There are many methods for identifying and isolating breast CSCs but they are quite expensive. Does anyone have any alternate idea for isolating true CSCs? I want to isolate it from patient tissue sample.
Dorothy,
I have not tried anything. But it is easy to guess the cost of the methods I have come across. They may work for sure but I can't afford them. So I need an economic one.
Dear Shakti,
your question is quite broad...kindly make it specific...
from where you would like to isolate CSCs: directly from patient tumors or cell lines?
further, which cancer types...different cancer types has different markers for isolation...
there are some general, relative less expensive techniques as well.
My point is instead of restricting yourself due to budget and wasting time on cheap with no results, better to explore the better ways first. Time is also expensive.
Dear Sakti,
PLEASE NARROW DOWN YOUR CONCEPT ON QUESTION FOR BETTER RESULTS.
Dear Tushar,
I do understand your point. I want to isolate breast CSCs from patients sample. In spite of the popularity of the expensive techniques, I would prefer a simple and economic technique. Please suggest if you have come across any.
Thanks.
Dear Prof. Bisoyi,
I have edited my question with specific information.
Regards,
There is several ways to isolate cells for example Cells isolation kits from Miltney, STEM CELLs, Cells sorting using antibodies. What do you consider expansive? How much are you willing to spend on the isolation.
"Traditional" FACS is usually cheaper than the fancier magnetic beads. Though core labs may not be too happy to handle primary human cells. It boils down to how are you going to gate the population of breast CSC, CD44+CD24-, CD44+CD24-CD326+, or CD44hiCD24lo, etc.. FACS offers more flexibility.
May I ask the purpose or use of the isolated primary CSC?
Be precise and concise in asking a question to get a concrete answer from the experts.
Dear Abhimanyu,
It is precise. I want to isolate breast CSCs from patients tissue sample. That is it.
I guess you want to define which subpopulation of CSC you are talking about, then we can find the best way to enrich them. if you are looking for CD44+CD24-, then you can either use FACS or magnetic beads such as kits from Miltney or other vendors. If you are talking about Aldefluor+ cells, FACS is your only choice.
And I bet you know that no matter which method you go, you will get a "CSC-enriched" population, rather than a "pure" CSC population.
I agree with u Chung. FACS has always been the choice. I was searching for some alternative. Thanks for the answer.
Dear Shakti
Breast Cancer stem cells could be isolated using Anoikis resistance assay, based on the fact that the non-adherent conditions promote the survival of stem cells only and other cell subtypes die by anoikis (Harrison et al., 2010). In this assay, cells were cultured in polyhema coated flasks in mammosphere culture media for 12 hours at 37ºC and 5% CO2. Viable anoikis resistant cells were then harvested by centrifugation at 2500rpm for 5 minutes and counted using hemocytometer slide.
This method is cheap and easy but the number of cells not too many but enough to do experiment.
please read this paper http://www.ncbi.nlm.nih.gov/pubmed/20068161
Good Luck
Ebtesam Nafie
Hello Shakti,
I would suggest, start by harvesting cells from the patient's tissue in a serum medium (DMEM or RPMI should work) following the standard protocol you are using. Once you get attached cells, you might change the medium to the one which "selectively" supports the growth of stem cells. Such a medium is usually serum free and contains growth factors like EGF, PDFG and FGF. Also, you might start growing cells directly in such a medium and using ultra-low attachment dishes, right after isolating them from the tissue. However, you still need to do immuno-cytochemistry/histochemistry using stem-cell specific markers to find out if the obtained population is that of cancer stem cells.
Good luck!
I will be very careful to put primary tumor cells into serum-containing medium because it may select the so-called CSC subtypes. One paper argues that serum is really a reason why make CSC such a rare population in the established cell lines, as compared to a much higher CSC population in primary tumor. (PMID: 17692807).
Dear Shakti,
Sorry for late response after you clarify your question. As per my experience, FACS sorting using CD44+ and CD24-, is not always easy to find and sort that specific population.
I like the suggestion of Ebtesam Nafie to select the cells based on Anoikis resistance. However I would like to add another aspect in this assay which account for slow proliferation of CSC-like cells. You can use a cell membrane binding dye like PKH-26 to your cells and grow them in serum free non-adherent conditions and later sort them for PKH-26 positive cells after 7-10 days to enriched CSC-like cells. This method has been used in literature and it worked very well in my lab as well.:
http://www.ncbi.nlm.nih.gov/pubmed/20074520
If you need more info regarding this method, don't hesitate to contact me further.
All the best,
Tushar
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