Patent Application: US-201214124900-A

Abstract:
a device and method are disclosed to enable fluid manipulations without disturbing or removing particles or cells . by engineering assay compartment geometries to take advantage of fluid mechanical effects , the flow velocity may be diminished around particles or cells . this technology may be used to process particles or cells of various kinds that are suspended in liquids or gels , and is suitable for use in high throughput screening .

Description:
various terms are used herein to refer to aspects of the present invention . to aid in the clarification of the description of the components of this invention , the following definitions are provided . flow velocity is used herein to describe the velocity of an element of fluid at a given position and time . the flow velocity u of a fluid is a vector field : which gives the velocity of an element of fluid at a 3 - d position vector x and a time t . sheer force is used herein to describe a force vector that is perpendicular to the surface normal vector of the fluid element . micro - structures is used herein to describe features having at least one height , width , or length geometry in the range between 100 nanometers and 100 microns . such features may include , but are not limited to , posts ( cylindrical , elliptical , rectangular , polygonal cross - section ), wells ( cylindrical , elliptical , rectangular , polygonal cross - section ), walls , baffles , honeycombs , domes , cones , and pyramids . macro - structures is used herein to describe features having at least one height , width or length geometry in the range between 0 . 10 mm and 10 mm . such features may include , but are not limited to , posts ( cylindrical , elliptical , rectangular , polygonal cross - section ), wells ( cylindrical , elliptical , rectangular , polygonal cross - section ), walls , baffles , honeycombs , domes , cones , and pyramids . compartmentalization is used herein to describe a physical boundary between regions . patterned structures is used herein to indicate macro - or micro - structures arranged in a specific pattern , such as rectangular array ( 2 - d ), close - packed array ( 2 - d ), linear array ( 1 - d ), or randomized cluster with a specific range of spacing , such that the pattern has the desired fluid dynamic effects . the lowest surface of a well array is inside the wells . the lowest surface of a post array is in between the posts . wells is used to describe compartments within plates ( such as , but not limited to 6 -, 12 -, 24 -, 96 -, 384 - or 1536 - format plates ), slides , petri dishes or assay dishes which can contain fluid and have a physical boundary between nearby compartments . these wells may or may not include patterned structures . the invention will now be described in detail with reference to a few preferred embodiments , as illustrated in the accompanying drawings . in describing the preferred embodiments , specific details are set forth in order to provide a thorough understanding of the invention . however , it will be apparent to one skilled in the art that the invention may be practiced without some or all of these specific details . in other instances , well - known features and / or process steps have not been described in detail so as not to unnecessarily obscure the invention . in addition , like or identical reference numerals are used to identify common or similar structural elements . the devices and methods described herein exploit the fluid mechanical properties of patterned micro - or macro - structures to diminish movement and loss of particles or cells that have settled to the lowest surface of the patterned structures . while some or all of the particles or cells may in some cases adhere to the surface of the patterned structure , this device and method is particularly advantageous for particles or cells that remain non - adherent . in traditional assay devices , non - adherent cells are at risk of loss where no means are provided to protect the non - adherent cells from flow . while liquid is being dispensed inside a well , a positive pressure is created between the orifice of the pipette tip and the distal areas of the well . this pressure is equilibrated by flow as the fluid moves out to the distal areas . the pressure difference between two arbitrary points in the patterned structure region ( referred to as region r2 below ) has the potential to produce flow along the lowest surface of the microstructures . this flow is diminished by increasing the resistance to flow of the patterned structures . the resistance to flow at the lowest surface increases with decreasing spacing of pattern elements , increasing the height of pattern elements , and increasing fluid viscosity ; the flow velocity at the lowest surface decreases with decreasing dispensing flow rate . the device design and method parameters must take into account the desired particle and washing liquid properties as well as dispensing rate constraints for optimal performance . a shelf region ( referred to as region r1 below ) may be included to deflect the flow from the pipette before it reaches the patterned structure region . by including a shelf region , the flow velocity vectors will be perpendicular to the surface normal vector of the bottom surface of the well . although not necessary , this condition is optimal for diminishing flow velocity at the lowest surface of the patterned structures where the particles or cells reside . biological cells are denser than cell culture media and thus settle out of suspension . however , their density varies , and with that the rate of settling . the drag on a particle or cell increases with increasing liquid density and increasing size . the device design and method parameters must take into account the properties of the particles or cells and the washing liquid for optimal performance . in the instant invention , an assay vessel contains one or more assay compartments or wells ( e . g ., each well in a 96 - well plate is an assay compartment ), and each assay compartment has on its bottom surface one or more patterned structures , and optionally one or more shelves . for the purposes of illustrating the principles of the invention , a representative embodiment of a 384 - well microtiter plate format ( 11 ) with one possible design including a patterned structure is shown in fig1 . the bottom of the well ( 12 ) includes two regions : r1 , which generally corresponds to a shelf ( 13 ), and r2 , which generally corresponds to a recessed portion ( 14 ) of the well ( 12 ) having patterned structures such as posts ( 15 ). r2 is recessed relative to r1 and the tops of the posts ( 15 ) may or may not be level with the shelf ( 13 ). the region r1 may be a wide variety of different sizes and may also take various shapes and relationships relative to r2 . a representative embodiment may contain one or more shelves ( 13 ) at the bottom of the well ( 12 ), where the shelf or shelves ( 13 ) are elevated above the bottom surface of the recessed portion ( 14 ) of the well ( 12 ), where the shelf or shelves ( 13 ) are connected to the sidewall of the recessed portion ( 14 ) of the well ( 12 ), where the shelf or shelves ( 13 ) divide the recessed portion ( 14 ) of the well ( 12 ) into two or more segments , or where the shelf or shelves are not connected to the sidewall of the recessed portion ( 14 ) of the well ( 12 ). in one embodiment , the ratio of the area divided between the shelf or shelves and the patterned structures is in the range of 1 : 100 , 000 to 100 , 000 : 1 . in another embodiment , the ratio of the area divided between the shelf or shelves and the patterned structures is in the range of 1 : 100 to 100 : 1 . in still another embodiment , the ratio of the area divided between the shelf or shelves and the patterned structures is in the range of 1 : 20 to 20 : 1 . it is understood that fig1 is a structure shown for representative purposes only , and that the patterned structures may exist as a wide variety of number and shapes . furthermore , r1 or the shelf ( 13 ) may in some cases be eliminated , and the benefits of the current invention gained from the patterned structures in r2 alone . fig2 shows ten selected embodiments of the regions r1 ( 23 ) and r2 ( 24 ) from fig1 with various patterned structures ( 25 ). patterned structures ( 25 ) can be posts as in fig2 a , 2 c , 2 d , or 2 e ; linear baffles / walls as in fig2 f , 2 g , or 2 h ; intersecting walls as in fig2 i ; or curved baffles / walls as in fig2 j . region r1 , which includes the shelf , can also take various shapes and relationships relative to r2 . region r1 can isolate region r2 to a corner as in fig2 a , 2 b , 2 g , 2 h , 2 i , and 2 j ; divide the well in half as in fig2 e and 2f ; be centrally located as in figure c 23 ; or alternatively the patterned structures in the region r2 can be sufficient to obviate the need for a separate region r1 as in fig2 d . alternatively , region r2 may be shaped such that the need for additional patterned structures within the region r2 is unnecessary as in fig2 b . it is understood that these geometries are representational only , and that the design of r1 and r2 may have a wide variety of different geometries . in one embodiment , the macro and micro structures within the recessed portion ( s ) of the well occur as arrays in the forms of linearly or offset arranged posts , linear baffles or walls , intersecting walls , curved baffles or walls , or with microwells or holes . in one embodiment , the macro or micro structures within the recessed portion ( s ) of the well are comprised of an aspect ratio of height to width within the range of 1 : 1000 to 1000 : 1 . in another embodiment , the macro or micro structures within the recessed portion ( s ) of the well are comprised of an aspect ratio of height to width within the range of 1 : 100 to 100 : 1 . in still another embodiment , the macro or micro structures within the recessed portion ( s ) of the well are comprised of an aspect ratio of height to width within the range of 1 : 10 to 10 : 1 . in still yet another embodiment , the macro or micro structures within the recessed portion ( s ) of the well are comprised of an aspect ratio of height to width within the range of 1 : 3 to 3 : 1 . fig3 shows a view of three selected formats which patterned macrostructures can reside upon . cell biology experiments commonly utilize 384 - well microtiter plate ( 31 ), petri dish ( 32 ) and slide ( 33 ) formats . it is understood that these formats are representational only , and that the format upon which the features are patterned may have a wide variety of different geometries . in one embodiment , the patterned microstructures are contained within a well . it is understood that the patterned macrostructures may also be addressed in bulk without individual compartmentalization of patterned macrostructures . it is understood that the patterned features may be macrostructures or microstructures . it is understood that compartmentalization is not limited to a well and may include , but is not limited to , other embodiments such as the use of a droplet of liquid restrained by a hydrophobic barrier . in one embodiment , the well ( s ) range in size from those within a standard 3456 - well plate ( 0 . 5 - 2 mm length / width / diameter by 3 - 4 mm height ) to those within a standard bioassay dish ( 200 - 250 mm length / width by 20 - 30 mm height ). in another embodiment , the well ( s ) range in size from those within a standard 384 - well plate ( 3 - 4 mm length / width / diameter by 11 - 12 mm height ) to those within a standard petri dish ( 30 - 150 mm diameter by 12 - 20 mm height ). in another embodiment , the well ( s ) are square or round and are sized equivalent to those within a standard 384 - well plate ( 3 - 4 mm length / width / diameter by 11 - 12 mm height ). fig4 is a schematic demonstrating liquid handling in a well ( 41 ) with patterned structures ( 46 ). fig4 a shows a liquid dispensing pipette tip ( 42 ) carrying particles ( 44 ) in suspension in a liquid ( 43 ) when lowered within close proximity of region r2 ( 45 ) which contains patterned structures ( 46 ). it is understood that the dispensing tip ( 42 ) may have a wide variety of sizes and locations in 3 - d space within close proximity of region r2 ( 45 ). fig4 b shows that the dispensing tip ( 42 ) has dispensed the liquid ( 43 ) containing the particle in suspension ( 44 ) into region r2 ( 45 ) and surrounding the patterned structures ( 46 ). it is understood that the dispensing flow rate , volume , as well as r2 coverage , and well fill fraction may have a wide range of values . fig4 c shows the particles ( 44 ), after some amount of time , have settled toward the bottom of region r2 ( 45 ) to the lowest surface of the patterned structures ( 46 ) leaving liquid without suspended particles ( 43 ) toward the top of region r2 ( 45 ). fig4 d shows a liquid dispensing tip ( 42 ) dispensing liquid ( 47 ) in proximity of region r1 ( 48 ) adjacent to region r2 ( 45 ) which contains the particles ( 44 ) at the lowest surface of the patterned structures ( 46 ). regions r1 and r2 are designed and the dispensing method is carried out in such a way that the liquid flow introduces minimal disturbance and maximum retention of the particles as the liquid flows over the patterned structures ( 46 ). it is understood that the dispensing tip may have a wide variety of sizes and locations in 3 - d space within close proximity of region r1 . it is understood that the dispensing flow rate , volume , as well as r2 coverage , and well fill fraction may have a wide range of values . fig4 e shows the dispensed liquid ( 49 ) continuous with the liquid in which the particles were originally suspended . diffusion can occur between the bulk liquid and region r2 ( 45 ) which contains the particles ( 44 ) at the lowest surface of the patterned structures ( 46 ). fig5 is a schematic of liquid changes , or washing , in a well ( 51 ) with region r2 ( 53 ) containing particles ( 52 ) at the lowest level of the patterned structures ( 54 ). fig5 a is a well ( 51 ) with region r2 ( 53 ) containing particles ( 52 ) at the lowest surface of the patterned structures ( 54 ) where the well is filled with a liquid ( 55 ). such a state may be obtained by following the stepwise procedure illustrated in fig4 . fig5 b shows a pipette tip ( 56 ) located in proximity to region r1 ( 58 ) withdrawing liquid ( 55 ) such that the flow introduces minimal disturbance and maximal retention of the particles ( 52 ) as it flows over the region r2 ( 53 ) which contains the patterned structures ( 54 ). it is understood that the pipette tip ( 56 ) may have a wide variety of sizes and locations in 3 - d space within close proximity of r1 . it is understood that the dispensing flow rate , volume , as well as r2 coverage , and well fill fraction may have a wide range of values . fig5 c shows a low liquid level ( 55 ) with liquid still covering the particles ( 52 ) in region r2 ( 53 ) which contain the patterned structures ( 54 ). fig5 d shows a pipette tip ( 56 ) located in proximity to region r1 ( 58 ) dispensing liquid ( 59 ) such that the flow introduces minimal disturbance and maximal retention of the particles ( 52 ) as it flows over the region r2 ( 53 ) which contains the patterned structures ( 54 ). it is understood that the dispensing tip may have a wide variety of locations in 3 - d space . it is understood that the dispensing flow rate , volume , as well as r2 coverage , and well fill fraction may have a wide range of values . it should be appreciated that multiple cycles of removal and introduction of fluids illustrated in fig5 may be performed to , for example , introduce reagents into contact with the cells and / or particles , and subsequently remove the reagent and wash the cells and / or particles . again , the introduction and removal of liquids in the well may be performed with little disturbance to the cells and / or particles residing at the bottom of the patterned structure . once a liquid is presented in the main volume of the well , this liquid can diffuse into the recessed patterned region in a non - turbulent manner that does not disturb the cells and / or particles . fig6 shows a 384 - well microtiter plate format ( 61 ) containing wells with patterned structures ( 62 ) with a 384 - tip liquid dispensing unit ( 63 ) attached to a robotic liquid handler ( 64 ). it is understood that the liquid handler can be robotic or manual with a wide variety of tip number and configurations possible . fig7 shows four views of a single well from a 384 - well plate which contains regions r1 ( 73 ) & amp ; r2 ( 74 ). referring now to fig7 a and 7b , a white line schematic of the well wall ( 72 ), region r1 ( 73 ) and region r2 ( 74 ), which contains patterned macrostructures ( 75 ) in fig7 a , is overlaid on photomicrographs of the actual well with cells ( 76 ) dispensed to it . a series of ten phosphate buffer saline ( pbs ) washes was executed using a 96 - tip liquid dispensing robot . after the ten washes , the wells of fig7 a and 7b were imaged again in fig7 c and 7d , respectively . ten washes was chosen as it was representative of a standard immuno - cytochemistry protocol . it is understood that the exact washing protocol used may have a wide range of liquid dispensing and removal steps , and a variety of reagents in various sequences . actual dimensions of one embodiment are described later in the text . fig7 a shows a well containing cells ( 76 ) dispensed to region r2 ( 74 ) with some cells ( 77 ) located in region r1 ( 73 ). region r2 ( 74 ) contains patterned macrostructure posts ( 75 ). the background fluorescence is from cell tracker green in the suspension solution from cell efflux . fig7 b shows a well containing cells dispensed to region r2 ( 74 ) with some cells ( 79 ) located near the center of region r2 ( 74 ), some cells ( 76 ) near the edge of region r2 ( 74 ), and some cells ( 77 ) in region r1 ( 73 ). in this example , region r2 ( 74 ) does not contain patterned macrostructures . the background fluorescence is from cell tracker green in the suspension solution from cell efflux . fig7 c shows the same well as fig7 a following a series of ten washing steps . few cells ( 78 ) remain in region r1 ( 73 ), while the cells ( 76 ) within region r2 ( 74 ) amongst the patterned macrostructure posts ( 75 ) remain largely in place , showing negligible loss or movement . the background fluorescence is no longer detectable , indicating that the dissolved cell tracker green fluorescent substance has been removed which is indicative of successful washing . actual retention data from one of the embodiments is described later in the text . fig7 d shows the same well as fig7 b following a series of ten washing steps . few cells ( 78 ) remain in region r1 ( 73 ). the center ( 80 ) of region r2 ( 74 ) no longer has cells present as they have moved or been removed during the washing steps . the edge ( 81 ) of region r2 ( 74 ) shows retention of cells . the background fluorescence is no longer detectable indicating that the dissolved cell tracker green fluorescent substance has been removed , indicative of successful washing . in a representative experiment , twelve wells ( 3 . 3 mm × 3 . 3 mm × 11 . 5 mm , l × w × h ) containing a region r2 ( 2 mm × 2 mm ), and a region r1 ( 1 . 3 mm wide shelf along two walls of the well ) in a 384 - well plate had a series of liquid handling steps performed within them . in the layout presented in fig7 a and 7b the wells contained a region r2 within which was a patterned macrostructure consisting of 12 tapered posts of the following design : 0 . 32 mm diameter at the base , 0 . 25 mm diameter at the top , 5 degree draft angle , 0 . 4 mm in height , 0 . 2 mm minimum distance between the base of posts , 0 . 18 mm minimum distance to the edge of region r2 . a 1 . 6 μl , volume of cell suspension , pre - labeled with cell tracker green , containing on average 985 cells ( as counted by count nuclei using metamorph software ) was dispensed to region r2 as described in fig4 . during the dispensing , the tip was brought to about 0 . 100 mm above the top of the macro - structure posts . a series of ten washing steps using phosphate buffered saline in which 24 μl , was aspirated at 0 . 75 μl / sec flow rate and 20 μl , was dispensed at 0 . 75 μl / sec was executed above region r1 as shown in fig5 . during the dispensing , the tip was brought to about 0 . 200 mm above the top of the macrostructure posts . after the ten washes , another count was performed . on average , fifteen cells were lost with a standard deviation of eighteen cells . this is an average loss of 1 . 3 % across the entire ten wash protocol . after two washes , the background fluorescence of cell tracker green was reduced by forty - nine fold , or approximately a seven - fold reduction per wash cycle . after two washes , the fluorescence was below the detection range of our camera and microscope . under parallel conditions , analogous wells without the patterned structures lost the vast majority of cells as a result the same liquid dispensing routine . fig8 shows the retention of cells following immunocytochemistry . a ) retention of cells following immunocytochemistry . acute myeloid leukemia cells ( plb - 985 ), a non adherent cell line , were loaded into the cellwasher plate after labeling with celltracker green ( live cells ); treatment with ethanol followed by dapi following ( dead cells ) and imaged . live and dead cells were subject to a full immunocytochemistry protocol including 13 wash steps and imaged again . retention percentage is equal to the number of cells at the end of the immunocytochemistry protocol divided by the number prior to it . b ) immunocytochemical staining of plb - 985 cells . a standard immunocytochemistry protocol for β - actin , with 13 wash steps was used . shown is an overlay of β - actin ( green ) and dapi ( blue ). c ) cell movement during immunocytochemistry . plb - 985 cells were labeled with celltracker green , loaded into the cellwasher plate and imaged before and after six washes with pbs . shown is an overlay of initial ( blue ) and final ( red ) cell positions . in one embodiment , the instant invention may be used to wash cells or particles by : providing a volume of liquid to the well ( s ) containing particles or cells ; providing an additional liquid volume ; allowing the particles or cells to settle to the lowest surface of the macro or micro structures by gravity , centrifugation , affinity or magnetism ; removing excess fluid volume ; and replacing this fluid volume once or multiple times . in one embodiment , the rate of dispensing and removing fluid volume is in the range of 10 nl / sec to 1 ml / sec . in another embodiment , the rate of dispensing and removing fluid volume is in the range of 50 nl / sec to 100 μl / sec . in another embodiment , the rate of dispensing and removing fluid volume is in the range of 100 nl / sec to 10 μl / sec . in one embodiment , the loss of cells or particles following each wash cycle is less than 10 % of the cells or particles present . in one embodiment , the loss of cells or particles following five wash cycles is less than 10 % of the cells or particle present . in one embodiment , the loss of cells or particles following 10 wash cycles is less than 10 % of the cells or particle present . in one embodiment , the loss of cells or particles is less than 10 % of the cells or particle present following 11 , 12 , 13 , 14 , 15 , 20 , 25 , 30 , 35 , 40 , 45 or even 50 wash cycles . in one embodiment , the concentration of a dissolved substance present after a wash cycle is less than 50 % of the concentration of that same substance before the wash cycle . the device and method described here may be utilized to carry out immuno - cytochemical staining of non - adherent cell lines ( e . g ., hl - 60 ). the cells would be cultured in the usual manner , suspended in cell culture media and dispensed to the device . the cells would be allowed to settle to the lowest surface the patterned structures . a series of wash steps would be executed using an automated ( or manual ) dispenser / aspirator . first , the cells would be fixed ( e . g . using formalin ), then permeabilized ( e . g ., using detergent such as triton x - 100 ), next the cells would be incubated with an appropriate blocking buffer ( e . g . serum ), followed by the primary antibody which recognizes the antigen of interest , and then the secondary antibody which carries the fluorescent dye ( or enzyme ). the last three steps are each followed by one or more washes with buffer ( e . g ., phosphate - buffered saline ). in this example , the device and method facilitate the automation of the cell staining by eliminating the need to spin the cells down in a centrifuge after each wash step . another embodiment , of this application may be utilized to carry out assays with adherent cells without the loss of cells rendered non - adherent . this is important in many cell biology experiments , such as live / dead assays where quantification of programmed cell death is desired as it is well known that adherent cells become non - adherent when they undergo programmed cell death , or apoptosis . here , the adherent cells would be seeded and grown on the lowest surface of the patterned structures using traditional cell culture methods . at the end of the assay , the cells could be stained with histochemical or immune - cytochemical reagents without loss of dead cells that were no longer adherent . another embodiment of this application involves the processing of particles suspended in a gel . here , the posts would provide a scaffold which would protect the particle - containing gel from shear forces applied by liquid washing . the particles could be cancer cells , and the gel could be extra - cellular matrix ( ecm ) of a particular composition . protocols involving fragile ( e . g ., low density ) ecm compositions would benefit particularly from this invention . yet another embodiment of this application would involve partitioning the region r2 of the surface into two sub - regions , one containing patterned microstructures and the other not . this would simultaneously enable some particles or gels to be exposed to shear force and others not . yet another embodiment of this application would involve partitioning the region r2 of the surface into two sub - regions , each containing patterned microstructures , and separated by a gap or wall . this would enable co - culture of two different cell types either in two dimensions on the lowest surface of the patterned structures , or suspended in a gel . region r2 could be further divided into an arbitrary number of subregions . another embodiment of this application would involve two distinct shelf regions r1a and r1b located adjacent to region r2 . region r2 could further contain a number of regions r1 adjacent to it . another embodiment of this application would involve patterning region r2 with various different patterned marco or micro structures . for example , a portion of region r2 may contain patterned macrostructures and another portion may contain patterned microstructures . another embodiment of this application would involve patterned structures in region r1 . this includes , but is not limited to , the various micro and macro structures discussed above and below in the context of region r2 . another embodiment of this application would involve subdividing the bottom surface of the assay well such that different sub - areas may be at different heights relative to one another ; in this embodiment , regions r1 and / or r2 could be each cover one or more sub - areas . a further embodiment may include a sub - region within region r2 which is designed to enable retrieval of non - adherent cells once the vessel is tilted ( e . g ., 45 °) by aspirating the liquid in that sub - region with pipette tip . the region r2 would be resistant to flow when liquid is dispensed and aspirated in region r1 . region r2 would be designed with one continuous lowest surface and have a sub - region within r2 shaped such that when the vessel ( e . g ., microtiter plate ) is tilted , the cells would roll over or be otherwise transported to the sub - region within r2 which could accommodate a pipette tip for aspiration of the cells . it is contemplated that a device of the kind described herein may be formed by injection molding . one embodiment of the fabrication process of a device containing the described features would be injection molding of all but the well walls and subsequent joining of the molded piece to another piece containing the well and tray / dish walls . one embodiment of the fabrication process of a device containing the described features would be injection / compression molding of all but the well walls and subsequent joining of the molded piece to another piece containing the well walls . one embodiment of the fabrication process of a device containing the described features would be hot embossing molding of all but the well walls and subsequent joining of the molded piece to another piece containing the well walls . one embodiment of the fabrication process of a device containing the described features would be machining the device out of a piece , or pieces of material ( s ). one embodiment of the fabrication process of a device containing the described features would involve using stereolithography to build the part via 3d printing processes . one embodiment of the fabrication process of a device containing the described features would involve casting the device using soft lithography techniques . an embodiment of the device that requires joining of two pieces could employ thermal bonding , adhesive bonding , ultrasonic welding , laser welding , plasma activated surface joining , solvent bonding , amongst other joining processes involving entanglement , covalent bonding , electrostatic interactions and van der waals interactions . it is understood that this is not a complete list of joining processes and that other processes may be employed to join the components . one embodiment of device fabrication would have it fabricated in a thermoplastic such as polystyrene , polycarbonate , cyclo - olefin polymer , petg , pet . it is understood that there are many thermoplastics that could be used and that these are just a few examples of possible materials . one embodiment of the device fabrication would require surface treatment to change the surface energy of the material that the device has been fabricated from . example processes to change the surface energy can include coating the surface with a surfactant , coating the surface with a biomolecule , using corona discharge and using a plasma treatment system . it is understood that there are many surface treatment methods and that only a few examples of possible methods has been listed . one embodiment of the device fabrication would require patterning of surface treatment to provide a border in conjunction with or in place of a well . such patterning could be done via stamping , masking and applying surface treatment and laser ablation patterning . it is understood that there are many ways to pattern surface treatment and that only a few examples of possible methods has been listed . while the invention has been described with respect to a limited number of embodiments , those skilled in the art , having benefit of this disclosure , will appreciate that other embodiments can be devised which do not depart from the scope of the invention as disclosed herein . it is understood that the particular embodiments disclosed herein are illustrative only , and the invention embraces as such forms thereof within the scope of the following claims . also , recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling with the range and each endpoint , unless otherwise indicated herein , and each separate value and endpoint is incorporated into the specification as if it were individually recited herein . all methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context . the use of any and all examples , or exemplary language ( e . g ., “ such as ”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed . no language in the specification should be construed as indicating any non - claimed element as essential to the practice of the invention . preferred embodiments of this invention are described herein , including the best mode known to the inventors for carrying out the invention . variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description . the inventors expect skilled artisans to employ such variations as appropriate , and the inventors intend for the invention to be practiced otherwise than as specifically described herein . accordingly , this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable laws . moreover , any combination of the above - described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context . 6 . yeom j , agonafer d d , han j - h , and shannon m a , “ low reynolds number flow across an array of cylindrical microposts in a microchannel and figure - 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