Method of introduction and removal of high concentrations of cryoprotectants by vascular perfusion
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Publication Number
US-11659835-B2
Publication Date
2023-05-30
Expiration Date
Abstract
This invention relates to the preservation of vascularized tissues and organs by freezing or by vitrification; to organ and tissue cryopreservation or banking; and to tissue and organ perfusion with cryoprotective agents (also known as cryoprotectants).
Core Innovation
The invention provides a method for perfusing tissues, organs, organ systems, or vascularized organisms with cryoprotective agents by a perfusion process that uses a lower arterial flow rate limit. The method increases the concentration of cryoprotective agents in an arterial perfusate at a constant first arterial perfusion pressure until the rising viscosity causes the arterial flow rate to decline to become equal to the set lower arterial flow rate limit.
After the arterial flow rate declines to the set lower arterial flow rate limit, the method holds the arterial flow rate constant at that limit. It then allows the arterial perfusion pressure to rise from the first arterial perfusion pressure to a higher pressure equal to or below a preset maximum arterial perfusion pressure as a result of rising perfusate viscosity in combination with the constant lower arterial flow rate limit.
The process continues by either continuing to perfuse at the set lower arterial flow rate limit above the first arterial perfusion pressure if the arterial perfusion pressure does not reach the preset maximum, or if the arterial perfusion pressure reaches the preset maximum, reducing the arterial flow rate to below the set lower arterial flow rate limit so as to maintain the arterial perfusion pressure at the preset maximum. Documented examples relate to cryoprotectant loading and removal, including washout, to equilibrate tissues and organs while limiting damage from viscosity-driven flow decline.
The dependent refinements include reducing perfusate viscosity during continued perfusion by raising the temperature of the arterial perfusate and/or lowering the concentration of cryoprotectant. Further refinements include using a predetermined second maximum arterial perfusion pressure and then transitioning to the first arterial perfusion pressure while maintaining an arterial flow rate under the lower-limit constraint, and maintaining perfusion conditions longer than the time needed for flow to reach the lower limit with locking of arterial flow rate and arterial pressure until the first arterial perfusion pressure is reached.
Claims Coverage
The independent claim covers a perfusion method defined by controlling cryoprotective-agent loading under a set lower arterial flow rate limit, while allowing arterial pressure to rise up to a preset maximum and applying conditional continuation versus flow reduction to maintain that maximum. The inventive features are further narrowed by viscosity-reduction steps and additional pressure and holding constraints in dependent claims.
Setting a lower arterial flow rate limit
A method for perfusing tissues, organs, organ systems, or vascularized organisms with cryoprotective agents comprising setting a lower arterial flow rate limit for the perfusion process.
Increasing cryoprotective agent concentration at constant first pressure until viscosity limits flow
Increasing the concentration of cryoprotective agents in an arterial perfusate at a constant first arterial perfusion pressure until the rising viscosity causes the arterial flow rate to decline to become equal to the set lower arterial flow rate limit.
Holding arterial flow constant at the lower limit while allowing pressure to rise to a preset maximum
Holding the arterial flow rate constant at the set lower arterial flow rate limit and allowing the arterial perfusion pressure to rise from the first arterial perfusion pressure to a higher pressure equal to or below a preset maximum arterial perfusion pressure.
Conditional continuation at the lower limit or flow reduction to maintain preset maximum
Either continuing to perfuse at the set lower arterial flow rate limit if the arterial perfusion pressure does not reach the preset maximum arterial perfusion pressure, or if the arterial perfusion pressure reaches the preset maximum, reducing the arterial flow rate to below the set lower arterial flow rate limit to maintain the arterial perfusion pressure at the preset maximum.
Reducing viscosity by raising temperature and/or lowering cryoprotectant concentration
Reducing perfusate viscosity by raising temperature of the arterial perfusate and/or lowering cryoprotectant concentration.
Using a predetermined second maximum arterial perfusion pressure below the preset maximum
Using a predetermined second maximum arterial perfusion pressure and then transitioning to the first arterial perfusion pressure while maintaining an arterial flow rate under the lower-limit constraint.
Time-based hold with locking arterial flow rate and arterial pressure
Maintaining perfusion conditions longer than the time needed for flow to reach the lower limit with locking of arterial flow rate and arterial pressure until the first arterial perfusion pressure is reached.
Overall, the claim set centers on maintaining a lower arterial flow rate limit during cryoprotective-agent concentration increases under a constant first arterial perfusion pressure, permitting pressure to rise up to a preset maximum with conditional continuation versus flow-rate reduction. Dependent claims narrow the process by viscosity reduction via temperature and/or cryoprotectant concentration changes, by introducing an additional predetermined second maximum pressure level, and by adding a time-based hold with locking of flow rate and arterial pressure until the first arterial perfusion pressure is reached.
Stated Advantages
Limiting damage from viscosity-driven flow decline during cryoprotectant loading and removal, including washout, while equilibrating tissues and organs.
Documented Applications
Vascular perfusion cryoprotectant loading and removal, including washout, to equilibrate tissues and organs.
Whole rabbit and rabbit kidney examples, including reported outcomes such as urine output and concentration changes and reduced inner-medullary ice formation target.
Post-transplant serum creatinine viability as a documented outcome in the provided content.
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