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How can I separate my cell line from feeder cells


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#1 nemesis52

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Posted 14 August 2009 - 07:06 AM

Hi,

I am working with trophoblast stem cell lines and I grow them over STO fibroblast feeder cells. I harvest my dishes by scraping them. Now, My problem is : "How can I completely separate my stem cells from feeder cells ? and if there is a way how can I validate it ? ".

I tried to do couples of short passages (about 1 hour) on gelatin covered dishes after scraping to remove feeder cells before RNA and protein extractions but I am still not sure that I got rid of them 100%.

I am also trying to find a good bio-marker for fibroblasts that is not expressed in any other cells so I can use it as control in my PCRs and Western blots.

I appreciate for any guidance. :)

#2 cellcounter

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Posted 14 August 2009 - 10:53 AM

Hi,

I am working with trophoblast stem cell lines and I grow them over STO fibroblast feeder cells. I harvest my dishes by scraping them. Now, My problem is : "How can I completely separate my stem cells from feeder cells ? and if there is a way how can I validate it ? ".

I tried to do couples of short passages (about 1 hour) on gelatin covered dishes after scraping to remove feeder cells before RNA and protein extractions but I am still not sure that I got rid of them 100%.

I am also trying to find a good bio-marker for fibroblasts that is not expressed in any other cells so I can use it as control in my PCRs and Western blots.

I appreciate for any guidance. ;)

So far as I know, differntial adhesion that you are doing is the usual method, if not the only method, to remove the feeder cells from stem cells. And it results in pretty good cleaning up for most purposes. Even if you have 1% fibroblasts left, I would only worry about it if that results in dramatic changes in RNA or protein patterns than you expect.

Having a good biomarker would be a good idea, though. I would think mesenchymal markers like alpha-SMA, fibronectin, vimentin would be the ones that would be present in fibroblasts but not in trophoblast stem cells, however, that is not a sure answer.. almost sure.

#3 nemesis52

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Posted 14 August 2009 - 12:15 PM

Hi,

I am working with trophoblast stem cell lines and I grow them over STO fibroblast feeder cells. I harvest my dishes by scraping them. Now, My problem is : "How can I completely separate my stem cells from feeder cells ? and if there is a way how can I validate it ? ".

I tried to do couples of short passages (about 1 hour) on gelatin covered dishes after scraping to remove feeder cells before RNA and protein extractions but I am still not sure that I got rid of them 100%.

I am also trying to find a good bio-marker for fibroblasts that is not expressed in any other cells so I can use it as control in my PCRs and Western blots.

I appreciate for any guidance. ;)

So far as I know, differntial adhesion that you are doing is the usual method, if not the only method, to remove the feeder cells from stem cells. And it results in pretty good cleaning up for most purposes. Even if you have 1% fibroblasts left, I would only worry about it if that results in dramatic changes in RNA or protein patterns than you expect.

Having a good biomarker would be a good idea, though. I would think mesenchymal markers like alpha-SMA, fibronectin, vimentin would be the ones that would be present in fibroblasts but not in trophoblast stem cells, however, that is not a sure answer.. almost sure.



Dear cellcounter,

Thanks for your reply. You gave me very good idea about markers. I will check them.

#4 Stephan

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Posted 16 August 2009 - 09:11 PM

You could use flow cytometry or magnetic beads to purify your sample.




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