High Sub-Zero Preservation and Revival of a Small Mammal (CRYORAT)

CRYORAT (https://cryorat.com) is CryoDAO's most ambitious research project yet: the first-ever high-subzero cryopreservation and revival of a rat.

Watch the interview with Aschwin de Wolf: https://x.com/cryodao/status/1863975265258574092

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Introduction

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Demonstration of reversible whole-body vitrification in a mammal is a necessary condition for low-temperature human suspended animation. Whole-body vitrification would also permit in-situ multi-organ preservation for organ transplantation. Given the formidable technical challenges to achieve this goal we propose a realistic research project aimed at understanding whole-body cryoprotectant distribution, the effects of whole-body perfusion on viability of individual organs and attempting high sub-zero temperature whole-body resuscitation.

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Background

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Vitrification (i.e., cryopreservation without ice formation) is increasingly the default approach in the cryopreservation of cells, tissues, and organs. Agents such as M22 have been shown to achieve ice-free cryopreservation in various organs, including the brain. In principle, such agents can be perfused throughout the whole body to protect against ice formation at cryogenic temperatures. While this approach is practiced today in human cryopreservation, the demonstrated limit for whole-body resuscitation has not been changed since the 1960s when Audrey Smith and colleagues were able to resuscitate (hibernating) animals from high sub-zero temperatures (~ -2 degrees Celsius) through a protocol of supercooling and control of ice formation. While major advances have been made in the vitrification of isolated organs, these whole-body results have never been surpassed by other researchers. Successful attempts to go beyond these results would require a deep knowledge of cryobiology and cardiopulmonary bypass techniques. The consensus of cryobiologists in organ cryopreservation is that is not possible or responsible to attempt whole-body vitrification revival attempts until specific intermediate goals have been achieved.   

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Research 

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We propose a comprehensive whole-body small animal vitrification program that seeks to:

1. Identify optimal surgical and perfusion techniques to equilibrate the whole-body with vitrifiable concentrations of cryoprotectant. 
2. Obtain a detailed characterization of cryoprotectant equilibration in organs, tissues, and vessels.
3. Investigate the effects of whole-body cryoprotection protocols on organ and tissue viability.
4. Understand the effect of whole-body cryoprotectant equilibration on the structure and viability of the brain.
5. Attempt high sub-zero whole-body resuscitation when the necessary conditions for resuscitation and sustaining integrated function have been identified and achieved.

Milestones

To break down this project in realistic milestones, and permit monitoring incremental progress of this project, we suggest the following milestones to be achieved:

1. Identify an optimal surgical whole-body cryoprotectant perfusion protocol. 

Deliverable to CryoDAO: CT Scan images of the cryoprotected rat in-vivo

2. Slice viability results from essential organs, including the brain.

Deliverable to CryoDAO: Viability measurements for individual organs

3. In-situ structural and biological viability results (including EEG) obtained during whole-body cryoprotection.

Deliverable to CryoDAO: brain electron micrographs, EEG, etc.

4. Multi-organ viability testing

Delivery: Results that multiple organs can be cryopreserved with good viability through whole-body cryopreservation.

Progressively lower-temperature whole-body resuscitation attempt will be made throughout the whole project.

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Why The Project Needs a Micro-CT Scanner

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Successful resuscitation from sub-zero temperatures requires complete cryoprotectant perfusion of all essential organs of the experimental animal, including the lungs, heart, kidneys etc. Being able to observe cryoprotectant distribution in real-time and upon completion of the experiment is the only meaningful way to understand the effect of different cryoprotectant, perfusion, and surgical protocols. This is a monitoring technology that cannot be outsourced by, say, taking a perfused specimen to a contracting lab every day for practical reasons. 

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Team

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Advanced Neural Biosciences (ANB) is a neural cryobiology research company with more than 15 years of experience in in-situ cryopreservation of the brain and ischemia research. Founded by neuroscientist Chana Phaedra and human cryopreservation expert Aschwin de Wolf, ANB can draw upon its extensive experience in applied cryobiology, vascular surgery, and neuroscience methods to conduct this project. ANB also can draw upon the extensive cryobiology knowledge from its sister company 21^st Century Medicine, which employs some of the finest cryobiologists in the world, including the pioneer of modern vitrification technologies, Gregory Fahy. Our data sharing agreement will significantly accelerate the progress relative to other labs. We also aim to hire one dedicated staff member who will be solely responsible for this project.

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Project Advisory Board

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Given the ambitious scope of this project, a Project Advisory Board will be formed that will review and authorize the initial research project proposal to be released. Subsequently, the Project Advisory Board will review the results obtained in this project on a quarterly basis to ensure compliance with the research proposal and propose modifications if some intermediate goals are reached shorter or prove to be more challenging. ANB proposes a Board that includes at least Dr. Brian Wowk and Dr. Gregory Fahy of 21^st Century Medicine given their extensive experience in general and applied organ (brain) cryobiology.

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Scope and Timeline

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We propose a two-year project running from 2025 to 2026, which can be extended by mutual agreement if desired. 

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Deliverables

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Internal monthly reports will be produced to document the progress of the project. Public annual reports will be made available to funders of the project. Upon completion of the research, a research paper will be submitted to a peer reviewed journal. Papers with intermediate results can be produced if mutually agreed upon by ANB and the funders.

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Budget

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2025: $300,000 (labor, equipment, and consumables) *

2026: $300,000 (labor, equipment, and consumables)

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Total: $600,000.00

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*2025 (micro-CT scanner) Necessary equipment but funding being sought from other entities.

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