Methods for introduction and prolonged washout by organ perfusion of maximally vitrifiable cryoprotectant solutions at elevated temperatures
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Publication Number
US-11849719-B2
Publication Date
2023-12-26
Expiration Date
Abstract
The present disclosure relates to an improved process of organ perfusion with vitrifiable concentrations of cryoprotective agents. In one implementation, the method may include perfusing an organ with a first solution containing a vitrifiable concentration of cryoprotectant at a temperature at or above −10° C. and perfusing the organ with a second solution containing a higher concentration of cryoprotectant than the first solution. The first solution may be adapted to vitrify at a cooling rate of less than 20° C./min, and the second solution may be adapted to vitrify at a cooling rate of less than 5° C./min. The perfusing with the second solution may begin at or above −10° C. and cause the organ to decline in temperature to below −10° C.
Core Innovation
The disclosure relates to organ vitrification by perfusing an organ with a cryoprotectant solution in a temperature-controlled process. An organ is first perfused with a first vitrifiable solution that is less vitrifiable than VSMax at an arterial temperature T1 greater than Tmin, and then perfusion switches to VSMax while the organ is cooled to Tmin.
VSMax is described as the maximally vitrifiable cryoprotectant solution. VSMax perfusion begins when the arterial temperature of the organ is T1 and the organ’s temperature is above Tmin, where Tmin is the lowest perfusion temperature employed in the process and T1 is in a range of −10 °C and above.
The disclosure further describes reduced cooling and toxic injury when VSMax is introduced at higher temperatures and cooled concurrently. It also describes VSMax washout methods for removing VSMax using ramped cryoprotectant concentration decreases, including pRamp and mRamp, with altered renal flow and washout kinetics and reduced injury markers.
Claims Coverage
Independent inventive concept in one method claim: perfusion uses a first vitrifiable solution less vitrifiable than VSMax at an arterial temperature T1 (−10 °C and above), followed by switching to VSMax combined with cooling to Tmin. The claim set includes one independent claim with dependent claims refining VSMax removal, concentration adjustment phases, osmolytes, and a warming step.
Switching to maximally vitrifiable cryoprotectant during cooling
Perfusing an organ with a first vitrifiable solution that is less vitrifiable than VSMax at an arterial temperature T1 greater than Tmin, and then switching to perfusion with VSMax in combination with cooling to Tmin, wherein perfusion with VSMax begins when the arterial temperature of the organ is T1 and the organ’s temperature is above Tmin and causes the organ to decline in temperature to Tmin.
Thermal boundary for VSMax introduction
VSMax corresponds to the maximally vitrifiable cryoprotectant solution, Tmin is the lowest perfusion temperature employed in the process, and T1 is in the range of −10 °C and above.
Ramped VSMax removal by continuous concentration decline
Removing VSMax by initially perfusing it with a continuously declining concentration of cryoprotectant until an intermediate concentration between 3 and 6 molar is reached, where the concentration declines at a rate of 50 to 600 mM/min.
Abrupt lower-concentration step followed by continuously decreasing concentration
Abruptly removing VSMax by perfusing a solution that is 0.5–1.5 M lower in concentration than the VSMax solution, then continuing perfusion at constant concentration 0.5–1.5 M lower for 5–15 min, followed by continuously decreasing the cryoprotectant concentration until reaching an intermediate concentration between 3 and 6 molar, with the cryoprotectant concentration decreasing at 50–600 mM/min.
Abrupt lower-concentration step followed by continuous decline
Removing VSMax by abruptly perfusing a solution that is 0.5–1.5 M lower in concentration than VSMax, then perfusing a continuously declining concentration of cryoprotectant.
Osmolytes in lower-concentration VSMax removal solution
The solution is 0.5–1.5 M lower in concentration than VSMax and includes osmolytes whose total osmolal concentrations sum to 0.3–0.5 osmolal.
Warming before continuous concentration decline
Warming an organ to above −10 °C by warming the temperature of the arterial perfusate before perfusing it with a continuously decreasing concentration of cryoprotectant.
The claims cover an organ-perfusion vitrification method that introduces VSMax at an arterial temperature T1 (−10 °C and above) and combines switching to VSMax with concurrent cooling to Tmin. Dependent claims further define how VSMax is removed using continuous or abrupt-and-continuous cryoprotectant concentration declines, including intermediate concentration and concentration-decline rate ranges, osmolyte/osmolality constraints in one removal variant, and a warming step before a continuously decreasing concentration phase.
Stated Advantages
Reduced cooling and toxic injury when VSMax is introduced at higher temperatures and cooled concurrently.
Reduced injury markers, including reduced mean peak creatinine versus prior art.
Improved recovery.
Altered renal flow and washout kinetics associated with the ramped washout approaches.
Functional outcomes associated with reduced peak creatinine.
Documented Applications
Organ vitrification using arterial perfusion with cryoprotectant solutions that transition from a first vitrifiable solution to VSMax during temperature decline to Tmin.
VSMax washout and removal from vitrified organs using ramped cryoprotectant concentration decreases (pRamp and mRamp), including approaches incorporating osmolytes in certain steps.
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