How long do NCI-H1299 cells normally take to adhere to a plastic cell culture surface following thawing? and how can we improve the speed of adherence?
We received the cell line from the ATCC. We thawed them two days ago according to the recommendations by the ATCC, but till now they are still suspended in the medium. The cells look global and I can see many paired cells, giving me the impression that they might be dividing
Hi Naim - not entirely familiar with that line but I have grown a lot of adherent lines.
Generally attachment begins within 1-2 hours but cells which are going to attach should have attached overnight (18 hours).
Failure to attach could be due to several problems:
a) cells were dead upon receipt - it would be unusual if all cells were indeed dead - even vials that had been poorly frozen generally have some cells that attach (albeit sparsely). If they were stored incorrectly after receipt (i.e transferred to -80 from the shipping dry ice rather than LN2) this may be a cause.
b) The prepared media does not include FBS or FBS at too low a level.
Adherent cells do not attach to the plastic of the flask directly. ECM proteins in the FBS (such as Fibronectin) adhere to the plastic and the cells stick to these proteins.
Adherence in some lines may be increased by pre-coating the flask.
c) The wrong type of flasks are being used. You need to use TC treated plastic ware for the adherence of cells (to allow proteins in FB to sick down).
Personally I would check that the media contains 10% FBS and that the correct flasks are being used. If both of these things are correct I would contact ATCC. They are generally pretty good at replacing cells if a batch of cells fail to grow and you are using the recommended media and FBS).
Sorry I can't be more help!
Gary
Dear Gary,Thank you very much for your valuable hints. In fact, I used 10% FBS and TC-treated flasks, in addition the cells were stored in LN2 upon recipient. Moreover I preconditioned the culture surface with complete growth medium (containing 10% FBS) for 25 min before transferring the cells into the flask. One thing I would do now is transferring some of the cell suspension into another type of cell culture paltes.
Thanks again
Hi Niam -it seems to me that you have done everything correctly. I was thinking of recommending a different format (i.e. spin and re-plate in a 6 well dish).
However the lack of adherence is deeply suspicious so I would advise contacting ATCC anyway - it will do no harm and they may provide a better vial.
Upon receipt you could always spread the contents of the vial over some different formats (i.e. transfer majority of cells to T75/T15 and a small amount into 24 well/6 well).
Very rarely I have had problems with the flasks (we had a batch where no one could get cells to grow in the centre of the flask) so sometimes this could be the cause.
Good luck with your research!
Gary
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