13 replies to this topic

[Alinor19]

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....

Edited by Alinor19, 01 October 2009 - 04:08 AM.

[cotchy]

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?

Edited by cotchy, 01 October 2009 - 04:32 AM.

[Alinor19]

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

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?

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...

[rhombus]

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

[Alinor19]

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

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

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

[cotchy]

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.

[Alinor19]

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

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.

Great... Thank you again

[rustyshackleford]

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!

[Alinor19]

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

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!

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....

[KaurJ]

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.

[newbie101]

Wow..this is such a spot on for my problems. Thanks Alinor19 for posting up this Q. I am trying to set up a tissue culture project but due to some problems we are not able to have a CO2 incubator (well at least for few months i hope so..)

I have already heard about the CO2 independent medium (Gibco) and the Leibovitz L-15 media but the literatures that i found usually just used it to prepare their cells for thier particular experiments so that their cells will still be happy during the course of the experiments out of the CO2 incubator. But they still grow and maintained their cells in other medias (DMEM etc) in Co2 incubator.

So, base on the replies given in this discussions, does it means that i can grow and maintain my cells using either CO2 independent medium or L15 or add HEPES buffer in other medias in a normal incubator (without CO2 supply)?

Thanx!

[cotchy]

newbie101, on Oct 8 2009, 04:41 AM, said:

Wow..this is such a spot on for my problems. Thanks Alinor19 for posting up this Q. I am trying to set up a tissue culture project but due to some problems we are not able to have a CO2 incubator (well at least for few months i hope so..)

I have already heard about the CO2 independent medium (Gibco) and the Leibovitz L-15 media but the literatures that i found usually just used it to prepare their cells for thier particular experiments so that their cells will still be happy during the course of the experiments out of the CO2 incubator. But they still grow and maintained their cells in other medias (DMEM etc) in Co2 incubator.

So, base on the replies given in this discussions, does it means that i can grow and maintain my cells using either CO2 independent medium or L15 or add HEPES buffer in other medias in a normal incubator (without CO2 supply)?

Thanx!

Yes As far as i am aware i think this should work fine,
adding 25mM HEPES to most medias including DMEM/F12 or DMEM buffers them like CO2 does in a CO2
incubator

Cotchy.

[cotchy]

KaurJ, on Oct 7 2009, 04:52 PM, said:

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.

i don think Rhombus means actually sealing the flask, i think you just just use a normal culture flask - without a vent at the top, and keep the lid closed, this ensures a contant internal environment

Rhombus correct me if i am wrong.

Edited by cotchy, 08 October 2009 - 03:41 AM.

[niki]

I used to use flasks without filters in a CO2 incubator, we would slightly loosen the cap. Would you have a loosened cap with flasks outside or completely sealed?

This is a good topic as it is something I have a problem with too. One of my incubators has broken so I am left with only one CO2 incubator which isn't enough.
I am using CHO freestyle cells from invitrogen. However my collegue tried using HEPES but got really bad cell clumping and no cell growth. Conditions as follows:
25mM Hepes in the Freestyle CHO medium, shaking at 125rpm at 37 C in a 250 ml flask

These cells normally need to be at 8% CO2 and are unusually in suspension. I don't know if this makes a difference, as most of our cell lines are adherent and at 5% CO2.

Should I just try a lower concentration? Or could I use NaHCO3? (I have read about the use of that but have no clue how to do it)