Kallel H, Rourou S, Majoul S, Loukil H
Viral Vaccines Research and Development Unit, Institute Pasteur, 13 Place Pasteur, BP 74, 1002 Belvédère, Tunis, Tunisia.
Appl Microbiol Biotechnol. 2003 Jun;61(5-6):441-6. doi: 10.1007/s00253-003-1245-3. Epub 2003 Feb 25.
We studied BHK-21 cells growth in a 2-l bioreactor and investigated the effects of microcarrier concentration, type of growth medium, culture mode and serum concentration. The highest cell density reached was equal to 4x10(6) cells/ml and was achieved in minimum essential medium supplemented with Hanks' salts, non-essential amino acids and 5% fetal calf serum, using a perfusion culture mode and a microcarrier concentration of 4 g Cytodex 3/l. We studied rabies virus production (PV/BHK-21 strain) by BHK-21 cells grown at the optimal conditions determined previously. We analyzed the effects of multiplicity of infection (MOI) and type of medium used for virus multiplication in spinner-flasks and showed that the highest virus titer reached (when the cells were infected at a MOI of 0.3) in M199 medium supplemented with 0.2% of bovine serum albumin was equal to 8.2x10(7) Fluorescent Focus Units (FFU)/ml. When we grew the cells in a 2-l perfused bioreactor, we obtained a maximal virus titer of 3x10(8) FFU/ml. In addition, we scaled-up to a 20-l bioreactor and obtained similar results for cell density and virus titer. The experimental vaccine we developed meets WHO requirements for vaccine potency. Each run yielded about 40,000 doses of potent vaccine.
我们研究了BHK - 21细胞在2升生物反应器中的生长情况，并调查了微载体浓度、生长培养基类型、培养模式和血清浓度的影响。达到的最高细胞密度为4×10⁶个细胞/毫升，这是在补充了汉克斯盐、非必需氨基酸和5%胎牛血清的最低基本培养基中，采用灌注培养模式和4克Cytodex 3/升的微载体浓度实现的。我们研究了在先前确定的最佳条件下生长的BHK - 21细胞产生狂犬病病毒（PV/BHK - 21株）的情况。我们分析了感染复数（MOI）和用于在转瓶中进行病毒增殖的培养基类型的影响，结果表明，在补充了0.2%牛血清白蛋白的M199培养基中，当细胞以MOI为0.3进行感染时，达到的最高病毒滴度为8.2×10⁷荧光聚焦单位（FFU）/毫升。当我们在2升灌注生物反应器中培养细胞时，获得的最大病毒滴度为3×10⁸FFU/毫升。此外，我们扩大规模至20升生物反应器，并在细胞密度和病毒滴度方面获得了类似的结果。我们研发的实验性疫苗符合世界卫生组织对疫苗效力的要求。每次生产可产出约40,000剂有效疫苗。
