Hi Nefeli,

I solved this problem by preparing a prediluted suspension of cells in culture medium, which is then transferred to 24well plate.

By doing so you do not need to spread the cells at all, since they are evenly distibuted in the cell suspension. I usually adjust the predilution of cells to cell/ml and plate 1ml medium containing cells. (e.g. 100.000cells/ml=100.000cells/well)

Do not shake the plates at all, but immediately put them to incubator.

Best,

Kai

Hi there! Never shake them in a circular movement, otherwise they will settle in the center. Make sideways movement!

don't use circular motion. Move the plate forward and back, and side to side on a flat surface a couple of times. That should solve your problem.

Salih and Fernanda, thank you very much for your response. This is exactly what they did but the problem persists. Any other idea????

Once I received a new vial of HepG2 cells, and no matter what I did, the cells would stack in the middle of my 24-well plate in multiplayers plus the cells became difficult to detach. we determined that the problem was with the HepG2 cells, and ordered a new vial.

Once you shake the plate forward and backward, side to side do not shift the plate immediately to incubator, keep the plate for 5 min in LAF then transfer the plate to incubator. Before I used to face this same problem during culturing HepG2. This actually works.

Hi Nefeli,

I solved this problem by preparing a prediluted suspension of cells in culture medium, which is then transferred to 24well plate.

By doing so you do not need to spread the cells at all, since they are evenly distibuted in the cell suspension. I usually adjust the predilution of cells to cell/ml and plate 1ml medium containing cells. (e.g. 100.000cells/ml=100.000cells/well)

Do not shake the plates at all, but immediately put them to incubator.

Best,

Kai

Hi Nefeli,

I am really sympathetic with you, because HepG2 are really difficult to plate.

After many trials and errors, I set-up the following protocols: after cell detachment, I pipetted HepG2 until I obtained a single-cell suspension (checking an aliquot after 10 up-down pipetting). To allow cells forming an even distribution after plating, I used two kind of tricks, both based on the observation that cell-cell contacts re-form in HepG2 faster than cell-substrate contacts:

1) I plated the cells using 2 ml of medium per well, and I left the plate undisturbed on a flat surface for almost 1 hour. Then I checked by microscope that the cells have been attached to the well's bottom before placing the multiwell plate in the incubator. Be careful at this point, because a little shake would mess up the plated cells, allowing clump formation.

2) I pre-coated the wells with Collagen I. This renders the bottom of the well really sticky, thus allowing the cells to adhere to the surface before adhering each other. This protocol is really easy, but cells tend to spread more on ColI coated surfaces than they do on plastic, and reach confluence faster.

Hi Nefeli!

I also use Collagen type I, rat tail (Millipore) at a concentration of 7 micrograms/cm2 to help your cells to attach at the surface of your flask, as our collegue Michalangelo does.

Thank you very much! :)

Another thing to keep in mind is to make sure you allow adequate time for cell dissociation when you're passaging the cells. HepG2s take longer than many other cell types to come up off the plate, and once they're off the plate, you need to allow dissociation to occur for a little while longer (~1-2 minutes or so) so that the cells can separate from each other (as mentioned by Michelangelo, the cell-cell contacts are stronger than the cell-substrate contacts; pipetting up and down is another way to promote dissociation, but is not absolutely required with sufficient incubation times). One more point is that sparsely plated cells will grow in clumps rather than even monolayers. HepG2s don't spread out into even monolayers as well as many other cell types; as they grow, they tend to clump together. Good luck!

You have to adjust with cell number. It is should be not too low. Especially at initial passage cell grow very slow and we should have good patience.

If the cell line to too old (after about 20 passage), they create problem. So, better to use new stock.

Best wishes,

Badri

Hi Michelangelo, 

I am also having this trouble and found your answer very enticing. Would you be so kind as to share the protocol to pre-coat the wells with collagen type I? Is it possible for me to pre-coat well that are already tissue culture treated?