Harvesting Recombinant Aedes aegypti Densovirus from Infected Cells

Begin with a culture flask containing a culture of mosquito-derived epithelial cells infected with recombinant Aedes aegypti densovirus.

Using a glass dropper, pipette the media up and down to dislodge the cells from the flask surface.

Transfer the cell suspension into a tube.

Incubate at an ultra-low temperature to freeze the cells.

Thaw the cells and vortex them.

This results in lysis of the cells and the release of intracellular contents, including the viral particles.

Repeat the cycle of freezing and thawing multiple times to ensure complete lysis of all cells.

Centrifuge the lysate under cold conditions to pellet the cellular debris.

Collect the supernatant containing virus particles.

Pass it through a syringe filter to remove any residual contaminants.

Aliquot the filtrate containing virus particles and store it at an ultra-low temperature until further use.

Five days after transfection, use a five-milliliter glass dropper to dislodge and suspend the cells in the dishes by pipetting up and down with the culture medium.

Transfer the cell suspensions from each flask into their own sterile 50-milliliter tube.

To harvest high-titer recombinant virus, it is necessary to maintain the cells for five days after transfection.

Freeze the individual tubes at negative 80 degrees Celsius or in a dry ice-ethanol bath for 30 minutes. Then, quick thaw them at 37 degrees Celsius, and vortex the cell suspension for one minute.

Next, collect the supernatant from the centrifuged sample and discard the pellet. Then, pass the supernatant through a 0.22 micrometer disposable syringe filter. Aliquot and store the final purified virion stocks at negative 80 degrees Celsius.