Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus
| Autor: | Melissa R. Humphrey, Hong-My Nguyen, Samuel D. Rabkin, Naresh Sah, Dipongkor Saha |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
medicine.medical_treatment
General Chemical Engineering medicine.disease_cause General Biochemistry Genetics and Molecular Biology Article Cancer immunotherapy In vivo Neoplasms Chlorocebus aethiops medicine Animals Vero Cells Cytopathic effect Virus quantification Oncolytic Virotherapy General Immunology and Microbiology Chemistry General Neuroscience Herpes Simplex Virology Oncolytic virus Oncolytic Viruses Herpes simplex virus Cancer cell Vero cell Immunotherapy |
| Zdroj: | J Vis Exp |
| ISSN: | 1940-087X |
| Popis: | Oncolytic viruses (OVs), such as the oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer immunotherapy. OVs, including oHSV, selectively replicate in and kill cancer cells (sparing healthy/normal cells) while inducing anti-tumor immunity. Because of these unique properties, oHSV-based treatment strategies are being increasingly used for the treatment of cancer, preclinically and clinically, including FDA-approved talimogene laherparevec (T-Vec). Growth, purification, and titration are three essential laboratory techniques for any OVs, including oHSVs, before they can be utilized for experimental studies. This paper describes a simple step-by-step method to amplify oHSV in Vero cells. As oHSVs multiply, they produce a cytopathic effect (CPE) in Vero cells. Once 90-100% of the infected cells show a CPE, they are gently harvested, treated with benzonase and magnesium chloride (MgCl2), filtered, and subjected to purification using the sucrose-gradient method. Following purification, the number of infectious oHSV (designated as plaque-forming units or PFUs) is determined by a "plaque assay" in Vero cells. The protocol described herein can be used to prepare high-titer oHSV stock for in vitro studies in cell culture and in vivo animal experiments. |
| Databáze: | OpenAIRE |
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