Devices and methods for isolating and recovering target cells

Abstract

A cell isolating device and method is provided to concentrate or isolate cells with specific characteristics from a mixture of different cell types. One embodiment may comprise two subtypes of antibodies that are directly conjugated to biotin (Ab b ) and conjugated to a fluorescent molecule (Ab f ). The conjugated antibodies (Ab b +Ab f ) bind to the target cells in a mixed cell suspension. The cell suspension is then passed over an immobilized avidin or streptavidin substrate on a glass microscope slide. The biotinylated target cells adhere to the avidin/streptavidin substrate, while the unbound cells are washed off and collected in a wicking member. Captured cells on the avidin/streptavidin substrate may then be visualized directly using a fluorescent microscope or detected and enumerated via an on-board fluorescent detection device. Additional chemicals and/or physical manipulation may then be applied to the device to release viable target cells for subsequent analysis.

What is claimed:  
     
         1 . A device for recovering a target cell from a cellular matrix, comprising: 
 a housing having at least one wall and a base, said base having a plurality of openings;    a reaction surface positioned proximate to said plurality of openings; and    a fluid absorbing media in communication with said substrate and adjacent to said housing.    
     
     
         2 . The device of  claim 1  wherein said housing further includes at least one fastening member capable of engaging said reaction surface.  
     
     
         3 . The device of  claim 1  wherein said housing and said reaction surface are in spaced relationship.  
     
     
         4 . The device of  claim 3  wherein said reaction surface and said housing are separated by a distance of approximately 2 to 20 microns.  
     
     
         5 . The device of  claim 1  wherein said reaction surface comprises a bioactive coating selected from the group consisting of avidin, streptavidin, avidin derivatives, streptavidin derivatives, and blends thereof.  
     
     
         6 . The device of  claim 5  wherein said bioactive coating has a surface area of approximately 8 cm 2 .  
     
     
         7 . A method of recovering a target cell from a cellular mixture comprising: 
 exposing a cellular mixture to a targeting solution to form a target cell mixture;    delivering said target cell mixture to a reaction surface;    controlling the delivery of said target cell mixture so as to optimize the flow over said reaction surface;    retaining said target cells on said reaction surface;    delivering a releasing solution to said reaction surface; and    collecting said releasing solution, wherein said releasing solution includes said target cells.    
     
     
         8 . A method as set forth in  claim 7 , wherein the controlling of the delivery of the target cell mixture is performed by establishing a predetermined delivery distance for said target cell solution to traverse to said reaction surface.  
     
     
         9 . A method as set forth in  claim 8  wherein the predetermined delivery distance is approximately 2 to 20 microns.  
     
     
         10 . A method as set forth in  claim 8 , wherein the controlling the delivery of the target cell mixture further includes absorbing non-reacting amounts of said target cell solution from said reaction surface at a predetermined rate.  
     
     
         11 . The method of  claim 10  further comprising delivering another aliquot of the target cell releasing solution to said reaction surface, mechanically agitating said target cell releasing solution, and collecting said target cell releasing solution, wherein said target cell releasing solution includes said target cells.  
     
     
         12 . The method of  claim 7  wherein said targeting solution includes a marker compound comprising a binder molecule and a probe.  
     
     
         13 . The method of  claim 12  wherein said binder molecule is selected from the group consisting of antibodies, antigens, glycoproteins, lectins, hormones, cell receptors, vitamins, amino acids, sugars, lipids, fatty acids, liposomes, DNA probes, and RNA probes.  
     
     
         14 . The method of  claim 13  wherein said probe is selected from the group consisting of flurochromes, radiolabels, fluorescent agents, and chromophores.  
     
     
         15 . The method of  claim 7  wherein said reaction surface comprises a bioactive coating selected from the group consisting of avidin, streptavidin, avidin derivatives, streptavidin derivatives, and blends thereof.  
     
     
         16 . The method of  claim 7  wherein said releasing solution is a buffer solution having a pH ranging from about 3.0 to 6.0.  
     
     
         17 . The method of  claim 7  wherein said releasing solution includes a cell releasing agent selected from the group consisting of chymopapain, trypsin, chymotrypsin, V8 protease, Ethylenediaminetetraacetic Acid, cyanogen bromide, or 2-Nitro-5-thiocyanobenzoate.  
     
     
         18 . A method of isolating target cells from a cellular mixture comprising: 
 exposing a cellular mixture to a targeting solution to form a target cell mixture;    delivering said target cell mixture to a reaction surface;    controlling the delivery of said target cell mixture so as to optimize the flow over said reaction surface;    retaining said target cells on said reaction surface;    analyzing said target cells remaining on said reaction surface; and    delivering at least one target cell releasing solution to said reaction surface, and collecting said target cell releasing solution, wherein said target cell releasing solution includes said target cells.    
     
     
         19 . A method as set forth in  claim 18 , wherein the controlling of the delivery of the target cell mixture is performed by establishing a predetermined delivery distance for said target cell solution to traverse to said reaction surface.  
     
     
         20 . A method as set forth in  claim 19  wherein the predetermined delivery distance is approximately 2 to 20 microns.  
     
     
         21 . A method as set forth in  claim 19 , wherein the controlling the delivery of the target cell mixture further includes absorbing non-reacting amounts of said target cell solution from said reaction surface at a predetermined rate.  
     
     
         22 . A method as set forth in  claim 21  further comprising delivering another aliquot of the target cell releasing solution to said reaction surface, mechanically agitating said target cell releasing solution, and collecting said target cell releasing solution, wherein said target cell releasing solution includes said target cells.  
     
     
         23 . The method of  claim 18  wherein said targeting solution includes a marker compound comprising a binder molecule and a probe.  
     
     
         24 . The method of  claim 23  wherein said binder molecule is selected from the group consisting of antibodies, antigens, glycoproteins, lectins, hormones, cell receptors, vitamins, amino acids, sugars, lipids, fatty acids, liposomes, DNA probes, and RNA probes.  
     
     
         25 . The method of  claim 24  wherein said probe is selected from the group consisting of flurochromes, radiolabels, fluorescent agents, and chromophores.  
     
     
         26 . The method of  claim 18  wherein said reaction surface comprises a bioactive coating selected from the group consisting of avidin, streptavidin, avidin derivatives, streptavidin derivatives, and blends thereof.  
     
     
         27 . The method of  claim 18  wherein said target cell releasing solution is a buffer solution having a pH ranging from about 3.0 to 6.0.  
     
     
         28 . The method of  claim 18  wherein said releasing solution includes a cell releasing agent selected from the group consisting of chymopapain, trypsin, chymotrypsin, V8 protease, Ethylenediaminetetraacetic Acid, cyanogen bromide, or 2-Nitro-5-thiocyanobenzoate.  
     
     
         29 . A method of recovering viable target cells from a cellular mixture, comprising: 
 providing an apparatus of  claim 1;     exposing the cellular mixture to a targeting solution to form a target cell mixture;    adding the target cell mixture to the apparatus;    controlling the rate of contact between the reaction surface and the target cell mixture by establishing a predetermined delivery distance for said target cell mixture to traverse to the reaction surface;    analyzing the reaction surface for target cells; and    removing target cells from the reaction surface by delivering a releasing solution to the reaction surface; and    collecting the releasing solution, wherein the releasing solution includes the target cells.