膽固醇自主型鼠源骨髓瘤細胞的馴化與電穿孔條件的研究

哺乳動物細胞的大量培養是近代生技產業生產高複雜度蛋白質藥物的重要技術之一。由於蛋白質藥物如單株抗體、干擾素等的蓬勃發展，加上進入臨床前與臨床試驗的蛋白質藥物與日俱增，核准上市的蛋白質藥物也快速增加，如何增加產量並降低製造成本來滿足這些需求，成為相當重要的課題。常見的方法有培養基的改良、細胞株的馴化及篩選、細胞培養條件的改進等。本研究的主要目標是建立無需膽固醇也能生長的NS0細胞(cholesterol- independent)。NS0是一膽固醇缺陷型 (cholesterol-auxotrophic)的鼠源骨髓瘤細胞株，是生產蛋白質藥物常用的宿主細胞之ㄧ，因為NS0無法自行合成膽固醇，培養基需添加膽固醇，但是脂溶性的膽固醇，在培養基的配製上有溶解度與穩定性的問題，若能建立膽固醇自主型細胞株，對於培養基的配製及細胞培養的製程將有莫大的助益。我們成功地將來自ECACC的NS0細胞，從含血清的培養基逐步馴化至不含膽固醇的無血清培養基，並測定細胞株相關的生長特性，包括最低接種密度以及接種密度對生長速率與最高細胞密度的影響。為了使細胞能表現出所需要的蛋白質藥物，需要將表現載體(expression vector)送入細胞，但是如何將載體有效率地送入細胞，並維持細胞存活率是需克服的問題。本研究採用電穿孔(electroporation)技術，轉染馴化到無血清培養基的NS0細胞，主要探討的操作參數是外加電場的強度、時間與電場施加方式(單次或多次)，使用的DNA是一表現綠色螢光蛋白質的載體，轉染後我們量測細胞密度、存活率與生長狀況，並以流式細胞儀測量轉染效率，藉此找出可以接受的電穿孔條件

關鍵字

NS0 cell line ；電穿孔；細胞馴化

並列摘要

NS0 cells —a cholesterol-auxotrophic mouse myeloma cells —require cholesterol ,which must be supplemented to the growth medium for growth and has also proven to be an effective host cell for the expression of antibodies.But cholesterol is difficult to solubilize. Use of protein-free, cholesterol-free media would be beneficial in terms of process consistency, cost of goods, and ease of downstream processing.We have adapted NS0 from the European Collection of Animal Cell Cultures to serum free medium and growth characteristics including the influence of the lowest seeding cell density and growth rate. We need to introduce expression vectors to the cell in order to express the protein drug. Electroporation (or electropermeabilization) is a valuable tool in molecular biology and biotechnology. The permeability of cell membranes can be transiently increased to facilitate the entry of exogenous molecules such as DNA, RNA, and proteins when an external electric field pulse is applied. We do the test with voltage and duration time in Bio-rad Gene Pluser xcell , expression vector is amaxa pmaxGFP.Cells density and viability were assessed in the Cedex counter.Transfection efficiency were assessed in FACS.

並列關鍵字

NS0 ； electroporation ； adaption

參考文獻

[1] Spens, E. and L. Haggstrom, Defined protein and animal component-free NS0 fed-batch culture. Biotechnol Bioeng, 2007. 98(6): p. 1183-94.

[2] Hartman, T.E., et al., Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies. Biotechnol Bioeng, 2007. 96(2): p. 294-306.

[3] Birch, J.R. and A.J. Racher, Antibody production. Adv Drug Deliv Rev, 2006. 58(5-6): p. 671-85.

[5] Seth, G., et al., Large-scale gene expression analysis of cholesterol dependence in NS0 cells. Biotechnol Bioeng, 2005. 90(5): p. 552-67.

[6] Seth, G., M. Ozturk, and W.S. Hu, Reverting cholesterol auxotrophy of NS0 cells by altering epigenetic gene silencing. Biotechnol Bioeng, 2006. 93(4): p. 820-7.

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膽固醇自主型鼠源骨髓瘤細胞的馴化與電穿孔條件的研究

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