Computational Fluid Dynamics: Bioreactor Analysis

Human pluripotent stem cells (hPSCs) are capable of differentiating into all somatic cell types and hold great potential for future clinical applications.  Recent studies on cell culture in stirred tank bioreactors indicate that shear forces resulting from the interaction of the cells with the turbulent eddies significantly affect the differentiation propensity of stem cells and, furthermore, excessive shear stress can cause mechanical damage to the cells. Bioreactors used for the culture of these cells must limit the intensity of shear while still providing adequate mixing for uniform dispersion of cells throughout the reactor. The intensity of shear depends on the size of eddies that exist in the bioreactor relative to the microcarrier particles. It has been reported that cell damage can be avoided if the particle size is smaller than the size of the smallest eddy as characterized by the Kolmogorov length scale for turbulence. Thus, the Kolmogorov length scale is a key determinant of the differentiation outcome of cultured stem cells. Because there is significant spatial variation in shear intensity in stirred vessels, CFD modeling is essential to predict the precise shear conditions experienced by cells.

We use a synergistic combination of computational fluid dynamic (CFD)-based simulations and experiments to study the effects of the turbulent shear stress on the cell culture performance.* The effects of parameters such as the impeller speed, culture medium fluid properties and cell size on the steady-state shear stress acting on the cell-laden microcarrier particles in the bioreactor are studied using CFD analysis. This is used to predict the precise shear conditions experienced by cells and identify optimum operating conditions that prevent turbulent shear damage of the cells. In addition, the effect of shear on the pluripotency of hPSCs is studied by determining the percentage of cells carrying the pluripotency markers Oct4, Sox2 and Nanog using flow cytometry and quantitative PCR. The cell cycle of hPSCs under different shear stress conditions is studied to determine the doubling time and the length of the G phase of the cells.

The first animation above shows a 3D CFD simulation of the Corning’s bench-scale stirred-flask bioreactors running at 60 rpm. A state-of-the-art multiphysics CFD program Flow-3D (www.flow3d.com ) was used for this analysis. In the computational model, the reactor is filled with 50 ml of cell culture media and loaded with micro carrier particles that are 150 microns in diameter in a concentration of 0.5 g/ml. The simulation takes into account fully-coupled particle/fluid interactions wherein the particles move in response to viscous drag imparted by the flow field and their motion, in turn, alters the flow. These interactions can be visualized in the second animation, which provides a close up view of an inside portion of the reactor. Here, the particles are colored by their x-velocity. The third animation shows the distribution of shear stress acting on the particles in a cross-sectional plane of the reactor. Regions that experience stress that could cause cell damage can be readily identified. The fourth animation shows the Kolmogorov length scale distribution inside the reactor at the same plane. This length scale is a key criterion in determining fate of the stem cells for differentiation. It has been reported that cell damage can be avoided if the particle size is smaller than the size of the smallest eddy as characterized by the Kolmogorov length scale for turbulence. The average eddy size can be controlled by changing the stirring speed. In this way, we identify the optimum conditions to prevent the turbulent shear damage.

*Koushik Ponnuru et al. "Analysis of Stem Cell Culture Performance in a Microcarrier Bioreactor System." Proc. Int. NSTI Nanotech Conf., Washington DC, June 2014.