A guest blog by Mary Lee McKay , Technical Applications and Marketing Associate, InVitria

Despite the fact that xeno-free is a term most cell culture scientists hear regularly, there is considerable confusion around what that term actually means. Ever since the dawn of the effort to remove fetal bovine serum from media due to its complex and variable nature, there has been a variety of terms put forth to describe varying levels of animal or human components in media. While most of these terms began as a way to market new medium products and distinguish them from those containing serum, the result has been that without standardized definitions, it is difficult to know what components to expect in your media formulation. For example the terms serum-free, animal-component free, protein-free and chemically-defined all mean different things but can overlap and, depending on the media manufacturer, these terms can be used differently or even interchangeably. For example, if a media is animal-component free, can it still contain human serum derived ingredients? After all, humans are animals. If a media is protein-free, can it contain peptides? Do small proteins count? These are some of the many questions cell culture scientists face as they select new media. With these not-so-standardized definitions, it is critical that cell culture scientists read the ingredients to be sure that their understanding of the terms match the media supplier’s definition.

With so much confusion about what xeno-free actually means, you can see why it would be difficult for cell culture scientists to associate any significant value with that term. Understandably, without being able to identify significant value, scientists can’t be expected to change their protocols and adjust their processes to move away from culture that includes FBS. If these terms are well-defined and generally uniform throughout different sectors of the industry, it will help the scientific community to at least understand the value of, if not implement these ideas. The benefits of an animal-free production system are numerous and significant, so it is important to clarify these concepts.

So let’s focus on xeno-free, what does it mean?

What Xeno-Free does mean:

The term “Xeno” comes from the Greek “Xenos” meaning strange. Xeno-free (or Xenogeneic-free) would therefore mean free from “strange” components, or components from a “strange” species (strange being relative to the native species you’re working with). In terms of cell culture, this would mean human cell lines can be cultured using human-derived components (like human serum), and it is considered xeno-free, since there is no difference between species. This allows for the elimination of fetal bovine serum, the most commonly used serum type for the culture of mammalian cells. But what if you are culturing VERO cells (kidney epithelial cells extracted from the African Green Monkey)? What is a xeno-free VERO medium? According to the definition, medium that contains fetal bovine serum, human serum, or any serum or component derived from said serum that is not from the Chlorocebus sabaeus species would be xeno-free. However, when the term xeno-free is used, humans are typically considered to be in one category, and all other animals fall into the second category. This means that xeno-free media can contain human serum-derived components, but no components from animals other than humans.

What Xeno-Free doesn’t mean:

Xeno-free does not mean serum-free, as some contain human serum. Xeno-free does not even guarantee animal-free, as most scientists define humans as animals. Xeno-free does not mean blood component-free as human blood components are often included. So despite all the nuances, most standard definitions of xeno-free mean that the media contains human serum components, but not non-human animal components.

Why is the xeno-free definition important?

In any scientific endeavor it is important to have a clear understanding of all the variables involved. Not only is this important for reproducibility of one’s own work, but it is also important when others try to reproduce your work. If we are not all playing by the same terms or using the same definitions, then these efforts to reproduce studies or even conduct fair comparisons of media or methods becomes extremely difficult.

Not only that, but the removal of fetal bovine serum is a big deal, and when xeno-free is mistaken to mean animal-free as well, actual animal-free formulations are discredited. Xeno-free and animal-free are very different terms, and in order to make the best decisions about cell culture media, this needs to be considered.

Why the xeno-free definition is important to cell culture decisions?

An increasing number of cell culture scientists, particularly in Stem Cell Research and vaccine manufacturing applications, are moving to animal component free media formulations. They are moving in this direction because there are significant benefits in removing these components, including removal of potential contaminants and improved consistency in both performance and quality. As scientists make changes to remove these ingredients, it is important to understand the benefits and limitations of media that is xeno-free. While removing one of the main culprits of inconsistency and raw material quality issues, fetal bovine serum, the label xeno-free does not necessarily mean mitigation of all potential contaminants or high consistency from lot to lot.

Therefore, one could argue that xeno-free doesn’t really solve the problems that come with the use of fetal bovine serum. In fact, it is a common misconception in the industry that a xeno-free media would be free from all serum or all blood-derived components. Really, xeno-free media uses human blood serum or platelet lysates to replace the typically bovine-sourced ingredient. While this eliminates several possible contaminants (in theory), a few human contaminants that would cause problems in cell culture and downstream production come to mind. Not only the ABC’s of hepatitis, HIV, and the recently transfected Zika virus, but even new and emerging viruses we don’t know about yet. While expensive testing can be done to verify the absence of some of these viruses, there is no way to account for the viruses that are not currently known. Even the manufacturers and distributors of human serum recognize that “no known test method can offer complete assurance that products derived from human blood will not transmit infectious disease” right on their website.

Possible contamination aside, the degree of variability between suppliers, lots, even bottles of human serum is extremely high. There are so many factors that go into each bottle of human serum, from the number of patients to patient health, age, weight, diet, ethnicity, genetic history, and beyond. (That explains why the FAQ section of a human serum supplier’s website has ‘Why do different bottles of human serum sometimes look different from one another?’ as a top question). Some suppliers offer human serum from all male patients or qualify their product by saying X number of human blood samples were collected to create the lot, therefore the genetic diversity should be minimal, and to be honest, this is fine. This is no different than fetal bovine serum suppliers who offer serum in varying levels of quality like heat inactivated, gamma irradiated, Australia or New Zealand sourced, etc. If serum is the product you want to use, these characteristics can help to limit diversity and give you a better idea of what to expect.

If the research being conducted doesn’t require a high level of consistency or isn’t an application where contamination risks would be of concern, the fetal bovine serum or human serum in the media probably doesn’t matter and in those applications researchers would be just fine using serum. In those cases, the definition is probably not critical. But if the goal is to remove all animal-components (including human) then having a clear definition of xeno-free is crucial. Some cell culture scientists feel that replacing fetal bovine serum with human serum allows for a sigh of relief that all of the problems with serum have been eliminated. Except…we just learned that the problems aren’t completely resolved.

So, if xeno-free doesn’t solve all the challenges associated with serum, what can?

Animal-free (or Animal Component Free, Animal Origin Free) cell culture media formulations can provide the key elements needed for cell growth and performance equivalent to that of serum-containing media, without the disadvantages that come from using serum. Animal-free media formulations are well-defined and contain a finite list of traceable ingredients, causing the variability to go way down. Not only is the risk of prions and other viral contaminants mitigated, but this ultimately corresponds to regulatory advantages and increased speed-to-market. These benefits have been widely reported in CHO cell based biomanufacturing.

There are some cases where the use of animal components is part of the only option for providing life-saving treatments to patients, and in these cases, they are often approved by regulatory agencies. However, replacing animal-derived components with animal-free components should be incorporated into cell culture whenever possible. It is better to recognize potential issues and implement animal-free cell culture from the very beginning than it is to look back and think about what could have saved your time, your product, or potentially your company.

How to implement Animal-free Culture

The implementation of animal-free cell culture practices is a tool that can be used by scientists who value consistency, reproducibility, and effectiveness in their research and product manufacturing. In our previous blog, “Cell Culture Media Optimization: Making the Perfect Magical Potion”, we explained that it doesn’t take unicorns and fairy dust to develop an animal-free cell culture media that meets all of your upstream cell culture needs, and simplifies all of your downstream processing efforts. Creating a cost-effective, reliable, animal-free cell culture media is a very real opportunity for increasing consistency and mitigating risk without sacrificing performance. With well-defined, recombinant, animal-free ingredients like Cellastim (recombinant Human Serum Albumin), Optiferrin (recombinant Human Transferrin), and ITSE Animal-Free (Insulin-Transferrin-Selenium-Ethanolamine), there is really no reason not to switch. The regulatory advantages and increased speed-to-market of products made from animal-free systems make it difficult to justify a switch from FBS to xeno-free. To really obtain the benefits and advantages of eliminating fetal bovine serum, switch to an animal-free media formulation. For answers to questions about converting to an animal-free cell culture media, or for help with formulating a custom animal-free media, call 1-800-916-8311 or e-mail CellCultureInfo@InVitria.com.

For additional reading, please see:

Xeno-free, What Is It?