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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