We are trying to obtain a pure oligodendrocyte culture from p0 mice. After one week of mixed glial culture in ornitine-coated flasks, we purified the oligodendrocyte precursor cells (OPCs) by shaking. However, we find that our cultures are quite contaminated by mainly astrocytes. Do you have any idea of how to remove that cell type? We have tryied different rpms in the shaker.
Thank you in advance.
Carol
If you read B Barres old papers, they used a monoclonal antibody (A2B5) for immunopanning. IgG-coupled magnetic beads sorting may be an alternative but still you need an antibody. Astrocytes tend to be invasive so just playing with culture conditions will not be enaugh.
The mixed glial cultures followed by shaking is an OPC-enrichment method, it will always have some contaminant astrocytes. FACS sorting will do it. This lab does it: http://ki.se/en/mbb/goncalo-castelo-branco-group
I am not sure whether you have this instrument, but I would suggest Laser Dissecting Microscopy (LCM).
Have you though about immunoprecipitation using GFAP antibody which is predominantly astrocyte-specific.
Negative selection with GFAP antibody might be a way to enrich in OPCs, if the A2B5 antibody is not available, however, this may not be 100% efficient.
Have you try to collect the non-adherent cells for the oligodendrocytes and astrocytes have different ability of adherent. For example, the detached cell suspension was plated in non-coated dishes for 1h at 37°C.Repeat the step for one more time. Then collect the nonadhering cells and seed into poly-D-L coated dishes in complete medium at 37°C. I also think it is difficult to isolate these two type cells just only by shaking and I think you also can try to isolate by combining shaking and adherent.
The following link may help
http://www.jove.com/video/3324/derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron
In the face of financial constraint and when all else fails, cloning is another option. Isolate a few glial cells manually by scrapping out with Pasteur pipet, and expand each of many isolates separately, maybe in 96 well or 24 well plates. Laborious, but that was the way the original cell line was obtained to begin with.
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