WO2014046621A1 - Micro-fluidic device and uses thereof - Google Patents
Micro-fluidic device and uses thereof Download PDFInfo
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- WO2014046621A1 WO2014046621A1 PCT/SG2013/000412 SG2013000412W WO2014046621A1 WO 2014046621 A1 WO2014046621 A1 WO 2014046621A1 SG 2013000412 W SG2013000412 W SG 2013000412W WO 2014046621 A1 WO2014046621 A1 WO 2014046621A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
- B01D21/0087—Settling tanks provided with means for ensuring a special flow pattern, e.g. even inflow or outflow
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
- B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
- B01D21/265—Separation of sediment aided by centrifugal force or centripetal force by using a vortex inducer or vortex guide, e.g. coil
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
- B01F25/40—Static mixers
- B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
- B01F25/421—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions by moving the components in a convoluted or labyrinthine path
- B01F25/423—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions by moving the components in a convoluted or labyrinthine path by means of elements placed in the receptacle for moving or guiding the components
- B01F25/4231—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions by moving the components in a convoluted or labyrinthine path by means of elements placed in the receptacle for moving or guiding the components using baffles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03B—SEPARATING SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS
- B03B5/00—Washing granular, powdered or lumpy materials; Wet separating
- B03B5/62—Washing granular, powdered or lumpy materials; Wet separating by hydraulic classifiers, e.g. of launder, tank, spiral or helical chute concentrator type
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L2200/0652—Sorting or classification of particles or molecules
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0883—Serpentine channels
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0463—Hydrodynamic forces, venturi nozzles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/08—Regulating or influencing the flow resistance
- B01L2400/084—Passive control of flow resistance
Definitions
- the invention is generally directed to micro-fluidic devices having curved micro-channels with non-rectangular cross sections for particle focusing and mixing.
- the invention is directed to a micro-fluidic device that includes at least one inlet and a curvilinear microchannel having a trapezoidal cross section defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having a) the radially inner side and the radially outer side unequal in height, or b) the radially inner side equal in height to the radially outer side, and wherein the top side has at least two continuous straight sections, each unequal in width to the bottom side.
- the micro-fluidic device further includes at least one outlet.
- the micro-fluidic device includes two outlets.
- the micro-fluidic device includes a single inlet.
- the cross section of the micro-fluidic device can have (a) the height of the radially inner side larger than the height of the radially outer side, or (b) the height of the radially inner side smaller than the height of the radially outer side, or (c) the top side including at least one step forming a stepped profile, or (d) the top side including at least one shallow region in between the radially inner side and the radially outer side.
- the trapezoidal cross section can be a right trapezoidal cross section.
- the top and/or bottom sides of the trapezoidal cross section can be curved, with a curvature that can be convex or concave.
- the radially inner side and the radially outer side of the trapezoidal cross section can have a height in a range of between about 20 microns ( ⁇ ) and about 200 ⁇ .
- the top side and the bottom side of the trapezoidal cross section can have a width in a range of between about 100 ⁇ and about 2000 ⁇ .
- the curvilinear microchannel can be a spiral microchannel. In another aspect, the curvilinear microchannel can be a serpentine microchannel.
- the curvilinear microchannel can have a radius of curvature in a range of between about 2.5 mm and about 25 mm, and a length in a range of between about 4 cm and about 100 cm.
- the invention is directed to a method of separating by size one or more particles from a mixture of particles.
- the method comprises introducing the mixture into at least one inlet of a micro-fluidic device that includes a curvilinear microchannel having a trapezoidal cross section defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having the height of the radially inner side smaller than the height of the radially outer side, at a flow rate that isolates particles along portions of the cross-section of the microchannel based on particle size, wherein larger particles flow along the radially inner side of the microchannel to a first outlet and smaller particles flow along other portions of the microchannel to at least one other outlet, thereby size separating the particles from the mixture.
- the method can include collecting size separated particles from the first outlet.
- the flow rate can be in a range of between about 0.5 mL/min and about 7.5 mL/min.
- the particles can be cells,
- the flow rate can be about 2.5 mL/min
- the larger particles can have a diameter in a range of between about 18 ⁇ and about 40 ⁇
- the smaller particles can have a diameter in a range of between about 10 ⁇ and about 20 ⁇ .
- the flow rate can be about 1.5 mL/min
- the larger particles can have an diameter in a range of between about 15 ⁇ and about 25 ⁇
- the smaller particles can have a diameter in a range of between about 5 ⁇ and about 10 ⁇ .
- the flow rate can be in a range of between about 2.5 mL/min and about 3.0 mL/min
- the larger particles can have a diameter in a range of between about 25 ⁇ and about 40 ⁇
- the smaller particles can have a diameter in a range of between about 5 ⁇ and about 15 ⁇ .
- the mixture of cells can be a blood sample, and the larger cells can be circulating tumor cells (CTCs), and the smaller cells can be hematologic cells.
- CTCs circulating tumor cells
- he flow rate can be adapted to size separate about 7.5 mL of blood in about 8 minutes.
- the larger cells can be leukocytes, and the smaller cells can be hematologic cells.
- the mixture can be a bone marrow sample, wherein stem cells can be separated from
- the invention is directed to a method of concentrating cells from a mixture.
- the method comprises introducing the mixture into at least one inlet of a micro-fluidic device that includes a curvilinear microchannel having a trapezoidal cross section defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having the height of the radially inner side larger than the height of the radially outer side, at a flow rate that isolates the cells along the radially inner side of the cross section of the microchannel and directs them to a first outlet, thereby concentrating the cells from the mixture.
- the method can include collecting concentrated cells from the first outlet.
- the flow rate can be in a range of between about 0.5 mL/min and about 10 mL/min.
- the invention is directed to a method of filtering particulates from a mixture.
- the method comprises introducing a particulate containing mixture into at least one inlet of a micro-fluidic device that includes a curvilinear microchannel having a trapezoidal cross section defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having the height of the radially inner side larger than the height of the radially outer side, at a flow rate that isolates particulates along the radially inner side of the cross section of the microchannel and directs them to a first outlet, thereby filtering the particulates from the mixture.
- the mixture can be water.
- the method can include collecting particulates from the first outlet.
- the flow rate can be in a range of between about 0.5 mL/min and about 10 mL/min.
- the invention is directed to a method of distributing cells in a mixture.
- the method comprises introducing the mixture into at least one inlet of the micro-fluidic device that includes a curvilinear microchannel having a trapezoidal cross section defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having the top side that includes at least one step forming a stepped profile, at a flow rate that distributes cells along portions of the stepped profile, wherein cells do not impact the sides before, during, or after distribution to separate outlets, thereby distributing the cells in the mixture.
- the method can include collecting distributed cells from the separate outlets.
- the flow rate can be in a range of between about 2 mL/min and about 10 mL/min.
- the invention is directed to a method of mixing cells in a liquid.
- the method comprises introducing a liquid and cells into at least one inlet of the micro-fluidic device having a curvilinear microchannel having a trapezoidal cross section defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having the top side including at least one shallow region in between the radially inner side and the radially outer side, at a flow rate that mixes cells along the microchannel and directs the mixture to a first outlet.
- the method can include collecting the mixture from the first outlet.
- the flow rate can be in a range of between about 0.1 mL/min and about 2 mL/min.
- This invention has many advantages, including higher resolution separation than could be obtained with present micro-fluidic devices.
- the magnitude of the channel dimensions are normally >3 times the particle diameter, which not only makes the device free of clogging issues and high throughput, but also reduce the cost of fabrication.
- These advantages suggest a broad range of applications of curved micro-fluidic device in the future.
- the invention described here offers many distinct advantages over traditional rectangular curved micro-channels.
- particles are separated into two main streams along the inner and outer side according to their diameter and the flow rate. This type of device is able to achieve high resolution, high throughput separation, which is not feasible with traditional rectangular channels.
- FIGS. 1A and IB are schematic illustrations showing the top-view of two typical curvilinear micro-channels (FIG. 1A: spiral, FIG. IB: serpentine).
- the cutaway view of the spiral micro-channel is shown on the left.
- the width of the channels is typically larger than the depth.
- the Dean flows in a rectangular cross section channel have a main flow from inner side to outer side with the flows being parallel to the top and bottom wall. Under the influence of Dean flow and inertial lift, particles will focus at the inner half of the channel that have lower Dean flow.
- the positions of the particles are controlled by parameters such as channel dimensions, aspect ratio, radius of curvature, particle diameter, and flow rate.
- FIGS. 2a-2d are schematic illustrations of different cross sections of curved channels: FIG. 2a) Curved micro-channels with a deeper inner side (near curvature center) and shallow outer side. The two Dean vortex cores are skewed towards the inner side, trapping particles within them. This type of micro-channel has applications in particle concentration and filtration; FIG. 2b) Curved micro-channel having a shallow inner side and a deeper outer side. The two vortex cores are skewed towards the outer side and have the ability to entrairi particles of certain smaller diameter within them, which can be used for size based separation; FIG. 2c) in a curved microchannel with step-like cross-section, particles are trapped at the corners of the steps; FIG. 2d) Curved microchannels with sandwiched shallow regions create complex Dean flow and inertial lift profiles preventing particle focusing or trapping. Such channels can be used as a mixer.
- FIGS. 3 A and 3B are photographs of an actual trapezoid cross section spiral microfluidic device with a single inlet and two outlet tubes.
- the channels shown in FIG. 3A are filled with a dye for visualization.
- the device is made of two PDMS layers bonded via plasma. One of the layers that have a spiral pattern is cast from a micro milled PMMA mold.
- the cut view of the channel is shown in FIG. 3B.
- the width of the channel is 600 ⁇
- the inner height (bottom) is 80 ⁇
- outer height (top) is 140 ⁇ .
- FIGS. 4A-4H are top views showing the comparison of fluorescent beads distribution at the outlet of FIGS. 4A-4D) a 80 ⁇ height 600 ⁇ width rectangular cross section spiral microchannel, and FIGS. 4E-4H) a trapezoid cross section spiral microchannel as described in FIG. 1 A with flow rates increased from 0.5mL/min (left) to 7.5mL/min (right).
- the diameters of beads shown are FIGS. 4A & 4E) 5.78 ⁇ , FIGS. 4B & 4F) 9.77 ⁇ , FIGS. 4C & 4G) 15.5 ⁇ , FIGS. 4D & 4H) 26.25 ⁇ .
- FIG. 5 is an illustration of a computational fluid dynamics (CFD) simulation result of the Dean flow field across a trapezoidal cross section spiral channel compared with experimental results indicating the force balanced position of particles in the cross section of a spiral channel. Arrows indicate the direction and magnitude of Dean flow, map indicates the magnitude of Dean flow. Dots are positions of 26.25 ⁇ beads from experimental results.
- CFD computational fluid dynamics
- FIGS. 6A-6D are results and photographs showing separation of neutrophils from fresh human blood using a spiral channel with a trapezoidal cross-section.
- FIG. 6A Polymorphonuclear leukocytes (PNLs), mainly neutrophils, isolated from fresh human blood using Mono-Poly Resolving Medium (Catalog #1698049, MP
- FIG. 6B Blood samples with different hematocrit were spiked with isolated PNLs, which were stained with APC -conjugated anti-CD45 antibody and used as input samples to the spiral channel at 0.8 ml/min flow rate. The recovery of PNLs and RBC cell counts in output fraction of inner outlet were determined by flow cytometry analysis and by hemocytometer, respectively.
- FIG. 6C Giemsa staining for the output fraction of inner outlet when 0.1% hematocrit fresh human blood was used as input sample under 0.8 ml/min. Most of the cells were neutrophils.
- FIG. 6C Giemsa staining for the output fraction of inner outlet when 0.1% hematocrit fresh human blood was used as input sample under 0.8 ml/min. Most of the cells were neutrophils.
- FIGS. 7A and 7B are graphs illustrating the size distribution of MSCs collected from two outlets after separation with a 80 ⁇ -inner, 130 ⁇ -outer and 600 ⁇ -width trapezoid cross section spiral device, 100 cells are manually measured from each outlet collection.
- FIG. 7A Sample is pumped in with 2.2 mL/min flow rate.
- FIG. 7B Sample is pumped in with 3.0 mL/min flow rate.
- FIGS. 8 A and 8B are microscope images of MSCs collection from inner (FIG. 8B) and outer (FIG. 8 A) output of 80 ⁇ - ⁇ , ⁇ ⁇ -outer and 600 ⁇ - width trapezoid cross section spiral device at 2.5 ml/min flow rate.
- FIG. 9A is a schematic of a trapezoidal cross section channel illustrating the principle of particle focusing and trapping within the Dean vortices.
- FIG. 9B shows photographs of an actual PDMS cast trapezoidal cross section spiral microfluidic device with two outlet tubes removed. The cut view of the cross section is shown on the left. The radius of the spiral curve varies from 7.5 mm to 12.5 mm. The inner & outer heights of the channel cross section are 80 ⁇ and 130 ⁇ , respectively. The width of the channel is 600 ⁇ .
- FIG. 10A is a top view image showing the comparison of fluorescent beads distribution at the outlet of a 80/130 ⁇ inner/outer depth, trapezoidal cross section spiral microchannel, and a 80 ⁇ height rectangular channel with flow rates ranging from 0.5 mL/min to 7.5 mL/min.
- FIG. 10B is a CFD simulation of Dean flow field (inner/outer depth: 80/140 ⁇ , width: 600 ⁇ , flow rate: 3.5 mL/min, channel radius: 7.5 mm) combined with 26.25 ⁇ fluorescent beads distribution from the top view and the side view, indicating the force balanced position of particles. Black cones indicate the direction and magnitude of Dean flow. Gray circles are positions of 26.25 ⁇ beads at typical flow rates from experimental results.
- FIGS. 1 1 A, 1 IB, and 1 1C are graphs of FACS results of particle separation with 80/130 ⁇ inner/outer depth, 600 ⁇ width trapezoidal cross spiral
- FIG. 1 1 A Input with 16.68 ⁇ and 26.9 ⁇ particle of 0.665% volume to volume concentration (about 2.6xl0 6 /mL)
- FIG. 1 IB Inner side output
- FIG. 1 1C Outer side output.
- FIG. 12 is a schematic diagram illustrating the direction of forces on particles at different positions. Black circles indicate locations of unstable balanced point. White circles indicate stable force balanced points. White cones indicate the direction and logarithmic magnitude of Dean velocity.
- FIG. 13 is a schematic (not to scale) of a spiral channel with trapezoid cross- section of 500 ⁇ width, 70 ⁇ (inner) and 100 ⁇ (outer) depth illustrating the operating principle.
- WBCs white blood cells
- FL inertial lift force
- FD Dean drag force
- RBCs red blood cells
- FIGs. 14A- 14D are: schematic (not to scale) and average composite fluorescent images indicating the inertial focusing of 10 ⁇ (white) and 6 ⁇ (gray) beads in FIG. 14A) spiral channel with rectangular cross-section of 500 ⁇ x 90 ⁇ (WxH) under optimal flow rate: 1 ml/min (De - 4.31);
- FIG. 14A schematic (not to scale) and average composite fluorescent images indicating the inertial focusing of 10 ⁇ (white) and 6 ⁇ (gray) beads in FIG. 14A) spiral channel with rectangular cross-section of 500 ⁇ x 90 ⁇ (WxH)
- FIGS. 15A- 15C are top-down view images demonstrating the focusing behavior of fluorescent particles as a function of flow rate (Q) inside spiral channel with trapezoid cross-section of 500 ⁇ width, 70 ⁇ (inner) and 100 ⁇ (outer) depth.
- Dashed horizontal lines indicate the position of channel walls, while the inner channel walls were shown on the top side of the images.
- FIGS. 16A- 16B are normalized intensity line scans indicating the .
- FIG. 16A spiral channel with rectangular cross-section (500 ⁇ x 90 ⁇ ) under optimal flow rate ( 1 ml/min), or FIG. 16B) spiral channel with trapezoid cross-section (500 ⁇ x 70/100 ⁇ ) under optimal flow rate (0.8 ml/min).
- FIGS. 17A-17D are characterizations of blood cells in a spiral channel with a trapezoidal cross-section.
- FIG. 17B Single-pass recovery percentage of total WBCs, PMNs, MNLs and RBCs at different hematocrit. Recovery percentage of 1 % hematocrit (FIG. 17C) and 1.5% hematocrit (FIG.
- 17D input sample after processing by a trapezoidal cross-sectional spiral in a 2-stage cascade manner.
- the amount of RBCs was measured by Coulter counter, and the amounts of WBCs, PMNs and MNLs were based on FACS analysis of Hochest-positive, CD66b-positive cells, Hochest- positive but CD66b-negative cells, separately. Error bars indicate the standard deviation of results from three tests.
- FIGS. 18A-18B are illustrations of spiral processing of buffy coat obtained via differential centrifugation.
- FIG. 18 A A photo of healthy blood sample after centrifugation with Mono-Poly Resolving Medium. The first layer (FR1) consisted of MNLs, while the second layer (FR2) contained the majority of PMNs and some RBC residual. Cells from these two layers were re-suspended in the same volume of the original whole blood sample and further processed by the spiral microchannel with trapezoidal cross-section.
- FIG. 18B Size distribution of cells in input and output samples of the trapezoidal cross-section spiral microchannel.
- FIGS. 19A-19B are illustrations of comparisons of PMN activation by spiral and other RBC removal techniques.
- FIG. 19A Nitroblue-tetrazolium (NBT) test on WBCs isolated by differential centrifugation method (MPRM) or spiral processing under conditions with or without 1 ⁇ PMA. Scale bar: 10 ⁇ .
- FIG. 19B Nitroblue-tetrazolium (NBT) test on WBCs isolated by differential centrifugation method (MPRM) or spiral processing under conditions with or without 1 ⁇ PMA. Scale bar: 10 ⁇ .
- FIG. 19B Nitroblue-tetrazolium
- FIG. 20 is an illustration of balance of particles in a rectangular cross-section spiral microchannel.
- the black cones within channel cross-section are the CFD simulation result of the Dean flow field at a flow rate of 3.5 mL/min in a channel with radius 7.5 mm.
- the figure also shows the vector plot of the Dean drag force on the particle since the force is proportional to the Dean velocity.
- the experimental images of 15.5 ⁇ fluorescent beads distribution from the top view and side view are placed at the bottom and to the left side of the simulation profile. By combining the top- and side- view observations, the positions of 15.5 ⁇ beads at typical flow rate are drawn as gray circles in the channel cross-section.
- FIG. 21 is an illustration of balance of particles in a trapezoidal cross-section spiral microchannel with 80/140 ⁇ inner/outer depth and 600 ⁇ width.
- the black cones within channel cross-section are CFD simulation result of the Dean flow velocity (also Dean drag force) at a flow rate of 3.5 mL/min in a channel with radius 7.5 mm.
- the experimental images of 26.25 ⁇ fluorescent beads distribution from the top view and side view are placed at the bottom and the left side of the simulation. By combining the top and side view observations, the positions of 26.25 ⁇ beads at typical flow rate are drawn in gray circles in the channel cross-section.
- FIG. 22 is a top- view experimental observation of fluorescently labeled microparticles at the outlet of rectangular cross-section spiral microchannels with different channel depths and a trapezoidal cross-section spiral microchannel for increasing flow rates.
- FIG. 23 is a graph of the collection from the inner outlet (%) as a function of flow rate (mL/min) showing the collection ratio of particles from the inner outlet of trapezoidal cross-section spiral channel (80 ⁇ inner depth and 130 ⁇ outer depth, 600 ⁇ wide) at different flow rates for various particle sizes.
- FIG. 24A shows scatter plots captured using flow cytometer (Accuri C6, BD Biosciences, USA) showing the results of separations of particle mixtures in a 80 ⁇ inner depth, 130 ⁇ outer depth, and 600 ⁇ wide trapezoidal cross-section channel.
- FIG. 25 is a schematic illustration of the forces acting on the particles at several typical positions in a trapezoidal cross-section microchannel. Forces acting on particles at positions a indicate the imbalance at inner side at high flow rate. Forces acting on particles at positions b, c, d and e illustrate that these particles tend to be trapped near the Dean vortices centered at different points. White cones indicate the direction and logarithmic magnitude of simulated Dean velocity as well as Dean drag.
- FIG. 26 is a schematic illustration of the forces acting on the particles at several typical positions in a trapezoidal cross-section microchannel. Forces acting on particles at positions a indicate the imbalance at inner side at high flow rate. Forces acting on particles at positions b, c, d and e illustrate that these particles tend to be trapped near the Dean vortices centered at different points. White cones indicate the direction and logarithmic magnitude of simulated Dean velocity as well as Dean drag.
- FIG. 27A is a schematic illustration of a trapezoidal cross-section spiral microchannel illustrating the principle of particle focusing and trapping within the Dean vortices.
- FIG. 27B is an illustration of an actual spiral microfluidic device for side view focusing position measurement.
- the microfluidic channel is filled with dye for visualization. Samples are flowed from center loops to outer loops for the measurement.
- FIG. 28 A is a schematic illustration of the relative flow velocity around a particle from top view of a curved channel.
- FIG. 28B are graphs of the Dean velocity as a function of flow rate (mL/min) showing that the Dean velocity UD increases with Re according to simulation in a rectangular channel.
- the value of UD in the top curve is the magnitude of Dean velocity at 22% of channel depth (focusing position), while U D in the bottom curve is the value of Dean velocity at the center of the channel.
- FIG. 28C is a graph of the channel depth ( ⁇ ) as a function of the Dean velocity (m/s) showing the magnitude of the Dean velocity along the y-axis at different flow rates at the center line of channel width.
- FIG. 29 is a graph of forces (N) as a function of flow rate (mL/min) showing the magnitude of 3 major forces on a 15.5 ⁇ particle.
- F DD and F AB C are calculated based on the simulation of a rectangular cross-section channel; the particle is placed at the equilibrium position (22% of channel depth).
- F L is calculated following
- FIG. 30 is an illustration of the effect of slant angle on particle focusing in trapezoidal cross-section spiral microfluidic channel.
- the white band in the image indicates the focus band of 15.5 ⁇ fluorescent beads from the top view.
- FIGS. 31 A-31C are top view microscopy images of 15.5 ⁇ fluorescent particles focus band shift with flow rate under different geometry of channel cross- section.
- the width of the channels is 500 ⁇ .
- the inner depth is 75 ⁇ .
- Area of channel cross-sections is designed to be equal in all three channels, 5.0x10 " mm . Lines indicate the channel walls.
- FIG. 31 A Convex trapezoidal cross-section.
- FIG. 3 IB Normal trapezoidal cross-section.
- FIG. 31C Concave trapezoidal cross- section.
- FIG. 32A is an illustration of the operating principle of CTC enrichment by a spiral channel with trapezoid cross-section (80/130 ⁇ : inner/outer channel height).
- CTCs are focused near the inner wall due to the combination of inertial lift force and Dean drag force at the outlet while white blood cells (WBCs) and platelets are trapped inside the core of the Dean vortex formed closer to the outer wall.
- WBCs white blood cells
- FIG. 32B is a photograph of the workstation setup for CTC separation.
- the lysed blood is pumped through the spiral chip using a syringe pump where CTCs are separated from other blood components rapidly and efficiently.
- FIG. 33 are phase contrast micrographs of cultures of control (unsorted) MDA-MB-231 cells and cells enriched by spiral chip. The images indicate no significant differences between the morphology and proliferation rate of the cells suggesting high viability and sterility. Scale bar is 200 ⁇ .
- FIG. 34A is a graph of the number of WBCs/mL as a function of
- FIG. 34B is a histogram plot indicating a high separation efficiency of about 85% for different cancer cell lines tested.
- FIG. 34C are phase contrast micrographs of control (unsorted) and sorted MDA-MB-23 lcells stained using trypan-blue dye indicating high cell viability, and bar graph results that confirm that the shear exerted on the cells during sample processing did not compromise their viability, retrieving > 90% viable cells.
- FIG. 35 are photographs illustrating the viability of captured CTCs by trapezoidal cross-section chip. Captured cells are plated onto 2D polylysine coated substrates and allowed to spread overnight. Clustering of platelets with CTCs can be observed in vitro. Viability of CTCs was confirmed using PI staining.
- FIG. 36A Immunofluorescence staining of isolated CTC. CTC is identified by the following criteria: Hoechst positive, pan-cytokeratin positive and CD45 negative.
- FIG. 36B CTCs enumeration plot for healthy donors, breast cancer patients, and lung cancer patients.
- FIG. 36C Identification of cancer stem cells (CSCs) in breast samples using standard markers. No CD44+/CD24+ were detected. It was found that CD44+ cells are larger than CD24+ cells.
- FIG. 36D Staining for apoptotic cells. The absence of cleaved caspase-3 in the isolated CTCs. Majority of the cells (> 95%) expressing cleaved caspase-3 were CD45+.
- FIG. 37 are photographs of a library of CTC images displaying cell size and nuclei heterogeneity among them.
- the scale bar is 10 ⁇ .
- FIG. 38 is a graph of FLA-4 as a function of FLA-1 illustrating a flow cytometric analysis of isolated CTCs for cleaved caspase-3 protein. Only 9.9% of cells were positive for cleaved caspase-3, confirming that the high flow rates in the microfluidic chip do not affect cell viability and integrity.
- FIG. 39 are photographs illustrating the detection of Centromere of chromosome 17 (Cen- 17) and HER2 of enriched CTCs. Cells were amplified for HER2 if HER2/Cenl7 ratio is >2. MDA-MB-231 and SKBR3 breast cancer lines were used as controls. Merged images (DAPI, Spectrum: HER2 signal, Spectrum: Cen-17) are under 20X magnification. Scale bar: ⁇ .
- particles flowing in curvilinear channels are influenced by both inertial migration and secondary Dean flows.
- the combination of Dean flow and inertial lift results in focusing and positioning of particles at distinct positions for concentration and separation applications. .
- Described herein is a set of curved micro-channels with non-rectangular cross-sections which are introduced into a microfluidic device resulting in the alteration of the shapes and positions of the Dean vortices which generates new focusing positions for particles.
- a curved micro- channel with a deeper inner side (along the curvature center) and a shallow outer side generates two strong Dean vortex cores near the inner wall, trapping all particles irrespective of size within the vortex.
- Such a channel finds vast applications in particle and cell concentration applications, such as water filtration and purification at ultra-high throughputs.
- Ultra-high throughput is a flow rate in a range of between about 0.5 mL/min and about 1 L/min. Ultra-high throughput can be achieved by combining multiple channels in a variety of combinations. In some aspects, multiple channels can be combined into a single micro-fluidic device. In other aspects, multiple channels can be combined into a multiplexed micro-fluidic device.
- an ultra-high throughput flow rate can be about 0.5 mL/min, about 5 mL/min, about 10 mL/min, about 20 mL min, about 40 mL/min, about 50 mL/min, about 100 mL/min, about 200 mL/min, about 300 mL/min, about 400 mL/min, about 500 mL/min, about 600 mL/min, about 700 mL/min, about 800 mL/min, about 900 mL/min, or about 1 L/min.
- the vortex centers are skewed near the outer wall at the outer side which entrains particles and cells within the vortex.
- larger particles with dominant inertial force are focused near the inner channel walls, similar to rectangular cross-section channels.
- the threshold diameter determining whether a particle/cell is trapped within the Dean vortex or focused towards the inner channel wall is dependent on the flow rate. This enables such a device to achieve good separation resolution between mixtures having a wide range of particle sizes.
- the throughput is much higher than in rectangular channels where the particles are focusing near the inner side of the channel, toward the outlet collection branches placed at the inner side. This leads to low separation resolution, as well as carrying high risks of channel clogging. As shown herein, with a trapezoidal cross- section, higher particle/cell concentrations can be processed with minimal interaction between them to achieve ultra-high throughputs.
- the device described herein demonstrates separation of polynuclear leukocytes (PNLs) (diameters in a range of between about 10 ⁇ and about 15 ⁇ ) from red blood cells (RBCs) (diameters of about 7-8 ⁇ ), and small mesenchymal stem cells (MSCs) (diameters in a range of between about 14 ⁇ and about 20 ⁇ ) from large MSCs, (diameters larger than about 20 ⁇ ) and shows that the device achieved good separation resolution and high throughput for use, e.g., in clinical analysis and cell study.
- channels with stepped cross- section and sandwiched shallow regions can be used for particle trapping and mixing.
- Fluid flowing through a channel with a laminar profile has a maximum velocity component near the centroid of the cross section of the channel, decreasing to zero near the wall surface.
- the fluid experiences centrifugal acceleration directed radially outward. Since the magnitude of the acceleration is proportional to quadratic velocity, the centrifugal force in the centroid of the channel cross section is higher than at the channel walls.
- the non-uniform centrifugal force leads to the formation of two counter-rotating vortices known as Dean vortices known as Dean vortices in the top and bottom halves of the channel, which have a radially outward flow in the center and two inward flows near the channel walls as shown in FIG. 1 A.
- Dean vortices two counter-rotating vortices
- the interplay between the inertial lift force and the Dean drag force reduces the equilibrium positions to just two near the inner channel wall at low flow rate, and move outward with an increase in flow rate, each within the top and bottom Dean vortex.
- the two equilibrium positions overlay each other along the micro- channel height and are located at the same distance from the micro-channel inner wall for a given cell size, i.e. viewed as a single position across the micro-channel width.
- FIG. 5 illustrates the phenomenon by simulating the Dean flow field with computational fluid dynamics (CFD) software and observing particle position from top and side view. It can be seen that the particles were flowing along the low Dean flow area. When the flow rate was lower than about 3 ml/min, particles were focused at the inner side as in rectangular channels, and when the flow rate increased over about 4 ml/min, the particles were trapped in the Dean vortex and move towards the outer channel wall.
- CFD computational fluid dynamics
- FIGS. 4A-4H present the position of different size particles within the cross- section when viewed from the top view for increasing flow rates.
- this device there were two typical regimes of focusing based on the particle size, the inertial dominant and Dean dominant regimes.
- the large channel dimension prevented them from focusing and these particles got trapped in the Dean vortex even at low flow rate.
- the larger particles e.g., about 9.77 ⁇ particles also could not focus at the inner wall and were trapped within the Dean vortices at flow rates > about 1 ml/min.
- a flow rate of about 2.5 ml/min about 26.25 ⁇ ⁇ particles can be separated from a mixture of about 26.25 ⁇ and about 15.5 ⁇ particles.
- a low flow rate can be in a range of between about 0.5 mL/min and about 2 mL/min.
- a low flow rate can be a flow rate of about 0.5 mL/min, about 0.6 mL/min, about 0.7 mL/min, about 0.8 mL/min, about 0.9 mL/min, about 1.0 mL/min, about 1.1 mL/min, about 1.2 mL/min, about 1.3 mL/min, about 1.4 mL/min, about 1.5 mL/min, about 1.6 mL/min, about 1.7 mL/min, about 1.8 mL/min, about 1.9 mL/min, or about 2.0 mL/min.
- a high flow rate can be a flow rate in a range of between about 6 mL/min and about 10 mL/min.
- a high flow rate can be a flow rate of about 6 mL/min, about 6.5 mL/min, about 7.0 mL/min, about 7.5 mL/min, about 8.0 mL/min, about 8.5 mL/min, about 9.0 mL/min, about 9.5 mL/min, or about 10.0 mL/min.
- the invention relates to a set of curved micro- channels with non-rectangular cross-section that give rise to unique Dean vortices for varying applications in micro-fluidic field relating to particle focusing, separation, and mixing.
- the invention is directed to a micro- fluidic device that includes at least one inlet and a curvilinear microchannel having a trapezoidal cross section defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having a) the radially inner side and the radially outer side unequal in height, or b) the radially inner side equal in height to the radially outer side, and wherein the top side has at least two continuous straight sections, each unequal in width to the bottom side.
- the device further comprises at least one outlet.
- a spiral channel with a trapezoidal cross-sections consisting of a shallow inner side and deeper outer wall is used as a high resolution size based particle separat
- the micro-fluidic device includes a single inlet, 2 inlets, 3 inlets, 4 inlets, 5 inlets, 6 inlets, 7 inlets, 8 inlets, 9 inlets, or 10 or more inlets.
- the curvilinear microchannel 120 can be a spiral microchannel as shown in FIG. 1A. In another aspect, the curvilinear microchannel 120 can be a serpentine microchannel as shown in FIG. IB.
- the curvilinear microchannel 120 can have a radius of curvature in a range of between about 2.5 mm and about 25 mm.
- the curvilinear microchannel can have a radius of curvature of about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24 mm, or about 25 mm.
- the curvilinear microchannel can also have a length in a range of between about 4 cm and about 100 cm.
- the curvilinear microchannel can have a length of about 5 cm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 55 mm, about 60 mm, about 65 mm, about 70 mm, about 75 mm, about 80 mm, about 85 mm, about 90 mm, about 95 mm, or about 100 cm.
- the micro-fluidic device further includes at least one outlet.
- the micro-fluidic device includes two outlets, 3 outlets, 4 outlets, 5 outlets, 6 outlets, 7 outlets, 8 outlets, 9 outlets, or 10 or more outlets.
- the micro-fluidic device has two outlets for waste and particle collection, respectively.
- the micro-fluidic device has a single inlet and only 2 outlets.
- the width can be in a range of between about 100 ⁇ and about 2000 ⁇ , such as a width of about 200 ⁇ , about 300 ⁇ , about 400 ⁇ , about 500 ⁇ , about 600 ⁇ , about 700 ⁇ , about 800 ⁇ , about 900 ⁇ , about 1000 ⁇ , about 1100 ⁇ , about 1200 ⁇ , about 1300 ⁇ , about
- the outer depth can be in a range of between about 20 ⁇ and about 200 ⁇ , such as an outer depth of about 40 ⁇ , about 60 ⁇ , about 80 ⁇ , about 100 ⁇ , about 120 ⁇ , about 140 ⁇ , about 160 ⁇ , or about 180 ⁇ .
- the inner depth can be in a range of between about 20 ⁇ and about 200 ⁇ , such as an inner depth of about 40 ⁇ , about 60 ⁇ , about 80 ⁇ , about 100 ⁇ , about 120 ⁇ , about 140 ⁇ , about 160 ⁇ , or about 180 ⁇ .
- the radius of curvature can be in a range of between about 2.5 mm and about 25 mm, such as a radius of about 5 mm, about 7.5 mm, about 10 mm, about 12.5 mm, about 15 mm, about 17.5 mm, about 20 mm, or about 22.5 mm.
- the slant angle is the angle between the top of the channel and the bottom of the channel. The slant angle can be in a range of between about 2 degrees and about 60 degrees.
- the slant angle can be about 2 degrees, about 4 degrees, about 6 degrees, about 8 degrees, about 10 degrees, about 12 degrees, about 14 degrees, about 16 degrees, about 18 degrees, about 20 degrees, about 22 degrees, about 24 degrees, about 26 degrees, about 28 degrees, about 30 degrees, about 32 degrees, about 34 degrees, about 36 degrees, about 38 degrees, about 40 degrees, about 42 degrees, about 42 degrees, about 46 degrees, about 48 degrees, about 50 degrees, about 52 degrees, about 54 degrees, about 56 degrees, about 58 degrees, or about 60 degrees.
- the slant angle of the channel affects the focusing behavior in two ways: (i) the threshold flow rate required to trap particles in the Dean vortex as a function of particle size and (ii) the location of the Dean vortex core.
- a large slant angle (i.e., in a range of between about 10 degrees and about 60 degrees) will lead to strong Dean at the outer side and increase the particle trapping capability.
- a large slant angle can also decrease the threshold flow rate required to trap particles of a given size within the Dean vortex.
- the invention is directed to a micro-fluidic device 100 as shown in FIG. 1 A that includes at least one inlet 1 10 and a curvilinear
- microchannel 120 having a trapezoidal cross section 201 as shown in FIG. 2a or 202 as shown in FIG. 2b, defined by a radially inner side 210, a radially outer side 220, a bottom side 230, and a top side 240, the cross section having a) the radially inner side 210 and the radially outer side 220 unequal in height, or b) the radially inner side 210 equal in height to the radially outer side 220 as shown in FIG. 2d, and wherein the top side 240 has at least two continuous straight sections 240a and 240b, each unequal in width to the bottom side 230.
- the micro-fluidic device further includes at least one outlet 130.
- the cross section 201 of the micro-fluidic device can have the height of the radially inner side 210 larger than the height of the radially outer side 220 as shown in FIG. 2a.
- the height of the radially inner side 210 can be smaller than the height of the radially outer side 220 as shown in FIG. 2b.
- the top side 240 can include at least one step (241, 242, 243, etc.) forming a stepped profile 203 as shown in FIG. 2c.
- the radially inner side 210 can be larger or smaller than the height of the radially outer side 220.
- the top side 240 can include at least one shallow region 240c in between the radially inner side 210 and the radially outer side 220 as shown in FIG. 2d.
- the trapezoidal cross section can be a right (i.e., normal) trapezoidal cross section as shown in FIG. 3 IB.
- the top and/or bottom sides of the trapezoidal cross section can be curved, with a curvature that can be convex, as shown in FIG. 31 A, or concave, as shown in FIG. 31C.
- the radially inner side 210 and the radially outer side 220 of the trapezoidal cross section can have a height in a range of between about 20 microns ( ⁇ ) and about 200 ⁇ .
- the height of the radially inner side 210 can be about 20 ⁇ , about 40 ⁇ , about 60 ⁇ , about 80 ⁇ , about 100 ⁇ , about 120 ⁇ , about 140 ⁇ , about 160 ⁇ , about 180 ⁇ , or about 200 ⁇
- the height of the radially outer side 220 can be about 20 ⁇ , about 40 ⁇ , about 60 ⁇ , about 80 ⁇ , about 100 ⁇ , about 120 ⁇ , about 140 ⁇ , about 160 ⁇ , about 180 ⁇ , or about 200 ⁇ .
- the height of the radially inner side 210 can be about 70 ⁇ , or about 80 ⁇ , or about 90 ⁇ , and the height of the radially outer side 220 can be about 100 ⁇ , or about 120 ⁇ , or about 130 ⁇ , or about 140 ⁇ .
- the top side 240 and the bottom side 230 of the trapezoidal cross section can have a width in a range of between about 100 ⁇ and about 2000 ⁇ , such as a width of about 200 ⁇ , about 300 ⁇ , about 400 ⁇ , about 500 ⁇ , about 600 ⁇ about 700 ⁇ , about 800 ⁇ , about 900 ⁇ , about 1000 ⁇ , about 1100 ⁇ , about 1200 ⁇ , about 1300 ⁇ , about 1400 ⁇ , about 1500 ⁇ , about 1600 ⁇ , about 1700 ⁇ , about 1800 ⁇ , or a width of about 1900 ⁇ .
- FIG. 24A shows experimental results confirming the three dimensional particle focusing in spiral microchannels.
- the results indicate that particles form two bands along the depth symmetrically between the zero-lift force plane and the centers of the Dean vortex in spiral channels.
- a multi-loop microchannel was employed to calibrate the focusing of different size standard micro particles of about 5.78 ⁇ , about 9.77 ⁇ , about 15.5 ⁇ , and about 26.25 ⁇ diameter for flow rates ranging from about 0.5 to about 7.5 mL/min.
- spiral microchannels can comprise one or more loops.
- the multi-loop microchannel can be a 2 loop microchannel, a 3 loop microchannel, a 4 loop microchannel a 5 loop microchannel, a 6 loop microchannel, a 7 loop microchannel, an 8 loop microchannel, a 9 loop
- the multi-loop microchannel can be an 8 loop microchannel.
- the device can be an 8-loop spiral microchannel with one inlet and two outlets with radius of curvature decreasing from about 24 mm at the inlet to about 8 mm at the two outlets for efficient cell migration and focusing.
- the width of the channel cross-section can be about 600 ⁇ and the inner/outer heights can be about 80 ⁇ and about 130 ⁇ , respectively, for the trapezoid cross- section.
- the multi-loop microchannel can be a 4 loop microchannel.
- one or more micro-fluidic devices can be coupled, thereby generating a multiplexed device,
- the outlet of one micro-fluidic device can be connected to the inlet of one or more micro-fluidic devices.
- multiple channels can be integrated into a micro-fluidic device.
- the number of channels that can be multiplexed and/or integrated into a micro-fluidic device can be in a range of between about 2 channels and about 500 channels.
- the number of channels can be about 2 channels, about 5 channels, about 10 channels, about 20 channels, about 30 channels, about 40 channels, about 50 channels, about 100 channels, about 200 channels, about 300 channels, about 400 channels, or about 500 channels.
- the micro-fluidic device can further comprise other components upstream, downstream, or within (e.g., a multiplexed) a device.
- one or more micro-fluidic devices can further comprise one or more collection devices (e.g., a reservoir), flow devices (e.g., a syringe, pump, pressure gauge, temperature gauge), analysis devices (e.g., a 96-well microtiter plate, a microscope), filtration devices (e.g., a membrane), e.g., for upstream or downstream analysis (e.g., immunostaining, polymerase chain reaction (PCR) such as reverse PCR, quantitative PCR), fluorescence (e.g., fluorescence in situ hybridization (FISH)), sequencing, and the like.
- collection devices e.g., a reservoir
- flow devices e.g., a syringe, pump, pressure gauge, temperature gauge
- analysis devices e.g., a 96-well microtiter plate,
- An imaging system may be connected to the device, to capture images from the device, and/or may receive light from the device, in order to permit real time visualization of the isolation process and/or to permit real time enumeration of isolated cells.
- the imaging system may view and/or digitize the image obtained through a microscope when the device is mounted on a microscope slide.
- the imaging system may include a digitizer and/or camera coupled to the microscope and to a viewing monitor and computer processor.
- the invention is directed to a method of separating by size one or more particles from a mixture of particles.
- the method comprises introducing the mixture into at least one inlet (not shown) of a micro- fluidic device 900 that includes a curvilinear microchannel having a trapezoidal cross section defined by a radially inner side 910, a radially outer side 920, a bottom side 930, and a top side 940, the cross section having the height of the radially inner side 910 smaller than the height of the radially outer side 920, thereby defining a slant angle 945, at a flow rate that isolates particles along portions of the cross- section of the microchannel based on particle size, wherein larger particles 970 flow along the radially inner side 910 of the microchannel to a first (inner) outlet 950 and smaller particles 980 flow along other portions of the microchannel to at least one other (out
- Particles present in a variety of mixtures can be introduced into the device.
- mixtures include biological fluids (e.g., a biological sample such as blood, lymph, urine, and the like), liquids (e.g., water), culture media, emulsions, sewage, etc.
- biological fluids e.g., a biological sample such as blood, lymph, urine, and the like
- liquids e.g., water
- culture media emulsions
- sewage emulsions
- the biological sample is whole blood
- the blood can be introduced unadulterated or adulterated (e.g., lysed, diluted). Methods of lysing blood are known in the art.
- the volume to volume concentration of the particles as compared to other cells can be less than about 5%.
- the volume to volume concentration can be about 4%, about 3%, or about 2%.
- dilution of blood sample can be to a hematocrit in a range of between about 0.5% and about 2%.
- the hematocrit of a diluted blood sample can be about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%», about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.
- the invention is directed to a method of concentrating cells from a mixture.
- the method comprises introducing the mixture into at least one inlet of a micro-fluidic device that includes a curvilinear microchannel having a trapezoidal cross section 201 as shown in FIG. 2a defined by a radially inner side 210, a radially outer side 220, a bottom side 230, and a top side 240, the cross section having the height of the radially inner side 210 larger than the height of the radially outer side 220, at a flow rate that isolates the cells along the radially inner side of the cross section of the microchannel and directs them to a first outlet (not shown), thereby concentrating the cells from the mixture.
- the method can include collecting concentrated cells from the first outlet.
- the flow rate can be in a range of between about 0.5 mL/min and about 10 mL/min.
- the invention is directed to a method of filtering particulates from a mixuture (e.g., water).
- Particulates can include bacteria, fungi, parasites, floe, or other sedimentary aggregates present in water.
- the method comprises introducing particulate containing water into at least one inlet of a micro- fluidic device that includes a curvilinear microchannel having a trapezoidal cross section 201 as shown in FIG.
- the method can include collecting particulates from the first outlet.
- the flow rate can be in a range of between about 0.5 mL/min and about 10 mL/min.
- the invention is directed to a method of distributing cells in a mixture.
- the method comprises introducing the mixture into at least one inlet of the micro-fluidic device that includes a curvilinear microchannel having a trapezoidal cross section 203 as shown in FIG. 2c defined by a radially inner side 210, a radially outer side 220, a bottom side 230, and a top side 240, the cross section having the top side 240 that includes at least one step (241, 242, 243, etc.) forming a stepped profile, at a flow rate that distributes cells along portions of the stepped profile, wherein cells do not impact the sides before, during, or after distribution to separate outlets (not shown), thereby distributing the cells in the mixture.
- the method can include collecting distributed cells from the separate outlets.
- the flow rate can be in a range of between about 2 mL/min and about 10 mL/min.
- a method of mixing cells in a liquid includes introducing a liquid and cells into at least one inlet of the micro-fluidic device having a curvilinear microchannel having a trapezoidal cross section 204 as shown in FIG. 2d defined by a radially inner side 210, a radially outer side 220, a bottom side 230, and a top side 240, the cross section having the top side 240 including at least one shallow region 240c in between the radially inner side 210 and the radially outer side 220, at a flow rate that mixes cells along the microchannel and directs the mixture to a first outlet (not shown).
- the method can include collecting the mixture from the first outlet.
- the flow rate can be in a range of between about 0.1 mL/min and about 2 mL/min.
- fluid can be introduced into the micro- fluidic device in a variety of ways.
- fluid can be introduced into the micro-fluidic device using a syringe pump.
- fluid can be introduced into the micro-fluidic device using a piston pump, a gear pump, a peristaltic pump, a piezoelectric micropump, or using a controllable pressure regulator.
- the flow rate of fluid through the micro-fluidic device will vary depending on the use.
- the flow rate can be in a range of between about 0.5 mL/min and about 10 mL/min, such as a flow rate of about 1 mL/min, about 2 mL/min, about 3 mL/min, about 4 mL/min, about 5 mL/min, about 6 mL/min, about 7 mL/min, about 8 mL/min, or about 9 mL/min.
- a variety of particles can be separated using the micro-fluidic device.
- larger particles can be separated from smaller particles.
- Larger particles can have a diameter from about 18 ⁇ to about 50 ⁇ .
- larger particles can have a diameter of about 19 ⁇ , about 20 ⁇ , about 21 ⁇ , about 22 ⁇ , about 23 ⁇ , about 24 ⁇ , about 25 ⁇ , about 26 ⁇ , about 27 ⁇ , about 28 ⁇ , about 29 ⁇ , about 30 ⁇ , about 31 ⁇ , about 32 ⁇ , about 33 ⁇ , about 34 ⁇ , about 35 ⁇ , about 36 ⁇ , about 37 ⁇ , about 38 ⁇ , about 39 ⁇ , about 40 ⁇ , about 41 ⁇ , about 42 ⁇ , about 43 ⁇ , about 44 ⁇ , about 45 ⁇ , about 46 ⁇ , about 47 ⁇ , about 48 ⁇ , about 49 ⁇ , or about 50 ⁇ .
- Smaller particles can have a diameter from about 2 ⁇ to about 14 ⁇ .
- smaller particles can have a diameter of about 2 ⁇ , about 3 ⁇ , about 4 ⁇ , about 5 ⁇ , about 6 ⁇ , about 7 ⁇ , about 8 ⁇ , about 9 ⁇ , about 10 ⁇ , about 11 ⁇ , about 12 ⁇ , about 13 ⁇ , or about 14 ⁇ m.
- the flow rate can be about 2.5 mL/min
- the larger particles can have a diameter in a range of between about 18 ⁇ and about 40 ⁇
- the smaller particles can have a diameter in a range of between about 10 ⁇ and about 20 ⁇ .
- the flow rate can be about 1.5 mL/min
- the larger particles can have an diameter in a range of between about 15 ⁇ and about 25 ⁇
- the smaller particles can have a diameter in a range of between about 5 ⁇ and about 10 ⁇ .
- the flow rate can be in a range of between about 2.5 mL/min and about 3.0 mL/min
- the larger particles can have a diameter in a range of between about 25 ⁇ and about 40 ⁇
- the smaller particles can have a diameter in a range of between about 5 ⁇ and about 15 ⁇ .
- the particles can be cells, such as stem cells.
- the cells can be present in a biological fluid (e.g., blood, urine, lymph, cerebrospinal fluid, and the like).
- the cells are present in a blood sample, wherein the larger cells are circulating tumor cells (CTCs), and the smaller cells are hematologic cells.
- the CTCs are cancer cells (e.g., metastatic cancer cells) from a (one or more) breast cancer, colorectal cancer, kidney cancer, lung cancer, gastric cancer, prostate cancer, ovarian cancer, squamous cell cancer, hepatocellular cancer, nasopharyngeal cancer and other types of cancer cells. Because this approach does not require initial cell surface biomarker selection, it is suitable for use in different cancers of both epithelial and non-epithelial origin.
- the methods described herein can further comprise collecting and isolating the separated particles (e.g., cells).
- the method can further comprise downstream analysis such as immunostaining, qRT-PCR, FISH and sequencing.
- the method can further comprise conducting a heterogeneity study.
- the capture efficiency of particles can be in a range of between about 60% and about 100%, such as about 62%, about 64%, about 66%, about 68%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, about 98%, and about 99%.
- the capture efficiency can be an average recovery of 80%, or 85%, or 87%.
- detection of HER2 amplification in CTCs can identify high-risk breast cancer patients who may benefit from HER2 associated therapeutic strategies.
- the micro-fluidic device described herein can process milliliter quantities of fluid, e.g., blood, in minutes.
- the micro-fluidic device having a trapezoidal cross section can process 7.5 mL of blood, (e.g., lysed red blood cells) in about 8 minutes, allowing enrichment of viable CTCs, and can process smaller quantities of blood, such as 4 mL in about 5 minutes, and can process larger quantities of blood, such as about 20 mL in about 15 minutes, or 40 mL in about 30 minutes, or 60 mL in about 45 minutes, or 80 mL in about 60 minutes, or larger quantities in more than 1 hour.
- blood e.g., lysed red blood cells
- larger quantities of blood such as about 20 mL in about 15 minutes, or 40 mL in about 30 minutes, or 60 mL in about 45 minutes, or 80 mL in about 60 minutes, or larger quantities in more than 1 hour.
- the larger cells can be leukocytes, and the smaller cells can be hematologic cells.
- the mixture can be a bone marrow sample, wherein stem cells can be separated from hematologic cells.
- Size based cell separation is a challenging requirement in cell study for the isolation of certain types of cells from cell mixtures.
- cancer metastasis mortal consequence of tumorigenesis, accounts for about 90% of all cancer related deaths.
- viable tumor-derived epithelial cells circulating tumor cells or CTCs
- CTCs circulating tumor cells
- CTCs which are typically about 20 ⁇ in diameter
- RBC blood cells
- leukocytes about 10-15 ⁇
- neutrophils which are key effectors of the innate immune response against bacterial infection; over-exuberant response could lead to systemic inflammation and organ dysfunction in sepsis. Therefore, neutrophils themselves have been recognized as a potential target in controlling sepsis. In animal models of sepsis, studies showed that depleting neutrophils or antagonizing their activities helps to maintain organ function. It's plausible to hypothesize that deletion of circulating neutrophils in sepsis patients' blood might help to control
- PNLs polymorphonuclear leukocytes
- monocytes and lymphocytes with cell diameter in a range of between about 7 ⁇ and about 8 ⁇
- erythrocytes with a disk diameter in a range of between about 6 ⁇ and about 8 ⁇
- a size-based separation technique might be helpful in fractionating blood into different blood components.
- FIGS. 6A-6D show the performance of an exemplary device in separating PNLs from fresh human blood.
- the device achieved >90% PNL recovery for a 2% hematocrit blood sample, while maintaining -75% RBC removal, in a continuous and high-throughput manner, allowing the selective transfusion of neutrophil-depleted blood when being coupled. Giemsa staining of the output sample further confirmed that 98% of isolated PNLs were neutrophils.
- the NBT test on both the input and output sample demonstrated that the isolated neutrophils remained alive and non-activated after being processed by this device. Therefore, the subsequence clinical and molecular diagnostics tests on the isolated neutrophils should reveal the initial state of the input sample.
- an early passage MSCs cell line was diluted to about lOk/ml and tested in a 80 ⁇ inner and 130 ⁇ m outer height, 600 ⁇ width 8 loops spiral. After experiment, 100 random cells were measured manually from each outlet. The size distribution results are shown in FIGS. 7A-7B and 8A-8B. As expected, cells are separated into two subgroups according to their size. At 2.2 mL/min flow rate, the majority of cells collected at the inner outlet are 18-30 ⁇ cells (about 30% of the test MSCs), while the cells at the outer outlet range from 15 ⁇ to 19 ⁇ (about 70% of the test MSCs).
- Microfluidic channels were cast from a polymethy methacrylate (PMMA) mold made by a precision milling process (Whits Technologies, Singapore).
- PMMA polymethy methacrylate
- the design consists of a single inlet, two-outlet spiral channel with multiple loops and curvature radius of about 10 mm.
- the patterns were cast with Sylgard 184 Silicone Elastomer (PDMS) prepolymer mixed in a 10: 1 ratio with the curing agent. After curing, the PDMS mold with patterns was peeled and plasma bonded to another 3mm thick PDMS layer. Input and output ports were punched prior to bonding.
- PDMS Sylgard 184 Silicone Elastomer
- the device was cut along the output section of the channel with about 2 mm distance and then a second cast was made by keeping the device vertical to a flat bottle container. Tubings were connected to the ports before the second cast to prevent PDMS mixer flow into the channel.
- the microfluidic device was placed on an inverted microscope (Olympus X71) and fluorescence images were captured with a Phantom V9.1 camera (Vision Research Inc. USA) near the end of the channel.
- Input samples were made by diluting 1% solid fluorescent particles (Bangs Laboratories, Inc. USA) of different sizes with DI water and pumped into the channel under different flow rates with a NE-1000 syringe pump (New Era Pump Systems, Inc. USA) to observe the focusing positions.
- NE-1000 syringe pump New Era Pump Systems, Inc. USA
- FIG. 10A shows the focusing bands of different sized particles with increasing flow rates as viewed from the top.
- the results clearly show the separation principle, with particle streams of different sizes shifting from the inner wall (inertial regime) to the outer wall (Dean regime) at different flow rate.
- particle streams of different sizes shifting from the inner wall (inertial regime) to the outer wall (Dean regime) at different flow rate.
- particle streams of different sizes shifting from the inner wall (inertial regime) to the outer wall (Dean regime) at different flow rate.
- particles with >15.5 ⁇ diameter can be separated from smaller ones by collecting from the inner and outer outlets.
- increasing the flow rate to 2.5 mL/min enables the separation of particles with >26.25 ⁇ diameter from smaller ones.
- FIGS. 1 lA-11C present the separation efficiency of two different size particles (16.68 ⁇ and 26.9 ⁇ ) at an optimized flow rate of 3.4 mL/min. The purity of both outlets collection are over 96%, while throughputs of 8.85xl0 6 /min can be reached, which is 1.33% volume to volume concentration (equivalent to hematocrit number in blood samples).
- FIG. 12 illustrates the forces that act on particles at different positions in thechannel cross section.
- Position #2 all the forces are in the channel width direction, but a slight disturbance along the channel depth direction will make F L change direction and increase in magnitude, which renders this point an unstable balance point.
- Position #3 is a stable force balance point at a low flow rate (0.5 mL/min).
- a trapezoidal cross-section spiral microfluidic channel has been developed for size based particle separation.
- the experimental results show that the channel is able to achieve high resolution and high throughput cell separation. Particles were successfully separated between 16.68 ⁇ and 26.9 ⁇ particles at 1.33%
- Inertial microfluidics has recently drawn wide attention as an efficient, high- throughput microfluidic cell separation method.
- the achieved separation resolution and throughput, as well as the issues with cell dispersion due to cell-cell interaction have appeared to be limiting factors in the application of the technique to real-world samples such as blood and other biological fluids.
- a novel design of spiral inertial microfluidic (trapezoidal cross section) sorter with enhanced separation resolution is presented herein and its ability is demonstrated in separating / recovering polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes (MNLs) from diluted human blood (1-2% hematocrit) with high efficiency (>80%).
- PMNs polymorphonuclear leukocytes
- MNLs mononuclear leukocytes
- Red blood cells or erythrocytes are the most abundant cell component in many biological fluids, including blood (where it makes up about 45% of the volume), bone marrow aspirate and peritoneal aspirate. Depletion of contaminating RBCs from those samples is often an indispensable sample preparation step before the application of any scientific, clinical and diagnostic tests due to various reasons.
- blood where it makes up about 45% of the volume
- bone marrow aspirate and peritoneal aspirate.
- peritoneal aspirate peritoneal aspirate.
- Depletion of contaminating RBCs from those samples is often an indispensable sample preparation step before the application of any scientific, clinical and diagnostic tests due to various reasons.
- inadvertent lysis of RBCs releases hemoglobin, leading to chemical interference and deteriorating the PCR-based test performances.
- WBCs white blood cells
- leukocytes which play a key role in carrying out and mediating the immune response to various pathogens.
- the information extracted from the isolated leukocytes would be meaningful to facilitate disease prognosis only when the key features of leukocytes' original state are not masked by sample preparation artifacts.
- a "deterministic lateral displacement (DLD)" microchannel containing an array of microposts leads to differential lateral displacement for particles above or below the critical hydrodynamic diameter as a result of the asymmetric bifurcation of laminar flow around the microposts.
- DLD dynamic lateral displacement
- PFF pinched-flow fractionation
- the parabolic velocity profile of laminar flow within the contraction region leads to the alignment of particles near the channel sidewall in a size-based manner, so that large particles with comparable diameter to the channel width of the contraction region stay closer to the middle streamlines, but smaller particles have their center closer to the channel sidewall.
- This difference in lateral positions of particles with varying size is further amplified upon entering the expansion region, resulting in efficient separation.
- Both techniques have the high resolution required for separating RBCs from other cell types but are severely limited in their practical application on clinical samples by the low processing throughput.
- the separation resolution of curvilinear microchannels has been improved, while maintaining the high- throughput feature, by modifying the channel cross-section to be trapezoidal rather than rectangular, and its ability is demonstrated below for efficient RBC depletion from a human blood sample with negligible effects on PMN immuno-phenotype.
- the current design can directly process the diluted whole blood sample when the blood sample volume is on the order of a microliter, (e.g. fingerprick), and as a
- the trapezoidal cross-sectional spiral microchannel described herein can be used as a generic, highthroughput method for removing RBCs and enriching target cells from other biological fluids, such as harvesting mesenchymal stem cells (MSCs) from bone marrow aspirates.
- MSCs mesenchymal stem cells
- the trapezoid cross-sectional spiral channels were made of
- PMMA poly(methyl methacrylate)
- PDMS prepolymer mixed with curing agent in a 10: 1 (w/w) ratio was then cast on the PMMA template master and cured under 80°C for 2 hours to replicate the microchannel features.
- the cured PDMS molds were peeled off from the PMMA master and punched for the inlet and outlet reservoirs using a 1.5 mm- diameter biopsy punch.
- the PDMS molds were irreversibly bonded to another flat 0.5 cm-thick PDMS sheet following oxygen plasma treatment (Harrick Plasma Cleaner/Sterilizer, Harrick Plasma, Inc., USA)
- the resulting PDMS devices were cut at four different locations and the cross-sections were measured under microscope to determine the exact dimensions of the devices.
- the rectangular cross- sectional spiral channels were also fabricated in PDMS polymer, but by using a double molding process from an etched silicon wafer master (See Bhagat, A.A.S., et al., Lab on a Chip, 201 1. 1 1(11): p. 1870-1878). Briefly, positive AZ4620 photoresist was first patterned on a 6-inch silicon wafer to define the microchannel features. Then, the patterned wafer was etched to the desired depth using deep reactive ion etching (DRIE), followed by residual photoresist removal using oxygen plasma treatment.
- DRIE deep reactive ion etching
- trichloro(lH,lH,2H,2H-perfluorooctyl)silane (Sigma- Aldrich, USA) was coated on the etched wafer for 1.5 hours (h) to assist PDMS mold release.
- PDMS liquid mixture with 5 parts of prepolymer and 1 part of curing agent was then poured onto the silicon wafer and cured under 80 °C for 2 hours.
- the resulting PDMS mold had channel features protruding from the surface and served as a master for subsequent PDMS molding.
- the silane treatment and PDMS curing was repeated with this PDMS master to get a negative replica.
- the negative replica with inlet and outlet reservoirs punched, was bonded to another PDMS substrate by standard plasma-assisted bonding.
- fluorescent polystyrene particles (1 wt. % solid content) with a diameter of 6 ⁇ (5.518 ⁇ ⁇ 0.122 ⁇ ), 10 ⁇ (10.3 ⁇ ⁇ 0.4 ⁇ ) (Polysciences, Inc., USA), or 15.5 ⁇ (15.5 ⁇ ⁇ 1.52 ⁇ , Bangs Laboratories, Inc.) were diluted in deionized water (0.1% volume fraction) containing 0.025 mg/mL PEG-PPG-PEG Pluronic® F-108 (Sigma- Aldrich, USA), respectively, serving as the input sample.
- the device was mounted on an inverted phase contrast/epifluorescence microscope (Olympus 1X71, Olympus Inc., USA) equipped with a 12-bit CCD camera (C4742-80-12AG, Hamamatsu Photonics K.K., Japan). Samples were loaded within a syringe and pumped through the microchannel at varying flow rates using a syringe pump (Harvard Apparatus PHD 2000, Harvard Apparatus Inc., USA). To prevent the particle/cell sedimentation, a small magnetic stir bar placed inside the syringe was agitated during sample processing. Using ImageJ ® software, the positions of fluorescent particles within the channel cross-section were determined by taking the average fluorescence intensity of the image series.
- Both the input sample and the output samples from two outlets were collected and analyzed on BDTM LSR II flow cytometer (BD Biosciences, USA) to determine the WBCs (Hoechst-positive cells) and PMNs (CD66b-positive cells) in each sample.
- WBCs Hoechst-positive cells
- PMNs CD66b-positive cells
- MNL count was based on the number of Hoechst-positive but CD66b-negative cells.
- the RBC concentration was further measured by Z2 Coulter counter (Beckman Coulter Inc, USA).
- WBCs isolated by centrifugation with MP-RM were stained for surface marker, CD66b, and nucleus.
- the stained WBCs were then resuspended in sample buffer with the same volume of the initial whole blood volume and processed by the device.
- the size distribution of cells in the sample was measured by Z2 Coulter counter and a flow cytometer was used to analyze the sample composition.
- the gates for activated PMNs were drawn based on PMNs treated with 30 minutes of 1 ⁇ phorbol 12-myristate 13- acetate (PMA; Sigma- Aldrich, USA) under 37°C (complete activation achieved), followed with
- WBCs isolated by the spiral process with 1 % hematocrit input sample were resuspended in sample buffer to a final concentration of about 1 million cells/mL.
- 40 ⁇ _, of each cell sample was deposited onto Poly-L-lysine coated glass slide (Sigma- Aldrich, USA), respectively, where the sample region had been circled using Hydrophobic Barrier Pen (ImmEdgeTM Pen, Vector Laboratories, Inc., USA).
- the assay buffer for NBT test was freshly prepared and consisted of lx Ca 2+ /Mg 2+ -containing DPBS buffer (Dulbecco's Phosphatase Buffered Saline;
- the assay buffer also contained 1 ⁇ PMA. After incubation, 40 ⁇ . of assay buffer was added onto the slide for 20 minutes of incubation at 37 °C. Lastly, the cell sample was observed under phase contrast microscope (Olympus CKX41, Olympus Inc., USA) and color images were taken by a DSLR camera (Canon EOS 500D, Canon, USA) with a 60x objective under microscope using a NDPL-1 (2x) connecting lens (Vivitar® Sakar International, Inc., USA).
- Inertial lift forces include the shear- induced lift force resulting from the parabolic velocity profile of flows in a confined channel (See Di Carlo et al., 2007) and the wall-induced lift force arising from the disturbed rotational wake around the particles when close to the wall (See Zeng, L., S. Balachandar, and P. Fischer, Journal of Fluid Mechanics, 2005. 536: p. 1-25).
- a p IDh 0.07
- D h —— is the microchannel hydraulic diameter
- a and P are the area and perimeter of channel cross-section, respectively
- the interplay between shear-induced and wall-induced lift forces leads to lateral migration of the initial randomly distributed particles to stable equilibrium positions around the microchannel periphery.
- the magnitude of the vortex flow can be expressed using the non-dimensional Dean number (De) and the viscous force, known as Dean drag force (F D ), experienced by the particles can be quantified by assuming Stokes drag.
- microchannel can be applied as a possible size-based particle/cell separation device.
- the modified velocity field of a spiral with a trapezoidal cross-section leads to a greater shift for small particles towards the outer wall without affecting the focusing position of large particles, thus providing a greater difference in equilibrium positions between them, resulting in higher separation resolution, as shown in FIGS. 14A-14D.
- the trapezoidal cross-section also has an impact on the size- and flow-rate- dependence of particle focusing.
- particles with a p /D h ⁇ 0.07 initially focus near the inner channel wall at low Re c , and then move towards the outer wall as Re c increases.
- Re c is sufficiently high, Dean drag force dominates the particle behavior leading to de focusing of particles.
- the results presented herein indicate that instead of D h , the channel depth at the inner wall (D inner ) serves as a better critical channel dimension to determine whether particles of a certain diameter can form a focused stream near the inner wall. This was confirmed by using trapezoid channels satisfying a D outer I D inner ⁇ 1.5 criterion as shown in FIGS.
- the optimized PDMS device for RBC removal developed herein consists of a 1 -inlet, 2-outlet spiral microchannel with a trapezoidal cross-section of 500 ⁇ width (485.00 ⁇ ⁇ 2.31 ⁇ ), 70 ⁇ (inner wall, 72.84 ⁇ ⁇ 1.16 ⁇ ) and 100 ⁇ (outer wall, 102.65 ⁇ ⁇ 3.55 ⁇ ) depth. Near the outlet region, the 485 ⁇ wide channel was split into two outlet channels with a channel width ratio of 3 : 7 (inner : outer), while their channel lengths were adjusted to be equal with each other.
- the inner outlet was defined to be the WBC outlet with RBC-depleted sample ⁇ i.e., PMNs MNLs) and the outer outlet to be the RBC waste outlet.
- PMNs and MNLs isolated via centrifugation using MP-RM were injected through the device separately to determine their equilibrium positions inside the channel, shown in FIG. 17 A. Under optimal flow rate, PMNs formed a focused stream at a distance of about 75 ⁇ away from the inner channel wall in the top-down view, and MNLs occupied a similar lateral position but had a slightly wider stream width presumably due to the smaller cell size.
- 17B shows the recovery of blood components from the WBC outlet of the present device after a single pass, where optimal performance was achieved for a 0.5% hematocrit blood sample with about 95% RBC removal and 98.4% of total WBC recovery (99.4% PMN recovery and 92.4% MNL recovery).
- the device's throughput translates to about 10 of whole blood (45% hematocrit) per minute which is significantly higher than other microfluidic leukocyte isolation devices, such as "hydrodynamic filtration” with about 29 fold WBC enrichment at 20 ⁇ !7 ⁇ for 10-fold diluted blood (See
- the device can still achieve 86.8% RBC removal and 96.2% of total WBC recovery.
- a 2-stage process where the output sample from the WBC outlet of the first device was used as the input of the second device without any dilution, achieved high RBC removal while maintaining good WBC recovery for 1%-1.5% hematocrit samples, as shown in FIGS. 17C and 17D. Since WBCs collected from the first stage were
- the device can also be used as a secondary step of differential centrifugation, whose performance was subjected to variance of blood source and manual transfer of different cell layers to different tubes. It is often the case that some RBC residuals stay with the isolated WBCs after the first 30-min centrifugation and additional slow centrifugation washing steps or RBC lysis step are required for further RBC removal.
- the RBC removing ability of this device was demonstrated in processing buffy coat in a case where notable amounts of RBCs were isolated along with WBCs by centrifugation (FIGS. 18A and 18B). Based on the size distribution of cell sample, it was observed that the WBC percentage (cell diameter: 6.6 - 15 ⁇ ) among the whole population increased from 30% to 91 % after processing by this device.
- a novel, high-throughput RBC removal technique has been developed using a trapezoidal cross-sectional spiral, which provides higher resolution separation as compared to a rectangular cross-section with similar dimensions, as shown by an experimental demonstration where the asymmetry velocity field within a trapezoidal spiral channel affects the inertial focusing phenomenon, indicating the feasibility of using channel cross-sectional geometry (other than width and depth) as a parameter for optimization of a curvilinear inertial microfluidic sorter.
- This size -based separation technique eliminates the need for long-term exposure of blood cells to nonphysiological conditions and thus minimizes artificial alterations on cellular phenotypes during separation.
- the spiral microchannel described above functions at a high operational flow rate (in the mL/rnin range) with large channel dimensions accommodating the abundant RBCs (up to about 2% HCT), and thus possesses high throughput and is amenable to process blood samples.
- the highly repeatable performance and ability in enriching WBCs to >90 of total cell population also makes it a good choice to completely deplete RBCs from various biological fluids when used alone or in combination with differential centrifugation. Further optimization of channel cross-section and other structural features is possible to apply this technique to many other primary cell separation problems.
- FIG. 20 shows the focusing positions of 15.5 ⁇ particles from the top and side view in a spiral channel with a 80 ⁇ x 600 ⁇ (H x W) rectangular cross- section.
- the focusing position of the particles moves gradually from the inner wall towards the outer wall with increasing flow.
- two clear bands are observed along the depth direction, indicating two distinct focusing positions near the top and bottom walls.
- the focusing position along the depth direction is largely independent of the flow rate, and remains fixed at 22.0 ⁇ 1.1% of channel depth from the top and bottom walls for flow rates ranging from 0.5-7.5 mL/min. The result is in line with previous simulation and
- FIG. 22 shows the flow rate dependence of the focusing position of fluorescent particles in a spiral channel with rectangular and trapezoidal cross- sections.
- the diameters of the particles were 5.78 ⁇ , 9.77 ⁇ , 15.5 ⁇ , and 26.25 ⁇ respectively.
- the cross-sections of the rectangular channel were 80 ⁇ x 600 ⁇ (H x W) and 120 ⁇ x 600 ⁇ (H x W) respectively.
- the width of trapezoidal channel was fixed at 600 ⁇ , and the depths at the inner and outer side of the channel are 80 ⁇ and 130 ⁇ , respectively.
- the results shown in FIG. 22 demonstrate that in the rectangular channels, particles were focused near the inner channel wall at low flow rate, and then the focusing position started to gradually move towards the outer wall as the flow rate increases.
- the ideal scenario is for particles of different sizes to focus at positions as far as possible from each other. This will not only increase the separation resolution, but also allows one to process samples with higher particle concentrations by minimizing the interaction between particles of different sizes (e.g., high hematocrit cell solutions in the case of blood separation).
- the results in FIG. 22 demonstrate that the trapezoidal cross-section channel met these requirements.
- FIG. 23 presents the collection of different standard National Institute of
- FIGS. 24A-24B The high throughput separation capability of trapezoidal channels is presented in FIGS. 24A-24B with fluorescently labeled particles which have different mean diameters.
- the scatter plots in FIG. 24A indicate two groups of particles and their separation efficiency.
- the separation results of 15.5 ⁇ and 18.68 ⁇ beads at 1.61xl0 7 /min (1.87% volume concentration, equivalent to 'hematocrit number' in blood separation) throughput indicates over 92% separation efficiency.
- the separation results of 18.68 ⁇ and 26.9 ⁇ at an optimized flow rate of 3.4 mL/min show that the purity of both outlets was over 96%, with a total throughput of 8.85xl0 6 /min, which is 1.33% volume concentration.
- a microscope image demonstrating the separation between 18.68 ⁇ and 26.9 ⁇ particles is shown in FIG. 24B.
- the high-speed image indicates the separated particle streams near the opposite channel walls at the outlet.
- Cells are different from rigid particles in terms of the deformability and shape. Hur et al. have reported that the shape of particles does not have an obvious influence on the focusing position in inertial microfluidics, but the hydraulic diameter of particles is the key factor, while the deformability has an evident effect on the focusing position of particles/cells, which makes the focused band of particles/cells slightly shift away from the channel wall as compared to that of rigid beads of the same size. If the device is employed in cell separation, the variation of cell deformability may affect the separation efficiency. But as shown in Example 2 above, the trapezoidal spiral is capable of producing comparable separation between deformable leukocytes and red blood cells, perhaps aided by the large distance between the inner focusing and outer trapping positions.
- a coordinate system (x, y, z) is defined as shown in FIGS. 20 and 21.
- the direction along the channel curve (main flow direction) is along the x axis.
- the direction along the channel depth is the y axis, and the radial direction along the channel (the width direction), is the z axis.
- FDD is primarily acting parallel to the z-axis. In the region between 28 ⁇ 0.5% and 72 ⁇ 0.5% of the channel depth, FDD points to the negative direction of the z-axis, while at regions near the top/bottom wall, it follows along the positive direction of the z-axis.
- a trapezoidal channel with shallow inner and deep outer cross-section causes the main flow to shift towards the outer side of the channel cross- section. This generates a stronger Dean flow at the outer side and a weaker Dean flow at the inner side (FIG. 26).
- F DD has two components here, a component along y-axis pointing toward the center of channel cross-section near "zero Dean flow plane", termed as Fo y and the corresponding component along z-axis, termed as FD z - From FIG.
- the resultant force of F D D and FL will push the particle to an equilibrium position close to the center of the vortices.
- the forces acting on the particle near vortex center are illustrated in FIG. 26.
- the trapping is caused by a dynamic balance of these two forces, which rely on many parameters, such as the slant of channel, the flow rate, and the diameter of particle. For example, under the resultant force of FDD and FL, a particle at position b will tend to cross the minimum lift plane and migrate towards position c, where a strong FDD can then push the particle towards position d.
- a trapezoidal cross-section with a deeper inner wall compared to the outer wall will have strong vortices formed at the inner side, resulting in all the particles being trapped despite varying particle size and flow rate.
- Such geometry is not applicable for size based separation.
- Microchannels were also fabricated with the top wall having a concave, convex or just a regular slanted top wall and their effect on particle focusing and trapping was studied. The experimental comparison of these three patterns is discussed below.
- the slant of the channel affects the focusing .behavior in two ways: (i) the threshold flow rate required to trap particles in the Dean vortex as a function of particle size and (ii) the location of the Dean vortex core.
- a large slant angle will lead to strong Dean at the outer side and increase trapping capability of particle.
- Large slant angle can also decrease the threshold flow rate required to trap the particles of a certain size within the Dean vortex.
- the tolerance of the pattern is controlled to within 10 ⁇ in the x-y direction and 2 ⁇ in the z-direction with a surface roughness of Ra of about 0.8 ⁇ .
- the mold was carefully inspected and its dimensions were measured accurately before use.
- the microchannels were then made by casting Sylgard 184 silicone elastomer (PDMS) prepolymer mixed in a 10: 1 ratio with the curing agent. After curing, the PDMS was peeled from the mold and plasma bonded to another 3 mm thick flat PDMS layer. Input and output ports were punched prior to bonding.
- PDMS silicone elastomer
- the device was cut along the periphery of the spiral channel with about a 2 mm gap between the channel and the edge of the PDMS part.
- the PDMS mold with the microchannel pattern was then placed vertically in a flat-bottomed petri-dish and a second cast of PDMS was poured to hold the chip vertically (FIG. 27B).
- Tygon tubing was connected to the ports before the second cast to prevent PDMS from flowing into the channel.
- the device is placed on an inverted microscope and the image of the straight section is captured using fluorescent particles. Since PDMS is an elastic material the cross-section of the channel would undergo pressure induced
- a spiral channel with a low aspect ratio rectangular cross-section was fabricated.
- the microchannel was 600 ⁇ wide and 80 ⁇ deep, with the aspect ratio of 7.5 (width/depth). If the refractive index difference between the fluid and PDMS channel is large, the imaging of fluorescent particles within the channel through a thick piece of PDMS is challenging due to significant refraction at the interface.
- dimethyl sulfoxide (DMSO) was mixed with ethanol in a 1: 1 volume ratio, which produces a mixture with refractive index of 1.42, density of 0.9805 g/ml and viscosity of 0.978 mPa » s at 298.15K.
- the refractive index of the mixture is similar to that of PDMS (1.43) and enhances imaging by elimination of refraction based dispersion.
- the solution was shown to dissolve the polystyrene (PS) particles (Bangs Laboratories, Inc. USA) and Tygon tubing after prolonged immersion of 1 week. However, for the short duration of the experiments, the fluid mixture had no effect on both the particles and the tubings, making it an ideal replacement to water for the PS particles (Bangs Laboratories, Inc. USA) and Tygon tubing after prolonged immersion of 1 week. However, for the short duration of the experiments, the fluid mixture had no effect on both the particles and the tubings, making it an ideal replacement to water for the
- the Dean flow field of the fluid in curved channel was simulated using commercial computational fluid dynamics (CFD) software COMSOL 4.2a
- a particle flowing with surrounding fluid is subject to the following known forces: the drag force F D , the centrifugal force F c , the buoyancy force, i.e., the pressure gradient force F B , two unsteady forces due to a change of the relative velocity, the added mass force or virtual mass force F A , and the Basset History force F H , the gravitational force FG, and the inertial lift force FL.
- Drag force FD The drag force on the particle is in the direction of relative flow velocity U R with respect to the particle.
- the magnitude of F D can be given as
- D is the (hydraulic) diameter of tube
- co e are the slip angular velocities of the particle at a position with relative distance d from the center of the tube's cross-section and equilibrium position respectively.
- ⁇ ⁇ / ⁇ ⁇ is a function of particle position d and Reynolds number Re
- FL is zero at the equilibrium position, which is around 20% of D from the wall, and changes its direction when the particle moves across the equilibrium position.
- the magnitude of FL near the equilibrium position, according to above equations, is approximately proportional to U m U s ⁇ 0.05U ⁇ .
- p p and V p represent the density and volume of the particle, respectively.
- Buoyancy force F B - In a fluid with constant pressure gradient, particles are subjected to a buoyancy force pointing to the center of the channel curvature, i.e., along the z-axis direction,
- Basset history force FH - The Basset force describes the force due to the lagging boundary layer development with changing relative velocity (acceleration) of bodies moving through a fluid. It is difficult to calculate accurately and is commonly neglected for practical reasons.
- the resultant force FABC is pointing to the center of the channel curvature, and the magnitude is proportional to U U s , i.e. proportional to 3 ⁇ 4 ⁇ It is one or two order smaller than FDD, as shown in FIG. 29.
- the effect of FABC on the focus position is thus not dominant.
- the slant of the channel affects the focusing behavior in two ways: (i) at lower inner side, the increase of channel depth breaks the balance of the lift and drag force at high flow rate resulting in particle migration to the outer side and trapped at the vortex core, i.e., determines the threshold flow rate; and (ii) the location of the Dean vortex core.
- a large slant angle will lead to strong Dean at the outer side and increase trapping capability of particle.
- a large slant angle can also decrease the flow rate to drag out particles from the inner side. Particles will switch to the outer side at lower flow rate for a large slant angle channel, which is confirmed by experimental observation shown in FIG. 30 as well as the observation shown in FIG. 22.
- FIGS. 31A-31C normal trapezoidal cross-section with constant slant angle, top wall convex cross-section that have a large slant angle at the inner side but a small slant angle at the outer side, and a concave cross-section with only the outer half having a large slant angle.
- CTCs circulating tumor cells
- a spiral microfluidic device with trapezoidal cross-section is described herein for ultra-fast, label-free enrichment of CTCs from clinically relevant blood volumes.
- the technique utilizes the inherent Dean vortex flows present in curvilinear microchannels under continuous flow, along with inertial lift forces which focus larger CTCs against the inner wall.
- Using a trapezoidal cross-section as opposed to a traditional rectangular cross-section the position of the Dean vortex core can be altered to achieve separation.
- This approach can surmount the shortcomings of traditional affinity-based CTC isolation techniques as well as enable fundamental studies on CTCs to guide treatment and enhance patient care.
- the trapezoidal channels only need a single inlet for the sample, in which the sample can be introduced, e.g., using a single syringe pump, and two outlets for waste and enriched cell collection, respectively, during operation.
- the sample can be introduced using a piston pump, a gear pump, a peristaltic pump, a piezoelectric micropump, or using a controllable pressure regulator.
- a microfluidic device with this newly designed microchannel, enrichment of a high number of CTCs (3-125 CTCs/mL) from peripheral blood of patients with metastatic breast and lung cancer has been demonstrated.
- This device can process 7.5 mL of red blood cell lysed blood in about 8 min, allowing enrichment of viable CTCs with relatively high purity and yield.
- the trapezoidal spiral channels can be produced at extremely low-cost and with high resolution using conventional micro- milling and PDMS casting, and can be operated using a single syringe pump, which facilitates easy automation.
- This strategy can be utilized for large-scale processing of clinical samples in order to enrich sufficient amount of CTCs for various detailed molecular analyses as well as clinical monitoring of individual patients undergoing therapy.
- the device is well suited to process even larger quantities of blood if required (20 mL in about 15 min), to satisfy a growing need for obtaining large number of CTCs for multiple downstream tests.
- the device design consists of an 8-loop spiral microchannel with one inlet and two outlets with radius increasing from 8 mm to 24 mm for efficient cell migration and focusing.
- the width of the channel cross-section is 600 ⁇ and the inner and outer heights were optimized at 80 ⁇ and 130 ⁇ , respectively, for the trapezoid cross-section.
- the mold with specific channel dimensions was designed using SolidWorks software and then fabricated by conventional micro-milling technique (Whits Technologies, Singapore) on polymethyl methacrylate (PMMA) sheet for subsequent PDMS casting.
- the microfluidic device was fabricated by casting degassed PDMS (mixed in a 10: 1 ratio of base and curing agent, Sylgard 184, Dow Corning Inc.) on the mold and subsequent baking inside an oven for 2 hours at 70 ° C. After curing, the PDMS was peeled from the mold and access holes (1.5 mm) for fluidic inlet and outlets were punched with a Uni-CoreTM Puncher (Sigma- Aldrich Co. LLC. SG) and the PDMS devices were irreversibly bonded to another layer of cured PDMS using an oxygen plasma machine (Harrick Plasma, USA) to complete the channels. The assembled device was finally placed inside an oven at 70 C for 30 minutes to further enhance the bonding. Cell culture and sample preparation
- GFP green fluorescent protein
- the cells were seeded into coated T25 flasks (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and cultured with high-glucose Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) and 1% penicillin- streptomycin (Invitrogen, USA).
- DMEM Dulbecco's modified Eagle's medium
- FBS fetal bovine serum
- penicillin- streptomycin Invitrogen, USA
- the culture was kept in a humidified atmosphere at 37 °C containing 5% (v/v) C0 2 and harvested at 80% confluence for spiking.
- Sub-confluent monolayers were dissociated using 0.01% trypsin and 5.3mM EDTA solution (Lonza, Switzerland).
- the spiral biochip was initially mounted on an inverted phase contrast microscope (Olympus 1X71) equipped with a high speed CCD camera (Phantom v9, Vision Research Inc., USA).
- the biochip was primed with a priming buffer (lx PBS, 2 mM EDTA supplemented with 0.5% BSA) using a syringe pump (PHD 2000, Harvard Apparatus, USA) for around 2 minutes at a flow rate of 2 mL/min.
- a priming buffer lx PBS, 2 mM EDTA supplemented with 0.5% BSA
- PPD 2000 Harvard Apparatus, USA
- cancer cells and blood sample were filled in a 10 mL syringe and pumped through the device using a syringe pump connected to the microchannel through flexible Tygon® tubing.
- the flow rate was set to 1700 ⁇ 7 ⁇ for all the experiments.
- High speed videos were captured at the channel outlet using the Phantom Camera Control software and then analyzed using Image
- CTCs were identified by staining with FITC-conjugated pan-cytokeratin (CK) (1: 100, MiltenyiBiotec Asia Pacific, Singapore). Cells staining positively for pan-CK and Hoechst (nuclei stain) and negatively for CD45 with characteristic morphology of cancer cells (i.e., high nucleus to cytoplasm ratio) are identified as CTCs. Cells staining positively for CD45 and Hoechst and negatively for pan-cytokeratin are identified as leukocytes.
- CK pan-cytokeratin
- MDA-MB-231 and MCF-7 GFP-tagged cells mixed with blood from healthy donors were processed through the spiral microfluidics, and cell viability was assessed via trypan blue (or Propidium iodide (PI)) exclusion assay and through long-term re-culturing.
- Isolated CTCs were seeded onto polylysine-coated 2D cell culture substrates and cultured overnight as described. Cells were then stained with propidium iodide (PI) stain in situ. Cells were imaged and enumerated for PI positive staining to determine the percentage of cell viability after lysis and processing. The cell viability numbers were compared with cells obtained after lysis without spiral biochip processing.
- Clinical samples were compared with cells obtained after lysis without spiral biochip processing.
- Fluorescence in situ hybridization was performed on SKBR3 (amplified HER2 signals) and MDA-MB-231 (non- amplified HER2 signal) cells lines as well as isolated CTCs according to the manufacturer's protocol. Cells were spun onto slides using a Cytospin centrifuge (Thermo Scientific, USA) at 600 rpm for 6 minutes. Slides were fixed in 4% PFA at room temperature for 10 minutes and dehydrated via ethanol series (80%, 90%, and 100%). For FISH analysis, slides were treated with RNase (4 mg/mL) (Sigma, USA) for 40 minutes at 37 °C, washed with lx PBS/0.2% Tween 20 (Sigma, USA) thrice and denatured with 70%
- FITC fluorescein isothiocyanate
- CK pan-cytokeratin
- FITC fluorescein isothiocyanate
- FIG. 32B shows a picture of the experimental setup during sample processing.
- the continuous collection of CTCs facilitates coupling of the device with conventional 96-well plate or a membrane filter for subsequent downstream analysis such as immunostaining, qRT-PCR, FISH and sequencing.
- An optimal technology for CTC isolation must aim to isolate the maximum number of viable cells with acceptable degree of purity (i.e., depending upon the contamination tolerance of downstream molecular assays) without relying on specific markers (e.g., EpCAM) with minimum sample processing steps.
- a conventional chemical RBC lysis approach was utilized to boost the throughput of the system while maximizing the number of enriched CTCs.
- Extensive characterization was performed to find the optimal device design by studying the effect of various parameters, including channel aspect ratio, flow rate and sample concentration using both latex particles and healthy blood samples spiked with cancer cell lines.
- WBCs and platelets concentrations are relatively high (> 3%) in the lysed blood, their complete removal is pivotal for achieving meaningful enrichment.
- processing of blood was carried out under different nucleated cell concentrations.
- Initial 7.5 mL whole blood samples collected from healthy donors were lysed chemically using ammonium chloride and the nucleated cell fraction was re- suspended back to 15 mL (0.5x concentration), 7.5 mL (lx concentration) and 5 mL (1.5x concentration) using PBS buffer for processing.
- FIG. 34 A shows the total cells count (DAPI+/CD45+) collected from the CTC outlet at different sample concentrations.
- This graph shows that this device perform best when cell concentration is below lx (about 3.5-4xl0 6 WBC/mL) where minimum contamination of WBCs is observed (mean, 500 WBCs/mL of lysed blood; range, 400 to 680 WBCs/mL).
- 0.5 x concentration was selected as being optimal for processing of clinical samples which translates to a total processing time of around 8 min for a 7.5 mL blood sample. It is believed that this is the highest throughput achieved by a microfluidic platform for CTC isolation reported to date.
- the processing time can be further decreased by multiplexing of biochips together.
- CTCs are extremely rare in blood stream, it is crucial to isolate the maximum number of target cells in a blood sample for various downstream assays.
- three different cell lines i.e., MCF-7, T24 and MDA-MB-231
- MCF-7, T24 and MDA-MB-231 were employed in this study to quantify the performance of the trapezoidal spiral biochip for CTC isolation and recovery. These cell lines were chosen to ascertain the versatility of the technique in the detection of CTCs.
- the aforementioned cells have an average diameter in a range of between about 10 ⁇ and about 50 ⁇ .
- the model system is constructed by spiking a known number of cells (about 500 cells) into 7.5 mL of blood obtained from healthy donors.
- the isolated cells were re- cultured onto 2-D culture substrates where they attached and proliferated under standard culture conditions (see FIG. 35).
- the viability of cells before and after processing was also validated using functional assays including staining with propidium iodide (PI) and/or Trypan blue.
- PI propidium iodide
- the results demonstrate high viability of captured cells confirmed by their minimal staining ( ⁇ 10%) with Trypan blue (see FIG. 34C). Further morphological analysis of cancer cells also confirmed that cells remain relatively unchanged during multiple steps of processing (data not shown).
- a 7.5 mL of blood sample was obtained from each of (i) 5 healthy individuals (control), (ii) 5 patients with metastatic breast cancer (MBC) and (iii) 5 patients with non-small cell lung cancer (NSCLC) (Table 1).
- Presence of isolated CTCs was determined by immunostaining with Hoechst (DNA), FITC-pan-cytokeratin (CK) antibodies (cancer/epithelial biomarker), and APC-anti-CD45 antibodies (hematologic biomarker) (see FIG. 36A).
- Hoechst+/pan- C +/CD45- cells were scored as CTCs.
- Data presented in Table 1 demonstrates the clinico-pathological characteristics of the breast and lung cancer patients, as well as the CTC counts obtained from the spiral biochip.
- CTCs were detected in 10/10 patient samples (100% detection) with counts ranging from 6-57 CTCs/mL for MBC samples and 3-125 CTCs/mL for NSCLC samples (FIG. 36B).
- Epithelial cells positive for cytokeratin were also detected in healthy volunteers (1-4 per mL), but a distinct detection threshold can be drawn in comparison with that of patient samples. Threshold analysis demonstrated 3-4 CTCs per 7.5 mL of blood sample as the optimal cut-off value for predicting metastatic disease.
- Enriched CTCs are highly heterogeneous as previously reported in various studies (see FIG. 37). Staining of cancer stem cell markers CD44 and CD24 reveals distinct populations of CTCs which are mostly either CD44+/CD24- or CD44- /CD24+ (FIG. 36C). CD44+/CD24- cells are evidently larger in size than the CD44- /CD24+ cells. It should also be noted that a portion of CTCs are likely apoptotic. This is demonstrated by staining for cleaved caspase-3 marker which plays an integral role in the apoptotic process of mammalian cells.
- EpCAM-/pan-CK+ cells were detected in the isolated CTCs.
- the population of EpCAM-/pan-CK+ cells were much lower among lung cancer samples as compared to breast samples, indicating significant limitation of EpCAM based approaches for accurate detection and enrichment of putative CTCs.
- the ability to capture viable CTC is demonstrated by overnight culture of the isolated cells. In this study, isolated cells, shown in FIG. 35, were seeded onto polylysine coated well culture plates overnight under culture conditions as described above. Viable cells were able to spread onto the substrates and appeared negative for propidium iodide (PI) when stained.
- PI propidium iodide
- HER2+ CTCs may be observed in about 30% of samples obtained from HER2- origin.
- DNA fluorescence in-situ hybridization FISH was carried out to evaluate HER2 status in isolated CTCs.
- HER2 signals in isolated CTCs were compared against control breast cancer cell lines MDA-MB-231 (non- amplified HER2 signal) and SKBR3 (amplified HER2 signals) as shown in FIG. 39.
- Amplified HER2 expression is determined when the ratio of HER2/centromere of Chromosome 17 (Cenl7) signals in single nuclei is > 2. A range of HER2/Cen-17 signal was observed.
- cells with amplified HER2 signals were also detected, indicating the definite presence of CTCs. This is in accordance with previous findings that heterogeneity of HER2 status is evident in CTC as compared to the primary tumor.
- the "Holy Grail" of cancer medicine is the establishment of personalized therapies, where treatments shift from fixed regimes to therapies tailored to individual patients' tumor conditions. Circulating tumor cells have been shown to be a good alternative to primary tumor biopsies, carrying similar genetic information. Detection of CTCs in the peripheral blood of cancer patients at different disease stages has shown promise as a prognostic marker for treatment efficacy and patient survival, indicating strong clinical relevance. However, systematic characterization of CTCs in vitro via downstream assays has been delayed by the lack of reliable and sensitive methods to detect and enrich these cells. Despite the rapid advancement in microfluidic technologies, the isolation of CTCs with high throughput, high purity and high cell viability remains elusive.
- CTCs Viability of isolated CTCs was also retained after processing which will allow potential culture and expansion studies.
- the majority of isolated CTCs from peripheral blood of breast cancer patients are viable but non-proliferative after days in culture, suggesting the requirement of new therapeutic approaches that targets cells in dormancy.
- the continuous collection of CTCs facilitates coupling of the device with conventional 96-well plate or a membrane filter for subsequent downstream analysis such as immunostaining, qRT-PCR, FISH and sequencing.
- the precision and recovery rates at low cell spiking levels given by the inertial microfluidic system were high. Because this approach does not require initial cell surface biomarker selection, it is suitable for use in different cancers of both epithelial and non-epithelial origin.
- CTCs are reported to be highly heterogeneous and variable in EpCAM and cytokeratin expression, biomarkers used for CTC enrichment in many microfluidic devices.
- the selection criteria of cell size will overcome this limitation and capture a wider proportion of CTCs.
- Cells with lower expression of specific cytokeratins will still be identified with immunostaining due to the use of pan-cytokeratin antibodies.
- the sensitivity of the system was analyzed by determining the recovery rate of GFP-tagged breast (MCF-7 and MDA-MB-231) and bladder (T24) cancer cell lines spiked into blood obtained from healthy volunteers at the concentration of 500 cells/7.5 mL of blood.
- CTCs CTCs isolated with the trapezoidal spiral biochip were treated by PAP stain and revealed high nuclear to cytoplasmic (N/C) ratio, which is characteristic of cancer cells.
- N/C nuclear to cytoplasmic
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Also Published As
Publication number | Publication date |
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CN104797340A (en) | 2015-07-22 |
EP3628401B1 (en) | 2021-06-23 |
EP2897730A4 (en) | 2016-04-27 |
US20150238963A1 (en) | 2015-08-27 |
AU2013318647B2 (en) | 2017-10-26 |
EP2897730A1 (en) | 2015-07-29 |
JP6265508B2 (en) | 2018-01-24 |
EP3628401A1 (en) | 2020-04-01 |
KR20150061643A (en) | 2015-06-04 |
SG11201501837TA (en) | 2015-04-29 |
AU2013318647A1 (en) | 2015-04-09 |
US9789485B2 (en) | 2017-10-17 |
JP2015535728A (en) | 2015-12-17 |
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