Impact of Serum-Free Culture on HEK293 Cells: Growth and Metabolic Changes

News 10 9 月, 2024

Animal-derived serum, particularly fetal bovine serum (FBS), is commonly used as an additive in cell and tissue culture media, providing essential nutrients such as growth factors, adhesion factors, vitamins, transferrin, hormones, and trace elements that support cell growth in vitro. However, FBS presents challenges, including batch variability, risk of viral contamination, and the introduction of unwanted substances. As a result, the production of vaccines and therapeutic proteins ideally relies on serum-free culture systems.

Serum-Free Cultures and HEK293 Cells

Human embryonic kidney (HEK293) cells have become widely used in recent years for both basic research, such as protein interaction and signal transduction studies, and applied research, including recombinant protein production and viral vector development. HEK293 cells are naturally adherent, but suspension culture applications are also well-documented. However, the impact of serum-free and suspension culture on HEK293 cells’ metabolism has not been extensively studied, despite growing attention to how culture media affect cell metabolism, phenotype, and function.

Researchers at the Norwegian University of Science and Technology, led by Mi Jang, explored these effects in their study published in Frontiers in Bioengineering and Biotechnology titled “The Impact of Serum-Free Culture on HEK293 Cells: From the Establishment of Suspension and Adherent Serum-Free Adaptation Cultures to the Investigation of Growth and Metabolic Profiles.”

The study describes the transition of HEK293 cells from serum-based to serum-free culture and adaptation to suspension growth. It investigates the metabolic differences under various culture conditions, adaptation methods, and phenotypes.

Transition to Serum-Free and Suspension Culture

Researchers used Freestyle 293 Expression Medium and applied two adaptation methods—sequential serum reduction and direct serum reduction (as shown in the figure below)—to adapt HEK293 cells to serum-free culture. When cell confluence reached 70%, cells were passaged sequentially. Cells undergoing adjacent adaptation stages were cultured simultaneously, and if cell viability fell below 80%, the process was reverted to the previous adaptation stage. To ensure stable adaptation, cells were passaged 3-5 times in serum-free conditions before proceeding to suspension culture adaptation.

Serum-Free Adaptation Process

During serum-free adaptation, HEK293 cells maintained their morphological characteristics when serum content was above 5%. As the serum was reduced, cells became rounder, and adhesion weakened. Following serum-free adaptation, cells were gradually adapted to suspension growth in low-speed (80 rpm) shaker flasks with anti-clumping agents, achieving single-cell suspension after three generations.

Five culture systems were established:

• CGM (Complete Growth Medium) Serum Culture Group (CGM)
• Sequential Adaptation Adherent Group (Seq. ad)
• Sequential Adaptation Suspension Group (Seq. sus)
• Direct Adaptation Adherent Group (Dir. ad)
• Direct Adaptation Suspension Group (Dir. sus)

Metabolic Changes in Serum-Free HEK293 Cells

Intracellular metabolite analysis revealed that suspension cells generally had lower metabolite concentrations compared to adherent cells, except for methylbutyric acid and succinic acid. Cells cultured in serum-free media exhibited higher lactate levels and lower amino acid concentrations than those cultured in serum-containing media. The results indicated that the differences in metabolic profiles were more pronounced between culture modes (adherent vs. suspension) than between media types. Differences due to the adaptation process were minimal, with only slight metabolic variations observed between Seq. sus and Dir. sus groups.

Conclusion

The study successfully established adherent and suspension culture systems for HEK293 cells in serum-free Freestyle 293 medium. Adherent cells are traditionally used for viral production and clinical research, while suspension growth serves as a platform for therapeutic protein production. The establishment of these systems in serum-free media opens up broader applications and provides a valuable reference for serum-free adaptation in other cell types.