September 23, 2020

Chill out: from cryotherapy to cryopreservation

By Conor McKechnie and Dodi Axelson

Chill out: from cryotherapy to cryopreservation

What do cryotherapy, endangered species, and cancer patients have in common? You might want to grab your woolly jumper before joining Dodi and Conor in this week’s episode.

DODI: Conor, I want to start today's episode with a little anecdote and a little pop science from a mutual friend of ours. In fact, she's more than a friend. She is the executive producer of Discovery Matters.

CONOR: Aha, Andrea, what's she been up to?

DODI: Well, the one and only Andrea Kilin. And she'll tell us herself.

ANDREA: So, me and my partner went to Amsterdam for a weekend.

DODI: They went there to visit some friends and watch a concert. And when they got there, her friend started talking about cryotherapy.

ANDREA: So we quickly searched online. We found that it showed some promising results for people with chronic migraines, which is what my partner is unfortunately suffering from for the past almost two years now. We said yeah, we tried everything from MRIs to CT scans to acupuncture and all the shamanism that you can think of, so let's go to cryotherapy. We went there expecting nothing. And you get there. You're told what to do. You're given very fluffy socks. You enter a cabin with at maximum four people. You get earmuffs, so you don't get frostbite on your ears. You get gloves. You get a mask, so you can breathe, because you're going to go in -110°C. That’s -166°F. In the first chamber, it's -50°C.

CONOR: So, this is basically the opposite of a sauna. You walk into a room where it's just freezing. Just trust the Nordic to invent this stuff.

ANDREA: You wear nothing but your undergarments. You go into a room where it's -50°C. And you spend 20 seconds there, you start getting used to the cold, and then somebody knocks on the door. And then, you know, you have to open the other refrigerator door. So we walked in and Alex started shaking like crazy. She started shaking really, really bad. And she said, ”No, I can't do this. I'm leaving.” So I grabbed her by her two hands, and I said ”Andas in, andas ut.” In Swedish that means breathe in, breathe out. And since my partner is half Swedish, I thought that might get to her faster. And she managed to focus on her breathing. We stayed the whole three minutes inside, and we're still alive. We felt very, very relaxed after it. A sense of calm overwhelms you almost immediately. We felt like we had a new body.

DODI: She went from a very cold freezer to a very, very, very, very cold freezer. And this apparently resulted in a feeling of relaxation and revitalization. And Andrea said that those who worked there were almost addicted to this therapy.

CONOR: Hmm. And so would Andrea do it again?

ANDREA: Oh, yes, absolutely. I loved it. Alex barely survived. I'm proud of her. But if it was closer, I would go twice a week for sure.

DODI: So, after chatting with Andrea, I thought, well, let's find the real science in this. Because on Discovery Matters we love the real science, not just the pop science. And you know what cryotherapy does with cells and organisms is pretty important. I mean, we freeze them to take care of them in a way.

CONOR: So, we're getting away from the pop science of cryotherapy and looking at the real science and application of cryogenics. I guess that's what matters in today's episode.

DODI: That's right.

DODI: Now, the notion of cold is usually kind of a negative. You know, we go on vacation to warm beaches, we think of the sun, and we smile and we think of John Denver. But for some people out there it is the complete opposite.

GUEST: I think it almost depends on what your background is, because one thing that I've been looking at not as part of work but just in general, is the creation myths of different peoples, and it depends what they really hate. To a lot of the Nordic ones hell is a very cold place. And that sort of agrees with what you expect from very cold countries. But if you look at the other religions that come out, say from the Middle East, like Christianity, hell is traditionally a big fire, a very hot place.

I think it depends on really just what your background is. If you come from a very hot place, maybe you want somewhere colder, because the heat's the problem. But for us in Northern Europe, often it's too cold. So we want somewhere warm, not so much because it's warm but just because it's different, and it's not what we have day to day.

CONOR: And who is this?

DODI: This is Peter Kilbride. And along with his colleague, Julie Meneghel, we chatted about all things cryo.

PETER: The first thing that we have to remember is that human cells do not like the cold. Human cells are used to being at a very controlled 37°C inside the body for their whole life. And lowering your core temperature even by a little bit can be extremely dangerous and just very uncomfortable. It's what we see when we're shivering or we go outside. And in extreme cases, such as with polar explorers or mountaineers, you can see their extremities get very cold.

They can end up having a condition called frostbite, which is essentially the tissue freezing and then becoming permanently damaged. It never recovers even after rewarming. So, I think our relationship with so-called cryopreservation is really the science of tricking cells into not realizing that they don't like low temperatures. And then trying to use even some of the things that normally are bad for cells at low temperatures, somehow to our benefit and to the benefit of the cells.

CONOR: So what exactly is cryogenics doing to cells, and why is this good?

JULIE: Temperature is going to impact the rate of every reaction taking place in a cell. So, the colder it gets, the slower these reactions and diffusion rates will become until a point where they stop completely. They will come to a halt. And this is where organisms will be in a suspended animation. At that point they can be stored theoretically forever until temperatures warm up again and biological reactions resume.

GUEST: What is really common to all these organisms is the physical events that take place.

CONOR: But hold on. Who have we got here?

DODI: That is Fernanda Fonseca, research director of the French National Research Institute for Agriculture, Food, and Environment. And I chatted with her to get another side of the cryo story.

FERNANDA: Our utmost ambition is to find general principles for rationalizing the cryopreservation of biomaterials, of cells, of organisms. So, when you are going to cryopreserve all these organisms you necessarily go through cooling, forming ice. And according to the cooling rate, you will have vitrification. And again back to higher temperatures, you will have the reverse reactions, so the vitrification and going to a viscous state and melting. And you have your stuff again in an ambient temperature. We have found out that there are still things to understand about how it works, how it works inside organisms. And that's our work today within cryopreservation.

DODI: Fernanda also told me about how she lives the science every day by reminding her kids that this transition of going from one state to another happens even when they warm things up in the microwave oven. And when you have your breakfast. Talk about having a scientist as a mother.

FERNANDA: I talk to my children on glass transition, on vitrification, and they know that when they are going to the microwave with their bread, they're making a transition. That will give you an idea of the point about vitrification that amazed me. And when you have your cornflakes in your box, it's still open for some days and it's not very crispy and you also had a glass transition that happened. When you have your gums in your mouth, you have glass transition, and so on. So, that is something that with cryopreservation you work on all time is vitrification, that occasion of going from a glass state to a viscous state and reverse reaction. And that is our life.

CONOR: Okay, hang on a second. I might have to get my woolly jumper on here, because I'm starting to get a little bit cold. So, hang on a second.

DODI: Just the thought of cold makes you cold.

CONOR: It's just too much cold. I mean, okay. Right, that's better. So, how do you make sure it's cold enough, but not too cold, and it doesn't destroy everything because you know, like, I put sloes in the freezer to break their skins when I make sloe gin. And so why aren't we breaking everything when we freeze it so, so hard?

DODI: Well, I'd love to talk more about that sloe gin. But let's ask Julie about this.

JULIE: The problem with frostbite in human bodies, for example, is related to the fact that here we're talking about tissues. So it's a 3D structure of many different cell types that are all functioning together. As this structure freezes, there will be ice crystals forming that may disrupt all this structure and even break cells. So, this is something that we are trying to avoid when cryopreserving biological samples. It is something that we can do even if it requires work and optimization. We can manage to do this for cell suspensions for a few specific types of tissues. But for larger organs or even whole organisms, this is not something that we can successfully manage to do today.

CONOR: Okay, but the reality is, right now, it only happens on long space flights or in weird movies in Hollywood, correct?

DODI: That's right. That's right.

CONOR: Okay, like Alien. That was a good one. They send the whole crew into space, they get cryopreserved.

DODI: And it didn't turn out so well, did it?

CONOR: So yeah, the best thing about that movie is really good, right? It's the movie where nobody listens to the smart lady and they all die.

DODI: That's right. These are some really smart ladies. That's what we're coming around to this entire episode.

CONOR: Listen to them, or you will die. So, what do Peter and Julie think when they see scenes like that? I mean, so they're real cryobiologists, they just look at that and shake their heads and sit and think well, what are we putting into the popular imagination here? Or, you know, might it happen?

PETER: I think to follow on with what Julie was saying, in some respects we can do organs to a limited degree. So, if you have a deceased donor, the organ can't survive in them for very long. However, it you remove that organ, whether it be heart or liver or kidney, and then transport it just above freezing point, you get many more hours where it can be used for transplant. Still, it's not very long. And actually there's a huge number of organs that don't get transplanted just because the length of time it takes the say, heart, to get from the donor to the recipient it gets too far damaged. I think that really one of the key aims in cryopreservation in general is: Is there a way we can preserve whole organs for maybe even just a few days or a few weeks, and that way you could really properly match organs between different donors and recipients and have almost no waste in that process?

I think when it comes to doing anything more than a whole organ, that's science fiction and might be for a while. Each organ type and cell type has very different colds, so you have to treat them in very different ways. It's science fiction for now and I think it will be for a long time, perhaps by the time you can freeze a whole organism. There'll be so much more advanced science and biology, people won't really be dying in the first place, perhaps not far away. So I think day to day this is not something we'd work on.

CONOR: Okay, since Peter mentioned humanity and preserving humanity, what does it look like for artificial insemination and preserving populations?

DODI: Well, in fact that's actually where one of the first parts of cryobiology existed.

PETER: Cryobiology's bigger successes are in reproductive medicine. So, people having in vitro fertilization (IVF) will be able to freeze sperm. They can freeze eggs, they can store embryos, and one of the great successes of the past few years is people who are undergoing cancer treatment that can then make them infertile. Eggs can be taken before treatment starts and be frozen, so that people who historically would not have been able to have a child can then have children. And that's one of the big successes.

Cryobiology has also been used a lot more when it comes to endangered species. Quite a lot of zoos around the world now have things called frozen zoos. And if they have an endangered species and say it dies, they'll take some tissue samples from that and cryopreserve them. And even if the species goes extinct, you’ve still got a small amount of biological material from them. And hopefully one day that can be used to then bring back a species that's gone extinct in this period of time when many species are going extinct.

CONOR: Okay, so what are some examples of this working, or potentially working in the future?

DODI: Well, let's think about something ancient and massive.

CONOR: Something ancient and massive...T. Rex, Jurassic Park.

DODI: Almost. There's a legend about bringing this animal back to life.

CONOR: Are we talking about the woolly mammoth?

DODI: We are.

PETER: So, the woolly mammoths are really quite a good example of how cryopreservation can work. Woolly mammoths existed in northern latitudes thousands of years ago before they were killed off by early human hunters. But luckily because they lived in such far north latitudes some of them were frozen, because in those days it was very cold. And nowadays, you can actually sometimes find woolly mammoths that still have intact soft tissue.

Of course, the cells in that system are not alive. They're not something we'd freeze in the lab ourselves, but they do still have some DNA. So, there has been some thought that we could extract that DNA and then use it to recreate a woolly mammoth, for example, perhaps by getting some DNA and then inserting it into the egg and sperm of an elephant, which is one of the closest species currently alive that is like a woolly mammoth. I think that is something I wouldn't say is impossible. It's certainly incredibly challenging because with species nowadays you've got sperm, you've got eggs, you've got the reproductive tissues that you need.

When it comes to woolly mammoths the DNA is normally very badly damaged, and the closest species to a woolly mammoth is an elephant. but it's actually quite different. And you need an elephant's living uterus to have these animals reproduce. So, I think that certainly it will be possible, and I expect that sometime in the future it will be done. I think before that stage we're probably looking at bringing back species that humanity has caused to go extinct, just because we have much more material from them. And because they were frozen using modern science as opposed to just fortunately dying in cold climates where some of the soft tissue was preserved.

DODI: And you know, when I was chatting with Fernanda she brought up the often-forgotten side of cryobiology. And that is the environmental impact.

FERNANDA: Until now, we were aware about quality of the products and the cost of our production processes. Now we have to think about multicriteria approaches and consider the impact of the whole chain, the whole cycle our product is having. And what is really amazing is that we can no longer work and research without many multidisciplinary approaches. So, in these cryopreservation teams you need to have around you biologists, physicists, biochemists, and mathematicians, and people concerned with the environment helping to assess the best strategy.

CONOR: So, if we bring it back to right now, what's going on today and patients... Where exactly is this finding the most use? Where is it helping?

DODI: Let's go back to Julie and find out.

JULIE: The area that has attracted much attention in recent years is cell therapies, and especially CAR T cell therapies with a chimeric antigen receptor. It is a cell therapy that has been developed for patients with some specific types of blood cancer. Their own T lymphocytes are harvested from their bloodstream. They’re genetically modifed to make them stronger to attack cancer cells within that person’s own body. The cells are amplified to create billions and billions of them, and then reinfused back to the patient to help him or her fight the cancer. Cryopreservation is a critical step during this process, because cryopreservation allows for more flexibility in terms of patient treatment.

PETER: And I think another area as well that's quite common, and many listeners may have heard of, will be called blood cryopreservation. That's something Julie and I have been working on quite a lot recently as well. So, when babies are born, you have the umbilical cords. And the blood within them contains a lot of early-stage cells that can be really good for treating people with specific diseases later on. So, one area that a lot of scientists have gone into and is still being improved starts with extracting some of that early blood from the umbilical cord of newborns. And then you can freeze it and have very large biobanks with huge numbers of different blood from different babies that you can then match to people who may need some sort of cell therapy in the future.

I think that's quite a good example of cryopreservation’s potential because often, in those cases, you'll need to store the blood for months, years, often decades before it becomes useful. And of course, you can never go back to a baby to get umbilical cord blood twice. When the baby's born, you have one shot and that's it. So that's one area where cryopreservation is really quite common. And we do research where we try and improve the outcome even further after decades of being stored.

CONOR: It's just extraordinary, isn't it, being able to reduce the temperatures to really, really cold are so fundamental to understanding things. Remember, we talked in our episode on quantum biology, about needing very, very cold temperatures to see quantum effects, and that meaning you wouldn't be able to see it at warm temperatures. And people thinking that didn't exist. Like being able to get things really cold is really important for science.

DODI: But I was also thinking about the episode we did about sense about science and what is real and what is pop science. And what is beneficial for human health and advancing therapeutics. So, I think this has been a really interesting journey from talking about Andrea and her experience walking into a super cold room and how that made her feel relaxed and rejuvenated, to when we actually take cells and freeze them in order to rejuvenate them later and make them useful in therapeutics.

CONOR: It's a kind of interplay between sort of what we read about science and what we want science to drive us towards and how we feel about innovation in areas like consumer health, and then people's imaginations driving science forward. I mean, people take science fiction ideas like the communicator in Star Trek, and it becomes the mobile phone. There's this really interesting interplay between pop science, science fiction, and real science and where the world can go. It feels like we're in an age now where if you can imagine it, there's probably someone somewhere working to make it real. And that might be a good thing. And it could also be pretty difficult.

DODI: So, I think in the last about 20 minutes, you know, we hope that this episode has answered some questions about cryogenics but also raised some questions, because it's important that science is constantly raising the right questions. Am I right, Conor?

CONOR: You're absolutely right. Look, every day is a school day. And the best thing about science is being proven wrong.

DODI: Every day's a school day. We got there. Thank you for listening.

CONOR: Thank you for listening.

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