Viral antigen production in cell cultures on microcarriers: Bovine parainfluenza 3 virus and MDBK cells

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• 作者：M.M. Conceiç ; ã ; o ; A. Tonso ; C.B. Freitas ; C.A. Pereira
• 关键词：MDBK cells ; PI-3 virus ; Microcarriers
• 刊名：Vaccine
• 出版年：2007
• 期刊代码：89_0264410x
• 类别：imm
• 出版时间：7 November 2007
• 卷：25
• 期：45
• 页码：7785-7795
• 文件大小：1550 K

摘要

Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin–Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90%MCs with confluent cell monolayers) and cell growth (2.5 × 10⁶ cells/mL and a μ_x (h⁻¹) 0.05). Since cell cultures performed with lower amount of MCs (1 g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8–10) × 10⁵ cells/mL at 120 h) and cell loading (210–270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7–9 log₁₀ TCID₅₀/mL and 1.5–2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24 h and those of PI-3 antigen increased after 24 h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2 L were infected and the PI-3 virus production in 12 L attained 12 log₁₀ TCID₅₀. Other than establishing a protocol for PI-3 production in MDBK cell cultures on Cytodex 1, the experiments are proposed as a basis for approaching the development of a virus production protocol in mammalian cells cultivated on microcarriers in bioreactors.