Hello all,
I'm currently working with PBMCs which had been processed from human blood samples and preserved in freezing media (20% DMSO in RPMI 1640 media [plus FBS, pen/strep, and l-glut]) and stored in a liquid nitrogen cryofreezer. This work was done a couple of years ago by a former post-doc and not too effectively at that.
I've been attempting to thaw one patient's vial of cells and perform proliferation assays by first separating out monocytes for differentiation to dendritic cells. However, the number of starting cells immediately post-thaw is barely 0.5x10^6. I need to wash once, preferably twice, before I can even hope to select, and despite being as painstakingly careful as possible when aspirating off media by pipet, I still lose enough cells that I'm down to about 0.2x10^6; and after monocyte selection, I'm scarcely at 25,000 total--just enough for ONE replicate of dendritic cell differentiation and later proliferation with the next, and last, patient vial's 0.2x10^6 cells.
Is there any way I can retain more of my starting cells? We're spinning at 1200rpm for 10-15 minutes at 4C between washes. A better way to siphon off media? A better way to trap the pellet?
These samples are really precious and I dare not attempt another go with another patient's sample pair unless I'm sure I can retain more cells to make the assay worthwhile. Thanks in advance!
Dear Nick,
that sounds like a tough task. I have only heard people using frozen huPBMCs for FACS marker analysis later on because with these procedures you loose so many cells already.
Centrifugation should be fine, you could reduce it to 5 mins, to reduce the time cells undergo the whole process.
Further, when taking off the supernatant, do not be too exact and rather leave a bit volume in, you have outdiluted the DSMO anyway already and (I assume you CD14 MACS bead sort monocytes) then you resuspend the monocytes in your separation buffer.
I understand that these samples are precious, you could use normal blood, threeze the same amount and just get the technique up and running before you use the patients samples.
And at last, to be honest, if it doesn't work well, you should rethink if you can exploit these samples in a different way rather wasting half of it and then having n numbers which won't tell you any significant differences. Sorry to be a bit negative, but working with human samples is tricky and painful at times...
Maybe someone else has some better ideas.
Best of luck,
Axel
Dear Nick,
Try spinning at 1500rpm for 15-20 minutes at 4C between washes in tubes with conical bottom. I am sure this should resolve your problems.
best of luck.
hmm, another question. Do you "loose" the cells or are they dead? Not sure what you trypan blue count ratio is.
hmm, another question. Do you "loose" the cells or are they dead? Not sure what you trypan blue count ratio is.
@Axel, we actually have been doing an adherence step for monocyte selection (leave in RPMI at 37C for 2h, then wash off the non-adherent cells). We do have access to a MACs, but as the beads are so expensive and we have a large number of patients to get through usually, we only use it for a few highly select patients. In any case, I had worked out this protocol using the voluminous amounts of cells we regularly pull from members of the lab, but that means I typically start out with 10+ times the # cells per vial of those folks than our patients; so, unfortunately, not indicative of the amounts I have been getting once I started with our real samples.
I will try leaving a bit more media, which I agree, should help some. Was worried about leaving any trace of DMSO. Speaking of, the dead cells tend to constitute between 25-50% of the cells post thaw. Ergo, I've been rapidly thawing and transferring to RPMI as quickly as my hands can fly. None of the lab folk samples I process and freeze have ever come out more than 10% dead post-thaw (and it's rare I get even that many). The precious samples were most likely left at RT by the post-doc who processed them, so a good washing is essential.
@Mohd, thanks for the suggestion. I was worried to spinning at too high a speed, but can try some test cells and see if that substantially increases number of damaged cells.
hm, ok, I see the price issue, but maybe worthwhile calculating that, because of the low cell numbers you won't need so many beads... Not sure what the recovery with small cell numbers is though (maybe worthwhile contacting a rep?).
Another idea to get every CD14+cell would be cell sorting if you have access to such a facility, however, the procedure is long and might affect cell viability (you could also do dead cell exclusion with 7-AAD), just another brainstorming suggestion.
I agree, starting with more cells makes live much easier...
tough project man. Sorry that I can't help more :(
@Axel, we're going to use the CD14 beads for another project coming up, so maybe I can convince others to perform it in such a way as to mimic my own conditions and see how many I could expect to pull out. Nevertheless, thanks for the suggestions. Always appreciated.
Your welcome, Good luck, fingers crossed and keep us uptodate with progress, so we all learn from that ;-)
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