There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37degreesC and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37degreesC in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24degreesC in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4degreesC for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37degreesC. Adherent cultures of CHO-DG44 cells paused for 2 days at 4degreesC in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4degreesC or 24degreesC in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4degreesC, 12degreesC, or 24degreesC in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells. (C) 2004 Wiley Periodicals, Inc.