mouse fibroblast culture: what medium? - (Apr/29/2007 )

I am new to cell culture and I want to work with mouse fibroblast cells. Unfortunately, I am confused. I hope someone would enlighten me on the following matters.

What is the best DMEM for mouse fibroblasts? For instance, if I wanted to use the protocol of the Thomson Lab (http://ink.primate.wisc.edu/~thomson/pdfs/harvesting_MEF.pdf), what should I use?

One of the catalogs I have read listed down several types of DMEM
i. e.
DMEM (w/ sodium pyruvate, glucose, sodium bicarbonate and L-glutamine)
DMEM (w/ sodium pyruvate, glucose, sodium bicarbonate; w/o phemol red and L-glutamine)
DMEM (w/ glucose; w/o L-glutamine, L-leucine, L-methionine and sodium bicarbonate)
etc.
And should I work with the liquid medium or the powder medium?

Also, is the PBS required for the culture the Dulbecco's PBS (without Ca/Mg) or just plain PBS? And is the concentration of trypsin and EDTA standardized? Is it only 0.25% trypsin/0.02% EDTA?

Please help.

Thanks

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Hi

It depends on what kind of fibroblasts you use. Is it a cell line (which) or a primary culture? Is it embryonic? What would you like it to use for (differentiation or other)?

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I am going to use it for primary culture and they're going to be embryonic. I want to test the effect of certain bioactive compounds on them.

Thanks!

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We use DMEM 10% FCS +piruvat+ 2mM glutamine +_ vitamine + pen\strep. Works well with lung embrio fibroblasts

About powder or liquid - it depends on have you acess to deionised water or not. Usage of powders is cheap.

when you only start cultivate fibroblasts from source i think it will be better to use DMEM with 4,5g\l glucose

About trypsin - we never use 0,25% commercial we dilute it 5 fold till 0,05% and use it as working solution

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Hi

We've worked with home-made primary embryonic fibroblasts and we used DMEM high glucose (Sigma, I guess 4.5 g/L ) + 10% FCS (Euroclone) + Pen/Strep (antibiotics). The medium contained glutamine. If you use glutamine free medium, you need to supplement your medium with it and we usually added fresh glutamine after 2 weeks.

If I remember well, at the beginning we probably used non-essential amino acid, too but then it just did not seem to be necessary

What we always tested was the FCS, we used one batch that worked well.

We used home-made as well as Sigma trypsine.
Also, we used both powder and liquid. However, when you use liquid, the quality of your water is essential (we had problems with this). MIllipore MQ water we used. Sometimes extra filtration was required depending on the system.

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Hi clockwatcher

We use mouse embyonic fibroblasts (MEFs) routinely in our lab.
Our growth media is MEM (Gibco) which contains Earle's salts, L-glutamine, and nonessential amino acids. We order in powder form (cat no. 41500-018), add the specified amount of sodium bicarb and make up in milliQ water- then filter sterilise. We have used liquid form also and have found that either is fine.
We generally culture out MEFs in this media with 8% NCS added, and antibiotics (Gentamicin 40 mg/ml
Penicillin 60 mg/ml).

You will definitely need to use PBS that is Ca and Mg free. We make our own. I have put the recipe we use below- its for a 1L 1x solution.

Na²HPO4 0.92g
KH²PO⁴ 0.20g
KCl 0.20g
NaCl 9.84g

We also use trysin at a working conc of 0.25%, however we make ours in PBS and don't add any EDTA.

Hope this helps

Laura

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Hi! Many thanks to everyone who replied.

My dilemma now revolves around this: should I choose the medium with or without sodium pyruvate? And is sodium bicarbonate absolutely important?

From the protocol I have found, the MEF culture medium consists of 450 mL DMEM high glucose + 50 mL FBS + 5 mL 100x pen/strep/L-glutamine. Is it okay if L-glutamine is already added to the DMEM (like the media mentioned in the preceding paragraph)?

To Laura, I will prepare the PBS as you mentioned. But why are the amounts of the reagents I found at this site: <http://www.bio.com/protocolstools/protocol.jhtml?id=p2152> different? Do I still have to adjust the pH of the solution using your specifications?

Hoping for your favorable and much needed responses.

-clockwatcher-

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oh yeh sorry, forgot to say about pH!! Our recipe says to adjust to pH of 7.3 with NaOH, however I never need to make any adjustments when I make it, the pH is always about right. As far as I am aware, there are a variety of ways to make PBS. The recipe we use is actually called MOBS (mouse osmolarity buffered saline) and works just fine for all the mouse cell lines we work with.

about the media, if L-glutamine is already in the media you buy- thats good and you won't need to add any more. But once your media is more than a month or so old (if in liquid form), I would top up with fresh L-glut as it degrades even at 4 degrees.
about sodium bicarbonate- I think it is necessary for buffering??. If you buy a powder media without it, the packaging will instruct you to add it when making up your solution.
not sure about sodium pyruvate- my understanding is that only some cell lines need it- and our MEFs grow excellent without it so I would imagine that you don't need to worry about it for yours.

goodluck

Laura

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