained NF JAM cultures at constant viable cell numbers with virtual absence of thymidine-3H labeling . Similar results were obtained with WI-38 cultures, but WI-38VA13A cells continued active DNA synthesis and mitosis in medium with 0
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