I have been trying for a while to get lung cancer stem cells (from tumours) and the percentage of success has been close to zero. In some, we obtained nanospheres of 60-100micros but then they are difficult to disassociate (by mechanical means) and to maintain.
Hi Christopher. Thank you for your answer.
Regarding the issues that you mentioned: our nanospheres might be smaller when we make the passage, however this shouldn't interfere with the disassociation (by mechanical means). It could interfere in terms of maintaining the cells, however in our case the cells take up to 10-12days to reach the 60-100microns and if we let more time the primary cells of the core will become darker maybe due to hypoxia.
Our principal difficulty is to disassociate from P1 and obtain enough cells to proceed.
I have another question. I used MACS Cd133 MicroBead Kit to separate human lung stem cells from tumor. The problem is in both separated stem cells and leftovers from this process I noticed really active growth of fibroblasts. My question is it normal? Are they going to grow over my cancer cells?
Another question, what medium do you use for lung cancer stem cells? Right now I use DMEM and try to culture them adherently. It's just one bottle of them and there are places in it that are fine and grow, but the rest doesnt seam to proliferate. How fast stem cells proliferate?
We don't separate the lung cancer stem cells using stem cell markers because we are afraid of selecting a subpopulation of cancer stem cells. Has you know there are several subpopulations of cancer stem cells and there is no consense which cancer stem cell marker is the best, at least for lung. What we do is plating the cells obtained from the tumor in low attachment plates. Generally the fibroblasts will still attach and we will get rid of them through consecutive passages. The media that we use that is also available in papers is: Defined Keratinocyte-SFM serum-free medium supplemented with 2% B27, 0.25ug/mL Amphotericin, 20ng/mL EGF, 10ng/mL FGF, 1% L-glutamine and 1% Penicillin-Streptomycin
So what you do is just trying to culture them in spheres ?
Do you have any paper about this subpopulations of cancer stem cells. We asked some other researchers and they said that there are no clear arguments against usage of such stem cells separators (for now) :).
About fibroblasts now. The are finally detaching from the bottle after 4 days incubation with geneticin in concentration of 100 ug/ml.
Do stem cells grow adherently in this Defined K-SFM ?
And I wanted to ask again what is doubling time for your stem cells more or less?
If you try to culture the stem cells in low attachment plates you will avoid biased selection of stem cell population. Alternatively you can also inject directly in mice. So by common sense i will say that if there are several sub-populations and if you are selecting them you will end up doing your studies in these sub-populations.
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology
- Cancer Cells
- Cancer Stem Cells