Current and future research topics

Research & Development Roadmap

Research Objectives

On a high level Tomorrow’s R&D initiatives have two objectives:
1) What can we do today to improve the quality of a cryopreservation that might happen tomorrow? (This is mostly the Development side of R&D)
2) What R&D projects can we start now or soon to fundamentally advance the field in the long-term? (This is mostly the research side of R&D)Initially, we will mostly concentrate on the lower hanging fruit on the development side.
The reasoning for that is two fold. Firstly, as an organization with significant in-house medical expertise and capability we’re set up to produce substantial improvements in the short and mid-term. And second, there is still significant existing knowledge that can be implemented into cryopreservation procedures (especially in Europe). Research projects on the other hand will initially be conducted in collaboration with others or just funded by us.

Perfusion & Procedures

Perfusion Protocols (Whole-Body Field Cryoprotection)

Problem: In most cases, either field washout (i.e. no on site perfusion of cryoprotective agent) or a neuro-focus CPA perfusion is used. This means that the patient is at a warmer temperature at the end of standby procedures, leading to more degradation before vitrification occurs at the long-term care facility.

Development: Protocols and equipment targeted at whole-body field cryoprotection/perfusion allows for local cooldown to dry ice temperatures, enabling longer transport times without degradation and higher quality cryopreservations.

Perfusion Technology

Problem: Perfusion equipment differs significantly between countries and organizations — from gravity-feed perfusion and embalmer pumps to medical-grade perfusion circuits . Historically, Europe has been rather rudimentary in this regard. Employing professional perfusion equipment and procedures is an important step to improve quality.

Development: For our whole-body cryoprotection procedure we will use professional medical-grade pumps and circuit setups (practically heart-lung machines) to improve quality with ideal pressure control, bubble-avoidance, pulsative flow, overpressure protection, etc. This setup will be enhanced continuously and additional local teams will be trained to perfect response times.

Support, Training, and Guidance for Local Teams

Problem: A good cryoprotection needs speed and skill. Speed to start cooling as soon as possible after circulatory arrest (and legally speaking after pronouncement) and skill to perform a high-quality cryoprotection. Unfortunately, member numbers even for the largest organizations are not large enough yet to allow for multiple professional teams that can be at the patients site without significant delay.

Development: For now, a combination of local teams to allow for fast initial cooling combined with centrally positioned professional teams is the best solution. In most cases those local team are part-time and volunteer organizations. To support them as best as possible, we’re organizing trainings, offer hands-on support and advice and developing extensive digital support tools to allow good standby even in remote locations.


Cooling Technology

Problem: Cooling technology is well established in lab or hospital settings, but complexity comes from field application. Techniques likes liquid ventilation, gastric lavage or fast extracorporeal bypass (before significant cooling) all require significant skill, comprehensive training and last but not least procedures and equipment that is realistically and reproducibly usable.

Development: Apart from implementing robust external and internal cooling (via cooled perfusate), novel cooling methods promise faster cooling rates leading to less warm ischemia.

Quality Metrics

Problem: To improve in a goal-driven fashion, comprehensive outcome metrics are required. Similar to those in medicine such as 5-year survival rate in cancer treatments or re-hospitalization and complication rate in operations.

Development: While some quality metrics exist (extent of dehydration and ice formation measured by CT scan), significant more work is required. Establishing new and improving existing metrics is a short/mid-term focus for us.

Toxicity Reduction and Markers

Problem: Some of the ingredients of CPAs are toxic. Understanding toxicity better (by establishing markers) and reduction of toxicity are important topics to limit the amount of cellular damage that needs to be repaired.

Development: Toxicity can be reduced for example by combining ingredients that in combination are less toxic than they would be individually.

Improvements & Ischemia

Improvement of Cryoprotective Agents (CPA)

Problem: The quality of cryoprotective agents is one of the most crucial factors determining overall quality.

Development: Our primary focus is the optimization for “real-world situation” as opposed to lab-settings. Lab settings are usually the well controlled and ideal, real world situations are less. Topics include the addition of blood-brain-barrier openers, optimization for transport times, edema reduction, optimization for different tissues, etc.

New CPAs

Problem: Creating new CPAs has been tried in the past by multiple organizations without much/no improvement over existing options.

Development: Similar to improving existing CPAs, creating new ones purpose-built for non-ideal (non-lab) situations is a valuable endeavour. There is interesting and promising basic research but translating it might pose significant challenges.


Problem: Ischemia is one of the fundamental problems of today’s cryopreservation practise. It leads to diverse issues such as perfusion impairment, edema, pressure increase, etc.

Development: Our research projects focus on improving the handling of non-ideal cases with different degrees of ischemia. Approaches include different CPAs, perfusion techniques,  decompressive craniotomie, medication, etc. We also work on logistical optimization to reduce ischemia in the first place.


Whole-body ITS (Intermediate Temperature Storage)

Problem: Most patients in cryopreservation are in “immersion storage”, this has been the standard storage option for many years. While it's well understood and comparatively easy to do and maintain it brings some downsides. One of the most talked about ones is “fracturing”. During cool-down to -196°C, even if done very slowly, thermal stress develops in larger structures, this leads to fracturing of the structures. This creates an additional need to repair once revival technology is possible.

Development: Cooling down less but still below the glass transition temperature would bring the same protective qualities of low temperatures without the same degree of thermal stress and in turn significantly less fracturing. Intermediate temperature storage is doing just that. We plan to implement the first whole-body ITS solution for cryopreservation patients.

Warming & Restoration of Life

Warming Protocols

Problem: Warming cryopreserved tissue, especially, when larger volumes are involved (such as organs or the brain) exacerbates the complexities involved in cryopreserving the tissue. Ice nucleation and formation, for example, is much harder to control while warming from cryogenic temperatures than when cooling down to them.

Development: Specific warming protocols and methods are required. This will first be researched on smaller animals and then increasingly on larger more complex organisms.

Re-Perfusion Protocols

Problem: To wash out CPAs and reestablish circulation the tissue needs to re-perfused and resupplied with oxygen. This brings its own set of complexities such as re-perfusion injury.

Development: Fundamentally the concepts to do perfusion after cryopreservation needs formulated. This will first be researched on smaller animals and then increasingly on larger more complex organisms.

Concepts for Repair

Problem: Everything that is done during the cryopreservation process is done to reduce the amount of cellular and sub-cellular damage incurred due to active and passive processes started after circulatory arrest and by the procedures themselves. Nevertheless, damage is still accumulating.

Development: In total four types of damage will need to be repaired: 1) damage from before circulatory arrest (e.g. due to diseases or general degradation), 2) damage occurring after circulatory due to ischemia (e.g. apoptotic and necrotic processes) , 3) damage from the cryopreservation itself (e.g. toxicity, ice nucleation, etc), and 4) damage from the warming and re-perfusion procedures (e.g. ice nucleation).Some of the repairs probably need to be done at sub-zero temperatures, further complicating the issue. Needless to say, significant basic research is required to understand what is required to perform these repairs.

Restoration of Life

Problem: There are preliminary ideas for restoration of life, but there is no experimental evidence yet. Significant research is needed to understand how restoration of life might work conceptually and practically.

Development: Once warming, re-perfusion and repair is understood and done, all procedures come together in a kind of “resuscitation” similar to how cardiopulmonary resuscitation is made up of different parts leading to the “restoration of life” in the case of heart attack. Much conceptual and theoretical groundwork needs to be done before more applied research projects make sense.
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