By growing cells in unrealistic liquids, scientists may have skewed results

I attended a small local conference a few years ago at which a presentation on practical low oxygen cell culture was given. It seemed like the perfect way to pep up our fetal hippocampal neuron cultures. But it never happened because of, um, "research culture"

I've used specialist media for endothelial cells, and stem cells of various types (human and mouse, embryonic and mesenchymal). It could be up to £200 for 500ml of culture media, with a shelf life of just a week once you'd added all the frozen supplements. This makes things very costly once you scale things up.

But culture media are only the beginning of the problem. Another source of big problems are the culture conditions. Standard incubator conditions used everywhere are 37.5°C, 5% CO₂, 100% humidity. However, this is not remotely realistic, and is actively damaging to most cell types. The 5% CO₂ keeps the pH in balance (the media contains buffers). But the other 95% is regular atmosphere containing ~20.9% O₂, so the effective concentration is ~19.9% O₂. But the physiological oxygen concentration in the body's tissues is much less, a fraction of a percent.

One culture phenomenon is that "primary" cultures of isolated cells don't proliferate much, but after several "passages" they eventually become "established" and more proliferative (essentially, somewhat cancerous, and losing some of the characteristics of the original cell type). There is also a limit on the total number of passages the cells can withstand (all except the most aggressive cancer lines). This is largely down to cumulative DNA damage from oxygen. If you culture the same cells under low oxygen conditions, the cells don't become established, retain their characteristics, and can be cultured nearly indefinitely. Much more realistic. This can be done with an incubator with additional gas feeds and an oxygen sensor, which can purge the excess oxygen with N₂ then add CO₂ to 5%.

Another problem is that the culture medium is static. The cells use up its nutrients and then sit in their own waste. It needs regular replacement. But this cycle of nutrient excess and deficit and waste accumulation isn't realistic either, and may also have profound effects upon cellular physiology. Tissues in the body are continually perfused by blood plasma, maintaining nutrient levels and removing waste. This is something else which researchers should be developing; in fact, developing such systems was a part of my PhD (an unsuccessful part, I should add; it's actually quite technically difficult to perfuse patterned 3D scaffolds). But doing this for flasks and plates is a much simpler problem.

So these three major areas (media, conditions, perfusion) all contain major deviations from physiological conditions which all have the potential to grossly affect research outcomes. The fact that we have all collectively stuck with what worked for over 50 years is somewhat outrageous, particularly when for the first two there are commercial solutions which could be used today, but most people ignore. That's lazy. None of it is a "money-making scheme", though there would certainly be opportunities for media and equipment vendors.

http://advances.sciencemag.org/content/5/1/eaau7314#sec-10

And the ingredients are here:

http://advances.sciencemag.org/content/suppl/2018/12/21/5.1....

The idea that scientists from the Beatson, an illustrious research institute run by a charity, would be hawking some snake oil is absurd. I suggest you tell your cancer researcher friend that they need to have a word with themselves.

This is precisely the reason "50% of cancer research is irreproducible" is a conservative statement.

If we had started with 'full' media from the beginning we'd still be describing and making media variants rather than getting any cancer research done. And we've done a hell of a lot of good cancer research since these media were defined. It's a little like the 'Standards' xkcd comic - if you make too many standards, the standards cease to be useful as a standard.

Further, the effect of the media in this case here is likely more profound when doing 'biology' rather than 'biochemistry' experiments. And until recently, the cells themselves were toeholds onto real human biology; HeLa cells are very unusual, but we used them because we could reproduce the work between labs because the cells proliferated indefinitely. Who cares about a little unnatural media when the genomes of the cells are completely messed up. (And again, the use of HeLa cells taught us a LOT even if they were very not-normal). Normal human cells just don't last outside of a body - so if one insisted we only use normal human cells there would be zero research, and even less reproducible research because no one could use the same stocks.

It's only and precisely because we do have a (admittedly flawed) standard that such a different and likely more natural media even can be objectively compared against.

Biology has a lot of variables. You can't hold everything constant the way you'd like. So you pick just those that you expect will make the biggest differences. And we should certainly go back and check ourselves every once in a while to ensure we don't get lazy and assume a particular protocol should remain the same in the face of changes in the field. But I don't think that's the problem here. We're just better scientists now - and we're learning that our early assumptions can now be improved on.

The article, especially in its final paragraphs is actually quite even handed. The title, however, is very much hyperbole.

Media are formulated for various reasons. The first reason is to get something to grow in culture at all. This is hard. Most cells from mammals won't survive more than a few hours outside the body. Most bacteria won't grow in liquid culture. So the first thing a medium is formulated for is to get something to grow. Often you fail at this entirely. Leprosy is still cultured in armadillo footpads because we don't have a medium it will grow in. Penny Boston is probably the best in the world at this, and her artistry is basically thinking of what weird thing might put the right bits in medium to get some funny bug to grow. Rusty nails, for example. Getting a culture medium is a big deal.

Next is getting a repeatable culture medium. Old microbiology papers regularly used beef heart broth. That's made by boiling beef hearts and adding a few things to it. The amounts in it are completely uncontrolled. If you try the same experiment two weeks later with stringier beef hearts, and you get different results, is that the broth? Maybe. So we get defined media, media that are repeatable. Weirdly, that may still involve ground up critters. Luria-Bertani broth, which is ubiquitous, includes ground up yeast. We know how to culture yeast into a repeatable ingredient, though.

Then you have specialized broths. Can this organism produce its own ascorbic acid? Try to grow it in a medium without ascorbic acid. Once you get through a bunch of experiments like that, you end up with a minimal medium. Minimal media are incredibly useful for metabolism studies. Let me rephrase: they are essential for metabolism studies.

And then there are selective media, such as 7H9 used for mycobacteria. Tuberculosis grows in it okay. Very little else does. That's really helpful because almost every culturable organism grows faster than tuberculosis, so without a selective medium, you pretty much always get contaminated cultures.

And there are logistical issues. I know that if I grow my culture in a rich medium and then take cells from it and grow them in a minimal medium, I will get different results than if they were passaged through a minimal medium. That's not genetic, it's just physiological adaptation. But in tuberculosis, rich medium takes weeks to grow a culture in. Minimal medium takes months. Better to do a bunch of experiments in the rich medium to minimal medium, see if I find anything, then check if it's an artifact being held over from the rich medium. Cell culture studies aren't as long, but the same argument applies.

Plus every time I start working in a new medium, I have weeks or months of experiments to calibrate it, make sure I know how it affects what I'm studying, and get the data to be able to compare it to my previous media. It's a big investment, so most labs will have a handful that they use.

Meanwhile the organisms are also evolving under the selection pressures you're applying by growing them in these crazy environments. Formulating lab experiments that remain relevant to the world beyond is a difficult, detail driven subject and can go horribly wrong in many fascinating, mind bending ways. So there's a lot of wasted money in research, but it's not because of this stuff. This is table stakes, because biology is hard.

The sigh is because the result is trivial, or because it should be a priori false?

Billions drink alcohol and coffee, so whether any "amounts of" them are good or not is important to study.