本文關鍵詞：CHO細胞在Tubespin中的培養(yǎng)工藝及其代謝分析　出處：《暨南大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： 旋轉管 轉瓶 細胞培養(yǎng) 蛋白表達 無血清培養(yǎng)基 細胞代謝

【摘要】：本研究以應用一種新型高通量生物反應器Tubespin(旋轉管)進行細胞培養(yǎng),并與傳統(tǒng)的轉瓶進行比較,證明Tubespin的優(yōu)勢,然后對Tubespin培養(yǎng)CHO細胞的條件進行優(yōu)化和代謝分析,旨在通過上述實驗選擇一種高通量的細胞培養(yǎng)方法,并優(yōu)化得到細胞生長和蛋白表達的最佳條件,并獲得自主知識產權的無血清培養(yǎng)基配方,并以此細胞為模型為動物細胞大規(guī)模培養(yǎng)平臺技術的建立提供實驗基礎。 我們分別使用轉瓶和旋轉管培養(yǎng)CHO細胞,對其細胞密度、活力、溶氧度、蛋白表達量,PH值進行比較,然后通過非浸入式溶氧電極對這兩種反應器的體積傳遞系數(kLa)進行測定,結果表明,旋轉管細胞培養(yǎng)效果明顯優(yōu)于轉瓶,在分別培養(yǎng)CHO DG44和CHO TNF兩種細胞系時,旋轉管中最高細胞密度分別達到了6.0×106 cells·ml-1和8.5×l06 cells·ml-1,溶氧在培養(yǎng)過程中始終保持在80%以上,培養(yǎng)結束時PH值為6.7,而在轉瓶培養(yǎng)時兩種細胞的最高密度都只能到4×106 cells·ml-1,而且培養(yǎng)過程中溶氧分別跌至1.9%和0%,而培養(yǎng)結束時PH值低至6.2,蛋白表達量也低于旋轉管培養(yǎng)。測定體積傳遞系數發(fā)現：旋轉管具有極高的kLa,達21.5 h-1,而轉瓶只有2.7 h-1。接著,我們對旋轉管培養(yǎng)的培養(yǎng)條件進行優(yōu)化,得到最佳培養(yǎng)條件為搖床轉速180 rpm,培養(yǎng)體積10 ml,此時細胞密度,蛋白表達量都處于最佳水平。應用旋轉管最佳培養(yǎng)條件對氯化鈉促進細胞重組蛋白表達條件進行優(yōu)化,發(fā)現氯化鈉對蛋白表達促進作用明顯,在濃度為60 mM時能使重組蛋白表達量提高一倍。 通過試驗比較,選定DMEM/F12培養(yǎng)基為基礎培養(yǎng)基,進行CHO無血清培養(yǎng)基優(yōu)化,對EAA, NEAA, Lipids, Vitamins進行正交設計優(yōu)化,得到化學成分明確培養(yǎng)基SFM1,在進行蛋白胨優(yōu)化實驗得到無血清培養(yǎng)基SFM2,對SFM2進行細胞培養(yǎng)和蛋白表達驗證,結果優(yōu)于商業(yè)化培養(yǎng)基CD DG44,接近商業(yè)化培養(yǎng)基P8,但成本節(jié)約一半以上。 對CHO細胞培養(yǎng)過程中的主要代謝進行測定分析,發(fā)現葡萄糖作為細胞主要碳源,其消耗率達80%以上；氨基酸中消耗率最大的是胱氨酸,達100%,其耗竭亦可能是最終營養(yǎng)限制的主要原因；代謝過程中主要代謝產物乳酸和氨的含量會隨著培養(yǎng)的進行不斷增加,濃度都不會達到抑制濃度；培養(yǎng)過程中培養(yǎng)基中溶氧變化與細胞密度相對應,在細胞密度最高時,培養(yǎng)基中溶氧最低；攝氧率的變化與細胞代謝狀態(tài)相近,在遲滯期,攝氧率逐漸增大,在對數期時達最高值,然后逐漸下降。
[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.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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