Going Serum-Free: The How and Why of Removing Serum From Your Media
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Serum is a highly variable product, and the variations between batches can alter cellular processes, impacting your results. Serum can also be a potential source of contamination. It is possible to either remove serum completely from your media as some cells with grow happily without it. Alternatively, you can replace serum with synthetic additives.
Culture medium is probably the most critical aspect of cell culture, and many media recipes and commercial media preparations are the results of years of refinement. One often-used supplement in cell culture media is serum, commonly fetal bovine serum (FBS). But do you know why you add this, and, more importantly, can your cells survive without it?
We discuss the pros and cons of using serum in cell culture media and how to remove it if you want to eliminate it from your workflow.
What Is the Importance of Serum in Cell Culture?
Culture media contain vitamins, sugars, and buffering agents to maintain the health of your cells. And you will often add serum, such as FBS. This is one of the main ingredients in cell culture media because it contains hormones, lipids, and growth factors that are essential for cell proliferation and growth.
You will notice a big difference if you remove the serum from your culture medium. Deliberate serum starvation is used to induce cell cycle synchronization, but take care when doing so, as starving your culture of serum for too long will also lead to reduced cell survival and increased apoptosis.
Why Should I Consider Going Serum-Free?
While using serum in cell culture can keep your cells healthy and happy, there are some notable downsides.
1. Serum Is Highly Variable
For starters, serum is an animal-derived natural product, and each new batch will vary. While serum does contain factors that are important for the health of your cells, it also contains things like immune complement proteins that can harm your cells.
To get around this, serum is usually heated to inactivate harmful proteins. Of course, heat will denature beneficial proteins too, which can increase the variability between media preparations.
While this might not be a big issue for some types of research, variability in factors that are critical for cell growth and survival can confound your results if you are running, for example, a cell-based assay measuring the effect of a growth factor or drug on a given cellular function.
In this case, one of the biggest reasons for going serum-free is to increase the standardization of culture conditions for more consistent and reproducible results. This type of standardization is even more critical in regulated laboratories, and using a defined medium allows greater control over your cell culture and your experiments. 
2. Serum Can Be a Source of Contamination
Another potential issue with serum in cell culture is viral or bacterial contamination. Again, lot-to-lot variability is in play here, and heat inactivation may not wholly neutralize microbial pathogens. Adding contaminated serum to your culture will affect the health and growth of your cells and render them unusable for experiments.
3. Serum Is an Animal Product
Finally, another consideration for going serum-free is to support the 3R’s—refinement, reduction, and replacement—for the use of animals or animal products in research.
What Are the Negative Effects of Going Serum-Free?
Maybe you are convinced that going serum-free is the best approach for your cell culture experiments. But like many things in science, this comes with trade-offs, and one of them is finding exactly the right mix of growth factors to support your culture.
The growth requirements of different established cell lines are varied and complex. Optimal culture conditions can even change between different passages of cells! Throw in primary cultures or cultures in suspension, and you will have quite the task in front of you.
Fortunately, you are not alone, and there are many expert sources online to guide you.  Furthermore, various manufacturers of culture media have developed specific serum-free formulations for standard immortalized cell lines like Chinese Hamster Ovary (CHO) cells.
Besides the difficulty in getting the serum-free conditions just right, other adverse effects of going serum-free include slower cell growth and cell clumping when passaging. Continued optimization of culture conditions can help with the reduced rate of growth, and gentle repeated pipetting of cell clumps should be enough to disperse them.
How to Go Serum-Free
Now that we’ve covered the pros and cons of going serum-free, let’s explore how to do it in practice.
Note that some cell types can be cultured in the complete absence of serum, while others will need other factors added to replace the role of serum in the media. Several serum alternatives are available to purchase that you can try as an alternative to completely removing serum; however, some cell types will have more unique needs that require adding specific factors to the media.
The FCS-free Database provides details of additives noted in the literature for specific cell lines to be cultured without FBS. 
1. Direct Adaptation
One approach to going serum-free is switching from serum-containing medium to serum-free medium. A guide from ThermoFisher suggests starting with a mid-log phase culture with >90% viability and sub-culturing when the cell density increases after several days of culture in the serum-free medium. 
Going serum-free will be relatively painless if you work with a cell line that can tolerate the direct switch.
2. Gradual Adaptation
If your cells can’t quit serum cold turkey, then you will have to sub-culture them in a mixture of serum-supplemented and serum-free media, gradually increasing the fraction of serum-free medium with each passage until you passage into 100% serum-free medium.
Another approach is to supplement serum-free medium with a conditioned medium. If you are not familiar with this term, conditioned medium is the medium that the cells were grown in for one full passage. You can recycle this and mix it with increasing ratios of serum-free medium until the cells are 100% adapted.
A Final Note on Serum in Cell Culture
There is no doubt that serum is one of the most common supplements in cell culture media, but it’s also the most inconsistent. The reagent industry has developed serum-free media to solve these problems. But going serum-free isn’t always practical or possible. Most serum-free formulations only work on a specific cell type, which can strain research budgets if your work involves multiple cell lines.
Hopefully, you have found this article helpful. Please let us know if you are considering or have gone serum-free, and share any tips you have below in the comments.
Originally published in April 2020. Reviewed and updated in October 2022.
- Baker, M. Reproducibility: Respect your cells! Nature 537, 433–435.
- Fawcett, F. Serum-Free Conditions. Cell Culture Dish. Accessed: 04 October 2022.
- FCS-free database. 3Rs-Centre Utrecht Life Sciences. Accessed: 04 October 2022.
- Adaptation of Cell Cultures to a Serum-Free Medium. Thermo Fisher Scientific. Accessed: 04 October 2022.