Hi everyone!

Now that our facility has been running for a couple of years, we are starting to have large numbers of virus stocks.  I am wondering how others are dealing with long-term storage of viruses?  I know Sabine is using BIIC's.    We had a few BIIC preparations that gave very low yields after thawing (though I cannot rule out that we messed something up) and therefore have not adopted it as standard yet.  What are other people's experiences and how are you storing your viruses?  THanks!

best,

Peggy

Hi Peggy,

My previous lab (Berger lab@EMBL-Grenoble) are using BIIC routinely for long-term virus storage and it works fine. Below is our protocol (based on the MultiBac tutorial of Berger lab):

Freezing BIIC (50mL of culture for 5 aliquots of 1mL of BIIC):

1.    Infect a 50 mL culture of Sf21 cells (0.7 - 1.0 x 10⁶ cells/mL) with V1 virus

2.    Maintain the culture at a denity of 0.7-1.0 x 10⁶ cells/mL until date of proliferation arrest (dpa)

3.    Pellet the cells by centrifuging at 100 g for 10 minutes

4.    During centrifugation, prepare a cryoprotectant solution (5 mL) as follows:

Dissolve 0.05 g BSA powder in 4.5 mL media and filter sterilize the solution

Add 0.5 mL sterile DMSO to the solution under a sterile hood                                                

(Final concentrations: 90% media, 10% DMSO, 10 g/L BSA)  

5.    After centrifugation, remove the supernatant and resuspend cell pellet with the cryoprotectant solution (final density: 0.7-1.0 x 10⁷ cells/mL)

6.    Prepare five 1 mL aliquots and store them in a -20 degree freezer for 1 hour

7.    Transfer the aliquots into a -80 degree freezer and store them for 24-48 hours

8.    Transfer the aliquots in liquid nitroge for long-term storage

Test the BIIC stocks by infecting 0.4-1.0 L Sf21 culture (0.7 - 1.0 x 10⁶ cells/mL) with one aliquot of BIIC. You can scale up the BIIC preparation if desired.

Some parameters might need to be adjusted according to the setting of your insect cell expression facility. Further comments/ideas are very welcomed.

Cheers and Happy Expression!

Yan

Dear Yan,

Thanks!  We were using a similar protocol but we didn't add BSA to the cryoprotectant, we will give it a try again.

best,

peggy