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Abstract

Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

“…Actively metabolizing cells maintain their growth arrest through inhibiting growth mechanisms. In addition to an increase in productivity induced by growth arrest, cultivation of cells under mild hypothermic conditions offers other relevant advantages: extended culture times (lower cell populations reduce overall nutrient uptake and waste production) [24], decreased O 2 demand [25], reduced intermolecular product aggregation [26], increased sensitivity to pH changes [27,28], and a decreased sensitivity to pro-apoptotic agents [29,30]. Protein sialylation [24], acidic glycoforms [31], and antennary structures [31] are relevant quality parameters that are improved in cells cultured under hypothermic conditions.…”

The combined effect of sodium butyrate and low culture temperature on the production, sialylation, and biological activity of an antibody produced in CHO cells

“…Conversely, reduced culture temperature influences q p in a different fashion. It has been suggested that lowering culture temperature causes a shift in the proportion of cells from S to G1, which is partially responsible for increased q p [28,32]. It is well known that cells at G1 phase do not need to devote cellular resources to biomass production, thus subsequently can be employed for recombinant protein synthesis.…”

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions

“…The suspension and the Cytodex 3 culture remained viable for a significantly longer period in the biphasic cultures than in the single-temperature cultures, leading to higher integrated cell densities. Moore and coworkers reported that low temperature culture leads to growth arrest in the G 0 /G 1 phase of the cell cycle and delays the onset of apoptosis (Moore et al 1997). Slikker and co-workers reported that hypothermia enhanced expression of the antiapoptosis protein, bcl-2, and protected against oxidative stress-induced cell death in CHO cells by increasing the activity of anti-oxidant enzyme, glutathione peroxidase (GSH-Px) (Slikker et al 2001).…”

“…The benefits of mild hypothermia include improved cell viability, reduced cell lysis from dead cells, reduced nutrient consumption rates, and often, improved productivity (Yoon et al 2003;Trummer et al 2006a). Despite the potential usefulness of mild hypothermia, there are significant limitations including G 0 /G 1 arrest (Moore et al 1997) and suppression of the cell growth, leading to low volumetric productivity. Biphasic culture, in which cells are cultivated at 37°C until they reach the maximum viable cell density, followed by a reduction in temperature to prolong cell longevity, presents a possible strategy to alleviate the disadvantages of hypothermia.…”