My suspension cells are clumping together. What is going on?

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Answer

There are a number of factors that can contribute to this:

1) Presence of calcium and magnesium ions. Wash cells in a balanced salt solution without calcium and magnesium. Gently pipette cells to obtain a single cell suspension.

2) Mycoplasma contamination. Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.

3) Cell lysis and release of DNA resulting from overdigestion with proteolytic enzymes. Treat cells with DNase I.

4) Certain suspension cell type try to aggregate when they are not under optimal agitation speed and optimization is required.

5) Certain suspension cell type try to aggregate in bioreactors and it is recommended to use Anti-Clumping Agent (cat # 0010057AE).

Answer Id: E3985

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62ece66b1a9302d885a13328c5d9d074_FAQ

Are there any recommendations for preventing or dispersing cell clumps in a suspension culture?

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Answer

When cells meant to be grown in suspension are grown in static culture, they may form clumps. These clumps will severely limit transfection efficiency and protein expression. It is suggested that FreeStyle™ 293 cells in FreeStyle™ media and CHO-S cells in CD-CHO or CHO-SFM are grown in agitated suspension to reduce the appearance of clumps. However, if they do form you can try the following protocol to break up the clumps:

1) Collect all cells in a 50 ml conical tube. Spin at 500 X g for 10 minutes. Remove all media.
2) Resuspend the cells in 5 mls Trypsin-EDTA (0.5% Trypsin, 5.3mM EDTA*4Na). Titurate cells to break up clumps.
3) Spin cells at 500 X g for 10 minutes. Remove all trypsin and resuspend cells in 5 mls CD-CHO media. Titurate cells to break up clumps. Add an additional 20 mls CH-CHO media.
4) Spin cells at 500 X g for 10 minutes. Remove all media and resuspend cells in 5 mls CD-CHO media. Titurate cells to break up clumps. Add an additional 20 mls CH-CHO media.
5) Spin cells at 500 X g for 10 minutes. Remove all media and resuspend cells in 5 mls CD-CHO media. Titurate cells to break up clumps.
6) Count cells and return to culture. Because there is little protein in CD-CHO media it is necessary to remove all trypsin from the cells before returning the cells to culture.

Answer Id: E4097

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f7eb48b3fac4c765e510b2a5cbdaeda5_FAQ

How much Anti-clumping agent should I use?

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Answer

Use anti-clumping agent at a dilution of 1:1000 in culture medium.

Answer Id: E6411

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b9b1e31e5a884b505105af35f01c9cdc_FAQ

I am using your FreeStyle™ Max 293 Expression System and my cell viability is at most 87-90%. Is this normal? Do you have any tips to increase the cell viability? I am using 125 mL and 250 mL non-baffled, polycarbonate shaker flasks with vented caps. Will using baffled flasks help? Should I add Pluronic® reagent to the medium?

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Answer

Culture viability should be in the high 90% range for the seed stock that you are maintaining and growing for the transfections. After transfection, the viability will slowly decrease to the lower 90% to 80% range by about 3-6 days post-transfection and can drop more dramatically at 7 days or longer. This is when using our recommended protocol, which does not suggest any media changes or supplementation post-transfection.

A few parameters we recommend checking to assure high culture viability are: pH at ~7.0, no visible clumping of the cells, CO2 levels at approximately 7-8% (this should be adjusted to achieve the appropriate pH), volume of culture at 40% or less of nominal flask capacity, and incubator temperature at 37 degrees C. If you have a shaker platform setup in your incubator, the platform may generate sufficient heat to increase the temperature of the incubator. We recommend using a thermometer attached to the shaker platform. Use of baffled flasks may help, if insufficient oxygenation of the cultures is suspected. We also recommend handling and storing the culture media properly, as light exposure can result in breakdown of certain media components. If you are using FreeStyle™ 293 Expression Medium, supplementing with Pluronic® reagent is not necessary.

Note: The two main causes of clumping are 1) allowing the cells to grow to too high a density in culture, and/or 2) not providing sufficient agitation. Hence, clumping can be prevented by sufficient agitation of the culture, regular cell passage schedule, and maintenance of cells at recommended densities. If clumping is observed, it is suggested to discard the culture and start with a fresh vial of frozen cells. Vigorous vortexing for 10-30 seconds may be required at each subculture for a number of passages until the cultures grow predominantly as single cells. Additionally, at each passage, one can weed out the clumped cells by allowing the clumped cells to settle. One can increase the rpm of the spinner and also make sure to passage the culture each time before it grows to over 1.5-2 x 10e6 cells/mL. As long as cell viability remains above 95%, the rpm of the spinner can be increased. At the time of transfection, decrease the rpm for at least the first 2 to 4 hours post-transfection to make sure the DNA-transfection reagent complexes attach well and enter into the cells. One can also add Anti-Clumping Agent (Cat. No. 0010057AE) at a dilution of 1:1000 (1:200 maximum) for the first two passages, then subculture without Anti-Clumping Agent prior to transfection. Do not add Anti-Clumping Agent at any time during the transfection because it will interfere with the transfection. However, Anti-Clumping Agent can be added to the cultures several hours post-transfection.

Answer Id: E9268

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c79cdb2b2f88458e33b8a1b4d3bfe144_FAQ