Media Selection, Optimization, and Prep

Cell culture media has evolved to meet the demands of “working” cells that produce therapeutic proteins, cell and gene therapies, and vaccines. As cell types and end uses have multiplied, so do options related to media composition, optimization goals, and physical formats.

The starting point is always the same: Use a medium that is best suited to the cell type. Investigators typically begin with essential medium for adherent cells and RPMI-1640 for suspension cells, followed by tweaking to maximize expansion, productivity, etc.

To sustain growth and productivity, high-performing cell growth media should mimic the production cells’ natural environment, whose nutritional milieu includes a carbon source (e.g., glucose), vital nutrients (carbohydrates, lipids, amino acids, and vitamins), hormones and growth factors, and salt for pH and osmotic pressure regulation.

“In classical medium formulations, serum supplies these components,” notes Isabelle Robert, Ph.D., Research and Development Scientist at Proteogenix. “But due to its animal origin, classical media also carry substantial risks such as high batch-to-batch variability, contaminants, viruses, and toxins.”

Newer cell growth technologies overcome these shortcomings by replacing serum with chemically defined nutrients but even these, when selected sub-optimally, can lead to low yields and slow cell growth.

Aside from the most basic requirements for cellular nutrition and growth, optimizing culture media formulations involves experimentation. Media that are optimal for one expression system, cell line, or production requirement may perform poorly under different conditions or circumstances. That is why cell line and expression system development often go hand-in-hand with specially formulated media to support the specific needs of the production platform.

The XtenCHO™ expression system from Proteogenix, designed for high-yielding transient protein expression, is just one example. XTenCHO includes a novel, eponymous cell line, expression system, and culture medium. XTenCHO achieves, in transiently-transfected cells, expression levels that would have been considered "pretty good" for stably-transfected CHO cells 20 years ago.

Just-in-time

Overnight shipping and cold chaining ensure that cells reach their destinations on time to meet a project’s objectives. These logistical concerns play into the overall economics of media preparation as well—for example whether to receive shipments of powdered, fully constituted, or concentrated media, or whether to invest in an onsite media formulation plant to deliver media on demand.

“Media plays a strategic role in the successful transportation of cell cultures, whether traveling across the state or to the other side of the country,” says Fang Tian, Ph.D., Director of Biological Content at ATCC.

In addition to shipping cells frozen in vials, ATCC ships live cell culture in flasks filled with media to allow users to begin working as soon as the culture arrives. “We overnight-ship our cell cultures and media at room temperature to reduce the chance of damaging the cultures,” Tian tells Biocompare. “Further, we add penicillin or streptomycin to all flasks, prophylactically, to prevent microbial contamination during transit, but we caution customers against reusing the transit medium after the cultures arrive. Fresh medium is recommended if shipment time exceeds two days or the shipping medium has yellowed.“

Since contamination is a significant contributor to inaccuracy and irreproducibility, media preparation requires stringent sterility control, which ATCC recommends throughout media preparation and delivery.

The reason this is required, even when protocols are strictly followed and environmental conditions maintained, is that with culture media appearances can be deceiving. “Media and cultures do not always show signs of compromised sterility when antibiotics are used,” Tian explains. “While antibiotics such as penicillin or amphotericin B reduce the likelihood of contamination we advise against their prolonged use since they may, over long time periods, harm the cells. In some instances, overgrowth can affect normal cell processes and even induce changes in gene expression. That is why we recommend comprehensive sterility testing of cells and media to detect the presence of bacteria, fungi, or mycoplasma at the earliest stages. If strict aseptic technique is employed during cell growth, contamination should not occur.”

Aseptic processing is essential

Media is used or supplied in several standard formats, each with its pros and cons. Media in powdered form weighs the least and takes up the least space, a factor that positively affects transportation costs and logistics. But these benefits come at the cost of greater effort (and complexity) in reconstitution.

“The key here is maintaining an aseptic environment while hydrating the powder, and assuring homologous mixing of the components,” says Vivek Mallick, Product Line Manager for Custom Media at Corning Life Sciences. “This is a critical step, as even the slightest mishandling may risk contamination of the entire lot. This operation is best suited for clean rooms that allow addition of water in a closed, aseptic environment. Concentrates readily mix with the solvent but require proper storage conditions to prevent precipitation. Liquid formats are the easiest to work with directly as they are directly applicable to cells in a controlled environment. Liquid media carries the lowest risk since it comes directly from cGMP facility that conducts its formulation in a controlled environment.”

Many media formulations require addition of serum to boost cell proliferation. “But since it is animal-derived, serum shows variations that are not easily controlled,” Mallick explains. Serum-free media has a more chemically defined formulation that allows repeatable growth of cells without dependence on animal components.

When they were first introduced, animal component-free and chemically defined media were presented as compromise alternatives to serum-based products: easier to characterize and formulate, but lacking a certain something. That is no longer true, as these media types have been optimized for various end results. For example, Corning^® hybrigro™ SF defined, animal component-free medium is optimized for antibody production, and is superior in terms of protein yield to serum-containing medium. Researchers looking to lessen the impact of serum but who are not ready to give up that component entirely might look into Corning^® transfectagro™ Reduced Serum Medium, which allows at minimum a 50% reduction in serum use with minimal adaptation.