One or more properties of a cell line are likely to change during an extended period of continuous passage. In order to ensure consistency of the properties of a cell line or of a product derived from it, it is, therefore, necessary to cryopreserve and characterize a reference stock of cells. Thereafter, throughout a research, development, or manufacturing program cell cultures are discarded after a defined passage interval and the cryopreserved stock used to reinitiate fresh cultures. Although a small number of ampoules in a single bank may be sufficient for a small research program, to ensure continuity of supply of a cell line from a culture collection or for the manufacture of a biopharmaceutical, it is usual to prepare a two-tiered cell bank system. This consists of a master cell bank (MCB) of 50–400 ampoules and manufacturer’s working cell banks (WCB) each derived in an identical manner from one ampoule of the MCB (Table 1). In this chapter the methods of preparation, storage, and characterization of such cell banks are described.