In my experience I have found the newer protocols, especially those published in Nature Methods to be inferior to older, established methods. We and others comprehensively evaluated washing/feeding frequency etc. so all of that data is published. In our lab we isolate MSCs from bone marrow, plate at 1.4 million/cm sq, and wash (not vigorously) at 72 hrs post-plating. We then feed infrequently and perform immuno-depletion with streptavidin beads. Even if you do not grow cells in low oxygen, this approach will yield several million cells, which can be expanded for a few population doublings in 21% oxygen. We use standard alpha-MEM without ribonucleosides or ribonucleotides (and differentiate cells in high glucose DMEM) with 10% lot selected sera and no other supplements. I do not believe supplements are the problem but rather the way you are handling the cells. Mouse MSCs are quite finicky and need careful maintenance (even re-feeding can be a stressor) unlike human cells. If you grow cells long-term to remove hematopoietic lineages you inadvertently select for immortalized clones so some form of positive or negative selection is required.

The concept that all non-clonal populations of connective tissue cells are MSCs is quite pervasive but not substantiated by rigorous data. Demonstrating the ability to generate a mineralized matrix or accumulate lipid in vitro does not make a cell functionally equivalent to bone marrow-derived MSCs, which themselves are heterogeneous. My question is why you would expect two cells from anatomically distinct regions of bone marrow to be functionally equivalent. Although I appreciate (probably better than anyone) the difficulty in growing marrow-derived MSCs from mice, you should be cautious in choosing the source of cells. Collagenase digestion can be very tricky and hard to reproduce from sample to sample, which makes the isolation of cells directly form bone a bit tricky. We tried the 2009 protocol several times without success and have not spent time on trying to improve the method.

Best of luck.

Culture success depends upon the tissue source of the MSCs and the conditions used to propogate cells. MSCs derived from mouse bone marrow are acutely sensitive to atmospheric oxygen (see Boregowda et al. Stem Cells 2012; 30:975 and Krishnappa et al. Cytotherapy 2013; 15:536). Be advised that cells isolated from compact bone exhibit different properties and are not equivalent to bone marrow-derived cells. We attempted to isolate cells from the protocol you cited several times without success. Why have you chosen this protocol over other more established methods? In our experience media replete with nucleosides and nucleotides is less favorable. Was your alpha-MEM media low glucose as well? I don't believe NEAA has any impact, we never used this supplement.

Before this protocol I tried isolating MSCs from bone marrow (Soleimani et al, nature Protocols, 2009) which involved initial changing of media every 8 hours for 3 days to get rid of cells of hematopoietic origin. I could get cells but they took a long time to grow. This method worked well for me every time (11 times) and was not as time consuming as isolating from the earlier mentioned protocol. Also it's a tight funding situation which means I would not be able to use MACS beads to remove hematopoietic contamination. I aim to utilize a method to avoid or minimize hematopoietic contamination and therefore isolation using compact bones seemed to me the best.

I, through papers from your lab and others, am very well aware that atmospheric oxygen can drastically affect the proliferation and differentiation potential of MSCs but unfortunately we do not have a hypoxic chamber in place to do that.

Dr. Brenton Short, in his paper (Prospective Isolation of Mesenchymal Stem Cells from Mouse Compact Bone) have shown that these compact bone derived cells are MSCs. This was another reason to use compact bones for isolating MSCs. What kind of differences do you see between the BM derived MSCs and compact bone derived MSCs?

I was using Alpha MEM media that had low glucose levels. Would that affect? After a lot of literature survey I realize people use either Alpha MEM low glucose or DMEM low glucose. I was told that high glucose medium is generally used for differentiation of MSCs.

Dr. Phinney, in your expert opinion what method and media formulation should I use to isolate MSCs? Do you think I should follow the classical method of plating the BM cells and then changing media after 3 days to get rid of hematopoietic contamination?

In my experience I have found the newer protocols, especially those published in Nature Methods to be inferior to older, established methods. We and others comprehensively evaluated washing/feeding frequency etc. so all of that data is published. In our lab we isolate MSCs from bone marrow, plate at 1.4 million/cm sq, and wash (not vigorously) at 72 hrs post-plating. We then feed infrequently and perform immuno-depletion with streptavidin beads. Even if you do not grow cells in low oxygen, this approach will yield several million cells, which can be expanded for a few population doublings in 21% oxygen. We use standard alpha-MEM without ribonucleosides or ribonucleotides (and differentiate cells in high glucose DMEM) with 10% lot selected sera and no other supplements. I do not believe supplements are the problem but rather the way you are handling the cells. Mouse MSCs are quite finicky and need careful maintenance (even re-feeding can be a stressor) unlike human cells. If you grow cells long-term to remove hematopoietic lineages you inadvertently select for immortalized clones so some form of positive or negative selection is required.

The concept that all non-clonal populations of connective tissue cells are MSCs is quite pervasive but not substantiated by rigorous data. Demonstrating the ability to generate a mineralized matrix or accumulate lipid in vitro does not make a cell functionally equivalent to bone marrow-derived MSCs, which themselves are heterogeneous. My question is why you would expect two cells from anatomically distinct regions of bone marrow to be functionally equivalent. Although I appreciate (probably better than anyone) the difficulty in growing marrow-derived MSCs from mice, you should be cautious in choosing the source of cells. Collagenase digestion can be very tricky and hard to reproduce from sample to sample, which makes the isolation of cells directly form bone a bit tricky. We tried the 2009 protocol several times without success and have not spent time on trying to improve the method.

Best of luck.

Thanks a lot for lot for these excellent and very helpful suggestions. One question that I would like to ask is that by what passage do you see cells becoming immortalized? I understand that population doubling is more meaningful that passage but to generalize one should try finishing the experiments between what passages? I plan to finish mine before passage 8.

I do agree that 2009 protocol has a lot of problems with it. Unfortunately the authors did not respond to my queries. I took the matter further to the editor of nature protocols to whom the authors have failed to reply yet. The authors from 2010 protocol also have never reverted back to me with my queries. I greatly appreciate you taking time to answer my queries.

It is difficult to gauge exactly when immortalization occurs but may be visible in culture by the appearance of rapidly growing foci in cultures that exhibit slow growth. The problem is that repeated passage homogenizes the population so if there are immortalized clones they are distributed throughout and therefore are impossible to discern. Following growth kinetics may be helpful as you should see slow growth over a period of 1-2 months followed by an acceleration in population doubling time. Therefore, if 8 passages is beyond 6-8 weeks I would be concerned that cells are immortalized. You can look at p53 levels, which are very high in immortalized lines. In our lab we immuno-deplete 7-10 day old plastic adherent cultures, expand cells for 1-2 more population doublings (in 21% oxygen or up to 6-8 doublings in 1-5% oxygen) and then use for experiments.

In C57B6 mice we had much better results with compact bone/collagenase protocol then anything else. We used NEAA addition and it seems it was making cells grow a bit better. Also, Dr. Rai, pay attention what stain of mice you are using. B6 have very sensitive and tricky MSC, while BALB/c and 129 has much more stably growing cells. In our experience the moment of the cell passaging is important (so called 80% confluence), so it is convenient to have several dishes and passage them at slightly different confluency state and passage further the one that is better growing and so on.

Thanks a lot Dr. Andrei! What happens I one splits the cells if it has reached 100% confluence? Do the cells start undergoing apoptosis or there is an increase in senescence?

The second option, they will stop proliferating...and loose stemness.

So thanks Dear colleagues, for your good information that mention in your discussion. my question is that about what is best culture media can use for mouse bone marrow and adipo derive msc from mouse (c57 bl/6)?

Dr. Phinny, Which protocol would you suggest? I recently tried the Nature Protocol aforementioned and it did not work for me as well.

Andrei is correct. There are significant strain specific differences in yield of MSCs from bone marrow (and compact bone), which has to do with variations in total bone mineral density. We published on this many years ago. I am curious why there is so much interest in isolating cells from compact bone vs. bone marrow. Our immuno-depletion protocol coupled with expansion in low oxygen is yielding very high numbers of high quality, non-immortalized MSCs from bone marrow. See Boregowda et al., Stem Cells. 2012 May;30(5):975-87 and Phinney DG Methods Mol Biol. 2008;449:171-86

Thank you

DMEM already contains Glutamine