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iayork t1_ixkhr8v wrote

It’s actually relatively easy to freeze cells and recover them. It’s harder to freeze tissues and have them come back.

Cell lines are routinely frozen and stored for decades. The American Type culture Collection (ATCC) has some guidelines and explains “Most cell cultures can be stored for many years, if not indefinitely, using cryopreservation.” In general this also applies to egg cells.

Tissues are harder (though not necessarily impossible) because the structure is easily disrupted and because it’s harder to evenly freeze the cells that are part of it. The smaller the tissue, the better the chance of successful freeze and recovery. Embryos are tissues, with multiple cells, but they are frozen when they are very tiny - at an early-enough stage (100 cells or fewer) that they can be readily permeated by cryoprotectants.

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Magnetic_Syncopation t1_ixktt0n wrote

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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thephantom1492 t1_ixl5yx1 wrote

There is also the fact that on the lot some will survive. For tissues, even if a small portion die, it still render the tissue useless. If a few eggs die, well, you have others.

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RNnoturwaitress t1_ixme2xc wrote

Eggs don't freeze as well as embryos for some reason. Also, some women have very few eggs, so they might not have many more. To the majority of those undergoing fertility treatments, every egg and sperm are precious.

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Lady_Nimbus t1_ixn9cpe wrote

Eggs don't freeze as well because it's one cell vs. an embryo that is multiple cells. An embryo may survive the death of a couple of its' cells. An egg won't because it's just one cell and that's all there is.

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Aleuna t1_ixnt1cl wrote

This is actually not entirely true for eggs/embryos. If some cells from an embryo die at thaw, the remaining ones can often fill in for the dead ones and reform a complete embryo. Eggs on the other hand are single cells so if one dies it’s gone. Yes, you may have other eggs, but they are more fragile and less resistant to cryodamage due to their unicellular nature.

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Primary-Signature-17 t1_ixl67uc wrote

I wonder if the 30yo embryo babies are going to be watched to see if there are any problems as they grow? Are they the oldest embryos ever born?

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triffid_boy t1_ixlbujy wrote

There's probably no need to specifically watch/screen people born from older embryos. it'll be part of their doctor/patient interactions and there's no real reason to think that stuff stored in liquid/vapour phase of nitrogen would biologically age much at all.

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Primary-Signature-17 t1_ixldjsa wrote

I suppose we'll hear about it in the years to come if something is different. Saw on the news that adopted children are not only curious about who their biological family is but, they want the medical history of them, too. I wish them all a great and healthy life.

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_googlefanatic_ t1_ixl4s5m wrote

Why can tissues be not frozen and 'stored' ?

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Ishana92 t1_ixlj0dr wrote

You will lose some cells after thawing. The bigger the tissue, the larger the damage. And for tissue every bit of damage has an impact.

If you take cell culture, its basically asuspension of independent cells. You can lose 50% of them and the rest will repopulate everything. But if you lose 50% of cells in your tissue that tisdue is no longer functional and wont survive.

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Terr_ t1_ixlkxhy wrote

Another metaphor:

Suppose you have a thousand planks of wood in nice stacks. You can store them and still be okay if a random 10% become rotted and must be thrown out.

Now imagine you store a house made out of a thousand planks of wood, and again 10% of it rots. It might become dangerously weak, and it will be very expensive to check and replace the bad parts.

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_googlefanatic_ t1_ixlmglt wrote

But I don't understand , why does a few cells destroyed in a tissue affect the tissue , the cells can re divide , right? If not then why so ?

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wedontlikespaces t1_ixlnbah wrote

Because when they're independent cells they are independent but when they're in a tissue they're part of a larger structure, so if that larger structure is damaged the whole tissue becomes nonviable.

If 10% of a heart dies, then the entire heart no longer works.

Going back to the house example, if a structural support beam is one of the 10% of wooden beams that rots, then the whole house is going to fall down. It's not a perfect analogy because obviously there's some bits of wood which are more important than others, but you get the idea.

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Terr_ t1_ixlnrjg wrote

Many of the cells in your body have stopped dividing, they've settled into specialized roles and positions and will not "fill in the gaps" by duplicating to replace missing neighbors. The shape of everything else around them and the signals from their neighbors cause them to act in certain narrow ways. When they don't do that and start dividing again, that's often cancer.

New cells are usually generated from deeper spots, from special cells with the job of just churning out certain kinds of gradual replacements.

So if you choose a random zone of tissue and kill some cells in it, those gaps or weaknesses may persist in that area until everything around it is pushed out and replaced by the march of fresh cells from further in.

If you damage one of those special zones where new cells are still being made, the damage could be permanent, like getting a scar on your skin that lasts even when almost all of the cells have been replaced over time.

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_googlefanatic_ t1_ixlu4ze wrote

Got it , but It should be rare right ? Like when we damage the exact location ....

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Ishana92 t1_ixm22tp wrote

If you had time to repair all the damage it would be fine, probably. But those cells that died are releasing all sorts of messages to neighbouring cells. It's not just that they died and now there is a hole to fill, it's that now there is a hole and neighbouring cells are also freaking out and not doing their job. And then if you have living organism the immune response is going to kick in and just go crazy at site that is damaged. You get the whole cascade when cells are all dependent.

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TEW20 t1_ixmthod wrote

Not necessarily, some structures never regenerate and scar tissue forms where the damage happened, so that part of the tissue never recovers it's function. That's why damage to some organs cannot be repaired.

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_googlefanatic_ t1_ixlmgyn wrote

But I don't understand , why does a few cells destroyed in a tissue affect the tissue , the cells can re divide , right? If not then why so ?

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Ishana92 t1_ixm2pue wrote

Its because of complexity. Lets say you freeze a kidney then you thaw it and try to reassimilate it. Cells thst died will severely disrupt its function. In many cases replacing those cells is a slow and gradual process and not something that can be done quickly (whereas in cell culture you usually only have one cell type and they are functionally all the same).

Then you have the tissue and system response. Cells that die during freezing die messy. They burst and that releases toxins into your blood stream. Then immune cells come via blood and start inflammation which further damages the tissue. In the end you have a string of failures.

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GreatBayTemple t1_ixne205 wrote

So you need nanobots that can connect to cell tissue and each other, mimic the cell structure and send signals to neighboring cells to function as usual while cells are cultured into place on the organ? Like a special film?

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Ishana92 t1_ixnjkrq wrote

If you had all that then you wouldn't need to freeze anything, just grow it from scratch. This is the basis of 3d organ printing.

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_googlefanatic_ t1_ixlmcka wrote

So you mean that If we lose 50% of individual cells , it doesn't matter as we can mitosise them. But if we lose 50% cells of a tissue , the tissue may stop working?

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CMxFuZioNz t1_ixmr0w7 wrote

No. If you freeze 50 eggs and 25 of the eggs are damaged by freezing/thawing that doesn't affect the other 25 eggs which weren't damaged.

If you freeze a heart and half of it is damaged, the whole thing will no longer work when you put it in someone.

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ButtersLeopold09 t1_ixnf25h wrote

But if water expands when frozen, how does the cell not rupture the membrane or break the integrity of the cell? Are they simple enough?

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CrateDane t1_ixngpm3 wrote

You freeze cells slowly, which gives water time to diffuse through the membrane and equalize the pressure.

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