Due to technical issues, we had to leave spleens overnight in PBS at 4C (fridge) and isolate cells the next morning for FACS analysis of CD4CD25FoxP3 - we took 1 freshly removed spleen as a comparison - there was almost no difference between fresh and stored spleen samples in terms of staining and numbers of cells. Fantastic information

It will probably work (some cells always survive), but I wouldn't do it. The immunologists next door in our lab usually do night shifts, when they do experiments with mice. And we also used it directly when it wasn't possible to freeze them.

Hi Christian, in principle I think exactly that way myself!

Only now, we might have to decide if to do it with an over night storage or not at all. It's really about making as much use of an animal as we can, and if we use many different organs we may not be able to process all of them at once. Another problem we face at the moment is that our animal colonies are kept 2h travel on public transport away from the lab, so sometimes we may also come back to the lab late evenings and only start processing then. Nightshifts are very common among immunologists and I'm the last person to drop everything at 5, but knowing your options can be a lifesaver sometimes.

I agree completely. The best way would be running a comparision, is there any chance for you doing this?

I was hoping to scrounge other people's experiences, but if that's not possible, running some inital comparisons will certainly be necessary.

The major concern is of coure that different cell types won't die equally fast. E.g. lymphocytes may be tougher than monocytes and so the total population may not reflect the ex vivo composition of cell types within the population correctly anymore.

Immune cells have potential to induce vigorous oxidative stress which is not at all good for culture. Storing organ over night will definitely induce oxidative stress on them. Again as stated earlier running a comparison is advisable.

Most important thing is weather you planning to culture these cells following any treatment or you planning to do experiments on them following culture. It makes a lot of difference if you store them over night (my opinion).

If you are keeping whole organ and isolating cells afterwards, this is working, but yield of cells is definitely less. I think, the reason is that cells inside the organ can not be supplied properly, because blood flow is off and PBS or even complete medium can not diffuse inside of the organ efficiently. Therefore, cells die.

Usually I preferred to do everything at the same day but if I could not do that, then I preferred always to prepare single cell suspension. In this case everything was fine. Even functional experiments were working. Once I tested function of OT-I splenocytes after 48hr in at 4C and they showed nice proliferation. Although if you have myeloid cells, for example, neutrophils, I would recommend you to do everything as soon as possible, because the functional activity and number will be definitely changed.

My experience is one night at most. Keep the cells on ice at 4C until use the next day. Good luck!

I agree with Tamar Kapanadze. If you can get them into single cell suspension you will be more likely to have surviving cells. We homogenize the tissue by pushing it through a 100 um cell strainer using a syringe plunger. After that the cells (in our case T cells) are pretty stable for several days although I think there are some mouse strain differences. Obviously it is easy to use the cell strainer with softer tissues (spleen, thymus, lymph nodes, liver) without the need for collagenase digestion but not so with harder ones (heart, lung etc.). Could you process the tougher ones the same day and just homogenize the easy ones for further processing the next day?

Good Luck!

organ store will destroy immune cells disproportionately. Better option is to a quick harvest and store before leaving lab. but it also depends on which type of cells, you will use down the road.

It seems like you already appreciate this point but just to emphasize it for anyone else in the future....you will lose cells this way. With that in mind, it is absolutely possible to do this and you should be able to culture them the next day with little difficulty. I've even isolated bone marrow for bone marrow derived macrophages after a couple days and it has been generally OK. I usually will keep the tissue in a media similar to culture media if I need to keep them happy overnight in the fridge. I have always suspected this works better than keeping them as a single cell suspension, but that's almost entirely anecdotal and I haven't really tested it so others might be right about that point.

Hi Jack,

You are right to emphasize that cells will be lost.

I have tried keeping a spleen intact for about 5 hours before homogenizing and comparing it to a spleen that was homogenized right away. Homogenizing right away was vastly better than keeping it intact in terms of how many live cells were recovered. Now this is only the spleen so maybe I'm wrong to extrapolate this to other tissues. Has anyone tried this with any other tissues?

Jack, yes I do. I'm really looking for a 'worst case scenario-escape' option as alternative to throwing tissue away. Thanks for the info on the bone marrow. If this still works, I have hope for the other tissues.

Gisela, that's very interesting! Did you by any chance check which cell populations were affected most? I would assume myeloid cells rather than lymphocytes?

Gisela, the spleen seems to be far more sensitive than many other tissues. In one of my labs we had a really nice automated cell counter and as such we had extremely reproducible viability data on many cell and tissue type and in my experience it is hard to get a viability above 75-80% on a spleen even under the best circumstances (and sometimes it is closer to 60%) but on, say, a kidney, bone marrow, or peritoneal exudate if you ended up below about 85% you had probably done something wrong.

Hi.

It really depends on what you are looking for, and what you intent to do with the cells afterwards. I have tried with spleens and lungs as a single cell suspension, kept in media at 4 degrees O/N (by the why. I did not use any collagenase for the lung tissue. You can mash it through a net with a plunger from the surringe. Just like the soft organs. We do it on a regular basis and it is deffinately durable. You might get a bit tired in your hands, but it's durable). Anyway, if you are looking at specific temporary surface markers, I would strongly recommend you to do the analysis instantly, so get a more 'in vivo like' picture. However, if you just look for Ag specificity + lineage markers it shouldn't be a problem. At least not with T cells.

Good luck!!

Best

Benjamin

@ Susanne: I just did viability by cell count, so no info on what cell type.

@Jack: I work mostly with spleen so your info about other tissue is great! Thanks!

It depends. Spleen remains OK after O/N storage in the fridge in full RPMI. Colon though is almost completely digested. I would not suggest to even store unfixed, in formalin , isolated cells. The question is always if the phenotype of the isolated cells remains the same as it was in the fresh tissues. So better to isolate the cells stain them and fix them for overnight storage

We have no problem with keeping a tissue in the ffridge overnight to isolate immune cells next day. I f you want to isolate cells from blood for FACS, we may keep them overnight in the fridge but it may be better with a gel pack an not id direct contact with the cells, but covering the gelpack with some cloth.

Due to technical issues, we had to leave spleens overnight in PBS at 4C (fridge) and isolate cells the next morning for FACS analysis of CD4CD25FoxP3 - we took 1 freshly removed spleen as a comparison - there was almost no difference between fresh and stored spleen samples in terms of staining and numbers of cells. Fantastic information

hi all,

i learned a lot from this discussion..

may i know, is it possible to keep the primary cell that i obtained from cell isolation overnight?

im going to enrich the macrophage from primary cells and do flow cytometry..