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I am working with HepG2 cells, and they really like to form clumps. Pipetting up and down in TrypLE does not seem to be very effective in breaking them up.

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  • $\begingroup$ Rob: can this question be answered by Google? If it can, please come back and post an answer to your question. Here's a hint: hepg2.com/index.html $\endgroup$
    – Thomas Ingalls
    Dec 22, 2011 at 3:53

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I find that I only get clumps (with HeLa and similar cell types) if I leave them in Tryple too long. Generally I leave them in room-temp (not hot) Tryple for ~8 minutes, then I angle the dish, lid off, so that I can see the "sheen" of cells on the dish (this happens in the hood, of course). Then I blast the sheen with Tryple using a 1000ul pipetter, which de-adheres the cells. These are generally much less clumped than if I let the Tryple de-adhere the cells.

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In some protocols for setting up primary cultures (for example from mouse bone marrow or rat endometrium), there is a step that requires pushing cell suspension through a large needle to get rid of clumps. Of course, this carries a high risk of damaging the cells, so sometimes collagenase is used instead.

It might also be helpful to wash your cells with PBS without ions before passaging and use tripsin with EDTA. That should get rid of at least some calcium from the plate, so that action of adhesion proteins would be inhibited.

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Cell clumps that not dissociate with rigorous pipetting might very well be caused from free DNA in your cell suspension (i.e. if you trypsinized for too long). Free DNA attracts cells which bind altogether forming clumps.

Two possible ways to get rid of these clumps:

  1. Centrifuge your cell suspension at 5000 rpm for 2 minutes with acceleration on (at 4) and brake (at 3) allowing the way heavier molecules to precipitate. Then aspirate ~0.5ml from the top of the suspension and transfer to a new flask. Repeat if needed.
  2. Use DNAse at concentrations from 20-100µg/ml (concentration resulted from personal use) as long as you will not intend to carry out DNA related experiments.

You can always go back and buy a fresh ampule of the cell line you want clumps free :)

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