Need to grow in the absence of CO2 - (Oct/01/2009 )

Hallo
I looked around the forum to check for a possible answer to my problem,
but I did not find anything.
It is possible that I missed something and, if this is the case, I beg your pardon in advance.
I'm not an expert in cell culture, and I have a problem.
We are setting up an experiment in which we want produce lentivirus from 293 cells.
We do have a special room dedicated to the use of lentivirus, but, unfortunately,
in this room we have an incubator in which it is not possible to inflate CO2.
So I wonder if there is any trick (suggestion) that could allow me to grow the cells for
about 24-48 hours (the time required before the harvesting of the virus) in absence of
CO2. I know, for example, that Invitrogen has a product, called CO2 indipendent medium,
that they suggest for this kind of application, but they give very little informations
about the composition of the buffering system (I add down here what they wrote to me)


PRODUCT DESCRIPTION

This medium contains a non-HEPES proprietary buffering system that maintains
stable pH for extended periods of time under atmospheric CO2.

PROTOCOL AND APPLICATION NOTES

Cell Lines Successfully Cultivated:

AE-1, Murine Hybridoma, BHK-21, adherent CHO, CHO in suspension, HT-29,
adenocarcinoma, MDBK, VERO, WI-38, works very well for primary fish cells
CO2-Independent Medium was designed for use in an air-equilibrated environment.

CO2-Independent Medium requires supplementation with both serum and L-glutamine prior to use.

Growth of various cell lines indicate that cells cultured in CO2-Independent Medium
proliferate at rates equal to, or greater, than cells cultured in control media.
Without cells, the pH will be maintained for long periods of time (months, years).

Benefits:

1. Long term pH stability and strong buffering capacity.

2. Maintenance of normal cellular function during culture procedures.

3. May be utilized in open or closed culture systems, depending on the cell line.

4. Ideal for use in toxicological or virus procedures where there is a risk of aerosol contamination or infection.

5. Maintenance of tissue function during transport.

6. Allows for total utilization of product (product does not become alkaline).

7. Phosphate buffer, not HEPES.


Thank you for your patience and for any suggestion you will be able to give me....

Does the experiment have to take place fully in the absence of CO2?

If not why not culture the cells in a flask without vented lid (only Co2 present is in the flask)

this way the CO2 will soon be used up and then your experiment can starrt?

Also if not a big problem why not just disconnect the CO2 or turn it off ro a couple of days?

cotchy on Oct 1 2009, 02:32 PM said:

Thank you for your interest, but probably I didn't managed to make myself clear....
If I could, the experiment SHOULD BE DONE IN NORMAL CO2 CONDITION, but unfortunately,
I'm forced to work in a room were I have not the possibility to provide it to the cells for 24-48 h.
Therefore, I guess, the cells are going to soffer from this unnatural way of growing.
I'm looking for a medium that can MINIMIZE this soffering... Special kind of buffering that can
compensate the missing CO2.
I hope that this time I menaged to explain better my problem.
Excuse me for the misunderstanding...

Dear Alinor19,

The answer to your problem could be very simple. BUT there is one stipulation...the flasks, tubes or vessels that you are growing the cells in have to be SEALED ie. no 0.2uM filter in the caps or dishes/multi-well plates.

All you need is the media, RPMI/DMEM or any other basic media that is buffered by 25mM Hepes.

In the old days (30 years ago), before the use of 0.2uM Filters, the only way to protect the cells from the potentially contaminated growing environment was to use sealed flasks and Hepes buffered media. The cells will be perfectly happy in this media, WITHOUT CO2.

Hope this is useful.

Rhombus

rhombus on Oct 1 2009, 03:05 PM said:

Dear Rhombus
thank you a lot for your suggestion (that seems to arrive from long time.. ).
I think that your experience will be, as you hope, of great help for us...
Thank you again

Alinor19

Hi Alinor yes i couldnt make out yopur exact query but rhombus has hit the nail on the head here.

HEPES is used to buffer media where CO2 is not present however what he doesnt say is you can use any media you have yourself and just add HEPES in to it to make a final concentration of 25 mM

so if you use a different media just buy in some HEPES (all cell biology material supplierss stock this) and it to your own media.

cotchy on Oct 1 2009, 03:53 PM said:

Great... Thank you again

Hi Alinor19,

All cells in our lab are cultured using Leibovitz's L-15 media, which does not require a CO2 environment for buffering.
Please see this Invitrogen link for more details:
http://www.invitrogen.com/site/us/en/home/...ulation.80.html

We've never had any problems using this media in the absence of CO2.

Hope this helps!

rustyshackleford on Oct 2 2009, 04:14 AM said:

Hi rustyshackleford

Thank you for your suggestion that may really reveal usefull. One more question: which kind of cells did you successfully used with this medium. At Invitrogen site they report just a little number of cell type. I wonder if it can fit also with our 293 cells....

Hi

to: Rhombus - why do the cells have to be sealed from environment when using HEPES?

I am currently trying to use HEPES to grow cells in 96 well microtiter plates and after removing cells from CO2 environment the medium turns alkaline (phenol red goes purple), since I did not seal the plate I was wondering whether this might be the reason. I couldn't find any mention of the need to seal the cells in literature.