Dear colleagues, I'm planning an experiment, and to buy an appropriate amount of reagents, I need to find out the number of HepG2 cells per 1 cm^2 of a culturing flask. Please help.
The density of these cells will depend very much on what you would do with them. In some ways they are easy to culture, in other ways very difficult. They have a tendency to form small piles rather than a pure monolayer after a few passages and this is where difficulties in reproducibility can occur. If you have to keep them sub confluent then max density would be approx 8 x 10^4 cells / cm2. I think that's OK as a rough guide for you as you will need to check seeding and harvesting densities empirically anyway. I would recommend that you either purchase several vials of hepG2, (recommend ECACC), and / or make sure that you establish a master bank with at least 10^6 cells per vial as early as possible. The phenotype changes that accompany 'piling up'are either difficult or impossible to reverse. I recommend that you make sure you have adequate FBS, passaging reagents etc from single batches to cover all your work. If the HepG2 phenotype or response you are looking for is sensitive you obviously want to limit variables. Good luck.
Hi Peter,
If you can trypsinize the cells (for example while passaging them) you will just need to use a cell counting chamber (e.g. Neubauer). If this is not possible, I would take pictures of the cell culture, calibrate your microscope and count cells in the view fields, which will give you a rough estimate of the number of cells per surface area.
Hope this helps.
Best regards,
Jochen
Dear Jim, Jochen and Gregor, thank you for considering my problem.
Hello Peter,
It depends on the seeding density because after cells adhere, their growth rates and area depend on contact inhibition. A typical well of a 96-well plate has an area of about 0.32 cm^2. Seeding 10000 HepG2 cells/well after 24h form about 20% confluence and 80000 cells/well - about 80% confluence. Here you can see the curve of the relationship confluence/cell seeding density: https://www.academia.edu/40969549
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