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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机译：除先前描述的细胞类型外（Kruse等人，1963年，J。Nat。Cancer Inst。> 31： 109； Kruse和Miedema。1965年。J。Cell Biol。> 27：< / strong> 273）通过灌注培养技术发现形成多层培养。包含43 x 10 6 胚胎大鼠肌肉（NF-ER）细胞，23 x 10 6 二倍体人扁桃体（NF-JAM）细胞，77 x 10 6 人胸腔积液（RPMI 2650）细胞，35 x 10 6 胚胎二倍体人肺（Flow 2000）细胞，21 x 10 6 牛肺（FB4BM）细胞，108 x 10 6 蝙蝠肺（Tb1Lu）细胞和81 x 10 6 SV-40病毒转化的胚胎二倍体人肺（WI-38VA13A）在6至14天之内从T-60或T-75烧瓶中的稀释接种物中获得细胞；这些分别相当于大约4、3、3、4、2、4和8个单层。用培养基加10％的小牛血清灌注6周的NF-ER培养物，其细胞密度相当于12个单层细胞（每个T-75烧瓶140 x 10 6 个细胞）。用胸苷- 3 H脉冲90分钟后，该培养物从下至上显示出随机的核标记。培养基加上0.1％的血清将NF-JAM培养物维持在恒定的活细胞数，几乎没有胸苷- 3 H标记。 WI-38培养物获得了相似的结果，但是WI-38VA13A细胞在含0.1％血清的培养基中继续进行活跃的DNA合成和有丝分裂，形成16-20层细胞（191-239 x 10 6 细胞每个T-75烧瓶）在27天之内。 WI-38VA13A细胞在无血清培养基中停止增殖并迅速失去活力。
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