I culture glial cells from the spinal cords of neonatal wistar rat pups. The problem is, however I try to remove the meninges from the spinal cord, I find it tricky to be able to completely get rid of fibroblasts. Since the fibroblasts grow vigorously, it poses a great problem to the purity of the culture. I have been using 100 micron nylon mesh to filter those cells out, but does not seem to be working. Should I go for smaller pore size- 40 or 20 micron that is to say? or is there any other method to solve the issue?
Also, I have noticed that within 9-12 DIV often the cultures grow confluent in an uneven fashion- while there are some patches absolutely confluent, while in some places the cells grow scarcely. I wondered what may be causing this difference. I have tried increasing the density, but to no avail.
Thank you Dr Thomas.
Magnetic beads may not be feasible to me in the present or near future. i wondered if using conventional culturing procedures with specific modifications i could still obtain a pure mixed glial culture?
Dear Dr. Thomas,
Thanks again. May be i should go for the dynabead selection.
Sure Dr Thomas.
That sounds reasonably good. The only problem would be the feasibility, which i will try to resolve. Thanks again.
Fibroblasts are notoriously annoying when working with endothelial cultures, as the presence of only a small number of these will usually lead to the fibroblasts outgrowing the endothelial cells after some time in culture. A good single cell suspension and rigorous washing steps are important to avoid fibroblasts. Large cell clumps that could consist of both fibroblasts and endothelial cells will inevitably lead to co-isolation of fibroblasts. Non-specific isolation of fibroblasts (and other cells) caused by free DNA in the sample is also a possibility, and this could possibly be improved by including a DNase treatment step prior to isolation.
Fibroblast depletion
For fibroblast depletion I would recommend using the Dynabeads Pan Mouse IgG with a Mouse anti-Human Thy-1 antibody (CDw90) to target the fibroblasts. There are two recommended antibodies:
1) Mouse anti-HumanThy-1 (CDw90) antibody (clone F15-42-1, cat.no MCAP90). Serotec, Oxford, UK.
2) Mouse anti-HumanThy-1 (CD90) antibody (IgG1, clone AS02, Cat. no. Dia 100). Dianova, Hamburg, Germany.
fibroblast depletion from single cell suspension of other tissue origin:
S. Fernández-Shaw, S. C. Shorter, C. E. Naish, D. H. Barlow, and P. M. Starkey. Isolation and purification of human endometrical stromal and glandular cells using immunomagnetic microspheres. Hum.Reprod. 7 (2):156-161, 1992.
Do you sterilise the spinal cord in ethanol before cleaning from meninges? To my experience (I prepare glial cell cultures from neonatal murine brains), the shorter time you leave the brain in ethanol (1-2 secinds are enough), the easier disposing of meninges is.
Well Tomas! That sounds brilliant! i never did that but do not see why not!! The effect of ethanol on meninges is immediate, i have experienced it myself. One more question, it is the routine 70% ethanol, or the absolute one? Thanks for the answer...
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