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How to seed adhering cells (HepG2) evenly in 24-well plates? - (Apr/19/2005 )

Please help me with my problem: the adhering cells (HepG2) form a belt in the middle of the wells of a 24-well plate after seeded. Because of this, cells are overcrowded within the belt, but there are only few cells outside the belt.

I add 0.5 ml media with cells to each well either by using a 10ml disposable pipet or a 1ml pipetor. I have tried swaying back and forth and sideways or doing nothing before putting the plate into the incubator. There is no difference.

With 100mm plate, swaying back and forth and sideways make the cells seed evenly.

Thank you!

Rosi sad.gif



Shaking the plates a few times should have sperad the cells quite nicely, you will need to be quite vigorous for this to work with 24 well plates. To check the spread you can always look at the plate under the microscope before leaving them to attach.

I find that tipping them from side to side works quite well also.



As you are using 24-well plates you could put in 0.5ml of media before you add the cell suspension then try to give the plate a couple of gentle shakes in opposite dirrections to help disperse the cells more freely.

Also, HepG2 cells are notorious for clumping and this may be part your problem, if they have not been seperated well before plating. How do you separate them? I have used either repeated aspiration through a syringe needle (yellow- can't remember gauge) or extending the time used to trypsinize cells to 10 -15 mins depending on just how confluent they were in a flask before repeatedly asprating them with a 5 ml pipette. (I always found the latter to work best). You can them have a look at some cell suspension under the microscope to see if you have any clumps and if so aspirate again as they are very hard to get into single cells for seeding.

Hope this is of some use.



I agree with Kelly that ensuring your cells are well suspended before seeding into the 24-well plate is important ... may I recommend extended trypsin exposure with pipetting up and down to mix thoroughly. Going thru a needle can shear the cells and decrease viability if not done correctly.

Also, shaking the plates do not necessarily spread your cells but pipetting will. If you use a 1 ml pipette, aliquot all the cells and then tilt the plate and pipette the cells up and down from each well. This will evenly distribute the cells in the media and therefore they should settle more evenly.

Have fun biggrin.gif


I usually trypsinize the cells for only 5 mins.
If trypsinized for too long, does it affect the cells viability?
Usually how long will you trypsinize the cells?


Hi Rosi,

I would use 1 ml medium rather than 0.5 ml and mix the suspension using pipette. Because its a small diameter well, moving the plate back and forth on bench causes them to concentrate on one side.

Hope it helps.