Copyright © IFAC Computer Applications in Biotechnology, Osaka, Japan, 1998 MODELING OF MULTIPLE STEADY STATES IN MAMMALIAN CELLS. A CYBERNETIC APPROACH M. J. Guardia', A. Europa', A. Gamhhir', R. Ramakrishna 2, D. Ramkrishna 2 and W-S. Hu.' I Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455-0132. 2School of Chemical Engineering, Purdue University, West Lafayette, IN 47907 Abstract: Mammalian cells utilize glucose and glutamine as the two key substrates. These two nutrients are complementary and substitutable in nature and, in cellular metabolism, can be oxidized completely, or be converted to excreted metabolites. The ability of cells to grow at the same rate at different physiological states gives rise to multiple steady states in continuous culture. Experimental results indicate that the steady state reached is strongly dependent on the initial condition of cultivation, or the reaction path. However, it has been elusive to develop a correlation between initial conditions and the resulting steady states. We have used a cybernetic model as the conceptual framework to explore the effect of initial conditions on the behavior of continuous cultures. The results of simulation demonstrate that a cybernetic model is capable of illustrating the effect of the history of a culture on the resulting steady state. Copyright © 1998 IFAC Keywords: Multiple steady states, Cybernetic model, Mammalian cells, Initial conditions 1. INTRODUCTION A typical mammalian cell culture medium consists of glucose and glutamine as the main sources for carbon and energy. The metabolism of glucose and glutamine in mammalian cells are related as illustrated in the simplified metabolic chart shown in Figure 1. Both provide the carbon skeleton for cellular molecules and are catabolized for energy generation. They are often referred to as partially substitutable and partially complementary substrates. Mammalian cells are capable of utilizing varying proportions of glucose and glutamine, and metabolize them differently under different chemical environments. Glutamine becomes a predominant source of energy at low glucose concentrations. The metabolism of glucose is also greatly affected by glucose concentration (Zielke et at. 1978). At high glucose levels, glucose is consumed at a faster rate; however, the majority of that consumed is converted to lactate. At low glucose concentrations, the consumption rate is lower and a larger proportion is completely oxidized to CO 2 . Glutamine is taken up by cells at a rate far exceeding what is needed for incorporation into biomass (as cellular protein or nucleotides) and protein products. Under normal 455 growth conditions, excess nitrogen is excreted as some amino acids, including NH3 and alanine. However, under the condition of low glucose concentration, glutamine also serves as an energy source and the source of carbon skeleton for other cellular constituents. It has been shown previously that the metabolism of the cells can be altered in fed-batch cultures under low-glucose conditions, while maintaining a constant growth rate. In this experiment, on-line measurements were employed to estimate the metabolic demand of nutrients and the feeding rate of concentrated medium (without bulk salts) in fed- batch culture to maintain glucose concentration at a low level throughout the exponential growth period (Zhou et aI., 1995). Lactate production was reduced to near zero towards the end of the cultivation. At the beginning of the cultivation, 85% of the glucose carbon was excreted as lactate; each mole of glucose consumed was accompanied by one mole of oxygen consumption. Towards the end of cultivation almost no glucose carbon was excreted as lactate and the stoichiometric ratio between oxygen to glucose increased to six. With the lower degree of accumulation of lactate, a high cell concentration of