Optimization of the amount of TPCK trypsin adding to MDCK cell suspension for culturing H7N9 avian influenza virus

Ziyan Meng; Jiayou Zhang; Chen Liu; Zhegang Zhang; Ran Qiu; Dan Luo; Jinrong Shi; Wei Zhao; Zhiwu Xia; Xiaoming Yang

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Optimization of the amount of TPCK trypsin adding to MDCK cell suspension for culturing H7N9 avian influenza virus

VernacularTitle: 悬浮MDCK细胞培养H7N9流感病毒时TPCK胰酶添加量的优化研究
Author: Ziyan MENG ^{1
,

2} ; Jiayou ZHANG ^{1
,

2} ; Chen LIU ^{1
,

2} ; Zhegang ZHANG ^{1
,

2} ; Ran QIU ^{1
,

2} ; Dan LUO ^{1
,

2} ; Jinrong SHI ^{1
,

2} ; Wei ZHAO ^{1
,

2} ; Zhiwu XIA ^{1
,

2} ; Xiaoming YANG ³
Author Information

1. National Engineering Technology Research Center of Combination Vaccines, Wuhan 430207, China
2. The Second Laboratory of Viral Vaccine Research, Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China
3. National Engineering Technology Research Center of Combination Vaccines, Wuhan 430207, China；China National Biotechnology Group, Beijing 100029, China
Publication Type:Journal Article
Keywords: MDCK cells; H7N9; Avain influenza virus; TPCK trypsin; Optimization study
From: Chinese Journal of Microbiology and Immunology 2019;39(11):835-839
CountryChina
Language:Chinese
Abstract: Objective:To investigate the best amount of TPCK trypsin in Madin Darby canine kidney (MDCK) cell suspension for the culture of H7N9 avian influenza virus.
Methods:Different concentrations of TPCK trypsin were added during the periods of cell growth and virus production. Their effects on cell growth, viability, glucose and lactate metabolism, and hemagglutination titer were monitored every 12 h. Inter-batch differences were analyzed. The amount of trypsin added in the cell growth phase was 0, 1 μg/ml, 2 μg/ml, 4 μg/ml, 6 μg/ml, 8 μg/ml, 10 μg/ml and 15 μg/ml. The amount of trypsin added during the virus production period was 0, 0.5 μg/ml, 1 μg/ml, 1.5 μg/ml, 2 μg/ml and 2.5 μg/ml. When the hemagglutination titers were same, the adding amount was further optimized at different multiplicity of infection (MOI) of 0.001, 0.005, 0.025 and 0.05.
Results:No significant linear effects of TPCK trypsin concentration on cell number, viability, and glucose and lactate metabolism were observed. No toxicity to cell growth was observed when TPCK trypsin concentration reached 15 μg/ml. After the inoculation of H7N9 avian influenza virus, the hemagglutination titers in the 1 μg/ml, 1.5 μg/ml, 2 μg/ml and 2.5 μg/ml TPCK trypsin groups reached the peaks at 48 h, which were 1∶2^6.5. At 60 h, the hemagglutination titers of the latter two groups decreased faster than those of the former two groups. When the MOI was 0.005, the hemagglutination titer of the 1.5 μg/ml group at 48 h was 2^6.5 higher than 2⁶ in the 1 μg/ml group under the same condition. There were differences between different batches of TPCK trypsin.
Conclusions:Adding 1 μg/ml and 1.5 μg/ml of trypsin could better promote the proliferation of H7N9 avian influenza virus, and 1.5 μg/ml of trypsin had a wider range of MOI applicability.