To amplify an adenovirus, you typically infect a permissive cell line, like HEK 293 cells, with the adenovirus at a suitable multiplicity of infection (MOI), allowing the virus to replicate within the cells until significant cytopathic effects (CPE) are observed, then harvest the infected cells and collect the viral particles from the cell lysate, which can be further purified and stored for future use; essentially “growing” the virus within the host cells to increase its quantity.
Key steps in adenovirus amplification:
- Cell preparation: Grow a monolayer of susceptible cells (usually HEK 293) in a suitable culture flask until they reach near confluency.
- Infection with adenovirus: Add the desired amount of adenovirus stock to the cell culture, ensuring an appropriate MOI to achieve efficient infection without overwhelming the cells.
- Incubation and monitoring: Incubate the infected cells at the optimal temperature (usually 37°C) and monitor for CPE, which indicates successful viral replication.
- Harvesting infected cells: Once significant CPE is observed, collect the infected cells and the cell culture supernatant by detaching them from the plate.
- Cell lysis: Lyse the harvested cells using a freeze-thaw cycle or other methods to release the viral particles.
- Clarification: Centrifuge the cell lysate to remove cell debris and obtain a clarified supernatant containing the amplified adenovirus.
- Purification (optional): For high-quality viral preparations, further purification steps like CsCl gradient centrifugation can be employed.
Important considerations:
- Cell line selection: HEK 293 cells are the most commonly used cell line for adenovirus amplification due to their high susceptibility to adenovirus infection.
- MOI optimization: The MOI should be carefully chosen to balance efficient infection with minimizing cell damage.
- Viral titer determination: It is crucial to determine the viral titer of the amplified adenovirus stock using a plaque assay or other methods to accurately quantify the viral concentration.
- Storage conditions: Adenovirus stocks can be stored at low temperatures (-80°C) with the addition of a cryoprotectant like glycerol to maintain viability.