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Fetal bovine serum is this rather quite pretty reddish-amber colour, but most scientists don't think about its color much, except along one of two lines of thought: 1) "yep, checks out, it's a blood product" or 2) "this FBS looks weird, what's going on?"
We run into this when I drop off bottles of FRS Pioneer to research institutes for their regular FBS batch testing ritual. FRS is clear, so unfortunately it's not exactly a blinded head-to-head comparison. The colour of FBS comes down to hemoglobin and its breakdown products. These heme-derived pigments carry over from blood collection and processing, which can vary from batch to batch. Because it’s commonly used as a processing quality indicator, certificates of analysis often flag FBS hemoglobin content. The same is not true of most sources of variability in FBS. These variability sources range from the well-known, for example growth factor concentration variability, but extends to protease inhibitors, oxidant capacity, miRNA-containing extracellular vesicles, and dozens of other parameters which modulate cellular biology in completely unaccounted for ways that most scientists never ever think about, let alone record in the methods section of xyz journal. So you've got some options. Moving to 100% chemically defined media eliminates this variability entirely, but FBS reduction also has a meaningful impact. The research institutes we work with are typically considering reduction of standard FBS concentrations down to 1–2%, and at that level you've already cut a significant fraction of the batch variability. Plus much as we all love our ritual FBS batch testing processes, wouldn't it be even lovelier if it happened once a decade? As always, journal articles below if you're keen to dive deeper! FBS batch variability - https://www.sciencedirect.com/science/article/pii/S3050620425000429 Protease inhibitor deep dive and sources - https://www.linkedin.com/posts/kathleenbashantday_most-scientists-dont-think-about-this-but-activity-7416587209304203264-XT5T?utm_source=social_share_send&utm_medium=member_desktop_web&rcm=ACoAABxyS3cBZRJoB-cKhs2ybXkT-p4uHHtCnJk miRNA extracellular vesicles deep dive and sources - https://www.linkedin.com/feed/update/urn:li:activity:7431801402559209472/?originTrackingId=MQz2KBX26vsc%2FLqOCAlJsA%3D%3D
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Behind the scenes of building a biotech company: It’s been about 6 weeks since we’ve started shipping out our chemically defined FBS replacement every Monday.
We ship, I refresh my tracking app incessantly throughout the week, and we learn new things about new countries' import processes every single week. On the upside, I came into this accustomed to Australian import (one of the more complicated bars), so things have been smooth sailing more often than not. The real world humbles us but also enthuses us. User guides get improved to prevent mistakes during onboarding. We publish new data on our website showing the limits and capabilities of the product. Folks have success with new cell lines and we add them to our tally. In the background, we pull together application notes for fully chemically defined primary cell culture and operationally easy adherence in serum-free systems. We fall into a regular manufacturing and QC cadence; it’s boring and predicable, just the way I like my manufacturing processes. In a few months, we’ll know even more about product performance and edge cases. We’ll start sending product to distributors globally, to make FRS more accessible to folks outside Australia. We’ll scale up the size of each manufactured batch. And I guess that’s how a “real company” is born. Only took a few years! Trends I’m seeing in the cell culture space lately:
→ AI meets biology is accelerating the already-in-progress trend towards chemically defined research tools. This is logical. If you’re training models using cell culture data, you want the inputs as clean and consistent as possible. For example, this makes the variability introduced by FBS more noticeable as well as problematic. → Completely anecdotal but there seem to be some bad batches of FBS circulating. I’m hearing from scientists who have used fetal bovine serum for years, but suddenly - despite certificates of analysis and in-house batch testing - their latest batch is testing positive for problematic viruses or has heavy precipitation/flocculants. → Cell culture media optimisation platforms are becoming more and more advanced. The result is highly specialised cell culture media built for specific cell types allowing for faster proliferation rates, cheaper costs per liter, and higher densities than ever. → Despite these advanced cell culture media, there are plenty of scientists looking for “this is easy to use and it grows the six different types of cells that my lab grows” instead. Knock out serum replacement (KSR) seems to be the best known "serum replacement", which is kind of interesting because it was designed primarily for pluripotent stem cells and usually doesn’t substitute well for serum in most other cell types. → In this vein: there’s increasingly a natural split between “exploratory biology media” and “production media.” Highly optimized, specialised media are necessary for high densities or cost optimisation at huge scales. But for exploratory work, operational simplicity is important; a single formulation that reliably grows a variety of cells and is sufficiently forgiving of handling variation is valuable. → Adherence is a sticking point (hah, I amuse myself anyway). In most serum-free media systems, coating plates with adherence proteins is the standard. This is operationally annoying relative to culturing adherent cells with serum-containing media. (Let’s do something about this, why don’t we? 😉) Hey New Zealand scientists, FRS Pioneer is travelling across the ditch! This year, Beyond Animal Research is supporting NZ scientists in accessing Media City Scientific’s FBS replacement. The first shipments have already landed on NZ soil and we’ll be working together to develop additional case studies showing how scientists can easily replace FBS with a chemically defined, animal-origin-free alternative for cell culture or cryopreservation. Being animal free AND being able to reproducibly control exactly what’s in your cell culture media makes this a win-win for everyone. Thanks Tara Jackson and the BAR team for making this happent! Are you a NZ scientist keen on using FRS Pioneer in your research or teaching? You can get involved by emailing [email protected]. Cheers ☺️  Most cell culture troubleshooting focuses on what you might be doing wrong, while less attention goes to what FBS is correcting for in the background. That's something worth understanding when a batch behaves unexpectedly, or when you try to replace FBS entirely.
FBS is a remarkably effective antioxidant buffer. Not because it was human-designed to be so; it's a biological fluid, it just comes that way. But that function matters and it's almost never replaced when scientists DIY a serum-free cell culture media. Most scientists think of FBS mostly as a source of growth factors and nutrients, but it also contains a sophisticated antioxidant pool that buffers the oxidative environment your cells are living in. Most serum-free formulations replace some of this, usually in the form of albumin, transferrin, and a selenium source, but there's considerably more to antioxidant buffering than that. Strip out the wider pool without replacement and subtle shifts in ROS levels start affecting mitochondrial function, altering proliferation rates, and in sensitive cell types pushing cells toward senescence prematurely. These aren't dramatic phenotypes and you probably won't notice them immediately under the microscope. Instead, they usually show up as variability. I frequently joke that FBS covers all manner of sins. It's forgiving in part because its antioxidant buffering absorbs a lot of handling variation: trypsin left on slightly too long, feeding with cold media, a suboptimal seeding density, media that's been in the fridge a month. But that buffering capacity isn't consistent between batches. Albumin, transferrin, selenium, tocopherols, and others - these are all biological and fluctuate with the source animal's condition, age, and processing. It's also a parameter that's not usually measured during batch testing. Defined media tends to be more honest, because it reflects exactly what you give it. This is a real advantage, but only if the antioxidant pool has been engineered carefully. Get it wrong and you've traded one source of variability for another, potentially more confusing one. This is why we spent a full year in external pilot testing before bringing our chemically defined FBS replacement to market, including trialing with students. We saw what happened when different passaging reagents, seeding densities, experience levels, and protocols were applied to one reagent. Some things broke. We made the product more robust wherever we could, and where specific handling processes were necessary, we documented it clearly. A formulation that can't handle real-world variation has no business replacing something as forgiving as FBS. Takeaway: antioxidant buffering is one of the most overlooked variables in cell culture, whether you're troubleshooting batch-to-batch inconsistency in FBS or building out a serum-free formulation. Something to pay attention to! And as always, more reading below if you want to dive deeper 😊 Roche M et al. (2008) covers the antioxidant properties of serum albumin - https://pubmed.ncbi.nlm.nih.gov/18474236/ Barry Halliwell has written extensively on ROS, Fenton chemistry, and biological antioxidant systems. His work is definitely some to check out if you’re interested in this space! One of his papers to get you started: https://www.sciencedirect.com/science/article/pii/S0014579303002357 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/ Something that annoys me to an arguably irrational level about the “serum-free media” space is how confusing the vocabulary gets once you start digging.
I’ve lived and breathed this stuff for the better part of a decade, and it still sometimes takes me a beat to understand a serum-free supplement. You see these words everywhere: Serum-free. Animal-free. Chemically defined. Reproducible alternative to FBS. A quick Google and you’d be forgiven for thinking there are a wide variety of equivalent, high-quality serum replacements out there. But these words can be layered together in ways that blur real scientific differences. First, some formulations simply replace FBS with another complex biological supplement. This is a reasonable strategy; for example, replacing FBS with human platelet lysate in cell therapy manufacturing to reduce cross-species risk. But you still have batch-to-batch variation. ”Animal-free” removes animal origin risk but can include other undefined extracts. Love this for ethics or regulatory positioning. It's less useful for having control over what’s in your media or batch reproducibility. “Reproducible alternative to FBS” has given me the ick several times. I’ve seen this wording, dug into the supplement, then realized it isn’t chemically defined in the slightest. There may be process consistency or tighter QC than raw serum but if the inputs are undefined, reproducibility has a ceiling. “Chemically defined” is the gold standard. Every component and concentration is known. This one tends to be safer, but there are levels. You’ll sometimes find purified bovine serum albumin or similar components, despite the fact that albumin is serum-derived and carries residual batch variability depending on purification and lipid loading. To be clear, this is how the category evolved rather than a criticism of any specific product, and perfect shouldn't be the enemy of good. People come to me because they’re over FBS for a lot of different reasons: Ethics. Reproducibility. Regulatory risk. Cost. Most existing serum replacements were designed to tackle one or two of these, not eliminate all of them simultaneously - fair. Still, when we built our replacement, we were deliberate about the vocabulary: fully chemically defined and fully animal-free, designed for reproducibility. I am regularly challenged on these terms by people who’ve been burned before - by products that were "reproducible" but not defined, or animal-free at the product level but not across the supply chain. And frankly, I love it, because I know our language matches the technical reality. The goal has always been to provide an alternative that can realistically compete with FBS on price, sustainability, regulatory AND experimental control/reproducibility across time, labs, and geographies. This vocabulary, and how we use it, matters a lot if we as scientists want to achieve that. To help prospective customers quickly understand the breadth of what FRS Pioneer can do, we've put together this helpful diagram. As always, please get in touch if you have any questions about using FRS Pioneer in your laboratory.  There’s a hidden modelling assumption baked into many AI- or ML-enabled biological datasets: If your models are trained on cell culture data, your media formulation is part of the model.
Here’s how a default choice I’ve seen teams make early on, accidentally becomes expensive to unwind later: When companies using cell biology to train predictive models are first established, cell culture processes are often given relatively minimal thought. Many companies treat this as basic infrastructure, while the critical part of the model sits downstream in protein folding, functional phenotypes, or biological performance metrics. Unfortunately, by starting from standard cell culture processes, many of these companies begin using processes that rely on fetal bovine serum or other undefined, variable cell culture media inputs. These model datasets are intended to become moats, but they’re only as robust as the conditions under which they were generated. Serum and other animal-derived nutritional supplements influence growth rate, shape stress responses, and impact metabolism, signaling, phenotypic baselines, and every facet of cell biology. From a modelling perspective, this means your training data can encode variability that isn’t obvious until conditions change. Unsurprisingly, this shows up in the data set as unexplained variability or performance drift. The major challenge is that this risk is easy to miss early on, when your model data is fairly limited, e.g. it’s been collected in one laboratory, using one batch of FBS, under “pretty constant” conditions. Usually, this risk rears its ugly head when datasets grow. Suddenly, you’ve used up your batch of FBS and you’re switching to a new one, or you’re transferring your findings to a different lab/company/site and the model’s results aren’t holding up. At that point, performance drift is sometimes blamed on “biology” or “model issues,” when it’s at least partially due to the “basic” cell culture processes that were popped in place three years ago. Reality is, if your experimental system isn’t controlled, your training data isn’t either. This will have implications for your model. My suggestion here is simple; think about this early! Media formulation should be a deliberate modelling decision, not a background reagent choice that we just roll into because “oh yeah, the literature says DMEM + 10% FBS so let’s go for it.” Teams that commit early to chemically defined, stable culture conditions are less likely to face costly or time-consuming surprises when models are applied, transferred, or scaled. Stable inputs in, more reliable models out, and a healthier data moat over time. That tends to keep everyone happy! 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/ |
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