Low-temperature pausing of cultivated mammalian cells.
There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees 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 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees 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 degrees C, 12 degrees C, or 24 degrees 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.
Hunt
Lisa
L
Swiss Federal Institute of Technology Lausanne, Faculty of Basic Sciences, Institute of Biological and Chemical Process Sciences, CH1015 Lausanne, Switzerland.
Hacker
David L
DL
Grosjean
Frédéric
F
De Jesus
Maria
M
Uebersax
Lorenz
L
Jordan
Martin
M
Wurm
Florian M
FM
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D015925:Cryopreservation / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000502:physiology*; D013696:Temperature; D013997:Time Factors