My endothelial cells formed light brown color clumps a day after I plated it. It looks like cells clumped together but I am not sure. The cells have reached only up to some 60% Confluency and I suspended these cells in the media very well. How to get rid of clump formation?
The aggregations are still present after the second day? Can you exclude a mycoplasma contermination or a bacterial contamination?
With EA.hy926 cells you should not get these big clumps. There are a couple of possible explanations
1) these are just clumps of ea cells .
if this is the case , when using trypsin to release cells from the dish/plate do not shake tap or bang the plate. Just leave them in the incubator for 5-10 minutes check them regularly to assess whether they have been released. To check, rock the plate so that you can see the trypsin move and look for the cells floating in suspension. Also look with low magnification on a microscope to ensure there are not lots of clumps of cells.
When diluting the trypsin with Fbs dmem pipettes multiple times to disrupt any remaining clumps.
2) watch to see if the clumps expand, or the media changes colour.
in this case then it is likely that the culture has been contaminated. In this case throw out the cells and media. Then Clean your incubator before thawing new cells.
EA.hy cells should not form brown clumps. I would check for contamination first, as mentioned by Neil.
Also, for any endothelial cell culture, I would recommend not diluting them too much when you split them (I like a 1:3 ratio once or twice/week, max 3x/week). They grow much better and keep a more normal phenotype when they stay in contact with many other cells. This ensures a nice monolayer formation, without crowding or "clumping".
I work with EAhy926 cells since a number of years but never observed such a phenotype, obviously in agreement with the other authors. How sure are you that you really have EAhy? They should be positive for vWF, CD31 and NO production for instance - can you measure this? Or check their identity by other means, eg cell typing?Honestly, I would consider to throw them away and use a new batch instead of characterizing a mystery. Anyway, I wouldnt keep them in culture for no longer than 20 passages either.
There is another endothelial cell line available, which responds to shear stress with increased eNOS expression and NO formatioon, which EAhy926 in our hands do not: human umbilical vein endothelial cells (HUE; ATCC CRL-1730). I would use these cells instead of these EAhy with this strange phenotype. Like Sven I would throw these cells away.
If the cells keep clumping after passage into fibronectin coated plate then you may have real problem with them, Some endothelial cells need suppot of extracellular matrix to grow on. Another option is a dilute coating of the plate with collagen or matrigel
Hi Sriram,
Have you changed the serum batch? In my experience, if adherent cells dont attach well they will from clumps. The serum afects the cell attachement.
My first guess would be a sub-optimal trypsinization. At the next passage, increase the trypsin incubation and monitor your cell dissociation. Once you clearly singularized cells, proceed to the wash steps and replating as usual. These cells are an hybridoma of HUVECs and lung carcinoma cells, giving them important cell-cell junctions.
Otherwise, have you ever have problem with them before or it just appeared in your culture?
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