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MITOTIC AND NONMITOTIC MULTIPLE-LAYERED PERFUSION CULTURES

P. Kruse,W. Whittle,E. Miedema

Published 1969 in Journal of Cell Biology

ABSTRACT

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 106 embryonic rat muscle (NF-ER) cells, 23 x 106 diploid human tonsillar (NF-JAM) cells, 77 x 106 human pleural effusion isolate (RPMI 2650) cells, 35 x 106 embryonic diploid human lung (Flow 2000) cells, 21 x 106 bovine lung (FB4BM) cells, 108 x 106 bat lung (Tb1Lu) cells, and 81 x 106 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 106 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% 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 0.1% serum to form 16–20 layers of cells (191–239 x 106 cells per T-75 flask) in 27 days. WI-38VA13A cells ceased proliferation and became nonviable rapidly in serumless medium.

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