Platelet-Targeted Microfluidic Isolation of Cells

Abstract

Methods and systems for isolating platelet-associated nucleated target cells, e.g., such as circulating epithelial cells, circulating tumor cells (CTCs), circulating endothelial cells (CECs), circulating stem cells (CSCs), neutrophils, and macrophages, from sample fluids, e.g., biological fluids, such as blood, bone marrow, plural effusions, and ascites fluid, are described. The methods include obtaining a cell capture chamber including a plurality of binding moieties bound to one or more walls of the chamber, wherein the binding moieties specifically bind to platelets; flowing the sample fluid through the cell capture chamber under conditions that allow the binding moieties to bind to any platelet-associated nucleated target cells in the sample to form complexes; and separating and collecting platelet-associated nucleated target cells from the complexes.

1 - 22 . (canceled) 
     
     
         23 . A two-stage microfluidic system for isolating platelet-associated nucleated target cells from a sample fluid comprising:
 a first chamber comprising
 a microchannel having an inlet, a waste outlet, a product outlet, and 
 an array of microposts arranged between the inlet and the outlets, wherein the microposts are arranged in rows and spaced apart by a distance that enables red blood cells and unbound platelets to flow through the device to a waste outlet and to cause platelet-associated nucleated target cells to be laterally displaced by the array of microposts to a product outlet, wherein the microposts in each subsequent row are offset laterally from microposts in a previous row by a distance less than the spacing between the microposts within the row; 
   a second chamber comprising
 a microchannel having an inlet and an outlet, wherein fluid flows from the inlet to the outlet through the microchannel, and binding moieties fixed to at least one internal surface of the microchannel, wherein the binding moieties specifically bind to platelets; and 
 a fluid connection between the product outlet of the first chamber and the inlet of the second chamber. 
   
     
     
         24 . The system of  claim 23 , wherein the microposts are spaced apart within a row by a distance of about 30 microns to about 60 microns, and subsequent rows are spaced apart from a previous row by a distance of about 5 microns to about 15 microns. 
     
     
         25 . The system of  claim 23 , wherein the first chamber and the second chamber are both located on a single substrate. 
     
     
         26 . The system of  claim 23 , wherein the first chamber and the second chamber are located on separate substrates and are in fluid connection via the conduit. 
     
     
         27 . (canceled) 
     
     
         28 . The system of  claim 23 , wherein the binding moieties are bound to nanostructures that comprise a first member of a binding pair, wherein one or more internal surfaces of the second chamber are bound to a layer of gelatin functionalized with a plurality of second members of the binding pair, and wherein the nanostructures are bound to a top layer of the gelatin by a binding interaction of the first and second members of the binding pair. 
     
     
         29 . The system of  claim 23 , wherein the binding moieties comprise antibodies that bind specifically to platelets. 
     
     
         30 . A microfluidic system for isolating platelet-associated nucleated target cells from a blood sample, the system comprising:
 a blood debulking chamber configured to remove a number of red blood cells and unbound platelets from the blood sample;   a cell capture chamber comprising
 a microchannel having an inlet and an outlet, wherein sample fluid flows from the inlet to the outlet through the microchannel, and binding moieties fixed to at least one internal surface of the microchannel, wherein the binding moieties specifically bind to platelets; and 
 a fluid connection between the product outlet of the blood debulking chamber and the inlet of the cell capture chamber. 
   
     
     
         31 . The system of  claim 30 , wherein the blood debulking chamber comprises curved microfluidic channels to differentially focus and sort cells based on their sizes by centrifugal or inertial forces. 
     
     
         32 . The system of  claim 30 , wherein the blood debulking chamber comprises a hydrophoretic filtration system configured to sort cells of different sizes. 
     
     
         33 . The system of  claim 30 , wherein the blood debulking chamber comprises an acoustic standing wave system configured to sort cells of different sizes. 
     
     
         34 . The system of  claim 30 , wherein the cell capture chamber further comprises a plurality of grooves defined in and arranged on an internal surface of one or more walls, floor, and ceiling of the microchannel to create microvortices within the sample fluid. 
     
     
         35 . The system of  claim 30 , wherein the blood debulking chamber comprises
 a microchannel having an inlet, a waste outlet, a product outlet, and   an array of microposts arranged between the inlet and the outlets, wherein the microposts are arranged in rows and spaced apart by a distance that enables red blood cells and unbound platelets to flow through the device to a waste outlet and to cause platelet-associated nucleated target cells to be laterally displaced by the array of microposts to a product outlet, wherein the microposts in each subsequent row are offset laterally from microposts in a previous row by a distance less than the spacing between the microposts within the row.   
     
     
         36 . The system of  claim 34 , wherein the microposts are spaced apart within a row by a distance of about 30 microns to about 60 microns and subsequent rows are spaced apart from a previous row by a distance of about 5 microns to about 15 microns. 
     
     
         37 . The system of  claim 30 , wherein the blood debulking chamber and the cell capture chamber are both located on a single substrate. 
     
     
         38 . The system of  claim 30 , wherein the blood debulking chamber and the cell capture chamber are located on separate substrates and are in fluid connection via a conduit. 
     
     
         39 . The system of  claim 30 , wherein the binding moieties are bound to nanostructures that comprise a first member of a binding pair, wherein one or more internal surfaces of the second chamber are bound to a layer of gelatin functionalized with a plurality of second members of the binding pair, and wherein the nanostructures are bound to a top layer of the gelatin by a binding interaction of the first and second members of the binding pair. 
     
     
         40 . The system of  claim 30 , wherein the binding moieties comprise antibodies that bind specifically to platelets.