Cell culture media demystified for performance, quality, and innovation

Cell culture media optimization can improve your cells’ growth and production. But a cell culture medium can seem like a black box. It’s a mystery solution that you put your cells in that (hopefully) allows them to thrive. But many people don’t know what’s in the media and how it affects their cell line’s performance. So where does one even start with media optimization? And how can you be confident in your media’s quality? Read on to find out what four cell culture specialists have to say.

Understanding cell culture media

In biotherapeutic development and manufacture, cells are used to produce a variety of biologics including monoclonal antibodies (mAbs), recombinant proteins, and viral vectors (VV). Cells do this hard work within a medium, which is any liquid or gel that supports the growth of cells outside of an organism.

Media is therefore designed to provide the cells with both a hospitable environment and the tools they need to do their jobs. Glucose to feed the cells. Amino acids to use to build proteins. Buffers to stabilize pH. Antibiotics to prevent bacterial and fungal growth. Growth factors and hormones to support cell function. And so much more. A medium may contain 60 to 100 different components!

Fig 1. Cell culture media may all look similar, but their composition can vary widely.

Many types of media exist. Some media are natural such as biological fluids, tissue extracts, and clots, but synthetic media are most commonly used for developing and manufacturing therapeutics. Synthetic media can be broadly categorized into five groups.

• Classical media: contain amino acids, vitamins, salt, glucose, and other nutrients cells need; often used as a starting point; requires 5% to 10% serum supplementation to be complete; examples: Dulbecco’s modified eagle medium (DMEM) and minimum essential medium (MEM)
• Serum-free media with animal-derived components: contain no serum for greater reproducibility but still contain animal derived components; example: Cytiva SFX-insect and SFM4CHO media
• Animal-derived-component-free (ADCF), serum-free media: contain no animal-derived components for greater reproducibility and lower variability; safe from zoonotic diseases; contain hydrolysates and/or yeastolates
• Chemically defined media: ADCF, serum-free, and hydrolysate-free media that contain known quantities of all components; provide greater control and even greater reproducibility
• Custom media: designed and optimized for a specific cell line or a specific use; designed to meet customer requirements (e.g. ADCF, protein-free, chemically defined, etc.)

Media’s impact on cell growth and productivity

“I think the biggest challenge people face when working with HEK293 cell lines is to find the right medium that allows the cell to grow nicely with a short population doubling time, keeps aggregation levels low, provides high peak cell densities, and maintains transfectability and infectivity at scale. You need to find a medium that combines all of the above and does it all well,” says Pär Larsson, Cell Culture Field Application Specialist at Cytiva.

Pär also highlighted the importance of having a medium that works for the entire process. Sometimes you might find that a particular medium is good for cell growth for your cell type, but another medium is better for production. Switching between the two media would require adapting the cells to the new medium, which is a complex process that adds time to your schedule. Sticking with one medium streamlines your process. “The best possible medium is the one that fits your cells, your needs, your process, and finally also the product,” Pär explained.

One way you can improve your cell culture medium on your own is to add boosters or enhancers to your media. Boosters and enhancers can either increase the number of cells in the culture, increase the productivity of individual cells, or increase both attributes to achieve higher productivity. These products can be helpful but may not be enough to allow your cells to reach their full potential. To achieve that, you may need a custom media developed specifically for your cell line.

Using custom media to optimize your cell culture

Custom cell culture media is media designed specifically for your cell line and your desired product. A media manufacturer will collaborate closely with you to fine tune the medium to optimize cell growth and productivity. Off-the-shelf media seem like black boxes because their formulations are often closely guarded trade secrets. Having custom media offers the possibility of access to the media formulation and the chance for further optimization of the formulation that work best for your cells.

“Cells are very different,” says Zhou Jiang, Director of Cell Culture Media Services. “That means cell lines actually require very different nutrients. Certain cell lines may require a particular nutrient more than other cell lines.” Zhou explained that his team adjusts nutrients to maximize cell growth and uses analytics on metabolites such as lactate to monitor the health of the cells. But the team’s methodology is more than just balancing nutrients. “We look at the product quality attributes. We look into trace elements.” These elements are present in very small concentrations but are critical to the function of the cells and have a significant impact on the resulting protein’s quality attributes. Performing analytics on cell culture media is key for optimization. These studies allow you to understand the nutritional requirements of your cell line so you can extend cell viability and increase their productivity. For example, spent media analysis can check leftover amino acids and carbohydrates to determine possible limiting factors (Fig 2).

Fig 2. Performing a variety of spent media analyses can help you optimize your cell culture performance.

When Zhou’s team works with a client, even the collaboration process is tailored to the client (Fig 3). The collaboration starts with meetings in which our scientists listen to the client to learn their processes, challenges, and desires. If you’re willing to send your cells to us, the team will use the cells to first replicate your processes and data and then test the cells in different media.

Because a medium may contain 60 or more components, it is not feasible to test them individually. Zhou’s team uses a variety of techniques to create your medium. They use design of experiments (DoE), high-throughput screening, and advanced analytics to develop media formulations. After using these tools to design a starting medium, they will then tweak certain components to optimize the medium even more. The team’s knowledge and experience help with this fine-tuning stage.

Successful media optimization can have a big effect on your cells’ performance. Depending on your original process and medium, you could see a two- to four-fold (or more!) increase in productivity.

Fig 3. Custom media development often has similar parameters, analysis, and testing techniques, but how the process is conducted is varied to suit your requirements.

Quality, scalability, and consistency in media development

When selecting media, you might have concerns about the manufacturer’s ability to produce the media at scale. This concern is amplified for custom media. Can a manufacturer produce large quantities of a custom medium with the same quality as its initial formulation? Drue Kunz, Sr. Director of Bioprocess Cell Culture Media understands this question. He explains, “when you're in the developmental phase, you're typically working with very small quantities. And you must have equipment that produces that effectively. But then when you take that to 6500 kg of powder versus 10 g of powder, that's a very different proposition.”

“You have to make sure that your formula is sound to scale from development quantities and research quantities up to full manufacturing quantity. That's crucial. But you've got to have the equipment to be able to produce it and render that same product in a consistent way,” continued Drue. He explained how our manufacturing facilities are set up with equipment of various sizes that can consistently produce the same product at any scale. Furthermore, the various Cytiva manufacturing facilities use the same equipment and processes so that clients can obtain the same product no matter where it was produced. “The equipment is managed, maintained, and validated all in the same way to yield consistent results.”

Quality and consistency of the raw materials used in media is also important to achieving batch-to-batch and within-batch consistency. A media manufacturer should check the quality attributes of the raw materials as well as the quality attributes of the final product.

Surprisingly, the packaging of the media also affects the quality and performance of the media. The right packaging protects the media from humidity, contaminants, and damage during transport and storage. The right packaging also allows the user to access the media without compromising the consistency of the product. For example, you might want a container that you can connect directly to your bioreactor. Or you might want one that lets you easily add additional ingredients to the media before use. By customizing your packaging, a good media manufacturer can protect the quality of your optimized media.

Future trends in cell culture media optimization

As the bioprocessing and biotechnology fields grow, the need for customized media is also growing. “CHO cells have diverse metabolic needs and important variations exist within each CHO cell line that need to be understood in order to optimize performance. For example, the nutritional requirements for a CHO-GS line will be different from a CHO-K1 cell line,” stated Jeron Larsen, Senior Scientist in Cell Culture Media R&D. As a result, the desire for faster custom media development is high.

According to Jeron, companies are now exploring the use of machine learning and artificial intelligence (AI) in media development. “We are very interested in using machine learning to speed up development timelines and increase performance. We’re using that technology to help our customers be as efficient as possible,” he said.

One possible use of machine learning is to help developers understand which molecular pathways are most important to the productivity and efficiency of a cell line. A cell is very complex and doing analytics of every aspect of its function is not feasible.

Another trend being explored in media development is the use of predictive models. The idea behind these models is to use early results of a fed-batch to predict how the cells will perform further down the line. Time will tell if these models will help media developers reduce their development cycle, which would in turn allow faster results for them.

All these ideas sound exciting, and Jeron is eager to see which ones will pan out. “Right now, I think the ability of AI is comparable to what can be done with traditional DoE and metabolic analysis. But, what’s coming is going to be really exciting!”