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Extending Viability in CHO Cells

Diagram showing viability over time.

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?

Key features of the CHOgro® High Yield Expression System.


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).

Summary of protocol options for use of CHOgro® High Yield Expression System.


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

Graphs showing antibody titer along with CHO cell viability over two weeks post-transfection at 32 and 37 °C with and without the CHOgro® Titer Enhancer.

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.

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