Media Selection for Mammalian Cell Cultures

Florian Wurm of Switzerland's Polytechnic Institute created waves in the mid-2000s by attributing the rising productivity of Chinese hamster ovary (CHO) cell cultures almost exclusively to advances in culture media. Hardly anyone argues against that claim today, as monoclonal antibody (mAb) titers continue rising thanks to improvements in media, feeds, supplements, and general manufacturing methods.

A step-by-step guide to critical factors affecting selection of mammalian cell culture media would take up many volumes. This article therefore focuses on general strategies involved in media selection for mammalian production cultures, plus two applications that were in their infancy during Wurm's heyday: stem cell cultures and 3D cultures.

Focusing on cell stage

”In mAb manufacturing, cells function as protein production factories with a level of productivity and consistency required for therapeutic quantity and quality. While safety is an issue in mAb production, downstream purification isolates the process from patients" says Sasha Aleksic, Marketing Manager for Cell and Gene Therapy at FUJIFILM Irvine Scientific.

"By contrast, in cell and gene therapy manufacturing the cell itself is the therapy. Because media selection focuses on generating cells with the desired functionality and potentiality for direct-to-patient therapy, the need is great for risk-based management of raw materials."

Cell culture media contains nutrients, growth factors, and hormones, and helps control pH and osmotic pressure. Media that contains undefined components or serum can cause undesirable effects on cells during expansion or differentiation. Using a chemically defined medium provides more control and consistency in therapy production and reduces the risk of patient exposure to unknown components.

"This is why high-performance, chemically defined media are essential to the future of advanced cell-based therapies. Using raw materials that are strictly controlled and traceable helps smooth the transition from development to clinical applications."

Streamlining workflows

“Utilizing a single medium and feed, with minimal supplementation throughout clone selection, expansion, and production, allows more simplified workflows," according to Erica R. Wehling, Research and Development Manager at Thermo Fisher Scientific. "This approach can reduce expansion timelines as it eliminates the need for adaptation."

An initial screen for these ingredients, Wehling says, would focus on their concentrations, going from lean to rich formulations. "Spent media analysis, statistical modeling, principal component analysis, metabolomics, and proteomics are then applied to identify components which, when added or removed from the lead mixture, affect key responses such as cell growth, titer, and quality."

Depending on a clone's metabolic needs and the expansion method, a single medium may be suitable for both expansion and production without any changes to medium components or concentrations.

Then, when all targets are met for expansion and production, the clonal selection medium is developed using the lead expansion/production medium as a template. A number of components that support cloning (e.g., cell attachment) may either be removed, reduced in concentration, or added back as supplements.

"Utilizing this stepwise development approach improves the likelihood of success and reduces the number of media/supplements for the different stages of the upstream process," Wehling says. "This aids in developing a cell-line development workflow that will include a seamless adaptation or that can eliminate the need for adaptation altogether, reducing the COGs and simplifying the workflow.”

Quest for consistency

All decisions related to bioprocessing these days aim first at quality—which incorporates product homogeneity, predictability, and patient safety—and only when those criteria are satisfied, on productivity (with implications for yield and cost of goods). It therefore makes sense, at least to consider using SF and CD media during all development and production stages.

Although serum is essential for some applications, demand for CD and SF medias is growing, says Eva Nokes, Ph.D., Scientific Support Manager at Corning Life Sciences. She cites the usual reasons, primarily consistency and safety. "Users select these products to reduce lot-to-lot variability and to control for factors that may affect experimental outcomes or cell growth."

CD and SF media are often not plug-and-play but starting points. "They require optimization of supplements and their concentrations," Nokes says. The selection process begins with a classical medium or combinations of multiple classical media, to which trace elements, vitamins, alternate protein sources, and other factors are added. The availability of specialty basal media and supplements has greatly advanced the state of the art, particularly for complex biological drugs originating from cultured hepatocytes, hybridomas, stem cells, insect cells, etc.

Choice of supplier is a recurring theme in cell culture media selection, and all-important given that cells go through several distinct production stages such as clone selection, expansion, more expansion, and the expression of proteins or some other phenotype.

"There is sometimes a need to use multiple media types during the course of growth, such as in cell differentiation," Nokes says. "Here Corning recommends starting with an established protocol and optimizing to the specific cell type(s), which requires understanding which factors cells need at which stages." Often, one base media is used throughout, the differences being which supplements are added, when, and in what quantities.

The decision tree for 3D cell cultures differs somewhat from large-scale fed-batch CHO cultures, but the selection strategies are similar. With 3D cultures the goal is not expansion or productivity, but maintenance of the phenotype for the duration of the experiment.

Given the correct microenvironment, organoids will self-assemble from stem cells or progenitor cells into microscopic versions of parent organs. "To recreate this specialized functionality requires selecting media and reagents designed specifically for organoids, their component cells, and of course the all-important downstream applications," Nokes tells Biocompare. "Organoids have now been created for many organs and applications, and protocols that fully describe media, feed, and supplement requirements are available in the literature. Typically one will need an extracellular matrix (ECM) like collagen or Corning Matrigel for scaffolding, and appropriate growth factors and supplements."

Because 3D cultures are used for testing, not therapy, considerations for animal component-free or chemical definition in growth media are less urgent than in CHO/antibody manufacturing.

"There are many recipes in the literature for creating 3D culture microenvironments," Nokes says. "Users often begin with a classical media and add appropriate ECM, growth factors, and supplements specific to the organoid type and application. But the more complex the environment (as with 3D co-cultures), the more challenging the selection process. Fortunately, many suitable classical media are commercially available, as well as published protocols for using them as a starting point."

Custom Media?

Much of the progress in bioprocessing results from standardized processes and equipment including, to a degree, cell culture media. Thus, it is a reasonable assumption that one can optimize media by adding whatever magic sauce is known to work to an off-the-shelf complete media.

"This approach poses challenges," says Brad N. Taylor, Ph.D., VP of Marketing at Nucleus Biologics, which specializes in custom-developed media. Nucleus uses an AI-driven platform to arrive at optimal components and concentrations.

"First, the formulas of off-the-shelf complete media are often guarded, so the exact components and concentrations are unknown. It is a 'one size fits all' model that fails to account for the highly variable nature of cell culture. Optimization requires manual addition and, in some cases, depletion and extensive testing. Customers should always know what's in their media."

Both reproducibility and scalability suffer due to lack of transparency.

"Second, determining the exact components to add, deplete, or adjust to achieve a critical quality attribute requires extensive research and expertise."

By the saying, "you are what you eat," media transparency takes on new meaning for emerging cell-based therapies. "Changing one component of CAR-T cell culture media has been shown to affect potency corresponding to a full log reduction in tumor burden. Other positive benefits noted post-optimization include yield improvement and higher transfection efficiency."

Taylor makes a parting comment on the importance of supplier reliability, validation, vendor qualification, and the potential to source media at scale for the duration of a project. He cautions that manipulating an off-the-shelf medium is often a source of variability but with custom formulation the buyer owns the medium and can easily track ingredients and gauge quality, which equates to simpler validation and qualification.

"If the global pandemic has proven anything it is that therapy providers cannot be dependent on one source for their media. Owning your media formula is the best strategic plan to build an optimized and scalable system."