Kruse P F, Whittle W, Miedema E
J Cell Biol. 1969 Jul;42(1):113-21. doi: 10.1083/jcb.42.1.113.
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 (Tb1Lu) 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-(3)H. Medium plus 0.1% serum maintained NF-JAM cultures at constant viable cell numbers with virtual absence of thymidine-(3)H 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 10(6) cells per T-75 flask) in 27 days. WI-38VA13A cells ceased proliferation and became nonviable rapidly in serumless medium.
除了先前描述的细胞类型（Kruse等人，1963年，《国家癌症研究所杂志》31:109；Kruse和Miedema，1965年，《细胞生物学杂志》27:273）外，还发现通过灌注培养技术可以形成多层培养物。在T - 60或T - 75培养瓶中，从稀释接种物开始，在6 - 14天内获得了高密度细胞群体，其中包含43×10⁶个胚胎大鼠肌肉（NF - ER）细胞、23×10⁶个二倍体人扁桃体（NF - JAM）细胞、77×10⁶个人胸腔积液分离物（RPMI 2650）细胞、35×10⁶个胚胎二倍体人肺（Flow 2000）细胞、21×10⁶个牛肺（FB4BM）细胞、108×10⁶个蝙蝠肺（Tb1Lu）细胞以及81×10⁶个SV - 40病毒转化的胚胎二倍体人肺（WI - 38VA13A）细胞；这些分别相当于约4、3、3、4、2、4和8个单层。用含10%全小牛血清的培养基对NF - ER培养物进行6周的灌注，产生的细胞密度相当于12个单层（每个T - 75培养瓶中有140×10⁶个细胞）。在用³H - 胸腺嘧啶脉冲标记90分钟后，该培养物的细胞核从底部到顶部呈现随机标记。含0.1%血清的培养基使NF - JAM培养物的活细胞数量保持恒定，几乎没有³H - 胸腺嘧啶标记。WI - 38培养物也得到了类似的结果，但WI - 38VA13A细胞在含0.1%血清的培养基中继续活跃地进行DNA合成和有丝分裂，在27天内形成了16 - 20层细胞（每个T - 75培养瓶中有191 - 239×10⁶个细胞）。WI - 38VA13A细胞在无血清培养基中迅速停止增殖并变得不可存活。
