Amino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis

Abstract

Amino acid compositions of cell culture media are empirically designed to enhance cell growth and productivity and vary both across media formulations and over the course of culture due to imbalance in supply and consumption. The interconnected nature of the amino acid transporters and metabolism suggests that changes in amino acid composition can affect cell physiology. In this study, we explore the effect of a step change in amino acid composition from a DMEM: F12-based medium to a formulation varying in relative abundances of all amino acids, evaluated at two amino acid concentrations (lean LAA vs. rich HAA). Cell growth was inhibited in LAA but not HAA. In addition to the expected effects on expression of the cell cycle, amino acid response and mTOR pathway genes in LAA, we observed an unanticipated effect on zinc uptake and efflux genes. This was accompanied by a lower tolerance to zinc supplementation in LAA but not in the other formulations. Histidine was sufficient but not necessary to prevent such zinc toxicity. Additionally, an unanticipated downregulation of genes in the cholesterol synthesis pathway was observed in HAA, accompanied by an increase in cellular cholesterol content, which may depend on the relative abundances of glutamine and other amino acids. This study shows that changes in the amino acid composition without any evident effect on growth may have profound effects on metabolism. Such analyses can help rationalize the designing of medium and feed formulations for bioprocess applications beyond replenishment of consumed components.

Leveraging machine learning to dissect role of combinations of amino acids in modulating the effect of zinc on mammalian cell growth

“…CHO cells routinely maintained in HDFE medium were inoculated into HAA media. 6 Briefly, HAA medium is formulated identically to HDFE with the exception of cysteine at 0.05 mM, cystine at 0.3 mM and all other amino acids at 1 mM concentration. Both HDFE and HAA media are formulated with 2.25 μM ZnCl 2 .…”

“…We chose 30 μM ZnCl 2 based on our previous work where this concentration induced toxicity in low amino acid concentrations but not in HAA or HDFE. 6 The concentration of each of the 20 amino acids was varied in at least one of the experimental conditions. The exact amino acid formulations evaluated in this study are indicated in TablesS1 and S2.…”

“…This is not surprising since histidine has been reported to have a protected effect from the adverse effect of zinc under LAA conditions. 6…”

“…This is consistent with our previous conclusion that the effect of zinc was predicated on the abundance level of at least some amino acids in the medium. 6 The component with the second highest score was histidine.…”

“…investigate the previously reported dependence of zinc toxicity on amino acid availability 6 and use machine learning to identify higherorder interactions between amino acid availability and zinc toxicity.…”