Methods of cell separation

Abstract

The present invention relates to the use of a combination of: (i) a macromolecular erythrocyte sedimentation enhancer, and (ii) dimethyl sulphoxide (DMSO), dimethylglycine (DMG) and/or valine; to recover non-erythrocyte blood cells from a blood cell-containing sample and/or to prime non-erythrocyte blood cells to protect their integrity in subsequent cryopreservation step(s).

1 . A method for priming non-erythrocyte blood cells for cryopreservation, said method comprising contacting the cells with DMSO, DMG and/or valine, wherein when the cells are contacted with the DMSO, DMG and/or valine, the concentration of DMSO, DMG and/or valine does not exceed 5% v/v. 
     
     
         2 . A method according to  claim 1 , wherein the non-erythrocyte blood cells are present in the form of a non-erythrocyte blood cell fraction that has already undergone a treatment(s) to remove erythrocytes. 
     
     
         3 . A method according to  claim 1 , wherein the non-erythrocyte blood cells are present in the form of a blood cell-containing sample which comprises said non-erythrocyte blood cells. 
     
     
         4 . A method according to  claim 3 , wherein the sample is whole blood. 
     
     
         5 . A method according to  claim 1 , wherein the non-erythrocyte blood cells comprise white blood cells. 
     
     
         6 . A method according to  claim 1 , wherein the method is for increasing the proportion of viable white blood cells recovered following a subsequent cryopreservation. 
     
     
         7 . A method according to  claim 1 , wherein the non-erythrocyte blood cells are taken from a mammal. 
     
     
         8 . A method according to  claim 1 , wherein the non-erythrocyte blood cells are taken from a human. 
     
     
         9 . A method according to  claim 1 , which comprises contacting the non-erythrocyte blood cells with a combination of:
 (i) a macromolecular erythrocyte sedimentation enhancer, and   (ii) the DMSO, DMG and/or valine;   wherein when the blood cell-containing sample is contacted with components (i) and (ii), the concentration of component (ii) does not exceed 5% v/v.   
     
     
         10 . A method for priming a non-erythrocyte blood cell fraction for cryopreservation, said method comprising contacting the cell fraction with a combination of:
 (i) a macromolecular erythrocyte sedimentation enhancer, and   (ii) DMSO, DMG and/or valine;   wherein when the blood cell-containing sample is contacted with components (i) and (ii), the concentration of component (ii) does not exceed 5% v/v.   
     
     
         11 . A method according to  claim 10 , comprising contacting the cell fraction with a composition comprising components (i) and (ii), wherein the cell fraction and the composition are mixed at a ratio of 10:1 to 1:10, preferably 5:1 to 1:5, more preferably 2:1 to 1:2. 
     
     
         12 . A method according to  claim 9 , comprising contacting the non-erythrocyte blood cells with a composition comprising components (i) and (ii), wherein the composition comprises 0.1-10% w/v of component (i) and 0.1-10% v/v of component (ii). 
     
     
         13 . A method according to  claim 9 , wherein, once the non-erythrocyte blood cells have been contacted with components (i) and (ii), the concentration of component (i) is 0.25 to 5% w/v, and the concentration of component (ii) is 0.25 to 5% v/v. 
     
     
         14 . A method according to  claim 9 , wherein component (i) is dextran and component (ii) is DMSO. 
     
     
         15 . A method according to  claim 9 , wherein component (i) is dextran having a molecular weight of at least 50 kDa. 
     
     
         16 . A method according to  claim 9 , wherein component (i) is dextran 500 and component (ii) is DMSO. 
     
     
         17 . A method for preparing non-erythrocyte blood cells for cryopreservation, which method comprises (a) a method as defined in  claim 1 , and (b) adding a cryoprotectant to the thus obtained non-erythrocyte blood cells. 
     
     
         18 . A method for the cryopreservation of non-erythrocyte blood cells, which method comprises (a) a method as defined in  claim 1 , (b) adding a cryoprotectant to the thus obtained non-erythrocyte blood cells, and (c) cryopreserving the non-erythrocyte blood cells. 
     
     
         19 . A method for the cryopreservation and subsequent recovery of non-erythrocyte blood cells, which method comprises (a) a method as defined in  claim 1 , (b) adding a cryoprotectant to the thus obtained non-erythrocyte blood cells, (c) cryopreserving the non-erythrocyte blood cells, and (d) thawing the non-erythrocyte blood cells. 
     
     
         20 . A method according to  claim 17 , wherein the cryoprotectant comprises DMSO. 
     
     
         21 . A composition comprising (i) a macromolecular erythrocyte sedimentation enhancer, and (ii) DMSO, DMG and/or valine, which composition is suitable for use in recovering non-erythrocyte blood cells from a blood cell-containing sample and/or priming non-erythrocyte blood cells to protect their integrity in subsequent cryopreservation and thawing steps, wherein if component (ii) is DMSO, then the concentration of DMSO in the composition is 10% v/v or less. 
     
     
         22 . A composition according to  claim 21 , wherein component (i) is dextran and component (ii) is DMSO. 
     
     
         23 . A composition according to  claim 21 , wherein component (i) is dextran having a molecular weight of at least 50 kDa. 
     
     
         24 . A composition according to  claim 21 , wherein component (i) is dextran 500 and component (ii) is DMSO. 
     
     
         25 . A composition according to  claim 21 , wherein the concentration of component (i) is 1-5% w/v and the concentration of component (ii) is 1-5% v/v. 
     
     
         26 . An apparatus comprising a composition as defined in  claim 21 , which apparatus is a bottle, a blood bag, a pre-filled syringe for injection into a blood bag, or a kit comprising a composition as defined in  claim 21  and a blood collection vessel.