Hi Everyone,
I thawed a new vial of Caco-2 cells from ECACC, I've done everything according to protocol, but the cells grow very slow and some of them are detaching and dying.
I have also observed a strange looking filaments (like on attached pictures), but only in a few cell groups, rest of them looks OK.
I've done PCR test for bacteria and fungi contamination, but I got a negative result.
Medium is clear and slowly changing color to orange-red.
Can anyone suggest what is wrong with my cells?
I would appraciate any help.
Hi Frida Gorreja ,
thank you for your answer!
As for now, I use two types of media in two separate bottles, just to check which one is better: EMEM +10%FBS+1%NEAA+1%L-glutamine+1%P/S+1% Aphotericin B and DMEM+20%FBS+1%NEAA+1%L-Glutamine+1%P/S+1%Aphotericin B.
The one with EMEM looks slightly better, but also can see a few floating cells.
I thawed them as in Sigma protocol - in water bath in 37degrees for about 1 minute then transfer to tube with 5 ml of medium and cetrifuge at 150 x g for 5 minutes.
Now I am on third passage. Maybe there is something wrong with passaging... it is almost a week after passage, I change medium every other day, but still there is not even 70% confluent.
I remember using DMEM for my Caco-2 culture, but I also remember having to add sodium bicarbonate because the cells needed a specific pH. Have you come across this in papers using Caco-2?
I haven't found any information about adding sodium bicarbonate to the medium. DMEM and EMEM I use already have sodium bicarbonate in the composition.
I will try to find some more info about pH for Caco-2 culture.
Thank you Vanesa G Martinez !
I used MEM plus HEPES and sodium bicarbonate, I´m trying to find out exactly how much I added of each but it was a long time ago!
Hi,
I'd recommend you to check data on Caco-2 cells on ATCC web page as follows: https://www.atcc.org/products/all/htb-37.aspx#culturemethod
There you will find all necessary data on culture, subculturing, doubling time (62 h by the way) and even a few pics of culture morphology.
Maybe it will help you. There is too little inf on your questioning in order to help you further.
At least I didn't see signs of Mycoplasma spp. in your cultures. But you may check it anyway by hoescht or PCR.
Maybe it will help you. There is too little info on your questioning in order to help you further.
Hope it helps.
Hi Aleksandra,
We don't use antibiotics with our Caco-2 cells and culture them in MEM with added HEPES, 10% FCS and L-glut.
Part or your problem may be that you are feeding them with fresh medium every day. From my experience with Caco-2, with routine growing, they are slow the first day but grow progressively faster with time...probably some autocrine factor production....so if you feed every day, they will not produce sufficient factors for their growth.
Another point to watch is the confluency. If they are allowed to become too confluent they will spontaneously start to differentiate. From about day 6 in culture, some of the cells, particularly those in the center of colonies will start to differentiate even if there are still areas without cells. Natoli et al., 2010 published a paper on good cell culture practices for Caco-2 and that group recommends splitting them when 50-70% confluency is reached or 6 days. This is what we routinely do with our Caco-2s.
Hope this is of help
Hi, Aleksandra.
Very nice support from Dr Joanne Keenan and it agrees to the ATCC rules for culture. So, two responses giving your support for 6 -7 days of culture and only 1-2 changes in medium (MEM + FCS 10-20%).
All the best for you!
Thank you all for your help!
I will keep it in mind and won't let them grow too confluent.
As regards to the doubling time, I found the SOP for thawing, propagating and Cryopreserving Caco-2 where PDT is approximately 32 h, that's why I was so impatient. Anyway the SOP is from 2012, so it might be a little out of date.
I also wanted to ask: when I'm changing a media, should I wash cells with PBS, or just aspirate medium and add fresh one?
I´ve never really washed them when changing the media, just aspirate and add fresh. If anything, replacing only part of the media could leave some cell-produced factors floating around and get cells to grow quicker.
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