Extending Viability in CHO Cells

One common practice to maintain higher viability is to shift cell cultures to mild hypothermic conditions, e.g. 37°C to 32°C. Here, we discuss a hot (er… cold?) tip involving the CHOgro® High Yield Expression System, which also supports additional flexible protocol options for researchers who cannot shift cultures to 32°C or who desire extended cellular viability beyond one week following transfection. With the CHOgro® High Yield Expression System, researchers can choose the protocol that matches their viability and protein quality requirements. 

High protein titers are obtained with the CHOgro® High Yield Expression System due to a combination of superior growth medium for suspension CHO cells, a proprietary transfection reagent formulation and our novel CHOgro® Titer Enhancer. The system features a quick transfection and titer enhancing workflow that can be completed in as little as 1 hour on Day 0. A common question we encounter is: how can I extend CHO cell viability through the process of protein production?

Options to Extend Viability

Researchers following the primary CHOgro® High Yield Protocol (Option 1) can expect cell viability above 70% up to approximately 7-8 days post-transfection. This protocol incorporates Day 0 addition of the CHOgro® Titer Enhancer and temperature shift from 37°C to 32°C, which are beneficial for both protein titer and viability. Researchers who prioritize speed over maximum yield may choose to follow the 37°C Protocol (Option 3), which is our fastest protocol at a single incubation temperature. Additional protocols are designed for extending viability of cultures at either 32°C (Option 2) or 37°C (Option 4) and include addition of a feed 18-24 hours post-transfection (feed not included in the CHOgro® High Yield Expression System). Instructions for a cell culture feed addition to extend cell viability are listed below and in the protocol (PDF).

Addition of Cell Culture Feeds to Extend Cell Viability

Maximum yields are achieved if cultures are shifted to mild hypothermal conditions (32°C) immediately after addition of the CHOgro® Titer Enhancer. Cells maintained at 37°C are generally less productive and experience a decrease in viability at earlier timepoints post-transfection. The following steps can be performed to extend the viability of cells post-transfection for cultures incubated at either 32°C or 37°C:

Complete Steps A-D as described in the CHOgro® High Yield Expression Protocol (PDF).

1. At 24-48 hours post-transfection, add 15% (v/v) culture volume of EX-CELL® Advanced CHO Feed 1 (with glucose) (Sigma Cat. No. 24367C), e.g. 3 ml of EX-CELL® Advanced CHO Feed 1 (with glucose) for a 20 ml culture. Note: Prepare a fresh solution of EX-CELL® Advanced CHO Feed 1 (with glucose) before each use as the prepared solution is unstable. The optimal amount of feed to add may vary from 10-20% (v/v) culture volume and should be empirically determined for each culture system.
2. Maintain cultures at 32°C (or 37°C if necessary) for the duration of the expression experiment. Whenever possible, monitor cultures for viability and protein titers at several timepoints post-transfection (e.g. 5, 7 and 10 days post-transfection), as sufficient titers are often achieved at earlier time points with the CHOgro® High Yield Expression System.
3. Harvest cells and/or supernatant and assay as required.

Experimental Data

Extend CHO Cell Culture Viability and Achieve High Titers with the CHOgro® High Yield Expression System. Human IgG1 was produced in Freestyle CHO cells (Thermo Fisher Scientific) using the CHOgro® High Yield Expression System (Mirus Bio). Cultures were shifted to either 32°C or maintained at 37°C, shaking, immediately post-addition of the transfection complexes and the CHOgro® Titer Enhancer. Cultures were analyzed daily for titer (bars) and viability (solid line) post-transfection over the span of 2 weeks. The last day at which viability is above 70% is highlighted. Supernatants were assayed using a standard sandwich human IgG ELISA and viability was measured using a Guava® easyCyte™ 5HT flow cytometer (EMD Millipore). Error bars represent the standard deviation of triplicate technical replicates.