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Going Serum-Free: The How and Why of Removing Serum From Your Media

A bottle of serum.

Culture medium is probably the most critical aspect of cell culture, and many media recipes or commercial media preparations are the results of years of refinement. One common additive for 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’ll discuss the pros and cons of using serum in your cell culture media and how to remove it if you want to eliminate it from your workflow.

What Is the Importance of Serum in My Media?

Media contains vitamins, sugars, and buffering agents to maintain the health of the cells. [1]  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 essential for cell proliferation and growth.

If you remove the serum from your culture medium, you will notice a big difference. 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 serum can keep your cells healthy and happy, there are some notable downsides.

Serum Is Highly Variable

For starters, serum is an animal-derived natural product, and each new lot of material will vary.  While serum does contain items 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. [2].  Of course, heat will also 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 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 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.

Serum Can Be a Source of Contamination

Another potential issue with serum 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.

Serum Is an Animal Product

Finally, one other consideration for going serum-free is to support the three R’s – refinement, reduction and replacement – for the use of animals in research.

Are There Negative Effects of Going Serum Free?

Maybe you are convinced that going serum-free is the best approach for your cell culture.  But like many things in science, it comes with trade-offs, and one of them is finding exactly the right mix of growth factors to support your culture.

The needs 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 culture 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. [3]  Various manufacturers of culture medium have developed specific serum-free formulations for standard immortalized cells 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 foetal calf serum. [4]

Direct Adaptation

One approach to going serum-free is simply to switch from serum-containing medium to serum-free medium.  In a guide from ThermoFisher, they suggest 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. [3]  If you are working with a cell line that can tolerate the direct switch, going serum-free will be relatively painless.

Gradual Adaptation

But, if you can’t quit serum cold turkey, then you will have to sub-culture your cells in a mixture of serum-supplemented and serum-free media, increasing the fraction of serum-free medium with each passage until you passage into 100% serum-free medium.

Another approach is to supplement mix serum-free medium with 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.

Hopefully, you have found this article helpful.  Please let us know if you are considering or have gone serum-free and please share any tips you have below in the comments.


  1. Lynnea Waters. What’s in Your Cell Culture Medium? Bitesize Bio. 25 October 2017.
  2. Lian Chee Foong. Heat Inactivation of Serum for Tissue Culture – Is it Necessary? Bitesize Bio. 6 May 2015.
  3. Timothy Fawcett. Serum-Free Conditions. Cell Culture Dish. 23 April 2013
  4. FCS-free database. 3Rs-Centre Utrecht Life Sciences. Accessed: 07 March 2020.
  5. Adaptation of Cell Cultures to a Serum-Free Medium. Thermo Fisher Scientific. Accessed: 02 February 2020.
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