Device for cell separation and analysis and method of using

Abstract

A microflow device 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 which are arranged across the width of a collection region in an irregular or set random pattern so as to disrupt streamlined flow. Sequestering agents, such as Abs, are attached to all surfaces in the collection region via a hydrophilic permeable hydrogel coating. The collection region is formed as a cavity in a body molded from PDMS, which flexible body is sandwiched between a glass slide or comparable flat plate and a rigid top cap plate, both of which are pressed into abutting relation with the PDMS body by a heat-shrunk polymeric sleeve. Following cell separation and washing, cells can be released from the sequestering agents and the device centrifuged to force said cells to collect adjacent the hydrogel-coated slide or plate. Slitting the polymeric sleeve allows the body to then be peeled from the slide or plate, using an integral tab, to expose the separated cells on the top surface thereof for ready microscopic examination.

1 . A microflow device for separating biomolecules, such as cells, from a sample of a bodily fluid or other liquid, which device comprises:
 a body having a flow path formed therein 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 which includes a collection region extending between said inlet and outlet means, which collection region is formed as a cavity in a flat bottom surface of said body and includes a plurality of transverse separator posts that protrude from a base surface of said cavity,   a flat, rigid closure plate having a top surface that is in abutting contact with said flat bottom surface of said body and closes said flow path cavity,   said posts being integral with said base surface of said collection region and extending to the top surface of said closure plate,   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   a polymeric sheet wrap encircling said body and said flat rigid plate and pressing same into surface to surface contact with each other to seal said flow path against leakage,   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 via sequestering agents attached to said surfaces in said collection region, including said top surface of said flat plate, and   whereby removal of said wrap, following separation of target biomolecules from such a sample, allows smooth dissociation of said flat rigid closure plate with said biomolecules exposed on said top surface thereof from said body and permits ready microscopic examination and/or analysis of the separated biomolecules.   
   
   
       2 . The device according to  claim 1  wherein said body is molded from a flexible polymeric material and wherein said wrap encircles said body and said plate throughout at least the entire longitudinal length of said collection region. 
   
   
       3 . The device according to  claim 1  wherein said body is molded from a flexible polymeric material in substantially the shape of a parallelepiped and wherein a flat cap is superimposed atop said body so that said encircling wrap sandwiches said body between said flat cap and flat closure plate. 
   
   
       4 . The device according to  claim 3  wherein said body is molded with a tab extending from one longitudinal end thereof which facilitates separation of said body from said flat closure plate after the separation of target biomolecules has been effected. 
   
   
       5 . The device according to  claim 3  wherein said cap is formed with a pair of openings which extend transversely therethrough, which openings are aligned respectively with said inlet means and said outlet means in said body. 
   
   
       6 . The device according to  claim 5  wherein said encircling wrap is a heat-shrunken sleeve of polymeric material having a pair of spaced apart, generally circular apertures, which apertures are in alignment with said openings in said cap. 
   
   
       7 . The device according to  claim 6  wherein said cap has a width which is less than the width of said body and has longitudinal sides that are beveled. 
   
   
       8 . The device according to  claim 3  wherein said body has a width equal to between about 50% and 80% of the width of said flat closure plate. 
   
   
       9 . The device according to  claim 8  wherein said cap has a width, at its widest dimension, equal to between about 75% and 100% of the width of said body. 
   
   
       10 . The device according to  claim 1  wherein said inlet means and said outlet means comprise passageways of substantially circular cross section, which passageways have axes which are respectively aligned at between 120° and 150° to said flat bottom surface of said body. 
   
   
       11 . The device according to  claim 10  wherein said axes lie in a vertical plane substantially perpendicular to said flat bottom surface of said body and are angularly aligned at between 80° and 100° to each other. 
   
   
       12 . The device according to  claim 10  wherein said inlet passageway is of generally conical shape and wherein a reservoir is received in said inlet passageway which has an interior volume of at least about 0.2 ml and serves as a well from which a liquid sample can be drawn so as to flow through said collection region as a result of the application of vacuum to said outlet passageway. 
   
   
       13 . The device according to  claim 1  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. 
   
   
       14 . The device according to  claim 1  wherein a hydrophilic permeable hydrogel coating at least about 1 micron thick is formed on all said surfaces in said collection region from an isocyanate-functional prepolymer that is a reaction product of PEG, PPG or a copolymer thereof and polyisocyanates, to which hydrogel coating said sequestering agents are directly or indirectly bound. 
   
   
       15 . A method of separating and examining biomolecules, such as cells, from a sample of a bodily fluid or other liquid, which method comprises the steps of:
 a. causing a sample containing target biomolecules to flow along a flow path in a body of a device, the flow path including a microchannel arrangement which includes a collection region formed as a cavity in a flat bottom surface of said body wherein a plurality of transverse separator posts protrude from a base surface of said cavity, 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, said body having inlet means and outlet means connected to said microchannel arrangement,   said device including a flat, rigid closure plate having a top surface that is in abutting contact with said flat bottom surface of said body and closes said flow path cavity, and   said device also having a polymeric sheet wrap encircling said body and said flat rigid plate and pressing same into surface to surface contact with each other to seal said flow path against leakage,   whereby disruption of streamlined flow throughout said collection region occurs as a result of said irregular pattern of said posts and creates effective capture of target biomolecules via sequestering agents attached to said surfaces in said collection region, including said top surface of said flat plate,   b. removing said wrap following separation of target biomolecules from such a sample,   c. smoothly dissociating said flat rigid closure plate, having said biomolecules exposed on said top surface thereof, from said body, and   d. subjecting the separated biomolecules on said closure plate to microscopic examination and/or analysis.   
   
   
       16 . The method according to  claim 15  wherein microchannel arrangement containing said captured biomolecules is washed prior to said dissociation. 
   
   
       17 . The method according to  claim 16  wherein said collection region is treated with a chemical reagent to release said captured biomolecules from said surfaces following washing and prior to said dissociation. 
   
   
       18 . The method according to  claim 17  wherein, following said release, said device is subjected to centrifugal force to cause said released biomolecules to collect upon said closure plate surface prior to said dissociation. 
   
   
       19 . The method according to  claim 15  wherein said body is made of flexible polymeric material and is peeled from said plate using a tab provided at one longitudinal end thereof. 
   
   
       20 . The method according to  claim 15  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.