Methods and compositions for the cryopreservation of organs

Inventors

Fahy, Gregory M. • Wowk, Brian

Assignees

21st Century Medicine Inc

Abstract

Methods and compositions are provided for the cryopreservation of human organs and tissues. In certain embodiments, Step 1 comprises perfusion with a vitrifiable cryoprotectant solution at a temperature above −10° C. for a time insufficient for the approximate osmotic equilibration of the organ with the solution, followed by cooling the organ to below −10° C. by perfusion with said solution at a reduced temperature. In certain embodiments, Step 2 comprises increasing the concentration of cryoprotectant further at a temperature from −10 to −40° C. In certain embodiments, Step 3 comprises cooling and vitrifying the organ, rewarming it, and perfusing the organ with a vitrifiable concentration of cryoprotectant whose temperature is either raised gradually or is held at ≧−15° C. Compositions are provided that allow safe organ perfusion with vitrifiable media at >−10° C. and almost complete avoidance of chilling injury at −20 to −25° C. and that allow slow warming after vitrification without freezing.

Core Innovation

The invention provides a method for cooling an organ from a temperature above -10° C. to a target temperature below -10° C. by continuous vascular perfusion with minimal injury. The method includes perfusing the organ with a vitrifiable first cryoprotectant solution having a first cryoprotectant concentration, where the perfusion period is insufficient for the difference between the first cryoprotectant concentration and the venous cryoprotectant concentration to decline to 200 mM or less.

At the end of that period, the temperature of the first cryoprotectant solution is lowered to or below the target temperature while continuously perfusing, and perfusion continues until at least a portion of the organ reaches the target temperature. The first cryoprotectant solution is sufficiently concentrated to be vitrifiable at a cooling rate of ≤20° C./min, and the disclosure characterizes vitrifiable concentration and ice-free upon vitrification/rewarming behavior.

In addition, the invention addresses rewarming and solution transition by reperfusing with a second cryoprotectant solution at a perfusate temperature equal to or above the target temperature and replacing the second cryoprotectant solution with the first cryoprotectant solution. The replacement lowers the arterial perfusate cryoprotectant concentration by 0.5–1.5 molar while the temperature is raised from below -20° C. to above -10° C., with conditions designed to maintain an ice-free state upon vitrification and rewarming.

Claims Coverage

The independent claims cover two main method categories: continuous vascular perfusion cooling of an organ below -10° C. using a vitrifiable first cryoprotectant solution under a non-equilibration constraint and a vitrifiability cooling-rate criterion, and a post-rewarming transition in which a second cryoprotectant solution is reperfused and then replaced by the first cryoprotectant solution with a defined concentration drop during warming. Two inventive features are identified.

Overall, the claim coverage centers on continuous vascular perfusion cooling using a vitrifiable first cryoprotectant under a non-equilibration constraint and a defined reperfusion-and-replacement transition that produces a specified arterial cryoprotectant concentration drop during warming through the -20° C. to above -10° C. range.

Stated Advantages

Documented Applications