支持高密度BHK-21细胞培养和高产FMD病毒的无血清培养基开发与优化

doi:10.13560/j.cnki.biotech.bull.1985.2020-0343

摘要/Abstract

摘要：

优化无血清培养基,以实现BHK-21悬浮细胞的超高密度生长和口蹄疫病毒高效扩增。依据BHK-21细胞的代谢特点和动力学分析识别无血清培养基中的关键营养成分,优化组分浓度,对比优化前后细胞的生长和产毒能力,并在生物反应器中验证。优化后的无血清培养基支持批培养模式下最高活细胞密度达到1.78×10⁷cells/mL,代谢副产物积累的情况得到改善;口蹄疫病毒TCID₅₀与含低比例血清的培养基相比具有相似水平,约为7.25 lgTCID₅₀/0.1mL;146s含量与低血清培养基相比提高50.7%,达到15.6 μg/mL。以细胞的生长需求和代谢规律为基础,优化营养成分和降低代谢副产物积累能有效解除“细胞密度效应”,实现超高细胞密度接毒和高产口蹄疫病毒。

关键词: BHK-21悬浮细胞, 无血清培养基, 高细胞密度, 高产口蹄疫病毒

Abstract:

Based on the high-density suspension culture of BHK-21 cells,this study is to optimize the serum-free medium in order to improve proliferation of the BHK-21 cells and amplification of foot-and-mouth virus（FMDV）. Firstly,the key nutrients in the serum-free medium were determined based on the metabolic characteristics and dynamics analysis of the BHK-21 cells. Then,the composition of the medium was optimized. Finally,the growth and FMDV-producing ability of the BHK-21 cells between before and after the optimization were compared and verified in bio-reactor. The maximum viable cell density reached 1.78×10⁷ cells/mL during batch culturing of the BHK-21 cells in optimized serum-free medium（Opt-SFM）,and the situation of accumulating metabolic by-products was improved. Compared with non-optimized medium containing 1%（V/V）serum, TCID₅₀ of the produced FMDV in the Opt-SFM showed a similar level at 7.25 lgTCID₅₀/0.1 mL,while the yield of 146s particals increased by 50.7% to 15.6 g/mL. In conclusion,based on the requirements of cell growth and metabolic characteristics,optimizing nutrients and reducing metabolic by-products accumulation may effectively eliminate “cell density effect” and achieve ultra-high cell density and high yield of foot-and-mouth virus.

Key words: BHK-21 suspended cells, serum-free medium, high-density cell culture, high yield of foot-and-mouth virus

陈敏, 刘旭平, 赵亮. 支持高密度BHK-21细胞培养和高产FMD病毒的无血清培养基开发与优化[J]. 生物技术通报, 2020, 36(10): 62-71.

CHEN Min, LIU Xu-ping, ZHAO Liang. Development and Optimization of Serum-Free Medium for High-density Culture of Suspended BHK-21 Cells and High-yield of FMD Virus[J]. Biotechnology Bulletin, 2020, 36(10): 62-71.

图/表 10

表1 氨基酸浓度优化的理论添加量

添加物质	接种后第3天剩余比例/%	0-3 d比消耗速率 mmol/（10⁹ cells?day）	推荐增加的浓度比例/%
Asp	6.95	0.12	32.92
			69.17
Asn	11.48	0.20	26.46
			60.94
Gln	14.09	0.17	22.73
			56.20

表2 氨基酸浓度优化的实验设计

实验组	Asp浓度	Asn浓度	Gln浓度
S1-SFM-a	+	+	+
S1-SFM-b	+	-	+
S1-SFM-c	+	+	-
S1-SFM-d	+	-	-
S1-SFM-e	-	+	+
S1-SFM-f	-	-	+
S1-SFM-g	-	+	-
S1-SFM-h	-	-	-
S1-SFM-i	0	0	0

表3 铜锌离子浓度实验设计表

实验组	铜离子浓度/（mmol?L^-1）	锌离子浓度/（mmol?L^-1）
S2-SFM-a	0.04	0.2
S2-SFM-b	0.04	0.02
S2-SFM-c	0.04	0.002
S2-SFM-d	0.004	0.2
S2-SFM-e	0.004	0.02
S2-SFM-f	0.004	0.002
S2-SFM-g	0.0004	0.2
S2-SFM-h	0.0004	0.02
S2-SFM-i	0.0004	0.002
S2-SFM-j	0	0

图1 BHK-21细胞在P-SFM批培养过程中的生长和代谢情况 A：VCD（活细胞密度）、葡萄糖、乳酸、氨;B：氨基酸

图2 无血清培养基的优化 A：葡萄糖浓度对细胞前3 d总IVCC和第2天乳酸浓度的影响;B：氨基酸浓度对第2天乳酸浓度的影响;C：铜锌离子浓度对细胞前3 d总IVCC的影响

图3 基于摇瓶体系,BHK-21细胞在Opt-SFM中的生长和代谢 A：0-3 d内细胞总IVCC;B：传代时葡萄糖比消耗速率、乳酸和氨比生成速率;C：BHK-21细胞的稳定传代;D：细胞形态（标尺=200 μm）

图4 基于反应器体系,BHK-21细胞在Opt-SFM中的生长和代谢 A：细胞的活细胞密度、葡萄糖消耗、乳酸和氨累积;B：0-3 d内葡萄糖比消耗速率、乳酸和氨比生成速率

图5 细胞密度和接毒方式对病毒产量和单细胞产毒能力的影响

图6 BHK-21细胞在Opt-SFM中接种口蹄疫病毒细胞生长与病变情况 A：BHK-21细胞的活细胞密度和接毒后的病变率;B：接毒0 h细胞形态;C：接毒12 h细胞形态（标尺=200 μm）

图7 培养基优化前后病毒效价TCID50和146s完整病毒颗粒的含量

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