what is the best way to eliminate clumping suspension cells used for transfection. Anti clumping agents interfere transfection and hence can't be used. Though maintaining the cell density low helps, but doesn't help in the long run.

  • $\begingroup$ changing the media can help - for example, chemically-defined media designed specifically for protein expression might work better than media containing serum, which can promote the expression of cell adhesion proteins $\endgroup$
    – MattDMo
    Sep 13, 2013 at 14:20
  • $\begingroup$ Thanks for the reply. I am currently using invitrogen's CHO S SFM which, though not completely defined chemically, is serum free. $\endgroup$ Sep 18, 2013 at 16:14
  • $\begingroup$ I had clumping issues with HepG2 cells for a long time, I had to split them every 2 days and trypsanize them for 30-45 minutes at 37C to really break them up. Then I would split them at 2 or 3 to 1 ratios so that they formed a more uniform distribution. Less density seemed to cause more clumping, probably because each cell grew into a clump. These aren't CHO cells, but might be helpful. $\endgroup$
    – user137
    Jul 27, 2014 at 6:16

1 Answer 1


After looking around for a bit, I came across a few potential solutions however they are not a definitive answer to your question and requires testing under your experimental conditions to see which works best.

This page contained one excellent suggestion and that is to select for cells that do not clump during the passage, by pooling the cells in 15ml of media in a tube and letting the clumped cells to settle down by gravity for 5 minutes and afterwards taking the top 1ml and passaging it and after 20 passages using the exact same technique, you should be able to get single cell suspension. The main caveat is the heavy selection for non-clumped cells, which might harbour certain properties that might be undesirable for the procedure you are interested in so it might be nice to do some RNA expression profiling to observe the differences and perhaps even publish it! Amongst the suggestions on this page included using different concentrations of Mn2+, Mg2+, and Ca2+ in your (custom?) media since cell adhesion is regulated by integrins, cadherins, and selections and in general, Mn2+ increases ligand affinity, Mg2+ promotes adhesion to cells, and Ca2+ decreases cell adhesion as referened here. You can also try heparin, as tested by this paper on CHO cells and it impotent to note that in the study, heparin did not reducing cell growth, antibody secretion, and antigen-binding activity although I'm unsure as to whether heparin would be classified as an anti-clumping agent and whether it interferes with the ability of the cells to get transfected but that can be simple tested by a GFP transfection and looking at the transfection efficiency of the heparin treated and control cells treated with whatever heparin is dissolved in.


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