We want to purify a colon cancer cell line from primary culture but there are lots of fibroblasts in the clone. So, how can I isolate them?
I am not quite clear. You mention your source of cells as being a "clone" which by definition is derived from a single cell. It should not have multiple cell types ( unless possibly cells in different stages of differentiation).
Are you in fact referring to a colon cancer tissue explant - a piece of tissue consisting of a mixture of primary cells (including fibroblasts) and cancerous cells (epithelial cells?).
Have you tried to see if different adhesion onto tissue culture plates makes a difference? eg trypsinisation followed by a short adhesion time on plates to enable fibroblasts to stick and then replating the cells which do not adhere as quickly - this may have to be repeated several times. Have you tried soft agar cloning or selective removal or enrichment by antibody beads targeted at surface markers? Ultimately to get a cell line you will have to do single cell cloning for eg. in 96 well plates . This will of course enable you to distinguish between fibroblasts and epithelial cells. Either way it is likely to be quite time consuming !!
http://www.nature.com/aps/journal/v34/n6/full/aps201356a.html
http://ac.els-cdn.com/S0002961099801354/1-s2.0-S0002961099801354-main.pdf?_tid=70d4222e-290b-11e4-a450-00000aacb361&acdnat=1408609164_bf699acd3435ef81f377cc037b84ab02
There is a thread on RG regarding this problem:
https://www.researchgate.net/post/How_can_we_stop_fibroblast_growth?_tpcectx=qa_overview_following&_trid=53f63555d3df3e1a538b45a9_
It is possible to eliminate fibroblasts by their expression of CD90. At least in skin explants one can use this to purify primary endothelial cultures from fibroblast contamination. Check whether your fibroblasts express CD90 which should not be on the epithelial cells. See PMID:9369534
Hi,
There is a very simple method. Just plate your cultures for 10 minutes on BD Primaria plates, the fibroblasts adhere rapidly, simply remove the floating cells and plate on your regular TC plates. I used to do this for isolating fibroblasts from cardiomyocyte populations.
sir, did u isolate the colon normal cells as Adam Pickard mentioned ???. did it work out well, i am also planning to do that. please suggest the method to isolate colon normal cells
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