Help with adherent cell culture - (Mar/16/2013 )

We regularly use adherent cell culture in our lab for cells of human origin for cytogenetic studies. Often we see attachments within the first 2-3 days but for some flasks, the attachments do not develop into colonies even after regular feeding. I wonder what might be the reason for this, when a duplicate culture flask grows without any issues.

It could possibly be due to presence of trypsin/EDTA or bad quality of cell culture flask.

Dear Rajpoot,

Thank you for your reply.
We use sera in our culture medium, so Trypsin shouldn't be active I suppose. and culture flasks are of a good make. Like I said, the duplicate culture seems to grow without any issues.

Cells like to grow with other cells, so I guess it is possible that the seeding density is too low to encourage good growth in some conditions?
You could try the same number of cells in a smaller flask?

Dear Leelee,

Thank you for you suggestion.

We do use T-25 flasks, so guess there isn't a flask smaller than that.
But seeding density, might be an issue. We do get some flasks with low cell density.
Since we cant use smaller flasks, guess we will have to resuspend them in lesser media.
Is there anything else that we can do.

(Sorry to ask basic questions, but I have no idea about tissue culture)

Also, any suggestions on what else we could to to promote growth of attached cells?

Thanks,
Ameya

A smaller volume won't really help, as you will still have the same amount of cells for the same surface area.

What volume are you currently using?

And you said in your first post that you have a duplicate flasks that grow well- so do you take an aliquot of cells and split it evenly between two flasks? Or are they duplicate samples from the same source?



No need to apologise for basic questions, everybody has to start somewhere

If not the case - you can try to preliminary coat the plates with collagen type I. Depending on the culture flasks and cells you're using this might be beneficial.

Dear leelee & 2xzwei,

we suspend the cells in 4 ml of media, which is enough to cover the entire surface area of the flask.
and yes, using lesser media won't make a difference (doh)
We set up duplicate cultures from the same aliquot of cells, so some times, when we get very little sample, cell density reduces drastically, and we tend to get a few attachments, but they do not grow.

Reg, sample type, it could be amniotic cells, placental, skin, or other tissue. All our culture flasks come pre-treated for adherent cell culture from the manufacturer. Would collagen help these cells as well?