Abstract
BackgroundHigh proliferative and differentiation capacity renders embryonic stem cells (ESCs) a promising cell source for tissue engineering and cell-based therapies. Harnessing their potential, however, requires well-designed, efficient and reproducible expansion and differentiation protocols as well as avoiding hazardous by-products, such as teratoma formation. Traditional, standard culture methodologies are fragmented and limited in their fed-batch feeding strategies that afford a sub-optimal environment for cellular metabolism. Herein, we investigate the impact of metabolic stress as a result of inefficient feeding utilizing a novel perfusion bioreactor and a mathematical model to achieve bioprocess improvement.Methodology/Principal FindingsTo characterize nutritional requirements, the expansion of undifferentiated murine ESCs (mESCs) encapsulated in hydrogels was performed in batch and perfusion cultures using bioreactors. Despite sufficient nutrient and growth factor provision, the accumulation of inhibitory metabolites resulted in the unscheduled differentiation of mESCs and a decline in their cell numbers in the batch cultures. In contrast, perfusion cultures maintained metabolite concentration below toxic levels, resulting in the robust expansion (>16-fold) of high quality ‘naïve’ mESCs within 4 days. A multi-scale mathematical model describing population segregated growth kinetics, metabolism and the expression of selected pluripotency (‘stemness’) genes was implemented to maximize information from available experimental data. A global sensitivity analysis (GSA) was employed that identified significant (6/29) model parameters and enabled model validation. Predicting the preferential propagation of undifferentiated ESCs in perfusion culture conditions demonstrates synchrony between theory and experiment.Conclusions/SignificanceThe limitations of batch culture highlight the importance of cellular metabolism in maintaining pluripotency, which necessitates the design of suitable ESC bioprocesses. We propose a novel investigational framework that integrates a novel perfusion culture platform (controlled metabolic conditions) with mathematical modeling (information maximization) to enhance ESC bioprocess productivity and facilitate bioprocess optimization.
Highlights
Embryonic stem cells (ESCs) have the potential to self-renew limitlessly and differentiate into any somatic cell type, which make them a promising cell source for use in tissue engineering & regenerative medicine and drug discovery applications [1]
The mathematical model captures the kinetics of batch culture well with the apparent discrepancy of the peak cell density at end of the exponential phase at day 4
It has been hypothesized that accumulated metabolic toxicity due to the mode of bioprocess operation, even in the presence of sufficient growth factors, results in loss of embryonic stem cells (ESCs) pluripotency
Summary
Embryonic stem cells (ESCs) have the potential to self-renew limitlessly and differentiate into any somatic cell type, which make them a promising cell source for use in tissue engineering & regenerative medicine and drug discovery applications [1]. Such applications require bioprocessing methodologies that are efficient and cost-effective [2]. High proliferative and differentiation capacity renders embryonic stem cells (ESCs) a promising cell source for tissue engineering and cell-based therapies Harnessing their potential, requires well-designed, efficient and reproducible expansion and differentiation protocols as well as avoiding hazardous by-products, such as teratoma formation. We investigate the impact of metabolic stress as a result of inefficient feeding utilizing a novel perfusion bioreactor and a mathematical model to achieve bioprocess improvement
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