I am using some primary human cells for my experiments, But the activity/proliferation rate of the cells in their late passages (>P8) are very low and I can not use them for long like cell lines.
(a) What is the factor affect the decreased proliferation?
(b) Is it due to aging ? or Trypzinisation?
(c) If trypzinisation is the factor!! can I use cell scraping instead?
Please help me to clarify
Thanks
Nimal
In addition to the correct comments above, I might add that primary cells should not be grown and treated as if they are a cell line. "Primary Cell Line" is a contradiction in terminus, just like "dry water" and "cold fire".
It should not be a surprise that primary cells start to become difficult to handle after eight passages! Beware that cell lines also have a limit to passage numbers and start to change after many passages according to the same principles. Cell lines are just easier to grow in vitro compared to primary cells, but that does not mean they remain the same after many passages. I would not recommend culturing through 8-10 passages for any cell line. I hope this helps.
I agree with Harald Kühnel that it's senescence. We don't use primary cells beyond P5 for that reason. Not only because of (strongly) reduced proliferation but also because of altered expression profiles in general. The cells no longer represent their origin acurately.
That's also why immortalized cell-lines (with their own pros and cons) are in use...
I had similar issues with primary HUVEC culture. Immortalizing primary cells will give you extra mileage if you really want to stick to the same cells throughout your experiments. Otherwise use newly isolated primary cells.
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