Conventional cell expansion strategies for the production of biopharmaceuticals and biologics typically involves a working cell bank cryovial (at 1-5×10 7 cells/mL) as a starting point. This method of cell expansion involving open aseptic manipulations using multiple vessels is time consuming, labor intensive and presents significant risks in terms of possible contamination. This study describes the successful development of a large volume cell culture inoculum in cryobags that can be used to mitigate the potential disadvantages of conventional cell banks and to provide a fully closed cell expansion method. Issues related to bag design and validation, methods for producing high cell density cultures (batch centrifugation, continuous flow centrifugaiton, hollow fiber cell concentration, and perfusion culture), choice and concentration of cryoprotectant, large scale freezing studies and optimization of thawing conditions are addressed. The results demonstrate that cell growth characteristics and adenovector production using this new methodology are comparable to the conventional cell expansion strategy.