Propagation and Harvesting of Oncolytic Herpes Simplex Virus in Mammalian Cells

Take a culture of mammalian cells in a growth medium. Remove the medium and wash with a buffer.
Add the oncolytic herpes simplex virus suspension and agitate for even distribution.
During incubation, the viral glycoproteins bind to the host receptors, enabling adsorption.
Remove the unadsorbed viruses and add the medium.
Upon incubation, the viral envelope fuses with the host membrane, releasing the nucleocapsid into the cytoplasm.
The nucleocapsid delivers the viral genome into the nucleus, where the host machinery is hijacked to produce progeny viruses.
The rising viral load causes structural damage to the cells.
Collect the conditioned medium and scrape to detach the cells. Add the conditioned medium, transfer them to a tube, and spin down the cells.
Discard the supernatant. Resuspend in a buffer and the conditioned medium to maintain virus stability.
Snap-freeze the suspension and store at ultra-low temperatures to preserve the virus structure.

Begin by washing the T-150 square centimeter flasks with two times high glucose DPBS supplemented with 1% IFCS. Aspirate the DPBS and add seven milliliters of virus inoculum per flask.

Gently rock the flasks for five minutes and incubate at 37 degrees Celsius for 1.5 to 2 hours. Then, remove the inoculum and add 25 milliliters of DMEM supplemented with 1% IFCS to each flask. After incubation at 37 degrees Celsius and 5% carbon dioxide for two to four days, collect 20 milliliters of the culture supernatant from each flask into 50-milliliter centrifuge tubes.

Using a cell scraper, scrape the cells from the bottom of the flasks. Add approximately 15 milliliters of the previously collected culture supernatant to each flask to bring the volume up to 20 milliliters, then use a 10-milliliter sterile serological pipette to gently wash the bottom of the flasks a few times. Collect the cells with medium into 50 milliliter conical centrifuge tubes placed on ice.

Centrifuge the tubes at 300 times g for 10 minutes at four degrees Celsius. Resuspend each pellet thoroughly in 1.25 milliliters of virus buffer, or VB, and 1.25 milliliters of previously collected culture supernatant. Mix all resuspended pellets into one 50 milliliter conical centrifuge tube. Snap-freeze the resuspended cells using dry ice and 100% ethanol and store at minus 80 degrees Celsius.