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So, you've moved your cells to a serum-free media and now they look bad. Been there 🙃 Here's what to check first, because your cells' disapproval might have nothing to do with the cell culture media formulation you've chosen.
1. Remove or reduce antibiotics by 5-10x FBS binds and sequesters common cell culture antibiotics, so the activity level in culture is reduced compared to serum-free cultures. "1X" antibiotics in serum-free culture might impact your cells more than you'd expect. 2. Gentle passaging wins FBS covers all manner of cell culture sins. Serum-free cultures often require gentler handling; minimise exposure to passaging reagents, keep cells and wash media warm, and try Accutase or TrypLE over trypsin. Trypsin also isn't inactivated by most serum-free media, so something to keep in mind! 3. Adaptation has an impact Weaning cells gradually into a new media is gentlest on the cells and typically recommended. If using a direct adaptation process, move the cells into their new media when they aren't facing any other kind of passaging or thaw-related stress. For adherent cells, this means letting the cells attach to the plate and enter the exponential growth phase before performing a 100% feed to new media. 4. Adherent cells lifting up from the surface, looking like a viability issue? Rule out an adherence issue before playing with the media formulation. Impaired attachment or spreading can very, very easily read as a viability issue, but requires a different trouble-shooting approach. Look for debris, check viability empirically, and consider eliminating FBS for the first time mid-passage, so (as much as possible) you can decouple the impacts of the media on viability/cell growth versus on attachment. Not totally comprehensive, but the check list I wish I had when I started working in serum-free media dev ~6 years ago!
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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 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! |
What's been happening?Sharing the Media City journey has been important to us because we want to encourage the next generation of scientists to establish companies that will advance scientific research. Check back regularly for the "building in public" updates on what it looks like to establish a scientific company. Archives
April 2026
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