培养基中硫酸葡聚糖对水疱性口炎病毒滴定的影响。

Impact of dextran sulfate in culture media on titration of vesicular stomatitis virus.

机构信息

Department of Bioprocess Engineering, National Research Council Canada, Building Montreal, Montréal, Canada.

Department of Bioprocess Engineering, National Research Council Canada, Building Montreal, Montréal, Canada; Department of Bioengineering, McGill University, Montréal, Canada.

出版信息

J Virol Methods. 2020 Jan;275:113758. doi: 10.1016/j.jviromet.2019.113758. Epub 2019 Oct 31.

DOI:10.1016/j.jviromet.2019.113758

PMID:31678047

Abstract

Viral vectors derived from vesicular stomatitis virus (VSV) are important vectors for the development of vaccines and for the treatment of cancer. The efficiency of therapy based on VSV is dependent on the dose of virus used. Therefore it is essential to measure accurately and reproducibly the amount of functional vectors in the samples to be tested. Two common methods used to measure the titer of VSV are TCID and plaque assay. In the current study, we compared these two titration methods by using a recombinant VSV expressing the green fluorescent protein (VSV-GFP) as a model virus. Some culture media developed for suspension mammalian cells contain dextran sulfate. We observed that plaque assay, but not TCID, can underestimate the virus titer up to 10 fold when VSV-GFP was produced in culture media containing dextran sulfate. Dextran sulfate is commonly used in serum-free culture media to reduce cell aggregation in suspension culture. The inhibitory effect of dextran sulfate on the titration of VSV-GFP was confirmed by supplementing the culture medium with this compound during virus production. Our results also demonstrated that extending the incubation time during plaque assay and TCID increases virus titer.

摘要

源自水疱性口炎病毒（VSV）的病毒载体是疫苗开发和癌症治疗的重要载体。基于 VSV 的治疗效率取决于使用的病毒剂量。因此，准确且可重复地测量待测试样品中功能性载体的数量至关重要。测量 VSV 效价的两种常用方法是 TCID 和噬斑测定。在本研究中，我们使用表达绿色荧光蛋白（VSV-GFP）的重组 VSV 作为模型病毒比较了这两种滴定方法。一些为悬浮哺乳动物细胞开发的培养基含有硫酸葡聚糖。我们观察到，当在含有硫酸葡聚糖的培养基中生产 VSV-GFP 时，噬斑测定而不是 TCID 会低估病毒效价多达 10 倍。硫酸葡聚糖通常用于无血清培养基中，以减少悬浮培养中的细胞聚集。通过在病毒生产过程中向培养基中添加该化合物，证实了硫酸葡聚糖对 VSV-GFP 滴定的抑制作用。我们的结果还表明，延长噬斑测定和 TCID 的孵育时间会增加病毒效价。