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.
Highlights
Biotechnological products are obtained by treating cells as little factories that transform substrates into products of interest
One must notice that at variance with the standard formulation of Dynamic Flux Balance Analysis (DFBA), the terms involving the dilution rate in the right-hand side of both equations enable the existence of non-trivial steady states which are impossible in batch
Note that Eq 13 depends on X and D only through the ratio 1/ξ = D/X, such that ξ is the number of cells sustained in the culture per unit of medium supplied per unit time
Summary
Biotechnological products are obtained by treating cells as little factories that transform substrates into products of interest. A classical example of continuous cell culture is the chemostat, invented in 1950 independently by Aaron Novick and Leo Szilard [7] (who coined the term chemostat) and by Jacques Monod [8]. In this system, microorganisms reside inside a vessel of constant volume, while sterile media, containing nutrients essential for cell growth, is delivered at a constant rate. Only a fraction 0 φ 1 of cells are carried away by the output flow D This variation of the continuous mode is known as perfusion culture
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