Lechner J F, Haugen A, McClendon I A, Pettis E W
In Vitro. 1982 Jul;18(7):633-42. doi: 10.1007/BF02796396.
Defined culture conditions for routine clonal growth of normal human adult bronchial epithelial cells have been developed. Serum and feeder cell requirements were abrogated by: (a) optimizing the calcium concentration in nutrient medium, MCDB 151; (b) supplementing with purified factors (epidermal growth factor, 5 ng/ml; insulin, 5 micrograms/ml; transferrin, 10 microgram/ml; hydrocortisone, phosphoethanolamine and ethanolamine, each at 5 x 10(-7) M; and trace elements); and (c) coating the surface of the culture dish with a mixture of fibronectin, collagen, and bovine serum albumin. Endothelial cell growth supplement (100 micrograms/ml) and retinoic acid (3 x 10(-10) M) further enchanced growth, whereas cholera toxin was nonmitogenic and serum supplementation (greater than 2%) markedly reduced the growth rate. Using the defined system, dissociated cultures of bronchial epithelial cells, obtained from more than 15 donors, have been subcultured at clonal densities with a colony forming efficiency of 3 to 4%. In addition, high density cultures have been subcultured more than five times with four to six population doublings per passage. The features of this system permit pathobiologic investigations of bronchial epithelial cells, e.g., aging, differentiation, and carcinogenesis using conditions that isolate the results from the influence of serum, feeder cells, and other undefined factors.
已开发出用于正常人成年支气管上皮细胞常规克隆生长的特定培养条件。血清和饲养细胞需求可通过以下方式消除：(a) 优化营养培养基MCDB 151中的钙浓度；(b) 添加纯化因子（表皮生长因子，5 ng/ml；胰岛素，5 μg/ml；转铁蛋白，10 μg/ml；氢化可的松、磷酸乙醇胺和乙醇胺，均为5×10⁻⁷ M；以及微量元素）；(c) 用纤连蛋白、胶原蛋白和牛血清白蛋白混合物包被培养皿表面。内皮细胞生长补充剂（100 μg/ml）和视黄酸（3×10⁻¹⁰ M）进一步促进生长，而霍乱毒素无促有丝分裂作用，血清补充（大于2%）则显著降低生长速率。使用该特定系统，从超过15名供体获得的支气管上皮细胞解离培养物已以克隆密度传代培养，集落形成效率为3%至4%。此外，高密度培养物已传代培养超过五次，每次传代有四至六次群体倍增。该系统的特性允许使用能将结果与血清、饲养细胞和其他未定义因素的影响隔离开的条件，对支气管上皮细胞进行病理生物学研究，例如衰老、分化和致癌作用研究。
