Hi,
I used to work with T cells which I was told are much more resistant to cryopreservation. B cells on the other hand are apparently more sensitive and the viability after thawing is poor, particularly for CLL samples.
The frozen samples are obtained from collaborators so I do not know how they were frozen and I will not have control over that.
What I plan to do is aliquot 1mL of pre-warmed media into a Falcon tube. Thaw the cells in 37C quickly then transfer them into the media. Top up until 10mL and centrifuge at 1200rpm, 5 minutes, RT. Resuspend in 1mL, top up media to 10mL, repeat wash, transfer to T25 and incubate.
I also want to prevent as much aggregates/debris in the samples as I would need to use them for FISH experiments. If anyone has extensive experience working with B cells, could you tell me if slow/fast addition of media works better and what protocols you may have to achieve the best recovery. What is the % viability you would normally get from your methods?
Hello Darellynn!
Although I do not have much experience culturing B cells I can say that just eliminating the centrifuging step could improve the viability. You can change the media may be a few hours later. And fast addition of media is always good, as freezing should be slow and thawing fast.
The reason I am asking is because there are very few protocols that alter the thawing procedures for different cell lines. For neutrophils, I have read that HBSS is preferred over media as they are sensitive and prone to activation.
Protocols that I have found online for B cells tend to involve slow thawing. However, they include additional steps to prevent aggregation. I would like to ensure I have high recovery at the same time low aggregation.
I have done both slow and fast before depending on the cell lines/researcher I worked for.
Links here:
http://www.zen-bio.com/pdf/Human-CD19B-Cell-Manual.pdf
https://cdn.stemcell.com/media/files/pis/29273-PIS_1_0_0.pdf
Article Optimal Thawing of Cryopreserved Peripheral Blood Mononuclea...
This is an interesting topic. Waiting for some more inputs from other researchers. Good luck!
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