Method for selectively separating blood cells by using lectin

Abstract

The present invention provides a method for selective and high-yield separation, concentration, and recovery of desired cells from a blood sample. The method of the present invention is characterized in that the blood sample is caused to interact with lectins under conditions in which the cell membranes are inactive and cell-lectin complexes/non-complexes are formed, the sample containing these cell-lectin complexes/non-complexes is incubated together with a substrate, the surface of which is covered with polymers having carbohydrate chains which are specifically recognized by the lectins, and the cells are immobilized on the surface of the substrate via the lectins, and subsequently, the liquid layer and the solid phase are separated, and the desired blood cells are recovered from the liquid phase and/or the solid phase, and these lectins are present in such an amount that although the cells to be recovered from the solid phase bind to the solid phase with the polymer, the cells to be recovered from the liquid phase do not bind to the solid phase with the polymer.

1 . An apparatus for separating and recovering hematopoietic cells and/or erythroblasts from a blood sample containing differentiated mature cells, immature hematopoietic cells and erythroblasts comprising: 
 (1) a device in which said sample interacts with lectins to form cell-lectin complexes/non-complexes under conditions in which the cells are rendered inactive,    (2) a substrate on which incubation of said sample containing said cell-lectin complexes/non-complexes is carried out under said conditions, said substrate having a surface that is covered with a synthetic glycoconjugate polymer having carbohydrate moieties that are specifically recognized by said lectins, said cells being immobilized on the surface of said substrate via said lectins, and    (3) a device that separates a liquid phase from a solid phase and in which desired blood cells are recovered from said liquid phase and/or said solid phase, wherein said lectins are present in an amount such that they bind to the cells recovered from said solid phase and immobilize these cells on the surface of the substrate, but do not immobilize the cells recovered from said liquid phase to the surface of said substrate.    
     
     
         2 . An apparatus according to  claim 1  further comprising a device in which accelerating and stabilizing the immobilization of cells is carried out by centrifuging said substrate and cells simultaneously prior to or after the incubation or in which stabilizing the immobilization of cells is carried out by centrifuging the substrate on which the cells are immobilized during recovery of the desired blood cells.  
     
     
         3 . An apparatus according to  claim 1 , wherein said device in which said sample interacts with said lectins operates under said conditions under which said cells are rendered inactive comprising: low temperature conditions of 0° C. or above but less than 37° C., or conditions in which a pharmaceutical agent is added which suspends cellular respiration.  
     
     
         4 . An apparatus according to  claim 1 , wherein the concentration of said lectins is within a range of 20 mg/ml or less per cell.  
     
     
         5 . An apparatus according to  claim 1 , wherein said device in which said sample interacts with said lectins is set to permit said interaction for a time period within a range of 0 to 120 minutes, and said incubation on said substrate is carried out for a time period in a range of 10 to 120 minutes.  
     
     
         6 . An apparatus according to  claim 1 , wherein the substrate is selected from the group consisting of dishes, flasks, plates, cuvettes, films, fibers, or beads made of glass, polystyrene, polycarbonate, polysulfone, polyurethane, or vinyl copolymer, and that carbohydrate components capable of bonding with said lectins are treated on the substrate.  
     
     
         7 . An apparatus for separating and recovering hematopoietic cells and/or erythroblasts from a blood sample containing differentiated mature cells, immature hematopoietic cells and erythroblasts comprising: 
 (1) means for causing said sample to interact with lectins to form cell-lectin complexes/non-complexes under conditions in which the cells are rendered inactive,    (2) a substrate on which incubation of said sample containing said cell lectin complexes/noncomplexes is carried out under said conditions, said substrate having a surface that is covered with a synthetic glycoconjugate polymer having carbohydrate moieties that are specifically recognized by said lectins, said cells being immobilized on the surface of said substrate via said lectins, and    (3) means for separating a liquid phase from a solid phase and recovering desired blood cells from said liquid phase and/or said solid phase;    wherein said lectins are present in an amount such that they bind to the cells recovered from said solid phase and immobilize these cells on the surface of the substrate, but do not immobilize the cells recovered from said liquid phase to the surface of said substrate.    
     
     
         8 . An apparatus according to  claim 1  wherein said glycoconjugate polymer is selected from the group consisting of: 
 Poly(N-p-vinyl benzyl-[O-β-D-galactopyranosyl-(1→4)-D-gluconamide]) (referred to as PVLA);    Poly(N-p-vinyl benzyl-[O-α-D-glucopyranosyl-(1→4)-D-gluconamide]) (referred to as PVMA);    Poly(N-p-vinyl benzyl-[O-β-D-mannopyranosyl-(1→4)-D-mannamide]) (referred to as PVMan);    Poly(N-p-vinyl benzyl-[O-α-D-galactopyranosyl-(1α→6)-D-gluconamide]) (referred to as PVMeA);    Poly(N-p-vinyl benzyl-[O-6-carboxymethyl-β-D-galactopyranosyl-(1→4)—O-D-6-carboxymethyl-gluconamide]) (referred to as PVLACOOH);    Poly(3-O-4′-vinyl benzyl-D-glucose) (referred to as PVG);    Poly(N-p-vinyl benzyl-[O-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-gluconamide]); poly(N-p-vinyl benzyl-[O-D-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-gluconamide]); and mixtures thereof (all referred to as PVGlcNac);    Poly(N-p-vinyl benzyl-[O-β-D-glucopyranosyl-(1→3)-D-gluconamide]) (referred to as PVLam); and copolymers and combinations thereof.    
     
     
         9 . An apparatus according to  claim 1  wherein said glycoconjugate polymer is selected from the group consisting of: 
 Poly(N-p-vinyl benzyl-[O-β-D-galactopyranosyl-(1→4)-D-gluconamide]) having β-galactose residues obtained by polymerizing monomers synthesized from p-amino methyl styrene and lactose (referred to as PVLA);    Poly(N-p-vinyl benzyl-[O-α-D-glucopyranosyl-(1→4)-D-gluconamide]) having glucose residues obtained by polymerizing monomers synthesized from p-amino methyl styrene and maltose (referred to as PVMA);    Poly(N-p-vinyl benzyl-[O-β-D-mannopyranosyl-(1→4)-D-mannamide]) having mannose residues obtained by polymerizing monomers synthesized from p-amino methyl styrene and mannobiose (referred to as PVMan);    Poly(N-p-vinyl benzyl-[O-α-D-galactopyranosyl-(1α→6)-D-gluconamide]) having α-galactose residues obtained by the polymerization of monomers synthesized from p-amino methyl styrene and O-α-D-galactopyranosyl-(1→6)-D-glucose (referred to as PVMeA);    Poly(N-p-vinyl benzyl-[0-6-carboxymethyl-β-D-galactopyranosyl-(1→4)-O-D-6-carboxymethyl-gluconamide]) having carboxymethylated-β-galactose residues obtained by the carboxymethylation of PVLA which is obtained by the polymerization of monomers synthesized from p-amino methyl styrene and lactose (referred to as PVLACOOH);    Poly(3-O-4′-vinyl benzyl-D-glucose) having glucose residues obtained by the polymerization of monomers synthesized from p-chloromethyl styrene and glucose (referred to as PVG);    Poly(N-p-vinyl benzyl-[O-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-gluconamide]); poly(N-p-vinyl benzyl-[O-D-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-gluconamide]); and mixtures thereof having N-acetylglucosamine residues obtained by the polymerization of monomers synthesized from p-chloromethyl styrene and N-acetylglucosamine (all referred to as PVGlcNac);    Poly(N-p-vinyl benzyl-[O-β-D-glucopyranosyl-(1→3)-D-gluconamide]) having β1→3 glucose residues obtained by polymerizing monomers synthesized from p-amino methyl styrene and laminaribiose (referred to as PVLam); and copolymers and combinations thereof.    
     
     
         10 . An apparatus according to  claim 9  wherein said glycoconjugate polymer is a copolymer formed by polymerizing monomers one of which comprises an azide group.  
     
     
         11 . An apparatus according to  claim 10  wherein said copolymer is selected from the group consisting of: poly(3-azide styrene-co-{N-p-vinyl benzyl-[O-β-D-galactopyranosyl-(1→4)-D-gluconamide]}) (which is referred to as AZ-PVLA); poly(3-azide styrene-co-{N-p-vinyl benzyl-[O-α-D-glucopyranosyl-(1→4)-D-gluconamide]}) (which is referred to as AZ-PVMA); and combinations thereof.  
     
     
         12 . A device for separating and recovering hematopoietic cells and/or erythroblasts from a blood sample containing differentiated mature cells, immature hematopoietic cells and erythroblasts, comprising 
 cell-lectin complexes formed by interaction of lectins and said sample in which the cells are rendered inactive, and    a substrate having a surface that is covered with a synthetic glycoconjugate polymer having carbohydrate moieties, said cell-lectin complexes being immobilized to said carbohydrate moieties via said lectins,    wherein said lectins are present in an amount such that they bind to cells recovered from a solid phase that has been separated from a liquid phase, and immobilize these cells on the surface of the substrate, but do not immobilize the cells recovered from said liquid phase to the surface of said substrate.