犬肾细胞MDCK无血清贴壁及单细胞悬浮培养

Adherent and single-cell suspension culture of Madin-Darby canine kidney cells in serum-free medium

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?1,分别较有血清贴壁培养提高了3.6和1.6倍。此外,通过比较有血清贴壁培养、无血清贴壁培养和无血清单细胞悬浮培养三种体系中MDCK细胞的生长和基本代谢情况发现:无血清培养过程细胞生长快,代谢副产物产率低,尤其是单细胞悬浮培养方式能获得较高的细胞密度,更有利于大规模生产。该结果为MDCK细胞培养过程的优化和放大奠定了基础,也为其他动物细胞培养过程和疫苗等生物制品的工业化生产提供了借鉴。

In recent years,there are tremendous economic and social losses across the world because of virus-related diseases.It is well known that Madin-Darby canine kidney（MDCK） cells are easily handled,quickly amplified and efficiently infected with influenza virus.Therefore,they are considered as one of the most important cell lines for the production of influenza vaccine.In this work,we first developed a serum-free adherent culture process for MDCK cells with an in-house prepared serum-free medium MDCK-SFM.Next,we derived a cell line named ssf-MDCK,which was amenable for single-cell suspension culture in the serum-free medium.We found that during serum-free batch culture of MDCK cells,the peak viable cell density and maximum specific growth rate were 3.81×106 cells/mL and 0.056 h-1,respectively;3.6-and 1.6-fold increase compared with those in serum-containing adherent batch culture.In addition,we compared growth and metabolic characteristics of MDCK cells in serum-containing adherent culture,serum-free adherent culture and serum-free single-cell suspension culture.We found that less metabolic by-products were produced in both serum-free cultures.In serum-free single-cell suspension batch culture,the viable cell density was highest.These results are critical for establishing large-scale suspension culture of MDCK cells as subsequent well as large-scale influenza vaccine production.