Low-temperature pausing of cultivated mammalian cells

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Abstract
There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37°C and freezing in small batches. We investigated a third approach, the “pausing” of cells for days or weeks at temperatures below 37°C 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 6–24°C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4°C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37°C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4°C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4°C or 24°C 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 4°C, 12°C, or 24°C 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.