Chinese hamster ovary cells are often used in a pharmaceutical industry for the production of biopharmaceuticals. Part of the process is cell banking, where the cells are prepared for freezing and long-term storage to maintain the cell line in original form and condition. Ice formation during freezing is causing injuries to the cells, which can result in low viability after thawing. As a prevention we add cryoprotectants, which can be toxic for cells, depending on the concentrations and exposure time. In the work presented, we aimed to optimize the process of cell bank preparation by testing different concentrations of cryoprotectant DMSO, its exposure time of cryoprotectant before freezing and temperature of preparation. We also tested whether these factors affected titer of product at the end of fed batch bioprocess. Because of exposure to different temperatures during handling with cryovials, we also tested the effect of different temperatures on cells stored at ultra-low temperature on several cell lines. We conclude that low temperature during cell bank preparation can result in higher viability and allows longer exposure time to cryoprotectant. Effect of different temperatures during handling of cryovials after long term storage on viabilities after thawing is cell line dependent.