CHO细胞在Tubespin中的培养工艺及其代谢分析

发布时间：2018-01-10 07:17

本文关键词：CHO细胞在Tubespin中的培养工艺及其代谢分析　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

【摘要】：本研究以应用一种新型高通量生物反应器Tubespin(旋转管)进行细胞培养,并与传统的转瓶进行比较,证明Tubespin的优势,然后对Tubespin培养CHO细胞的条件进行优化和代谢分析,旨在通过上述实验选择一种高通量的细胞培养方法,并优化得到细胞生长和蛋白表达的最佳条件,并获得自主知识产权的无血清培养基配方,并以此细胞为模型为动物细胞大规模培养平台技术的建立提供实验基础。我们分别使用转瓶和旋转管培养CHO细胞,对其细胞密度、活力、溶氧度、蛋白表达量,PH值进行比较,然后通过非浸入式溶氧电极对这两种反应器的体积传递系数(kLa)进行测定,结果表明,旋转管细胞培养效果明显优于转瓶,在分别培养CHO DG44和CHO TNF两种细胞系时,旋转管中最高细胞密度分别达到了6.0×106 cells·ml-1和8.5×l06 cells·ml-1,溶氧在培养过程中始终保持在80%以上,培养结束时PH值为6.7,而在转瓶培养时两种细胞的最高密度都只能到4×106 cells·ml-1,而且培养过程中溶氧分别跌至1.9%和0%,而培养结束时PH值低至6.2,蛋白表达量也低于旋转管培养。测定体积传递系数发现：旋转管具有极高的kLa,达21.5 h-1,而转瓶只有2.7 h-1。接着,我们对旋转管培养的培养条件进行优化,得到最佳培养条件为摇床转速180 rpm,培养体积10 ml,此时细胞密度,蛋白表达量都处于最佳水平。应用旋转管最佳培养条件对氯化钠促进细胞重组蛋白表达条件进行优化,发现氯化钠对蛋白表达促进作用明显,在浓度为60 mM时能使重组蛋白表达量提高一倍。通过试验比较,选定DMEM/F12培养基为基础培养基,进行CHO无血清培养基优化,对EAA, NEAA, Lipids, Vitamins进行正交设计优化,得到化学成分明确培养基SFM1,在进行蛋白胨优化实验得到无血清培养基SFM2,对SFM2进行细胞培养和蛋白表达验证,结果优于商业化培养基CD DG44,接近商业化培养基P8,但成本节约一半以上。对CHO细胞培养过程中的主要代谢进行测定分析,发现葡萄糖作为细胞主要碳源,其消耗率达80%以上；氨基酸中消耗率最大的是胱氨酸,达100%,其耗竭亦可能是最终营养限制的主要原因；代谢过程中主要代谢产物乳酸和氨的含量会随着培养的进行不断增加,浓度都不会达到抑制浓度；培养过程中培养基中溶氧变化与细胞密度相对应,在细胞密度最高时,培养基中溶氧最低；摄氧率的变化与细胞代谢状态相近,在迟滞期,摄氧率逐渐增大,在对数期时达最高值,然后逐渐下降。
[Abstract]:In this study, a new high-throughput bioreactor, Tubespin (rotating tube), was used for cell culture, and compared with the traditional flask, the advantages of Tubespin were proved. Then the conditions of Tubespin culture of CHO cells were optimized and the metabolic analysis was carried out in order to select a high-throughput cell culture method through the above experiments. The optimal conditions for cell growth and protein expression were optimized, and the serum-free medium formulation of independent intellectual property rights was obtained. The model provides experimental basis for the establishment of large-scale culture platform for animal cells. CHO cells were cultured in flask and rotating tube, and their cell density, activity, oxygen solubility and protein expression were compared. Then the volumetric transfer coefficient (KLA) of the two reactors was measured by non-immersion dissolved oxygen electrode. The results showed that the cell culture effect of rotating tube was better than that of flask. Two cell lines, CHO DG44 and CHO TNF, were cultured respectively. The highest cell density in the rotating tube was 6.0 脳 10 ~ 6 cells 路ml-1 and 8.5 脳 10 ~ 6 cells 路ml-1, respectively. Dissolved oxygen remained above 80% in the culture process, and the PH value at the end of culture was 6.7, but the maximum density of both cells in flask culture was only 4 脳 10 6 cells 路ml-1. Moreover, dissolved oxygen decreased to 1.9% and 0 respectively during culture, and PH value was as low as 6.2 at the end of culture. The protein expression was also lower than that in rotating tube culture. The measurement of volumetric transfer coefficient showed that the rotating tube had an extremely high kLa-21.5 h-1, while the rotated bottle was only 2.7 h-1. We optimized the culture conditions of rotating tube culture. The optimal culture conditions were as follows: rotating speed 180 rpm, volume 10 ml, cell density. The optimal culture condition of rotating tube was used to optimize the condition of sodium chloride promoting the expression of recombinant protein in cells, and it was found that sodium chloride could promote protein expression obviously. At the concentration of 60 mm, the expression of recombinant protein was doubled. The DMEM/F12 medium was selected as the base medium and the serum-free medium of CHO was optimized. EAA, NEA, Lipids were optimized. Vitamins was optimized by orthogonal design to obtain SFM1 medium with definite chemical composition and SFM2 medium without serum was obtained by optimization experiment of peptone. The results of cell culture and protein expression verification of SFM2 were better than that of commercial medium CD DG44, close to commercial medium P8, but cost saving was more than half. The main metabolism of CHO cells was measured and analyzed. It was found that glucose was the main carbon source and the consumption rate of glucose was over 80%. The highest consumption rate of amino acids was cystine (100%), which may also be the main reason for the ultimate nutritional limitation. The contents of lactic acid and ammonia, the main metabolites in the metabolic process, would increase with the culture, and the concentration would not reach the inhibitory concentration. The changes of dissolved oxygen in culture medium correspond to cell density. When cell density is highest, dissolved oxygen in medium is the lowest. The change of oxygen uptake rate was similar to that of cell metabolism. In the delayed phase, the oxygen uptake rate increased gradually, reached the highest value at logarithmic stage, and then decreased gradually.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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