Device and method for pressurized cryopreservation of a biological sample

Abstract

A cryopreservation device ( 100 ) which is arranged for cryopreservation of a biological sample ( 1 ) comprises a pressure vessel ( 10 ) with a vessel wall ( 11 ) and an internal space ( 12 ) which is arranged to receive the biological sample ( 1 ), wherein the pressure vessel ( 10 ) is equipped with an actuating device ( 20 ) for cooling by lowering the temperature and raising the pressure in the pressure vessel ( 10 ) and configured for the cryopreservation of the biological sample ( 1 ). Said actuating device ( 20 ) is connected to the vessel wall ( 11 ) and comprises at least one pressure setting element ( 21 - 23, 31 ) and at least one of at least one cooling element ( 34 ) and at least one heat conducting element ( 35 - 38 ), wherein the actuating device ( 20 ) is configured for a time-dependent and/or location-dependent setting of the temperature and of the pressure in the pressure vessel ( 10 ). Methods are also described for cryopreservation of a biological sample ( 1 ), comprising biological cells ( 2 ) and a preservation medium ( 3 ), and methods for heating the biological sample ( 1 ) to maintain vitality.

1 . Cryopreservation device which is adapted for cryopreservation of a biological sample, comprising:
 a pressure vessel with a vessel wall and an internal space which is adapted to receive the biological sample, wherein   the pressure vessel is configured for cooling with a lowering of a temperature and an increase of a pressure in the pressure vessel and for the cryopreservation of the biological sample,   the pressure vessel is provided with an actuating device which is connected with the vessel wall and comprises at least one pressure-setting element and at least one of at least one cooling element and at least one heat conducting element, and   the actuating device is configured for a time and/or location dependent setting of the temperature and of the pressure in the pressure vessel,   
       wherein
 the pressure setting element comprises at least one of a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous expansion medium and communicates with the internal space, and a pressure clamp which acts from outside on the vessel wall. 
 
     
     
         2 . Cryopreservation device in accordance with  claim 1  in which
 the pressure setting element comprises a coiled section which acts on one end of the pressure vessel. 
 
     
     
         3 . Cryopreservation device in accordance with  claim 2  in which
 the expansion area comprises at least one hollow duct which protrudes from the pressure vessel. 
 
     
     
         4 . Cryopreservation device in accordance with  claim 3  in which
 the at least one hollow duct has at least one of branches and protrusions to different directions from the pressure vessel. 
 
     
     
         5 . Cryopreservation device in accordance with  claim 1  in which
 the cooling element comprises a cooling line which is arranged in the pressure vessel. 
 
     
     
         6 . Cryopreservation device in accordance with  claim 1  in which
 the heat conducting element comprises at least one of a profile on an outer side of the pressure vessel, a profile on an inner side of the pressure vessel and heat conducting bodies in the inside of the pressure vessel. 
 
     
     
         7 . Cryopreservation device in accordance with  claim 1  in which
 the vessel wall of the pressure vessel has a shape of a tube, a sphere or a flat cylinder. 
 
     
     
         8 . Cryopreservation device in accordance with  claim 7  in which
 the vessel wall of the pressure vessel has the shape of a bent tube. 
 
     
     
         9 . Cryopreservation device in accordance with  claim 1  in which the at least one of the following is provided in the internal space of the pressure vessel:
 an inner vessel adapted to receive the biological sample, 
 a substrate which is adapted for adherent receipt of biological cells in the biological sample, 
 a segmentation of the internal space into internal space sections, 
 a sensor device with a least one pressure sensor and a temperature sensor, and 
 a substance reservoir which is adapted to release a substance into the internal space. 
 
     
     
         10 . Cryopreservation device in accordance with  claim 9  in which
 the substance reservoir comprises hollow spheres made of a pressure sensitive material which are arranged distributed throughout the internal space. 
 
     
     
         11 . Cryopreservation device in accordance with  claim 1  in which
 the vessel wall includes an optical unit which is adapted for a visual observation of the internal space of the pressure vessel. 
 
     
     
         12 . Method for the cryopreservation of a biological sample, comprising biological cells and a cryopreservation medium, with the steps:
 provision of the biological sample in a pressure vessel with a vessel wall and an internal space, and   cooling of the pressure vessel in a cooling device with lowering of a temperature and increasing of a pressure in the pressure vessel, wherein the biological sample is transferred at least partly into a cryopreserved state in a vitreous phase,   the pressure vessel is provided with an actuating device which comprises at least one pressure setting element and at least one of at least one cooling element and at least one heat conducting element, and   a time and/or location dependent setting of the temperature and of the pressure in the pressure vessel using the actuating device,   
       wherein
 the pressure in the pressure vessel is adjusted using a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous extension medium and communicates with the internal space, and/or a pressure clamp which acts from outside on the vessel wall. 
 
     
     
         13 . Method in accordance with  claim 12  in which the setting of the temperature and of the pressure in the pressure vessel comprises the following steps:
 increase in pressure in the pressure vessel with the pressure setting element, and then 
 lowering of the temperature in the pressure vessel with the cooling device. 
 
     
     
         14 . Method in accordance with  claim 13  in which
 the pressure in the pressure vessel is increased using a coiled section which acts on one end of the pressure vessel. 
 
     
     
         15 . Method in accordance with  claim 12  in which
 the expansion area comprises at least one hollow duct which protrudes from the pressure vessel, and 
 the pressure is increased in the pressure vessel by first the at least one hollow duct being immersed into a cooling bath of the cooling device followed by a remainder of the pressure vessel. 
 
     
     
         16 . Method in accordance with  claim 12  in which the setting of the temperature and of the pressure in the pressure vessel comprises the following steps:
 lowering of the temperature in the pressure vessel with the cooling device, and subsequently 
 increasing of the pressure in the pressure vessel with the pressure setting element. 
 
     
     
         17 . Method in accordance with  claim 16  in which
 the cryopreservation medium includes a stabiliser substance which is suitable to stabilise the vitreous phase of a supercooled melt preferably up to a transition to a liquid state, on increasing the temperature of the biological sample. 
 
     
     
         18 . Method in accordance with  claim 17  in which the stabiliser substance is at least one member selected from the group consisting of:
 long-chain uncharged polymers with a molecular weight greater than 500 g/mol, 
 monosaccharides, 
 ethylene glycol, 
 di- and oligo saccharides 
 polysaccharides, 
 starch derivatives, 
 sugar alcohols; 
 water-soluble polymers 
 colloids comprising nano particle dispersions, 
 dendrimers, 
 polycations, and 
 polyanions. 
 
     
     
         19 . Method in accordance with  claim 18  in which the stabiliser substance is at least one member selected from the group consisting of:
 saccharose epichlorhydrin copolymer, and 
 silica gel coated with polyvinyl pyrrolidone. 
 
     
     
         20 . Method in accordance with  claim 17 , wherein
 the stabiliser substance in the cryopreservation medium has a concentration which is lower than 10%.   
     
     
         21 . Method in accordance with  claim 17 , wherein
 the stabiliser substance in the cryopreservation medium is arranged outside the biological cells.   
     
     
         22 . Method for the cryopreservation of a biological sample, comprising biological cells and a cryopreservation medium, comprising the steps:
 providing a cryopreservation device of  claim 1 ,   providing the biological sample in the pressure vessel of the cryopreservation device, and   cooling of the pressure vessel with lowering of the temperature and increasing of the pressure in the pressure vessel, wherein the biological sample is transferred at least partly into a cryopreserved state in a vitreous phase,   wherein the pressure in the pressure vessel is adjusted using a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous extension medium and communicates with the internal space, and/or a pressure clamp which acts from outside on the vessel wall.   
     
     
         23 . Method in accordance with  claim 12  with the following step:
 storage of the biological sample whilst maintaining the increased pressure. 
 
     
     
         24 . Method for the heating of a biological sample such as to maintain vitality, comprising biological cells and a cryopreservation medium in a frozen state which is arranged in a vitreous phase in a pressure vessel, wherein a temperature of the pressure vessel and of the biological sample is increased and simultaneously an increased pressure above atmospheric pressure is maintained in the pressure vessel. 
     
     
         25 . Method in accordance with  claim 24  in which
 the increased pressure above the atmospheric pressure is maintained in the pressure vessel until the biological sample achieves a transition from the frozen or vitrified state to a liquid state. 
 
     
     
         26 . Method in accordance with  claim 25  in which
 on transition from the frozen or vitrified state to the liquid state the pressure in the pressure vessel is reduced. 
 
     
     
         27 . Method in accordance with  claim 26  in which
 on transition from the frozen or vitrified state to the liquid state the pressure in the pressure vessel is instantaneously reduced. 
 
     
     
         28 . Method in accordance with  claim 26  in which
 the pressure in the pressure vessel is reduced by a contraction of the biological sample at the transition from the frozen or vitrified state to the liquid state. 
 
     
     
         29 . Method in accordance with  claim 24  in which
 the increased pressure is at least one of at least 100 MPa and maximum 300 MPa. 
 
     
     
         30 . Method in accordance with  claim 24  in which
 the cryopreservation medium contains a stabiliser substance which is suitable to stabilise the vitreous phase of the supercooled melt on increasing the temperature of the biological sample, preferably up to the transition. 
 
     
     
         31 . Method of using a stabiliser substance for the cryopreservation of biological samples, including the steps of
 increasing the temperature of a biological sample, comprising biological cells and a cryopreservation medium, and   maintaining a vitreous phase of the biological sample by an effect of the stabiliser substance up to a transition to a liquid state.