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

  更多相關(guān)文章： 旋轉(zhuǎn)管 轉(zhuǎn)瓶 細(xì)胞培養(yǎng) 蛋白表達(dá) 無血清培養(yǎng)基 細(xì)胞代謝

【摘要】：本研究以應(yīng)用一種新型高通量生物反應(yīng)器Tubespin(旋轉(zhuǎn)管)進(jìn)行細(xì)胞培養(yǎng),并與傳統(tǒng)的轉(zhuǎn)瓶進(jìn)行比較,證明Tubespin的優(yōu)勢,然后對Tubespin培養(yǎng)CHO細(xì)胞的條件進(jìn)行優(yōu)化和代謝分析,旨在通過上述實(shí)驗(yàn)選擇一種高通量的細(xì)胞培養(yǎng)方法,并優(yōu)化得到細(xì)胞生長和蛋白表達(dá)的最佳條件,并獲得自主知識產(chǎn)權(quán)的無血清培養(yǎng)基配方,并以此細(xì)胞為模型為動物細(xì)胞大規(guī)模培養(yǎng)平臺技術(shù)的建立提供實(shí)驗(yàn)基礎(chǔ)。 我們分別使用轉(zhuǎn)瓶和旋轉(zhuǎn)管培養(yǎng)CHO細(xì)胞,對其細(xì)胞密度、活力、溶氧度、蛋白表達(dá)量,PH值進(jìn)行比較,然后通過非浸入式溶氧電極對這兩種反應(yīng)器的體積傳遞系數(shù)(kLa)進(jìn)行測定,結(jié)果表明,旋轉(zhuǎn)管細(xì)胞培養(yǎng)效果明顯優(yōu)于轉(zhuǎn)瓶,在分別培養(yǎng)CHO DG44和CHO TNF兩種細(xì)胞系時(shí),旋轉(zhuǎn)管中最高細(xì)胞密度分別達(dá)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(jié)束時(shí)PH值為6.7,而在轉(zhuǎn)瓶培養(yǎng)時(shí)兩種細(xì)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(jié)束時(shí)PH值低至6.2,蛋白表達(dá)量也低于旋轉(zhuǎn)管培養(yǎng)。測定體積傳遞系數(shù)發(fā)現(xiàn)：旋轉(zhuǎn)管具有極高的kLa,達(dá)21.5 h-1,而轉(zhuǎn)瓶只有2.7 h-1。接著,我們對旋轉(zhuǎn)管培養(yǎng)的培養(yǎng)條件進(jìn)行優(yōu)化,得到最佳培養(yǎng)條件為搖床轉(zhuǎn)速180 rpm,培養(yǎng)體積10 ml,此時(shí)細(xì)胞密度,蛋白表達(dá)量都處于最佳水平。應(yīng)用旋轉(zhuǎn)管最佳培養(yǎng)條件對氯化鈉促進(jìn)細(xì)胞重組蛋白表達(dá)條件進(jìn)行優(yōu)化,發(fā)現(xiàn)氯化鈉對蛋白表達(dá)促進(jìn)作用明顯,在濃度為60 mM時(shí)能使重組蛋白表達(dá)量提高一倍。 通過試驗(yàn)比較,選定DMEM/F12培養(yǎng)基為基礎(chǔ)培養(yǎng)基,進(jìn)行CHO無血清培養(yǎng)基優(yōu)化,對EAA, NEAA, Lipids, Vitamins進(jìn)行正交設(shè)計(jì)優(yōu)化,得到化學(xué)成分明確培養(yǎng)基SFM1,在進(jìn)行蛋白胨優(yōu)化實(shí)驗(yàn)得到無血清培養(yǎng)基SFM2,對SFM2進(jìn)行細(xì)胞培養(yǎng)和蛋白表達(dá)驗(yàn)證,結(jié)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(xì)胞培養(yǎng)過程中的主要代謝進(jìn)行測定分析,發(fā)現(xiàn)葡萄糖作為細(xì)胞主要碳源,其消耗率達(dá)80%以上；氨基酸中消耗率最大的是胱氨酸,達(dá)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(chǎn)物乳酸和氨的含量會隨著培養(yǎng)的進(jìn)行不斷增加,濃度都不會達(dá)到抑制濃度；培養(yǎng)過程中培養(yǎng)基中溶氧變化與細(xì)胞密度相對應(yīng),在細(xì)胞密度最高時(shí),培養(yǎng)基中溶氧最低；攝氧率的變化與細(xì)胞代謝狀態(tài)相近,在遲滯期,攝氧率逐漸增大,在對數(shù)期時(shí)達(dá)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(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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