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Tips from the Bench: Cell Culture Tip

Maintain Suspension HEK 293 Cells

Suspension HEK 293 cells are commonly used for protein and virus production because they are well characterized, grow heartily in a variety of cell culture media (e.g. serum-free conditions) and are amenable to scale-up in biomanufacturing processes. These workhorses of the cell culture world still require proper care for optimal performance in your next experiment, whether it be for basic science research or for production of biotherapeutics.

Routine Cell Splitting

When first culturing suspension 293 cells, we recommend performing cell counts and evaluating viability daily. Healthy cells double every 24 hours and are ≥ 95% viable by trypan blue exclusion. Ensure that the cells do not overgrow by routinely splitting cultures to a lower cell density (e.g. 5×105 cells/ml). Established suspension 293 cultures grow best when maintained between 5×105 and 4×106 cells/ml. Employing a consistent 3-day split schedule (i.e. Monday, Wednesday, Friday) to keep them within this range of cell densities will allow for optimal cellular growth and performance. Ensure that the cells are split to 5×105 cell/ml on Friday to prevent the cells from overgrowing during the weekend.

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Example Cell Splitting Schedule for HEK 293 Cells
Example Cell Splitting Schedule. Employing a consistent 3-day split schedule will ensure suspension HEK 293 cells are growing optimally. The day before you plan to transfect cells for virus production with TransIT-VirusGEN® Transfection Reagent, resuspend the cells in fresh medium at a density of 2×106 cells/ml (see Tip below).

There may be times when higher cell densities are necessary, such as for transfection. Allowing your cultures to reach densities of up to 6×106 cells/ml for transfection should not adversely affect the cells if they are provided with sufficient nutrients and if growth in high density is only experienced occasionally.

Preparation for High Density Transfection

In some circumstances—such as in high titer lentivirus production using Mirus Bio’s TransIT-VirusGEN® Transfection Reagent—you may need to transfect your suspension 293 cells at a high cell density (e.g. 4×106 cells/ml) to achieve maximal viral titers. To ensure your cells are growing and healthy at the high density that is required, you may want prepare the cells the day before transfection as follows:
  1. Approximately 24 hours before transfection, pellet suspension HEK 293 cells by centrifugation at 300xg for 5 minutes.
  2. Resuspend the cells in fresh medium at a density of 2×106 cells/ml.

This gentle concentration of cells and media change will guarantee that your 293 cells achieve the necessary growth for high density transfection the following day.

Serum-free Complete Growth Media

A variety of serum-free complete growth media are commercially available for suspension 293 cells. Each medium has unique attributes concerning its ability to support high or low cell density growth and transfection compatibility. Some serum-free media contain polyanions such as dextran sulfate or heparin, which can inhibit transfection. Culturing cells in polyanion-containing media is not recommended if transfection is to be performed. If use of polyanion-containing media is necessary in your workflow, perform the transfection in a medium without polyanions and replace it 4 to 24 hours post-transfection. For most applications, a media change should not affect transfection efficiency if performed at least 4 hours post-transfection.

Dealing with Cell Clumping

Aggregates of cells or “clumps” may form during normal cell culture depending on the growth medium and attributes of the specific 293 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 the cells in the interior of the cell clump. Polyanions, such as dextran sulfate or heparin, can help decrease clumping, but can also inhibit transfection (see section ‘Serum-free Complete Growth Medium’ above).

The Transfection Experts at Mirus Bio find clumping of suspension 293 cultures can usually be mitigated by adding Poloxamer 188 (0.1-0.2% v/v final concentration), which 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 293 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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