Method and Apparatus for Prevention of Thermo-Mechanical Fracturing in Vitrified Tissue Using Rapid Cooling and Warming by Persufflation

Abstract

A method and apparatus are disclosed for avoiding fracturing, e.g., thermo-mechanical fracturing, in vitrified biological systems via rapid cooling and/or warming persufflation techniques, by reducing the domain size of fracturing and by reducing thermal gradients. Also disclosed is a system adapted to rapidly cool and warm vitrifiable vascular biological tissue by persufflation, significantly reducing cryoprotectant toxicity from that of surface cooled tissue, in which the system is constructed and configured to use one or more of helium gas, hydrogen gas, neon gas, argon gas, krypton gas, xenon gas, oxygen gas, or various gaseous compounds. The system can he operated under pressure to increase the density and heat capacity of the gas relative to its density and heat capacity at atmospheric pressure and to cool the gas by one or more of mechanical action and by the phase change of a material such as a cryogenic gas or solid.

What is claimed is: 
     
         1 . An apparatus for cryopreservation of vascularized tissue or an organ under hyperbaric conditions using perfusion and persufflation through a vasculature, comprising:
 (a) apparatus for perfusing a liquid cryoprotectant through the vasculature, comprising:
 (i) a perfusate reservoir; 
 (ii) a circulating pump; 
 (iii) a heat exchanger; 
 (iv) arterial and venous cannula for access to the vasculture; 
 (v) an oxygenator; and 
 (vi) a return line; and 
   (b) apparatus to persufflating a gas through the vasculature under hyperbaric conditions, comprising:
 (i) a hyperbaric chamber; 
 (ii) a gas supply; 
 (iii) cooling and warming coils; 
 (iv) chamber and tissue pressure regulators; 
 (v) gas introduction and removal lines; and 
 (vi) vent and pressure relief values. 
   
     
     
         2 . The apparatus of  claim 1 , further comprising metering pumps, a recirculating loop, a sterilizing filter, a return reservoir, and a mixer, whereby the liquid cryoprotectant is added and removed. 
     
     
         3 . The apparatus of  claim 1 , further comprising at least one sensor to monitor vitrification parameters of the vascularized tissue or organ. 
     
     
         4 . The apparatus of  claim 3 , wherein the sensors are connected to a computer, and data collected with the sensors is used for computer control of the cryopreservation process. 
     
     
         5 . The apparatus of  claim 1 , further comprising temperature sensors, flow sensors and pressure sensors to monitor persufflation parameters, including gas flow rates into the chamber and tissue and gas pressures in the chamber environment and tissue. 
     
     
         6 . The apparatus of  claim 5 , wherein the temperature, flow and pressure sensors are connected to a computer, and data collected with the sensors is used for computer control of the cryopreservation process. 
     
     
         7 . The apparatus of  claim 1 , wherein the persufflating apparatus further comprises a system to stabilize the chamber temperature. 
     
     
         8 . The apparatus of  claim 1 , further comprising a radio frequency diathermy generator in the chamber, whereby the generator is used to increase rates of rewarming. 
     
     
         9 . The apparatus of  claim 1 , further comprising a reservoir, a filter, and a gas compressor for capturing and reintroducing gas used during persufflation. 
     
     
         10 . The apparatus of  claim 1 , wherein the heat exchanger is a counter-current heat exchanger.