Hello, everybody,
I'm interested in culturing monocytes isolated from human peripheral blood. I would like to know if it is as correct to use cryopreserved monocytes or if it is better not to freeze them previously and seed them right after isolating them. Do they respond differently to stimuli?
Thanks in advance,
Elena
Ideally, you should always culture immediately after blood collection and purification. It's not so much a difference in response but freezing cells and reveving primary cells always leads to a lot of cell death. So the state of the cells after cryofreezing versus culturing them immediately after collection will be different.
For all kind of cells (including monocytes) fresh material should be preferred over frozen samples if possible, as you significantly reduce the amount of viable cells. If this is not avoidable, cryopreservation of monocytes is not inferior compared to other blood cells.
If you have the option of using freshly isolated cells without prior freezing it would be better definitely. Freezing usually affect cell viability and immune response, but still working if you don't have fresh cells.
For monocyte cultures previously frozen cells do not work well from my experience. First, monocytes numbers in PBMC are relatively small (around 6%), cell death is high, and DMSO itself nonspecifically activates cells.
Whether it is ok to cryopreserve monocytes or not, it depends on your downstream goals. If you want to see monocyte activation, inflammasomes, using fresh monocyte would be the appropriate. If you want to differentiate monocytes to macrophages or DCs, cryopreservation is not any problem. I have been using cryopreserved monocytes for macrophage differentiation over years. Never had any issue.
To Abdullah: could you please share how do you cryopreserve monocytes and culture them for differentiation to macrophages afterwards? Do you cryopreserve PBMCs and isolate monocytes later or isolate monocyte and cryopreserve them? What ratio do you use for plating? I've been trying a lot without success. Even 1:30 ratio of plating after cryopreservation does not really work- too many cells are dead. I use standard cryopreservation: FBS+10% DMSO.
Natallia Mikhalkevich I sort monocytes using CD14 MACS beads and then freeze them in Liq N2.
Composition of Freezing media: 500ul RPMI (containing 10% FBS)+ 350ul FBS + 150ul DMSO. Obviously, I use Mr Frosty while freezing. They are good over years. When needed, I thaw them in the waterbath, quickly transfer the cell suspension into 9ml of RPMI+10% FBS to wash. And seed them for macrophage differentiation using GM-CSF.
Thank you very much for your response. 15% DMSO in 40% FBS medium looks unusual for good monocytes viability after freezing, but worth trying.Thanks a lot again.
Hello again,
I want to show fresh monocytes after 7 days of culture in 20% autologous serum.
What do you think?
Thanks a lot
Elena
For me look like a little bit too low numbers for the surface area ( I usually plate 1:10 = expected monocytes number: PBMC number and have higher confluency at 7d with M-CSF in the medium, so cells are closer to each other, look more differentiated- but it depends a lot on the area of the well- I usually have more cells on the edges and in the middle of the well for some reason, and less numbers in between), but in overall look good with very few floating (usually residual lymphocytes), occasional mesenchymal stem cell - one with long "tail", and almost no dead.
Thank you Natallia Mikhalkevich !! Yes, it was a trial and I agree that numbers are low. Thanks for your advice. It happened also the same, more cells in the middle and edges.
Elena Guillén Brighter cells are live cells, faded cells are dead cells. Irregular shape is a characteristic of macrophages.