How can I improve viability when thawing PBMCs with 20% DMSO? Is there a protocol that can separate the dead cells from the viable ones?
Dear Prof. Alessandra Peres
The freezing medium for PBMCs is 90% FBS + 10% DMSO.
A1: You may improve the viability when thawing PBMCs by following these steps during thawing.
1. You should follow a quick thaw process. Hold the cryovial on the surface of the water bath at 37 deg C and occasionally flick it gently during thawing. Do not leave the cryovial unattended during the thawing process because thawing takes only a minute or two.
2. You may add warm complete RPMI medium drop-by-drop into the cryovial containing the cell suspension, slowly over a 40-second period. The final volume should be around twice the volume of the cell suspension (for instance, add about 1 ml of complete RPMI medium to a cryovial containing 1 ml of cell suspension). Be careful not to exceed the capacity of the cryovial.
3. Transfer the diluted cell suspension into a 15ml centrifuge tube containing 8ml of warm complete RPMI media. For cells preserved in a freezing medium containing DMSO, it is recommended to wash out or dilute the DMSO immediately post-thaw because DMSO harms the cells at higher concentration.
4. Centrifuge the cell suspension at low speed 250g for 7 minutes to prevent cell damage.
5. Decant the supernatant and tap the cell pellet.
6. Resuspend the cells in a desired volume of warm complete RPMI medium and take the cell count.
A2: You may use density gradient centrifugation to separate dead cells from viable ones. In density gradient centrifugation depending on the density of the particles in the sample, similar substances will group together when exposed to rotational force. Because dead cells and cellular debris are fractured, they become less dense than living, healthy cells. Adding in certain separation reagents such as Ficoll can purify the sample by acting as a barrier that only one population can pass through.
For instance, in a 50 mL centrifuge tube, you may layer 18ml of your cell suspension onto 12ml of a Ficoll-paque combination. Centrifuge your tube for 15 minutes at 400x g. After centrifugation, you will note that the live cells will collect at the interface and the dead ones will form a pellet at the bottom of the tube.
There is another simple method which would include centrifuging the cell suspension at 150-200g for 10 mins. You may discard the supernatant which consists of cell debris and resuspend the cell pellet in fresh medium.
Regards,
Malcolm Nobre
Hi Malcolm, Thank you very much for your reply and help, I'll try it out. Best Regards, Alessandra
Hello Alessandra,
I also use a 10% DMSO/90% FCS freezing solution, to some extent the quality of the thaw correlates with that of the freezing process. I find that a cooling rate of ~-1˚C/min in a (Mr Frosty) or CoolCell in a -80˚C freezer, before transfer to liquid nitrogen for long term storage is best for sensitive cells.
Thawing very similar to Malcolm's protocol
1. allow appropriate culture medium to warm to room temperature.
2. thaw cells in 37˚C waterbath, with gentle mixing, until a only a single small ice crystal remaining.
3. immediately transfer cells to 50ml polypropylene conical tube.
4. add 1ml complete culture medium dropwise - mix by swirling, leave for about 30 seconds, to allow DMSO to diffuse.
5. add 2ml complete culture medium dropwise - mix by swirling, leave for about 30 seconds.
6. repeat adding doubling volumes until 31ml complete culture medium added.
7. centrifuge 7 minutes at 400g.
8. resuspend pellet in 30ml complete culture medium.
9. repeat centrifugation.
10. resuspend cells at desired density for use.
Malcolm has already pointed out not the importance of NOT leaving the cryovials unattended during the Thawing process.
Best wishes
Both protocols are excellent. A few comments:
1) Freezing in FBS can be challenging for subsequent studies as the foreign antigens can impact T-cell assays, as well as others. One commercial approach is to use CryoStor 10% or 5% DMSO. This is a freezing medium that can meet GMP requirements. Another solution that can be used with DMSO is Plasma-Lyte A. This is used in combination with the addition of 20% vol:vol of 25% human serum albumin.
2) Given that PBMCs frequently are contaminated with PMNs that lysis on freeze-thaw it can be useful to add DNAse to the cryoprotectant. Final concentration of 5ug/ml for recombinant Dormnse alfa.
3) On review of our vial thawing protocols I note that we spray the vials liberally with 70% EtOH. We include this as part of our protocol. I note that we always refill the water bath with sterile water prior to thawing.
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