I have a problem with clumping of HEK293 suspension cells, and this clumping problem decreases the cell viability. I will transfect the cells if I were to reach a favorable cell count.
What can I do to decrease this clumping issue in bioreactor? And also, how anti-clumping agents will affect the transfection step?
Ceren Pekşen My first concern is that HEK293 cells are semi adherent and are usually only in suspension when they are overgrown - possibly look at subculturing at a lower confluency.
Clumping of these cells often occurs when the cells are treated harshly (detachment with trypsin is too long). This generates dead cells which leak DNA, causing stickiness and clumping of cells.
My recommendation is to first wash your cells with PBS twice and simply detach cells by gently pipetting up and down with a P-1000 four or five times. Centrifuge at 300 x g for 5 minutes and plate 1 x 106 cells in a 60 mm dish (or 2 x 106 in a 100 mm dish).
If this does not work, try adding DNase to your media.
Best of luck!
Ceren Pekşen I agree with Jack Hopkins . HEK293 cells are not normally grown in suspension. Adherent cells would not serve your purpose? If not, try a lower percentage of trypsin such as 0.05%. Also, wash with PBS thoroughly to remove dead cells and debris which might lead to cell clumping.
Dear Jack Hopkins and Nabodita Sinha Thank you for your answers, but, the HEK293 cell line I am using is is suspension-adapted, I do not use trypsin at any step. So this may not be the reason.
Also, Jack Hopkins I didn't understand the following statement: "only in suspension when they are overgrown"
Ceren Pekşen how do you split your cells into single celled suspension for experiments? The issue with clumping is that the cells in the middle may not have access to your transfection reagents.
To clarify my statement, if HEK293 cells reach a high confluency, they tend to dissociate from the surface of the culture dish and float.
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