Challenges associated with undefined protease inhibitors in FBS

Most scientists don’t think about this, but if you’re culturing cells with FBS, you’re also including a wildly batch-variable, undefined mix of protease inhibitors with downstream consequences for cell biology. The presence of protease inhibitors in FBS is critical for passaging cells with trypsin; these proteins are responsible for deactivating trypsin (assuming you’re using FBS-containing media to quench the reaction, rather than a separate trypsin inhibitor). Unfortunately protease inhibitors are more problematic during the culture process than most people realize - but odds are you’ve never even thought about the impact they’re having on your cells. Protease inhibitors regulate processes like ECM turnover/remodeling, cell-matrix signaling, or migration, invasion, and differentiation. So adding an undefined mix of protease inhibitors to your cultures, courtesy of FBS, inevitably has an impact. This is particularly relevant for studies linked to fibrosis, cancer invasion, differentiation, or organoid/3D culture systems. Here’s where it gets even more interesting: many scientists assume growth factor variation drives most of FBS batch variation, and this does play a role. However, protease inhibitor variation is very much present, largely unappreciated, and has major implications for our research studies. These variable protease inhibitors control how much cells can cut up and remodel their surrounding matrix. If one serum batch inhibits proteases more than another, cells will migrate differently, invade differently, remodel ECM differently, and respond differently to exactly the same experimental signals. So when it comes to publishing research results using different batches of FBS, ECM-related results can vary substantially. That’s a big reason experiments involving migration, invasion, fibrosis, or 3D cultures can be so hard to reproduce. And this is also why many scientists whose experimental readouts depend on ECM dynamics can and should consider working with chemically defined systems.

As always, literature below if you're keen to dive deeper!

For more reading:
Page-McCaw et al. - for a review on how protease activity controls ECM remodeling, signaling, and cell behavior. https://www.nature.com/articles/nrm2125
Bonnans et al. - shows how protease regulation impacts cancer and fibrosis. https://www.nature.com/articles/nrm3904
Gjorevski et al. - A deeper dive into how chemically defined, tunable systems are important for reproducibility in the context of ECM composition and remodeling studies. https://www.nature.com/articles/nature20168
Not FBS specific, but one of the older papers on protease inhibitors in serum. Love a good study from the 1950s: https://pubmed.ncbi.nlm.nih.gov/14946322/