Fatty acids promote the expansion of NK-92 cells in vitro by improving energy metabolism.

Natural killer-92 cells (NK-92 cells) need to be efficiently expanded by serum-free culture in vitro to meet clinical requirements. Fatty acids mainly provide substrates for energy production, which is of crucial importance to meet the energy demands of highly proliferating cells. This study optimized the medium (EM) for NK-92 cells by designing an experiment to expand cells efficiently. EM, an in-house designed chemically defined serum-free medium, was used as the basal medium. Fatty acids as additive ingredients were screened and optimized by the experimental design method. Three additives, arachidonic acid, myristic acid and palmitoleic acid, were screened; therefore, the optimized medium was named EM-FA. The total cell expansion of NK-92 cells in EM-FA was 72.61±11.95-fold on day 8, which was significantly higher than the 28.55±8.67-fold expansion in EM. To explore the mechanism by which fatty acids promote NK-92 cell expansion, the cell growth kinetics and metabolic characteristics in EM-FA were analyzed. The results showed that NK-92 cells in EM-FA were rapidly expanded while maintaining their cell phenotype and cytotoxicity and enhancing the oxygen consumption rate and mitochondrial function. Fatty acids promoted ATP production to elevate the energy flux for better cell expansion. This study developed an expansion strategy of NK-92 cells in vitro to facilitate their clinical application. KEY POINTS: • Arachidonic acid, myristic acid and palmitoleic acid in serum-free medium were optimized by experimental design to enable the rapid expansion of NK-92 cells in vitro. • Fatty acids upregulated oxidative phosphorylation levels and improved the energy metabolism of NK-92 cells.

Vitamin combination promotes ex vivo expansion of NK-92 cells by reprogramming glucose metabolism.

Robust ex vivo expansion of NK-92 cells is essential for clinical immunotherapy. The vitamin B group is critical for the expansion and function of immune cells. This study optimized a vitamin combination by response surface methodology based on an in-house designed chemically defined serum-free medium EM. The serum-free medium EM-V4 with an optimal vitamin combination favoured ex vivo expansion of NK-92 cells. The characteristics of glucose metabolism of NK-92 cells in EM-V4 and the relationships between cell expansion and metabolism were investigated. NK-92 cells in EM-V4 underwent metabolic reprogramming. An elevated ratio of glucose-6-phosphate dehydrogenase/phosphofructokinase (G6PDH/PFK) indicated that NK-92 cells shifted towards the pentose phosphate pathway (PPP). An increase in the ratio of pyruvate dehydrogenase/lactate dehydrogenase (PDH/LDH) suggested that the cells shifted towards the Krebs (TCA) cycle, i.e., from glycolysis to aerobic metabolism. The enhanced ratio of oxygen consumption rate/extracellular acidification rate (OCR/ECAR) indicated that NK-92 cells were more reliant on mitochondrial respiration than on glycolysis. This shift provided more intermediate metabolites and energy for biosynthesis. Thus, EM-V4 accelerated biomass accumulation and energy production to promote NK-92 cell expansion by regulating the metabolic distribution. Our results provide valuable insight for the large-scale ex vivo expansion of clinically available NK-92 cells.