Sponsor note: the supporter of this video is rush.cloud. If you are at all involved with doing preclinical drug discovery and would benefit from computational tools, you should check out their platform + beautiful website here: rush.cloud.
If you’re at all interested in working together for future episodes, reach out!
Listen on Spotify/Apple Podcasts/Youtube:
Introduction
This is an interview with Hunter Davis, the CSO and co-founder (alongside Laura Deming) of Until Labs, which you may also know by its prior name, Cradle. They are a biotech startup devoted to organ-scale cryopreservation. They raised a $58M Series A back in September 2025, and are backed by Founders Fund (especially interesting!), Lux Ventures, and others.
In this interview, we mainly talk about the engineering and scientific difficulties in the cryopreservation field, including some background details on their September 2024 progress report on neural slice rewarming, how they characterize tissue damage in their attempts to do kidney cryopreservation, the potential economics of future cryopreservation protocols, and lots more.
One of the most interesting conversations I’ve had in a long time. If any of this work seems interesting, Until Labs is actively and aggressively hiring!
Enjoy!
Timestamps
[00:05:00] Why don’t we have reversible cryopreservation today?
[00:07:05] Why is freezing necessary at all for preservation?
[00:08:23] Let’s discuss cryoprotectant agents
[00:14:09] Until Lab’s 2024 progress report on neural tissue cryopreservation
[00:20:28] How do you measure cryopreserved tissue damage?
[00:22:34] Translation across species
[00:26:04] Why was the cryopreservation storage time so short in the progress report?
[00:30:47] Nuances of loading cryoprotectants into tissue
[00:37:03] Let’s discuss rewarming
[00:43:02] What scientific problems amongst vitrification and rewarming keep you up at night?
[00:45:58] Why are there so few cryoprotectants?
[00:48:11] How can you improve rewarming capabilities?
[00:53:03] What are the experimental costs of running cryopreservation studies?
[01:01:34] Cryopreservation and immune response
[01:03:25] How do you filter through the cryopreservation literature
[01:05:54] How much is molecular simulation used at Until Labs?
[01:10:04] What are the (expected) economics of Until Labs?
[01:14:49] How much does cryopreservation practically solve the organ shortage problem?
[01:17:04] Synergy between xenotransplantation and cryopreservation
[01:21:12] How much will the final cryopreservation protocol likely cost?
[01:21:58] Who ends up paying for this?
[01:23:28] What was it like to raise a Series A on such an unorthodox thesis?
[01:27:49] What are common misconceptions people have about cryopreservation?
[01:29:58] The beginnings of Until Labs
[01:34:07] What expertise is hardest to recruit for?
[01:39:27] What personality type do you most value when hiring?
[01:44:17] Why work in cryopreservation as opposed to anything else?
[01:46:26] Until Lab’s competitors
[01:49:30] What would an alternative universe version of Hunter worked on?
[01:51:33] What would you do with $100M?
Transcript
[00:01:50] Introduction
Abhi: Today I’ll be talking to Hunter Davis, the Chief Scientific Officer of Until Labs, previously known as Cradle, which is a startup working to build reversible cryopreservation for use in organ transport, and eventually medical hibernation. Prior to starting a company, Hunter received a PhD in physical chemistry from Caltech and got a postdoc in neuroscience at Harvard. Today we’ll be talking about the recent progress report that Until Labs put out, what cryopreservation problems keep Hunter up at night, and a lot more. Thank you for coming onto the podcast, Hunter.
Hunter: Thanks for having me.
Abhi: So just to set the stage, I’d love if you could give me a very general overview of what cryopreservation exactly is and the primary roadblocks to outright solving the problem.
Hunter: Sure. If we look back to the origins of using temperature as a knob, you can go all the way back to 1776. At this point it was observed that sperm under a microscope could be cooled down to hypothermic temperatures and then motility would decrease. And then when it was rewarmed, it would come back. And this led to some hypothesizing about what might be causing this. Fast forward to around 1950, and people discovered that not only could you cool things to hypothermic temperatures, you could add cryoprotectants into these molecules and you could cool them entirely. Go down to the point that you completely arrested molecular motion. Fast forward again to more contemporary processes. And what you’re trying to do is slow down molecular motion inside of cells in a variety of different contexts. So one that you might envision is for a hypothermic use case. You have a patient who needs a surgery. In many of these cases, the ischemic time is far too short to complete the surgery at 37 degrees.
One example of this is aortic arch surgery. This is a heart surgery that can’t be bypassed and by default what would happen is the brain would go through ischemia because you can’t actively perfuse oxygenated solution into the brain during the surgery. So what the surgeon will do is they’ll take the patient and they’ll cool them down to around 15 degrees Celsius, and then they can complete the heart surgery and then rewarm the patient. And it shows no long-term neurological side effects. Then you can look at something like an organ. Here we might want to slow down the metabolism of an organ during transport to be able to increase its viability window. Here we can take the organ and literally just put it on ice. And what this does is it just decreases the rate of all these metabolic processes that are happening inside of the organ or in the case of the patient, inside of the body. Just reduces all of those by reducing the temperature.
. You can think of it as between zero and minus 130 is a danger zone where ice can form; below minus 130 is safe perpetually.
So what does the process look like in practice? You could take an organ, you load it up with some cryoprotectant molecules that reduce the rate of ice formation during this danger zone. Then you rapidly cool your sample from zero to below minus 130, store it there, rewarm, and then unload the cryoprotectant from the organ or tissue. And then you can bring it back up to 37 degrees and biological function will resume.
[00:05:00] Why don’t we have reversible cryopreservation today?
Abhi: What, why don’t we have this today?
Hunter: Yeah. There’s a lot of challenges with scaling this up. We do have it today in really simple systems. One example of this would be cryopreservation of embryos for in vitro fertilization. Here you have something that’s very small. Normally they’re operating on embryos at this stage where it’s four to six cells. And here you can take the embryo, dip it into DMSO, dimethyl sulfoxide, and then quickly dip it into liquid nitrogen. This rapidly cools it through this danger zone and embryos can be stored for decades in this state and then rewarmed and implanted and using the contemporary methods, the viability of embryos that are going through this process is a little over 95%. So we have seen that the live birth rate that occurs after vitrified embryos has started to exceed those of fresh implantation. And the reason for this is the allowance for additional genetic testing while the embryo is cryopreserved.
So this does exist for really simple systems. There’s also been some proofs of concept of bringing it up to more complex systems, like a rat kidney. The John Bischof lab at the University of Minnesota has shown that you can take a full rodent kidney, vitrify it, bring it down below this minus 130 degree state, bring it up, reimplant it into the rodent, and then it’ll support life for this rat. The challenge is that as you scale up, as you try to go up to something that’s the size of a human kidney, all the thermal transport becomes much more complicated. If you try to imagine cooling something that’s the size of an embryo, I can go really fast. If I do something that’s the size of a human kidney, which is around 150 grams, that’s going to be much slower. So then in this competition between you getting down to the safe zone, minus 130, and the rate of continuous ice formation in that zero to minus 130 range, you just start to lose out because you can’t cool fast enough. And similarly, rewarming becomes a challenge.
[00:07:05] Why is freezing necessary at all for preservation?
Abhi: When you mentioned earlier about how you can’t just perfuse a transplant organ with oxygenated blood to prevent ischemia, intuitively, why is that the case? Why do you need to freeze to preserve the cellular state of the organ and why can’t you just continuously pump oxygenated blood through?
Hunter: Yes, that’s a great question. There are technologies that exist on the market right now that will perfuse hyper-oxygenated fluid. And they do this in either a cold perfusion context traditionally. There’s a bunch of these products that exist on the market that can increase the viability of the organs that will then be donated. But all of them still have time... organs still time out.
At some point.
Abhi: I guess what are the specific reactions?
Hunter: What are the mechanisms that can account for here?
So one thing is that now the organ is in isolation. So let’s think of a kidney in isolation. Now I don’t have a liver. So any toxins that need to be cleared by the liver, the circuit isn’t going to be able to take care of this. We don’t have dialysis for a liver, for example. So I think that part of this is that you have taken the organ out of the context of being in a multi-organ system that is responsible for clearing a bunch of the toxins that are generated from the metabolism of any one organ outside of the concert of the other.
[00:08:23] Let’s discuss cryoprotectant agents
Abhi: That makes sense. And moving a little bit onto the cryopreservation step. First you have this vitrification step, second you have the rewarming step. Focusing on the vitrification part. You mentioned that you physically stop the water molecules from interlocking with one another and one way you can do this is by freezing really fast, such that they don’t have time to lock into one another. Another way is adding in cryoprotectant agents, which prevent the water molecules from doing that. What are cryoprotectants doing beyond that very simple mental model?
Hunter: Yeah. Yeah. That’s great. I think there’s a couple things to keep in mind here. Most cryoprotectants that you’re going to add are not going to completely prevent the formation of ice in perpetuity. You can think of it as reducing a reaction rate. If you think of the liquid to solid water as a first order rate equation. And this is the there’s this thing called classical nucleation theory. It predicts the rate of conversion from liquid water to solid ice.
Cryoprotectants increase the activation energy of that liquid to solid transition. There’s still a very molecular question of how do they do that?
There’s a couple of different mechanisms of action here. So one that you can look at is direct hydrogen bonding of water molecules. You can imagine the rotational tumbling that’s required for water molecules to align into hexagonal ice. If I can just slow that down by hydrogen bonding to the water, then I’ll reduce the rate of that alignment and buy myself more time to be able to cool down below minus 130. Other things is just generally increasing the viscosity of the solution. If you look at the Stokes-Einstein model for diffusion in a fluid, it’s obviously inversely proportional to the viscosity of the fluid. So these cryoprotectant molecules tend to directly hydrogen bond water and be viscous.
There’s also some interesting alternative mechanisms of action that you can explore. Looking to nature, for example, there are these things called antifreeze proteins. So instead of directly hydrogen bonding to liquid water, what they do is they preferentially bind to solid-phase water. So they allow some ice nuclei to form, and then by cooperatively hydrogen bonding, these macromolecules will stick to the ice surface and prevent it from extending. So there’s a few different mechanisms of action, and you could think to exploit all of them together in a cocktail.
Abhi: And I imagine the type of cryoprotectant agent that Until is most concerned with is the former category of directly binding to hydrogen and you don’t care too much about the antifreeze proteins.
Hunter: Yeah, I think that we’re open to exploring all of these mechanisms of action. I think in the end, when you want to do vitrification, the thing that’s going to have to do the dominant work is going to be these colligative agents. These are the small molecules that are actually directly interacting with liquid water. If you think about the antifreeze proteins that nature uses, most of the time, always, these organisms don’t care about surviving down to minus 130. What they care about is surviving in equilibrium at minus five degrees. These are very different processes. To try to prevent the extension of ice in a supercooled state, just below zero, is just a fundamentally different problem than trying to survive all the way through down to minus 130 and back. So yeah, the primary focus is on these colligative agents.
Abhi: When you look at the cryopreservation literature, has there been any evidence to suggest that using antifreeze proteins and being okay with just mildly arrested, or mild cryopreservation, is good enough in some cases or there hasn’t been too much work in that direction?
Hunter: Yeah, so not specific to antifreeze proteins though. I think that there are some applications here where people have been trying to use either things inspired by antifreeze proteins is mostly it. But there is a whole stack of products that will hit the market that are these supercooled organ solutions. I think of this as an extension of hypothermia where you can go instead of to four, now you can go to minus four. And there’s a few different tricks that people have used here. All of them have their trade-offs. But I would imagine that similar to how Until will be bringing a vitrification product to market, I would imagine there will be some near-subzero storage products as well that will have their own trade-offs. And in the end, I think the thing that’s going to matter for what wins out and becomes the dominant way of transporting organs is going to be the thing that gives the patients best outcomes.
Abhi: Yeah, that makes sense. Amongst the cryoprotectant agents that you guys are actively developing, how much work goes into improving those agents versus improving the next step, which is rewarming?
Hunter: Yeah. So we view this as a very multifaceted problem, and I think that one of the things that originally fascinated me about it is that it’s one of the few scientific problems that I’ve seen before that brings together things from applied physics, chemistry, transplant, biology, all the way to hardcore electrical engineering and power electronics. And we work on all of these things simultaneously. I view them as relaxing each other’s constraints. If you have devices that are good at rewarming quickly, then my molecular agent doesn’t need to be quite as performative and vice versa, right? If I have the killer molecular agent, then maybe I need a very facile, easy device. In the end I think these constraints are pretty hardened, so it’s going to be some combination of these solutions in the middle. But yeah, we work pretty intensively on both improving the cryoprotectant agents and on improving the devices that do the cooling and rewarming.
[00:14:09] Until Lab’s 2024 progress report on neural tissue cryopreservation
Abhi: Okay. I’m going to wrap back to these questions about cryoprotectants and rewarming in a bit. But what I really wanted to talk about, what actually started this conversation to begin with, was you guys released a progress report in September 2024, alongside your series A announcement, that described how you recovered electrical activity from a slice of mice cerebellum, I think, that were frozen, rewarmed, and you observed some level of electrical activity in the neurons there. This is obviously incredibly impressive work. But you did mention in the paper, in the progress report, about how there is more to neural functionality than simply recovering neural activity. What else is there?
Hunter: Yeah, so I think that, first of all, I think it’s maybe worthwhile to go into why we did that as our first POC. When Lauren and I met and we were talking about the idea of reversing, reversibly pausing biological function eventually for a whole organism, we had the initial conversation of what are the falsifiable hypotheses here? What are the experiments that we could do that would prove that this will not work? I think one of the first ones that we wanted to do was look at these very delicate pieces of tissue, being neural tissue. And so the easiest thing that we could come up with was doing these cerebellar slices. It’d be very easy to load these diffusely. We didn’t need to do any perfusion. You could cool and rewarm them very quickly. And the cerebellum is known for having very periodic firing. So you would be able to know if you got, at least on the single cell level, you would be able to tell if you were getting any action potentials that made sense. And so we performed that experiment and like you were saying, we saw the recovery of some electrical activity of action potentials in the slice.
But yeah, there’s the question of what comes next? Actually, a piece of very impressive work that developed along this axis came out of Alex German’s lab in Germany this year. And he was able to show using a very similar protocol that you can recover long-term potentiation from these acute slices. So this is... you take one canonical example would be like take the Schaffer collateral in the hippocampus. Here there’s a bundle of axons that are all synapsed in a very similar location in the hippocampus, and you look for potentiation of those synapses. This is like a bit flip for memory. And what he was able to show was that you can recover LTP in these slices. So this is all really interesting and I think it’s useful as a micro-circuit level problem, but if you were to talk about what does it take to preserve full neural function? This is a much more complex question than the micro-circuit inside of a slice. And eventually you have to go to the brain as a whole and to the organism as a whole. But this is a very deep challenge that I think will take quite a bit of time to get to the point that you can make traction against it, is my presumption.
Abhi: Yeah. One pretty shocking thing I found after we had our initial conversation a while back, is that the ultimate goal, or at least the short-term goal, of Until Labs right now is not whole brain preservation. Right now it is, I think you said kidney preservation. Why switch to kidney? When I read the report, I thought, okay, this is one step to whole brain cryopreservation.
Hunter: Yep.
Abhi: Why move to a different organ?
Hunter: Yeah. So I think the other thing that we have been interested in as the moonshot from the beginning is reversible cryopreservation of an entire patient. And this would involve allowing someone to pause their biological time as a whole. There are some really useful things about going through the process of doing this on an organ-by-organ basis. First one, this allows us to get therapies to patients in need in a very immediate way. You can deliver care to people who need it and use cryopreservation to do it. There’s also a natural scientific roadmap that’s built out of this, where you can start to learn technologies on isolated test beds where you can learn about how to preserve kidney, heart, lung, these kinds of things. And we view this as a natural foundational platform on which we can build towards our eventual goal of being able to do hibernation of an entire person.
Abhi: Why choose kidney over anything else?
Hunter: Yeah. So we’re actually pretty organ-agnostic.
Abhi: Okay.
Hunter: I think that kidney is the one that’s commonly talked about and I’m happy to discuss nephrology as a particular application. But I think one thing that’s nice about vitrification is because it leverages physics instead of going after very specific biomolecular pathways, it’s somewhat organ-agnostic. Okay. Which is, I think, exceptional compared to some other chemical strategies for preservation.
Abhi: Yeah. Returning back to the progress report, just because I imagine a lot of the questions, at least external people may have about cryopreservation, are probably answered or at least raised by the report. In the report, you did cryopreservation across four mouse samples and found, I think the primary results were over one mouse, but I think there were discussions over the other three as well. But I did want to ask how much heterogeneity was there in your success in being able to recover electrical activity from all four rats?
Hunter: Yeah. We were able to see electrical activity recovered from all four rats. There was a large degree of heterogeneity in the amount of electrical activity that we recovered. The traces that were placed into the report, I think are representative of the group. But particularly in that iteration of the device, I think we were very early and I definitely think that our QC was not as dialed. So there was quite a bit of heterogeneity in things like the cooling rates and the rewarming rates that were coming out of this little cartridge that we had.
Abhi: Why? Would you imagine the primary axis of variability... how much of it is just an experimental batch effect versus some rats are perhaps better at being cooled than others?
Hunter: I wish that I could tell you that it was down to the rats. I’m pretty sure at this point, at least particularly at the point that we were filing the report, I would chalk it up to experimental variability.
[00:20:28] How do you measure cryopreserved tissue damage?
Abhi: Okay. That makes sense. Returning back to the kidney cooling and rewarming moonshot, what is your metric for... you freeze a whole kidney, you rewarm it back up. How do you tell whether this kidney is good?
Hunter: That’s a great question and this is something that actually I didn’t appreciate until we started working on it, which is that there’s this whole literature and an entire field of organ evaluation in isolation, outside of a body. These are normothermic machine perfusion. So this is for the electronics people. This is like a test bench for your organ. You can hook up fluidic circuits into, if you imagine a kidney has one inlet and two outlets.
It’s got its arterial inlet and it’s got a ureter and a venous outlet. I can press certain fluids into the arterial inlet and then measure the fluids that are coming out of both the ureter and of the venous effluent side.
This allows you to measure things like what is the uptake of glucose in the organ. You can measure things like what is the lactate concentration in the stuff that is coming out of the venous side. So there’s a whole host of these biomarkers that have been established by the transplant community and I think that one marker of good cryopreservation work, as you guys are looking through the literature, is how well do they reproduce the metrics that have been established and known to work as predictors of transplant outcomes, which there’s a whole literature on this.
Abhi: How... are these metrics for kidney damages established by the transplant community? Are they fully dialed in? This is as good as it gets. Or there’s still room to improve there.
Hunter: I would argue that there’s still room to improve. It’s still an active area of research, improving the correlation of these NMP assays to transplant outcomes. I think it’s established that it is possible to correlate these things. I think that no one would claim that we are done
Abhi: Okay.
Hunter: With how to draw the best conclusions.
[00:22:34] Translation across species
Abhi: And right now are you still doing mice kidneys, or are you moved on to other species?
Hunter: I think you need to move up to pig as the standard preclinical model, and it’s for the reason of the size. So many of these constraints have to do with matching the size of the organs that you’re interested in.
Abhi: At what point... maybe this is an unanswered question in the transplant field, but how much do what you learn from pig kidneys transfer to human kidneys?
Hunter: Yeah, so this is a classic question of the translatability of any of these assays. And I think that particularly for something like vitrification, we’re going to have to see and we’re going to have to be intelligent with our trial design. I think that there are ways to access things like human organs that would otherwise be discarded to check for some of these questions. But this is a standard translatability question and particularly because we’re going to be pressing vitrification through for the first time, I think that we’re going to get to learn the answer to that question.
Abhi: At what point in time... are you still right now operating on... I’ll just ask the question. Slices of kidneys or are you doing the full kidney at any given run?
Hunter: I think you need to be doing the full kidney.
Abhi: Gotcha.
Hunter: And the reason for this... so there’s ways to chop this up and do these things on tissues, but in the end, vasculature is a really critical part of this process. I think this is probably something that I should have gone through in more detail as I was describing the original protocol, but the only reason this is possible is because of vasculature.
You need to be able to do mass transport of this cryoprotectant deep into tissue. The heat diffusion that I was complaining about previously because this kidney is 150 grams, the same logic would apply for loading the cryoprotectant if you weren’t to use vasculature. If I were to try to load it from the outside, it would take forever. But we are perfusing through the vasculature. So I think a lot of the development that needs doing is on how do you efficiently transport cryoprotectant agents into a kidney or another organ.
Abhi: How much of the... where do most of the ideas in the room usually come from? Are they from the nephrologists? Are they from the electrical engineers? Are they from physics people? What plays into pushing forward the kidney preservation goal?
Hunter: Yeah. The only reason that we’re able to make any progress is that the answer is all of them. Okay. I could give you... there are examples of times where I think really great ideas have come from unexpected places. So for example, Andrew Ted, who’s our head of applied physics was previously running battery materials research at Tesla.
Abhi: Interesting.
Hunter: And has a very interesting perspective on material discovery, which is critical to what we’re working on. Yeah. And then there’s stuff that comes from Gerald Brander who just joined as our Chief Medical Officer who has decades of experience in transplant. And he has obviously his own lens on the way that these things need doing inside of organs. So I think that the magic that I have seen always happens when these people get in a room and they have conversations and they can relax each other’s constraints. You have this specter of another field where it’s a black box and it’s oh, this seems quite challenging or hard because you have some perhaps overestimation of the level of constraint that collaborator has.
We just get these people in a room and they have a conversation. I think that’s where you can unlock a lot of upgrades to protocol.
[00:26:04] Why was the cryopreservation storage time so short in the progress report?
Abhi: How much... returning back to the report? You said you kept the neural tissue at negative 196 degrees centigrade for about a minute. I imagine you’re going to do the exact same for kidneys as well. Why not extend that out to a day, a month, a year? Why choose a minute? Was it just you don’t expect anything to change after that point or...
Hunter: Yeah, so that’s a great question. For the tissue slices, it was a very simple thing of just, this is the way that we built the device. I literally cannot express to you how quickly this device was thrown together to be able to do this report. And it took a lot of optimization on the protocol side. But literally we had this idea for this device and then threw it together mostly with thinking towards screening, not towards, Hey, we need to go get this milestone. And be able to report it out. Mostly to try to screen cryoprotectants. But for the kidney, I imagine that we’ll store it for longer. There’s a natural question that I think is at the core of what you’re getting at though, which is why does it matter or not matter?
Abhi: Yeah.
Hunter: And I would contend that it actually doesn’t matter.
Abhi: Okay.
Hunter: You can store it down there to be able to prove out the theory. But the reality is, because water is 13 log units more viscous down in this vitrified state than it is at room temperature. There’s a massive time dilation. Think seconds at room temp to millions of years in vitrified state for equivalent diffusion distances. So for any reasonable timetable, think days, months, years, doesn’t really matter because nothing’s going to have moved in this glass state.
Abhi: Yeah. One instinctive thought I would have is the reason you may care about a day or a month is... when you’re freezing something, you remove all of the blood from it and replace it with cryoprotectant. At least that’s my mental model of it. The reason you may care about testing it out for a day is what if you left a little bit of blood in there? And that blood will continue to gather, accrue damage over time. Is that at all...
Hunter: I don’t think so. So actually if you got... so let’s say I left some random red blood cells inside of the vasculature. It’s what’s going to happen? first of all, the water inside of those red blood cells is still going to exchange. Sure. So they would still equilibrate with all the cryoprotectant that you’re perfusing in there. And also, let’s say that maybe there’s a water pocket inside of this. if I got any ice on the way down and let’s say I induced damage as a result of that ...that damage is now there.
Abhi: Okay.
Hunter: it’s... there’s no period of time. Because even if I have ice that is nucleated it’s still not extending below minus 130. It really is the case that all the damage is getting done in this minus 130 to zero range.
That definitely doesn’t mean that you’re okay. if you have nucleated ice on the way down, there’s actually an annoying/interesting asymmetry in the physics here where you tend to form more nuclei as you’re cooling and then you tend to extend them while you’re warming. And that extension is really what kills the tissue because that’s where you start to tear through quite a bit of cells. yeah. And tear things up.
Abhi: One question I initially had when we first had this conversation was, let’s say centuries from now, we’re all cryo-frozen on a ship going through the stars. How much do we need to worry about background radiation of the universe still affecting our genomes?
Hunter: Yeah. I can’t prevent the DNA from getting nicked from radiation as a result of it being vitrified. But I think that at the time that we have all of the technology available for vitrification and deep space exploration, I would sincerely hope that we could figure out how to line the ship with lead or something.
Abhi: Yeah. Yeah. Yeah. That makes sense. Returning back to the question of metrics of damage you’re looking for when you are cryopreserving an organ. For the brain, it’s neural activity, it’s LTP. For kidneys, it’s this platform you just talked about. Do you think you’ll have to custom make this for every single organ or pretty much every organ in the human body has some sort of well-established protocol as to how you assess damage?
Hunter: I think that at least the donor organs, Okay. which are the first ones that we’ll be interested in, Yeah, I think there’s pretty canonical ways of evaluating their function. I think the things that we have to build out bespoke are things that are not so much for proving out a milestone of, is this thing healthy or not? There we can always reference back to the transplant community.
Abhi: Yeah.
[00:30:47] Nuances of loading cryoprotectants into tissue
Hunter: I think the thing where we’re both working on our own and also looking to the cryobiology community as well, is how do you assess things that are very specific to the cryo process? There are damage mechanisms that you want to be looking for and optimizing against that are specific to vitrification and cryoprotectant loading.
Abhi: What’s an example?
Hunter: So an example here might be figuring out the time constant that you can load the cryoprotectant in. So obviously you would like to get the cryoprotectant in there very quickly.
You don’t want to put it in too quickly.
Abhi: Why not too quickly?
Hunter: Yeah. That’s a great question. So I want you to, let’s do a liberally simple thought experiment first. Okay. So I have a cell in a test tube, by itself, just a cell. And to start it is in a solution that is isotonic with the interior of the cell. So water is exchanging into the cell just as quickly as it’s exchanging out of the cell. A key part about cell membranes is they’re really good at exchanging water. So there’s a protein that is in the membrane called aquaporin. And its entire job is being just a water-specific transporter. Just water freely diffuses through it in and out. So now what happens is, if I take, let’s say half the water out of the extracellular space, and I replace it with cryoprotectants.
Even very permeable cryoprotectants are still going to be, 100x slower at diffusing across the cell membrane compared to water. So what invariably happens is initially you have an influx of water... that water runs out of the cell. And then cryoprotectant can slowly get into the cell.
So the cell immediately shrinks initially and then starts to re-expand. if I shrink too much, then I die of osmotic shock. And similarly on unloading, and unloading actually is an even stiffer constraint. If it’s loaded with cryoprotectant and then I add a bunch of water on the extracellular space, then the cell inflates like a balloon and then pops. So there’s that constraint on not loading or unloading too quickly. You can imagine extending this very simple thought experiment of a single cell up to that first layer of the vasculature, that first endothelial layer of the vasculature. It’s seeing that maximum osmotic shock as you’re trying to increase the cryoprotectant. So when you’re loading this, you don’t just go from zero, isotonic solution, to full-blown cryoprotection. The protocol looks like a ramp. Gotcha. Where you’re linearly increasing the cryoprotectant as a function of time.
Abhi: Is there established theory as to what the slope of this line should look like or is it empirically determined?
Hunter: Yeah, so you can calculate what it should be. The thing that is challenging is that you don’t necessarily know all the transport metrics that you need to be able to establish. But I think it’s still useful to go through the first principles of how you would think about this. And it’s a balancing between the relative permeability of the cryoprotectant compared to the water. And then you also need some transport model of how is this cryoprotectant getting through the vasculature and perfusing along the flow path.
If you have these together, then you can establish a rough heuristic of what it should look like. Things that make this really challenging are that, yeah, not having the permeability coefficients for all the cryoprotectants in formulation for the cell types that it’s going to be seeing. That really ends up mattering. The other thing is that for some of these cryoprotectants, the permeability is actually a function of the cryoprotectant concentration, and then it’s just incredibly hard to model. So I think transport... one of the interesting biophysics questions that’s left in cryopreservation is good transport models for the cryoprotectant into the tissue.
Abhi: How in practice, how much do you use models versus just empirically determine that?
Hunter: Yeah, so I think that you want to use both, definitely don’t want to just write it down on pen and paper. Definitely the biologists in the company would prefer that I used a whiteboard marker less than I do. But I think that oftentimes you can make these things sing together. So for example, we really prioritize making simple assays, not just for how well are we doing, but also to try to establish these metrics, try to build models because you can’t proceed with, for example, how do I know what slope to load the organ? We’re pretty committed to having a first principles, let’s say hypothesis that’s built off of some simple cellular model. Some simple reduced experiment that can be done at high throughput. And then we make a best guess and adjust from there. But it always requires adjustment in the context of an organ because it’s so complex.
Abhi: How much do different cell types matter? Are all human cells, let’s say, all happy with the exact same slope or are some cell types especially sensitive to osmotic pressure and they will almost always pop if you...
Hunter: Yeah. Yeah. I think so there’s... talking like a physicist, I would say, to first order, you can consider them as all the same. And then there’s corrections. And one way of thinking about this is it is the case that aquaporin is the dominant way that water gets into and out of all cells.
So that story that I told you, that’s true across all the cells.
Abhi: Yeah.
Hunter: But the specific permeability of a given cryoprotectant is going to be different for different cell types because, for example, they use things like... they tend to hijack things like urea transporters to get into the cell. Obviously the density of urea transporters is going to be different in different cell types. So the relative permeability of these cryoprotectants in different cell types is going to be different.
Abhi: Yeah. How much do... if you move beyond organs and you start to consider, I’m trying to think of something that isn’t an organ... like blood vessels. For example. There probably are blood vessel transplants.
Hunter: Yeah.
Abhi: Transplantation. But it doesn’t sound super trivial to just perfuse blood through and see what pops out the other side. It’s more of a morphology question. Are there good metrics there? And if not, do you have to generally create your own?
Hunter: Yeah. So fortunately, again, if you want to do... so vessel cryopreservation is its own field.
Abhi: Oh, okay.
Hunter: It exists. Yeah. And there are things like H&E staining is really useful. This is also a place where you can lean on the medical field and look at established protocols for histology. And look to what is the inter-lumen supposed to look like? There’s a bunch of these structural assays that you have to lean on. Because you’re right. You can’t do the same thing of asking the question of, is the organ functional? Yeah. ‘Cause it’s just a pipe, so it’s not going to give you any information.
[00:37:03] Let’s discuss rewarming
Abhi: Yeah. We’ve spent a long time talking about the freezing process itself. Moving on to the rewarming side. What does that actually look like in practice?
Hunter: Yeah. So I think that, first let’s start with why do you need to rewarm quickly? We already talked about this, but we want to outcompete the formation of ice on the way up. And this is particularly challenging because ice nuclei that have been formed on the way down but are very small, are going to extend really quickly during the rewarming. So this placed a huge pressure on the field to create methods that would homogeneously and rapidly rewarm the tissue. So you can envision a few different ways that you could try to rewarm something that’s the size of a kidney. Maybe the most naive one is I just take it and I’ll just put it in warm solution. Here again, we have the problem of a heat diffusion equation. Everything tries to diffuse in from the outside. It’s going to be way too slow. Maybe you’d be able to warm the surface really quickly, but the core is going to stay cold. Similarly, maybe you could think of, okay, throw it in a microwave. But there again, you get cold and hot spots. If you’ve ever tried to rewarm something that’s very extended in your microwave, the surface gets warm, but the core does not.
So one of the innovations that came out of the Bischof lab at the University of Minnesota was they figured out that you can use biocompatible iron oxide nanoparticles that are perfused into the vasculature of the kidney. Fill it with metal and then put the entire kidney into an alternating magnetic field. And what this does is it rewarms the kidney, somewhat homogeneously, much like an induction heater in your kitchen. You’re flipping the magnetic dipoles back and forth. That generates some heat that tends to heat the organ relatively homogeneously. As it would happen, this was actually the topic of the second half of my doctorate. Not for the applications of organ rewarming, specifically. I was studying it in the context of cancer hyperthermia. It was the application I was looking at. But it’s a full circle moment for me that we’ve circled back to this. So this is one thing you could think to use is alternating magnetic field heating. This is one thing that we’re studying at Until. We’re also studying some other methods for volumetric rewarming. But this is the canonical one that the field is settling in on, is this idea of using alternating magnetic fields in combination with magnetic nanoparticles.
Abhi: Just to set the order of operations. The cryoprotectant agent and iron oxide nanoparticles are both given at the same time.
Hunter: So yes, you think of it as if we have this ramp of cryoprotectant that has to go into the organ, imagine the last part of that ramp, you also dope in the iron oxide nanoparticles into your solution. So it’s a colloidal suspension of iron oxide nanoparticles inside of cryoprotectant.
Abhi: Is there no interaction between the iron oxide and how the cryoprotectant actually works?
Hunter: That’s a great question. Iron oxide nanoparticles can be ice nucleating agents if they’re not coated properly. So the surface chemistry here becomes really interesting ‘cause you need things that are colloidally stable in cryoprotectants, which tend to be very high salt and kind of messy. And then you... I should say, high concentration of non-water substances. And you also need something that’s not going to nucleate ice. You don’t want to create surfaces that tend to allow for this nucleation process to occur.
Abhi: That makes sense.
You mentioned earlier, and this is something I hadn’t naively thought of, was as you freeze, I can vaguely understand water molecules interlocking with one another to create these ice crystals. How come during rewarming, there’s also a chance for nucleation to happen? It just feels...
Hunter: Yeah, it’s deeply counterintuitive, right? Yeah. But you get more ice that forms on rewarming. We can maybe walk through why.
Abhi: Sure.
Hunter: Basically between zero and minus 130, the chemical potential energy of ice, solid water, is lower than liquid water. This is true in that temperature range, whether you’re warming through that range or cooling through it. But there’s something that’s this asymmetry that we’ve been getting at. I guess we can go into a little more detail. What ends up happening is as you’re cooling, the temperature at which you get maximum nucleation, that’s the formation of these tiny nuclei, is actually colder than the maximum rate of extension of nuclei.
So it ends up happening is on the way down, you go through the temperature zone of maximum extension, then you get to the temperature of maximum nucleation.
So you go through extension, but there’s nothing to extend. And then you nucleate. On the way up, I nucleate a little bit more, and then I extend everything that I’ve nucleated on the way down and on the way up. So there’s this constraint where because you go through on the way up the nucleation and then this maximum extension phase, which is relatively warmer, you produce the majority of the volume of ice on rewarming, assuming you do symmetric cooling and warming rates.
Abhi: Okay. So if you have no ice crystals in your solution and you rewarm, there’s no chance for further crystals to form.
Hunter: Oh no, you can. Sorry. Just to clarify, the, if you think of it as the rate that you’re warming or cooling is separate from the question of just is the equilibrium of water, ice, or liquid at a given temperature, at a given absolute temperature. Not at a dT/dt, not at a change. Just think about in terms of the physics here, think of it as if there’s a water molecule that is at minus 80 degrees, it doesn’t know or care if you are currently cooling it or warming it.
So it is equally likely to do nucleation or do extension during the cooling or rewarming phase. You can think of just running a path integral over this entire curve to get the amount of ice that is formed.
[00:43:01] What scientific problems amongst vitrification and rewarming keep you up at night?
Abhi: Combining these two areas of... it seems two very difficult problems to solve. Building very good cryoprotectant agents and also building very good ways to rewarm a frozen tissue or organ. Which of those two axes do you consider hardest, easiest, or what problems in both keep you up at night?
Hunter: Yeah, both keep me up at night. I think that one thing that is nice about the rewarming system is we can bring to bear some pretty strong power electronics expertise that we have brought into the company to be able to build out a few different technologies there. And I think that we’re pretty committed to building out a wide technology platform. So we look at a few different mechanisms of action here. On the molecular side, I think there’s some give and take here because the reality is that we’ve been using... we, the field, have been using the same cryoprotectants... same few dozen cryoprotectants have been used since the 1950s. Like glycerol and then DMSO were quickly discovered. A lot of people still use glycerol and DMSO.
Abhi: Yeah.
Hunter: There’s obviously a very large chemical space that you could search for cryoprotectants.
And I think that you can find things that work better, but the question is how much better and how much less toxic can you get? And so because these things relax each other’s constraints, I don’t think of one as necessarily being harder than the other because if I were able to get traction on it and make progress on it, then I would just demand more of it.
Abhi: Sure.
Hunter: Yeah. And that actually builds a really cool team environment. Practically it builds a very cool team environment because there is no... for our applied physicists who are thinking about these molecular interactions, there is no “we’re done.” It just... you just keep chasing
Abhi: Yeah.
Hunter: continuously better versions of this. For the people who work on our cooling systems, there is no “we’re done” with improving the homogeneity and rate of cooling. I think this is true of the field writ large. This is why I think this is a relatively... yeah. This is why this is a relatively interdisciplinary, multidisciplinary field as a whole, is that everybody can find their angle through which their expertise can help with cryobiology.
Abhi: Yeah. With the development of better cryoprotectants. I think the one that you used in the September 2024 report was a well-established one with one particular ingredient removed. Yep. I’m curious about the rationale. It was just... you just wanted to get the report out of the way and there wasn’t that much work on fiddling around with the cryoprotectants. Yep. yeah.
Hunter: Yeah. So the report actually came out before we had a molecular development team.
Abhi: Okay. Okay.
Hunter: So we now have a molecular development team that looks at novel cryoprotectants. So we were working off of a sheet of stuff that had been previously used.
[00:45:58] Why are there so few cryoprotectants?
Abhi: When you say that only a handful of cryoprotectant agents have been developed since the 1950s, is that because finding better ones has been too difficult or more because it’s been pretty high-hanging fruit that people haven’t really been attracted to solving?
Hunter: it is a little bit complicated for me to understand the relative social milieu that has driven this, because I think that there actually has been quite good work on screening new cryoprotectant agents. I’ve been thinking about people like the Toner lab, the Higgins lab. There are labs that have been really focused on how to screen better cryoprotectant agents.
Abhi: Yeah.
Hunter: And yet everybody’s still using ethylene glycol, propylene glycol, formamide, DMSO. These are... there are various combinations of these. And so you’ll see like VS3, VMP, all these things thrown around in the literature. But fundamentally we’re just mixing around a few different agents and occasionally there’ll be a new one that will come in or a new additive that will come in. Some of them are quite toxic. But yeah, it is surprising to me because there has been really quality academic work on screening these things.
And yet people are still using the same stuff. So it’s an interesting contradiction.
Abhi: Is it potentially because there’s no... there’s not that many groups focused on actively translating this to a full actual organ?
Hunter: I think it’s a big lift to do this in whole organs. There are examples of people doing the full stack. So there’s great work coming out of a few different labs to try to scale this up. I think that John Bischof’s lab at UMN would be a good example of this. The Toner and Tessier labs at Harvard would be another good example.
Abhi: On the flip side of how do you improve the rewarming process? Is iron oxide like... will we ever get better than that?
Hunter: Yeah. I mean there are examples of... or is iron oxide it, meaning is magnetic warming it or...
[00:48:11] How can you improve rewarming capabilities?
Abhi: yeah. Is magnetic warming it, even where... even the agent as to how you do the magnetic warming, is that also as optimized as it could be?
Hunter: No, I think that you can continue to make better versions of this. And the... John Bischof’s group has continued to do work on improving the core compositions. We have done our own work on improving core composition materials. There’s a whole field obviously that pre-exists for how to do really good coupling of magnetic fields with nanoparticles. This is not specific to the problem of cryobiology. I think honestly, as with many questions inside of cryobiology, one need only look outside of the field, and this is probably true of any discipline in science, look outside of the narrow aperture and you will find someone else who has solved a similar problem. This is a good example of that.
Abhi: I guess even outside of magnetic induction, is there any other upcoming or perhaps already well-established approach that you view as particularly promising?
Hunter: Something that I was interested in that came out recently was the use of electric fields and microwave... actually very tens of megahertz frequency electric fields for rewarming a rabbit kidney. This was Greg Fahy was the person who led this study, and I just don’t see how this scales up.
Abhi: Okay.
Hunter: I think that maybe one day it will be doable, but it is quite challenging to think of how do you scale this from something that’s rabbit-sized up to something the scale of a human organ?
Abhi: Is it just because you mentioned earlier about how microwaving has the chance to introduce such large thermal gradients or something else?
Hunter: It... there’s there’s a bunch of... and some of the physics here is a little bit subtle, but yeah, there’s a tendency for hotspots to form inside of the organ when you’re using electric fields. Which couple... The way of thinking about this very intuitively is in one case I’m trying to directly couple to water, the thing that is everywhere inside of the organ. And so by definition, my energy is going to get attenuated just a little bit as I’m trying to go through. Whereas for the iron oxide, this is an exogenously introduced material that has a very high coupling coefficient that is very localized to the particle itself. So that... there’s some arguments for increasing the penetration depth using that.
Abhi: For the magnetic stuff, what are the waves actually interacting with? If you replace all the water...
Hunter: What are the waves interacting with? With the magnetic fields? Yeah. Yeah. So they’re interacting with the nanoparticles.
Abhi: Oh, okay.
Okay. They’re still... they’re still using the iron oxide or something...
Hunter: In the... sorry. In the case of any magnetic field stimulation, you’re going to have to use iron oxide nanoparticles.
Abhi: Okay. Okay. Yeah.
Hunter: For electric field stimulation, you can directly excite water molecules themselves. Gotcha.
Abhi: Okay. One thing that has been you’ve hinted at this, or perhaps explicitly mentioned at one point. All this inherently depends on the vascular system. Distributing everything nicely and equally. How does this work for tissues that don’t perhaps, or are not perhaps as heavily vascularized? I think even within an organ, I’m sure not every single little bit of it is... there’s potentially fascia that does not have that much vasculature attached to it. How much work goes into thinking about that? Is that kind of a long-term thing? Like we don’t need to worry about that right now? Or that’s on your mind?
Hunter: Yeah, I mean it is on the mind insofar as we are continuously thinking about the entire scientific program and not just what is right in front of us, but it is not right in front of us. In fact, yeah. There are areas of the kidney that are less vascularized than others, but all of them are sufficiently vascularized that you can, with some intelligent protocol development, get the cryoprotectant where it needs to go. And get the heating power where it needs to go. Yeah. But yeah, you’re right that the vascular density even inside of a given organ is not homogeneous.
Abhi: How are... you’ve mentioned a few metrics you use thus far for measuring function of the organ post-warming. Are there metrics to establish how well the perfusion process works in the first place?
Hunter: Yeah, so there’s a few different ways of doing this. So you can do... there’s a variety. One is I can just look at when I cool the entire thing down and vitrify it. This is a midpoint assay. You can actually use micro-CT to figure out, are there ice nuclei somewhere inside of this organ.
Abhi: That’s interesting.
Hunter: Because you can look for the crystals themselves. There’s also some crazy applications of MRI where you can look for chemical exchange with your cryoprotectant agent inside of the MRI. And that can give you an idea of local concentration of a given molecule. Actually, Alex German, the guy that I referenced previously, was a radiologist first before becoming a cryobiologist. He published a really cool paper on using something called CEST imaging to look for the concentration of given cryoprotectant species deep in extended tissue.
[00:53:03] What are the experimental costs of running cryopreservation studies?
Abhi: Okay. And one question I had while reading the September 2024 report was this... this experiment was done on four mice, which is a relatively low N. Yeah. And that made me think, how expensive was this whole process? How many shots on goal do you have when you’re running Until Labs? Do you do... you guys all sit in the room, you think very hard and then you run a million-dollar experiment? Or is it more there’s a bunch of intermediate assays you can do to get sanity checks?
Hunter: Yeah. Yeah. This is great. So definitely the mice are less expensive than the larger preclinical models for sure. But the way I like to think about this is as a funnel. And at the top of the funnel you have in silico models. You want to do as well as you can with in silico models because they are very cheap to run.
Abhi: Sure.
Hunter: Compared to everything that’s going to come downstream in this pipeline. Then below the in silico models, you have things that you can do in a test tube or things that don’t require any interaction with biology. A canonical example of this would be things like differential scanning calorimetry. So here you can take a cryoprotectant that is in water, you can put it in a tiny little sample and cool it down to minus 130 degrees. And you can look for an exothermic peak. This is heat flow out of your sample during the cooling or during the rewarming process. If you see this exothermic peak, that’s indicative of ice formation. The liquid-to-solid transition in that temperature range is exothermic. So you’ll be able to tell, oh, I got ice in this little solution, and the solution still hasn’t seen cells yet.
You can then imagine screening many of these such compounds in cultured cells.
Abhi: Yeah.
Hunter: And that gives you some idea of toxicity. And then from there, I think that this is where a lot of our work is done, it’s what is the gap? How do you bridge between doing cells and going all the way to an organ? Because setting up organ experiments, like you were saying, is both expensive in terms of capital, but mostly it’s that they’re very time-expensive. These are very long protocols. And I’m incredibly proud of our team for their ability to work through some of these arduous protocols, but you don’t want to waste that.
Abhi: Yeah.
Hunter: So I think that there’s a lot of focus on how can we move stuff up that funnel to more translational assays.
Abhi: To give some sense of how expensive this protocol is from a time perspective. What did it take to get these rat cerebellums frozen and rewarmed?
Hunter: Yeah. So the rat stuff, the rat acute slices, that’s not so bad.
Abhi: Okay. Okay.
Hunter: That’s not so bad. The challenge is when you want to do a preclinical model of an organ. And that obviously requires actually taking the organ out of the animal or getting it. And then you’re going to have to do things like flush the blood out of the organ. You need to load with cryoprotectant, you need to do the vitrification process, do the rewarming process, unload the cryoprotectant, and then do the assay.
And the assays themselves take several hours to be evaluating the function of the organ. So it’s just, it’s a very long process if you want to get down to an entire organ as an evaluation.
Abhi: That makes sense. One question that immediately popped up in my head was, I imagine you want to rewarm uniformly to prevent these thermal gradients and prevent mechanical stress. Is there any world in which you have a molecule that you can inject alongside the cryoprotectants to help with that? Are there such things as cryoprotectants that help prevent mechanical stress?
Hunter: Yeah, so I think mechanical stress is one that would be interesting to look into. I haven’t looked specifically at that, but what we have looked at... there’s a set of molecules that would help with not necessarily blocking ice, but help alleviate any damage that would be induced.
Abhi: Yeah.
Hunter: And we’re definitely not the only ones who are looking at this. I think the field as a whole is looking for these additive molecules that can help with things like the shock that is induced by going down and coming back up again. There are also some cryoprotectant agents whose mechanism of action is strictly what I would call biomechanical. That instead of blocking ice, what they do is they actually interpolate into the biological membrane and strengthen it. So you can actually have these, they’re polymer molecules that Medevelop developed that will actually sit in the membrane and will strengthen the biological membranes. This obviously works much better in small cells or embryo context. Pretty hard to get that to work at the scale of an organ, but it is an active area of research.
Abhi: Why is it difficult to get to work at the scale of an organ? Just...
Hunter: Loading things that are that large homogeneously outside of the vasculature becomes challenging. The transport here is one of the key limiting factors for any of these approaches.
[00:57:49] What happens to the cryoprotectants and iron oxide nanoparticles after the organ has been thawed?
Abhi: I think when we first talked, one of the most interesting questions I had and most interesting answers you gave was, you fill up this organ with cryoprotectant and iron oxide nanoparticles and you rewarm it back up. What... how does the body deal with all this stuff that’s left over?
Hunter: Yeah. Yeah. So you really don’t want to leave anything behind at 37.
Abhi: Yeah, for sure.
Hunter: And so I think a lot of the work that we do is making sure that you’re able to effectively clear all these cryoprotectants. I think that there is sometimes a misunderstanding of a beautiful symmetry here. So when you go down to around four degrees, which is where we load the cryoprotectant into the organ, obviously the metabolism is much slower.
Abhi: Yeah.
Hunter: And as a result, the toxicity of any of these molecules is substantially reduced. As long as you’re loading down at four degrees. And we load between four and 10. So you hold there, you load your cryoprotectant up. Similarly, when you want to go back and you want to unload, I think that oftentimes there’s this misconception. It’s oh, I need to add a chelator or something to take the cryoprotectant out. But you can unload the cryoprotectant the exact same way that you loaded the cryoprotectant,
Abhi: Just flush...
Hunter: Just slowly reducing the concentration of the cryoprotectant in what’s being flowed through the vasculature. And then you get passive diffusion that starts to take it out.
But yeah, critically we don’t want to leave cryoprotectant behind and then warm the organ back up to 37 degrees Celsius. Because then we eat all of this toxicity that’s going to be a result of being warm and loaded with cryoprotectant.
Abhi: Yeah.
Hunter: Similarly on the nanoparticle side, I think a lot of work in the field has been focused on passivating the surface of the nanoparticles to make sure you’re not leaving iron oxide behind in the kidney.
Abhi: You’ve mentioned a few times in the past about, you don’t want your cryoprotectant agent to be too toxic, but why does toxicity actually matter if it’s so cold that no reactions are actually occurring?
Hunter: Oh, interesting. Yeah. So the thing is that some reactions are occurring at four.
Abhi: Okay.
Hunter: That’s the trick. Gotcha. Yeah. It doesn’t matter if it’s toxic once you’re down at minus 130.
Abhi: Okay.
Hunter: For sure. And there have been some strategies that have been brought up previously where you can actually have very toxic cryoprotectant agents that can be loaded even colder. So one example of this would be M22, which is a formulation that must be loaded at minus 22 degrees. That’s where it got its name. Very effective cryoprotectant, also very toxic.
Abhi: What does toxicity usually... yeah. What does toxicity usually manifest as?
Hunter: Yeah, there’s a variety of different... when you say manifest as, it’s like in cultured cell context or...
Abhi: In full organ context, or I guess has there ever been a particularly toxic cryoprotectant used in organs?
Hunter: I think one tends to not use a really toxic cryoprotectant in whole organs. Obviously, the outcomes here are going to be different based on different organs.
Abhi: Yeah. That makes sense.
Moving... perhaps relatedly, there was this kidney... mouse kidney transplant thing that you discussed earlier. How did that go? When they... everything was an autologous thing. Where they took the kidney out, froze it, rewarmed it, put it back in. Was the mouse just perfectly fine or... I think, was it a rabbit or mouse?
Hunter: It was a rat.
Abhi: Rat. Okay. A rat. Okay. Did the rat live?
Hunter: Yes. The rat in fact lived. Yeah. It was a cool study. They showed that you can actually regain normal function in the kidney. I think that if you look at the recovery time, it took some time for the organ to recover, a matter of weeks for the organ to fully recover normal function. But they were able to get a viable kidney through the cryopreservation and rewarming process.
[01:01:33] Cryopreservation and immune response
Abhi: Gotcha.
And I remember you mentioned at some point that there was this concern of an immune epitope exposure as a result of the rewarming and the freezing and rewarming process. Would you be able to walk through that again? Because I thought that was a very interesting anecdote.
Hunter: Yeah. I think that anytime that you’re going to induce cell lysis in tissue, you can invoke an immune response. And so there’s actually been some examples of this in human pediatric cases. Where you can take some heart tissue out of a person, it can go back into the person afterwards. So obviously there’s no self versus other response. But the strategy that you use for cryopreservation affects the outcomes of these very tiny little tissue extracts and then reimplantation. And one hypothesis for the mechanism of action here is that because you get some cellular lysis during the cryopreservation process, you expose a bunch of the intracellular molecules that are then going to induce some immune interaction. And can cause rejection of the graft, even though it’s an autologous transplant.
Abhi: I imagine there haven’t been so many cryopreservation transplant studies done such that it’s pretty clear when this will happen and when it won’t happen.
Hunter: Yeah. I think that this is such a nascent field that I wouldn’t say... The thing that is not nascent is embryo cryopreservation.
Abhi: Okay.
Hunter: There, we really, I think, have many N on that process. Right now, I think this year there will be something like 150,000 embryos... 150,000 live births this year in the US alone that were previously vitrified as embryos, which is a wild number to me. And you can store for... they can get stored for 30 years, which is crazy to me.
[01:03:24] How do you filter through the cryopreservation literature
Abhi: Cryopreservation is a nascent field. It does seem like it’s ramping up in recent years. And now that there’s this probably deluge of cryopreservation papers consistently popping up all the time. I’m curious, what is your own filter when you’re reading through these papers? What marks a paper as particularly high quality and you pay attention to this versus something that you can probably just skim and not super pay close attention to?
Hunter: Yeah. I think that there are fortunately a whole host of groups that are doing things that would meet this bar. But I think that the bar that I’m particularly interested in for when you’re doing things that are going to translate into an organ is to ask the question, are the assays that are done to demonstrate the viability of this organ in concert with what the field that they’re trying to interact with has established? So for example, if they’re doing kidneys, are they doing standard assays that would be accepted in nephrology to evaluate the viability of this organ? And I think that if you go all the way down to the molecular study, I think the molecular studies that I’m particularly interested in are ones that don’t do random scattershot screening, but instead really get into what’s the mechanism of action of the interaction of this cryoprotectant with the water molecule. Water is an under-hyped molecule. It’s actually incredibly subtle and complicated to interact with it. I’m biased, but it is currently becoming my favorite molecule. And I think that you can write really deep interrogations of the interactions of these cryoprotectant agents with the water molecules. I think that those can be deeply informative for follow-on study.
Abhi: And sorry, these are papers specifically trying to create new cryoprotectants or even in the context of no novel cryoprotectant work, you still want to know what the interaction of the cryoprotectant was?
Hunter: I think I learned stuff even when people just look at DMSO interacting with water. And people have done great things of studying the Raman spectroscopy of water and DMSO interacting to try to figure out the actual coordination, like how is this hydrogen bond actually coordinating? I think that sometimes these things are academic and can’t be translated into better engineering, but I think oftentimes we can lay better foundations for our mental models of coming up with better cryoprotectants and definitely we can come up with more interesting metrics we can be calculating in silico. I think that’s where a lot of these physical chemistry studies become really helpful is it’s oh, how can I think about simulating that? How can I think about driving this onto a chip?
[01:05:54] How much is molecular simulation used at Until Labs?
Abhi: On the topic of in silico simulation, how much molecular dynamics, molecular simulation goes on internally at Until?
Hunter: Yeah. I think we think a lot about how to do in silico screening well, and I think that there’s a variety of different ways that you can do this. One that is quite common is you can think about molecular dynamics simulations where you’re trying to look at what is the extension rate of an ice nucleus that is placed inside of some solutions. These are coexistence simulations. The way that you can think about this is just set up a box. This box has hexagonal ice in one half of it, and the other half it has some mixture of cryoprotectant and water. And you can look for, does the ice tend to extend or does it tend to melt?
And this is a good way of measuring the equilibrium between the liquid and solid state, given some cryoprotectant mixture. And we found that this is somewhat interesting to look at for in terms of ice formation.
Abhi: I’m obviously not at all a molecular simulation expert. My interpretation was that phase changes, especially of water in molecular simulation, is really gnarly and not very well modeled. Does it... do you see that? And even if you do see that, you still think these simulations are pretty predictive of what happens in real life.
Hunter: Yeah. I think one nice thing about trying to be an engineer here and not trying to be a scientist is that you just want to find things that correlate.
Abhi: Okay.
Hunter: And we have the thing in the lab. It is true, the thing that you’re getting at, which is a real problem for the field as a whole, is it’s quite hard to simulate actual ice nucleation. So this is a very... the thing I just pitched you is super contrived, right? I literally just set up a boundary of ice and I’m trying to look at the thing that is interacting with it. This obviously has some deep limitations on its translation to the actual test tube that I’m doing the experiment in, and it’s because I haven’t allowed for nucleation. Nucleation has been taken completely out of the equation.
And that’s the whole thing that you’re trying to get at, is how to suppress the nucleation. The reason nucleation is hard to simulate is that it’s actually... it’s too fast for us, but it’s too slow for molecular dynamics. Because if your time steps are one femtosecond and you need to simulate for a nanosecond or something to be able to see nucleation, for every cryoprotectant that you want to interrogate, this is not tractable. So I think that it’s a challenging thing to get around is what is the right thing to simulate? And I think the things that our applied physics group spends a lot of time thinking about is what are the right things to simulate to be able to have some predictive power around what is the efficacy of this cryoprotectant going to be?
Abhi: How much do you personally or perhaps anyone in the company pay attention to the neural network potential research that’s coming out?
Hunter: Yeah, I think that this is actually really helpful. There’s some versions of this that are more useful than others, as it would always be the case. Some of the neural network potentials are still very costly to simulate compared to classical force fields. And so I think you want to pick and choose which ones you use and in what context you use them. It’s I think there’s no, at least in our hands, there is no skeleton key for, oh, you do this exact simulation and it works. I think it’s more of an intelligent ensemble of simulations to try to get some interpretation. But yeah, I think the work that’s been coming out of ML-related tools for better simulating interactions is going to affect cryopreservation, just like it affects drug discovery. This is fundamentally improving our ability to model the physical world in silico. And I think that is great, and I would encourage those academics to continue to push hard.
Abhi: Maybe a naive question, how much does QM matter for these sorts of simulations? Or is molecular mechanics fine.
Hunter: Yeah. I think that there are some things that you really care about quantum mechanics for, and this is where the neural network-based potentials can be helpful is the actual hydrogen bonding of the stuff to water obviously involves things that are not well simulated just with a standard Lennard-Jones potential. Sure. So there, I think that it is useful to think through some more complex interactions than just a simple Lennard-Jones system.
[01:10:04] What are the (expected) economics of Until Labs?
Abhi: Okay. Yeah.
That makes sense. Moving on to almost non-scientific questions about Until. One immediate question I had upon learning about you guys when you were named Cradle was, what are the economics of this setup? My mental model of organ transplantation is that there is this organ waiting list. When your name gets called, you go and get your organ. There’s no direct-to-consumer setup. Who are buying these organs?
Hunter: Yeah. So the transplant pipeline is very complex as I have been learning. And the thing is there is no direct-to-consumer. I can’t call up and say, “Hey, I need a kidney.” That’s not how this works. As you were mentioning, there is a transplant list. There are the organ procurement organizations who are responsible for facilitating the transaction of organ comes out of this donor, goes into this recipient. And there are a host of companies that specifically handle the logistics of transporting the organ from one place to another viably. A really big player in this space would be TransMedics, a publicly traded company, who literally... they have a suite of private jets that will fly around and pick up the organ from the donor and bring it back to the recipient. This is all very heavily coordinated and the logistics are certainly not trivial.
Abhi: We were just talking about this before we started this conversation. I had this question about, right now putting an organ on ice is incredibly cheap. Perfusing with oxygen is incredibly cheap. What’s the value proposition to go for something like Until Labs? A potentially much more expensive protocol. And you mentioned that you’ll need to rely on this incredibly expensive transport chain less heavily.
Hunter: Yeah. I think that one of the nice things about doing something like organ vitrification is that because it takes urgency out of the process, it just relaxes all of these logistical constraints. So for example, I don’t need to have a private jet to go get the organ anymore. I also don’t need to wake up a transplant surgeon at 2:00 AM because that’s when the organ became available. Yeah. We now have in our vision a process that can be much more, let’s say, disciplined about bringing the organ from the donor to the recipient. And this has a bunch of knock-on effects.
So one for example is that it could increase testing. If you look at the outcomes for living versus dead donors for kidneys, if you look at 10-year graft survival rates, the 10-year graft survival rate in the US for a kidney recipient from a dead donor is around 50%. From a living donor, so this would be you get it from your brother or something. Sure. It’s about 60%. So literally just the increase in immune matching of getting it from a living donor... and it may also have some other logistical constraints there of you can literally do it on the table that’s next to the person. But there’s that much benefit to get just from improved matching in the biological sense.
If you look at the reference that I made previously about the fact that in vitro fertilization of embryos... you now have a higher chance of getting a live birth from a cryopreserved embryo than from a freshly implanted embryo. And the reason for this is, again, increased ability for testing. So we think one, the cryopreservation process can lead to better outcomes for patients because we have this time that we have bought to be able to improve matching. We think that it can improve the equality of organ allocation by allowing us to respect the transplant list more, and have fewer open calls where the organ just needs to go to someone because we don’t want it to get wasted. And then, yeah, no private jets required because we can get them there. No surgeons woken up in the middle of the night.
Abhi: When it comes to what the supply chain would look like if Until Labs ruled the entire system. How careful do you need to be with a vitrified organ? Can I put it in a truck and just have the truck go? Or does it need to be in a very specific, very special container?
Hunter: So I think we would manufacture a container that was sufficient such that it could go on a truck. I think the things that are significant is you don’t want thermal gradients to be able to come in. You don’t want to thermally cycle the organ ever up to above minus 130 degrees. There’s some stuff around tight temperature control.
But I think that these are all highly solvable problems. And I think the marginal cost of doing this on a program basis is pretty trivial.
[01:14:49] How much does cryopreservation practically solve the organ shortage problem?
Abhi: Yeah.
And again, in this hypothetical of Until Labs is everywhere. How much... is the organ shortage problem solved overnight?
Hunter: No. Okay. Unfortunately not. I wish that were the case. But in the end, this is still a supply-limited market.
Abhi: What gets better? Let’s say 10% of people who need... 10% of names are crossed off every year from the organ transplant list and everyone else dies. What does that number rise up to, at least for kidneys?
Hunter: Yeah. So I think that what’s going to end up happening is you’re going to have initially, let’s say, a few thousand organs, which would be the ones that should be going into patients, would be viable, but get lost due to logistics. It’s like the organ is on the plane, needs to get de-iced, the organ expires while it’s on the plane.
Abhi: Does that happen?
Hunter: Yeah.
Actually, Laura, my co-founder, was literally talking to a transplant surgeon the other day that was recounting this exact story. I’m not making this up. This is an actual thing.
Abhi: And this is not a particularly rare incident.
Hunter: I think that this particular annoyance of plane de-icing maybe is rare. But I think that the idea... if you can get a few thousand additional organs, if you reduce logistical constraints. And these are publicly available figures.
Abhi: Yeah.
Hunter: The thing that is an interesting unlock in the long term is if you can start to relax the supply constraint.
Things like being able to get organs that are from... there’s a concept of DCD versus DBD donors. A DCD donor is death by a cardiac event.
These are more, let’s say challenging logistically to get out. The time constraints are tighter.
Abhi: And just because ischemia happens immediate...
Hunter: Okay. Exactly. It’s an ischemia issue. The clock starts earlier. And so as a result, that is a nascent field where people are trying to push into these DCD organs to try to increase organ availability. The other in the long term that I’m particularly excited about is xenotransplantation.
[01:17:04] Synergy between xenotransplantation and cryopreservation
Abhi: I was going to ask about that.
If we had sufficiently good xenotransplantation, do we need cryopreservation?
Hunter: I think sufficiently good xenotransplantation mandates cryopreservation, but that’s my perspective.
Abhi: What’s the rationale there?
Hunter: I want to envision a future for xeno that is maybe more aggressive than has previously been talked about. I want to envision a world where someone who has a heart attack and maybe would not even have time to get a heart transplant, can now get a heart transplant. Where you go into an ER and there is a liquid nitrogen dewar sitting there that is filled with hearts that are ready to go, they can be transplanted in. Now all of a sudden, organ donation is no longer something for chronic conditions. It is now something that is also for acute conditions. And this is the thing that will dramatically increase the availability of organs is if xenotransplantation could get solved. I think that there is some reasonable, let’s say, skepticism about the timetable on which xenotransplantation will come online. But yeah, I am, let’s just say I’m cautiously optimistic that those guys will make progress. And I think that cryopreservation would be a natural fit for their logistics supply chain.
Abhi: I think bringing acute conditions onto the table is really fascinating. I had never really thought about it that way. What do you think of the xeno... I’m not super familiar with it. I just know there was that pig heart that was CRISPRed to be a little bit more humanized. It was implanted into a human patient. The patient ended up dying, I think, but potentially for reasons unrelated to the heart.
Hunter: Yep.
Abhi: Do you think that field is going to rapidly mature over the next five years or there’s some big insurmountable problems there?
Hunter: Yeah. I should clarify. I’m certainly not an expert on the process or the progress of that field. And I think that if you ask five people, you might get six opinions on the future of xeno. I’ve heard everything from, “Oh yeah, it’s right around the corner.” To one transplant surgeon told me, “Xeno is the future of transplantation and it always will be.”
Abhi: It’s perpetually five years away.
Hunter: Exactly. It’s perpetually... Yeah. I think that was his perspective. Okay. So I think that there’s a variety of perspectives on that one.
Abhi: One thing I was really curious about, this X number of people die per year because they are unable to receive that organ. How has X changed if Until Labs really succeeds? I’m not sure if... the answer you gave was, oh, several thousand because we’re still figuring things out. I’m not sure if you had an exact... there’s been almost models drawn up as to how much can we put a dent in the organ shortage crisis if this really takes off.
Hunter: Yeah. This is a really, unfortunately, it’s a super complicated question to answer. And really a complicated, even more complicated question to answer well. And the reason for this is that the data here is just, it’s very challenging. Yeah. I imagine I should tease out what is the counterfactual of the organ going into a person or not going into the person. So we have some statistics on the expiry of organs during transit, which is where I got the few thousand organs metric. You can actually look at a pie slice of what’s the outcome of various organs. And the ones that are basically, were viable but expired in transit... if you add that pie slice with some other ones that are clearly logistically related, that’s where you arrive at a few thousand organs a year.
Abhi: Gotcha.
And so it’s not necessarily the case that, let’s say Until succeeds, 10 years goes by, we’ll have a surplus of organs, like every organ that’s currently in transport right now will be given to someone.
Hunter: Yeah. I think, that for the time being, this is still going to be a supply-limited problem.
Abhi: Gotcha. Okay.
Hunter: So it will still be the case that there will be a waiting list. Unfortunately, it’ll still be the case that there will be people on dialysis in this country. And I think that it will have to be cryo plus some other technology that will need to come online for that to not be the case anymore.
[01:21:12] How much will the final cryopreservation protocol likely cost?
Abhi: Yeah, that makes sense. How much do you envision the Until Labs protocol costing? Is it an undecided figure or...
Hunter: It’s an undecided figure. Partially because we don’t know what the Until Labs protocol will be.
Abhi: That’s fair.
Hunter: I think it’s... we have such a large suite of potential technologies that are brought to bear, but what I can say is that I don’t see any obvious place where this is going to be a gene therapy that costs a million dollars a patient. That’s not what we’re talking about here. And I think that the parts that are expensive are primarily the devices.
Which will be amortized over many, organs. These perfusion devices that we’re talking about, you’ll have a disposable component to it for sure. But that’s not the expensive... the expensive part is the part that’s reusable.
[01:21:58] Who ends up paying for this?
Abhi: When it comes to companies like TransMedics, who’s paying? Are they primarily contracting with insurance companies and you are also planning on contracting with insurance companies? Yeah. Who... yeah. Whose job is it to ensure almost there’s alignment between what the patient wants and the money that you expect to get from this.
Hunter: Yeah. So there are Medicaid reimbursement rates that are set up for organs, basically. And so there’s a public payer. Obviously that’s setting some market baseline.
And then yeah, there’s insurance companies that you have to be able to figure out what they’re willing to compensate the transplant centers for. The customers that you’re actually working with though, these are transplant directors. And you’re going to be working with some OPOs. That’s the people who you’d be directly interacting with.
Abhi: And what’s the approval process for this? Because it’s not a drug, it’s almost a procedure more than anything else.
Hunter: Yeah.
Abhi: Is it a... what is it classified as exactly?
Hunter: Yeah. So we don’t know yet. But I think that medical device is probably how it’ll be classified. Yeah.
Abhi: How... and I imagine there’s not that much precedent for something like cryopreservation or would... was the ice stuff also... that had to go through its own approval process?
Hunter: Yeah, I think there’s a different... there’s going to be different approval processes for each of these. I think that there is some precedent if you look at things like hypothermic machine perfusion. I think that could reasonably set some precedent for the vitrification process. But again, we’ll have to leave that to the FDA to decide.
[01:23:28] What was it like to raise a Series A on such an unorthodox thesis?
Abhi: Yeah, that makes sense. Moving on to the actual raising journey for Until Labs. What was the series A again?
Hunter: We did 58 million.
Abhi: Okay. Okay. Until Labs is a little bit of a strange thesis for a company. It’s a biotech, but it’s not a therapeutics company. It’s serving transplantation, which I had not conceptualized as, oh, there could be a for-profit company really playing and doing innovative biomedical research here. And as far as I can tell, you guys are one of the very few people playing in that area. What was the fundraising journey like?
Hunter: Yeah, so I think that, first of all, I would say that while it may appear that we are one of the few people working in the area, my guess is that is not for long.
Abhi: Okay.
Hunter: And in terms of the fundraising journey, I think it started about a little over a year ago, not where we were actively looking to raise. We had just completed our seed and the neural slice paper came out right after we announced our seed.
Abhi: So what neural... the German lab?
Hunter: The one... no, the one you were referencing.
Abhi: Gotcha.
Hunter: The one where we did the rat cerebellum. We used that and announced the seed at the same time. And that was 15 million for the seed. So, yeah, about a year, a little over a year ago, we had this idea that maybe we wanted to start playing around with this donor organ problem.
And as I said, there had already been some of this work from the University of Minnesota, which we had been looking at. And I think it just became clear that this was going to be on the roadmap anyways, this is going to be on the scientific trajectory that we would want to be on and we get to start helping patients. So Isla, who’s currently our director of preclinical research, came to me and pitched this whole cloth, was saying, look, we have this long-term roadmap, there’s this obvious use case in getting this into patients, we should go after this seriously, and we should start scaling right now to go into preclinical models. And so that kicked off this journey where we started to initially just kick the tires on, could we take our protocols and move it over? Could we take our engineering team, task them off to this? And now this is the dominant focus of the company is how to get this translated.
One thing that we wanted to be able to do there is raise to be able to accelerate that process of getting this done and towards the point of doing a first-in-human trial. And so I think that the primary purpose of the raise is to bring on additional capital to be able to parallelize a lot of these processes and get things to market quicker.
Abhi: Was... what was raising like? How many questions did you get over the economic thesis versus the scientific thesis versus some other thesis?
Hunter: Yeah. I think we got questions along both axes. Okay. And I think that we are very fortunate to have excellent partners. Excellent capital partners who I think understand the thesis really deeply. I think actually in one of the pre-conversations you had asked me, what advice would you give to people who are in similar positions? And I think I didn’t give you a particularly satisfying answer because I don’t think that I have one. And the reason for that is I think that we were really fortunate in that the people who... so the raise was led by Founders Fund with Field Ventures and Lux joining.
And all three of these groups are able to do exceptionally detailed diligence on their own. Having technical conversations with their technical team was like you and I sitting here talking about science, plus on the level of depth that was required. But it wasn’t some story that needed to be packaged. For them, it was just an actual conversation about the technical risk.
Abhi: So it wasn’t necessarily that, oh, we want to do brain preservation and we’re moving over to kidney preservation because we’re bringing in outside investors...
Hunter: Yeah. No, that was... it was very much the reverse process there. It’s like we went to go get more capital to accelerate being able to get the donor stuff to market.
[01:27:49] What are common misconceptions people have about cryopreservation?
Abhi: What are common misconceptions that people have about the cryo field, especially people who are external to the field, and perhaps not even laymen, but people who you consider smart and what their misconceptions?
Hunter: Yeah, I think that I had a misconception when I originally jumped into this, which was that it was not doable because of ice. That you would always get ice that would form and irreversibly damage tissue. And in reality, you have this minimum temperature for ice formation at minus 130 degrees Celsius where water turns to glass, not ice. So if you can traverse this danger zone between zero to minus 130, then the tissue will be safe and you can rewarm it without damage.
Abhi: But you did mention about how these cryoprotectants have existed since the 1950s.
Hunter: Yeah.
Abhi: And so there, I imagine there should have been time for the rest of the scientific field to be aware that, oh, ice nucleation is a solvable problem. Why do you think there still is this misconception that it’s fundamentally unsolvable?
Hunter: I think that part of it probably has to do with the fact that when we actually do cryopreservation of things like cultured cells in the lab every day, we use a completely different process that does allow ice to form, that would not be compatible with tissue. So you imagine, take some cultured cells and you want to store those in vapor phase storage.
Abhi: Yeah.
Hunter: There you use a process called slow freezing. You’re allowing ice to form in the extracellular space and it slowly expands and hyper-concentrates the cryoprotectant elsewhere and then forms the glass. I think intuitively people understand this should not work for tissue. Because you’ll tear up the extracellular matrix. Yeah. So I think that probably has facilitated a misunderstanding of fundamentally how the cryopreservation process works when you want to go to these more tissue-specific or organ-specific processes.
Abhi: Does... it sounds like people don’t actively think of the vascular system as a very good transportation medium. Is that fair to say?
Hunter: So I think that’s also a common misconception. It’s oh, if I need to diffusely load this object, isn’t it going to take forever? Because they think about the equivalent of, okay, I have in vitro fertilization, I have a tiny little embryo that’s six cells that needs to load cryoprotectant. People can envision in their head, okay, I load the cryoprotectant, I cool really fast. That makes sense. And I think that a key unlock is hijacking the vascular system for mass transport.
[01:29:58] The beginnings of Until Labs
Abhi: Why... at the very early beginning when you and Laura first founded this company, was the plan, oh, we’re just going to push forward on this brain thing until it’s done?
Hunter: Yeah. So when Lauren and I met... that’s a funny story. So Lauren and I met via her cold emailing me while I was a postdoc in Adam Cohen’s lab at Harvard.
And she hit me with a very open-ended question, which was if I thought it was possible to reversibly pause biological function. And my initial response to her was the same response that any reasonable person has, which is basically, are you kidding me? Of course not.
Abhi: Yeah.
Hunter: And there was a couple of days after there where something was eating at the back of me, which is... I was a physicist by origin. And it’s okay, if you have this kind of response to someone who has this history, right? Laura had a very established history at that point of making strong scientific bets.
Abhi: Yeah.
Hunter: It’s like, okay. I should be questioning my assumptions here. And so I went back and looked at it and was like, oh, this problem is fascinating. And I think that there’s real traction that’s been made recently and I have some ideas about how we might be able to continue to do that.
And so initially it was very much this curiosity that drove me into wanting to join up with Laura and get this going. And that curiosity manifested in a slowly expanding way where I realized the places that this could help. As a bit of personal context, in 2016, my father-in-law, Mark, was diagnosed with a terminal case of cancer and was given a six-month prognosis. And he lived almost exactly six months. And near the end of his life, a clinical trial for Keytruda came online for his disease. But he was too sick to qualify for it. And I think in these conversations with Laura, something that hit me was this is a technology that could help people like Mark.
People like Mark and their families who they don’t need some hundred-year jump into the future. They need six months.
They need a year. And this continued conversation with Laura led me to understand, oh, Mark’s case is not isolated. This is not some exceptional thing. It is the case that oncology drugs are forever getting better and survival rates are increasing. Things like pandemics, think about AIDS. Okay. In the 10 years between the onset of the AIDS pandemic and the creation of combination antiretroviral therapies, 9.7 million people died. 10 years, 9.7 million people. And I think there’s just this overwhelming sense of... there were no other things that I had touched before scientifically that had this kind of a lever arm on them to be able to affect this many people just by time. It was a very skeleton key solution to healthcare. So yeah, I flew out to meet with Laura and basically, flew back, quit my job, wrapped things up and moved out. And I think initially we really were committed, and still remain very committed to this day, to this hibernation as the long-term goal.
And this is the idea of taking an entire person, taking an entire patient who would be terminal with some disease and giving them access to cures that are right around the corner.
We started with that in mind and that was why the neural stuff, that was the first thing we wanted to do because it’s like the hypothesis is, oh, that’s going to be the hardest thing. Yeah. That’s going to be the thing that for sure breaks.
These are insanely delicate tissues. In the lab we would have challenges just keeping them viable on their own, much less cryopreserved. So yeah. That’s what we came out to originally do.
Abhi: I think prior to even learning about Cradle existing, whenever I thought of cryopreservation, I just assumed, oh, that’s thermodynamically impossible to do. It’s not... no one’s ever going to crack the problem. Everyone working in it are grifters.
Hunter: Yeah.
[01:34:07] What expertise is hardest to recruit for?
Abhi: Yeah, it... I think when I read that progress report in preparation for this interview, I was like, oh my God. That’s obviously small scale, how do you scale it up, but still a crazy achievement. One question I had was, you’ve mentioned over and over again about how multidisciplinary cryopreservation is as a field. What expertise is hardest to recruit for?
Hunter: Yeah, I think that again, you want top talent across everything. And so getting top talent in any field takes a disciplined approach. And I think that you want to show people that this is for real and show them how they can contribute. I think the thing that practically is very hard to onboard is medical talent and expertise.
And I think that this is primarily driven by the labor and economic incentives of being a doctor in the US. No one wants to stop doing their clinical practice, which means that it is really challenging to be able to get good medical talent to come and work on it. We’ve been very fortunate to recently have some awesome talent on, not necessarily even from MDs, but from PhDs who’ve been working at Hopkins for example. So Dr. Amanda Lofton, who has recently joined us, she’s a PhD at Hopkins who studied things like the perfusion of these donor organs at hypothermic temperatures. So there are... you can find it. It takes a disciplined approach. And you’re definitely looking for personalities that have a high degree of, let’s say, openness to change.
Abhi: How important is it that the people that you recruit for these positions have actual... have spent several years in an organ transplant clinic versus they have done an MD and know the biology?
Hunter: Yeah. so I think that we tend to across the board recruit for people who understand the platform, understand the biology as a platform and not very specific... So I think hiring too specifically in a company like this is a deep mistake.
Abhi: Really? Why? Why is that?
Hunter: And so I think, for example, you could go and say, oh, we’re just going to hire... let’s, if we shifted over to the molecular development side. Sure. You’d say, oh, we’re exclusively going to hire people who have developed cryoprotectants before. Yeah. I think that it’s reasonable to hire people who have developed cryoprotectants before. But I think that those are certainly not the only people who are capable of contributing well to this problem.
And so I think our approach on recruiting has been to go find not people who specifically have thought about this problem, but have thought about the facet of the problem that we need, maybe from a different lens. So Andrew maybe is a great example of this, very talented material physicist, both previously thinking about x-ray scattering and...
Abhi: This is the Tesla guy?
Hunter: This is... Yeah, exactly. He was doing battery research previously. He’s... you wouldn’t naturally think thinking about lithium ions is very similar to cryopreservation, but he’s been a very effective leader for that group. And I think that is just one of many examples of highly agentic people coming into our group and being able to make real progress against this problem.
Abhi: I imagine the mental leap to finding Andrew out in the wild working at Tesla and thinking, oh, maybe he’d be really useful for a cryoprotectant group is a big jump. Did he reach out to you first? Did you reach out to him first?
Hunter: Andrew and I go way back, so that was helpful. We used to race bicycles together actually when we were at Chicago. So that was helpful. And honestly, that was one of the benefits of having come up through a scientific background that put me in academia for a long time. Yeah. Is that I have the benefit of knowing who is very good from having seen them work before. And Andrew always struck me as someone who was both exceptional at physics, but also was deeply operationally minded. Very no-BS problem solver. And he was a natural early hire for the company.
Abhi: How much of your recruiting process is reaching out to people who seem hyper-talented in this orthogonal discipline to cryopreservation and saying, Hey, have you ever thought about working at Until versus them reaching out to you and saying, I want to turn my talents into this field I know nothing about.
Hunter: Yeah. I think that in the beginning it was almost all the former. Because we’re in stealth mode. And no one knows who we are. I think that we are slowly seeing it shift to be the latter where... Laura and I are getting cold emails from some thermodynamics expert who’s “Hey, I saw this.” Yeah. “And this is crazy. Can you tell me more about the glass transition in water?”
And I think that’s part of why I want to have conversations like this. This is part of why I think it’s my job to go around and talk about what we’re working on, is that I do think that there are likely very many intelligent physicists, biologists, chemists who would think this problem is interesting, but maybe don’t see themselves as being able to contribute to it meaningfully and I think I would want to make the vociferous pitch that is not the case.
Abhi: Okay. That’s an open call to everyone watching this to apply to Until.
Hunter: Indeed.
[01:39:27] What personality type do you most value when hiring?
Abhi: What, amongst all the talented people who end up in the recruitment pipeline for Until, is there a particular personality archetype that you think is most useful to have around?
Hunter: Yeah, I think that the first thing that I’m looking for is a highly agentic personality. And what I mean by this is a willingness to take on a high degree of responsibility for solving a really hard problem. I think that’s one. The other that I always look for is people who understand failure deeply. Because I think the one thing we pride ourselves on at Until is failing really fast. One of the things about building out a really wide tech platform, the way to do that effectively is if there’s an idea that seems good, design the experiment that proves that it’s bad, and do that at a very high rate. And I’ve found that people who have simultaneously a high degree of agency and a low enough ego to be able to design something to prove themselves wrong, those are the people who end up actually being able to, I think, move really quickly and contribute really meaningfully to the problem.
Abhi: You had, I’m not sure if I’m hallucinating this, but I think you mentioned your postdoc advisor or your PhD advisor had this one quote about, you should strive to treat everything as the same field or something along those...
Hunter: Oh, okay. Adam. Yeah. Okay. So I, this is... I have deep respect for the people that I’ve gotten to work for. I actually started... my first research job was with an ultrafast optics person, a professor at the University of Chicago, Greg Engel. Greg was great and gave me a job when I knew less than nothing. You’re an undergrad and you think you know something, so you’re actually less useful than a complete naive human being. And then in grad school, I got to work for Mikhail Shapiro at Caltech, exceptional scientist. And was the one that converted me from physics over to biology. And then my postdoc, I actually got my dream postdoc, which was to go work for this guy named Adam Cohen. And Adam is both an exceptional physicist and a great biologist and neuroscientist. And there’s this interdisciplinary way that his mind worked that just fascinated me. I had been following his publication history from even before he was a professor. And I’m looking at this guy as someone who had been able to traverse these discipline boundaries.
And so when I showed up, I asked him, “How do you do this? How is it possible that you seem to be pressing things all the way from single-molecule biophysics all the way up to solving neural circuits to building crazy microscopes?” I was an optics... I thought of myself as an optics expert and I’m looking at the microscopes that are in this guy’s lab and it’s completely blowing me away. The answer that he gave has stuck with me so deeply, which is that nature does not care. Nature doesn’t respect these boundaries. There is no such thing as biology to nature, or chemistry or physics. This is all just fundamentally the same at its core. And I think he and I share a physics background, so I think at his core it’s just physics at the core. With more scaffolding built up around it. And I think oftentimes when physicists say that, they’re trying to give themselves priority or something, but that’s very much not how he meant it. What he meant was these discipline boundaries are just purely human-imposed. Yeah. Yeah. So yeah, that has totally stuck with me. And Adam’s mentorship in particular was incredibly helpful in setting up for what I’m doing now.
Abhi: Do you think the only... the primary way one can imbibe that mindset is just to simply learn about many different fields? Or... I’m curious, if you were mentoring someone how do you encourage them to think about the scientific process?
Hunter: Yeah. I think that in terms of learning many different fields, first of all, just get comfortable with being an idiot about different disciplines, particularly early on because you have to be deeply, comfortable with the idea that if you’re going to move through a discipline with which you are unfamiliar, you will be a fish out of water in that space for a while. But I think that what... and this is one of the things that I particularly love about physics, is that it sets this beautiful foundation through which you can attempt to apply analogy to a whole host of different disciplines. And this isn’t always functional. This obviously breaks down in some very complex systems. But it gives you a language for thinking about the physical world. But I’ve also seen people who are very good biologists use their own version of analogy to try to understand nature. And have similar, conversations. Mandy, this person who I was talking about, who does a bunch of these organ perfusions for us.
She and I can have a conversation. And it’s interesting because we will find a way to find common language to describe something from completely different backgrounds. Yeah. So that’s how I think about it.
[01:44:17] Why work in cryopreservation as opposed to anything else?
Abhi: And you’ve mentioned you’ve worked in a lot of different fields. I went through your Google Scholar and I saw papers on biophysics, neuroengineering, bioimaging, molecular engineering, and nanotechnology. Why... you did mention this thing about your father and the field that Laura pitched to you as especially interesting, but I’m curious why start a cryonics company as opposed to any other company?
Hunter: Yeah, critically the stuff that we do, we consider slightly different than cryonics, which is a different process. But why start a cryopreservation company as opposed to any other company? I think a few things as we’ve gone over previously, I think one of them comes down to impact. I had actually not even thought about starting a company. I thought I was going to be an academic when Laura and I met. I was looking forward to starting my own lab, becoming a professor. I think that it was really a pull and not a push. I enjoyed doing academic research. But there was the sense of, oh, the scale at which we can have impact with this problem, the level of neglect that it has had in terms of being able to amass the amount of capital required to really go after it hard. These things were really contributing.
And then there’s this... and again, I learned this next part from Adam. Adam always applies a three-part test to figure out if he should work on something. So the first one is, do I find it interesting? Do I think that it’s something that I want to work on? Do I have the skills that are required to execute it well? And then the final one was the removal test. If you deleted me from the Earth, is someone else likely to do it? And I applied the same criteria here in my own way. And I think for me it passed those... that three-part test of should I pivot into this? I don’t think that I could have appreciated what it would look like when I did. I really was casting myself into an unknown. But yeah, that was fine.
[01:46:26] Until Lab’s competitors
Abhi: One thing I perhaps should have asked earlier is, I kind of view Until Labs as existing as perhaps one of the very few cryopreservation companies. What does the landscape of competitors look like? Do you view TransMedics, the current dominant organ supplier company, as competition? Or do you view other cryopreservation companies as competition?
Hunter: That’s a great question. I think that in the end, there’s going to be a marketplace for all of these technologies to coexist and there will be a question of which ones are used in what context. That’s all going to come down to what are the relative viabilities of all of these strategies. But yeah, there’s a whole host of companies that are coming up for doing near subzero storage. These store at minus five. There’s already established... there’s a lot of already established devices that do zero degrees up, like four degrees, hyper-oxygenated machine perfusion at cold temperatures. It’s very well established all the way up to normothermic machine perfusion. So this is at 37 degrees actively perfusing the organ during transplant. These all have different trade-offs. Some of them are organ-specific trade-offs. So yeah, it’s hard to say yet who our competition is because it’s hard to say yet who is going to be able to press those viability metrics as high as possible.
Abhi: If you view what Until is working on as, you can store an organ infinitely You can leave it for centuries and it’ll come back just as fine. Yeah. And the very top is, the organ needs to immediately go into a patient, otherwise it will go ischemic and just die. Yes. What does the time look like for each? For putting an organ on just pure ice. How long does that organ last versus if you go a little bit up or down the hierarchy, how long does it last?
Hunter: Yeah, so it’s obviously very organ-dependent. Okay. And so for something like a kidney, kidneys are incredibly robust actually to ischemia. So you can keep a kidney on ice for 72 hours and still it will think back up and recover. If you go for tissues like the lungs, that is not the case. You have, a few hours to be able to get it transplanted because of really short ischemic windows. So it’s going to be a very organ-specific question.
Abhi: And even if you have this hypothermic blood diffusion, it’s still max 72 hours.
Hunter: Yeah. I’m not actually sure if there’s an established machine for hypothermic machine perfusion of lungs. If there is, I’m not familiar with it.
Abhi: Oh, even for kidney?
Hunter: Oh, for kidney?
Abhi: Yeah. You’re saying it’s extending beyond 72?
Hunter: So I think that there’s no... yeah, I think there’s not a ton of research on pressing kidneys out beyond the 72 hours. There is, but in terms of established places where people are really looking hard at perfusion, it’s mostly actually liver is something that’s had quite a lot of perfusion work done for it. But all these things, you’re trying to press out those time windows to make it progressively longer. Yeah. But in the end, if you get to vitrification, then that just... you take time completely out of the equation. Yes. Yeah. Yeah.
[01:49:30] What would an alternative universe version of Hunter worked on?
Abhi: That makes sense. I’m curious what... yeah, you’ve worked in a lot of different fields. What would an alternative Hunter have done, if not for cryopreservation?
Hunter: Yeah. It’s a challenging question because it was such a sliding doors moment for my life that sometimes it’s hard to look back and think about it. But I guess in a complimentary world, things that I was interested in at the time that I was working with Adam. A lot were mostly revolved around two questions. One was advanced sensing and the other was neural computation. I think I was drifting very much towards advanced concepts of sensing. So something I did in my PhD was building out small-scale devices to look at really tiny magnetic fields inside of cells.
Which was mostly an academic study at the time that I did it. But I think that there are some interesting downstream applications for being able to do something like MRI on a single cell. To try to look at diffusive transport of tiny little molecules label-free inside of a cell. I think that this is one of those things where it was beautiful and academic and I think that the relative impact was not quite as high as what I could be doing outside. Yeah. And I think it’s a really natural fit and I feel really blessed to have had it walk into my life.
Abhi: Do you think if this Until Labs opportunity did not pop up, you would’ve felt pretty content not doing a ton of translation work throughout the rest of your career, or you think there was always something in the back of your head thinking, I should turn this into something that’ll reach a patient?
Hunter: Yeah. I think that was one of the things that was always challenging for me in academia actually, was that there was always this biting thing in me that was a need for a large impact. I think all of us want this, right? Sure. All of us want to make a larger impact. And I’m not alone in being an academic that’s I wish that I could find some way of translating this work. I also think a lot of academics do a really good job of eventually finding that in their career.
[01:51:33] What would you do with $100M?
Abhi: But yeah, that opportunity was just presented itself to me directly.
Yeah. That makes sense. And I think that perhaps the last question I have is if someone handed to you a hundred million dollars equity-free. You can spend it on Until or you can spend on some other scientific field that you’re very interested in. Yeah. Where would you allocate the money?
Hunter: Right now? Right now I’d allocate it directly into Until. Okay. And I’m not saying that as a cop-out. I actually think that having seen the interior of the organization, it’s like... for me, this is the obvious place where we can make rapid advancements for humanity.
And yeah, that’s where I would want to spend it. I think that in terms of where we would allocate it inside of Until, I think parallelizing more to be able to go after more kinds of organs and get these things to clinic faster. Because I think there is some real urgency to try to... there are a hundred thousand people right now in this country waiting on organs on a waiting list. And I think that I have... my personality is very, let’s say I have a bias to urgency.
Abhi: When it comes to actually using the hundred million dollars, what is the primary bottleneck that the money will be used to help solve?
Hunter: Yeah. So I think that the primary bottlenecks are a couple fold. One is in vivo experiments are hard. It’s not even in vivo experiments, organ experiments are complex, require lots of people and are capital intensive. And to just... in the end, some of these things don’t translate unless you scale up to there. So it’s a deep focus on that translational research. I also think that it allows us just to explore more of these fundamental questions. Work with academics to explore more of these fundamental questions, and build out the foundation that actually predates Until substantially around some of these more fundamental scientific questions, to try to figure out what is the best avenue for us to build off of as we go forward. Because I think that even the solution of doing this on isolated organs does not solve the long-term problem. I think that the capital injection is also quite helpful for helping us lay a foundation to be able to eventually do this on an entire organism.
Abhi: Sure.
Is the... does it make sense to say that... the way that you’ve explained this sounds like both sides of... you need people to figure out the theory for everything and then you also need a small army of RAs to actually do the experiments.
Hunter: Yep.
Abhi: Would you allocate the money equally or you think just the empirical animal studies are infinitely more valuable than people theorizing?
Hunter: I think that it’s a weird cost equation because it’s I wouldn’t say that one is more valuable than the other, but empirically one is much more expensive than the other.
Abhi: Yeah.
Hunter: That’s... and it is simply the case that doing preclinical model experiments is much more expensive.
Abhi: Okay. Cool. Hopefully someone gives you a hundred million dollars equity...
Hunter: I think we’re pr... I think we’re pretty good on capital for a while and thanks. If you’re showing up with equity-free checks, I’ll take them.
Abhi: Okay. Thank you so much for coming onto the podcast, Hunter.
Hunter: Thank you.
Abhi: Okay. Think we’re good.
