FBS contains bovine extracellular vesicles, loaded with bovine-derived miRNAs!

Here’s something fairly uncomfortable to consider if you regularly culture cells with fetal bovine serum: there is a whole lot of cow protein and genetic material in that golden elixir, and the implications of this scientific reality are still being worked through.

Let’s zoom in super niche for a moment: FBS contains bovine extracellular vesicles, loaded with bovine-derived miRNAs. When you culture cells in FBS-containing media, those vesicles can be taken up by your cells. In some systems, their miRNA cargo has been shown to alter gene expression.

Here’s why: miRNAs are potent post-transcriptional regulators. A single miRNA can suppress a whole network of targets. Many bovine and human miRNA sequences are similar in the specific stretch of sequence that determines which genes they bind to. As a result, bovine miRNAs have been shown in some systems to engage human gene regulatory pathways after uptake from serum-derived vesicles. How strong or consequential that effect is in your specific model is almost never validated when changing batches of FBS.

For most routine cell culture, this is arguably background. But there are contexts where it may matter more: RNA biology experiments, EV research, gene expression studies, or any work where you’re trying to attribute a transcriptional phenotype to a specific treatment. In these cases, you may have an additional source of regulatory RNA entering your cells from your media. Unfortunately, it's one rarely flagged in the methods section.

The extracellular vesicle (EV) research community has been aware of this issue for some time; EV-depleted serum became common practice in that field for good reasons, even though depletion methods don’t fully eliminate bovine EVs. The implications may extend beyond EV workflows, and yet they’re almost never discussed outside that context.

This is one dimension of what “FBS batch variability” can mean biologically. Scientists usually focus on growth factor concentrations or adhesion proteins varying between lots. But variability extends to differences like this as well - differences most scientists have never even thought to consider, much less one quantified across batches.

Just something to think about, particularly if you’re validating a new lot of FBS and running sensitive transcriptional experiments. Exosome-depleted FBS can help reduce this variable, or a chemically defined alternative can eliminate it altogether.

Anyway. One more reason "the same experiment, same results" thing doesn't always pan out 🙃.

As always, literature below if you want to dive deeper!

Wei et al. demonstrates RNA present in fetal bovine serum contaminates extracellular RNA analyses and can be misattributed to cultured cells.
https://www.nature.com/articles/srep31175?

Beninson & Fleshner show experimental evidence that exosomes present in FBS can influence cell behaviour in vitro, specifically demonstrating suppression of macrophage inflammatory responses. https://www.sciencedirect.com/science/article/abs/pii/S0165247814002387?via%3Dihub

Urzì et al. is a great review detailing how FBS-derived extracellular vesicles and RNA complicate EV studies. https://pubmed.ncbi.nlm.nih.gov/36214482/