Problem Statement and Roadmap

We’re building a pause button for biology: a technology which halts molecular motion across long timescales and restarts it on demand. We will use this to help patients in three time-sensitive areas of medicine.

1. Preclinical Translatability - Due to difficulties in procuring human brain tissue samples, neuroscientists primarily use rodent neural tissue for both basic research and drug development. The short ischemic window after a surgeon resects the tissue is one of the main constraints blocking access to human brain tissue. Banking cryopreserved slices of resected brain tissue would allow neuroscientists to order a human neural tissue sample at any time, accelerating neuroscience research and improving translatability for drug development.

   
   

2. Organ Donation - Thousands of organs are rejected every year due to insufficient time for testing and matching during the viability window immediately following the organ’s excision from the donor. Pausing molecular motion in donor organs after excision would remove biologically-imposed time constraints upon testing and matching procedures, improving outcomes by reducing rejection rates amongst recipients. 

   
   

3. Medical Hibernation - Despite the ever-increasing frequency of medical breakthroughs, many patients won't live to see a cure to their disease. By reversibly pausing the biological function of a patient, we can extend the critical window of care for those without other treatment options. For example, in the decade between the onset of the AIDs epidemic/pandemic and the widespread availability of combination antiretroviral therapies, more than four million afflicted patients died (source UNAIDS (2023)). In 1950, a patient with cystic fibrosis would have died in infancy, while those born today with the condition have a life expectancy that often extends into middle age. Finally, patients today still regularly die of cancers that could have proven treatable were they afforded the innovations provided by a few more years of rigorous medical research. Medical hibernation technology could help these patients pause their biological time and access cures that are right around the corner. 

Each of these objectives presents its own unique challenges, but their solutions share the simple insight that the rate of molecular motion and chemical reactions can be controlled with a single knob — temperature. Cooling to deep hypothermic temperature (20°C ) is a common strategy for protection from ischemic damage during cardiac surgery [1] . For in vitro fertilization (IVF), embryos can be stored for decades before implantation by cooling to cryogenic temperatures (below -130 °C) where the viscosity of water dramatically increases and all molecular motion stops [2]. This phase transition from liquid to glass–in this case referring to any amorphous solid–is known as vitrification. Molecular motion is too slow in this state for water molecules to rearrange into ice crystals, which would otherwise damage tissue. Over one hundred thousand babies are born each year from cryopreserved embryos or eggs stored using this vitrification [3].

Scaling cryopreservation technology from something embryo-sized to a tissue, donor organ, or whole patient requires sufficiently fast cooling and rewarming rates to prevent ice formation between 0°C and -130°C while simultaneously avoiding rates so fast that they cause the tissue to fracture from thermal gradients. Ice crystals nucleate at a very high rate in water cooled below -20C (up to 1023 cc-1 s-1) [4], so without any chemical modifications, tissue cannot be cooled at a rate that will outrun this process. Fortunately, preparing the system in advance by perfusing molecular cryoprotective agents (CPAs) while the tissue, organ, or patient is held at hypothermic temperatures can dramatically reduce ice nucleation and extension rates during the cooling phase.

Attaining the right combination of CPAs, engineering systems, and biological preparation is a considerable challenge, however recent findings have shown interesting proofs of concept. Last year, a study from the University of Minnesota demonstrated reversible preservation of a whole rat kidney, significantly derisking cryopreservation as a viable preservation strategy for donor organs [5]. This February, we preserved electrical activity in an acute slice of rat brain tissue, a first milestone for both our research tissue and whole patient objectives. 

There’s a long road ahead to achieving our research goals, but here are our next major milestones:

• Recovery of electrical activity from cryopreserved and rewarmed acutely resected rodent neural tissue. (Complete! See whitepaper.)

• Demonstration of maintained synaptic function in a cryopreserved and rewarmed slice.

• Demonstration of maintained long term potentiation (LTP) in a cryopreserved and rewarmed slice.

• Functional preservation of long-range neuronal projections in a small animal model (ex vivo)

• Functional preservation and rewarming of a whole organ isolated from a large animal model

• Successful human organ cryopreservation clinical trial

• Reversible whole-body cryopreservation of a small animal model

Each of these milestones will require advances across multiple scientific and engineering disciplines. Below, we’ve curated a short list of the problem domains and their corresponding technical approaches.

Engineering Systems

Our engineering and applied physics group creates devices for vascular perfusion, vitrification, rapid rewarming, high throughput screening, and cryoprotectant material characterization. Some key upcoming challenges for this group include:

1. Volumetric Rewarming – Ice forms faster during warming than during cooling, placing stringent requirements on warming rates. As we scale from small tissue samples to whole organs, warming power needs to be well distributed throughout the volume of the organ to prevent thermal gradients in the tissue.

2. High throughput screening systems – Cell and tissue-based screening platforms that can scan the combinatorial sample space of cryoprotectant solutions.

3. Material Physics Instrumentation and modeling – We’re building new models and instruments to dig deeper into the physical principles behind ice formation and vitrification. 

Cryoprotectant Molecules

Our molecular development group screens novel molecular compounds and solutions for improved cryoprotectant efficacy, biocompatibility, and biodistribution. Some key upcoming challenges for this group include:

1. Enhanced colligative cryoprotectants – Colligative cryoprotectants work by dramatically increasing the viscosity of the tissue at cryogenic temperatures, facilitating the transition of water to an amorphous state and preventing ice formation.

2. Biocompatible cryoprotectants – Current cryoprotectants are toxic to delicate tissue. Even highly redundant organs like kidneys require weeks to recover after successful cryopreservation [5]. Through high throughput screening in cultured cells, we’re exploring which molecules have the right balance of cryoprotectant efficacy and minimal toxicity.

3. Enhanced Biodistribution – Organs and organisms can be perfusively loaded with cryoprotectants using the native vasculature for mass transport. Unfortunately, diffusion across biological boundaries like the blood brain barrier slow the diffusion of perfused cryoprotectant molecules out of the vasculature into the volume of the tissue.

Neuroscience Assays

Our neuroscience team develops assays to measure viability and electrical function of neural tissue following cryopreservation and rewarming. Some key upcoming challenges for this group include:

1. High throughput functional assay development – To screen thousands of candidate formulations and cooling strategies, we need a way to rapidly assay neural tissue for cellular viability and electrical activity. 

2. Measuring Global Brain Activity – Cell-resolved interrogation of long range connectivity and activity in the brain remains a significant challenge. We’re pioneering optical and electrical techniques to measure these parameters in an intact organ. 

Surgical Protocols

Preparing organs and whole organisms for cryopreservation requires robust fluidic access to vasculature with tight perfusion control systems to maintain vascular integrity. Some key upcoming challenges for this group include:

1. Donor Organ Protocols – Cannulation and ex-vivo support of isolated organs for donors requires specialized perfusion hardware and monitoring tools. 

2. Neurosurgical Protocols – Surgeries for the isolation of neural tissue requires particular handling considerations.

Citations:

[1] Gocoł, R. et al. The Role of Deep Hypothermia in Cardiac Surgery. International Journal of Environmental Research and Public Health 2021, 18 (13), 7061. https://doi.org/10.3390/ijerph18137061.

[2] Nagy, Z. P.; Shapiro, D.; Chang, C.-C. Vitrification of the Human Embryo: A More Efficient and Safer in Vitro Fertilization Treatment. Fertility and Sterility 2020, 113 (2), 241–247. https://doi.org/10.1016/j.fertnstert.2019.12.009.

[3] 2020 National ART Summary https://www.cdc.gov/art/reports/2020/summary.html

[4] Laksmono, H. et al. Anomalous Behavior of the Homogeneous Ice Nucleation Rate in “No-Man’s Land.” J. Phys. Chem. Lett. 2015, 6 (14), 2826–2832. https://doi.org/10.1021/acs.jpclett.5b01164.

[5] Han, Z., et al. Vitrification and nanowarming enable long-term organ cryopreservation and life-sustaining kidney transplantation in a rat model. Nat Commun 14, 3407 (2023). https://doi.org/10.1038/s41467-023-38824-8