Can buffering strength of media reduce with time ? - (Sep/11/2010 )

Hi everyone...

I have been growing primary HUVECs for 3 years in my lab and never had any problems..
I grow then in M199 with 20% FBS, 1% glutamine, pen-strep and ECGS. we buy the powder from sigma and
reconstitute it ourselves..

Well, right now, Im a different lab for 6 months to do some expts and Im having a hell time to grow my cells.
Nobody in this lab grows HUVECs. So, i brought my own cells as well as media from my lab.
When i start with a new bottle of media, things are fine.
But then when it reaches around half, the cells are fine for a day --they are all adhered nicely. But then
on the second day they all come off and just float if they are in plates,. If they are in flasks with filters
i can see the cells starting to come off at the neck region, and if i replace this media with new media from a full bottle , it stops,
and they are fine again!!!!

WHat is going on over here?? I know the media becomes slightly alkaline as we use it from the bottle and it gets exposed to air,
but it was never an issue in my old lab..
could there be some problem with the CO2 incubator?? everyone else here grows cancer cells and they dont seem to have any probs..
Any ideas how to overcome it!!!!!!!!

raaann...

Hola, This powder medium has to be suplemented with 2.2 g/l of sodium bicarbonate to buffering with CO2. Good luck

Hi there,

If you are worrying about the buffer issue, HEPES serves as a better buffering system than Na2CO3.

In between, since you are culturing HUVEC cells, are you culturing them on fibronectin coated tissue culture flask? Actually, I am working with human brain microsvascular endothelial cells (HBMEC). Recently, I was given a vial of new HBMEC, but they didn't really grow well after sitting in the flask, drinking my medium happily for almost 2 months. I am thinking if fibronectin will make any difference to the cell growth. My old HBMEC were completely happy without fibronectin and endothelial growth factors, but these new cells are very fussy.

Good luck and thank you.

Hi there,

Well, my medium has Na2CO3 added into it during preparation.. and on top of it I also added 10mM hepes.
But can Hepes help the cells in long term, like for more than 24 hrs??

Also, I always coat my dishes with gelatin so that they can stick better..
I use fibronectin, and vitronectin too as a matrix coating , but only for expts.
Gelatin is the most inexpensive one, and i use to grow up my cells.

Right now, Im going to put my cells simultaneously in another incubator too..
and also check the ph before and after..

hopefully i get the answer to my problems..............


and thankyou for the replies...

Dear Raaann,

You need to check the CO2 Incubator with a Fyrite type device that measures the internal concentration of a CO2 Incubator. It's the only way to get a true measurement.

Kindest regards.

Uncle Rhombus