(404a) Mixing Issues in Stem Cell Culture Bioreactors Using Microcarriers

Though in the early 1980s animal cell culture was generally based on attaching cells to microcarriers, little work was done to optimise their suspension. Partly this was because, around this time, free suspension culture was established where cells were much less likely to be damaged by fluid dynamic stresses. For example, Croughan et al (1987) showed that mean specific energy dissipation rates had to be < ~1x10^-3 W kg^-1 to prevent damage during cell culture with 180 mm microcarriers whilst values up to 0.25 W/kg are possible in free suspension (Nienow, 2006). However, the optimisation of vessel/agitator configurations to achieve low mean specific energy dissipation rates suspension (W/kg)_S for were not investigated and some geometric recommendations (for example, the use of hemispherical bioreactor bases to aid suspension (van Wezel, 1985)) were counter to the general findings on particle suspension (Nienow, 1985, 1997). Now with the increasing importance of regenerative medicine and the need to grow stem cells on microcarriers at large scale for allogeneic usage, establishing efficient bioreactor geometries for microcarrier suspension has become important. Clearly, microcarriers suspension is essential if the well-mixed features of the stirred bioreactor are to be achieved. The minimum speed, N_JS, and mean specific energy dissipation rate at that speed, (W/kg)_S, has been measured for many geometries but still little work has been done on microcarriers. The most relevant study (Ibrahim and Nienow, 2004) investigated N_JS using  Cytodex 3 microcarriers with different diameter Chemineer HE-3 hydrofoils, a pair of Ekato InterMIG impellers and a six-blade, 45°-pitch turbine impeller in a baffled vessel of 19.2 L operating volume containing phosphate buffer saline solution. Flat and modified tank bases were used and N_JS values were observed to be in the range of 50 to 90 rev min^-1. The use of Zwietering’s correlation with geometric suspension parameters, S, from the earlier literature would have predict N_JS values up to 50% higher. The low N_JS values obtained were attributed to the very small particle–liquid density difference (40 kg m^-3), which eased the lifting of the particles from the tank bottom, compared to those used in non-microcarrier studies. With these microcarriers, the three-blade hydrofoil HE-3 impeller of D/T = 0.39 in a cone-and-fillet based tank was marginally the most efficient; that is, it had the lowest (W/kg)_S of ~ 0.5 x 10^-3 W kg^-1. However, (W/kg)_S < ~1 x 10^-3 W kg^-1 in most cases with the different size HE-3 hydrofoils, which implies that these geometries should be suitable for shear-sensitive microcarrier cell culture systems. When cells are attached to microcarriers, then damage might occur to cells from impacts between impeller and microcarrier, microcarrier and microcarrier and microcarrier and vessel internals (as in secondary nucleation (Nienow and Paul, in press)); or due to cells being stripped off them and direct stress from turbulent eddies. Thus, it is important to minimise (W/kg)_S. However, it must also be remembered that the mean specific energy dissipation rate is a critical parameter and needs to be sufficient to achieve the required rate of oxygen and CO₂ transfer and give an adequate homogenisation. Thus, it is important to consider all parameters which are dependent on it when developing the stirred bioreactor. This approach has led to the successful culture of mesenchymal stem cells in a 5L bioreactor.