A perfusion-based high cell density (HD) cell banking process has been developed that offers substantial advantages in time savings and simplification of upstream unit operations. HD cell banking provides the means to reduce the time required for culture inoculum expansion and scale-up by eliminating the need for multiple small to intermediate scale shake flask-based operations saving up to 9 days of operation during large-scale inoculum expansion. HD perfusion cultures were developed and optimized in a disposable Wave bioreactor system. Through optimization of perfusion rate, rocking speed and aeration rate, the perfusion system supported peak cell densities of >20 x 10(6) cells/mL while maintaining high cell viability (>= 90%). The cells were frozen at HD (90-100 x 10(6) viable cells/mL) in 5-mL CryoTube vials. HD cell banks were demonstrated to enable direct inoculation of culture into a Wave bioreactor in the inoculum expansion train thus eliminating the need for intermediate shake flask expansion unit operations. The simplicity of the disposable perfusion system and high quality of the cell banks resulted in the successful implementation in a 2000 L scale manufacturing facility. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 824-829, 2011