Berry J M, Barnabé N, Coombs K M, Butler M
Department of Microbiology and Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
Biotechnol Bioeng. 1999 Jan 5;62(1):12-9. doi: 10.1002/(sici)1097-0290(19990105)62:1<12::aid-bit2>3.0.co;2-g.
Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.
两种呼肠孤病毒毒株在静止培养或微载体培养的Vero细胞中增殖。在T型瓶中单层生长或在Cytodex - 1或Cultispher - G微载体的旋转培养物中生长的Vero细胞可被1型朗株呼肠孤病毒（T1L）和3型迪林株呼肠孤病毒（T3D）感染。在减少培养体积的情况下进行间歇性低速搅拌的方式对于确保微载体培养中的细胞被病毒感染至关重要。在150小时的培养期内，病毒滴度增加了3至4个数量级。在所有培养物中，T3D呼肠孤病毒毒株的滴度比T1L毒株的滴度高（43%）。就任何一种呼肠孤病毒类型而言，T型瓶和Cytodex - 1微载体培养物中的滴度显著高于Cultispher - G培养物中的滴度。微载体培养物中的病毒生产力取决于感染复数（MOI）和感染时的细胞/微珠比例。高MOI（5个空斑形成单位/细胞）和高细胞/微珠负载（Cytodex - 1大于400，Cultispher - G大于1000）的组合导致每个细胞的病毒生产力较低。然而，在低MOI（0.5个空斑形成单位/细胞）时，在两种微载体类型的培养物中，高细胞/微珠负载下每个细胞的病毒生产力显著更高。在Cytodex - 1培养物中，低MOI（0.5个空斑形成单位/细胞）和细胞/微珠负载为1000时获得了最大病毒滴度（8.5×10⁹个空斑形成单位/毫升）。这些培养物中每个细胞的病毒生产力为4000个空斑形成单位/细胞。Cultispher - G微载体培养物中较低的病毒产量归因于被包裹的细胞接受病毒感染的可及性降低。Cytodex - 1培养物中Vero细胞的高病毒生产力表明这是开发呼肠孤病毒科病毒疫苗生产系统的合适体系。
