6 replies to this topic

[East_YYH]

Currently I culture Calu-3 and BEAS cells in my lab for 3 times but they are all dead every time.(can only survive 1 week). The cells seems not to be contaminated but float up while culcured in the incubator. At the beginning, cells attached to petri dish but 3-4 days later some of them were detached and float, which happened for all cells after 1 week.
   The items inside incubator have been autoclaved and cleaned with 70% ethanol. Today I calibrate the CO2 concentration, it showed to be 6.4% and now I have set it to 5%. So is it the CO2 level resulting in the floating problem? Or it's because of fungal contamination or something else? My project is just inhibited by this issue,sigh..

Edited by East_YYH, 24 January 2011 - 09:50 AM.

[Eimerian]

There is no way that a mere 1,4 % excess of CO2 can have such a drastic effect.

You use petri dishes? You don't use cell culture flasks?
This might be the problem: Cell culture flasks are coated in order to be high-binding for cells.
If you use "normal" petri dishes (like for microbiological agar) some cell lines might be unable to attach properly.

http://www.lennox.ie...from_Iwaki.html

[East_YYH]

Thanks a lot for your assistance. However, what I used is the cell culture dish that is also coated for attachment. Now the case is, cells grew quite well in the same dish in another lab and attached tightly at the beginning. Then I brought them to my lab, which takes about 15 min outside, and then cultured in incubator. The cells can survive for about 1 week but floating cells get more and more until almost are detached. Is there possiblity that some problem exists during the transfering process?

Eimerian, on 24 January 2011 - 11:54 AM, said:

There is no way that a mere 1,4 % excess of CO2 can have such a drastic effect.

You use petri dishes? You don't use cell culture flasks?
This might be the problem: Cell culture flasks are coated in order to be high-binding for cells.
If you use "normal" petri dishes (like for microbiological agar) some cell lines might be unable to attach properly.

http://www.lennox.ie...from_Iwaki.html

Edited by East_YYH, 24 January 2011 - 12:58 PM.

[bob1]

How often are you changing the medium and/or passaging/subculturing the cells?

Edited by bob1, 24 January 2011 - 02:37 PM.

[East_YYH]

bob1, on 24 January 2011 - 02:37 PM, said:

How often are you changing the medium and/or passaging/subculturing the cells?

I change media every 2 days.

[bob1]

And passaging at what confluence?

[anasus]

I have a similar problem. A collaborative lab send me the cells by mail as had send some other cell lines. But this Calu-3 appears to have a slow growth. The flask is in the incubator for 2 weeks and there is only less then 50% confluence. The cells divide but most of it remain detached in the medium and are removed during the medium replacement...
The medium used is the same that in the other lab. DMEM:F12 with 10% FBS, 1%L-glutamin, 1% NEAA and 100 mg:
ml penicillin/streptomycin. How long did normally Calu-3 cell line to get confluent?