Method for Freezing and Storing Cells

Abstract

The invention relates to a method for freezing and storing cells in a vessel. Said method comprises the following steps: a) the cells are frozen to a storage temperature; and b) the cells are stored at said storage temperature. The inventive method is characterized in that the cells are stored in a gas-tightly sealed manner and/or at a partial oxygen pressure which is lower than the atmospheric pressure. The invention further relates to frozen cells in freezing medium in a vessel which is gas-tightly sealed and/or in which the partial oxygen pressure is lower than the atmospheric pressure.

1 - 21 . (canceled)  
   
   
       22 . A method of freezing and storing cells in a container comprising the steps of: 
 a) freezing a cell suspension comprising said cells down to a storage temperature;    b) storing said cell suspension at said storage temperature;    wherein the storing is performed under gas tight sealing and/or under reduced oxygen partial pressure as compared to atmospheric pressure.    
   
   
       23 . The method of claim  1 , wherein the reduction of the oxygen partial pressure is done by reducing the pressure and/or by gassing with a protective gas.  
   
   
       24 . The method of claim  1 , wherein the reduction of the oxygen partial pressure is done prior to freezing.  
   
   
       25 . The method of claim  1 , wherein the oxygen partial pressure in the container is reduced to between about 52.5 mbar and 0.21 mbar.  
   
   
       26 . The method of  claim 25 , wherein the oxygen partial pressure in the container is reduced to between about 26 mbar to 0.21 mbar.  
   
   
       27 . The method of  claim 26 , wherein the oxygen partial pressure in the container is reduced to between about 13 mbar to 0.21 mbar.  
   
   
       28 . The method of claim  1 , wherein the gas tight sealing of the container is made by shrink-wrapping in a gas tight film tube or by applying a gas tight adhesive film.  
   
   
       29 . The method of claim  1 , wherein a residual volume of air remains in the container after gas tight sealing of the container, wherein said residual volume is up to about 17.5 times the volume of the cell suspension in the container.  
   
   
       30 . The method of  claim 29 , wherein said residual volume of air is up to about 15 times the volume of the cell suspension in the container.  
   
   
       31 . The method of  claim 30 , wherein said residual volume of air is up to about 7 times the volume of the cell suspension in the container.  
   
   
       32 . The method of  claim 31 , wherein said residual volume of air is equal to the volume of the cell suspension in the container.  
   
   
       33 . The method of claim  2 , wherein the protective gas is selected from a group consisting of nitrogen, helium, and argon.  
   
   
       34 . The method of claim  1 , wherein the cells are of animal or of human origin.  
   
   
       35 . The method of claim  1 , wherein said freezing of said cell suspension down to said storage temperature is with a cooling rate of at least 20° C. per minute.  
   
   
       36 . The method of  claim 35 , wherein said freezing of said cell suspension down to said storage temperature is with a cooling rate of at least 40° C. per minute.  
   
   
       37 . The method of  claim 36 , wherein said freezing of said cell suspension down to said storage temperature is with a cooling rate of at least 64.5° C. per minute.  
   
   
       38 . The method of  claim 37 , wherein said freezing of said cell suspension down to said storage temperature is with a cooling rate of at least 80° C. per minute.  
   
   
       39 . The method of claim  1 , characterized in that the storage temperature is not lower than about −85° C.  
   
   
       40 . The method of  claim 39 , characterized in that the storage temperature is not lower than about −65° C.  
   
   
       41 . The method of  claim 40 , characterized in that the storage temperature is not lower than about −60° C.  
   
   
       42 . The method of  claim 41 , characterized in that the storage temperature is not lower than about −50° C.  
   
   
       43 . The method of claim  1 , wherein said freezing is achieved by contact of at least one surface of the container with a material of suitable thermal conductivity that has been pre-cooled to a suitable temperature.  
   
   
       44 . The method of  claim 43 , wherein said material is not metal.  
   
   
       45 . The method of  claim 43 , wherein said material is pre-cooled to a temperature between about −90° C. and −50° C.  
   
   
       46 . The method of  claim 43 , wherein said material is pre-cooled to a temperature between about −85° C. and −65° C.  
   
   
       47 . The method of claim  1 , wherein said storing of said cells is performed without the use of liquid nitrogen.  
   
   
       48 . The method of claim  1 , wherein the container is selected from the group consisting of 6-well, 12-well, 24-well, 48-well, 96-well, 384-well and 1,536-well plates.  
   
   
       49 . A composition comprising frozen cells in freezing medium in a container under gas tight sealing and/or under an oxygen partial pressure that has been reduced in comparison to the atmospheric pressure.  
   
   
       50 . The composition of  claim 49 , wherein the oxygen partial pressure in the container is between about 52.5 mbar and 0.21 mbar.  
   
   
       51 . The composition of  claim 49 , wherein the oxygen partial pressure in the container is between about 26 mbar to 0.21 mbar.  
   
   
       52 . The composition of  claim 49 , wherein the oxygen partial pressure in the container is between about 13 mbar to 0.21 mbar.  
   
   
       53 . The composition of  claim 49 , wherein the cells can be used directly after thawing in freezing medium in the container in a cell-based assay without passaging.  
   
   
       54 . The composition of  claim 53 , wherein the cell-based assay is done in the form of a high-throughput-screening (HTS).  
   
   
       55 . The composition of  claim 49 , wherein after storage at a storage temperature lower than or equal to about −65° C. for at least one month, said frozen cells have a vitality of above 70% of the vitality of cells of a non-frozen control container.  
   
   
       56 . The composition of  claim 55 , wherein said frozen cells have a vitality of above 80% of the vitality of cells of a non-frozen control container.  
   
   
       57 . The composition of  claim 56 , wherein said frozen cells have a vitality of above 90% of the vitality of cells of a non-frozen control container.  
   
   
       58 . The composition of  claim 55 , wherein said storage at said storage temperature is for at least 2 months.  
   
   
       59 . The composition of  claim 55 , wherein said storage at said storage temperature is for at least 4 months.  
   
   
       60 . The composition of  claim 55 , wherein said storage at said storage temperature is for at least 12 months.