WO2011119962A2 - Enrichissement par voie microfluidique de populations cellulaires choisies - Google Patents

Enrichissement par voie microfluidique de populations cellulaires choisies Download PDF

Info

Publication number
WO2011119962A2
WO2011119962A2 PCT/US2011/029999 US2011029999W WO2011119962A2 WO 2011119962 A2 WO2011119962 A2 WO 2011119962A2 US 2011029999 W US2011029999 W US 2011029999W WO 2011119962 A2 WO2011119962 A2 WO 2011119962A2
Authority
WO
WIPO (PCT)
Prior art keywords
blood cells
white blood
array
channel
buffer
Prior art date
Application number
PCT/US2011/029999
Other languages
English (en)
Other versions
WO2011119962A3 (fr
Inventor
Mehmet Toner
Alan Fishman
Ronald G. Tompkins
Ravi Kapur
Original Assignee
The General Hospital Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The General Hospital Corporation filed Critical The General Hospital Corporation
Publication of WO2011119962A2 publication Critical patent/WO2011119962A2/fr
Publication of WO2011119962A3 publication Critical patent/WO2011119962A3/fr

Links

Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • 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
    • 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
    • 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
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

Definitions

  • This document describes technologies related to manipulation of cells, for example, cells obtained from whole blood.
  • the current standard of clinical care constitutes sterile withdrawal of 50 ml of peripheral blood into an anticoagulant containing syringe, time based sedimentation through hydroxyethyl starch to deplete red blood cells, washing white blood cells to remove residual plasma, incubating enriched white blood cells with tracer dye, washing the cell suspension repeatedly to remove unbound tracer dye, and measuring an aliquot to ensure labeling performance prior to re- infusion back into the patient.
  • the invention relates, at least in part, to an automated, continuous, stand-alone processing unit that resides at point of care and can be used to process blood drawn from a patient (e.g., into a Vacutainer® or a syringe) through a microfluidic device to provide an output of a labeled population of white cells, with very low carryover of red cells, platelets and plasma for reinfusion back into the patient.
  • a patient e.g., into a Vacutainer® or a syringe
  • a microfluidic device e.g., a microfluidic device to provide an output of a labeled population of white cells, with very low carryover of red cells, platelets and plasma for reinfusion back into the patient.
  • one aspect of the subject matter described herein is methods of labeling white blood cells by obtaining whole blood drawn from a patient, separating the white blood cells from the whole blood by flowing the whole blood through a
  • microfluidic device that includes one or more arrays of obstacles configured to deflect the white blood cells in a direction away from the direction of remaining components of the whole blood, and collecting the separated white blood cells.
  • a buffer containing a labeling agent with an affinity for white blood cells is mixed with the white blood cells either before or after separating the white blood cells from the whole blood resulting in labeled white blood cells.
  • Flowing the whole blood through the microfluidic device can include flowing the whole blood through the microfluidic device that includes a first channel extending from one or more inlets to one or more outlets.
  • the first channel can be bounded by a first side wall and a second side wall located opposite from the first side wall.
  • a first array of rows of obstacles can be disposed within the first channel and can be bounded by the first side wall and a first array boundary formed by ends of the rows of obstacles in the first array.
  • a second array of rows of obstacles can be disposed within the first channel and bounded by the second side wall and a second array boundary formed by ends of the rows of obstacles in the second array.
  • a bypass channel can be defined by a region devoid of obstacles between the first array boundary and the second array boundary. The first array and the second array can be configured to deflect the white blood cells toward the bypass channel and separate the white blood cells from remaining components of the whole blood.
  • the methods can further include infusing the labeled white blood cells into the patient.
  • the labeling agent can include a radioactive material.
  • the collected labeled white blood cells in the buffer can be flowed through a microfluidic concentration device that includes at least one channel at a flow rate that focuses the labeled white blood cells in the at least one channel into one or more localized stream lines.
  • Each stream line can define a width that is substantially equal to or greater than a size of the focused labeled white blood cells to increase a concentration of the labeled white blood cells in the buffer relative to a concentration of the labeled white blood cells in the buffer collected from the outlet of the bypass channel.
  • the labeling agent can be mixed with the white blood cells before or after the white blood cells are flowed through the microfluidic concentration device.
  • the microfluidic concentration device can include a second channel that includes an inlet coupled to the outlet of the bypass channel to receive the white blood cells, e.g., labeled white blood cells, in the buffer, a first outlet through which substantially all of the focused white blood cells in the buffer are flowed, and a second outlet and a third outlet through which buffer that is not flowed through the first outlet is flowed.
  • the labeled white blood cells in the buffer can be flowed through the first inlet at the flow rate, and the labeled white blood cells of the higher concentration can be collected from the first outlet.
  • the microfluidic concentration device can include a spiral channel that includes a first inlet coupled to the outlet of the bypass channel to receive the labeled white blood cells in the buffer, a first outlet through which substantially all of the focused labeled white blood cells in the buffer in the buffer are flowed, and a second outlet through which buffer that is not flowed through the first outlet is flowed.
  • the labeled white blood cells in the buffer can be flowed through the first inlet at the flow rate, and the labeled white blood cells of the higher concentration can be collected from the first outlet.
  • the microfluidic concentration device can include a second channel arranged in parallel with the first channel.
  • the second channel can include an inlet coupled to the outlet of the bypass channel to receive the labeled white blood cells in the buffer, a first outlet through which substantially all of the focused labeled white blood cells in the buffer are flowed, and a second outlet and a third outlet through which buffer that is not flowed through the first outlet is flowed.
  • the labeled white blood cells in the buffer can be flowed simultaneously through the second channel, and the labeled white blood cells of the higher concentration can be collected from the first outlet of the second channel.
  • the labeled white blood cells of the higher concentration can be infused into the patient.
  • a first portion of the whole blood mixed with the buffer and the labeling agent can be flowed through a first inlet located closer to the first side wall than to the bypass channel and through the first array of obstacles.
  • a remaining portion of the whole blood mixed with the buffer and the labeling agent can be flowed through a second inlet located closer to the second side wall than to the bypass channel and through the second array of obstacles.
  • the first portion and the remaining portion can be flowed simultaneously.
  • Flowing the first portion of the whole blood mixed with the buffer and the labeling agent through the first inlet can include flowing the first portion at an angle relative to the first array such that white blood cells bound to the labeling agent in the first portion are displaced out of flow streamlines upon encountering an obstacle in the first array.
  • the labeling agent not bound to white blood cells in the buffer can be separated from the labeled white blood cells in the buffer collected from the outlet of the bypass channel. This can be done by flowing the labeled white blood cells in the buffer through a third array of obstacles disposed within the first channel between the first side wall and the second side wall, wherein the third array is configured to deflect the labeling agent not bound to the white blood cells in the buffer in one direction and the labeled white blood cells in the buffer in another direction.
  • the first array of obstacles can include multiple first obstacles that form a network of gaps and includes at least first and second rows of the obstacles.
  • the second row can be displaced relative to the first row so that a fluid passing through a gap in the first row is divided unequally when flowing around an obstacle and into two gaps on either side of the obstacle in the second row.
  • the second row can be displaced laterally relative to the first row.
  • aspects of the subject matter described herein are devices to perform the methods described herein. Yet other aspects of the subject matter described here are methods for separating monocytes from neutrophils and lymphocytes.
  • Implementations of the subject matter described in this document can provide one or more of the following potential advantages over current standard of clinical care.
  • the devices and methods described here can decrease (and nearly eliminate) the loss of white blood cells that are separated from whole blood. Consequently, less patient blood needs be used for imaging and opens up wider adoption of the test in pediatric applications.
  • the time required to obtain labeled white blood cells for reinfusion in a patient can be decreased. Consequently, both the quality of cells and quality of clinical care for the patient is positively impacted.
  • the labeled white blood cells can be of higher quality than those obtained by standard processes described above. Consequently, the in-vivo imaging can have better diagnostic quality.
  • the devices can enable using large blood volumes in high throughput devices.
  • the devices and methods can be implemented for all patient populations served by current clinical test, and in addition enable new patient populations (e.g., sickle cell anemia patients) that need a point-of-care device that can separate white blood cells without contamination (e.g., by red blood cells).
  • new patient populations e.g., sickle cell anemia patients
  • a point-of-care device that can separate white blood cells without contamination (e.g., by red blood cells).
  • the devices and methods described can provide a fully automated process in which whole blood is provided to the device as input and white blood cells in plasma- free cell-friendly medium are obtained as output.
  • the white blood cells can be bound to a labeling agent, concentrated, and provided for re-infusion to the patient in injectable saline.
  • Use of the devices and methods can reduce labor required relative to other approaches which can require up to several (e.g., 21) discrete steps of manual sample manipulation to obtain a similar output.
  • the devices and methods do not require centrifugation, reduce sample handling, and can reduce cell clumping that can result from multiple centrifugation steps and that can result in focal accumulation of radioactivity in lungs which lead to false positive results.
  • the devices and methods can provide very low red blood cell carryover. For example, maximum red blood cell carryover can be limited to 0.001% ( ⁇ 5 million from 50 ml of processed blood) in comparison to centrifugation-based methods that typically have 1% carryover. Lower red blood cell carryover reduces red blood cell and hemolysis product contamination.
  • the devices and methods can retain white blood cells with high efficiency (e.g., more than 99%) decreasing losses in white blood cells in the whole blood. These loss levels are lower relative to centrifugation methods in which the white blood cell loss can be range between 20%> and 40%>, and which can require, for example, larger volumes of whole blood to compensate for the white blood cell loss. Consequently, the amount of whole blood required when implementing the methods described here can be decreased.
  • the devices and methods can isolate white blood cells or sub-populations of the white blood cells in a physiological shear stress environment without exposure to chemicals (e.g., Ficoll®, hydroxyethyl starch, and the like), thereby providing high quality cells that can be labeled more effectively with a labeling agent.
  • chemicals e.g., Ficoll®, hydroxyethyl starch, and the like
  • Such labeled cells can migrate to site(s) of infection.
  • the devices and methods can deplete plasma, along with the red cells and platelets to reduce interference in downstream labeling from carryover of plasma.
  • Some implementations of the devices and methods described here can enrich monocytes relative to lymphocytes and neutrophils in whole blood.
  • the monocytes can be retained with high efficiency, and as such, the relative percentage of the total monocyte population can be increased.
  • FIG. 1 is a flowchart of a process for manipulating whole blood to obtain labeled white blood cells.
  • FIG. 2 is a schematic of one embodiment of a system to manipulate whole blood to obtain labeled white blood cells.
  • FIG. 3 A is a schematic that shows an example of a multiplex microfluidic device.
  • FIGs. 3B-E are schematics of scanning electron microscope images that show microfluidic channels and arrays of obstacles through which fluid is flowed.
  • FIGs. 4A-E are schematic representations of an example of a multiplex microfluidic device.
  • FIG. 5 A is a schematic that shows an example of a microfluidic concentration device.
  • FIG. 5B is a schematic that shows an enlarged portion of an end of one of the channels shown in FIG. 5A.
  • FIG. 5C is a graph that shows a line cut image of a focused stream created using the focusing device of FIG. 5 A.
  • FIG. 6A is a schematic that shows an example of a microfluidic focusing device including multiple straight focusing channels arranged in parallel.
  • FIG. 6B is a schematic that shows an enlarged portion of an end of one of the channels shown in FIG. 6A.
  • FIG. 7A is a schematic that shows an example of a first stage of a two-stage microfluidic separation device.
  • FIG. 7B is a schematic that shows an example of a second stage of a two-stage microfluidic separation device.
  • FIG. 7C is a schematic that shows an example of the two-stage microfluidic separation device.
  • FIG. 8 is a schematic that shows the different elements of a system to manipulate whole blood to obtain labeled white blood cells.
  • FIG. 9 is a schematic that shows an example of a microfluidic separation device including a spiral channel for concentrating the cells in the sample fluid.
  • FIGs. 10A and 10B are schematics that show two different multiplex microfluidic devices to separate leukocytes from blood (FIG. 10A) and to preferentially separate monocytes from lymphocytes and neutrophils (FIG. 10B).
  • FIGs. 10A and 10B are schematics that show two different multiplex microfluidic devices to separate leukocytes from blood (FIG. 10A) and to preferentially separate monocytes from lymphocytes and neutrophils.
  • a microfluidic-based approach is implemented to separate leukocytes from the other components of human whole blood (red blood cells, platelets and serum).
  • microfluidic devices described with reference to the following figures are automated, continuous, stand-alone processing units that can process whole blood drawn from a patient (e.g., in a Vacutainer®) to provide an output of white blood cells, with very low carryover of red cells.
  • the separated white blood cells can be collected in a cell-friendly medium and concentrated using a microfluidic concentration device described below.
  • the white blood cells can be bound to a labeling agent resulting in labeled white blood cells, and can be further processed using another microfluidic device to remove any labeling agent that is unbound to the white blood cells.
  • the labeled white blood cells can then be provided for re-infusion into the patient to image infection sites to which the labeled cells migrate.
  • FIG. 1 is a flowchart of a process 100 for manipulating whole blood to obtain labeled white blood cells.
  • Whole blood drawn from a patient is obtained (105).
  • 40 mL of blood is aseptically drawn into sterile 50 mL syringes containing 10 mL of acid citrate dextrose anticoagulant (22.0 g/L trisodium citrate, 8.0 g/L citric acid, 24.5 g/L dextrose) with the use of a 20G needle. Samples are drawn and transported immediately to the laboratory for processing.
  • acid citrate dextrose anticoagulant 22.0 g/L trisodium citrate, 8.0 g/L citric acid, 24.5 g/L dextrose
  • microfluidic device that separates white blood cells from remaining portions of the whole blood (110).
  • a microfluidic device can be implemented as a multiplex device including multiple arrays of obstacles, each of which operates under the principles of deterministic lateral displacement described in U.S. Published Application No. 2007/0026381 (entitled “Devices and Methods for Enrichment and Alteration of Cells and Other Particles," the entire contents of which are incorporated herein by reference).
  • the device has been developed and validated with over 1000 blood samples and used to isolate leukocytes from maternal blood for prenatal diagnosis.
  • whole blood is introduced into an array of obstacles (for example, microposts) under laminar flow conditions.
  • the large white blood cells are displaced out of the flow streamlines upon encountering an obstacle.
  • the enucleated components of the blood remain unperturbed by the array and follow the flow streamlines.
  • the displacement of the white blood cells out of the flow streamlines separates these cells from remaining components of the whole blood.
  • multiple arrays of obstacles are arranged with bypass channels that are devoid of obstacles to increase a throughput of white blood cells separated from whole blood.
  • the separated white blood cells can then be collected (e.g., in a Vacutainer®) and provided to a microfluidic concentration device configured to increase the concentration of the white blood cells in solution.
  • the fluidic pathway of the device and associated buffer solutions will be sterile, endotoxin free components.
  • the container in which the separated white blood cells will be collected is filled with a collection buffer (e.g., injectable saline solution) and sealed.
  • a collection buffer e.g., injectable saline solution
  • the required connections are made between the microfluidic device and the various reservoirs and containers.
  • a synthetic blocking buffer 1% Pluronic® F68
  • This blocking buffer passivates the blood-contacting surfaces to minimize protein adsorption and associated leukocyte
  • the sample reservoir is replaced with a fresh reservoir and injectable grade saline is flushed through the system to removed excess Pluronic® F68.
  • the product container is decoupled from the system and available for radiolabeling. All consumables are then disposed of using standard universal precautions. Exposed instrument surfaces are decontaminated using 70% ethanol or equivalent. The separated white blood cells can be flowed through a microfiuidic
  • the microfiuidic concentration device that increases a concentration of white blood cells relative to concentration of white blood cells collected from an outlet of the microfiuidic device (115).
  • the microfiuidic concentration device can be implemented as a flow-based focusing device that operates under the principles of inertial focusing described in U.S. Published Application No. 2009/0014360 (entitled “Systems and Methods for Particle Focusing in Microchannels,” the entire contents of which are incorporated herein by reference).
  • the microfiuidic concentration device can include a straight channel through which the white blood cells in solution (for example, buffer or labeling agent solution) can be flowed at flow rates that cause inertial forces to act on and focus the white blood cells into one or more stream lines.
  • the straight channel can include multiple outlets (e.g., three outlets) - one to collect the focused white blood cells, and the others to collect waste (i.e., the remaining solution).
  • the microfiuidic concentration device can be implemented in a spiral channel having one or more inlets and one or more outlets, as described below.
  • multiple concentration devices can be arranged in series or in parallel or both. The dimensions of the concentration devices can be same or different.
  • the decrease in the solution in which the white blood cells are flowed results in an increase in white blood cell concentration. Examples of microfiuidic concentration devices that yield 4X or higher increase in concentration are described below.
  • applications of the microfiuidic devices described here include re -infusion of labeled white blood cells (i.e., white blood cells bound to a labeling agent) into a patient so that the labeled white blood cells can be imaged upon migration to an infection site.
  • labeled white blood cells i.e., white blood cells bound to a labeling agent
  • An agent that has an affinity for a selected cell population i.e., white blood cells
  • the labeling agent includes a radio- labeling agent, such as, e.g., Tc-HMPAO (Ceretec, GE Healthcare), Indium 111
  • the labeling agents can be conjugated to lipohillic moieties, e.g., Tc-HMPAO and Oxyquinoline respectively, to enable uptake by cells.
  • Other examples of labeling agents include FDG (used to image primary tumors) by active uptake through glucose transport receptors.
  • the white blood cells can be labeled with nanoparticles for imaging by Magnetic Resonance Imaging (MRI).
  • MRI Magnetic Resonance Imaging
  • any in-vivo imaging agent can be used as is or conjugated to cellphillic agents for cellular uptake as a labeling agent for labeling the white blood cells.
  • imaging reagents that are not conducive to modification for cellular uptake can be electroporated into the white blood cells.
  • the labeling agent can be mixed with the white blood cells (113) at one of multiple instances.
  • the labeling agent can be mixed with the whole blood drawn from the patient and incubated for a duration that is sufficient to result in labeled white blood cells.
  • the labeling agent can be mixed with the white blood cells that have been separated using the multiplex device.
  • the labeling agent can be mixed with the white blood cells that have been concentrated using the microfluidic concentration device.
  • the labeled white blood cells can be separated from the remaining components of the blood using the multiplex device and then concentrated using the microfluidic concentration device.
  • the labeled white blood cells can further be processed to separate unbound labeling agent from the labeled white blood cells (120).
  • the labeled white blood cells can be flowed through another microfluidic separation device that includes one or more arrays of obstacles arranged in series, each of which is similar to one of the arrays of obstacles in the multiplex device.
  • the microfluidic separation device can be configured to separate labeled white blood cells from unbound labeling agent, for example, by the principles of deterministic lateral displacement.
  • the labeled white blood cells can be flowed through one or more microfluidic concentration devices under flow conditions that further concentrate and separate the labeled white blood cells from the unbound labeling agent.
  • the labeled white blood cells thus separated can be collected in a suitable container, e.g., a syringe, and provided for re-infusion in a patient (125).
  • FIG. 2 shows a schematic to implement a system 200 to manipulate whole blood to obtain labeled white blood cells.
  • the system 200 includes the micro fluidic devices described above.
  • Whole blood 205 obtained from a patient is flowed through a multiplex microfluidic device 210 described herein with reference to FIGS. 3 A to 4E that separates white blood cells from remaining components of the whole blood.
  • the separated white blood cells can be collected, for example, in a container 215 and provided as input to a microfluidic concentration device 220.
  • System 200 shows a microfluidic concentration device 220 that includes multiple concentration devices 222 arranged in series and coupled to another concentration device 224 in series, e.g., as described with reference to FIGS. 5A to 9.
  • the concentrated white blood cells can be collected in another container 230 and provided as input to
  • microfluidic separation devices 235 and 240 configured to separate unbound labeling agent from the white blood cells.
  • the separation devices 235 and 240 can be similar to either the multiplex device 210 or the concentration devices 222 or 224 or combinations of them.
  • the white blood cells can be mixed with a labeling agent that binds to the white blood cells at one of several stages, for example, before the whole blood 205 is flowed through the multiplex microfluidic device 210, before the white blood cells separated by the multiplex microfluidic device 210 are flowed through the microfluidic concentration device 220, or after the white blood cells have been concentrated using the concentration device 220.
  • a labeling agent that binds to the white blood cells at one of several stages, for example, before the whole blood 205 is flowed through the multiplex microfluidic device 210, before the white blood cells separated by the multiplex microfluidic device 210 are flowed through the microfluidic concentration device 220, or after the white blood cells have been concentrated using the concentration device 220.
  • the system 200 is configured to process 60 mL of whole blood 205.
  • the multiplex microfluidic device 210 can harvest 99% of white blood cells with less than 0.1% carryover of red blood cells and nearly no platelets or proteins.
  • the white blood cells separated by the multiplex microfluidic device 210 can be collected in 60 mL of PBS in the container 215.
  • the microfluidic concentration device 220 can include a five- channel concentration device 222 arranged in parallel to have a throughput of 2.5 mL/min coupled in series to a one-channel concentration device 224.
  • the five-channel concentration device 222 and the one-channel concentration device can result in a 5X concentration of white blood cells.
  • the concentrated white blood cells can be collected in 2.5 mL of PBS in the container 230.
  • the labeling agent can be added to the container 230. After an incubation period (e.g., 15 minutes), the labeled white blood cells can be flowed through another 5 -channel micro fluidic separation device 235 coupled to a one- channel microfluidic separation device 240 that separates unbound agent from the labeled white blood cells.
  • the entire process can span approximately 3.5 hours (2 hrs to separate white blood cells from whole blood using the multiplex microfluidic device 210 can span approximately two hours, 30 minutes to concentrate the separated white blood cells, 30 minutes to label the white blood cells, and 30 minutes to separate unbound agent from the labeled white blood cells).
  • FIG. 3A shows an example of a multiplex microfluidic device 210.
  • FIGS. 3B-D show scanning electron micrographs of arrays of obstacles included in the device 210.
  • the device 210 includes one or more arrays of obstacles that allow deterministic lateral displacement of components of whole blood.
  • the array includes rows and columns of obstacles (e.g., microposts) arranged to separate particles according to size in a network of gaps formed between the obstacles.
  • a fluid passing through a gap is divided unequally into subsequent gaps.
  • the array includes a network of gaps arranged such that fluid passing through a gap is divided unequally, even though the gaps may be identical in dimensions.
  • the gaps are formed by an array of obstacles that deterministically direct particles having a hydrodynamic size above a critical size in one direction and particles having a hydrodynamic size below the critical size in a different direction.
  • the spacing between any two adjacent obstacles in the same row can be equal (e.g., 21 ⁇ from center-to-center).
  • each row of obstacles can be shifted horizontally with respect to a previous row by a distance that is a fraction of a center-to-center distance between the posts (e.g., 1/60 ⁇ the center-to-center distance between two adjacent obstacles in the same row).
  • the ratio of the fraction to the center-to-center distance determines the ratio of flow bifurcated to the left of the next obstacle.
  • flow bifurcation can be achieved based on a cross- section of each obstacle in the array. For example, as shown in FIG.
  • each obstacle can have a substantially elliptical (e.g., tear-drop) cross-section in which a curvature of a first edge of the obstacle is different from a curvature of a second edge that is opposite the first edge in a direction transverse to fluid flow.
  • flow bifurcation can be obtained by flowing fluid at an angle to a first row of the array of obstacles.
  • the rows need not be horizontally offset from one another, and the obstacles can be of any cross-sectional shape (e.g., cylindrical, teardrop).
  • whole blood can be flowed at a small angle ( ⁇ ) relative to the array, such that the white blood cells, which are large with respect to the flow
  • the critical cutoff diameter (D c , above which cells are deflected and below which cells pass through unperturbed), is defined by the following relationship:
  • ⁇ is the row shift fraction (shift of each row relative to the obstacle spacing)
  • g is the gap between obstacles
  • ⁇ is a variable parameter that corrects for flow non-uniformity in the gap.
  • size can be thought of more as a "hydrodynamic size," a combination of size, shape, and flexibility.
  • erythrocytes which have a discoid shape, separate based on their small axis ( ⁇ 2 ⁇ ) even though their widest dimension (4-6 ⁇ ) overlaps with typical leukocyte dimensions. Accordingly, deterministic lateral displacement can be used to effectively distinguish leukocytes from erythrocytes as described above. Referring to FIGS.
  • the multiplex micro fluidic device 401 is a multi-stage multiplex device in which multiple multiplexes are arranged in series.
  • the device 401 can be a silicon device multiplexing fourteen 3 -stage duplex arrays.
  • the dimensions of this particular device are 90 mm x 34 mm x 1 mm.
  • Each array is a duplex array with a single bypass channel and includes flow resistors for flow stability.
  • the device arrays and channels can be fabricated in silicon using standard photolithography and deep silicon reactive etching techniques with an etch depth of 150 ⁇ .
  • Through holes for fluid access can be made, e.g., using KOH wet etching.
  • the silicon substrate can be sealed on the etched face to form enclosed fluidic channels, e.g., using a blood compatible pressure sensitive adhesive (9795, 3M, St Paul, Minn.).
  • the device can be mechanically mated to a manifold, e.g., of plastic, with external fluidic reservoirs to deliver blood and buffer to the device and extract the generated fractions.
  • An external pressure source can be used to apply a pressure, e.g., in a range of 1 to 35 psi, e.g., a pressure of about 2.4 psi, to the buffer and blood reservoirs to modulate fluidic delivery and extraction from the packaged device.
  • the multiplex micro fluidic device 210 shown in FIG. 4B includes multiple duplex arrays (FIGS. 4C-E) that are connected by a common inlet 400 and a common outlet 402.
  • each duplex array includes a channel 403 extending from one of the inlets 400 to one of the outlets 475, and is bounded by a first side wall 404 and a second side wall 406 located opposite the first side wall 404.
  • a first array of obstacles 405 is disposed and bounded by a first array boundary formed by ends of obstacles in the first array 405 and the first side wall 404.
  • a second array of obstacles 410 is disposed within the channel, and bounded by a second array boundary formed by ends of obstacles in the second array 410 and the second side wall 406.
  • a bypass channel 415 defined by a region devoid of obstacles is formed between the first array boundary and the second array boundary.
  • the first array 405 and the second array 410 are configured to deflect the labeled white blood cells in the buffer toward the bypass channel 415.
  • the first array 405 and the second array 410 are formed as mirror-images of each array other so that each deflects white blood cells in the direction of the other resulting in the white blood cells being collected in the bypass channel 415. The collected white blood cells flow toward the outlet of the bypass channel.
  • the obstacles in the array may continue up to and run into the side wall.
  • another region devoid of obstacles can be formed immediately adjacent the side wall and the array boundary.
  • the physical boundary of the bypass channel may be defined by the array boundary on one side and a sidewall on the other side. If the array were infinitely large, the flow distribution would be the same at every gap. In practice, the boundaries of the array perturb this infinite flow pattern. Portions of the boundaries of arrays may be designed to generate the flow pattern of an infinite array. Boundaries may be flow-feeding, i.e., the boundary injects fluid into the array, or flow-extracting, i.e., the boundary extracts fluid from the array.
  • a flow-extracting boundary widens gradually to extract flux from each gap at the boundary. For example, the distance between the array and the sidewall gradually increases to allow for the addition of flux to the boundary from each gap along that boundary. The flow pattern inside this array is not affected by the bypass channel because of the boundary design.
  • a flow- feeding boundary narrows gradually to feed flux into each gap at the boundary. For example, the distance between the array and the sidewall gradually decreases to allow for the addition of flux to each gap along the boundary from that boundary. Again, the flow pattern inside this array is not affected by the bypass channel because of the boundary design.
  • a flow-feeding boundary may also be as wide as or wider than the gaps of an array. A wide boundary may be desired if the boundary serves as a bypass channel, e.g., to allow for collection of particles.
  • a boundary may be employed that uses part of its entire flow to feed the array and feeds flux into each gap at the boundary.
  • bypass channels can be designed, in conjunction with an array to maintain constant flux through a device.
  • bypass channels are designed to remove an amount of flow so the flow in the array is not perturbed, i.e., substantially constant.
  • Such a design may also be employed with an array duplex.
  • the center bypass channel may be shared between the two arrays in the duplex.
  • FIG. 4D shows a third array of obstacles 420 and a fourth array of obstacles 425 formed within the channel in series with the first array of obstacles 405 and the second array of obstacles 410, respectively.
  • a bypass channel 430 devoid of obstacles is formed between the third array 420 and the fourth array 425.
  • the outlet of the bypass channel 415 is connected to an inlet of the bypass channel 430.
  • Spacing between the obstacles in the first array 405 and the second array 410 can be the same as each other, and can be different from spacing between the obstacles in the third array 420 and the fourth array 425.
  • a fifth array of obstacles 435 and a sixth array of obstacles 440 can be disposed within the channel in series with the third array 420 and the fourth array 425, respectively.
  • a bypass channel 445 devoid of obstacles can be formed between the fifth and sixth arrays, and connected to the bypass channel 430.
  • the device described with reference to FIGS. 4C-D represents a three-stage duplex microfluidic device.
  • Several such duplex devices can be coupled in parallel (FIG. 4B) to form the multiplex microfluidic device 210.
  • Table 1 shows an effectiveness of a multiplex microfluidic device 210.
  • the multiplex microfluidic device 210 can support a flow throughput ranging to 30 mL/hr (within physiological shear stress) to separate white blood cells from red blood cells, platelets, and plasma with a reliability of greater than 97%.
  • the bio-burden of product obtained by separating particles in the multiplex microfluidic device 210 were tested based on endotoxin levels assayed via a Limulus Amoebocyte Lysate (LAL) test.
  • a surrogate medium e.g., lx iDPBS
  • the endotoxin levels were measured in three components flowed through the device 210— input buffer, waste output, and product output.
  • the bio-burden of the product output was assayed by measuring the Total Aerobic Microbial Count of the product.
  • the assay results showed that each of the input buffer, the waste output, and the product output had less than 0.05 EU/mL mean endotoxin value indicating no microbial growth.
  • white blood cells separated from whole blood using the multiplex micro fluidic device 210 were enriched (as observed by Wright-Giemsa Staining), viable (as observed by 1 ⁇ g/mL Calcein AM cell Staining and 1.5 ⁇ g/mL CellTracker® Orange Cell Staining), and motile (average neutrophil velocity of 18 ⁇ 3 ⁇ / ⁇ ).
  • white blood cell separation using the multiplex micro fluidic device 210 offers a white blood retention of 98%, red blood cell retention of 0.02%, with no platelets retained.
  • Human white blood cells obtained from the multiplex micro fluidic device 210 were bound with In-111-Oxyquilone and re-infused in the patient to image E. coli muscle infection and Pseudomonas muscle infection. Similarly obtained human white blood cells were bound with Tc99-HMPAO and used to image cecal ligation. Thus, the human white blood cells enriched with the multiplex micro fluidic device 210 and labeled with In-111-Oxy migrate to site of infection. Such labeled white blood cells are absent from inflammation control (injury model without infection) demonstrating that localization is specific to presence of infection.
  • white blood cells in whole blood can be separated using the multiplex microfluidic device 210.
  • the cells can be collected in a buffer (e.g., PBS) and optionally mixed with a labeling agent.
  • the concentration of the cells in the buffer can then be increased using the microfluidic separation device 220.
  • FIG. 5 A is a schematic that shows an example of a microfluidic concentration device 220 into which the white blood cells separated from whole blood can be flowed to concentrate the white blood cells.
  • the concentration device 220 can include a straight channel 500 formed in a substrate (e.g., silicon substrate) and can have an inlet through which the white blood cells (labeled or unlabeled) are flowed through the buffer solution.
  • the flow rate can be selected such that inertial forces established in the flowing fluid act upon and focus the white blood cells into one or more localized stream lines.
  • Each stream line can define a width that is substantially equal to or greater than a size of the focused labeled white blood cells.
  • a stream line can have a width that is lesser than the cross-sectional width of the channel 500 and can include a white blood cell within.
  • Channel 500 can have various geometries and cross-sections for focusing the white blood cells suspended within the buffer.
  • the channel 500 can have a rectangular cross-section with an aspect ratio of substantially 1 to 1.
  • White blood cells flowing within such a channel geometry will be separated, ordered, and focused into four streamlines corresponding to four equilibrium points or potential minimums at a distance from each face of the four channel walls.
  • the channel 500 can have a rectangular cross-section with an aspect ratio of substantially 2 to 1.
  • White blood cells flowing within such a channel geometry can be separated, ordered, and focused into two focused streamlines corresponding to two equilibrium points or potential minimums along top and bottom walls across the width of the channel.
  • an aspect ratio of 1 to 2 can also be used.
  • the channel can have a circular, triangular, diamond-shaped, or hemispherical cross-section.
  • an annulus or arc of focused particles can be formed within the channel.
  • particles can be focused into streamlines corresponding to equilibrium positions at a distance from the flat faces of each wall in the geometry.
  • the channel 500 can have a hydraulic diameter and a ratio of a size of the white blood cells to the hydraulic diameter that is greater than or equal to about 0.07.
  • the ratio of white blood cell size to hydraulic diameter can be less than or equal to about 0.5.
  • a Reynolds Number of the fluid flow during focusing can be greater than or equal to about 1 and less than or equal to about 250.
  • a particle Reynolds number for the whole blood moving through the channel can be greater than or equal to about 0.2.
  • the one or more focused stream lines can have a width that is less than or equal to about five times, four times, three times, two times, and/or 1.05 times a size of the focused particles.
  • the whole blood flow through the channel 500 can be laminar. Focusing can produce a localized flux of enriched white blood cells or another whole blood component based on the component size.
  • a white blood cell diameter divided by a hydraulic diameter of the channel can be greater than or equal to about 0.07 and the white blood cell diameter divided by the hydraulic diameter of the channel can be less than or equal to about 0.5.
  • the channel has a rectangular cross-section, a height, a width, a hydraulic diameter is equal to 2*height*width/(width+height).
  • the rectangular cross-section can have an aspect ratio of between approximately 0.3 and 0.8 and/or approximately 1 to 2.
  • Two dimensionless Reynolds numbers can be defined to describe the flow of particles in closed channel systems: the channel Reynolds number (Rc), which describes the unperturbed channel flow, and the particle Reynolds number (R p ), which includes parameters describing both the particle and the channel through which it is translating.
  • Rc channel Reynolds number
  • R p particle Reynolds number
  • FIG. 5B is a schematic that shows an enlarged portion of an end of one of the channels shown in FIG. 5A.
  • the stream of white blood cells can be flowed through the channel 500 at volumetric flow rates of approximately 100 ⁇ / ⁇ .
  • the channel 500 can include three outlet branches - one to receive the focused stream of white blood cells and the other two to receive the buffer that is not in the focused stream.
  • each of the outlets can be coupled to an inlet of a corresponding channel to transport the white blood cells and the buffer that is not in the focused stream.
  • Such channels can be sized to transport the white blood cells at a volumetric flow rate of 20 ⁇ / ⁇ , and to transport the separated fluid at flow rates of 40 ⁇ each.
  • the channel that transports the focused stream of white blood cells can be longitudinally coupled to the channel 500, and the channels that transport the separated buffer can be coupled transversely to the channel 500.
  • the channel 500 can be 80 ⁇ wide, 30 ⁇ tall, and support a throughput of 100 ⁇ / ⁇ .
  • 3.22 M WBC/mL in 6.08 mL were flowed.
  • the focused stream of white blood cells that were collected included 12.7 M WBC/mL in 1.33 mL resulting in an overall yield of 87% representing a 4.6X reduction in volume and approximately 4X increase in concentration.
  • the white blood cells were focused within a stream spanning approximately 30 ⁇ within the 80 ⁇ width of the channel 500.
  • FIG. 6 shows an example of a microfluidic concentration device 600 including multiple channels arranged in parallel.
  • ten straight focusing channels similar to channel 500, each having a width of 80 ⁇ , a height of 30 ⁇ , and supporting a throughput of 1 mL/min were coupled in parallel.
  • the channels were arranged to have a common inlet for the white blood cells in the buffer and a common outlet for the focused stream of white blood cells. Two outlets were formed for each channel to separate the buffer from the focused stream.
  • Table 2 The concentrated white blood cells obtained using the microfluidic concentration device 600 shown in FIG. 6 are presented in Table 2.
  • Table 2 shows that, on average 89%> of the white blood cells flowed into the microfluidic separation device 220 were recovered, 7% of white blood cells were not focused, 3.8%) were lost (due to experimental errors - CBC counting, white blood cells in dead volume, white blood cells retained in input syringe), resulting in an average volumetric reduction of 4.7X and an average white blood cell concentration of 4.3X.
  • FIGS. 7A and B are schematics of an example of a two-stage microfluidic concentration device 700 that includes a first concentration device coupled in series to a second concentration device.
  • the first concentration device (FIG. 7A) is similar to the device described with reference to FIG. 6B, and includes five straight focusing channels, each having a width of 80 ⁇ and a height of 30 ⁇ .
  • the focused stream of white blood cells from the first separation device is provided as input to a second concentration device (FIG. 7B) that includes a single straight channel having a width of 80 ⁇ and a height of 30 ⁇ .
  • the throughput of the two-stage microfluidic separation device (FIG. 7C) 220 is 500 ⁇ / ⁇ .
  • the focused white blood cells can be collected in a container 230. If the labeling agent has been mixed with the white blood cells prior to flowing the cells through the microfluidic separation device 220, then the container 230 contains labeled white blood cells. If not, then the labeling agent can be mixed with the white blood cells in the container resulting in labeled white blood cells.
  • the labeled white blood cells can then be flowed through the microfluidic separation devices 235 and 240 to separate the labeled cells from unbound agent.
  • the microfluidic separation devices 235 and 240 can be similar to the multiplex microfluidic device 210 or the microfluidic concentration device 220 or combinations of them.
  • the microfluidic separation device can include a single array of obstacles configured to flow unbound agent in an axial direction and to deflect labeled white blood cells in a direction lateral to the axial direction.
  • FIG. 8 shows a schematic to implement a system 800 to manipulate whole blood to obtain labeled white blood cells.
  • the system 800 is similar to the system 200 described with reference to FIG. 2, and includes a multiplex microfluidic device 210 through which whole blood is flowed to separate white blood cells collected in a container 215.
  • the system 800 includes a microfluidic concentration device 805 that includes a spiral channel including one or more inlets and at least two outlets.
  • the separated white blood cells (labeled with a labeling agent or unlabeled) are flowed through the inlet with a buffer.
  • the spiral channel of the concentration device 805 is configured to separate the white blood cells from the buffer, thereby increasing a concentration of the white blood cells.
  • FIG. 9 shows an example of a microfluidic separation device 805 including a spiral channel.
  • the spiral channel can be an expanding spiral shaped channel having a rectangular cross-section with an aspect ratio of substantially 2 to 1 , although the aspect ratio can vary.
  • the width of the spiral channel can range from about 2000 ⁇ to 772 ⁇ , and the height can be about 50 ⁇ .
  • the input volumetric flow rate can be about 3 mL/min.
  • the focused white blood cells can be collected through two outlets, and the fluid that is not in the focused stream lines can be collected through a waste outlet. In such implementations, the white blood cells are focused into a single stream line a distance away from an inner wall of the channel corresponding to a single equilibrium point or potential minimum within the channel. A reduction in volume of between about 4X and 10X and an increase in concentration of between about 4X and 6X were measured.
  • the location of the focused stream of white blood cells within the channel can depend upon inertial forces and Dean drag forces acting on the cells. The location can further depend upon centrifugal forces acting on the cells.
  • a Dean number for flow through the channel can be less than or equal to about 20.
  • the curvature in the spiral channel introduces Dean drag that will push the white blood cells in different transversal directions depending on position. For example, a particle located in the center will be pushed towards the outer wall and recirculated through the outer edge roof or bottom until the particles reach the equilibrium position near the inner wall where the Dean forces are superimposed to the inertial forces from the inner walls.
  • the main forces impacting the white blood cell focusing in the channel height direction may be inertial lift forces, while the Dean forces have a strong influence on the lateral positioning of cells.
  • a cell can remain focused as long as the Dean force trying to push the particle away from the inner wall is balanced by the inertia lift force from the inner wall trying to push the cell towards the inner wall. This results in cells focusing in single-stream lateral positions in two parallel symmetric streams along the height of the channel.
  • the cells are ordered in uniform spacing in the direction of the flow.
  • FIGs. 10A and 10B are schematics that show two different multiplex microfluidic devices to separate leukocytes from blood (FIG. 10A) and to preferentially separate monocytes from lymphocytes and neutrophils (FIG. 10B).
  • the system 200 can be configured to separate monocytes from lymphocytes and neutrophils.
  • the critical cutoff diameter (D c , above which cells are deflected and below which cells pass through unperturbed) is defined by the following relationship:
  • ⁇ € 2 ⁇ ⁇ ⁇ , where ⁇ is the row shift fraction (shift of each row relative to the obstacle spacing), g is the gap between obstacles, and ⁇ is a variable parameter that corrects for flow non-uniformity in the gap.
  • Large flexible cells like neutrophils are able to deform enough to behave like effectively smaller cells. This phenomenon can be exploited to enrich monocytes relative to lymphocytes and neutrophils.
  • two devices with the same basic device architecture were produced, each having a different D c (Table 3).
  • the critical cutoff diameter can be further optimized to identify the maximal possible monocyte enrichment allowable.
  • Table 4 The concentrated white blood cells obtained using the microfluidic separation device 805 shown in FIG. 9 are presented in Table 4. Table 4
  • the whole blood can first be flowed through a microfluidic concentration device under laminar flow conditions in which the white blood cells are focused in one or more localized stream lines.
  • the whole blood with focused white blood cells can be flowed into a multiplex microfluidic device to separate white blood cells from remaining components of the whole blood.
  • the microfluidic concentration device can focus the white blood cells into streams that include some red blood cells (e.g., 10%>).
  • the multiplex microfluidic device can remove the red blood cells from the white blood cells, which can then be incubated with the labeling agent.
  • the labeled white blood cells can be flowed through a second microfluidic concentration device to separate unlabeled labeling agent from the labeled white blood cells.
  • the microfluidic concentration device can be used upstream of the multiplex microfluidic device. Because the upstream concentration device provides concentrated white blood cells from which the multiplex microfluidic device can remove residual red blood cells (and other whole blood components), the white blood cells can be separated from a small volume of whole blood (e.g., 30 mL) and labeled in a short duration (e.g., one hour). Also, the number of interactions that the multiplex microfluidic device needs to deflect the white blood cells away from the residual red blood cells is decreased as the microfluidic concentration device has already concentrated the white blood cells into localized streams. Consequently, the throughput of the multiplex microfluidic device can increase while its size can decrease.
  • the upstream concentration device provides concentrated white blood cells from which the multiplex microfluidic device can remove residual red blood cells (and other whole blood components)
  • the white blood cells can be separated from a small volume of whole blood (e.g., 30 mL) and labeled in a short duration (e.g., one
  • the cells can be mixed with the labeling agent, and flowed through the multiplex microfluidic device.
  • the multiplex microfluidic device can additionally separate unlabeled labeling agent from the labeled white blood cells. Such a device can further increase the throughput, e.g., to process 30 mL in 30 minutes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Dispersion Chemistry (AREA)
  • Ecology (AREA)
  • Clinical Laboratory Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des procédés, dispositifs et systèmes d'enrichissement par voie microfluidique de populations cellulaires choisies. Les procédés de marquage des globules blancs comprennent les étapes consistant à obtenir du sang total prélevé chez un patient, séparer les globules blancs du sang total en faisant circuler le sang total à travers un dispositif microfluidique comprenant une ou plusieurs matrices d'obstacles, configurées pour dévier les globules blancs dans une direction s'éloignant d'une direction des composants restants du sang total, et collecter les globules blancs séparés. Une solution tampon contenant un agent de marquage ayant une affinité pour les globules blancs est mélangée aux globules blancs, soit avant, soit après que les globules blancs aient été séparés du sang total avec pour résultat de marquer les globules blancs.
PCT/US2011/029999 2010-03-26 2011-03-25 Enrichissement par voie microfluidique de populations cellulaires choisies WO2011119962A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US31806510P 2010-03-26 2010-03-26
US61/318,065 2010-03-26

Publications (2)

Publication Number Publication Date
WO2011119962A2 true WO2011119962A2 (fr) 2011-09-29
WO2011119962A3 WO2011119962A3 (fr) 2012-01-12

Family

ID=44673890

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/029999 WO2011119962A2 (fr) 2010-03-26 2011-03-25 Enrichissement par voie microfluidique de populations cellulaires choisies

Country Status (1)

Country Link
WO (1) WO2011119962A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015024469A (ja) * 2013-07-26 2015-02-05 京セラ株式会社 切削工具
EP2971279A4 (fr) * 2013-03-15 2016-08-03 Univ Princeton Méthodes et dispositifs de purification à rendement élevé
EP2971287A4 (fr) * 2013-03-15 2016-08-03 Gpb Scientific Llc Traitement microfluidique intégré de particules
US10844353B2 (en) 2017-09-01 2020-11-24 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics
US10976232B2 (en) 2015-08-24 2021-04-13 Gpb Scientific, Inc. Methods and devices for multi-step cell purification and concentration
US11142746B2 (en) 2013-03-15 2021-10-12 University Of Maryland, Baltimore High efficiency microfluidic purification of stem cells to improve transplants

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040019300A1 (en) * 2002-07-26 2004-01-29 Leonard Leslie Anne Microfluidic blood sample separations
US20070059718A1 (en) * 2005-09-15 2007-03-15 Mehmet Toner Systems and methods for enrichment of analytes
US20070160503A1 (en) * 2003-06-13 2007-07-12 Palaniappan Sethu Microfluidic systems for size based removal of red blood cells and platelets from blood
US20080023399A1 (en) * 2006-06-01 2008-01-31 Inglis David W Apparatus and method for continuous particle separation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040019300A1 (en) * 2002-07-26 2004-01-29 Leonard Leslie Anne Microfluidic blood sample separations
US20070160503A1 (en) * 2003-06-13 2007-07-12 Palaniappan Sethu Microfluidic systems for size based removal of red blood cells and platelets from blood
US20070059718A1 (en) * 2005-09-15 2007-03-15 Mehmet Toner Systems and methods for enrichment of analytes
US20080023399A1 (en) * 2006-06-01 2008-01-31 Inglis David W Apparatus and method for continuous particle separation

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113512522A (zh) * 2013-03-15 2021-10-19 普林斯顿大学理事会 用于高通量纯化的方法和设备
EP2971279A4 (fr) * 2013-03-15 2016-08-03 Univ Princeton Méthodes et dispositifs de purification à rendement élevé
EP2971287A4 (fr) * 2013-03-15 2016-08-03 Gpb Scientific Llc Traitement microfluidique intégré de particules
US10324011B2 (en) 2013-03-15 2019-06-18 The Trustees Of Princeton University Methods and devices for high throughput purification
EP3569313A1 (fr) * 2013-03-15 2019-11-20 GPB Scientific, LLC Traitement microfluidique sur puce de particules
EP3608022A1 (fr) * 2013-03-15 2020-02-12 The Trustees of Princeton University Procédés et dispositifs de purification à haut rendement
US11493428B2 (en) 2013-03-15 2022-11-08 Gpb Scientific, Inc. On-chip microfluidic processing of particles
US10852220B2 (en) 2013-03-15 2020-12-01 The Trustees Of Princeton University Methods and devices for high throughput purification
US11486802B2 (en) 2013-03-15 2022-11-01 University Of Maryland, Baltimore Methods and devices for high throughput purification
US11142746B2 (en) 2013-03-15 2021-10-12 University Of Maryland, Baltimore High efficiency microfluidic purification of stem cells to improve transplants
JP2015024469A (ja) * 2013-07-26 2015-02-05 京セラ株式会社 切削工具
US10976232B2 (en) 2015-08-24 2021-04-13 Gpb Scientific, Inc. Methods and devices for multi-step cell purification and concentration
US11149251B2 (en) 2017-09-01 2021-10-19 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics
US11306288B2 (en) 2017-09-01 2022-04-19 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics
US10988734B2 (en) 2017-09-01 2021-04-27 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics
US10844353B2 (en) 2017-09-01 2020-11-24 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics

Also Published As

Publication number Publication date
WO2011119962A3 (fr) 2012-01-12

Similar Documents

Publication Publication Date Title
Warkiani et al. Large-volume microfluidic cell sorting for biomedical applications
Yu et al. Microfluidic blood cell sorting: now and beyond
CN102791616B (zh) 用于粒子过滤的系统和方法
Rafeie et al. Multiplexing slanted spiral microchannels for ultra-fast blood plasma separation
JP3299284B2 (ja) 粒子分離システム及び方法
SooHoo et al. Microfluidic aqueous two phase system for leukocyte concentration from whole blood
US6221315B1 (en) Apparatus for separation of biologic components from heterogeneous cell populations
WO2011119962A2 (fr) Enrichissement par voie microfluidique de populations cellulaires choisies
DE69332926T2 (de) Kontinuierliches zentrifugationsverfahren zum abtrennen von biologischen komponenten aus heterogenen zellpopulationen
WO2019047498A1 (fr) Système et procédé de séparation de plasma de sang total
US20170144159A1 (en) Combined sorting and concentrating particles in a microfluidic device
US20060118479A1 (en) Particle separating devices, systems, and methods
WO2013116523A1 (fr) Techniques et dispositifs de capture de cellules tumorales circulantes
Kim et al. A microfluidic device for continuous white blood cell separation and lysis from whole blood
CN111330656A (zh) 一种微米颗粒悬液体积浓缩微流控器件
AU2013204820B2 (en) A System and Method for Particle Filtration
Laxmi et al. Extracting white blood cells from blood on microfluidics platform: A review of isolation techniques and working mechanisms
JP6403190B2 (ja) マイクロ流路構造体及び粒子の分離方法
Iqbal et al. Recent advances in microfluidic cell separation to enable centrifugation-free, low extracorporeal volume leukapheresis in pediatric patients
CN111433350B (zh) 从生物样品分离颗粒的方法、系统和过滤单元
CN111690534B (zh) 一种基于黏弹性聚焦技术的肿瘤细胞多级分选装置
US20230347345A1 (en) Spiral inertial microfluidic devices and methods to remove contaminants
WO2022044600A1 (fr) Dispositif de préparation d'échantillon et système de préparation d'échantillon
Zhang et al. High throughput cell-free extraction of plasma by an integrated microfluidic device combining inertial microfluidics and membrane
Bhal Designing a continuous, high throughput flow-filter for whole blood fractionation and enrichment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11760313

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11760313

Country of ref document: EP

Kind code of ref document: A2