Currently I'm doing primary cell culture of carcinoma by ex-plant method. In the culture, I observed fibroblast cells along with carcinoma cells and fibroblast cells are growing faster than the carcinoma cells. Can anybody help me with ideas how I can separate fibroblast cells from carcinoma cells effectively?
We simply add a low concentration of Geneticin (G418) say 25 to 50 mircograms/ml final concentration. All the cancer cell lines we have established tended to be relatively resistant to G418 at these concentrations for a short time of about 3 - 7 days. Fibroblasts and other primary contaminating cells are sensitive to this level of this antibiotic and are usually killed during this incubation period. Another option is to use the G418 for 48 hrs replace fresh medium and repeat the treatment after 24 or 48 hr recovery period. A few cycles of this treatment may work as well. Also note that not all the fibroblast will immediately succumb to the G418. However, even after removal of the reagent the remaining fibroblasts will not recover. That is, the death pathways that are initiated can take some time to kill the cells. Good luck.
Could you try differential trypsinization?? For example you can first treat the co-culture with cold trypsin expecting the carcinoma to get flushed away and then once you pipettte out these the rest cells would be your fibroblast which are strongly adherent cells.
You can try to decrease the FBS concentration to slow down the proliferation of the cells. In our experiments the fibroblasts were more sensitive to the FBS concentration compared to other cell types.
Another way which is tedious and time-consuming is to physically scratch away the contaminating cells (place a microscope under the hood and scratch the fibroblast with a 200µl tip; we did that procedure for the immortalization of murine endothelial cells.)
Good luck
Dear Thomas Mohr, thank you very much for your good suggestion. But sorry to say that I don't know much about negative selection using either MACS or Dynabeads coated with anti CD90 antibody. It would be much appreciable if you explain it elaborately for me. How I can do that? Thank you once again.
Dear Sabyasachi Dash, thank you very much for suggestion. For your kind information, I need carcinoma cells not fibroblast cells. I tried differential trypsinization. Initially fibroblast cells flashed away as they are loosely attach cells. But this process is not giving me complete separation. Still there was almost 30% fibroblast left over and they are growing faster than carcinoma cells in subsequent sub-culture. What can I do further? Thanks.
Dear Tobias May, thank you very much for your good suggestion. I reduced FBS concentration from 20% to 10%. But still fibroblast cells are growing fast. Is it a good idea to use no FBS? Can carcinoma cells grow without FBS? What do you think?
I didn't try your second method. If I take the entire microscope under hood, will it produce any contamination? How can I avoid contamination in this method? Thank you very much.
I established many carcinoma cell lines, most of them grew together with fibroblasts in the primary cultures. In most cases you can seperate fibroblasts and epithelial cells by trypsin/EDTA treatment since they react differently - or are differently sensitiv to this treatment: Wash shortly with trypsin/EDTA, add about 0.5ml trypsin/EDTA to the culture and watch under the microscope until the first cells detach. Either these cells are the tumor cells or these cells are the fibroblasts. Transfer them together with trypsin/EDTA into a new flasc. Feed the old flasc with fresh medium.
In rare cases the tumor cells and the fibroblasts detach equally. Then try scraping away the fibroblasts.
Besides: don't worry too much about fibroblasts. In most cases it's just a matter of time when the tumor cells overgrow the fibroblast layer. Time means months!
Good luck!
We simply add a low concentration of Geneticin (G418) say 25 to 50 mircograms/ml final concentration. All the cancer cell lines we have established tended to be relatively resistant to G418 at these concentrations for a short time of about 3 - 7 days. Fibroblasts and other primary contaminating cells are sensitive to this level of this antibiotic and are usually killed during this incubation period. Another option is to use the G418 for 48 hrs replace fresh medium and repeat the treatment after 24 or 48 hr recovery period. A few cycles of this treatment may work as well. Also note that not all the fibroblast will immediately succumb to the G418. However, even after removal of the reagent the remaining fibroblasts will not recover. That is, the death pathways that are initiated can take some time to kill the cells. Good luck.
In my experience, a single method is not usually enough. The simplest method I tried was differential trypsinization followed by selection media. Incubate the cells in 0.025% Trypsin-EDTA for 2-3 min at 37C. Tap the plate/ flask on the bench to remove the loosely adherent cells, usually fibroblasts and stop Trypsin action by flushing with complete medium. After this is done you can use media specifically designed for selecting epithelial cells. One of such media is Eagle's MEM with D Valine modification. I used this media to establish primary cultures from both tumor and normal regions from Human renal specimens both in the cortical and in the papillary regions.
Thank you very much you all guys for helping me with different ideas. I'll try different options and see which one works better in my case. Wish you all a happy career and happy life as well.
I also have fibroblasts in my lung cancer and lung cancer stem cells cultures. I''m trying geneticin in concentration of 100 ug/ml. After one day ... no effects at all. But I hear it takes up to week to remove them with geneticin, so I guess it's nothing strange. They divide so fast...
Hello
Is G418 treatment working for your guys to eliminate fibroblasts?
Any positive results to be shared?
Andrew
Hi Guys,
I also have a problem about the purification of tumor cells from fibroblast cell. I am now trying 3D culture to get colony, and isolation the colony by 40 uM strainer. after these step, i use trypsin to digest the colony and culture in 2D condition. hope it helps.
Dear Silke Brüderlein
I though that differential trypsinization is used to enrichment of cancer stem cell and remove differentiated epidermal cells ( not to differentiate epidermal from fibroblast).
There is an article published in scientific reports that is mentioning this point. I wouild aapreciate if you follow the article
"""Low adherent cancer cell subpopulations are enriched in tumorigenic and metastatic epithelial-to-mesenchymal transition-induced cancer stem-like cells """ @Silke Bruderlein
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