体外培养和冷冻保存对微囊化细胞生长和内皮抑素表达的影响

Effect of the in vitro Culture and Cryopreservation on the Growth of the Microencapsulated Recombinant Cell and Endostatin Production

摘要:

微囊化基因工程细胞移植治疗肿瘤是一种新兴的肿瘤治疗方法，如果将此技术应用到临床研究，就需要制备大量的细胞活性良好、重组蛋白表达量高的生物微胶囊。体外培养和冷冻保存是生物微胶囊制备过程中两个重要的环节，因此需要考察体外培养和冷冻保存对微囊化重组基因细胞生长和蛋白表达的影响。以重组CHO细胞为模型，考察了体外培养时间和冷冻保存对微囊化细胞在动物体内生长和内皮抑素表达的影响及体外培养时间对微囊化细胞冷冻保存的影响。结果表明：体外培养时间对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性具有较大的影响，体外不培养和培养4d的微囊化细胞在小鼠腹腔内生长良好、内皮抑素表达量高，并且微囊稳定性好，而体外培养8d的微囊化细胞在移植后的第26天破裂。体外培养时间对微囊化细胞冷冻保存也具有较大的影响，体外培养4d和8d的微囊化细胞在液氮中冷冻保存40d，复苏后细胞生长良好、内皮抑素表达量高，而冻存前未经过体外培养的微囊化细胞，复苏后细胞几乎全部死亡。综上所述，生物微胶囊在体外比较适宜的培养时间为4d。并且冷冻保存对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性没有显著的影响。

Abstract:

Microencapsulated recombinant cells technology is a novel approach to tumors therapy. It is necessary to prepare a plenty of the microcapsules with better cell viability and higher endostatin production in order to bring this technology into the clinic. The in vitro culture and cryopreservation are very important parameters in the preparation of microencapsulated cells. In this work, we studied the effect of the in vitro culture and cryopreservation on microencapsulated recombinant cells growth and endostatin production and the effect of the in vitro culture on the cryopreservation of microencapsulated recombinant cells. The results showed that the time of in vitro culture potently affected microencapsulated recombinant CHO cells growth in vitro, endostatin production and the microcapsule stability. The microcapsule kept intact after 36 days of implantation when thein vitro culture time was under 4 days. The thawed microencapsulated recombinant CHO cells had better cell growth and higher endostatin production after 40 days of cryopreservation when the in vitro culture time was 4 days and 8 days. Therefore, the best in vitro culture time was 4 days according to the results of the in vitro culture and cryopreservation and the cryopreservation did not affect microencapsulated recombinant CHO cells growth in vitro, endostatin production and the microcapsule stability.