I'm trying to culture lung cancer cells. I get samples from surgeon and try to make it grow as a cell line. But i struggle two problems
1. I can't see any growth, cell division ... I have no idea if something is missing in medium or it's just because of tumor tissue. Medium is RPMI + geneticin/penicilin/streptomycin + FBS. Do you add any growth factors guys ? What are minimal requirements for them?
2. Only thing that actually grow in this bottles are fibroblasts. They overgrow lung cells. When I try to remove them with geneticin, also lung cancer cells peel of and I was trying really low concentration of G418 (100-25 ug/m) Is there any other way to remove fibroblasts without any harm for other cells?
Hi Monika, not all tumors will become cell lines. I am working with thyroid cancer and my success rate after 160 patient tumors is only 10%. To get rid of fibroblast, you can try the following: " After trypsin, label fibroblasts with anti-human fibroblast antibody attached to microbeads (Miltenyi Biotec) and remove fibroblasts by passing through an LS column under a magnetic field." 90% fibroblasts will be removed. You can also try to digest your cells with trypsin using different incubation times. Fibroblasts usually detach faster than cancer cells. Good luck!
Than you Ying, well we also work with glioma cancer and use same methods and almost every tumor starts to "work" after deriving single cells. That's why such low success rate annoys me with lung cancer. Bout fibroblasts, I tried yesterday the method with different times of detachment, labeling will be to expensive for me I think. We will see how it goes. Thank you very much for sharing your knowledge :)
We often use 2ME in our culture medium, some of our tumour cells will not grow without it. Have you tried using a different sort of media (i.e. DMEM)? Our samples (pleural fluid) often contain a lot of fibroblasts which die off after about 10 passages, but we often have a very low tumour cell count in these samples to begin with.
You could also try to isolate the tumour cells before culture? Though i am not sure what the effect of cell viability would be after isolation (via depletion or Percoll etc). This is not something I have tried personally but some others in our lab have.
Well we are generally interested in cancer stem-like cells and firstly we tried two kits. One of them was isolating only CSCs (finaly we also got fibroblasts between them, and cells number was so little that line didnt start to derive), and second was clonogenic assay-based kit separating cancer cells from others. It also didnt work. Number of cells was every time too small. So we decided to make any cell line first and than separate CSC. But still not big success.
Bout 2-ME now. What it does to the cell culture ? Why you're using it?
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