FBS-Based Media — Is It Time to Transition Between Traditions?

“Without our traditions, our lives would be as shaky as a fiddler on the roof.”

— Tevye, Fiddler on the Roof           

Cell Culture Media

A Tradition That’s Difficult to Scale-Up

The more the world becomes topsy-turvy, the more we humans tend to struggle to be grounded, traditional. Whether dancing on a roof in Fiddler’s village of Anatevka—or teetering on the bleeding edge of biotechnology—you probably can cite a few odd traditions. Maybe you poke fun at them, wrestle with them, debate their future utility. But please don’t claim you have none, especially when it comes to nurturing your precious cell culture! If you don’t stick with “tradition” in that regard, how can you reliably predict their quality in future clinical trials where safety and efficacy is at stake?

One “tradition” that has worked well enough for 65+ years is fetal bovine serum (FBS).[1] This ubiquitous reagent—a by-product of the meat industry—is the basis for 600 to 800 thousand liters of yearly media supplementation across life sciences including diagnostics, biotech research, protein biologics manufacture, cloning, stem cell research, and vaccine production. It was noted 2014 that approximately 80% of IND submissions to the FDA involving hMSCs (mesenchymal stromal/stem cells) describe FBS use for their manufacturing. [2] This tradition continues even today.

As an enriched blend of growth, survival, and adherence factors, FBS’ 20th Century breakthrough could enable culture of almost any cell type with the right balance of supplements. It’s no exaggeration that FBS has been foundational for $100s of billions in annual global economic activity, playing a role not unlike Haber-Bosch processed and/or pit-mined NPK fertilizers for the world’s breadbasket farmlands. Yet like NPK blends, [3] are there signs that regenerative medicine can less afford FBS as high-quality sources become scarcer? [4] Can “cultivators” of hMSCs and other cells transition to a new, future-facing “tradition” and maintain or exceed previously optimized quality attributes? You can probably guess that the answer to both these questions is yes. Here in this blog, we’ll show you why.

“Something’s Gotta Give”

Consider a “back of envelope,” round-number calculation. Not even counting experimental CAR-T therapies, doses of many adherent cellular therapies or their derived exosome/EVs can approach one billion cells per patient. 1 billion may seem high, but this number factors in overfilling, lot testing, failed batches, and pre-production development runs. Let us next generously assume that media productivity is as high as 500,000 cells per liter and that these are supplemented with 5% FBS. That could potentially supply 8 trillion cells, or 8000 doses per year. Wait!Not so fast! Only about 200,000 liters (not 800,000) of these FBS products are of GMP quality. It turns out that a very limited number of suppliers provide serum that has been obtained in suitable, International Standards Organization (ISO)-grade environments. As stated in a review article titled Peak serum: implications of serum supply for cell therapy manufacturing (2012), [4] “serum destined for the production of cell therapies has to undergo rigorous testing and be thoroughly documented in terms of place of origin, specific details of the collection facility, government-approved establishment number, and information regarding the amount and date of collection.” Thus, even if average cells per dose is counted tenfold less, the number of regenmed treatments that can be supplied by clinical-grade, FBS-containing media is likely in the low-10s of 1000s per year range.

~20,000 doses per year could perhaps meet the needs of a rarer indication such as GvHD—or improve the outcomes of the ~2500 patients (in the USA, alone) who would otherwise be implanted with an LVAD for heart failure. However, it would be nowhere near sufficient to assist the 10 million worldwide with Parkinson’s disease, or the more than 15 million suffering annually from strokes, or even the 100,000 who die annually in the USA’s ICU wards, alone, from acute respiratory distress syndrome (ARDS; prior to COVID-19). If demand suddenly increased with back-to-back approvals of several cell therapies directed at major disease indications, scarcity could surely follow. Calling to mind Frank Sinatra, he once sang, Something’s Gotta Give. Isn’t the whole point of industrialization in life sciences [5] to bring abundance, breaking the deadlock between Sinatra’s ♬ irresistible force ♫ (demand for better health) and an ♫ old immovable object ♬ (scarcity of raw materials). How do we do that?

Adherent cells like hMSCs have several basic needs. These include nutrient metabolites, a carbon source, and co-factors, delivered in an isotonic and buffered, pH-stable basal media. The cells also need signaling factors that confer survival (anti-apoptotic) and pro-mitogenic effects—often tailored for the kind of cell being expanded. And then they need a surface to grow on that not only facilitates “sticking” but also spreading to avoid anoikis, in such a way that the cell of origin’s morphology and identity are maintained [6]. For most cell types, FBS contributes mitogenic activity at higher percentages (%v/v), but it’s also sufficient to promote survival and adherence at much lower percentages. Unfortunately, heavy reliance on FBS does not guarantee genomic stability [7] of primary cells, ex vivo. Fortunately, there are solutions offered by RoosterBio and others that can greatly reduce or eliminate the use of “traditional” FBS in specialized culture media. Further, we have pioneered media efficiency to the extent where our cell expansion protocols do not require media exchanges; this not only reduces media use but also is more environmentally friendly. [8] Methods to eliminate bovine serum and increase cells per ml of media include the use of GMP-friendly sources of potent factor additives and/or optimized growth substrates.

Media Formulations Changing Hands

Sunrise, Sunset.” It can be difficult letting go of the past, but times change; new cell scientists choose their own ways that best fit their needs. Yet, looming scarcity aside, other compelling reasons explain why many regulatory agencies and major producers of cell culture media are advancing beyond FBS. Have you ever wondered why the conversion rate from Phase III to FDA approval is much lower for cell therapies (14.3%) than for other classes of pharmaceuticals (48.7%)? [9, 10] The conventional wisdom is that higher-powered studies from more patients fail to replicate efficacy. On the other hand, an equally valid reason is that many cell therapy products manufactured at small scale can longer replicate the same critical quality attributes (CQAs) demanded by larger-scale Phase III trials and the market. Can it be that the successful Phase II product is simply not the “same” as its failed Phase III counterpart? This may partly be due to batch-to-batch variability in lots of FBS, making it challenging to quality control biological output via this “black box.” [11]

Hence, it’s now sensible for many developers to override the variability of serum’s pleiotypic biofunctions and replace them with known components of known quality and activity. We’ve written previously on this topic, [12] showing that both our optimized commercial formulations in either low-FBS or “xeno-free” (XF) formats can clearly outperform competitor media that are FBS-based or serum-free. When “hidden costs” are posited, the new costs and timelines of therapeutically relevant expansions end up much reduced via high-performance media. [13] This is partly because these fine-tuned media enable lower doubling times to reach higher cell densities toward the target population doubling level (PDL). [14] With no media exchanges required, there’s also less media volume and lower risk of human error or culture contamination. [15] Further, these built-in cell engineering tweaks facilitate a more seamless transition to a higher production scale—such as the move from two-dimensional cell stacks to 3D bioreactor cultures. Although it’s well and good to be mindful of waste, the “green premium” associated with removing FBS is probably far lower than you think. [8]

Nudging New Traditions Toward One Health

The “One Health” concept [16] is closely linked to the adoption of lower-waste cell culture media. Recent experience reminds us that the biosphere, our food and companion animals, and human beings are profoundly interconnected. Not only is bovine serum linked to xenogeneic immune responses, [17, 18] there’s also the trouble with zoonosis via unknown or emerging microbial or prion diseases! [19] Although BSE is now tightly monitored and under control, it has been estimated that between 20-50% of commercial FBS is virus-positive. [20] This is not to say that most FBS lots have contaminants that are pathogenic to humans, but it may well caution that the next zoonotic hazards might not be ones that we can readily detect—or are even aware of.

Alternatives to replace FBS do exist and are in commercial/clinical use today. [21] Among them, human platelet lysate (hPL) is currently the most attractive option. It’s also conceivable that—with sufficient R&D—media and cell attachment substrates from recombinant or GRAS (“generally recognized as safe”) cGMP sources will one day compensate effectively for total serum-free conditions, without FBS or platelet lysate. In the case of immortal or transformed cell lines, some cells (e.g., CHO-K1 and HEK-293) can readily adapt to serum-free media and attachment-free growth with relatively few additives. For many adherent primary cell types, however, these so-called “chemically defined” media are “easier said than done” in terms of resulting cell quality output or cost. That is not to say that new innovations aren’t forthcoming. The multi-billion-dollar investment into so-called “clean meat” that is based on multi-layer, multi-cell type co-culture may initially sound like another “moonshot” out of stargazing Silicon Valley executives, but what happens when it finally reaches fruition? The spinoffs will surely reverberate into regenerative medicine, including its media.

Now, about that Fiddler on the Roof. The “everyman” character. Tevye says, “You might say every one of us is a fiddler on the roof trying to scratch out a pleasant, simple tune without breaking his neck…And how do we keep our balance? That I can tell you in one word: Tradition!”

Isn’t there a little bit of Tevye in each of us, especially cellular therapeutics manufacturers? We all need our traditions, but we don’t need to throw them away, merely modify them a little: replace one imperfect good with a greater good. Like electric lights in lieu of whale oil—or high efficiency, xeno-free media in lieu of FBS—we can still keep our balance, and in turn craft a superior art along the way.


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  12. RoosterBio. Comparability of hMSC Economic & Quality Attributes after Expansion in Bovine Serum-Containing vs Xeno-Free Bioprocessing Media Formulations. 2016; Available from: https://www.roosterbio.com/blog/comparability-of-hmsc-economic-and-quality-attributes-after-expansion-in-bovine-serum-containing-vs-xeno-free-bioprocessing-media-formulations/.
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  16. One Health. Available from: https://www.cdc.gov/onehealth/index.html.
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  22. Image adapted from M., L., 2011. Glasgow MEM cell culture medium. https://upload.wikimedia.org/wikipedia/commons/1/10/Glasgow_MEM_cell_culture_medium.jpg [Accessed 18 May 2022].

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