title: Improving embryonic stem cell expansion through the combination of perfusion and Bioprocess model design.
abstract: BACKGROUND
  High 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.
  
  
  RESULTS
  To 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
  The 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.
authors: Department of Chemical Engineering, Imperial College London, London, United Kingdom.
authors: David
authors: D
authors: Yeo
authors: Alexandros
authors: A
authors: Kiparissides
authors: Jae Min
authors: JM
authors: Cha
authors: Cristobal
authors: C
authors: Aguilar-Gallardo
authors: Julia M
authors: JM
authors: Polak
authors: Elefterios
authors: E
authors: Tsiridis
authors: Efstratios N
authors: EN
authors: Pistikopoulos
authors: Athanasios
authors: A
authors: Mantalaris
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D019149:Bioreactors; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation; D051379:Mice; D008954:Models, Biological; D010477:Perfusion; D039904:Pluripotent Stem Cells / Q000166:cytology