Tips from the Bench: Cell Culture Tip

Adapt Adherent CHO Cells to Suspension Culture

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Chinese Hamster Ovary (CHO) cells are a popular workhorse for production of recombinant therapeutic proteins. Amenable to both adherent and suspension culture, CHO cells offer versatility in growth platform that is favorable to the end user’s requirements. See Table 1 for factors that differentiate cultures in adherent versus suspension platforms.

Table 1. Factors Differentiating Adherent versus Suspension CHO Cell Cultures

Adherent Suspension
Simple media changes Fresh media can be supplemented during subculturing
 Ease of use at small scale Ease of scale up via dilution of maintenance cells
Growth limited by surface area Growth independent of physical surface
Cells must be dissociated for maintenance/scale up Do not require enzymatic dissociation

So why the switch? Simply put, growing CHO cells in suspension will allow for more manageable cell culturing at larger scales and ease in harvesting, which ultimately results in higher yields.

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TFTB Antibody Yield of Suspension-adapted CHO-K1
Antibody Yield of Suspension-adapted CHO-K1 Cells Equals That of Commercial Suspension CHO Cell Line. Human IgG antibody was produced by transient transfection with TransIT-PRO® Transfection Reagent (Mirus Bio) at a 1:1 reagent-to-DNA ratio (vol:wt) and 1 µg plasmid DNA per milliliter of culture in FreeStyle™ CHO-S cells (Thermo Fisher Scientific) and in-house suspension-adapted CHO-K1 cells cultured in CHOgro® Expression Medium (Mirus Bio) containing 4 mM L-Glutamine and 0.3% (vol:vol) Poloxamer 188 at a cell density of 4×106 cells/ml and grown at 37°C. All cells were split into non-tissue culture treated 6-well plates at the time of transfection. Day 7 and 14 supernatants were analyzed using a standard sandwich human IgG ELISA. Error bars represent standard deviation.

Protocol to Transition Adherent CHO Cells to Suspension with CHOgro® Expression Media

1. Maintain adherent CHO cells in basal media, typically Ham’s F12 or DMEM, containing 10% FBS until cells are doubling normally.

Adapt Cells to Suspension Growth in Serum-Containing Media

2. Trypsinize cells and dilute to 0.5×106 cells/ml using basal media containing FBS plus 0.3% (vol:vol) Poloxamer 188.
3. Incubate cells in a shake flask at an appropriate rpm (e.g. 125 rpm for a 1.9 cm orbital throw in a 125 ml Erlenmeyer-type flask) at 37°C in 5% CO2.
4. Check culture daily for growth and viability. Split with fresh media when needed, keeping cells between 0.5×106 and 4×106 cells/ml. When viability reaches > 90% in suspension culture, move on to adaptation in serum-free media if required for your processes.
5. Prepare complete serum-free CHOgro® Expression Medium by supplementing with 4 mM L-Glutamine and 0.3% (vol:vol) Poloxamer 188.

Reduce the Serum Percentage Slowly by Adaption to CHOgro® Expression Medium

6. Mix the culture media such that it is 10% serum-free complete CHOgro® Expression Medium and 90% serum-containing medium. Culture cells in this mixed medium until the cells return to normal doubling time and viability is ˃ 95%. This usually requires a few passages. Do not increase the percentage of serum-free medium if the viability is below 95%.
7. Increase the ratio of the serum-free medium stepwise until 100% of the medium is complete serum-free CHOgro® Expression Medium (i.e. culture cells in 50% serum-containing + 50% serum-free media, then 25% serum-containing + 75% serum-free media and finally 0% serum-containing + 100% serum-free media) as described in Table 2. Only increase the percentage of target medium when cells are exhibiting normal doubling time and viability is > 95%. Once cells are fully adapted to complete CHOgro® Expression Medium, adjust to growth in 8% CO2 if desired.
8. Cryogenically bank the cells that have been suspension-adapted to serum-free media at 1×106 cells/ml.

Table 2. Stepwise Adaptation Schematic

Basal Media with FBS and Poloxamer 188 Complete CHOgro® Expression Medium
90% 10%
75% 25%
50% 50%
25% 75%
10% 90%
0% 100%

Adapting adherent cells to suspension is a straightforward procedure that provides an alternative to purchasing commercially available cell lines. Implementing the use of suspension-adapted cells can expand growth and harvest workflows while simplifying the culturing process.

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