Kawada H, Shannon J M, Mason R J
Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
Am J Respir Cell Mol Biol. 1990 Jul;3(1):33-43. doi: 10.1165/ajrcmb/3.1.33.
We have developed a serum-free, hormonally defined medium for maintenance of differentiation of adult type II cells cultured on Engelbreth-Holm-Swarm (EHS) tumor basement membrane gels. This defined medium consists of 1:1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media supplemented with insulin, dibutyryl cyclic AMP, hydrocortisone, epidermal growth factor, selenium, and albumin/linoleic acid complex. Compared to cells cultured on EHS gels in serum-supplemented medium, type II cells cultured on EHS gels in this defined medium showed increased acetate incorporation into total lipids (10-fold) and an increase in the relative percentage of acetate incorporated into phosphatidylcholine (PC) (87.8 +/- 0.4% versus 78.5 +/- 1.0% [mean +/- SE]; P less than 0.01), saturated phosphatidylcholine (SPC) (61.4 +/- 0.5% versus 55.2 +/- 0.9%; P less than 0.01), and phosphatidylglycerol (PG) (5.3 +/- 0.3% versus 0.8 +/- 0.1%; P less than 0.01) and decreased acetate incorporation into neutral lipids (9.7 +/- 0.8% versus 62.6 +/- 1.9%; P less than 0.01). No response to this defined medium was seen when type II cells were cultured on tissue culture plastic. Type II cells cultured on EHS gels in serum-supplemented medium for 4 d had numerous neutral lipid droplets in their cytoplasm. In contrast, neutral lipid droplets were not commonly observed within the cytoplasm of the cells cultured in serum-free, hormonally defined medium on EHS gels. This morphologic finding was consistent with the result that cells cultured in serum-supplemented medium significantly increased the relative percentage of acetate incorporated into neutral lipids. These data indicate that adult type II cells cultured on a reconstituted basement membrane (EHS gels) can be maintained in synthetic culture medium without serum. These culture conditions permit the expression of a pattern of differentiated phospholipid biosynthesis and cell morphology more similar to normal type II cell differentiation.
我们开发了一种无血清、激素成分明确的培养基，用于维持在恩格尔布雷特 - 霍尔姆 - 斯旺（EHS）肿瘤基底膜凝胶上培养的成年II型细胞的分化。这种成分明确的培养基由Ham's F12和杜尔贝科改良伊格尔培养基按1:1（体积/体积）混合而成，并添加了胰岛素、二丁酰环磷腺苷、氢化可的松、表皮生长因子、硒以及白蛋白/亚油酸复合物。与在添加血清的培养基中于EHS凝胶上培养的细胞相比，在这种成分明确的培养基中于EHS凝胶上培养的II型细胞显示，乙酸盐掺入总脂质的量增加（10倍），且乙酸盐掺入磷脂酰胆碱（PC）的相对百分比增加（87.8±0.4%对78.5±1.0%[平均值±标准误]；P<0.01），饱和磷脂酰胆碱（SPC）（61.4±0.5%对55.2±0.9%；P<0.01），以及磷脂酰甘油（PG）（5.3±0.3%对0.8±0.1%；P<0.01），而乙酸盐掺入中性脂质的量减少（9.7±0.8%对62.6±1.9%；P<0.01）。当II型细胞在组织培养塑料上培养时，对这种成分明确的培养基无反应。在添加血清的培养基中于EHS凝胶上培养4天的II型细胞，其细胞质中有许多中性脂质小滴。相比之下，在无血清、激素成分明确的培养基中于EHS凝胶上培养的细胞的细胞质中，通常未观察到中性脂质小滴。这一形态学发现与在添加血清的培养基中培养的细胞显著增加乙酸盐掺入中性脂质的相对百分比的结果一致。这些数据表明，在重组基底膜（EHS凝胶）上培养的成年II型细胞可以在无血清的合成培养基中维持培养。这些培养条件允许表达一种更类似于正常II型细胞分化的分化磷脂生物合成模式和细胞形态。
