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Magnetic_Syncopation t1_ixktt0n wrote

Reply to comment by iayork in If freezing tissue generally damages the cells, how are we able to freeze human eggs and embryos for birthing later? by badblackguy

What are examples of cryoprotectants often used? Sugar?

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SemogAziul t1_ixkxkus wrote

There are two types of cryoprotectants: extracellulars and intracellulars. Extracelullar cryoprotectants are impermeable to the mebrane and protect the membrane of the cell and they can be sugar-based (glucose-based, lactose-based, others) or protein based (egg yolk, albumine, others). Intracellular cryoprotectants are permeable to the membrane and prevent against cryoinjuries (ice particules forming inside the cell and then breaking the membrane) and they can be glycerol, dimethylsuffoxide, dimethylacetamide, ethylene glycol, methanol, methylglycol. There are more cryoprotectants but these are the most common used

Edit: The cryoprotectants I've mentioned are all that I've used on sperm cells. Some might not be viable for embryos

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Magnetic_Syncopation t1_ixl0wo2 wrote

Interesting that methanol is safe in an embryo, but it's also a poison that can lead to blindness.

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GalFisk t1_ixl3k2x wrote

Methanol is toxic once you have a nervous system that can be affected by it, and a liver that can convert it to formic acid.

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pavlovs__dawg t1_ixn2xzt wrote

More important in this context is the concentration. Methanol at a certain concentration will 100% kill the cells but a high concentration isn’t needed for cryoprotection. As always, dose defines the poison.

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FabulouslyFrantic t1_ixni91d wrote

Your last sentence triggered a question: how low does a dose of something need to be for it to no longer be considered 'poisonous'.

Are there poisons that stop being poisonous on a molecular level?

And I'm considering 'actual' poisons such as arsenic, digitalis, etc. rather than overdoses of, say, opioids. (Me no science brain person, it's just a question)

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krista t1_ixns2f0 wrote

”poison” and ”toxic” are sort of blanket terms, like ”death” or ”dying”: there's a zillion different ways a poison or toxin can screw with your system.

for instance, opiates (drugs derived directly from poppy plants) aren't exactly toxic, but they might be considered a poison... if someone overdoses, they stop breathing, and die because they stopped breathing. if you were to put them on a ventilator, when the opiates were metabolized by their body, they'd be fine... minus the trauma of sticking a tube into their lungs for the ventilator.

digitalis screws with the balance of sodium and potassium... mainly around your heart. roughly, sodium causes a muscle to contract, potassium causes it to relax. those two, plus calcium, also work in the mitochondria to produce atp. a little bit of digitalis was one of the first heart medications for atrial fibrillation (the top part of the heart spasming)... too much really screws with the sodium/potassium balance in the entire body... and you die.

arsenic is a bit more of a full-body thing from the start... it disrupts atp production in the cells, killing your cells directly. this tends to destroy everything that gets access to the arsenic, which is usually your organs first. interestingly, arsenic was also used as a medicine quite a while back, and in small doses is a stimulant.

cyanide (a whole category of molecules) kills by binding to the iron in your body, disrupting your ability to transport oxygen... one of the reasons for its name (cyan is a blue-ish color): you turn blue when poisoned with it, similar to asphyxiation... except you can breathe, you just can't do anything useful with the oxygen.

hell, if you dive underwater (scuba), pure oxygen itself becomes toxic well before you get 33' (10m) deep. really deep prolonged divers breath a mix of gases, going sometimes as low as 0.8% oxygen. i don't exactly remember the mechanism that kills you here.

some things, like tylanol (acetaminophen, apap), aren't toxic directly, but as your liver depletes the stuff it uses to break the drug down, it starts using a secondary reaction to get rid of the excess... this secondary reaction produces a toxic substance that kills your liver... aaannnddd you die.

think of the body as a miraculous balancing act: there are hundreds (possibly thousands) of balances between two or sometimes lots more than two things. to an extent it trys to maintain a balance... but swing something too far in one direction and the whole thing collapses. because it's balanced in so many different ways, there's a lot of different ways you can screw with that balance.

edit/add: some of the most potent things are very similar to the signals your body uses to signal that one or more of those balances are off... or to signal your body to correct one of those balances somehow. a metaphor here would be tossing a bit of sand in the eye watching a crane do its work: a tiny bit of sand can lead to something catastrophic if the person running the crane is doing something delicate. same with your body: screw with the wrong signaling mechanism and your body will destroy itself trying to correct a problem that doesn't exist anywhere except the alert system telling your body something is wrong.

keep in mind, those signaling and sensing mechanisms in your body are also in balance.

some we call ”medicine”... some we call toxins or poisons... the difference is usually the dose and intent, not the substance.

there's a thing called a ”therapeutic index”, which is a measure of how big the difference between ”medicine” and ”poison” is. some things, like a lot of over the counter medicine, has a very high therapeutic index. other things, like digitalis, has a very low therapeutic index...

so therefore at a molecular level, there's nothing that i know that can kill you, a human, from having a few molecules of it in your body... unless you consider a prion a molecule, in which case a few of those in the wrong place can cause a horrible death in a number of years... or rna/dna, like in a virus... but those are generally considered disease vectors and not directly toxic.

in short, a few molecules of something won't kill you because you are made of a shitload of molecules. it can get a bit dicey, though, when you start getting into micrograms of things that are very finely balanced. keep in mind there are a lot of molecules in a microgram.

anyhoo, apologies for the braindump, (writing this on my mobile off the top of my head) and not having a simpler answer for you :(

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otterscotch t1_ixnvhzh wrote

This was a fascinating read. Very simple and clearly written. Thanks for the brain dump!

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clivehorse t1_ixnt479 wrote

Paracetamol (Tylenol) is fairly easy to accidentally kill/maim yourself with. Botox is the classic poison-as-medicine, along with lithium. Ethanol lulz.

According to google arsenic is a treatment for a very specific leukaemia . Digitalis is commonly used as heart medication https://www.nhs.uk/medicines/digoxin/. Warfarin (the blood thinner) is obviously great if you need your blood thinning and terrible if you get physical trauma, even in the same concentrations.

Pretty much anything can be poison in high doses. There's that one lady who killed herself with too much water in a radio competition.

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FogeltheVogel t1_ixl9uro wrote

Lots of cryoprotectants are toxic to the cells we add to. The trick is that, after adding it to the sample, the sample quickly goes in the liquid nitrogen to freeze. This stops biological activity, which is why the toxic properties of the cryoprotectant don't damage the cells.

After thawing a sample, the cryoprotectant is quickly replaced with regular media before it can damage the sample

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ebfortin t1_ixlvn08 wrote

What happen when you thaw the sample? How do you make sure it thaw relatively evenly? You can freeze pretty quickly but thawing is another matter.

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FogeltheVogel t1_ixlwx8v wrote

The cells won't instantly die. We're talking about a window of (up to) hours here. And we're also talking about vials containing 1 millilitre of liquid.

In general, you thaw such a sample by simply placing it in liquid water. It'll be thawed in minutes.

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aTacoParty t1_ixlx6cp wrote

Depends on the size of the sample. Cells are really small so the volume they are frozen in can be very small too. I work with cells (stem cells, neurons, and others) and cryopreserve them regularly often at 100,000 cells in 500 microliters (0.017 fl ounces) using DMSO as a cryopreservent. When I thaw them, I'll place the vial in a 37C water bath which will thaw them in about 30 - 45 seconds. Then the cells are quickly diluted in media without DMSO to reduce the concentration. The solution is spun so the cells all pellet in the bottom of the tube, the media with DMSO is removed and replaced with fresh media for plating.

Larger volumes are generally not used precisely for the reason that they do not freeze and thaw evenly (IE the interior freezes/thaws slower than the outside). It can be done with special plasticware that increases the surface area but I've never seen it done in routine tissue culture as there is no need.

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ebfortin t1_ixm0kad wrote

And a cell needs oxygen and fuel to stay alive. After you thaw them what happen? How do they stay alive? Are they in some kind of stopped state?

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Farts_McGee t1_ixm16ut wrote

So a single cell's oxygen requirement is trivial. Atmospheric diffusion is more than enough to supply the required amount.

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Saccharomycelium t1_ixmfysi wrote

Cells are ok in liquid cultures, as long as the amount is right. The majority grows fine at 5% carbondioxide and for some types, 20% oxygen. Typicalls the cells are kept in wide surface containers instead of tubes, so there can be some gas exchange passively. If the volume is too large, it may be insufficient. But also if there's too little liquid medium, the nutrients get exhausted faster and cell waste piles up faster, which is toxic and will kill cells again. And one of the main wastes is carbondioxide again.

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FogeltheVogel t1_ixmhkkp wrote

The media contains nutrients (fuel), that's (part of) its purpose. As for oxygen, it is dissolved in the liquid and the cells get their oxygen from that. Just like how, in our body, oxygen is dissolved in blood.

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aTacoParty t1_ixn3ctv wrote

The cells resume their cell functions almost immediately after thawing. You'll hear the term "media" a lot when talking about cell culture. Media is a general term for liquid food for cells. It contains macronutrients (carbs like sugar, protein usually in the form of amino acids, and fats), micronutrients (vitamins/minerals), some other components (hormones/growth factors), and a buffer to maintain a physiological pH.

There are a ton of different types of media for different cells but the most commonly used is DMEM (Dulbecco's modified Eagle's medium) supplemented with 2-10% fetal bovine serum (FBS) for additional nutrients. Often researchers will also add antibiotics such as penicillin and streptomycin to prevent bacterial growth.

The media provides the fuel while atmospheric dissolved oxygen provides oxygen. Once the cells return to 37C, they spend a little time recovering as generally cells undergoing stress such as temperature changes and exposure to organic solvents like DMSO will stop dividing. This recovery period can take between 2-48 hours depending on cell type. For commonly grown cells like HeLa (from Henrietta Lacks) or HEK293, they take about 6 hours to recover and begin dividing.

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Tiny_Rat t1_ixn2c85 wrote

You freeze in small volumes and thaw them as fast as possible. Partly thawing 1ml or so of media in a waterbath, and then adding saline to thaw it the rest of the way only takes a minute or two if you do it right.

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SemogAziul t1_ixl184t wrote

I should clarify that I wrote that for freezing in general, I've only worked with sperm cells and the list is sort of the less toxic to the most toxic

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ukezi t1_ixleac1 wrote

Methanol is a problem because it gets converted to formic acid in the liver. However the embryos we can safely freeze are still in the clump of cells stage and don't have anything like a liver, circulation system or neuronal tissue that can be damaged.

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King_Marmalade t1_ixlyb1c wrote

The cell lines I've generated and distributed to ATCC were all frozen in DMSO (which you mentioned) in fetal bovine serum. For me this included cell types from many tissues like skin, lung, liver, endothelial cells (from vascular tissue), etc. Even poorly differentiated glial cells are frozen in DMSO, but using a culture medium in place of serum so they don't differentiate.

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Lowtiercomputer t1_ixmop80 wrote

What is a differentiated glial cell?

Glial cells make the myelin sheath, right?

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Bax_Cadarn t1_ixmqkic wrote

I don't know what makes poorly differenciated glial cells any special but yes, gliala cells makes myelin sheath, as well as supporting cns tissues, and differenciation is the process of a cell turning from a basic, undifferenciated cell into one with a very specific function.

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Neurofish8 t1_ixn0sfn wrote

Depends on the glial cell: oligodendrocytes produce myelin in the central nervous system, but there are also microglia and astrocytes. Peripheral nervous system has Schwann cells that produce myelin.

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King_Marmalade t1_ixnl66s wrote

Glial cells encompass multiple types of mature cells with diverse functions (astrocytes, microglial cells, oligodendrocytes) you can think of them as sort of "helper cells" for neurons. In some types of cancers, cells can enter what is considered a "poorly differentiated" state, where they lose some characteristics of a mature cell and become more stem-like. Some of the cell lines our lab generated were from GBM (glialblastoma multiforme) samples.

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Supersnow845 t1_ixo131r wrote

All cell work I do uses DMSO and fetal bovine serum as well, it’s the standard for freezing cells down in -80 freezers

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cheezemeister_x t1_ixmvvyt wrote

> Extracelullar cryoprotectants are impermeable to the mebrane

You mean the membrane is impermeable to the extracellular cryoprotectants.....

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Chojenoe t1_ixkv2jc wrote

Glycerol- based solutions are the most common used in my field of tissue preservation. Most are proprietary.

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craigdahlke t1_ixl8wrv wrote

Lab I work in uses a mixture of RPMI (think a pH balanced salt solution with nutrients in it), fetal bovine serum, and DMSO (dimethylsulfoxide) for cryopreservation of human immune cells.

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Metalmind123 t1_ixmt77m wrote

Though the only cryopreservant in that is the DMSO. The rest is just a standard supplemented growth medium.

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SinXgularity t1_ixlcs7z wrote

While not necessarily used for embryos much, nonreducing disaccharides, like trehalose, are continuously at the forefront of CPA research.

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Metalmind123 t1_ixmtm0c wrote

That is one option, though the type of sugar matters.

As does the temperature you intend to freeze it at. Be it liquid nitrogen storage, a -80°C freezer, or your bog standard -20°C.

Common preservants would be DMSO, Glycerol or Trehalose (less used because of the cost), combined with appropriate media and buffer. In general, these together manage ice crystal formation (DMSO, Glycerol, Trealose), keep the pH in the right range (buffer), and reduce oxidation (DMSO).

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Westbrook_Level t1_ixmig0i wrote

We almost always use 5% DMSO. It works best in my experience.

https://en.wikipedia.org/wiki/Dimethyl_sulfoxide

Then frozen slowly in vials in something like this.

https://www.thermofisher.com/order/catalog/product/5100-0036

When thawing you went a fast thawing process so putting the vial in a water bath at body temp works well.

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merf_me2 t1_ixmxpm3 wrote

When choosing the name of a company that develops technology to freeze embryos "Mr Freezy" is not what I would have expected them to choose

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dardarBinkz t1_ixmwxf4 wrote

When I worked in tissue banking we would use rpmi and glycerol. Tissues can be stored for a number of years but it's very tissue dependent

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01-__-10 t1_ixnf8ax wrote

I used dimethylsulfoxide (DMSO) to freeze some mammalian cells just yesterday.

A few weeks ago I used glycerol for bacterial cells.

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