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How to passage primary prostate cancers?


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#1 MagdaG

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Posted 08 November 2013 - 02:17 AM

Hallo,

 

i have problems with primary prostate cells. They grew very well, but it was difficult to passage them with Accutase,and they were sticking to dish so I had to scrap them. And after passage they were growing in clusters and after 1-2 weeks begin to dye. It looked like they were not able to attach really to the dish.

Has someone some experinces with this kind of cultures? I would be gratefull for any advice.

 



#2 bob1

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Posted 08 November 2013 - 03:21 PM

Scraping is not a good option for passaging cells, it damages the cells too much.  The only prostate line I have worked with is the LNcap line, which is very sticky and requires a long time in trypsin to lift the cells - about 20 min.

 

Primary lines are also tricky as they often have growth limiting conditions, such as reaching confluence, length of time in culture (Hayflick limit), or require special additives to keep them growing (hormones, different concentrations of FBS, non-essential amino acids, etc.).



#3 MagdaG

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Posted 12 November 2013 - 05:41 AM

Thank you!  Ineed, scraping wasn't good idea, mostly fibroblasts survived :(. Anyway I will try tripsine longer keep them with tripsine. I was only afraid that it can be too harmfull for cells.



#4 bob1

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Posted 12 November 2013 - 12:06 PM

Yes, trypsin is also damaging to the cells, you need to balance the time with damage - check the cells frequently until you see them lifting off.  On the positive side, this should select against easier to trypsinise cells like fibroblasts - which should clump together with long trypsin treatment, and can then be removed relatively easily.






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