Does anybody have any views on the best methods for differentiating rat oligodendrocytes in vitro please? My feeling is that as soon as they start to mature passed the OPC stage a large subset begins to undergo apoptosis, even with added CNTF or n-acetyl cysteine. I'm mostly driving differentiation using T3.
The best (and, to my knowledge, only) to do that is to eliminate mitogens (FGF-2, eventually EGF) from the culture medium and wait. If you can send me the reference of the medium you use, I can gice you more details.
Please, take care: OPCs can be differentiated into myelin-forming oligodendrocytes, but immortalized cell lineage cannot.
The McCarthy-de Vellis method still works the best using Bottenstein medium to differentiate the rat oligodendrocytes. The cocktail of Biotin, insulin,T3, selenium, progesterone and BSA in serum-free DMEM with 4.5g/L glucose, glutamine and sodium pyruvate gives you homogeneously differentited oligodendrocytes in 4 days. our experimental window is usually 3-6 days. Unfortunately mice do not behave like this and seem to "differentiate and die asynchronously".
There are usually a few astrocytes and microglia around but for the most part the cultures are morphologically spectacular and myelin markers are prominent. Other methods will give you enough cells for a coverslip.
In the Barres lab, we did 2 things. One is to remove mitogens (PDGF and NT3, in our lab), and the other is to add T3 to promote differentiation from OPCs to oligodendrocytes. Rat and mouse OPC media (defined, serum-free) is slightly different, for the recipe I would look at Jason Dugas' and Ben Emery's papers from the Barres lab.
Hi everybody, thanks very much for your great advice! I might be in touch with you soon to talk oligodendrocytes again! Thanks, Marc
and...yes, expect quite a large amount of cell death..in my experience, between 30%-50& of OPCs die before differentiating..good luck!
Are these primary rat cells, or a cell line?
If primary, are they cortical, optic nerve, or another source?
I use Sato medium for primary cell differentiation: DMEM supplemented with 2mM glutaMAX-I, 1mM sodium pyruvate, 30nM 3,5,30-triiodothyronine, 30nM thyroxine, 1% N2 supplement, 50U/ml penicillin and 50 ?g/ml streptomycin. I usually culture cells for 7-12 days to get >80% MBP+ cells with highly complex morphologies, but you can see MBP+ cells start to appear much earlier.
The attached paper shows some of my oligodendrocyte cultures.
The protocol of our procedure for differentiation of murine and human OPCs towards myelin-forming cells is in the attachement.
Hi Everybody, thank you very much for your kind and excellent advice and for supplying your methods, I really appreciate it! (sorry for the delay in saying thanks, I've been on vacation!). Hope to exchange some more ideas soon.
Hi, Marc. I am wondering if you have solved the problem above. Recently I am trying to do mouse OPCs primary culture. I am OK with the isolation stage. I can get enough OPCs from the mixed-cultured glial cells after the centrifugation step. But I have been haunted by the situation that the cells cannot survive longer than 4 or 5 days after the isolation, which is just consistent with what you have described here as "a large subset begins to undergo apoptosis". I have seen a lot of vacuolar degeneration in my oligodendrocytes.
I know that mouse oligodendrocytes culture is kind of different from rat oligodendrocytes. But I still want to ask for your advice and experience on resolving this problem.
My proliferation medium is DMEM/F12, 20 ng/ml PDGF, 20 ng/ml bFGF and 2% B27.
My differentiation medium is DMEM/F12, 30 ng/ml T3, 20 ng/ml CNTF and 2% B27.
Do I miss any important factors here? Thanks for your help!
I know this thread is rather old but I am having the very same problem and is making it difficult to biochemically analyze my OL cultures due to the vast cell death. I am using the same method of differentiation using DMEM-SATO media and removing the PDGF/NT3 and supplementing with T3 to induce differentiation. All is going well the first 2 days post supplementation then without fail day 3 post T3 addition there is vast cell death and vacuolization. We are culturing in 10% CO2. I understand there will be some cell death to an extent however the past 3 DIV there seems to be very few OLs left alive if any. It just seems very odd considering they appear very healthy at the begining of differentiation. Any input would be greatly appreciated
- Badges
- Science topic
- Similar topics
- Psychology