
By growing cells in unrealistic liquids, scientists may have skewed results - lnguyen
https://www.theatlantic.com/science/archive/2019/01/cancer-culture-media-plasmax/579283/
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bsder
There is also an issue with growing cells in regular atmosphere (20% oxygen)
vs body atmosphere (<8% oxygen).

However, until you _show_ that these differences matter, nobody cares.

~~~
xkcd-sucks
[https://scholar.google.com/scholar?q=low+oxygen+cell+culture...](https://scholar.google.com/scholar?q=low+oxygen+cell+culture&hl=en&as_sdt=0,22)

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"

~~~
bsder
It's actually not that expensive to do:
[http://brincubator.com/](http://brincubator.com/) (Disclaimer: I have links
to these folks, so treat that as unsubstantiated marketing drivel until you
prove otherwise :) )

Those folks have lots of literature citations.

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YeGoblynQueenne
>> This red elixir, known as Eagle’s minimal essential medium (EMEM)...

It's just me, right? That doesn't sound like something out of a D&D
sourcebook?

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nurettin
Showed this to a cancer researcher;

He told me that this is a money-making scheme for selling different lab
equipment.

~~~
rleigh
That's an answer which hasn't had much thought applied to it. In the groups
I've worked in, the problems with culture conditions were long appreciated.
The factors entrenching the existing practices are largely down to:
standardisation between laboratories, being able to publish (non-standard
practices can be a large negative for reviewers), and cost (DMEM is cheap in
liquid or powder form).

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.

~~~
twic
I cultured immortalised and primary cells during my PhD, and i came away with
one firm decision about my postdoc: i would work on a model organism where i
didn't need to do this cringe-inducingly unphysiological practice!

There were groups at the university working on zebrafish and _Xenopus
tropicalis_ (a little frog), both of which are pretty sweet experimental
systems.

Zebrafish are really easy to raise, and are transparent, so you can do live
microscopy on them without having to do surgery or take explants. Someone i
knew worked on a project where you zap them with lasers to cause wounds, and
then watch immune cells respond to the damage.

Trops are more work to raise, and the embryo is not transparent, but they are
the only vertebrate for which we have powerful classical genetic tools - it's
naturally diploid (two copies of each chromosome, like humans, but unlike many
other animals you might consider using), but if you produce embryos using
sperm which has been zapped with UV light to destroy its DNA, they're haploid,
and then if you block the first cell division with a cold shock, they're
diploid, but with two identical copies of each chromosome [1], so sort of
functionally haploid in a way which lets you investigate the effect of
mutations really easily.

[1]
[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4273174/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4273174/)

~~~
pvaldes
uh? Zebrafish aren't transparent. They are white and stripped in metallic
blue. Am I missing something here?

~~~
jonlucc
As the other comment mentions, the embryos are, but also, you can get
amelanistic zebrafish.

This might be an interesting addition. You can't usually just buy zebrafish
from Petco and use them for research. There are standards in animal facilities
that determine whether your fish must be "specific pathogen free" or various
other levels, and so you have to generally buy them from one of a few sources
if you can't bum some from a beneficent lab. When we went to purchase embryos
to set up a zebrafish colony (at something like $400 for 2 females and 1
male), the facility in Europe had suffered a fire that required them to stop
production for 4-6 months.

~~~
pvaldes
As an aquarist I find this hilarious. For $399 you could hire an expert in
fish pathology to check that the fishes are free from diseases and spend the
other dollar into buying the real fishes, and put it in a quarantine tank
then.

> the facility in Europe had suffered a fire that required them to stop
> production for 4-6 months.

This sounds very strange, unless you want to artificially create a scarcity in
the supply of an animal that anybody can breed by thousands in their living
room. You don't need almost any space to breed this fishes, and aquariums can
be moved right?. 6 months seem too much.

I wonder why nobody has tried with another of the 100000 species of fish with
transparent embryos and bypass this money-grabbing structure.

~~~
Balgair
The issue is the genetic 'cleanliness' of the animals. Some zebrafish will
only glow red when you feed them special stuff, some glow green, some only
glow green in certain parts of their yolks/chorion. Sometimes this only
happens with every other generation of females.

It's _very_ complicated and quite 'messy' when it comes to the
phylogeny/genetic trees. Most of the time, researchers only need those 20 fish
or so once every few years, if that. Sometimes they need to interbreed these
different fish to glow green and red. There are literally tens of thousands of
types of fish that have to be kept. As an example: Jackson Labs has mice ready
to order to the specific genotype that you want and it gets _very_ specific
[0]; zebrafish aren't quite at that level yet, but are getting there.

But the effort is worth it. Zebrafish are a great animal for studying neuro
development and many other things. Many of the proposed treatments for ALS,
Alzheimer's, Parkinson's, etc. got their start in zebrafish and in studying
how those little vertebrates function. The effort will save countless human
lives.

[0][https://www.jax.org/strain/008199](https://www.jax.org/strain/008199)

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dekhn
In practice, this could be really bad for machine learning when applied to
cell images (I work for a company that grows modified human cells and collects
the images and applies DNNs to them as classifier). DMEM (or FBS, which we
use) would effectively cause a systemic bias that would be hard to correct
for.

~~~
nycthbris
Sounds interesting. Out of curiosity, what company do you work for?

~~~
dekhn
insitro.com. See also Recursion Pharma.

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msie
This makes me mad. Billions are being poured into medical research and I
wonder how much of it is being wasted. Several weeks ago I heard that some
study concluded that moderate amounts of alcohol and coffee are good for
you[0]...Argh.

[0] [https://abc7news.com/health/alcohol-coffee-could-be-key-
to-l...](https://abc7news.com/health/alcohol-coffee-could-be-key-to-living-
longer-study-finds/4969361/)

~~~
madhadron
How much is being wasted? A lot of it. But media is not one of the big
problems.

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_.

~~~
thunderbong
And all this while I used to think programming is hard!

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rezeroed
I haven't read the article yet, but the title sounds like "by putting salt
water fish in fresh water, scientists may have skewed mortality rates". I hope
it's not that bad.

~~~
rezeroed
Found the anal retentives.

