SERUM-FREE CULTURE OF FRACTIONATED BOVINE BRONCHIAL EPITHELIAL-CELLS
SERUM-FREE CULTURE OF FRACTIONATED BOVINE BRONCHIAL EPITHELIAL-CELLS
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DOI：
10.1007/bf02631078
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发表时间：
1992-01-01
影响因子：
2.1
通讯作者：
RENNARD, SI
中科院分区：
文献类型：
--
作者：
BECKMANN, JD;TAKIZAWA, H;RENNARD, SI
已经建立了牛支气管上皮细胞密度分离群体的无血清培养程序。通过蛋白酶消化分散的上皮细胞通过密度平衡离心分离，然后将小的基底样群体镀在I型胶原包被培养皿上。2 ~ 3代的1:4分裂富集了活跃分裂的细胞群，这些细胞可以储存在液氮中供后续使用。克隆生长试验显示，使用RPMI 1640培养基和LHC-9（人支气管上皮细胞培养基）1:1的混合物增殖最佳。通过添加RPMI小于50%的LHC-9培养基，可以方便地降低细胞生长速率，根据细胞制备的不同，细胞生长速率为每天0.6 ~ 1.3倍。与其他物种的气道上皮细胞培养相比，血清刺激了该系统中牛支气管上皮细胞的生长。然而，转化生长因子β -1抑制生长并诱导分化为鳞状表型。此外，与其他体系相比，牛细胞对100 nM的十四烷醇醋酸酯或1 μ m钙离子载体A23187的生长抑制具有抗性。磷酯与电离层的结合降低了有丝分裂活性，尽管没有观察到鳞状形态的诱导。因此，生长抑制和鳞状分化在该系统中并不是紧密耦合的。最后，由这些细胞沉积的生物合成基质刺激了生长速度。因此，该培养系统将有助于在没有血清的情况下评估可溶性因子和基质相关因子的活性。
Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split enriched for a population of actively dividing cells, which could be stored in liquid nitrogen for subsequent use. Clonal growth assays revealed optimum proliferation using a 1:1 mixture of medium RPMI 1640 and LHC-9, a medium employed for human bronchial epithelial cells. Cellular growth rate, which was 0.6 to 1.3 doublings per day depending on the cell preparation, was conveniently decreased by supplementing LHC-9 medium with less than 50% RPMI. In contrast to airway epithelial cell cultures from other species, serum stimulated the growth of bovine bronchial epithelial cells in this system. Transforming growth factor beta-1, however, inhibited growth and induced differentiation into a squamous phenotype. Also in contrast with other systems, the bovine cells were resistant to growth inhibition by 100 nM tetradecanoyl phorbol acetate or 1-mu-M calcium ionophore A23187. Combination of phorbol ester with ionophore decreased mitotic activity, although induction of squamous morphology was not observed. Therefore, growth inhibition and squamous differentiation were not tightly coupled in this system. Finally, biologically synthesized matrix deposited by these cells stimulated growth rate. This culture system will therefore be useful in assessing the activities of both soluble and matrix-associated factors in the absence of serum.