This paper reports the optimization of a perfusion bioreactor system previously reported by us (Chouinard et al., 2009). The implementation of a proportionalintegral (PI) controller algorithm to control oxygen concentration and pH is presented and discussed. P and I values used by the controller were first estimated using a First-Order-Plus-Dead-Time (FOPDT, Matlab Simulink) and then tuned manually. A new gas exchanger design compatible with the PI controller was introduced and validated to decrease interaction between the injected gases and overall inertia of the system. The gas exchanger was used to adjust both pH and dissolved oxygen (DO) concentration. This new bioreactor system allowed real-time PI control over pH and DO concentration at different flow rates (from 2 to 70mLmin-1). Cell viability and proliferation were investigated to validate the updated bioreactor design and performance. Biotechnol. Bioeng. 2012; 109:13051313. (C) 2011 Wiley Periodicals, Inc.