Hi Mohannad, 

I had a similar problem in the past and it was solved by reducing the amount of serum to 3-5%. In my case cells it seemed to be cause of cell overproliferation. Cells were dividing too fast and they tended to form very tight layers after a few days. These layers gained buoyancy, detached and float away. Did you see also some debris floating?

Also, if you have, you can test different types of plastics. Clump formation is often a sign that the cells interact better among them than with surrounding substrate.

Cheers,

Hello Sebastian 

Thank you so much for you help sebastian I have been trying to solve this problem for over than 2 weeks and every time the clumps become bigger. 

Yes actually I have been seeing a lot of floating cells and floating debris ... so if i passed those cells into new flask from different manufacturer and reduced my serum concentration to 5% that would do the drill? 

Sincerely,

Hopefully it will, otherwise at least it will help to narrow down some possible suspects...

I would agree with Sebastian about the plastic. We work with these cells on a daily basis and they grow best in these flasks or these types of plates (Sigma T175 flasks F7677-1CS,     BD 15cm2 dishes 353025). They should form a nice single cell monolayer after plating and if they are healthy none should detatch when you change medium. What concentration are you plating them down at? Possibly you are starting with too few cells. I wouldn't decrease the serum from 10% personally as from your picture they do not look like they are reaching confluency before they clump. Also, what concentration glutamine are you using? Have you run a mycoplasma test?

We also do multiple batch tests with different serums (companies will usually provide a 50ml sample to test for free) as these cells very much prefer some serums over others. 

regards,

Rachel

This is a well known issue. Hek293 cells adhere very weakly to plastic alone. In a flask they will usually be fine. In a 6-well plate they will also usually be fine. But you will almost always notice that when you change the medium you will get a sort of "flap" at the area that you change the medium where the cells come up. If this is very bad, then the cells will come up entirely. When they are detached from the substrate, they kind of clump/ball up together. Once they come back down you get the morphology that you see in that picture you posted.

Use normal flasks for culture. They will be fine. The medium for Hek293 should be EMEM + 10% FBS + 1% P/S, not DMEM (http://www.atcc.org/products/all/CRL-1573.aspx#culturemethod). 

For experiments, coat your plates with poly-L-lysine. The protocol is simple. Add sufficient volume of 0.01% PLL to cover the bottom of the well plate you are using. Incubate at RT for 1 hour. Wash 2x with ultra pure water. Dry under UV light for 30-60 minutes.

When you seed cells again on the PLL they will adhere very tightly and you will be able to do numerous medium exchanges over a period of days without losing any cells.

Dear Mohannad,

I am also struggling with this problem with my NIH3t3 cells. I have to let them grow for a period of 2 weeks, for Foci formation assay. Currently have started using 4% serum DMEM, the clumping problem has reduced significantly but not completely. If you got any other solution to this problem, pls share with me.

Thanks

Apoorva Verma

Hello Apoorva

Thank you for your suggestion. Yes I reduced the concentration of FBS to 5% and it worked just fine. HEK cells just grow super fast and reducing the serum conc. will slow them down a little bit and will reduce clump formation. 

Regards,

Mohannad

Looks like stressed/dead cell culture to me... If memory serves, Its been years since I've done culturing but, that, I've seen it before: Happens when pH media gets too acidic and nutrient level drops. Also, is this before or after a split? Cells are very sparse. HEK293 tends to have more healthy/characteristic morphology when cell count is relatively higher... Sometimes, you won't get a perfect population, but, that's why you have error bars for during data analysis. And, differences in morphoology may not always have significant affects on function when its coming from just a tiny subset of your whole sample.

Good luck future readers.