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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