The effect of in vitro culture conditions on cell metabolism

Thesis

Because cells have adapted to respond to their extracellular environment, the in vitro conditions under which cells are cultured have the potential to impact on cell metabolism. This study aims to investigate the effect of various components of culture medium on the metabolism of Chinese Hamster Ovary (CHO) cells.

Firstly, the effect of glucose and glutamine, two important carbon sources, was investigated. The impact of these nutrients on cell growth, viability, oxygen consumption rate (OCR) and steady state ATP levels was assessed. Glucose was found to have a suppressive effect on OCR, while under glucose limiting conditions, glutamine was able to enhance OCR. Although commonly considered a waste product, lactate can also act as a source of carbon and can be consumed by CHO cells. The effect of lactate on the above parameters was also measured, but no robust changes were observed.

Serum is added to the majority of cell culture media and is necessary for cell growth. However, since this product is animal derived, batch to batch variability is high and its composition is not well documented. The broad profile of components in serum means it has the potential to exert many effects on cells, but the effect serum of on cell metabolism is poorly understood. For this reason, the metabolic state of CHO cells in reduced serum was investigated using the aforementioned assays. Metabolite profiling was also performed using metabolomics. Cells cultured in 10% foetal bovine serum (FBS) were found to have a higher OCR than those cultured in both 1% and 0.1% FBS. This was supported by metabolite profiling which showed a greater abundance of tricarboxylic acid (TCA) cycle intermediates in these cells. The metabolite data also indicated increased oxidative stress in cells cultured in 10% FBS. Glucose metabolism also appeared to be affected, with a higher abundance of early glycolytic and glycogenesis intermediates in cells cultured in low (0.1%) serum.

This study shows that both the substrates available in culture media and the commonly used supplement, FBS, can influence metabolic pathways within mammalian cells, meaning their effects must be accounted for, particularly in studies of metabolism.

Authors: Hartley, F

• DOI: 10.5287/ora-n6v5ejnvr
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: Metabolism; Oxidative stress; Cell culture; Metabolomics; Mitochondria; Glucose
• Subjects: Metabolism; Cell biology