In May, scientists reported the successful reprogramming of T regulatory cells, transforming the normally immunosuppressive cells into inflammatory ones that both permit and magnify the antitumor immune response. And chimeric antigen receptor (CAR)-T cell therapy, which genetically alters a patient’s T cells to target tumor specific markers, continues to advance, offering a promising option for patients when other treatments fail. As such, the field of specialized immune cell research is revving up like never before. Choosing the right media is critical to optimizing T cell expansion, differentiation, and assays.

The T cell system is part of adaptive immunity. When exposed to antigens, T cells become activated, divide rapidly and secrete cytokines that regulate the immune response. They also differentiate into one of sundry subtypes. The system includes CD4+ T helper (Th) subsets (Th1, Th2, Th9, Th17, Th22, Treg(regulatory), and Tf(folicular)h) and CD8+ memory and effector subsets.

“There are many types of T cells that researchers are interested in culturing. The variety of applications are endless,” says Jessie Yu scientist at STEMCELL Technologies. “They can use ImmunoCult™ T cell differentiation supplements or use various cytokines and growth factors to study the biology particular subsets of CD4+ T cells, including Th1, Th2, and T regulatory cells. Researchers may also be interested in expanding T cells that have been genetically edited using technologies like our ArciTect™ CRISPR/Cas9 system.”

Anne Lodge, founder of Astarte, says they provide all types of T cells, from primary pan T cells to CD4+ and CD8+ lines. The company has also developed and characterized virus-specific and tumor antigen-specific T cells. “Customers can use these for evaluation of mediated cytotoxicity and T cell activation in thaw and use format.”

Media components

“T cell culture, in general, requires basic cell culture media components, including buffer system, protein, inorganic salts, trace elements, vitamins, lipids, and carbohydrates,” explains Yu. “The basic media requirements can work for most subsets of T cells.” These formulations differ in the details, like cytokines, growth factors, and energy sources, when it comes to specific subsets and assays.

Lodge thinks that proliferation can be sensitive to medium composition and says that T cells seem to grow pickier the longer they are in culture. CD4+ and CD8+ cells seem to have different requirements for optimal growth, “but what those requirements are is a bit of a mystery to me. What I do know is that given the same medium and the same stimulus, the two populations will grow at different rates.”

David Fiorentini, vice president of scientific affairs at Biological Industries, notes that, in general, media will look similar if serum is included in the recipe. But when serum-free is being used, “more defined differences between subsets of T cell medium requirements might be possible.” He explains, for example, that Th1 requires IL-12, Th2 needs IL-4, and Tregs, TGF-β1 and retinoic acid. And Yu says, “T cell culture protocols also typically require the addition of cytokines that support T cell expansion, the most common one being IL-2.” STEMCELL offers an immune cytokines wall chart that gives an overview of the cytokines required for the differentiation of various immune cells. The company’s ImmunoCult-XF T Cell Expansion Medium supports expansion of a variety of human T cells, and can be used alongside ImmunoCult T Cell Activators.

The concentration of cytokines must also be considered and depends on the protocol.

“Researchers should keep in mind that different sources of cytokines may have different specific activity, which may affect the required concentration. Vendors should provide product information sheets containing the specific activity of the cytokines they sell,” adds Yu.

Lodge advises using “the classic checkerboard approach” for evaluating cytokines, but admits that it can be a daunting task with three or more. “It can be done, though—96 well plates are made for this kind of thing. Run a series of cytokine concentration by rows and a second cytokine titration by ‘columns’ and choose your read-out carefully. If cell growth is what you want then measure the number of cells, if a phenotype is what you're after then look for that.”

What You Need to Know about T Cell Culture

  • T cells can be finicky and sensitive to media composition and density.
  • To function properly, T cells must be activated through antigen presentation. It is common practice to use IL-2 and CD3/CD28 antibodies in the form of coated magnetic beads, coated culture plates, or in soluble form.
  • T cells require specific cytokines for growth stimulation, survival, and differentiation. IL-2 is commonly used to support expansion, but a wide range of interleukins are utilized for specific subsets. Optimization experiments to determine cytokine concentrations are critical to successful T cell culture.
  • Many labs rely on antibiotics, but experts mostly agree that they should not be used since they can mask low level contamination and potentially interfere with cell metabolism. Cells grown under serum-free conditions are more fragile and should not be grown with antibiotics.
  • Serum is commonly used in research and can aid in supporting T cell growth. However, variability and potential for contamination remain concerns. Similarly, FBS may increase basal activation of T cells in antigen-specific assays. Clinical-grade cells rely on HS. Serum-free formulations can remove some variability from experiments, but may compromise cell robustness.

When it comes to the production of clinical grade cells, like CAR-T, the most important thing is that the medium be acceptable for FDA review, says Lodge. “Ideally the components of the medium are traceable back to their source and there is a Drug Master File available for the agency to review. Components derived from humans or animals would need to be tested for the presence of viruses.”

CAR-T cells have the same basic medium requirements. “However, media that are serum-free and specifically optimized for rapid T cell expansion can benefit the CAR-T cell manufacturing process. This allows CAR-T manufacturers to generate large amounts of T cells without the need for unknown or non-human components that can be safely used for adoptive cell therapy,” advises Yu. Medium should preferably be cGMP compliant. STEMCELL is currently working with GE Healthcare to develop a cGMP-grade version of their ImmunoCult™-XF T Cell Expansion Medium.

Serum considerations

Companies have gone to considerable effort to research, generate, and market serum-free formulations of media. By removing serum from the mix, scientists are also eliminating the murkiness inherent to it: undefined foreign components, inconsistent levels of hormones, proteins, and lipids that can affect performance and vary from lot to lot. And then there’s always the risk of contamination. Yet, still it’s used readily and often, especially for research. And certainly if mice are involved: “T cell cultures from mice have been done using traditional culture medium formulations supplemented by FBS [fetal bovine serum] for so long no one has bothered to move away from that,” says Lodge.

FBS is the biggest source of variability in DIY media recipes, as well. Yu says that this media must be filter sterilized and tested to ensure that it is contamination free. But she does not advise this approach since there are usually batch-to-batch inconsistencies.

“It would be wonderful to omit the use of serum, both human and bovine, from tissue culture. It is a black box that contains unknown amounts of growth factors and cytokines and we don't seem to care to know what those are doing for us. But serum still adds something good, on balance, to culture medium so we'll continue to use it until we know better,” says Lodge. Still, she says she prefers to work under serum-free conditions.

But FBS may increase basal activation of T cells in antigen-specific assays, cautions Yu. She says that in this case human serum (HS) is preferred. “Regardless, the use of serum can increase experimental variability, which is why we provide serum-free media such as our ImmunoCult-XF T Cell Expansion Medium.”

While HS is acceptable for clinical applications, Fiorentini says this will probably change. “In the future, the use of serum will not be accepted, and the use of serum-free, xeno-free media will be the only choice.”

Serum-free conditions, while desirable for many reasons, do make cells more fragile, according to Fiorentini and in this case, he does not recommend antibiotics. And Lodge says antibiotics should never be used. “If you have good sterile technique you don't need antibiotics. If you have bad technique and you don't use antibiotics you will start to learn better technique.”

Beyond media

Other aspects of culture beyond media can influence how T cells behave. STEMCELL found that maintaining T cells at lower cell density during early expansion improves their growth and viability. “Our study identifies day 3 as a key time point for culture volume adjustment,” explains Yu. The approach can lead to an up to 800-fold expansion of human T cells over two weeks in culture.

Getting into the nitty-gritty of media components and cell density may not be that glamorous, but these details make all the difference when dealing with T cells. Necessary, since the field shows no signs of slowing down. Says Lodge, “ I think it is important to be aware of the unique characteristics of these cells. Primary T cells have many different T cell receptors, and as soon as they are cultured the diversity of receptors probably declines.”