Characterizing steady states of genome-scale metabolic networks in continuous cell cultures

Abstract

In the continuous mode of cell culture, a constant flow carrying fresh media replaces culture fluid, cells, nutrients and secreted metabolites. Here we present a model for continuous cell culture coupling intra-cellular metabolism to extracellular variables describing the state of the bioreactor, taking into account the growth capacity of the cell and the impact of toxic byproduct accumulation. We provide a method to determine the steady states of this system that is tractable for metabolic networks of arbitrary complexity. We demonstrate our approach in a toy model first, and then in a genome-scale metabolic network of the Chinese hamster ovary cell line, obtaining results that are in qualitative agreement with experimental observations. We derive a number of consequences from the model that are independent of parameter values. The ratio between cell density and dilution rate is an ideal control parameter to fix a steady state with desired metabolic properties. This conclusion is robust even in the presence of multi-stability, which is explained in our model by a negative feedback loop due to toxic byproduct accumulation. A complex landscape of steady states emerges from our simulations, including multiple metabolic switches, which also explain why cell-line and media benchmarks carried out in batch culture cannot be extrapolated to perfusion. On the other hand, we predict invariance laws between continuous cell cultures with different parameters. A practical consequence is that the chemostat is an ideal experimental model for large-scale high-density perfusion cultures, where the complex landscape of metabolic transitions is faithfully reproduced.

A physical model of cell metabolism

“…Our model considers, in a simplified manner, the autocatalytic nature of cell growth 10 , 37 , the volumetric and biosynthetic costs of metabolic enzymes, ribosomes and other components 5 , and the existence of alternative metabolic pathways for the degradation of substrates (fermentation vs. oxidative phosphorylation) 5 . This represents an improvement over the “enzyme allocation” models that have appeared over the last decade to explain overflow metabolism 3 , 6 , 9 – 11 , 15 , 37 – 39 . The new addition is a mechanistic model for the energy demand of cell maintenance, which is included as an empirical constant in previous approaches.…”

“…Furthermore, larger enzymes imply a higher biosynthetic cost and, therefore, another measure of efficiency is the pathway rate per enzyme amino acid 10 , 11 . Today enzymatic costs are an integral component of cell metabolism models 12 – 15 .…”

Genome-Scale Metabolic Model Reconstruction and in Silico Investigations of Methane Metabolism in Methylosinus trichosporium OB3b

“…One potential explanation for the observed phenomenon is that different batch culture conditions (for example, different mixing rates) were used in different studies, making them quantitatively incomparable. Bacterial cultivation under methane-limited continuous culture conditions is expected to provide more reliable representation of the steady-state bacterial growth [60,61]. In the case of copper-free ammonium-supplemented medium, there is no reported values of specific growth rates for M. trichosporium OB3b.…”

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

“…Continuous culture systems allow for the long-term, controlled growth of microorganisms or cultured cells. Consequently, they have been used for many scientific and industrial uses, including producing biologics like small molecules [1–3] and recombinant proteins [4,5]; assessing the growth rate [6–9] or metabolism [10,11] of microorganisms or cultured cells under defined conditions; and for studying evolution [12–14]. Continuous culture systems typically operate in one of two modes: a chemostat, where a limited amount of nutrients are constantly added to the culture, or a turbidostat, where the culture density is kept constant [15].…”