Development and Optimization of Serum-Free Medium for High-density Culture of Suspended BHK-21 Cells and High-yield of FMD Virus

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

Abstract

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

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.

Figures/Tables 10

References 32

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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

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

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

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

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