Do you have experience with or are you aware of any studies that looked at whether culturing cells long-term affects their miRNA expression patterns?
We have experience in miRNA profiling in several kinds of cells, and I will tell you that the answer is YES. So, the expression profile of miRNAs is extremely dependent on the cell type, location, and environmental conditions. Longer culture times will exhaust nutrients in culture media and increase the concentration of secreted metabolic byproducts. In the case of primary cells you will accumulate senescent cells, and in fact the miRNA expression profile of these cells is completely different from the ones that are actively replicating.
We have experience in miRNA profiling in several kinds of cells, and I will tell you that the answer is YES. So, the expression profile of miRNAs is extremely dependent on the cell type, location, and environmental conditions. Longer culture times will exhaust nutrients in culture media and increase the concentration of secreted metabolic byproducts. In the case of primary cells you will accumulate senescent cells, and in fact the miRNA expression profile of these cells is completely different from the ones that are actively replicating.
Another issue to keep in mind is the confluence of your cells when you harvest them for miRNA profiling. An article published in PNAS several years ago (PNAS 2009, 106(17):7016-21) demonstrated that the level of miRNA dramatically increases with confluence. If you are not controlling for this variable, your expression patterns will be different making comparisons with culturing time difficult to quantify.
Hi Jaimee,
Francisco and John are absolutely correct. We have found substantial differences between freshly isolated and even P1 cells. The act of isolating cells is traumatic (for them) and what's more, we then drown them with excessive non-physiological quantities of mitogens, enzymes, inflammatory mediators, inorganics and fragments from dead and dying cells. We call this ex vivo cell culture.
I think that if I was a cell in similar circumstances, I too would respond by making wholesale changes to all of my core control systems.
It is simply another adaptive response to a changing environment.
I suspected that culturing cells would affect their miRNA expression (as I suspect it affects the expression of many proteins as well). Have any of you noticed any particular trends in the kinds of miRNAs that are upregulated and/or downregulated? For example, have you noticed that you see de novo expression of a particular miRNA not normally seen in your cell or tissue type?
Jaimee
Of course that the expected trend in any particular miRNA depends on the type of cells. In my hands, for example, human primary fibroblasts showed overexpression of miRNAs related with senescence along prolongued cultured times (mir-146a). Depending on the type of cell you will see different miRNAs up or down.
Regarding the expression of "new" miRNAs, I have no info about. Remember that miRNAs are reasonably well-known molecules and normally the expression of new miRNAs is related with pathological conditions. There are several examples in the literature.
Good luck. Cheers
Paco
I don't mean "new" miRNAs per se, I was more interested to know whether you might see expression of miRNAs associated with particular cell type (i.e. epithelial) when you culture a certain cell type for a period of time (i.e. muscle).
Thank you all for sharing your thoughts on this, I think this particular aspect of culturing cells is one that miRNA researchers really need to understand more thoroughly before being able to interpret certain experiments.
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