Caco-2 problems - caco-2 don't want to grow in adherence! (Jul/11/2005 )

Helle everyone!
Could anybody help me with Caco2 cell line! I received a Caco2 cell line from another lab. After 3 weeks that the cell line have been growing in 75ml flasks in adherence I tried to put them in a 6 wells plate. Now the cells are grwoing in suspension and they do not want to adhere to the bottom of the wells. Strangely when I split the 75 ml bottle in teo bottles, after 4 days I have a perfect confluent monolayer of caco-2 cells. Is it something wrong with my wells? Should I change plates? The cells are growing in DMEM 10% FBS.

Bye
Micky

ATCC recomends culture media compostion to be:

Minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, 80%; fetal bovine serum, 20%.

Try this.

can u discuss the trypsinisation details?

I tried this medium last time and I get even worst results as the cells became to dye! Moreover the other lab is working with this medium (DMEM 10% FBS) and everything is ok!
I used Trypsin 2.5 % in HBSS w/o Phenol Red w/o Calcium w/o Magnesium 1to 10 in PBS for trypsinization. Then 1000 rpm for 10 minutes, wash twice with PBS and resuspended in my medium. The one I putted in a flask are growing ok, the culture I put in wells are growing in suspension.........
Could someone helpme??


Micky

Hi, Mickey!
I´ve just been the same problems... my cells doesn´t grow at 24 or even 12 well plates... and normally grow at 25/75 cm flasks. I use the DMEM medium supplemented with 1% non-essential aminoacids and 10% BFS. What plates do you use? I was advised to change my plates, because they probably are not eletrostatic free...

very srange- normally they are very adherent - i didnt even think they would survive in suspension I also use 10% DMEM and have no problems but I am using the C2BBe1 clone which also requires transferrin to be present.........or they dont tend to stick to the flask (Ive been told - never tried it though)