Gertsch Group

Institute of Biochemistry and Molecular Medicine, University of Bern, Switzerland

Lipids in Fetal Bovine Serum (FBS) - Exploring the Black Box

Lipids in Fetal Bovine Serum (FBS) - Exploring the Black Box
Jurg Gertsch - Wed Sep 14, 2011 @ 07:29PM
Comments: 10


In science, a black box is a device, system or object which can be viewed solely in terms of its input, output and transfer characteristics without any knowledge of its internal workings, that is, its action is "opaque" (black). Almost anything might be referred to as a black box: an algorithm, a human mind, a transistor or a cell culture medium. 

When you are doing cell culture work you put one black box into another black box (cells into cell culture medium).

Anyone has probably experienced that the selection of fetal bovine serum for cell culture work is far from rational. Of course, it has got to be cheap and the cells should be happily growing, but apart from that, relatively little objective measures are applied.

Fetal bovine serum (FBS) (synonym fetal calf serum, FCS) is the sterile liquid that is obtained from the clotted blood of the bovine fetus. It contains numerous factors that are needed for the survival and propagation of mammalian cells in culture and for this reason was introduced early in cell biology research, subsequent to initial studies with hen and sheep sera (Carrel and Ebeling, 1922; Treadwell and Ross, 1963). Already in the 1950s profound differences for cellular growth between human serum and FBS were described and first attempts were made to culture cells in serum-free media (Treadwell and Ross, 1963; Evans and Boyant, 1956). Despite advances in the fabrication of standardized serum-free media over the last decades (van der Valk et al., 2010), FBS still remains the most widely used cell culture medium supplement in cell biology.

FBS is commercially available from numerous manufacturers and researchers typically chose their sera batches based on price, good viability and function of their cell cultures, or cloning efficiency. However, few researchers study the composition of their FBS batches and these sera therefore remain a major black box in cellular experiments. It was previously reported that different sera contain distinct compositions of fatty acids, including arachidonic acid (Lagarde et al., 1984), and that these lipids can directly influence cellular experiments (Stoll and Spector, 1984, Loomis et al, 1983). More recently, it was shown that FBS can contain unknown factors that are able to inhibit Toll-like receptor (TLR) activation under certain circumstances (Bas et al., 2010). Although it is generally accepted that the composition of FBS may directly influence the outcome of cellular experiments, relatively little is known about the impact of lipids.

In a recent study in our laboratory by Janine Marazzi et al. (J. Immunol. Methods) we were able to describe a new algorithm of the black box FBS. We show that the major endocannabinoid 2-AG is able to significantly affect cell culture work. Amazing, if you think about it that we have not managed to standardize our culture conditions to make experiments more comparable. More amazing even if we consider the fact that cell biology is highly descriptive and lacks scientific power (reproducibility, mathematical stringency, predictive value).

I suggest that we make an attempt to profile our cell culture media using state of the art metabolomics technologies - for the sake of insights and scientific progress.

A poem by Arielle Perkins:

I have in my hands two boxes,
Which God gave me to hold.
He said, 'Put all your sorrows in the black box,
And all your joys in the gold.'

I heeded His words, and in the two boxes,
Both my joys and sorrows I stored,
But though the gold became heavier each day,
The black was as light as before.

With curiosity, I opened the black,
I wanted to find out why,
And I saw, in the base of the box, a hole,
Which my sorrows had fallen out by.

I showed the hole to God, and mused,
'I wonder where my sorrows could be! '
He smiled a gentle smile and said,
'My child, they're all here with me..'

I asked God, why He gave me the boxes,
Why the gold and the black with the hole?
'My child, the gold is for you to count your blessings,
The black is for you to let go.'


Comments: 10


1. Ben Haskell   |   Wed Dec 07, 2011 @ 09:10AM

The ethnopharmacological screening of potential immunomodulatory agents usually involves RAW246.7 cells as the in vitro model. Typically, an antiinflammatory effect is observed at 10microMolar with RAW246 cells; this is hailed as some sort of proof for the validity of a traditional herb, and cultural pride in the wisdom of the ancestors (wounded from the onslaught of Modern Western Medicine) is restored. Yet it is quite difficult to achieve a plasma concentration of more than 1 microMolar for most phytochemicals, even with massive doses of herb.

Most immunomodulatory effects of Echinacea alkylamides are observed in the low microMolar range in the case of the RAW246 model; yet your lab observed immunomodulatory effects in the low nannoMolar range with an ex vivo whole blood model; this concentration is relevant the actual concentrations of Echinacea alkylamides achieved in human plasma with moderate doses of Echinacea extract.

This example of a 2-3 orders of magnitude difference in effective dose makes one ask: is the use of the in vitro RAW246 model for ethnopharmacological screening invalid? Is there something "wrong" with RAW246 cells themselves? For example, do they overexpress efflux pumps? Are murine immune cells less sensitive than human? Are viral-immortalized macrophages less sensitive than primary macrophages? Are macrophages less sensitive than other types of immune cells? I did see a paper in Journal Lipid Research in which there were subtle changes in the prostaglandin lipidomics when RAW246 and primary macrophage cultures were compared. But are any of these sufficient to account for 3 orders of magnitude in sensitivity?

Or are RAW246 cells OK per se, and is something present or lacking in the tissue culture media that dulls the sensitivity of the RAW246 model? Is it just that everybody (or nearly everybody) uses a bad medium?

I came across an interesting paper, in which it is shown that fetal calf serum is deficient in Selenium. Addition of 2 nannoMolar Selinite to RAW246 made them more resistant to stimulation by LPS. (Zamamiri-Davis, et al, 2002, Free Radical Biology & Medicine, 32(9):890-897. ) If selinite was added to RAW246 cultures, more LPS would be required to stimulate them, but would they become more sensitive to antiinflammatory phytochemicals?

Another difference between ex vivo whole blood and in vitro RAW246 cultures is that whole blood has lots of serum while most workers add about 5% serum to the cultural medium. One may think that adding more serum would reduce the sensitivity of the RAW246 model, because serum albumin would bind most phytochemicals in an "unavailable" form, and lower the "free" concentration, and hence slow down the absorption of phytochemicals. On the other hand, many phytochemicals have a tendency to aggregate (well studied in your lab for the case of Echinacea alkylamides), so that perhaps serum albumin would actually "monomerize" phytochemicals, and actually speed their absorption by cells in tissue culture. Lets go one step beyond mere "monomerization" of aggregates. Could albumin actually act as a sort of phytochemical chaperone, in which albumin docks onto external receptors (or transporter) so that the binding sites of albumin sits on top of the of receptor binding site, so that transfer of phytochemical to cell is actually catalyzed? Would increasing the serum level of RAW246 cultures lower the sensitivity to stimulation by LPS (by binding to LPS) or increase their sensitivity (by monomerizing or chaperoning LPS)?

Have you seen any paper in which the sensitivity of the RAW246 model (or any other type immune cell) is titrated against the concentration of serum in the culture medium?

2. Jürg Gertsch   |   Fri Dec 09, 2011 @ 04:41PM

Dear Ben,
Thank you very much for your thoughts - I totally agree with you. I don't think that the RAW246 cell line is suitable to elucidate mechanisms of action of bioactive compounds. I also get kind of worried seeing all this microM actions of compounds that probably never reach such concentrations in vivo. RAW246 cells may be used once an effect is already known, say as additional means of looking at some macrophage-mediate effect or a particular mechanism. Also, we find these cells to be very heterogenous and difficult to keep constant over several passages. Most interesting I find your suggestion that albumin is much more than just a serum protein in terms of having an impact on the biophysical behaviour of drugs, including natural products. We got interested in this now as we see 2-AG present in FBS, and it is surprisingly stable there - chaperone is a good hypothesis. The experiment you suggest should actually be made - we have done experiments without FBS and of course, the cells do not behave the same way - why this is remains to be elucidated.

3. Tim Carr   |   Thu Oct 11, 2012 @ 07:45AM

I'm glad I found this page and your article as I got some very strange effects between different media, using distinct FBS batches. The high 2-AG content may explain this. In most papers this point is not addressed at all and I wonder how other laboratories handle this issue.

4. E. Falkner   |   Sat Nov 24, 2012 @ 06:34PM

Congratulations to this important finding. I'm sury it will make a big impact! You should provide your article as open access to increase visibility. Best, E. Falkner

5. Jurg Gertsch   |   Fri Mar 07, 2014 @ 07:01PM

I think that most people simply ignore it - I guess for GPCR signaling this may shift the effect of some ligands to different signaling pathways

6. Steven   |   Sat Jan 24, 2015 @ 01:45PM

2-AG in the cell culture media is likely having effects in many cell culture applications. Thank you very much for this input!

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8. Kagawa   |   Thu Oct 22, 2015 @ 05:17PM

We also noticed that in some FCS there is high amounts of 2-AG and I wonder how one can eliminate this lipid - why is it stable in FCS?

9. Janet   |   Thu Feb 18, 2016 @ 11:45PM

Is there anywhere I can find a total fatty acid profile of FBS?

10. Jurg Gertsch   |   Mon Jan 23, 2017 @ 03:35PM

Hi Janet, to my opinion there is no such thing as a total fatty acid content of FBS. There are numerous lipids in FBS and they vary with batch and if you need to know them you have to measure them.

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