Why is there low yield adenovirus production with HEK293A cells?

04 August 2015 8 1K Report

I got some problem with producing CRE adenovirus. I bought this virus from company. When I tried to amplify it with 293A cells (MOI=10), I observed the Cytopathic effect (CPE). But this came very quickly, usually within 24h and all cells detached within 36h. The yield is very very low.

I read some protocols saying the CPE usually appears 4 days after infection. So is this means my 293A cells are not good (I use a very low passage, P4~P7)? Or is there any tricks to improve the adenovirus production? Thank you.

Leonard M Anderson

Hi Xue, it sounds like you added too much virus to the 293A cells and the virus did not have time to amplify enough in the cells before they lysed. If the media turns yellow before harvesting (if using phenol red media) your yield will be extremely low as well. Adenovirus amplification assays should be allowed incubate in the cells for at least 3 days before you start to see a significant CPE effect on the cells. The cells should be harvested once you observe ~70-80% CPE in the plate. You also might want to consider using less virus (maybe a dilution series), if you do not know the original titer of the virus. After harvesting, you should perform a titer assay to determine Adenovirus titer for MOI calculations for cell biology and in vivo experiments.

Xue Yuan

Thank you so much, Leonard. I will try to use less virus for the next transduction. I brought the virus stock from company, so I know the PFU. I read some protocol says using MOI=10. What is your suggestion?

My medium did not change to yellow. So I don't think pH is a matter here. When you say 70-80% CPE, do you mean 70-80% cells change to round shape or detach? Thanks again.

Panduranga Rao

Hi Xue,

If you are trying to make adenovirus stock, go with very low MOI (0.001to 0.01). The titer provided by the supplier might have gone down after a freeze thaw.

E1 deleted adenoviruses can replicate in 293 and when you are making your stocks use very low MOI's . People use high MOI to test protein expression in cell lines other than 293. In Non 293 cells virus cannot replicate and to infect all the cells at once high MOI is used and protein expression is analyzed 24-48hs after infection.

Xue Yuan

Thank you, Panduranga. Actually, I read the paper talking about low MOI infection yesterday. I plan to try it. However, the two paper I found is using suspension 293 cells (293s). So I am not sure what is the proper starting density for low MOI infection. Would you mind sharing your detail protocol to generate adenovirus stock with low MOI infection? Thank you very much.

Panduranga Rao

Use three MOI as per your stock (0.1, 0.01, 0.001).

Calculate quantity of virus required (number of cells /(virus titer*MOI)) for a given plate/flask.

Dilute the virus in required volume of media sufficient to cover the cells. Add media to cells, incubate with intermittent shaking (careful about uneven incubator racks if so some areas of cells may get dried and die) for an hour.

Add 0.1-0.2 ml of media per square centimeter.

Incubate and observe for CPE for 3-5 days

Select one MOI that gives you CPE around 4-5 days.

Abdelmalik Ibrahim Khalafalla

Hi Xue,

I am interested to know if you succeeded in otaining a high virus titer after reducing the MOI. An explanation to a low virus yield when usiing a high MOI is that defective intereferering virus particle might be produced that are not infectious due to truncation in their genome. Kindly let us know what happened to your virus.

Thanks

Maria Leonor Fernandez Murga

Dear Leonrad,

I am working with a Adenovirus and I have a question, because you commented if the medium is yelow the yield will be extremely low . Can you explain it? Please

Thanks!

Leonor

Yu Wa

Dear Esteemed Colleague,

Greetings. I hope this message finds you well and deeply engaged in your vital research involving adenovirus production using HEK293A cells. Your concern regarding low yield in adenovirus production is a significant one, as it impacts the efficiency and feasibility of viral vector-based experiments and therapies. Below, I address potential reasons for this phenomenon and suggest strategies for optimization.

Factors Influencing Adenovirus Production Yield in HEK293A Cells

Cell Health and Culture Conditions:The physiological state of HEK293A cells is crucial for optimal virus production. Cells should be in a robust growth phase, adequately nourished, and maintained at optimal confluence (typically 70-80%) at the time of transfection or infection. Suboptimal culture conditions, such as incorrect media pH, inadequate nutrient supply, or overconfluency, can significantly reduce viral production.

Transfection Efficiency:In scenarios where adenoviruses are produced by transfecting HEK293A cells with viral DNA, the efficiency of the transfection process is paramount. Low transfection efficiency can lead to insufficient viral replication machinery inside the cells. Optimization of the transfection protocol, including the choice of transfection reagent and DNA quantity, is essential.

Quality of Viral DNA:The purity and integrity of the adenoviral DNA used for transfection directly impact the success of viral production. High-quality, endotoxin-free plasmid DNA is recommended.

Cell Line Adaptation:HEK293A cells, like any cell line, may undergo genetic drift or adaptational changes over time and passages, which can affect their competence in viral production. Using low-passage cells and regularly refreshing cell stocks can help maintain production efficiency.

Viral Construct Design:The design of the adenoviral vector, including its size and the presence of any toxic genes, can influence the replication and packaging efficiency within HEK293A cells.

Strategies to Improve Adenovirus Production Yield

Optimize Cell Culture Practices:Monitor and adjust culture conditions to ensure cells are healthy and at the correct density before initiating virus production. Regularly check media pH and nutrient levels.

Enhance Transfection Methods:Explore different transfection agents and optimize the DNA-to-reagent ratio. Consider pulse electroporation as an alternative for higher transfection efficiency.

Use High-Quality Starting Material:Ensure the viral backbone plasmid is of high quality, with minimal contaminants that could impact cell health or transfection efficiency.

Adaptation and Selection of High-Producing Clones:Consider adapting HEK293A cells to select high-producing clones or utilizing other derivative lines of HEK293 that might offer higher viral production yields, such as HEK293T or HEK293FT.

Harvesting Time Optimization:Optimize the time of harvest post-transfection or infection, as premature or delayed harvesting can lead to lower yields. Typically, adenovirus is harvested 48-72 hours post-transfection, but this can vary.

Conclusion

Low yield in adenovirus production with HEK293A cells can result from a range of factors related to cell health, transfection efficiency, the quality of starting materials, and the viral construct design. By systematically addressing these aspects and optimizing protocols, you can significantly enhance adenovirus production efficiency.

Should you require further assistance or wish to discuss additional troubleshooting strategies, please do not hesitate to reach out. I am here to support your research endeavors and contribute to the success of your projects.

Warm regards.

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