The best choice should be application driven and purpose formulated
The vast majority of laboratory-scale cell cultures are initiated for pure research. Labs that use one cell line throughout a project acquire the cells, authenticate them, and expand them sufficiently for the lab’s purposes.
Before industrial biotechnology, standard practice was to do whatever was necessary to get the job done as quickly as possible—for example, producing enough protein for subsequent assays or characterization, or sufficient cells for cell-based assays. This “quick and dirty” approach often involved transient transfection, which leads to reliable phenotypes that last for one generation only. Cell culture media evolved toward this purpose, typically including bovine serum as the main ingredient.
Increasingly, companies involved in biomanufacturing and even research groups are switching from serum-based products to animal component-free preparations and chemically defined media, both for transient and permanent transfection.
“In many cases they’re already thinking of what comes next, perhaps a clinical phase, and opting for more consistent chemically defined media, even at the earliest stages,” says Chance Lai, associate director for product management at Thermo Fisher Scientific (Frederick, MD).
Thermo Fisher offers starter kits—essentially, off-theshelf media for transient expression, in which specific ingredients at specific concentrations replace serum.
In addition to consistency throughout a project and the potential to use similar media at large scale, chemically defined media often are less expensive than serum-based products. “Fetal bovine serum is very expensive, a by-product of the meat industry. It suffers from price fluctuations, and its performance varies from batch to batch,” Lai explains.
“Research groups often test serum ingredients when they’re delivered and, if it suits their purposes, will buy huge quantities to assure consistency throughout a project’s lifetime. Unfortunately, serum has a finite shelf life. With defined media, they know what they’re getting every time.”
In the details
According to Cory Card, principal scientist at GE Healthcare (Logan, UT), the principles that guide media development for bioproduction apply equally to lab-scale culture media. This includes a fundamental understanding of the metabolic needs of the cells, including trace elemental components—particularly metals—which can make or break cultures at any scale.
For example, sources of elemental iron may contain trace quantities of manganese, an element essential for glycosylation of proteins by animal cells. But effective concentrations fall within a relatively narrow range: too much or too little can be equally bad.
Card relates the story regarding the manufacturing of a well-known classical cell culture medium originally developed in the 1960s, which had become popular for not requiring serum supplementation. Due to its success, a leading vendor built a new plant to support increased demand. To the manufacturer’s disappointment, the product from the new plant did not adequately support cell growth without serum supplementation, thus defeating the medium’s major benefit.
The difference was water quality. Water delivered to the original plant contained impurities—metals that enhanced CHO cell growth, but were lacking in the “purer” water at the new facility.
Laboratory researchers are more cavalier about stringent specifications around cell culture performance, so they may not be as aware as biomanufacturers of sources of variation, including trace components, according to Card. “That is,” he explains, “until they find their cells don’t perform as expected.”
Stem cell media: Less is more
Media, feeds, and supplements appropriate for laboratory or production cells support cell growth, viability, and protein expression. Users begin with a stock, off-the-shelf medium, or a literature prep, and add ingredients to their liking, including serum. Formal medium development becomes more attractive for industrial or therapeutic biotech than in academic labs, where the objective is to grow the cells and do the experiment.
Relative to their somatic, primary, or immortalized counterparts, stem cells have unique media requirements based on their four principal functions: maintenance, expansion, differentiation, and downstream applications.
Maintenance involves keeping the cells in their original state long enough for experimentation or analysis. Through expansion, investigators seek to increase the numbers of cells for direct or downstream applications.
Inducing stem cell differentiation has been an empirical exercise. Turning embryonic, induced pluripotent, or mesenchymal stem cells into bone, nerve, or heart cells involves the addition of specific chemical messengers, cytokines, and supplements to coax cells into the desired lineage.
For these three applications, what’s absent from the culture medium is just as important as what’s inside. Stem cells represent a kind of moving target: they’re always trying to be something they are not. Culture media must therefore provide nutrients sufficient to maintain cells in the desired state but nothing more. “Stem cells are pluripotent by nature, which makes them unstable in culture,” says Angel Garcia Martin, CEO of StemTek (Bizkaia, Spain). A good deal of culturing involves maintaining the cells’ “stemness” until the right time.
Commercial media are now available for most stem cell types, including cancer stem cells, StemTek’s specialty. Major suppliers include BD Biosciences, Glycosan BioSystems, Lonza, MilliporeSigma, Miltenyi Biotec, and Thermo Fisher Scientific. A number of companies supply cells and either media or analysis tools.
Keeping in mind the law of unintended consequences, stem cell media should contain only what is necessary. According to Martin, that includes serum, “which is undefined and contains ingredients that induce differentiation.”
For example, embryonic and pluripotent stem cells are maintained in a cocktail of growth factors, including “knockout serum replacement”—a chemically defined ingredient mix that maintains pluripotency and prevents differentiation. Some stem cells thrive in serum-based media— for example, mesenchymal stem cells—but this practice demands vigilance. Users must test batches of serum individually to assure they promote growth but not differentiation.
Stem cells destined for therapy can never use serum due to safety concerns for adventitious species-specific viruses, prions, and other microbiological contaminants.
The discovery of cancer stem cells, which fuel tumor spread and recurrence, has led to discovery efforts for drugs that eliminate these cells (conventional chemo and radiation do not). StemTek’s Cell2Sphere™ product provides pharmaceutical companies with microplated cancer stem cell spheroids, which are three-dimensional structures designed to provide physiologic relevance.
StemTek did not provide details on the media composition for Cell- 2Sphere, but other suppliers of cancer stem cell media divulge some insights. Researchers have used serum-free media for immortalized research lines for 25 years, and this idea appears to have taken hold in cancer stem cell culture. PromoCell’s (Heidelberg, Germany) aptly named Cancer Stem Cell Medium, designed specifically for sphere-forming cell lines (as are most cancer stem cell media), contains growth factors and supplements but no antibacterial or antimycotic agents.
Given the plethora of research and production cells, selecting the right cell culture medium can be bewildering. Luckily, online guides for media selection are abundant, and vendors offer detailed guidance for specific cell lines.
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