We have in the past differentiated C57BL/6 bone marrow cells to dendritic cells using standard techniques (Current Protocols in Immunology), namely GMCSF without IL4. My question is about the best way to maintain the cultures if we need to postpone the experiment we were planning to use them for. If we continue to supply GMCSF beyond a week, preferably out to 10 or 11 days, will the phenotype be stable?
Any suggestions?
Thanks,
Ed Roy
University of Illinois at Urbana-Champaign
Extending the GM-CSF DC cultures to 10 or 11 days is indeed possible. However, you have to take care that you feed the cultures frequently with enough fresh medium and GM-CSF. Be careful to avoid too much acidification of the cultures. Phenotypically the resulting DCs will be more mature (e.g. higher expression of CD40, CD86).
Extending the GM-CSF DC cultures to 10 or 11 days is indeed possible. However, you have to take care that you feed the cultures frequently with enough fresh medium and GM-CSF. Be careful to avoid too much acidification of the cultures. Phenotypically the resulting DCs will be more mature (e.g. higher expression of CD40, CD86).
Perhaps this reference would help:
Winzler C., et al, 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. PMID: 9016880
In addition to the suggested paper above there is also a study from Manfred Lutz, 1999;223:77-92, J of Immunol Methods, which is of interest to you. I used to differentiate DCs from bone marrow by using medium which contained 30% conditioned medium from a cell line that produces GM-CSF, the "R1 cells". There is also a suspension cell line format of this but I've never used that. I did not add any recombinant IL4 and cultured the BMDC's for up to 14 days and they remained relatively "immature" (as far as you are allowed to say that they are really immature in an in vitro system). Make sure that you use serum with the lowest possible endotoxin levels. Testing a couple of serum batches can be really worthwhile for this type of cultures. I would be careful with bluntly removing all medium and to replace it with fresh medium. Rather replace part of the volume (like half) with fresh medium for preventing medium acidification and keeping the nutrients and GM-CSF on a good level. It can be stressfull for the cells when their external environment is suddenly replaced. They will quite abruptly be confronted with growth stimulants, etc. which were partially exhausted in the old medium. Furthermore, the cultures will develop islets which contain a lot of DCs. Disprupting these during culture is not advisible since such mechanical forces can activate DCs. So, try to exchange medium as gently as possible via the side of the dish. Expect that the BMDCs probably get an activation stimulus just by the harvesting procedure (in my case this was just a harsh rinse of the plate) so you might try to harvest and seed the BMDCs at least two days before you do your experiment.
If you need to postpone the experiments, the best thing to do is to start over. In our hands, after the 5th day, the antigen processing/presentation ability of these DCs start a continuous decline despite an apparently stable phenotype. In my opinion is easier and cheaper to start a new culture.
It is also possible to harvest the DCs at the usual time and cryopreserve them. This is certainly not ideal and you will obviously need to show that the cryopreserved and recovered DCs perform the function that you desire using appropriate controls. However, I would still personally use this approach rather than extending the culture time.
I think it's also possible to freeze bone-marrow cells just after recovery to postpone their differenciation in vitro, however I agree with Jose that it's better to start over if you can.
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