Metabolic flux analysis and population heterogeneity in mammalian cell culture

Abstract

Metabolic flux and population heterogeneity analysis were used to develop relations between mammalian cell physiology and specific culture environments and to formulate strategies for increasing cell culture performance. Mitochondrial characteristics associated with respiration, membrane potential, and apoptosis along with physiological state multiplicity involving both metabolism and apoptotic death played a key role in this research. Research involving the accurate calculation of metabolic flux and the analysis of cellular behavior occurring in continuous cultures set the stage for subsequent research on physiological state multiplicity. This phenomena was observed in continuous cultures when at the same dilution rate, two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations. Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the tri-carboxylic (TCA) cycle for energy generation. This metabolic adaptation was unlikely due to favorable genetic mutations and was implemented in subsequent research aimed at improving cell culture performance. A hypothesis stating that mitochondrial physiology and cellular physiology are correlated was tested and confirmed. A mammalian cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123. The MMP sorted subpopulations were subjected to apoptosis inducers, and the apoptotic death was characterized both morphologically through the determination of apoptosis related chromatin condensation and also biochemically through the measurement of caspase-3 enzymatic activity. The results showed dramatic differences in apoptotic death kinetics with the higher MMP subpopulations demonstrating a higher resistance to apoptotic death. These results were applied in the development of novel fed-batch feeding and operating strategies. The first strategy showed that overfeeding cells later in culture leads to an increase in culture viable cell concentration, viability, and productivity. The second strategy showed that cell populations with a higher mean MMP are able to resist apoptosis during fed-batch culture. These results indicate that mammalian cell populations have considerable flexibility in their ability to redistribute metabolic flux in central carbon metabolism. Furthermore, these cell populations contain subpopulations that vary in their resistance to apoptotic death. The analysis of mitochondrial physiology and metabolic flux led to these discoveries, and these areas will play a key role in future mammalian cell culture research.