We were thinking to culture mouse B cells from the spleen in vitro for a week or two. We need them to expand and produce cytokines. I did some research on the protocols, many groups use feeder cells that express CD40L. There are also protocols out there using recombinant CD40L. I would rather use recombinant CD40L rather than feeder cells if possible. But I don't know how good the system is. Would you guys share your experience and/or thoughts on this, pls?
Another question is - will the same protocol produce similar results for B cells of different activation states? -- If I want to culture B cells from immunized mice, is there anything to pay attention to in comparison to culturing naive B cells?
Recombinant CD40L usually gives a stronger activation with trimeric being rhe strongest. Feeder layers are cheaper and more suitable for longer treatments.
Recombinant CD40L usually gives a stronger activation with trimeric being rhe strongest. Feeder layers are cheaper and more suitable for longer treatments.
Hi Yu Qiao ,
I tried this many times and I agrees with Daniel Prieto and Christopher L Pinder .
You need to crosslink the CD40 somehow, therefore you need the CD40L stacked. this can be done by coating recombinant CD40L into wells, by using CD40L-His-tag and add an anti His tage antibody, or by coating with anti-CD40 (did also work well in may hands if used with other mabs such as anti CD49d etc.).
this can work nicely, but it depends on your other conditions.
can you use a high density of the cells in vitro then (do you have many mice and pool the splenocytes)?
is it only short term culture (this would be ideal for the use of the recombinat one and I would suggest that to avoid the very common in vitro artefacts)?
and what cytokine addition do you perform (should be of high quality from eukaryotic cells at best)? how often do you add them (I did always every 3d)?
or by using a cell line. For me iridiated feeder cell lines where always best for proliferation and long term culture. So if they dont harm your experiment, I would recommend to use a cell line. there are several as far as I know and some produce also helpfull other ligands (also integrines are very important to reduce apoptosis of your B cells) and cytokines.
But take care that those feeder cells do not produce cytokines which you want to measure! this might be a problem later with reviewers. :-)
which B cells do you use? If they are naive or only a bit stimulated, they might be very unstable and prone to apoptosis. If they are from the spleen of many times vaccinated mice, and hard memory type, they are more stable and to use recombinant CD40L might be better.
Before answering the two questions it should be noted that some applications for CD40L / CD154 are protected by numerous patents: whether CD40L expressed on transfected or soluble recombinant proteins cells. The most common application is the proliferation of B cells and cloning in order to produce specific antibodies.
As specified above, the shape of CD40L expressed by the feeder cells is the most efficient stimulus for B cells at any stage of maturity, as well as for dendritic cells.
However, the irradiation of these feeder cells remains a delicate point, because the inactivation of these cells is very little reproducible and can induce perverse effects, especially with the use of X-rays. On the other hand, it is relatively difficult to have a cell line that stably expresses CD40L in its functional trimeric form. To compensate for this you can increase the density cell culture, but it is not applicable to produce the antibodies in clonal form.
To answer the question about the state of the B cells, the CD40L has the performance that other activation systems do not have (eg CD20-CD22-CD44-TLR ...): it is active on all maturation stages: naive B cells, BM1, BM2, memory B cells, plasma cells ... However a surprising thing, it is very little active on B cells from spleen when the hyperimmune mouse receives a final boost intravenously three days before sacrifice. Finally there is a new formulation of CD154 (DDX-S2), consisting of cells expressing the trimeric form that have been lyophilized, thus giving great simplicity of use, stability and reproducibility of the results, with a performance allowing to cultivate less one hundred B cells per culture well.
Jean, Can you recommend me to literature that suggests that CD40L has very little active on B cells from spleen when the hyperimmune mouse receives a final boost intravenously three days before sacrifice ?
- Badges
- Science topic