• December 3, 2022

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Have you ever thought about what would happen if you suddenly need organ transplantation, but no one you know who is willing to donate is a match? An integral part of organ transplantation is, of course, donors and recipients, or people who donate the organs for matching people in need. They are registered within the Organ Procurement and Transplantation Network, an organization that arranges everyone on donor-recipient lists taking into consideration the severity of their illnesses. Their database contains all detailed information on blood and tissue types, organ sizes, medical urgency, and the geographical distance between the donor and the recipient. As soon as there is a newly available organ, a match is found throughout their database and shipped as soon as possible. Or at least that’s how the system aims to work.

But there is a hidden player – cold. From Ancient Greece and Rome to modern days, our society has utilized cold in many ways, mostly to preserve food. However, in modern medicine, cold was also found in quite a few applications, such as freezing human sperm and embryos in the process of in vitro fertilization. Intuitively, modern medicine also futuristically looks at cold as a useful agent that could save our lives many years ahead, in the sense of preserving (freezing) our bodies now, and reviving them once we find the cures for untreatable diseases that may have impacted us.

But, coming back to organ transplantation, cold plays a huge role in this process. Once the organ has been removed from the donor’s body, it needs to come to the recipient in the “exact same” functional state. Several external and environmental conditions can severely damage the organ until it’s no longer of use. One of the key factors is temperature, which needs to be low enough to slow down biochemical reactions happening in the organ after extraction to prevent further damage. To successfully transport and deliver organs, they need to be kept on ice (a term called hypothermic storage), with an average temperature of +4˚C. Unfortunately, the heart and lungs can “survive” on ice for only about 4-5 hours, after which they’re no longer usable. Human organ transplantation requires intense immunological screening of both the donor and the recipient, and this period is usually insufficient to perform it. Finally, 4-5 hours is not enough for an organ to travel from Europe to the United States, for example. It’s not even enough to travel within the United States, depending on the ending location, and in many cases, when paired with other logistical constraints, not even sufficient to travel from hospital to hospital. Therefore, geographical location plays a huge role in organ transplantation, and organs that cannot be delivered in a timely manner in optimal conditions will simply be lost. And that’s exactly what happens because about 28 thousand organs are wasted in the United States only per year, due to poor performance of currently available preservation methods.

The field of science that investigates the application of cold on biological samples is called cryobiology, whereas the process of using cold to preserve those samples is called cryopreservation. There are quite a few scientific groups, working both in academia and industry, that keep expanding the knowledge in these fields every day. The process of cryopreservation entails many steps, mainly cooling, storage, and rewarming. Each one of these steps can be divided into multiple reactions, and all of them could be performed in multiple ways. It is, however, vital that all of them are performed in an optimal way such that the biological sample that’s being preserved does not get damaged, or lose its functionality upon reviving. The main problem in cryopreservation is the formation of ice crystals, that can happen at any step of the way, but mostly when samples are being either cooled to or warmed from subzero temperatures. This is a major issue because the largest part of all biological samples is water. Therefore, many research groups in cryobiology are working on ways to avoid ice crystal formation.

If successful cryopreservation and reviving of complex biological samples, e.g. human organs, was made possible without the interference of ice crystals, organs could be easily transported throughout the world without considering the time it would take to get them to their final destination or be stored for a long time until somebody would need them, as opposed to discarding and losing hundreds of them on a daily basis. Similarly, even if their functionality could be prolonged to a few days instead of a few hours, tens of thousands of human lives could be saved every year. Some researchers dedicated their whole careers to making this happen, and today I will introduce you to one of them.

In my last article on cryopreservation, I had the pleasure of interviewing the group of Dayong Gao, that works on methods to improve reviving of frozen biological samples using single-mode electromagnetic resonance rewarming. Today, I’m interviewing Matthew J. Powell-Palm, an Assistant Professor of Mechanical Engineering and Materials Science at Texas A&M University, and a co-founder of BioChoric Inc. Following in the footsteps of his mentor Boris Rubinsky, he works on understanding the underpinnings of cryopreservation and manipulating the first major part of this process, i.e., freezing itself. The method they are establishing is called isochoric cryopreservation, a technique that could improve transplantation medicine immensely.

The History of Cryopreservation: Major Breakthroughs

By providing you a little bit of historical context, we’ll have a look over the major breakthroughs that happened in the field of cryobiology, and that instigated the modern use of cold in medicine. The start of the modern field of cryobiology is thought to have happened in 1948, when Christopher Polge discovered the cryoprotective effects of glycerol, a cryoprotective agent (CPA) that prevents ice crystal formation through the creation of bonds with free water molecules. Since then, a huge aspect of cryobiology and cryopreservation technologies was that we can modulate a given system’s chemistry by involving CPAs, which could, in theory, allow us to preserve a live biologic sample for a long time. Many more CPAs, like dimethyl sulfoxide (DMSO), appeared on the scene afterwards, revolutionizing the subfield of human sperm cryopreservation. In 1972, scientists Peter Mazur, Stanley Leibo, and David Whittingham published evidence of the first-ever successful cryopreservation of mammalian embryos using slow-freezing. Eleven years later, the first-ever human embryo was cryopreserved.

A turning point in cryobiology happened in the 1980s, the so-called golden era of cryopreservation. Building on seminal early work by Father Basile J. Luyet, a Catholic priest and professor who helped to establish the thermodynamic foundation of modern cryobiology, Gregory M. Fahy and William R. Fall introduced the process of vitrification to medical cryopreservation. Vitrification is a process of rapid cooling of liquid medium until it becomes a glass-like non-crystalline amorphous solid. It requires the protective effect of CPAs, which lower the freezing point of water, as a major part of biological systems. In its vitrified state, water is locked in place, preventing the formation of ice crystals, and the entire sample becomes a glass-like solid. Vitrification is used widely today in the cryopreservation of very small biological samples (specifically in in vitro fertilization and other reproductive applications), and many cryobiologists believe it could eventually be applied to freeze any biological materials, even organs and whole organisms.

Using vitrification, many research groups have already been able to successfully preserve and revive different cells and tissues, showing that there is major potential in cryopreserving and reviving organs as well. One of the major focus in cryobiology research is, in fact, centered around the process of vitrification and how much and which CPAs to add during this stage, or how to remove them in the rewarming stages. But, so far, CPA-aided vitrification only enabled the routine preservation of cells and cell suspensions and failed to produce any clinically translatable technique on how to preserve any complex biological systems like organs outside of the human body.

Isochoric Cryopreservation: Out With the Old, In With the New?

Methods in cryopreservation haven’t changed much in the last few years but there is a different approach currently available called isochoric cryopreservation. The term stands for cryopreservation of biological tissues at a constant volume, versus the more “traditional” way of cryopreservation that’s done at constant pressure, called isobaric cryopreservation. During isochoric preservation, the cooling process happens in a confined, constant-volume chamber, representing one of the biggest differences between isochoric and isobaric conditions. Another difference is minimized role of CPAs, which are very much needed in the classical isobaric cryopreservation, but not in several modes of isochoric cryopreservation. The advantage of isochoric freezing is that it completely avoids the question of the toxicity associated with CPA usage as well as the amount of CPAs needed to be present in the biological sample you might want to freeze. Even if there is a need to use CPAs, their concentrations would be dramatically decreased. Under isochoric conditions, a biological sample is confined within a container with high rigidity and strength, usually made out of titanium. The container is completely absent of the bulk gas phase, and is denied any access to the atmosphere, which changes both the thermodynamic equilibrium and the ice nucleation kinetics within the system inside.

Isochoric cryopreservation is a technique conceived initially by Boris Rubinsky, a Professor at the University of California at Berkeley. Prof. Rubinsky obtained his Ph.D. at MIT in 1981 and has been engaged in the field of cryobiology ever since. His major research interests include heat and mass transfer in biomedical engineering and biotechnology and, in particular, low-temperature biology, as well as the development of bio-electronics and biomedical devices for clinical purposes. He has also pioneered in the fields of medical imaging, cryoablation, and non-thermal electroporation. Prof. Rubinsky has been involved with more than 470 peer-reviewed scientific papers since the beginning of his career and holds more than 30 US-issued patents.

The aim of isochoric cryopreservation at Prof. Rubinsky’s group is not strictly preservation of biological samples (to be revived) per se, but rather about further developing the technique to offer the world a chance for a more successful general process of cryopreserving biological samples and decreasing the using toxic CPAs. Some of their latest research includes the creation of a quantitative approach to develop a general framework for the design of metastable supercooling protocols which incorporate the phase transformation and biochemical kinetics of the system. You can find the paper here. The group has also played with carbohydrate polymer protectants, as opposed to the small-molecular weight chemical ones mostly in use nowadays, and found that they can be used to manipulate the metastable-equilibrium phase change kinetics of the system at subzero temperatures. This approach has revealed that a carbohydrate polymer can be used to help modulate the stochasticity of ice nucleation in the supercooling system, which is important to designing supercooled biopreservation protocols for practical use. This research can be read here.

It seems the group is really striving to develop and optimize an application of supercooling and freezing techniques that could be used in biomedical devices already today. Some of Prof. Boris Rubinsky’s technologies were already used to treat tens of thousands of patients, and the companies he founded were acquired by the big fish, such as Cryomedical Sciences which became a $300 million NASDAQ company. A new name in the field of isochoric cryopreservation is eager to follow in these steps, and to further develop the field in his own way: Matthew J. Powell-Palm.

Future Players in Cryo-thermodynamics: Professor Matthew J. Powell-Palm

Matt Powell-Palm is one of Boris Rubinsky’s former PhD students and a leader in the field of isochoric cryopreservation. He is currently an Assistant Professor of Mechanical Engineering and Materials Science at Texas A&M University, and a co-founder of BioChoric Inc. (along with his former PhD supervisor), a medtech startup that is working on transforming transplant medicine by developing methods to prolong organ preservation. He obtained his Master’s degree in 2016 at Carnegie Mellon University under the supervision of Jon Malen, and his Ph.D. in 2020 at UC Berkeley.

Currently, a central focus of Matt’s research is within the field of isochoric thermodynamics and cryopreservation. His expertise revolves around the applications of isochoric supercooling and vitrification protocols and devices to improve organ preservation, conserve endangered marine animals, and improve global food storage and transportation. Even though he completed his Ph.D. only two years ago, he’s already established himself as one of the leaders in the field of isochoric thermodynamics and cryopreservation with more than 25 published peer-reviewed scientific papers and numerous patents. I was honored to share the online space for some time with Matt and pick his brain on all things cryo, plus ask some additional futuristic questions.

First, I wanted to see what Matt’s perspective was on different terms in cryobiology, and what he considers the differences between them.

Alex: Can you describe the differences between cryonics, cryobiology, and cryopreservation?

Matt: Cryopreservation is the application of cryobiology, and the biggest difference between it and cryonics is the end goal. The field of cryopreservation is not particularly interested in existential or societal aspects of life prolongation and is solving daily problems in medicine, conservation biology, agriculture, and in any application where the elongated shelf life is important. Cryonics is the application of cryobiology where the end goal is to prolongate a human life by freezing and reviving it in the future.

Alex: Can you talk about your current research and, specifically, the concept of isochoric cryopreservation?

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Matt: Looking back on the many successes and failures of modern cryopreservation, I have been asking myself the past few years if there are any new non-chemical ways in which we can manipulate the thermodynamic behaviors of water to achieve the goal of preventing ice crystal formation below the system’s melting point, which is the main problem in cryopreservation.

The umbrella technique the Rubinsky Lab has come up with leverages the effect of confinement or constant volume thermodynamic properties to manipulate phase transitions and equilibria of water. In the world around us, we are always in communication with the atmosphere as this constant and infinite pressure reservoir, and the core premise of isochoric cryopreservation processes is that we may be able to affect the phase equilibria and kinetics of water and ice by denying them access to this constant atmospheric pressure. When we do that, the natural variables that describe their existence are now constant volume and temperature, not pressure and temperature. When we confine the volume of a given system, it has a huge effect on the relationship between water and ice. We all know water expands almost 10% upon freezing, and we’ve all left a bottle of water or beer in the freezer only to come back and find it exploded. So let’s imagine what would happen if instead of having liquid in a glass bottle, we held it in an unbreakable titanium flask. Ice will form and try to expand, but now it can’t break the container or push the water out. What happens? Ice will start to expand, but the flask won’t break and will instead push back on the contents within, pressurizing the growing ice and the remaining water. As a result, only a small portion of the liquid will end up as ice, even at temperatures well below the freezing point.

And isochoric conditions affect not only the equilibrium between water and ice, but also the metastability of water, the vitrification process of water, and the ice nucleation and growth process. So we are working on a broad suite of thermodynamic techniques that aren’t dependent on chemical intervention but enable us to reach sub-zero temperatures without ice formation in a stored biologic, which opens up many new avenues for exploration in cryobiology.

Alex: Among the classical isobaric approaches used in cryopreservation with antifreeze agents, vitrification, and rapid reheating, how is isochoric preservation better?

Matt:

You can think of the isochoric effect as being a value-add to any system. Speaking generically, our data and research suggest that if you take any classical technique or system and conduct the same protocol not under atmospheric pressure, but instead under isochoric conditions, you will encounter a lower chance for ice crystal formation. For conventional vitrification for example, you need incredibly high concentrations of cryoprotectants, usually 7 to 10 mol/L, or up to 40-50 % of the weight ratio. By using isochoric conditions, we can relieve some of the work that the chemistry needs to do in aiding glass formation, facilitating the same process of vitrification using a lower concentration of cryoprotectants, but under isochoric conditions. Similarly we can supercool metastable systems with higher reliability by confining them, we can hold equilibrium systems in a passively pressurized ice-free state, and so on.

I’ll note too that a lot of the classical cryobiology literature and techniques have focused on ultra-low temperature preservation that targets months or years-long preservation, but there are all kinds of pressing medical cryobiology problems that don’t necessarily require that, the most obvious being full organ preservation, where shelf-life extension on the order of even a single day would be transformative. So there’s been a notable shift in the last decade towards what the community calls “high subzero” methods, which operate in the 0 – 20°C range and leverage processes that aim to be much less physically and chemically intensive on the biologic than something like vitrification. We’re finding that isochoric techniques can be particularly useful in this domain too, because you enter the realm where totally-CPA free isochoric supercooling or isochoric freezing protocols are very possible.

Alex: What about rapid reheating by using microwaves? How does the isochoric approach help with this?

Matt: Our goal is to build our protocol so that we ultimately won’t need rapid reheating, which is required to escape the high probability of ice crystal formation when rewarming biological samples. If we can decrease the probability of ice crystal formation across the board, we would decrease the need to use rapid reheating. For example, and although I can’t talk about it in too much detail, we are collaborating with the Smithsonian Conservation Biology Institute on vitrifying whole fragments of endangered corals under isochoric conditions, which has never before been achievable. In preliminary data, we are able to reheat the system without problems at a ballpark rate of 100s of degrees C per minute. The more sophisticated electromagnetic heating techniques achieves warming up rates of thousands of degrees and up in small systems, and those methods are indeed very cool, but so far unneeded for our systems. I’ll note too that another aspect of the rewarming challenge is heating the system without building up significant thermal stress, which can lead to cracking throughout the sample because of uneven heating. One advantage that the isochoric system appears so far to offer is that physically confining the volume can help stabilize the system against cracking. If your system is open to the atmosphere, as it warms, the outermost layer that’s open to the environment can expand freely, and cracking can happen easily. In the isochoric system, the boundaries of the sample are constrained, and it can help with reducing thermal cracking.

Matt’s answers really intrigued me. I have been looking at cryopreservation through the eyes of cryonics and improving medicine by being able to extend the time until we find cures for untreatable diseases, which would imminently save so many human lives. However, it seems one part of the field, which Matt is intensively developing with his colleagues, could help to save so many lives in the present time very soon. It seems like a real, graspable possibility.

However, this made me wonder about the field of cryopreservation I have been interested in for months now. We saw some major breakthroughs in the field a long time ago, but lately, it seems as if the progress has been really slow. Is it because the field has been focused on the complicated process of vitrification by using cryopreserving agents too much, or is there something else at play? I was interested in what Matt had to say about this.

Alex: Clearly, the field of cryopreservation has been around for quite soe time. Why did it not yet pick up?

Matt: This is a fascinating question that’s obviously affected by many different factors both historical and contemporary, but one of the biggest as always is funding, plain and simple. In the 90s and early 2000s, there was vanishingly little money available for research on cryopreservation, and what money there was was sort of narrowly focused. In the last decade however, cryopreservation, which we now include under the larger umbrella of “biopreservation”, has become something of a space race, and funders as varied as NIH, USDA, DOD, and even NASA are now giving out money for low-temperature biopreservation research. For example, NASA is looking for ways to protect astronauts in the theoretical manned missions to Mars. Even though using cryopreservation techniques to achieve goals like that seemed like sci-fi only a few years ago, we are now seeing more and more adventurous cryopreservation ideas getting funded, and funded well, and this has enabled the modern cryobiology field to start operating at the pace expected of a cutting-edge, super-impactful branch of science.

Alex: What happened in the last 5 years in cryopreservation research that may result in a major breakthrough in industrial applications?

Matt: Oh yeah, the last 5 years have been huge. I’m lucky to get to see watch this progress unfold from both the academic angle, as a professor, and the industrial and clinical angles, as a startup founder. The suite of core technologies driving cryopreservation these days has just exploded in the last half-decade or so, driven by key advances in our understanding of aqueous metastability and supercooling of bulk volume liquids, uses of electromagnetic effects and nanoparticles for rapid and uniform warming, new thermodynamic configurations like isochoric, and many more. These fresh approaches are driving work in all sorts of new applications, and bringing new interdisciplinary physical science angles to the field.

Supercooling alone is a potentially transformative technology for large clinical applications, e.g. to extend the shelf-lives of transplantable livers, hearts, kidneys, etc. I’d put my money on that technique seeing the light of day in the clinic within the next 5 years, as some kind of self-contained supercooling device. In my company, we have an isochoric supercooling technique that I think can be ready for pre-clinical trials very soon, though I can’t say too much there. But the potential public health benefit of stable supercooling is just tremendous. I mean, if you could extend the preservability of a heart by just 4-8 hours, you might save a thousand lives next year. Extend it by a day or two and you could potentially be saving tens of thousands of lives around the globe.

As a field, we don’t need technologies that will take ten more years to develop and will enable indefinite storage of a human heart—we need technologies that will take ten more months to develop and will enable storage of a heart for just long enough to get it from the donor to the recipient!

Although Matthew didn’t point it out now, he is also doing a lot of work on preserving and extending the shelf life of food, which is another pressing societal issue, given the rising problems of food waste in some regions of the world, and the lack of food in other regions at the same time. In one of the group’s latest research papers, isochoric supercooling and freezing have been applied to freshly harvested pomegranate, with its shelf-life being successfully extended for a month. You can read the publication here.

At his young age, Matthew is already wearing two hats (as he candidly points out), one of an academic professor and researcher, and the other as a co-founder and owner of a start-up company called BioChoric Inc. The company carries on with its research on isochoric preservation and aims at putting applicable devices and methods on the medical market as soon as possible, with everything being rooted in peer-reviewed and solid-proof research. Matt shared with me what the first days of starting the company looked like, and what their main future goals are.

Alex: When did you start BioChoric Inc. and what drove you to it?

Matt: We started the company in 2020 during the COVID pandemic. It was a spinout out of UC Berkeley, with me and Boris Rubinsky as founders, and the impetus was a crop of data we got on the effects of isochoric conditions on the supercooling of water, which suggested to us that an isochoric supercooling approach may be immediately applicable to organ and tissue preservation. We have a couple of integral patents and papers that describe the premise that, by confining the system, we can stabilize water in a metastable supercooled state, and predict the behaviors of this state in a rigorous quantitative sense, which has so far proven very difficult in unconfined systems.

The underlying philosophy of BioChoric Inc. is the obligation we feel to make rapid if incremental progress in full organ preservation. The degree of donor organ waste and the number of people dying on organ transplanting lists every day is huge, and that made us look at everything with a more clinical perspective. That’s what we’re pushing forward with BioChoric, even though the company is very small for now. One unique thing about the company is that it represents most of the thermodynamic expertise surrounding isochoric systems in the world today, and we rely heavily on interdisciplinary academic collaborations to help us further build the confidence and evidence we need to start pushing our techniques to clinical markets. We haven’t taken any outside funding and it’s fully internal equity, even though we’ve been approached by investors several times. We want to make sure we are scientifically sterling, peer-reviewed, bullet-proof before we start trying for the clinic.

One of the side hats BioChoric wears is also building isochoric biopreservation platforms and devices for other labs interested in advancing the science, and the small profit we generate from that helps to sustain our early R&D efforts.


It seems Matt is fully focused on improving human lives in the sense of prolonging the time transplantation-ready organs can be preserved, and that’s the main goal of BioChoric. However, Matt and Boris’s company is not the only one out there that offers cryo-products, although it may be the only one with a focus on isochoric cryopreservation, at least for now. Let’s see what Matt thinks about how his company compares to similar ones in the field of cryobiology.

Alex: How do you compare and compete with companies like Lorentz Bio or X-Therma? When do you think BioChoric Inc. will be ready to fundraise and go industrial-scale?

Matt: I think there are many great young companies popping up in this space, but I’m glad you bring up Lorentz Bio because it has sparked quite a bit of chatter in the community, and they’re taking an approach opposite to ours I think. My generic observation is that they have tackled raising the big money first, presuming they can fill in the scientific blanks later. In our case, it’s the scientists who have built the company, and built it on a core piece of new science, and we’re presuming we can fill in the money blanks later! Both fine ways to approach the problem. But personally, I’m not really in the business of speculation or gambling— I’m here to make sure we’re producing rock-solid, air-tight science, and the fundraising aspects don’t worry me as much. Maybe that’s just my academic side coming out. I think historically though, companies with really high checkbook-to-scientist ratios often end up coming to companies with really high scientist-to-checkbook ratios, like ours, to license our scientifically-established techniques and products. So suffice it to say, we’re focused on the science first and everything else second, and we’re shooting for both fundraising and expansion to industrial scale in the next two years.

As my final question, I asked Matt the same futuristic question I asked Dayong Gao’s research group at the University of Washington’s Center for Cryo-Biomedical Engineering and Artificial Organs in my first article on cryopreservation, which you can read here. Matt was brave enough to offer me a timeline in which we could see some real breakthroughs in cryonics, as opposed to only preservation.

Alex: When do you think we will be able to see isochoric cryopreservation being used to cryopreserve and revive a small mammal?

Matt: Interesting question! I would say within the next 5 years, we will certainly see isochoric preservation of endangered marine species. Marine biodiversity is such an unbelievably urgent problem, and we are thinking about expanding our research on coral to other marine organisms in the next few months. If things continue to go well, we may be looking at trying to deploy field-ready isochoric devices at every marine research station on Earth, as bombastic as that sounds! The problems there are just too pressing to wait. On the human organ scale, I think we will see the preservation of organs extended to at least single days within the next 5 years. And I also want to take this opportunity to give a shoutout to each and every research group working on this problem right now, because the many often divergent results from differing corners of the field each move us all forward.

I admittedly haven’t thought much about preserving small live mammals, so I can’t speculate in a properly scientific fashion, but I’ll speculate for fun! Current approaches would require us to preserve each organ of the mammal separately because the preservation process gets more complicated the more complex you go. Based on the progress in the last 5 years, we will probably see a supercooling approach to preserve every major organ separately within the next 5 years. I don’t know what happens at one step higher if you would want to preserve a multiorgan construct, and what would be different about it in comparison to just one organ. The relationship of these animals with air is also more complex than with marine animals that live submerged in a liquid anyway. But as a cop-out, I’ll go ahead and say that the timeline will once again depend really acutely on potential increases in funding, and it will depend on which aspects of the field will get the most funding. So, I would speculate we could see a small mammal preserved and revived in about 20 years if the funding goes in that direction. But in my opinion, there is much more pressing research to be done.

With such young and bright-minded scientists led by the field’s giants, like the combination of Matthew J. Powell-Palm and Boris Rubinsky, cryopreservation is definitely looking at several major breakthroughs coming from all areas of the field in the next few years. Also, as Matt also smartly pointed out, progress coming from different areas of cryopreservation actually helps developing all areas of cryopreservation, as the complex process of cryopreservation itself is made of various tightly-bound and regulated steps that cannot work alone.

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