Production and use of high pressure for cryopreservation and cryofixation

Abstract

Methods and devices are described for the concurrent delivery of elevated pressures and low temperatures to a sample, typically but not exclusively a biological sample. A medium that expands on cooling and/or freezing is employed with a sample immersed therein, typically but not exclusively encased in a sample container. Cooling the medium lowers the temperature and applies pressure to the sample such that reduced damage to a typical biosample occurs. Relatively long-lived metastable phases are also produced, including both metastable liquids and solids, without the need for very rapid cooling steps as required in conventional achievement of such metastable phases. Preliminary test data are also presented.

1 . A method of cryogenically storing a sample so as to reduce sample degradation comprising the concurrent isostatic application of elevated pressure to said sample and cooling said sample, further comprising:
 a) immersing said sample in a pressure-transmitting medium;   b) confining said pressure-transmitting medium in a sample container wherein said sample container transmits external applied pressure to said pressure-transmitting medium and thus to said sample;   c) immersing said sample container in a working fluid wherein said working fluid expands upon cooling;   d) confining said working fluid with said sample container immersed therein in a pressure-confining container such that expansion of said working fluid will result in elevated pressure being applied to said sample container; and   e) cooling said pressure-confining container thereby causing elevated pressure to be applied to said working fluid and said sample container and sample immersed therein.   
     
     
         2 . A method as in  claim 1  wherein said pressure-transmitting medium is water. 
     
     
         3 . A method as in  claim 1  wherein said working fluid is water. 
     
     
         4 . A method as in  claim 1  wherein said pressure-transmitting container is a substantially rigid tube having sliding gaskets on one or more ends thereof capable of transmitting elevated pressure from said working fluid to said pressure-transmitting medium. 
     
     
         5 . A method as in  claim 1  wherein said cooling of said pressure-confining container is performed preferentially on selected portions of said pressure-confining container. 
     
     
         6 . A method as in  claim 1  wherein said sample container can be moved to different positions within said pressure-confining container. 
     
     
         7 . A method as in  claim 1  further comprising two or more of said sample containers immersed in said working fluid confined in said pressure-confining container. 
     
     
         8 . A method of producing metastable supercooled liquid in a sample without the necessity of an ultra-rapid cooling step, comprising:
 a) immersing said sample in a pressure-transmitting medium;   b) confining said pressure-transmitting medium in a sample container wherein said sample container transmits external applied pressure to said pressure-transmitting medium and thus to said sample;   c) immersing said sample container in a working fluid wherein said working fluid expands upon cooling;   d) confining said working fluid with said sample container immersed therein in a pressure-confining container such that expansion of said working fluid will result in elevated pressure being applied to said sample container; and   e) cooling said pressure-confining container thereby causing elevated pressure to be applied to said working fluid and said sample container and sample immersed therein until a metastable liquid phase is produced in said sample.   
     
     
         9 . A method as in  claim 8  wherein said pressure-transmitting medium is water, said working fluid is water, said cooling is to a temperature of about −80 deg. C. thereby producing pressures on said sample approximately in the range from about 200 MPa to about 220 MPa. 
     
     
         10 . A method of producing a state of matter substantially similar to high density amorphous water in a water-containing sample without the necessity of an ultra-rapid cooling step, comprising:
 a) immersing said sample in a pressure-transmitting medium;   b) confining said pressure-transmitting medium in a sample container wherein said sample container transmits external applied pressure to said pressure-transmitting medium and thus to said sample;   c) immersing said sample container in a working fluid wherein said working fluid expands upon cooling;   d) confining said working fluid with said sample container immersed therein in a pressure-confining container such that expansion of said working fluid will result in elevated pressure being applied to said sample container;   e) cooling said pressure-confining container thereby causing elevated pressure to be applied to said working fluid and said sample container and sample immersed therein until a metastable liquid phase is produced in said sample; and   f) continuing cooling until a temperature at or below the glass transition temperature of said sample is achieved.   
     
     
         11 . A method as in  claim 1  further comprising:
 f) preferentially warming the sample side of said pressure-confining container thereby recovering said sample from cryogenic storage.