too much cell density can influence adhesion to culture plate - (Sep/08/2015 )

Dear all,

I need ur advice <<

For my experiment, I want to culture my cells in a very high density and the cells does not adhere well.

so if I collect the my Hela cells, and reculture it in a very high density for my experiment ( more than 100% confluent) in medium free of FBS , I see that most of cells are rounded.

However, when I use a part of remaining cells to make new culture plate ( in a lower density ) so it should give me 30% confluent ( medium with serum),  cells are really start to attach and adhere very well.

do you have any experience regarding seeding density and cell attachment.

what is known mostly that if cells are too diluted it may die, so what about if too concentrated.

do u think medium without serum can be responsible for such behaviour.

I would be very grateful if you have any experience in such topic and share it with me

Thank u

Madelin

Hi Madelin:

HeLa cells can be tricky. As you said, seeding them too light is detrimental, but yes, so is too heavy. Attachment shouldn't be a problem if you are using the proper flasks. You didn't seem to have a problem with attachment when you seeded at 30% confluency. I assume the cells stretched out from round to the usual "fibroblast" shape?

The round phenotype you saw with the overly heavy seeding density was probably due to overcrowding. The cells wouldn't be able to fully attach to the flask. I noticed when I grew HeLa's in a monolayer that they tended to pile on top of the monolayer when the culture started to get too heavy (dense). Seeding the cells at a heavy density should be avoided as the monolayer will get "sick" (cells starting to become necrotic, sheets of cells peeling away from the monolayer, etc). You could lose your entire monolayer that way.

As to the ideal culture conditions (concentration of cells for seeding), I recommend talking to the people at ATCC (www.atcc.org). The know their stuff when it comes to propogating the different cell lines in their archive.

Hope this helps.