CHO 工程細胞(11G-S)懸浮培養的無血清培養基的設計

劉興茂，劉紅，葉玲玲，李世崇，吳本傳，王海濤，謝靖，陳昭烈

軍事醫學科學院生物工程研究所，北京 100071

CHO 工程細胞(11G-S)懸浮培養的無血清培養基的設計

劉興茂，劉紅，葉玲玲，李世崇，吳本傳，王海濤，謝靖，陳昭烈

軍事醫學科學院生物工程研究所，北京 100071

以懸浮適應的表達重組尿激酶原(Pro-urokinase，pro-UK)CHO工程細胞系11G-S為對象，采用Plackett-Burman實驗設計及響應面分析法，設計支持 CHO工程細胞(11G-S)懸浮生長的無血清培養基。以細胞密度為評價指標，在單因素實驗的基礎上采用Plackett-Burman實驗設計對影響細胞生長的培養基添加成分進行考察，確定了3種對細胞生長明顯促進作用的培養基添加成分：胰島素、轉鐵蛋白及腐胺。繼而利用響應面法分析了這 3種添加成分的最佳水平范圍，設計了一種適用于CHO工程細胞(11G-S)懸浮培養的無血清培養基SFM-CHO-S。11G-S細胞在 SFM-CHO-S批次懸浮培養的細胞最大生長密度達到4.12×106cells/mL，pro-UK的最大累積活性達到5 614 IU/mL，培養效果優于商品化的同類無血清培養基。

CHO工程細胞，懸浮培養，無血清培養基，實驗設計

Abstract:With suspension adapted recombinant Chinese hamster ovary(CHO)cell lines 11G-S expressing human pro-urokinase(pro-UK)as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology.The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture.Among the 10 medium supplements,insulin, transferrin, and putrescine were identified as the most significant factors(P＜0.05).The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors.And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated.The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12×106cells/mL with a maximum pro-UK activity at 5 614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.

Keywords:recombinant Chinese hamster ovary cells, suspension culture, serum-free medium, medium design

CHO工程細胞是目前用于包括工程抗體和重組蛋白在內的生物技術藥物研發和生產的最重要的表達系統[1-2]。懸浮培養是哺乳動物細胞大規模培養研究和應用的重要模式和首要選擇[3-4]。對于 CHO工程細胞的懸浮培養而言，培養基是影響細胞生長和細胞表達產物生產的最重要因素之一。

受動物細胞表達產品生產過程優化和產品安全性要求的驅動，無血清培養已成為目前哺乳動物細胞培養技術的發展方向和應用趨勢[5]。……