PBMC resting after thawing

[Guest Jason]

Hi all,

I have a question about the thawing of cryopreserved PBMCs. According to the previous discussion of the forum, people suggest to rest the PBMCs for 1 hr or more after thawing and then plate the PBMCs in ELISPOT plate. In my case, I found there were some attached cells after the resting. Should I remove all the attached cells by Trypsin-EDTA or just leave them?

Thank you!

Jason 

 

[Christian@mabtech.com]

Dear Jason,

At Mabtech we do rest our cells for 1h in the incubator following thawing of cryopreserved PBMCs. Cells destined to die fall down to the bottom and you can continue seeding your ELISpot plate with cells that have a higher proportions of cells that will make it through the entire incubation. 

The dead cells that fall to the bottom of the tube often leak out DNA, that get tangled up with other cells and you get clumps. These should be removed in my opinion. Especially since you need single cells to go into the ELISpot plate, and not clumps of cells, because then you will get confluent blobs of spots. 

One thing that I have heard about is that you should "wash" the remaining clumps with new cell culture medium after you sucked off the "good supernatant". The thinking is that living cells have gotten "tangled" up in the mesh of dead cells, apoptotic cells and DNA strands. By adding fresh medium to the clumps, resuspending up an down a couple of times with a pipette, you can dissloge good cells for downstream use. Worth a try.

Using Trypsin-EDTA for the purpose of removing clumps I have personal experience with and not heard about. I have on the other hand been given recommendation to use DNAse in order to brake up the DNA strands from dead cells. This should inhibit clump formation. I have not tried it myself but sounds reasonable. 

  

[Guest Jason]

35 minutes ago, Guest Jason said:

 

Hi Chris,

Thank you very much for your quick reply!

Sorry for the confusing.  I plated the PBMCs in T25 flasks when resting and I could see some of cells attached to the bottom of flask when I removed the cell suspension. That's why I would like to recover those cells by trypsin-EDTA.  

It seems that you left the PBMCs in the conical tube during the resting? How did you separate the live and dead PBMCs? There must be some live PBMCs go down to the bottom of the tube after 1 hr of resting, right?

 

Thank you!

Jason 

 

 

[Christian@mabtech.com]

Ah, ok, you had the PBMC in a T25 flask! that is setup I have no experience with. Maybe then trypsin-EDTA is a better option, I do not know.

At Mabtech we use 15ml conical tubes for thawing the PBMC, yes. After resting, we gently stir the 15ml tube so that all clumps start spinning around and then let them fall to the bottom of the tube during 3-5 min. We then transfer the cell suspension without clumps using a normal pipette to an empty new 15ml tube. 

The remaining clumps and small amount of cell culture medium are then boosted with 2ml of fresh cell culture medium and I pipette the clumps up and down during like 30-45 seconds. I then wait for the clumps to fall to the bottom of the tube, and I again transfer the supernatant. In this way, you can "recover" viable cells that have "attached" to the DNA strands of the dead cells in the clumps. At least that is the theory.