Subcultures of primary cultures - would you use them for your experiments? - (Feb/14/2007 )

For real-time PCR , would you consider using only primary cultures to determine differences in mRNA expression between control and treatment groups or would you also use subcultures? Doesn't expression of receptors and other markers change each time you subculture, and the more passages you have, the further you are from the 'true' events? Thoughts please...

Dear Cepheus,

You are absolutely correct in your assumption that primary cells "change" with passage/subculture. However they can change just by sitting in a dish over 5-10 days in culture. This is why most "proof of principle" experiments are carried out on "Immortalised cell lines" , which in general do not change significantly over passage and time, and that control and treatment groups can be rigidly defined.

For example : inducible iNOS expression : J774.A1 are immortalised murine macrophages which can be induced with LPS/Interferon to express iNOS. You will get the same expression levels if you use passage 10 upto and including passage 217. In murine primaries expression levels of iNOS can vary over passage No. and time. It is particularly easy to express iNOS in nearly all animal cells. However when you look at Human primaries/immortilised cell lines, expression in general is difficult and can only be achieved using cocktails of cytokines.

Experimentation in the labs I have worked in follow this order :-

Animal cell lines, Animal primaries, Human cell lines, Human primaries and then Animal models of disease. The ease of experimentation is generally in the same order.

I hope this is useful.

Dear Rhombus,

Thank you, that WAS useful. Allow me to describe our experiments a little: we are culturing microglia from rats of varying ages to determine the effect of aging on microglia responses. This limits me to using primaries and not immortalised cell lines.

Therefore, for my experiments, should I insist on only using the primaries or can I prepare a cocktail of primary and subcultured cells? If I aim to obtain relatively uniform samples (i.e. each sample would include primary cells + passage 1 + passage 2), would that help 'even' things out a little?

Do scientists only ever use the primary cultures and never the subcultures? The fact that this matter is never mentioned in their publications gives me reason for doubt.

Dear Cepheus,

If I were doing those experiments then YES I would only use primary cells. The protocol for your experiments would depend on the numbers of cells you require for your RT-PCR.

In an ideal world, the Microglial cells would be isolated and characterised from A RAT/GROUPS of rats of the same age and then used straight away i.e. the same day as isolation. Passaging cells usually means that for what ever reason, you do not have enough cells at the start. But by passaging this can only INCREASE the variables you introduce into the experiment and therefore dilutes out any possible effects you might see.