动物细胞培养是细胞工程中的重要组成部分,广泛用于单克隆抗体、疫苗、重组蛋白等生物药物的生产,具有广阔的发展前景和市场潜力。开发适合动物细胞培养的载体及其相应反应器,是目前动物细胞培养工程中的重要内容之一。
胶原蛋白是一种天然蛋白,具有良好的生物相容性和理化稳定性,在医药、食品等领域中有着广泛的应用。本实验以天然胶原蛋白为原料,采用铬、甲醛、戊二醛改性试剂以及铬-戊二醛、铝-植物单宁复合改性试剂对其进行化学改性处理,继之以湿热灭菌处理改善胶原蛋白理化性质,特别是胶原蛋白的收缩温度和膜通透性, 制备动物细胞培养载体。然后,在不同载体上培养 CHO 细胞,通过载体的贴壁实验、细胞葡萄糖代谢速率以及目的蛋白——促红细胞生长素(Erythropoietin, EPO)浓度对不同培养载体的生物相容性进行评价;对培养过程中细胞代谢的主要参数——葡萄糖代谢速率以及 EPO 浓度进行监测,通过这两个参数的变化对细胞的生长周期及培养周期进行预测。
总结上述实验结果,并进行分析可得出如下结论:
1. 戊二醛改性胶原蛋白经热处理以后基本保持了原有性质,同时表面性状得到改善,更重要的是再经过湿热灭菌处理,其性状保持稳定,不发生变化,其上述性质使其可以用于动物细胞培养载体的制备。
2. 戊二醛改性胶原蛋白膜的通透性最好,对 BSA 为 2.47 mg·cm-2·h-1,对溶菌酶为 7.26 mg·cm-2·h-1,远高于天然胶原蛋白膜及其他改性剂改性膜对两种蛋白的通透性。
3. CHO 细胞在胶原蛋白载体上进行贴壁,3 小时内完成贴壁过程,之后延长贴壁培养时间对贴壁效果没有更大的影响,说明细胞在胶原蛋白载体上的贴壁比较牢固,不易从载体上脱落。
4. CHO 细胞在胶原蛋白膜载体上培养时,葡萄糖的代谢速率最高时达到 14.2mmol?ml-1?h-1,培养 10 天后 EPO 浓度达到 282 U/ml,相对于 C-DISK 培养细胞葡萄糖的消耗速率提高 16%,EPO 表达量提高近 30 %;相对于 A-DISK 葡萄糖的消耗速率提高了 13%,EPO 表达量提高近 18 %。
5. CHO 细胞培养过程中,不同的葡萄糖代谢速率,反映细胞的代谢水平和目 I的蛋白的表达强度,但这种反映并不是同步的,蛋白的代谢周期要比葡萄糖的代谢周期滞后 1-2 天,因此在细胞培养过程中,可以用葡萄糖的代谢速度对培养过程进行监控和预测, 对细胞的生长和目的蛋白的生产进行调控。
6. 胶原蛋白膜载体成本约 2.0 RMB/g, 仅为进口 A-DISK 载体价格的 5 %;自制 C-DISK 载体成本约 0.2 RMB/g,仅为进口 A-DISK 载体价格的 0.5 %。综合改性胶原蛋白膜载体在细胞生长及目的产物表达方面的优势,确定新型胶原蛋白膜载体性价比优于 C-DISK 及进口 A-DISK 载体。
Animal cell culture, by which many bio-products and bio-pharmacies aremanufactured such as monoclonal antibodies, vaccines, recombinant proteins and soon, plays more and more important role in biotechnology and bioengineering. For itsvital character and great profits, a great deal of currents and human resources areabsorbed and great rewards for their efforts have been made. Study on bioreactors andcarriers for animal cell culture has drawn great attentions for their vital status in thisfield. For its high bio-compatibility and stability to chemical and physical factors,collagen is widely applied in industries such as medicine and food.In this paper, the methods to manufacture new carriers with collagen areinvestigated. The main procedure is to denature natural casing by chemical reagents,just like chromium, formaldehyde, glutaraldehyde, chromium-glutaraldehydecomplex and aluminum-tannin complex etc, to improve its heat resistance, stability tochemical reagent, membrane flux and bio-compatibility. Then, the comparison andevaluation of performance and characteristic for culturing CHO cells by the newcarrier made by above modified natural casing, A-DISK carrier imported from U.S.A.and C-DISK carrier manufactured by ourselves have been made. Some estimation ontheir bio-compatibility is given for the new carrier made by above modified naturalcasing based on the experiment data of cell anchorage, glucose metabolic rate andEPO secreted concentration. Furthermore, the principle of cell culture is discussedaccording to metabolic rate of glucose and EPO concentration in medium.The following conclusions are drawn from all of the experiments:1. Natural casing membrane denatured by glutaraldehyde keeps its characters afterheating, and has better surface properties. Moreover, it could resist stream sterilizationand keep its performance stable, which makes casing membrane denatured byglutaraldehyde fit for cell carriers.2. Natural casing membrane denatured by glutaraldehyde show the highest flux toBSA and lysozyme, its flux to BSA is 2.47 mg·cm-2·h-1, its flux to lysozyme is 7.26mg·cm-2·h-1 , better than those nature and other denatured casing membranes. i3. The anchorage experiments of CHO cells on carriers show that natural casing membranedenatured by glutaraldehyde can be firmly stuck by CHO cells, also longer culture time doesno infection on cells’ anchorage.4. The highest metabolic rate of glucose in the culture procession on casing membranedenatured by glutaraldehyde is 14.2 mmol?ml-1?h-1, which is improved by 16 %higher than that of C-DISK and 13 % than that of A-DISK, EPO concentration is 282U/ml on casing membrane denatured by glutaraldehyde, which is improved 30 %higher than that of C-DISK and 18 % than that of A-DISK.5. The change of metabolic rates of glucose in cell culture reveals different metabolic level andprotein expression level, but they are not in synchronization. In our experiments, there is about 1-2days delay period between the change of glucose metabolic rate and EPO concentration, thisvacancy mean that we could inspect and detect the culture station.6. The cost of the carrier made by casing membrane denatured by glutaraldehyde isabout 2.0 RMB/g, which is about 5 % of A-DISK; the cost of C-DISK is 0.2 RMB/g,only 0.5 % of A-DISK.Although both denatured casing carrier and C-DISK carrierare much cheaper than imported A-DISK carriers, denatured casing carrier can still beregarded as the best carrier for their advantage in cell growth, protein expression.
