Low-temperature pausing of cultivated mammalian cells

Abstract

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.

Low temperature cell pausing: an alternative short-term preservation method for use in cell therapies including stem cell applications

“…Studies have demonstrated mammalian cells can remain viable at low temperatures (6-24°C) for up to seven days (Hunt et al 2005); supporting the potential of cell pausing as a short-term preservation protocol. Shipping of cells within the UK or globally can take hours or days depending on distance, means of transport and delays.…”

“…Notably, cell pausing has been achieved using standard media for recombinant CHO and HEK293 cells (Hunt et al 2005). It will therefore be valuable to examine the benefits/costs of the individual components of hypothermic storage media such as HTS and UW.…”

“…Assessment of viability and quality parameters after rewarming has shown mild to moderate hypothermia to be more successful than 4°C preservation (Hunt et al 2005). However, research to help improve cell storage at 4°C is a favorable strategy, due to current temperature controlled equipment used today.…”

“…More investigation into cell detachment and colony formation during pausing is needed to ascertain if cells can remain viable and functional if paused in suspension. Pausing cells in 3L bioreactors has been studied (Hunt et al 2005) using suspension adapted CHO cells and a modified storage solution has been patented which claims to preserve progenitor and stem cells in suspension at hypothermic temperatures (2-8°C) (Hope 2011). More detailed analyses of scalable pausing protocols using non-adapted cells (including stem cells) will be required if this technique can be used for regenerative therapies.…”

The role of biopreservation in cell and gene therapy bioprocessing

“…This includes hypothermic preservation (i.e. 'cell pausing') at either ambient [5][6][7] or refrigerated chilled temperatures [8,9]. Ultimately, the decision as to which biopreservation method is most appropriate will need to take into account which method has the least effect on the product's CQAs whilst remaining both commercially, clinically and logistically viable.…”