Cell separation using microchannel having patterned posts

Abstract

A micro flow device ( 11, 71 ) for separating or isolating cells from a bodily fluid or other liquid sample uses a flow path where straight-line flow is interrupted by a pattern of transverse posts ( 23, 81 ). The posts are spaced across the width of an expanded collection chamber region ( 17, 75 ) in the flow path, extending between the upper and lower surfaces thereof; they have rectilinear surfaces, being curved in cross-sections, e.g. circular or tear-drop shaped, and are randomly arranged so as to disrupt streamlined flow. The device is oriented so that its lower surface is aligned at about 45° to the horizontal. Sequestering agents, such as Abs, which are attached to surfaces of the collection region via a hydrophilic coating, preferably a permeable hydrogel containing isocyanate moieties, are highly effective in capturing cells or other targeted biomolecules while the remainder of the liquid sample exits horizontally.

1 . A method for separating biomolecules, such as cells, from a sample of bodily fluid or other liquid, which method comprises:
 causing such a sample containing target biomolecules to flow downstream along a flow path in a microflow device from an inlet to an outlet, which flow path comprises a microchannel arrangement that includes a collection region of expanded cross section, while orienting the device so that said flow path collection region is aligned at an angle of about 30° to about 60° to the horizontal,   separating target biomolecules from the flowing sample by
 (a) interrupting straight-line flow of the liquid through said collection region, as a result of blocking such flow with a plurality of separator posts located in said region, which posts are integral with an upper or lower surface of said microchannel and extend therefrom to the opposite surface thereof, said posts extending transverse to said flow path and being located in an irregular pattern that extends laterally across said collection region and prevents straight-line flow and streamlined flow therethrough, and all surfaces of said collection region including said posts having sequestering agents carried thereupon, and 
 (b) capturing target biomolecules found in the flowing liquid sample on surfaces in the collection region by binding the target molecules to the sequestering agents as a result of flow disruption by said irregular posts and force vectors that result from gravity, which vectors are aligned at an acute angle to said lower surface of said collection region, and discharging the remainder of the liquid sample through the outlet. 
   
     
     
         2 . The method according to  claim 1  wherein the sample is fed into a well at the inlet and drawn through said channel and out the outlet by vacuum. 
     
     
         3 . The method according to  claim 1  wherein the sample exits the device through the outlet in a substantially horizontal direction. 
     
     
         4 . The method according to  claim 1  wherein said device includes (a) a body having a flat surface wherein the collection region is formed as a cavity and (b) a flat plate which abuts said flat surface and forms a bottom wall. 
     
     
         5 . The method according to  claim 1  wherein said flat plate is optically transparent and wherein optical detection means which is positioned there adjacent monitors parameters of biomolecules captured in said collection region and provides an output signal corresponding to a monitored parameter. 
     
     
         6 . The method according to  claim 1  wherein said inlet passageway is aligned at an angle of about 40° to 50° to the flow path through the collection region. 
     
     
         7 . The method according to  claim 6  wherein said outlet passageway lies in substantially the same vertical plane as said inlet passageway and is oriented at an angle of about 90° to said inlet passageway in said vertical plane and wherein exit therethrough is in a substantially horizontal direction. 
     
     
         8 . The method according to  claim 1  wherein said sample is caused to flow through said collection region at an average liquid flow rate of about 0.2 to about 1 mm/sec. 
     
     
         9 . The method according to any one of  claims 1  to  8  wherein said surfaces in said collection region have a hydrophilic coating, which coating is a permeable hydrogel formed from an isocyanate-functional prepolymer that is a reaction product of PEG, PPG or a copolymer thereof and a polyisocyanate, and wherein said sequestering agents are indirectly bound to said hydrogel coating by coupling pairs. 
     
     
         10 . The method according to  claim 9  wherein said surfaces in said collection region are coated in situ by supplying an aqueous mixture containing said prepolymer thereto while said prepolymer undergoes final polymerization. 
     
     
         11 . A microflow apparatus for separating biomolecules, such as cells, from a sample of bodily fluid or other liquid, which apparatus comprises:
 a body having a flow path defined therein through which such a sample containing target biomolecules can be caused to flow, the body having an inlet passageway to said flow path, an outlet passageway therefrom, and a microchannel arrangement extending between said inlet and outlet passageways, and   a closure plate,   said microchannel arrangement includes a collection region having upper and lower surfaces, one of which is provided by said closure plate, and a plurality of transverse separator posts,   said posts being integral with one of said upper and lower surfaces of said collection region and extending laterally across said flow path to the other of said surfaces provided by said closure plate, said posts being located in an irregular pattern so as to interrupt straight-line flow and streamlined flow of liquid through said region,   said surfaces of said collection region, including said posts, carrying sequestering agents that will bind with target biomolecules, and   said inlet being aligned at an angle of between about 300 to about 60° to said flow path through said collection region,   whereby a sample can be fed substantially vertically downward through said inlet while said body is aligned with said flow path at about an angle of 30° to 60° to the horizontal and whereby said irregular pattern of said posts and force vectors resultant from gravity cause effective capture of target biomolecules in said collection region, particularly upon the lower surface thereof.   
     
     
         12 . The apparatus according to  claim 11  wherein said body is substantially flat and has a major flat surface in which said flow path is formed as a cavity and a substantially flat base surface in said cavity from which said posts extend, wherein said opposite surface is provided by said closure plate which abuts said major flat surface and closes said flow path, and wherein free ends of said posts are in contact with said opposite surface. 
     
     
         13 . The apparatus according to  claim 11  wherein surfaces of said posts and said collection region are coated with a hydrophilic, permeable coating, which coating is a permeable hydrogel formed from an isocyanate-functional prepolymer that is a reaction product of PEG, PPG or a copolymer thereof and a polyisocyanate, and wherein said sequestering agents are indirectly bound to said hydrogel coating by coupling pairs. 
     
     
         14 . The apparatus according to  claim 11  wherein said outlet passageway lies in substantially the same vertical plane as said inlet passageway and is oriented in said plane at an angle of about 90° to said inlet passageway. 
     
     
         15 . The apparatus according to any one of  claims 11  to  14  wherein said posts have at least about  3  different cross sectional sizes and wherein said posts are aligned substantially perpendicular to said flat base surface. 
     
     
         16 . A microflow apparatus for separating biomolecules, such as cells, from a sample of a bodily fluid or other liquid, which apparatus comprises:
 a body having a flow path defined as a cavity in a flat surface thereof through which such a sample containing target biomolecules can be caused to flow, the flow path having inlet means, outlet means, and a microchannel arrangement extending between said inlet and outlet means, which microchannel arrangement includes a collection region with a plurality of transverse separator posts located in said region, and   closure plate means having a flat surface that abuts said body flat surface and closes said flow path cavity,   said posts being integral with a base surface of said collection region and projecting therefrom so as to extend to the surface of said closure plate means,   said posts being arranged in an irregular pattern extending laterally across said flow path in said collection region so as to interrupt straight-line flow and streamlined flow of liquid through said region, and   all said surfaces of said collection region including said posts being coated with a hydrophilic permeable hydrogel and carrying sequestering agents that will bind with target biomolecules,   whereby disruption of streamlined flow throughout said collection region as a result of said irregular pattern of said posts creates effective capture of target biomolecules on said surfaces in said collection region.   
     
     
         17 . The apparatus according to  claim 16  wherein said posts have at least about 3 different cross sectional sizes and said posts are aligned substantially perpendicular to the base surface of said microchannel. 
     
     
         18 . The apparatus according to  claim 16  or  17  wherein said hydrophilic permeable hydrogel coating is at least about 1 micron thick and is formed from an isocyanate-functional prepolymer that is a reaction product of PEG, PPG or a copolymer thereof and polyisocyanates, and wherein said sequestering agents are covalently bound to isocyanate groups of said hydrogel. 
     
     
         19 . The apparatus according to  claim 16  wherein said sequestering agents on said surfaces in said collection region are coupled to said hydrogel coating through pairs of coupling agents. 
     
     
         20 . The apparatus according to  claim 19  wherein said hydrophilic permeable hydrogel coating is at least about 1 micron thick and is formed from an isocyanate-functional prepolymer that is a reaction product of PEG, PPG or a copolymer thereof and polyisocyanates, and wherein one of said pairs of coupling agents are covalently bound to the isocyanate groups of said hydrogel.