Hello

If you absolutely have to, you can keep the bones in DMEM in the incubator overnight before flushing them and isolating the cells.

BM cells can be painful to grow and maintain in culture (as is the case for most primary cells), even when harvested fresh and directly put in culture. I would not recommend freezing and thawing, as this would make recovery even harder. 

However, it is done in clinical settings (you could maybe look this up).

At any rate, I would make a trial with BM cells from murine bones, and establish best modality of use.

Plasticware will depend on culture purpose and subsequent assays.

Good luck

Jessica 

We use 10 cm petri dishes to culture BMDMs.

You cannot passage them as you would with a transformed cell line (if that's what you mean) as they don't proliferate to that extent. But as Jan above suggests you can replace them between days 6 and 10 for experiments.

You can indeed freeze the bone marrow in 10% DMSO, in my hands I have always gotten the best yields from frozen bone marrow when I have left the red blood cells intact prior to freezing as this acts as a buffer to the progenitor cells you wish to culture. You can lyse the red blood cells subsequently prior to beginning culture. This is probably better than trying to keep bones for longer than a day, though you will have a slightly reduced yield. 

Hope that helps :)

If you can't use fresh cells, yes you can freeze them, although you have to be careful because the DMSO can be toxic.

From my point of view fresh cells respond better. 

Hi Sarika,

Yes you can leave the bones inside culture media at 4 degrees for 1 day, maybe 2. But you will have less macrophages at the end of the differentiation period as some cells will die during the stay inside the fridge.

I prefere to freeze the bone marrow cells. For this you can flush the marrows, spin and ressuspend then in 95% FBS +5 % DMSO. One mouse's marrow (2 femures and 2 tibiae) can be freeze in 6 different cryovials and kept in liquid nitrogen.

Once you finish the differentiation (7 days or 10 days protocol) the cells are ready to be used. These macrophages are well known. These cells can be kept in culture for a long time, however they will not proliferate and they won't be the weel known macrophages anymore. So, I strongly recommend you to use the macrophages as soon as they are differentiated. 

If you need more cells that mice can provide you, consider using a cell line like RAW, J774....

Good luck

I think the best way to keep the bone marrow cells is using right a way. I used 10^e7 cells/well (in 24 well plate) added MCSF for around 5-7 days to develope macrophaegs then stimulated according to the plan. If I did not use then I frozen them 10^e7 cells/ml in either 90% FBS +10% DMSO or Recovery cell cuture freezing medium (both are good). When I developed BMDM from the frozen cells, did not experience difference from the fresh cells. The key is flushing the bone marrow cells in cold enviroment i.e. use ice cold PBS or HBSS to fulsh bone marrow cells and pre-chill the centrufuge etc. 

Good Luck.

Thank you all for your answers. That's a great help.

Jan and Daniel : do you mean after full confluency I can detach them with Trypsin and put some in another petridish to grow for 6 or more days and use rest for experiments?

Hi Sakira,

I have found that keeping bones at 4degC for more than 8 h really decreases the yield of cells. Oddly keeping them for longer at 37 actually makes it better. But teh quicker you do it the better the yield. 

Once flushed we freeze the bone marrow in 10% DMSO in FCS. Make sure to add the DMSO slowly along the side of the tube for best results and not pipette too vigorously after mixing. Transfer to a cryo tube quickly and put in a Mr. Frosty at 80degC.

From fresh BM differentiated with CSF-1 in petri dishes we get about 100 million from 1 mouse (4 bones - 2 femurs and 2 tibias). After freeze down (1 bone per vial) we get 15-20 million from 1 bone/vial.

Use petri dishes if possible as TC culture plastic makes them super adherent and difficult to remove from the plate later. 

Hope this helps.

Hi Sarika,

No that wouldn't work. Firstly scrape the cells rather than using trypsin. And secondly (and more importantly) once you've scraped them, you can re-plate them at desired cell concentrations for experiments but these must be done ideally 24h-48h later. Culturing them for another 6 days won't work: as with most primary cells, these cells cannot be passaged, you only get one go.

Hope that helps

You can keep BM in 4 degree for 24 h.

Freezing them in LN2 is totally fine. After freeze thaw is grow bit slowly for first few days.

You cannot go beyond 10-12 days with primary BMDM culture

Culture them in untreated 100 mm petri dishes (one used for suspension culture)

Thank you all. Few more questions please.

Which is the better source of M-CSF for growing  macrophages? L-929 conditioned medium or recombinant M-CSF? 

Also can we use the same protocol for mouse and rat macrophages ? 

Recombinant M-CSF is pure and expensive, L929 supernatant is "dirty" and cheap, you choose. In order to differentiate rat's macrophages you'll need to use rat M-CSF, or another source of RAT's M-CSF

Thanks Daniel !