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Cell Culture Advances for Vaccine Development and Production

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The COVID-19 pandemic has given rise to a number of scientific innovations, particularly in the production and testing of vaccine technology.

Since December 2020, a staggering 10 billion doses of SARS-CoV-2 vaccines have been administered worldwide – and while global vaccine equity remains a challenge to be overcome, some nations have even begun to experience SARS-CoV-2 vaccine surplus.

New and rapid production methods are carrying a record number of vaccines to clinics, a reality driven – at least in part – by the immunization demands of pandemic management and concurrent innovations in cell culture.

Cell culture – the process of growing cells outside of their natural environment under controlled artificial conditions – has been used widely in the development and testing of new vaccines since early methods were pioneered in the 1930s. Historically, vaccines have comprised either a whole or partial pathogen, inactivated for safe administration and grown within biological systems (such as chicken eggs or mammalian cells).

Today, the optimization of cell culture growth medium, the development of superior biological models and the reduced reliance on animal-derived components continues to drive the exciting and rapidly developing art of cell-based vaccine development.

Cell culture in exploratory vaccine testing

Cell culture methods are instrumental in the exploratory, testing and production aspects of vaccine development. Vaccine candidates must be proven safe and effective in cell culture experiments before they can enter the preclinical stages of testing and production.

Due to the limitations of in vitro models, a high failure rate in preclinical and clinical trials continues to prove a significant bottleneck to vaccine manufacturing. Increasing the accuracy and reproducibility of cell culture models remains one of the biggest drivers of methodological innovation in vaccine development today.

Adding another dimension to vaccine testing

In standard tissue culture practice, cells are grown in flat, two-dimensional (2D) monolayers – adherent to the plastic surface of the vessel in which they are grown. Despite its importance in the early stages of exploratory testing, 2D cell culture does not recapitulate the complex tissue architecture of in vivo systems and can fail to model the true infection cycle of a pathogen.

Three-dimensional (3D) cell culture models – which can include multiple cell types, extracellular matrix components and some microfluidic systems – have improved the therapeutic predictions of in vitro models with significant advantages to vaccine development. Bioprinting – the process of additively manufacturing biological structures composed of cells and extracellular matrix – now offers cell culture standardization with unprecedented accuracy and reproducibility. Through 3D bioprinting, complex multicellular structures can be created in vitro via the layer-by-layer addition of biological materials. Compared to 2D models, these 3D cultures can achieve more accurate responses regarding cell morphology, proliferation capacity and gene expression. They can also offer superior reproducibility and standardization with computer-aided design (CAD).

“One of the major advantages [of 3D bioprinting] is its reproducibility,” says Dr. Stephanie Willerth, associate professor of mechanical engineering at the Center for Biomedical Research, University of Victoria (Canada). Dr. Willerth forged a career in the field of stem cell bioengineering and 3D cell culture innovation.

“The same CAD file can be used with bioprinters all over the world to generate the same structures, provided they have access to appropriate cells and bio-inks,” adds Willerth. “Thus, an established tissue model could serve as a benchmark for screening potential vaccines or producing them.”

Optimal media for an optimal vaccine

Whether maintained as 2D or 3D structures, cells in culture must be supplied with an appropriate liquid growth medium. Cell culture media – usually a red or pink liquid in which in vitro cultures are submerged – is a cocktail of nutrients designed to support and maximize the growth of cells outside of their natural environment.

“The major expenses with 2D cell culture include the media and the labor required to produce the cells and resulting structures,” Willerth explains. “Thus, making media formulations that last longer and, in turn, reduce the amount of labor needed to generate a large number of cells is key.”

Cell culture media typically includes amino acids, vitamins, salts, sugars and supplementary nutrients. Animal serum is also sometimes added to provide supplemental growth factors and hormones within a tissue environment.

While widely used in cell culture practice, animal serums are often poorly characterized or standardized, if at all. Studies have revealed almost 1,800 proteins and 4,000 metabolites in a routinely used animal serum, with each causing potential variability in in vitro experiments. The removal of animal serum from the vaccine production pipeline has been an essential step in accelerating the delivery of therapeutics to market, speeding up exploratory testing and minimizing the number of safety checks needed for each candidate drug.

The supplementation of basal synthetic media – such as MEM, EMEM or DMEM – with additional lab-derived proteinaceous components and metabolites, now supports the scalability and reproducibility requirements for vaccine production. However, the optimization of cell culture media remains an ongoing process, as researchers continue to strive to mimic the complexity of each true tissue microenvironment.

Replacement, reduction and refinement

Animal products have played an essential part in the vaccine production pipeline for many years. However, with increasing public awareness of animal welfare and the scientific risks associated with animal product contaminants, alternative methods of cell-based production remain a priority for vaccine development.

Many human vaccines have been produced successfully and safely in animal cells. Vero cells – an immortalized cell line isolated from the kidney epithelium of the African green monkey – have been widely used in vaccine production, proving instrumental in the fight against SARS-CoV-2.

Many researchers argue that Vero cells do not offer the biological relevance for effective human vaccine development. However, human alternatives are also not without their controversies. In 2021, Johnson & Johnson (Janssen) faced skepticism for allegedly using human fetal cell lines in the production of their SARS-CoV-2 vaccine. While the cells used in the company’s pipeline were lab-derived and did not contain any fetal tissue, the public perception of human cell culture has caused some to refuse this vaccine preparation based on its cell-based production methods.

Going cell free

Messenger ribonucleic acid (mRNA) vaccines now represent a substantial portion of the global therapeutic market, promising to make the production process for vaccines faster still. The market share of mRNA vaccines is expected to reach a value of $15.49 billion within the next five years, raising questions about the future of cell culture in mass vaccine production.

In vitro synthesis methods for mRNA vaccine production can now bypass cells entirely, creating large volumes of therapeutic grade mRNA within bioreactor systems in a matter of hours. While the potential of this approach has been recognized for some time, developments in mRNA delivery systems have only recently made cell-free vaccines a possibility. According to some experts, mRNA vaccine technology may replace cell-based production methods sooner than we may think.

Dr. Namit Chaudhary is a doctoral candidate at Carnegie Mellon University (Pittsburgh), working on the development of lipid nanoparticles (LNPs) to deliver RNA-based, cell-free therapeutics. “RNA-based vaccines against infectious diseases, such as HIV, influenza, RSV, CMV etc., have already entered clinical trials. If they show positive results, we might have vaccines against several deadly pathogens in the near future,” he says. “Both Moderna and Sanofi already have candidates against the flu vaccine in clinical trials. Depending on the results we might have an mRNA-based flu vaccine soon.”

Like bioprinting, mRNA vaccines can come with high relative costs and infrastructure requirements. “One of the key challenges limiting the widespread use of RNA vaccines is their ultra-cold storage requirements,” Chaudhary adds. “Thermostable vaccines [like those produced using cell-based methods] that are stable at room temperature for extended periods of time will enable vaccine distribution in low-income countries that don't have ultra-cold supply chains.”

Cell-based vaccine production is here to stay

While all the licensed COVID-19 vaccines have shown success in clinical trials – and all have been tested using cell culture approaches – there remains room for optimization when it comes to cell-based vaccine production.

The availability of LNP technology for mRNA vaccine production might offer new, cell-free synthesis options, but cells remain necessary for post-production testing. “I think there is still space in the market for all technologies,” says Chaudhary. “Cell culture and animal models are still necessary to test the efficacy and immune response of vaccine candidates.”

Methods in 3D and serum-free cell culture will likely play an increased role in accurate and reproducible therapeutic testing. However, the high costs associated with lab infrastructure, training and reagents could possibly perpetuate the bio economical privileges of already wealthy nations. Further approaches are required to ensure that technological advancements in vaccine production can translate to global vaccine equity and public health.