Serum free medium

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Guy Sovak
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Serum free medium

Hi all,
I am working with BHK cells. For one of my experiments I need to use a DMEM/F12 without serum (usualy I use 5%FBS). Any one have got an expireance with this kind of studies?
How long can I grow the cells without serum.
Guy

macbride
macbride's picture
I've grown cells in low serum

I've grown cells in low serum media (1-2%) for a couple of days. I think I remember reading that serum starvation can be used to synchronize cells, so I probably wouldn't go longer than 2-3 days. I'm not sure what sort of experiment you're doing, but you could also try Opti-MEM, which is media optimized for low serum use.

Jason King
Jason King's picture
It depends on why you need to

It depends on why you need to be serum free and for how long.

When infecting cells in vitro with adenoviruses we usually remove the serum for the first hour or so to make infection more efficient (or to enable us to use less virus).

It is possible to slowly get your cells used to low serum levels. This is good if the cells need to be low or no serum for a few days (maybe because the cells are expressing and secreting a recombinant protein into the medium).

Otherwise there are serum substitutes (which are generally quite pricey) which get used for growing up hybridoma cells - again because the Abs are purified from the culture medium.

Guy Sovak
Guy Sovak's picture
I need to do an asay in which

I need to do an asay in which I use anti Insulin receptor Ab. The serum contains insulin so I dont want it inside the Medium for at least 5Hours.
Guy
parvoman wrote:

It depends on why you need to be serum free and for how long.

When infecting cells in vitro with adenoviruses we usually remove the serum for the first hour or so to make infection more efficient (or to enable us to use less virus).

It is possible to slowly get your cells used to low serum levels. This is good if the cells need to be low or no serum for a few days (maybe because the cells are expressing and secreting a recombinant protein into the medium).

Otherwise there are serum substitutes (which are generally quite pricey) which get used for growing up hybridoma cells - again because the Abs are purified from the culture medium.

R Bishop
R Bishop's picture
You will be fine. I

You will be fine. I routinely starve cells overnight (16hrs) in serum-free media. 5 hrs should be no problem.

RB

Fraser Moss
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I dont know what your
samm
samm's picture
gsovak wrote:Hi all,

gsovak wrote:

Hi all,
I am working with BHK cells. For one of my experiments I need to use a DMEM/F12 without serum (usualy I use 5%FBS). Any one have got an expireance with this kind of studies?
How long can I grow the cells without serum.
Guy

Hi! I've done HEK293Ts in serum free conditions (following washing off of 10% serum media) for 16 h with no problems - though cell cycle pogression comes to a near total halt by then.
Also, I have routinely transferred B cell hybridomas to serum-free media - but that has to be done in a phased manner (1:2 or 1:3 dilutions, until the serum is all gone) - and in "pure" SFM, the cells grow for 6-8 days before they deteriorate.
-sam

Guy Sovak
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Thank you all for the support

Thank you all for the support and help
Guy

asif123
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Dear frasermoss,

Dear frasermoss,
I am also working on BHK-21 cell line and want to adopt them in serum free media for the expression of protein of interest(not in the growth phase).
Do you have personal experience with any one of the said media?
I will be very thankful of your advice.
Regards
Asif

taotan
taotan's picture
Hi gsovak,

Hi gsovak,
 In our lab , we use Knock out DMEM (invitrogen). and We culture the cell in this medium without serum for 1-2days. After that, most of  cells (about 70%) were arrested in G1 phase. Hope this can help you!

Fraser Moss
Fraser Moss's picture
 @asif

 @asif

Sorry I was just providing the information on the major suppliers that I know of.

I have not had to work with those reagents myself

asif123
asif123's picture
Dear taotan and frasermoss,

Dear taotan and frasermoss,
Thanks for information, taotan your reply is very helpful for my work.
I want to know more detail about serum free, do you mean to say basal media (DMEM) without serum or serum alternatives.
In addition I want to keep the cells in serum free for more time, have you observed loss of cells after 2 days.
Thanks
Asif

taotan
taotan's picture
Dear asif,

Dear asif,
we used basal media without serum or serum alternatives. After 2 days treatment, more cells will die when compare to cells culturing in serum. We used PI to stain the cells and found the fraction of PI positive was increased.Hope this can help you!

asif123
asif123's picture
Dear Taotan,

Dear Taotan,
Many thanks for help, may I know the serum alternative you are using, because I find a lot of serum alternative available in the market and confused to choose any one of them.
Have you used NaOH solution for PH adjustment.
 
Regards
Asif

taotan
taotan's picture
Dear Asif,

Dear Asif,
We use GIBCO? Knockout? Serum Replacement from Invitrogen for stem cell culture. If we do not use serum alternative, we use NaOH solution for PH adjustment.Best regards!

samm
samm's picture
There are a bunch of animal

There are a bunch of animal component free defined serum replacements available now, many optimized to a specific cell type (e.g. fibroblasts). I have not had experience with your cell line.
Also, if this is for protein production, you can try the high glucose DMEM variant with glutamine, and wean the cells off to ~1% FBS for expression.

asif123
asif123's picture
Dear Taotan,

Dear Taotan,
I am very thankful of your valuable information, I will be very happy if I can support you in any respect.
One point I want to confirm from you, in the growth phase of the BHK cells I have developed the relation between expressions of the protein of interest and number of cells in the adherent culture. Because I do not have to add any inducing agent for the expression and I considered that the expression is directly proportional to the number of cells incase of growth phase (optimum condition for growth).
Could you please confirm my view, if you experience the same.
Thanks
Asif  

asif123
asif123's picture
Dear Samm

Dear Samm
Thanks for information, yes this is for protein production, could you please elaborate “high glucose DMEM with glutamine” because I experienced that in the case of serum free condition the glucose & glutamine consumption of the cells reduced by around 50%. And if I will change the media with serum free high glucose media then I afraid it will increase the lactic acid production.
Thanks in advance.
Asif

taotan
taotan's picture
Hi,

Hi,
 Asif. In our study, if the cells grow well, the expression level of your target protein is high. However, I have not much more details about this. Hope this can help you! Yours Tao

vanshita
vanshita's picture
I have grown cancer cell

I have grown cancer cell lines without serum, some of cell shown apoptosis . But when I culture cells in slowly lowering the FBS percentage then I found cells grown very well for longer time even without serum.

asif123
asif123's picture
Dear   Toaton,

Dear   Toaton,
Thanks, I always found you when ever I need help in the scientific field.
Again I am in need of your help, I want to adapt my cells in the suspension culture with serum, I do not have spinner flask, I am having conical flask, magnetic stirrer, CO2 incubator.
I have shaker  but can’t fit in to the CO2 incubator, could you please support me in this regards.
durraniasif1@rediffmail.com
 Thanks
Asif

MaryAnnS
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There are actually serum

There are actually serum subsitutes now that are actually reasonable cost wise. I don't know what your actual application is, but if you can be more specific, Ican  recommend some good supplements that can replace FBS in many different cell lines such as hybridoma, CHO, HEK293, etc.

The FFM
The FFM's picture
MaryAnnS

MaryAnnS

If you don't mind can you please take the time to list here which substituents can be used in place of FBS in medias for each different type of cell ine?

Thanks in advance

MaryAnnS
MaryAnnS's picture
Hello,

Hello,

Companies such as InVitria provide serum free alternatives without sacrificing productivity and without adding significant cost to your process. I have the most experience with ZAP-HYbridoma, InVitria’s animal free and IgG free FBS replacement supplement for hybridoma cells. This supplement does not require an adaptation period and it is very easy to use. It is already supplied as a sterile liquid supplement, so just add to your media and you are good to go. For CHO cells, InVitria also has a number of supplements to improve current commercially available serum free media formulations. You just add to your current media and it improves growth and productivity. In serum free cell culture, each cell line has a different requirement. It is best to visit InVitria’s website because it is a good resource for serum free cell culture of cell lines that I mentioned as well as other ones such as stem and primary cells. I hope this answers your question.