Pilot production of u-PA with porous microcarrier cell culture
Pilot production of u-PA with porous microcarrier cell culture
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DOI：
10.1023/a:1008127310890
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发表时间：
2000-07-01
期刊：
影响因子：
2.2
通讯作者：
Li, ZH
中科院分区：
文献类型：
--
作者：
Hu, XW;Xiao, CZ;Li, ZH
在30l Biostat UC搅拌槽反应器中，用Cytopore纤维素多孔微载体培养了一株分泌尿激酶型纤溶酶原激活剂（u-PA）的重组DNA CHO细胞系。培养26天后，用自旋滤器将细胞保留在生物反应器中，细胞密度可达1.33 × 10(7) ml（-1）。上清液中最大u-PA活性为7335 IU。ml(-1)，收集含有7.1 g u-PA的上清液204l。在0.1%胎牛血清培养基中培养100天后，用一种可向后冲洗的改良细胞保留系统代替自旋过滤器，以防止过滤器堵塞。细胞密度最大可达10(7)ml（-1）以上，上清液中u-PA活性最大可达6250 IU。ml(-1)，收获1604l含51 g - pa的上清液。与灌注培养相比，分批培养基替代培养可以提高培养基的利用效率，提高细胞比产率，改善37℃环境下不稳定的产品质量。细胞可以自发地从被细胞占据的种子多孔微载体移动到空的微载体，而不需要胰蛋白酶化，并继续生长，直到所有的微载体都含有细胞。结果表明，细胞孔多孔微载体是一种实用的微载体，可逐步扩大培养规模。
A recombinant DNA CHO cell line secreting urokinase-type plasminogen activator (u-PA) was cultivated with Cytopore cellulose porous microcarriers in a 30l Biostat UC stirred tank reactor. After 26 days of culture, using a spinfilter to retain cells in bioreactor, the cell density could reach 1.33 x 10(7) ml(-1). The maximal u-PA activity in supernatant was 7335 IU.ml(-1), and 204l supernatant containing 7.1 g u-PA was harvested. After 100 days of culture with 0.1% fetal bovine serum medium, a modified cell retention system which can be washed-out backward, substituted the spinfilter to prevent filter clogging. The maximal cell density was over 10(7) ml(-1), the maximal u-PA activity in supernatant reached 6250 IU.ml(-1), and 1604l supernatant containing about 51 g u-PA was harvested. Compared to perfusion culture, batch medium-replaced culture could raise utilizing efficiency of the medium, increase cell specific productivity and improve the quality of the product which was not steady in a 37 degrees C environment. Cells can move from seed porous microcarriers occupied by cells to vacant microcarriers spontaneously, without trypsinization, and continue to grow until all microcarriers contained cells. It shows that Cytopore porous microcarriers are very useful and convenient to scale up cultivation step by step.