纤维素多孔微载体的制备及其用于动物细胞培养

Preparation and Application of Cellulose Porous Microcarriers for Animal Cell Culture

将纤维素铜氨溶液喷洒至－４０℃的硅油：正己烷＝１：４的冷冻液中形成含冰晶的微球，用－３０℃、４０％的Ｈ２ＳＯ４再生纤维素，并用ＥＤＡＥ盐酸盐修饰其表面，制成适合动物细胞培养的纤维素多孔微载体。利用该微载体培养能分泌尿激酶原（ＰｒｏＵＫ）的重组ＣＨＯ细胞，在１００ｍＬ搅拌瓶中换液培养２５ｄ，细胞最高密度为６３×１０６／ｍＬ，尿激酶原最高活性为２３２５ＩＵ／ｍＬ，共获２８７ｍｇ产品。之后转入１０００ｍＬ搅拌瓶中培养，可观察到细胞可从种子微载体中自动转移到新微载体中生长繁殖直至所有微载体中都长有细胞。在２５ｄ二级培养中，细胞最高密度为７３×１０６／ｍＬ，尿激酶原最高活性为３１０８ＩＵ／ｍＬ，共获含３５３ｍｇ尿激酶原的上清１３７Ｌ。在培养后期换用无血清培养基培养，细胞生长及蛋白表达水平正常。

A cellulose cuprammonium solution was sprayed into the -40℃ frozen liquid containing 20% Silicone oil and 80% hexane to form microspheres contained fine ice crystals,then regenerated the cellulose with 40% H 2SO 4 cooled to -30℃,and the microspheres were modified with diethylaminoethylchloride,the cellulose porous microcarriers for animal cell culture were prepared.Semi continuous culture of recombinant CHO cells which can secret prourokinase(Pro UK) was carried out in a 100mL and a 1000mL spinner flasks using this kind of microcarriers.During the 25 days cultivating process in a 100mL spinner flasks,the cell density reached 6.3×10 6/mL,the maximal activity of Pro UK was 2325 IU/mL and the Pro UK production was 28.7mg.The seed microcarriers with rCHO cells cultivated in the 100mL spinner flasks were transferred into a 1000mL spinner.During 25 days subculture,it was observed that cells began to move from seed microcarriers into vacant microcarriers and continued to proliferate until all the microcarriers were occupied.The cell density reached 7.3×10 6/mL the maximal activity of Pro UK was 3100 IU/mL and 13.7L supernant which contained 353 mg Pro UK was harvested.During the later period of the cultivating process,the applying of serum free medium had no significant effects on the cell growth and protein expressing.