I am working with human brain tumor (grade IV: glioblastoma multiforme). The brain tumor has 70% glioma and 30% fibroblast cells in the culture. How can I avoid fibroblast growth and obtain a pure homogeneous culture of glioma cells?
Have you tried serial differential plating? Fibroblasts usually tend to attach to culture plates much faster than other cell types. You may plate the cells, wait for different times, say 30, 20, 10 minutes and then take out the supernatant containing non adherent cells and transfer to a fresh plate leaving the adherent cells. Do it serially 4 or 5 times or even the net day. You may be able to reduce the percentage of fibroblasts to a minimum. The only practical way to avoid fibroblasts completely is single clone isolation. If you are successful with my suggestion, please do share your success in this forum.
Even though I have not an answer for you, I am also very interested in this. Can you elaborate further on how you assess the glioma and fibroblast cells? Immunostaining?
Think about it. Are you having fibroblast in the brain?
I think not, this might be endothelial cells or astrocytes.
We perform isolation of brain tumor cells via mechanical and enzymatic methods. Cells assess by immunocytochemical staining.
You should think about what kind of cells you are having in the brain. There are no fibroblast in the brain.
Dear Mansoureh
I agree with Barbara regarding fibroblast. It is possible that you see stem cells. see next
J Neurosci. 2009 Jul 15;29(28):8884-96. BMI1 sustains human glioblastoma multiforme stem cell renewal.
Abdouh M, Facchino S, Chatoo W, Balasingam V, Ferreira J, Bernier G.
Stem Cell Res. 2012 Mar;8(2):141-53. Epub 2011 Oct 8. Bmi1 marks intermediate precursors during differentiation of human brain tumor initiating cells. Venugopal C, Li N, Wang X, Manoranjan B, Hawkins C, Gunnarsson T, Hollenberg R, Klurfan P, Murty N, Kwiecien J, Farrokhyar F, Provias JP, Wynder C, Singh SK.
To avoid astrocyte proliferation in culture we use AraC (cytosine arabinoside). But in your case this can affect the tumor 'cause AraC is a known chemotherapy agent
@ barbara Rath, I think, fibroblast contamination is quite normal in brain cell cultures...at least i had a problem with fibroblast contamination in primary astrocyte culture, if i dont remove the piamatter carefully ...any way one possible way to get ride of the fibroblast proliferation in culture is by replacing L valine with D valine which fibroblast cant utiulise..I hope .you can find some literature on this topic http://www.ncbi.nlm.nih.gov/pubmed/10826768
regards
shefeeq
@ mohammed: Its quite a rare event that gliomas growing on the surface of the brain close to the pia mater and they do not infiltrating the pia mater.
Have you tried serial differential plating? Fibroblasts usually tend to attach to culture plates much faster than other cell types. You may plate the cells, wait for different times, say 30, 20, 10 minutes and then take out the supernatant containing non adherent cells and transfer to a fresh plate leaving the adherent cells. Do it serially 4 or 5 times or even the net day. You may be able to reduce the percentage of fibroblasts to a minimum. The only practical way to avoid fibroblasts completely is single clone isolation. If you are successful with my suggestion, please do share your success in this forum.
What is the general consensus on fibroblasts being present in brain tumors?
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