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.
The most predominant factor should be age and presence of adequate nutrients or growth stimulants (e.g. growth factors) within your medium. At low concentrations, trypsin was even shown to facilitate a certain degree of cell proliferation in cancer cells but this might not hold true in all cell lines.
Article Autocrine Extra-Pancreatic Trypsin 3 Secretion Promotes Cell...
In my experience, trypsinization will always pose lesser damage to the cells compared to scraping especially in the case of passaging your cells. Although some might argue that leaving your cells in trypsin for too long might affect cell proliferation, it is still far lesser compared to the detrimental effects that cell scraping has on cell viability and proliferation rate. However, if you are considering cell harvesting methods for DNA/RNA/Protein extraction, you might want to look at your cell type again and whether your cells have membrane proteins which might be affected by trypsin. You can read more on that here:
Article Effects of different detachment procedures on viability, nit...
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 The cells no longer represent their origin acurately.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.
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.
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