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Tips from the Bench: Cell Culture Tip

Maintain Suspension CHO Cells

Suspension CHO cells have emerged as one of the most important cell lines for protein expression in biomanufacturing for their capacity to grow to high cell density in serum-free medium. Successful cell growth of these popular cells is dependent on several factors, which include media composition, culture format and appropriate cell maintenance.

Routine Cell Splitting

Suspension CHO cells grown in most modern media formulations, such as CHOgro® Expression Medium from Mirus Bio, can easily reach densities of 1-2×107 cells/ml. The doubling time is typically 24 hours for contact-inhibited adherent CHO-K1 cultures but can decrease to nearly half that for suspension CHO cells. This poses a challenge with regards to cell maintenance because cells will quickly consume nutrients and starve if not regularly split to lower cell densities and provided with fresh growth medium.

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Example Cell Splitting Schedule for CHO Cells
Example Cell Splitting Schedule. We recommend splitting suspension CHO cultures to a cell density of 2×106 cells/ml almost every day if the cells will be utilized for transient transfection experiments. To avoid coming into lab on the weekend, cells can be split to a lower density of 0.3×106 cells/ml on Friday. For best results, only cells that have been recently split to 2×106 cells/ml should be transfected.

The Transfection Experts at Mirus Bio recommend splitting suspension CHO cultures to a cell density of 2×106 cells/ml every day if the cells will be utilized for transient transfection experiments. To avoid coming into lab on the weekends, you can split to a lower density of 0.3×106 cells/ml on Friday; this will prevent overgrowth during the three days the cells are not passaged. Maintain cell densities between 0.3×106 and 8×106, and do not exceed 10×106. By following this splitting schedule, your suspension CHO cell culture should exhibit > 95%—often closer to 99%– viability at all times as measured by trypan blue exclusion.

Preparation for High Density Transfection

For optimal transient transfection and protein expression in suspension CHO cells, it is crucial that cells are split the day before transfection, preferably daily for two or more days before transfection. Transfecting suspension CHO cells that have not been passaged for multiple days may significantly reduce protein yields. We recommend splitting the cells to a density of 2×106 cells/ml the day before transfection such that a healthy, high density culture can be further split on the day of transfection to 4×106 cells/ml as specified for the CHOgro® High Yield Expression System. Dilution of the culture to the optimal density on the day of transfection is encouraged because it will provide fresh growth medium to the cells.

Dealing with Cell Clumping

Aggregates of cells, cell “clumps,” may form during normal suspension CHO cell culture depending on the growth medium and attributes of the specific CHO cell line. Clumps will not harm your culture but can aggregate into larger particles that are undesirable for downstream applications such as transfection, filtration or protein purification. Cell clumping also creates difficulties for accurate cell counting and uptake of nutrients and/or transfection complexes, which may not be readily available to cells in the interior of the cell clump.

Clumping of suspension CHO cultures can usually be mitigated through the addition of Poloxamer 188 to the CHOgro® Expression Medium (0.3% v/v final concentration). Importantly, 0.3% Poloxamer 188 will not interfere with transfection. If cell clumping persists, the following gentle decanting protocol can help to reduce the number of clumps in your culture:

  1. During normal cell maintenance, allow your suspension CHO cells to settle for 5 minutes without agitation. Large cell clumps should sink to the bottom of the flask. 
  2. Gently transfer the top half of the cell suspension into a new flask with a sterile pipette. Discard the clumped cells in the old flask.
  3. Add fresh media to reach your desired cell density.

This procedure may be repeated several times to decrease the cell clumps in a culture.

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Less Cell Clumping is Observed with the CHOgro Expression System

Less Cell Clumping is Observed with the CHOgro® Expression System. FreeStyle™ CHO-S cells were cultured in  CHOgro® Expression Medium or FreeStyle™ CHO Expression Medium and seeded into a 125 ml shake flask (20ml culture volume, Thomson) for transfection. Human IgG1 was produced by transient transfection using TransIT-PRO® (1:1) or FreeStyle™ MAX (1.25:1.25) transfection reagents according to the manufacturers’ protocol (reagent:DNA ratio). Transfections were performed using 1 µg or 1.25 µg plasmid DNA per milliliter of culture and cell densities of 2 x 106 cells/ml  or 1x 106 cells/ml  for the CHOgro® or FreeStyle™  System, respectively, at the time of transfection. Pictures were taken of representative flasks and cells (inset) 6 days post-transfection.

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