Ice Nucleation Formulations for Cryopreservation and Stabilization of Biologics

Abstract

This disclosure relates to ice nucleation formulations for cryopreservation and stabilization of biologics, and methods of use thereof.

1 - 64 . (canceled) 
     
     
         65 . A method of preserving a liver, the method comprising:
 perfusing or contacting, or both perfusing and contacting, a liver with a composition comprising a hydrogel particle and an ice nucleating agent, wherein the ice nucleating agent is enclosed in the hydrogel particle;   freezing the liver with the composition; and   cryopreserving the liver at an ice nucleating temperature between 0° C. and −15° C.   
     
     
         66 . The method of  claim 65 , wherein the liver comprises a mammalian liver. 
     
     
         67 . The method of  claim 66 , wherein the mammalian liver comprises a human liver. 
     
     
         68 . The method of  claim 65 , wherein the composition further comprises a cryoprotectant. 
     
     
         69 . The method of  claim 68 , wherein the cryoprotectant comprises any one or more of dimethylsulfoxide, ethylene glycol, propylene glycol, propane-1,2-diol, 1,2-propanediol, 3-O-methyl-glucose, or glycerol. 
     
     
         70 . The method of  claim 68 , wherein the cryoprotectant comprises any one or more of sucrose, trehalose, stachyose, raffinose, or a polymer. 
     
     
         71 . The method of  claim 65 , wherein the ice nucleating agent comprises proteins extracted from  Pseudomonas syringae.    
     
     
         72 . The method of  claim 65 , wherein the ice nucleating agent is silver iodide, a protein, a carbohydrate, or a phospholipid. 
     
     
         73 . The method of  claim 65 , wherein the concentration of the ice nucleating agent in the hydrogel particle is greater than 0.5 mg/ml. 
     
     
         74 . The method of  claim 65 , wherein the hydrogel particle comprises an agarose hydrogel particle or an alginate hydrogel particle. 
     
     
         75 . The method of  claim 65 , wherein the hydrogel particle has a diameter less than 5 mm. 
     
     
         76 . The method of  claim 65 , wherein the hydrogel particle has a volume less than 50 μl. 
     
     
         77 . The method of  claim 65 , wherein the hydrogel particle further comprises heavy water. 
     
     
         78 . The method of  claim 65 , wherein the ice nucleating agent is present in a sufficient amount within the hydrogel particle to increase the ice nucleation temperature of the liver to higher than −10° C. 
     
     
         79 . The method of  claim 65 , wherein the method minimizes ischemia-reperfusion damage of the liver. 
     
     
         80 . The method of  claim 65 , further comprising collecting a tissue sample from the liver and subjecting the tissue sample to a biochemical analysis. 
     
     
         81 . The method of  claim 65 , wherein cryopreserving the liver comprises cryopreserving the liver for more than 3 days. 
     
     
         82 . The method of  claim 65 , further comprising thawing the liver, wherein the liver remains viable after thawing. 
     
     
         83 . A method of preserving a liver sample, the method comprising:
 contacting a liver sample with a composition comprising a hydrogel particle and an ice nucleating agent, wherein the ice nucleating agent is enclosed in the hydrogel particle;   freezing the liver sample with the composition; and   cryopreserving the liver sample at an ice nucleating temperature between 0° C. and −15° C.   
     
     
         84 . The method of  claim 83 , wherein the liver sample comprises a liver tissue sample or one or more liver cells.