MDCK悬浮细胞制备及流感病毒敏感性研究

目的 建立无血清悬浮培养MDCK细胞的方法,分析其传代、线性放大过程中的稳定性及增殖流感病毒的敏感性。方法 运用逐步降血清悬浮驯化法将贴壁培养型MDCK细胞驯化为无血清全悬浮培养型MDCK细胞;进一步分析其生长动力学特性、连续传代稳定性,在5L生物反应器中扩大培养;接种流感和禽流感病毒,测定不同时间HA效价、CCID₅₀滴度,分析其病毒敏感性。结果 成功将ATCC引进的贴壁培养型MDCK细胞驯化为无血清全悬浮培养型MDCK细胞(命名为MDCK-XF02细胞)并冻存;不同接种密度培养MDCK-XF02细胞最大增殖密度均达13.0×10⁶ cells/mL以上,且差异无统计学意义(P>0.05);连续传代培养过程中细胞形态和生长状况稳定,比生长速率差异无统计学意义(P>0.05);三个代次的MDCK-XF02细胞以2.0×10⁶ cells/mL接种于5 L生物反应器,细胞生长状态良好,倍增时间小于21 h,在批培养中细胞浓度高达(14.57±0.47)×10⁶ cells/mL,比生长速率分别为(0.027±0.012)h^-1、(0.028±0.013)h^-1、(0.027±0.013)h^-1(P>0.05);接种甲型流感病毒H1N1和H3N2,HA效价达到(6~7)log₂HA/25 μL、CCID₅₀为(4.35~4.68)lgCCID₅₀/mL;接种乙型流感病毒B/P和BX-35增殖效果更好,HA效价达到(9~10)log₂HA/25 μL,CCID₅₀为(6.38~7.31)lgCCID₅₀/mL。接种重组禽流感病毒H5亚型Re-5、Re-6、Re-10均能很好的增殖,HA效价达到(7~9)log₂HA/25 μL、CCID₅₀为(6.21~6.96)lgCCID₅₀/mL。结论 本研究获得一株具有自主知识产权的流感/禽流感病毒敏感的无血清悬浮培养型MDCK-XF02细胞株,实现了生物反应器高密度放大培养,为我国开展流感疫苗研究和生产提供细胞基质,也为其他动物细胞悬浮驯化提供参考。

Establishment of a new MDCK suspension cell line and its sensitivity study on influenza virus

In order to establish a method for serum-free suspension culture of Madin-Daby Canine Kidney Cells(MDCK)cells, its stability during cell passage and linear amplification and the sensitivity of proliferation of influenza virus were analyzed. The adherent MDCK cells were acclimated into serum-free suspended MDCK cells by stepwise serum suspension and acclimation. The growth kinetics and continuous passage stability were further analyzed, and the culture was expanded in a 5L bioreactor. And influenza virus, HA titer and CCID₅₀ titer were measured at different times, and the virus sensitivity was analyzed. The adherent MDCK cells from ATCC were successfully acclimated into serum-free suspension MDCK cells (named MDCK-XF02 cells) and cryopreservation. The maximum proliferation density of MDCK-XF02 cells reached 13.0×10⁶ Cells/mL or more, and the difference was not significant (P>0.05); the cell morphology and growth status were stable during continuous subculture, and there was no significant difference in growth rate (P>0.05). The three generations of MDCK-XF02 cells with 2.0×10⁶ cells/mL was inoculated into a 5L bioreactor, and the cell growth state was good. The doubling time was less than 21 h. The cell concentration in the batch culture was as high as (14.57±0.47)×10⁶ cells/mL, and the specific growth rate was (0.027±0.012)h^-1,(0.028±0.013)h^-1,(0.027±0.013)h^-1 respectively, which showed no significant difference (P>0.05). Inoculated with influenza A virus H1N1 and H3N2, HA titer reached (6-7) log₂HA/25 μL, CCID₅₀ reached (4.35-4.68) lgCCID₅₀ /mL. Inoculation of influenza B virus B/P and BX-35 proliferation effect is better, HA titer reached (9-10) log₂HA/25 μL, CCID₅₀ reached (6.38-7.31) lgCCID₅₀/mL. Recombinant avian influenza virus H5 subtypes Re-5, Re-6, Re-10 can proliferate well, HA titer reached (7-9) log₂HA/25 μL, CCID₅₀ reached (6.21-6.96) lgCCID₅₀/mL. This study obtained a strain of serum-free suspension-cultured MDCK-XF02 cells with independent intellectual property rights of influenza/avian influenza virus, which achieved high-density amplification of bioreactor and provided cell matrix for influenza vaccine research and production in China. It also provides a reference for the suspension and domestication of other animal cells.