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WO2000040947A9 - Methode et dispositif permettant de separer des matieres biologiques et autres substances - Google Patents

Methode et dispositif permettant de separer des matieres biologiques et autres substances

Info

Publication number
WO2000040947A9
WO2000040947A9 PCT/US2000/000274 US0000274W WO0040947A9 WO 2000040947 A9 WO2000040947 A9 WO 2000040947A9 US 0000274 W US0000274 W US 0000274W WO 0040947 A9 WO0040947 A9 WO 0040947A9
Authority
WO
WIPO (PCT)
Prior art keywords
magnetic
coil
magnetic field
ofthe
target substance
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US2000/000274
Other languages
English (en)
Other versions
WO2000040947A1 (fr
Inventor
Leonard Sciorra
Joseph Zimnoch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rutgers State University of New Jersey
Rutgers Health
Original Assignee
University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
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 University of Medicine and Dentistry of New Jersey, Rutgers State University of New Jersey filed Critical University of Medicine and Dentistry of New Jersey
Priority to AU26016/00A priority Critical patent/AU2601600A/en
Priority to US09/869,741 priority patent/US7364921B1/en
Priority to CA002358069A priority patent/CA2358069A1/fr
Priority to EP00904229A priority patent/EP1151271A4/fr
Publication of WO2000040947A1 publication Critical patent/WO2000040947A1/fr
Anticipated expiration legal-status Critical
Publication of WO2000040947A9 publication Critical patent/WO2000040947A9/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • 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
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

  • the present invention relates to a method and apparatus for separating biological materials. Applicants claim the benefit of priority of the filing date 6 January 1999 of U.S. Patent Application 60/114,843, the entire contents of which are incorporated herein by reference.
  • the present invention is directed to a method for separating biological materials or other substances, comprising the steps of binding a substance of interest to magnetic particles to form a magnetic transfer complex, placing the magnetic transfer complexes onto a support medium, and subjecting the material to pulsed, variable strength magnetic fields which cause the magnetic transfer complexes to migrate across the surface ofthe support medium.
  • a further embodiment ofthe present invention is directed to an apparatus capable of creating pulsed, variable strength magnetic fields about a support medium.
  • highly purified cell sub-populations are isolated from small amounts of heterogeneous cell populations.
  • This embodiment uses magnetic carrier materials which have monoclonal antibodies specific to cell sub-populations which are desired to be separated from the heterogenous population placed on the surface ofthe magnetic carrier materials.
  • the magnetic carrier materials are introduced to the heterogenous population wherein the magnetic carrier materials bind to the desired cell sub-populations to form magnetic complexes containing members of the desired cell sub-populations.
  • the antibodies on the magnetic carrier materials can be specific to cell sub-populations to be removed from the heterogenous population (leaving the desired cell sub-population), in which case the magnetic complexes contain members of the sub-population to be removed from the heterogenous population.
  • the heterogenous population including the magnetic complexes, is then placed in an apparatus according to the current invention.
  • the apparatus comprises a support for a substrate medium which contains the heterogenous population, and a magnetic coil and power supply combination capable of producing pulsed, variable strength magnetic fields around the substrate medium.
  • the heterogenous population is placed in or upon the substrate medium.
  • the heterogenous population is then exposed to a laminar GSR magnetic field, whose direction, strength and duration can be controlled.
  • the magnetic field is pulsed, rather than constantly applied, about the sample ofthe heterogenous population.
  • the magnetic complexes then travel through or across the substrate medium and are separated from the remainder ofthe heterogenous population, because the substrate medium impedes the motion ofthe unbound materials.
  • the apparatus according to the current invention has the potential to increase quite significantly the specificity and sensitivity ranges now available in currently utilized magnetic bead separating devices.
  • the apparatus and method of the current invention is used to separate fetal blood cells from a mixture comprising maternal blood cells and fetal blood cells.
  • the fetal blood cells once separated by the apparatus and method of the current invention, can be subjected to various tests, as known in the art, to determine, among other properties, the potential for genetic defects in the fetus from which the blood cells originate.
  • Separation processes are based on the application of an external force on a mixture, wherein the external force reacts with a property of the components in the mixture. Separation methods can be based on component size (membrane filtration, dialysis, and screening), phase affinity (distillation, chromatography, sublimation, and crystallization), mass (centrifugation and spectrometry) or combinations of properties (gradient transport and electro osmosis).
  • Electrophoresis an apparatus separates components from a mixture via an electric field. Electrophoresis is the transport of electrically responsive particles in an electric field. Separation is based on different rates of migration of the components through a solution under the influences of an electric field. Particles in a solution create their own ionic environment, hence they create their own electrical field. The electric field ofthe apparatus causes the components to migrate by interacting with the electrical fields created by the components.
  • charge originates from the ionization of functional groups in the molecules. These charged molecules tend to adsorb oppositely charged particles near the phase boundary between the molecule and the solution in which it is placed. This collection of charged particles and the ions adsorbed thereon is referred to as the electrical double layer.
  • the electrical double layer is often larger in size and has a higher charge density than the original particles.
  • the electrical double layer creates an electric field about the particle.
  • the potential at the surface ofthe electric double layer, ⁇ 0 is defined as Q/( ⁇ a), where Q is the resultant charge on the electric double layer, sis the dielectric constant ofthe solution and a is the radius of the electrical double layer.
  • Q is the resultant charge on the electric double layer
  • a is the radius of the electrical double layer.
  • e is the electronic charge
  • n 0 is the bulk concentration of each ionic species
  • z is the valence ofthe symmetrical electrolyte
  • Tis the absolute temperature
  • k is the
  • the particles Upon application of an external electric field to the solution, the particles, including the electric double layer, are subjected to four forces: the electrophoretic attraction X the Stokes friction k 2 , the electrophoretic retardation ⁇ , and the relaxation effect K 4 .
  • K, QE (the product of the charge of the particle and the potential gradient);
  • the ability of particles to be separated by electrophoresis can be determined from knowledge ofthe intrinsic properties ofthe solution and the particles in the solution. Different types of particles will travel through the same solution, while being subjected to the same electric field, at different rates. This rate differentials allow for component separation.
  • the resolution provided by the rate differential is enhanced if an element of discontinuity is introduced into the electric field.
  • the mixture could be subjected to apH gradient, the sieving effect of high density gels, or the adherence of one ofthe substances in the mixture to a supporting medium.
  • Other factors include shape of the separation vessel and gravimetric effects introduced by running the separation in a vertical direction.
  • Magnetophoresis is the transport of magnetically responsive particles in a magnetic field.
  • a solenoid carrying current generates a magnetic field inside the core of the solenoid which is parallel to the axis ofthe solenoid.
  • a ferromagnetic core in one half side of the solenoid will generate a magnetic field with the same configuration extending the magnetic field of the pole linearly to the end of the solenoid.
  • the magnetic field generated by the solenoid causes responsive particles in the solution to become induced magnets, thereby creating their own magnetic field. These particles can be considered to be microscopic magnets.
  • the magnetic field ofthe apparatus causes the components to migrate by interacting with the magnetic fields created by the components.
  • the separating force supplied by the action of an external electric field on the electrical fields ofthe electrical double layer in electrophoretic devices and methods
  • the components desired to be removed via magnetophoretic devices and methods either exhibit magnetic fields of their own accord or by the attachment thereto of a magnetic particle.
  • Such particles may be iron particles which exhibit magnetic fields by magnetic induction.
  • the components to remain in solution either exhibit no magnetic field or exhibit a weaker a magnetic field and decreased transport properties than that of the desired component.
  • magnetic particles (with an affinity for the component desired to be separated from the solution) are introduced into the solution.
  • the affinity ofthe magnetic particles is often the product of having substances (which bind to the component desired to be separated from the solution with greater affinity than for any other components in the solution) placed on the surface ofthe magnetic particles.
  • the magnetic particles then bind to the component desired to be removed.
  • the usefulness of magnetic particles which have a biological affinity for a substance desired to be removed in such purification processes is well known in the art. After reactions occur between the substance on the magnetic particle surfaces and the desired component, the particles, with the component bound thereto, are magnetically separated from the solution.
  • the separation from the solution occurs by applying a magnetic field to the solution, thereby causing the magnetic particles, with the component bound thereto, to be transported through the solution toward the point of greatest (external) magnetic field strength.
  • the other components ofthe solution, having no, or a lesser, magnetic susceptibility are not transported through the solution by the magnetic field.
  • the particle-bound component can be recovered from the magnetic particles by methods known in the art for cleaving the bond between component and substance.
  • Fetal cells are used to obtain a wealth of information about the gestating fetus.
  • Fetal nucleated blood cells can be used as a source of DNA to determine the fetus gender, and to predict the likelihood ofthe occurrence of such genetic defects as Down's syndrome, ⁇ -thalassemia, phenylketonuria, cystic fibrosis, Duchene's muscular dystrophy, sickle cell anemia, and the like.
  • PUBS periumbilical blood sample
  • fetal red blood cells are present in the maternal whole blood supply as early as fifteen weeks into the gestation period. Therefore, maternal blood could be a source of fetal red blood cells. Drawing blood from the mother to obtain the supply of fetal red blood cells greatly reduces the risks associated with removing amniotic fluid from the placenta. Hence, it is desirable to develop a method and apparatus for purifying the fetal cell sub-population from the maternal blood sample. The desired method should obtain a high degree of purity while being minimally invasive to the mother and fetus. This would allow the performance ofthe useful tests on the fetal cells without the risks attendant with the removal of amniotic fluid.
  • Magnetic Activated Cell Sorting binds small iron beads covered with a monoclonal antibody, specific for the component desired to be removed from the solution.
  • the beads are introduced into the solution, where the antibodies react with the component to be removed, binding the beads to the component.
  • the solution is then applied to a magnetized surface.
  • the magnetic surface attracts the magnetic particles, thereby attracting the component bound thereto.
  • the surface is then washed to remove the non-bead-coated cells.
  • MACS has failed to provide the high level of separation achieved by the current invention. MACS suffers from non-specific adhesion of components to the metal filings and difficulty in removing magnetized components from the metal filings.
  • MACS requires a large initial sample to attain appreciable yields of the desired cell sub- population removed from the original sample.
  • the MACS apparatus creates areas having insufficient magnetic field strength to remove the desired sub-population from the sample.
  • a large number of washing steps are required to remove the bound cells from the magnetic surface. These washing steps not only decrease the yield of the desired sub-population, but decrease the concentration of the desired sub-population present in the washing step effluent.
  • Fluorescent Activated Cell Sorting an enrichment procedure, uses lasers to excite cells labeled with specific monoclonal antibodies as an enrichment means. These labeled cells are then sorted for further analysis.
  • This labeling method allows one skilled in the art to realize the presence of the desired component in solution, but does nothing to separate the desired component from the solution. Either before of after labeling, the desired substance is removed by other means.
  • FACS Fluorescence Activated Cell Sorting
  • NRBC nucleated red blood cells
  • This NRBC layer is then further purified by panning to remove CD 45+ cells (seen on almost all lymphocyte lineage cells, but not seen on NRBC). Markers comprising fluorescently labeled monoclonal antibody are then attached to the remaining cells.
  • the two markers with the best fetal cell specificity have been monoclonal antibody to the CD 71+ surface antigen and monoclonal antibody to the internal gamma chain of fetal hemoglobin.
  • the fluorescent labeling of the NRBC makes them identifiable for separation from the maternal blood supply by micro manipulators. (Bianchi, 1995).
  • Cheung et. al. (1996) attempt to isolate fetal cells present in the maternal circulation for genetic screening to search for molecular defects.
  • the method of Cheung et al. comprises the steps of density gradient separation of the maternal blood supply, subjecting the fetal cell enriched layer to MACS utilizing a CD71 -binding particle, applying the enriched portion from the MACS to a support, staining with anti -bodies specific for fetal or embryonic hemoglobin, and removing stained fetal cells with micro- manipulators.
  • U.S. Patent No. 4,241,176 to Avrameas et al. discloses a magnetic gel for use in separating materials. Optionally the gel can contain an antibody specific to the material which is desired to be separated from a solution.
  • the magnetic gel is placed along the inside walls of a column and held in place by a static magnetic field, as opposed to the current invention which uses pulsed, variable strength magnetic fields.
  • U.S. Patent No. 4,375,407 to Kronick discloses a high gradient magnetic separation device having a filamentary magnetic material in the interior chamber thereof. This reference discloses coating the filamentary material with a coating of hydrogel polymer. The device disclosed in this reference uses uniform magnetic fields. There is no teaching of the use of the pulsed, variable strength magnetic fields of the current invention.
  • U.S. Patent No. 4,594,160 to Heitmann et al. discloses a magnetic separator having a combination of screens and balls placed in the interior chamber thereof to intensify the magnetic field strength within the chamber.
  • the separator uses direct- current to produce a magnetic field of at least 1.5xl0 5 H at all times. Therefore, there is no teaching of pulsing the strength of the magnetic field, as utilized in the current invention.
  • U.S. Patent No.4,772,383 to Christensen also discloses a high gradient magnetic separator having permanent magnetic devices generating strong magnetic fields across a separating chamber, as opposed to the pulsed, variable strength magnetic fields utilized by the current invention.
  • U.S. Patent No. 5,004,539 to Colwell et al. discloses a magnetic separator having permanent magnetic elements which cause the separation chamber to be subjected to a permanent magnetic field.
  • the magnetic flux return paths are made of ferromagnetic materials (column 3, lines 13 -- 16), which by definition, permanently maintain their magnetic state.
  • the magnetic flux channels of the separator disclosed in this reference are incapable of producing the pulsed, variable strength magnetic fields utilized by the current invention.
  • U.S. Patent No. 5,122,269 to De Reuver discloses a magnetic filter wherein the magnetic gradient across the filter chamber is substantially constant.
  • the substantially constant magnetic gradient is required to maintain even filling, and reduced emptying, ofthe filter.
  • U.S. Patent No. 5,186,827 to Liberti et al. discloses a device and method for separating biological materials using magnets to produce magnetic fields about a contact surface.
  • the magnetic flux density quickly reduces in a direction away from the surface, thereby allowing the device to collect the desired biological component in a thin layer on the contact surface, preventing the undesired component from becoming entrapped in the desired material layer.
  • U.S. Patent No. 5,236,824 to Fujiwara et al. discloses an apparatus and method for quantitating the amounts of biological materials separated by high gradient magnetic separation. After separation, a light source, preferably a laser light, is radiated upon the separated material and the amount of returned (scattered, reflected) light is measured to determine the quantity of material separated. There is no teaching ofthe benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
  • U.S. Patent No. 5,275,933, to Teng et al. discloses material separation via a discontinuous gradient.
  • Test tubes are filled with three HISTOPAQUE solutions of different densities to provide a triple gradient layer in each ofthe tubes.
  • Whole maternal blood is added to the top of this triple gradient and the tubes are spun to yield layers into which cells of different densities have been partitioned.
  • the top layer is rich in lymphocytes.
  • the second layer contains NRBCs and the third layer is predominantly populated with granulocytes.
  • line 66 through column 7, line 5 the fetal cells are spread about over different layers in the test tube after centrifugation, not in a single discreet layer allowing for easy removal and analysis of the fetal cells.
  • the number of fetal cells yielded in this method is quite low and varies between different pregnant woman (between 1/10,000 to less than 1/1 ,000,000). Further, there is no teaching ofthe benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
  • U.S. Patent No. 5,279,936 to Vorpahl discloses a method of magnetically separating materials in solution.
  • the materials desired to be separated are bound to magnetic carriers.
  • a second solution, of different density than the material-containing solution is contacted with, without mixing, the material-containing solution.
  • the two fluid system is then subjected to static magnetic fields and the material bound to the carrier migrates across the solution interface.
  • U.S. Patent No. 5,340,749 to Fujiwara et al. discloses a method for collecting specimens comprising labeling the specimens with magnetic particles and subjecting the labeled particles to a permanent gradient magnetic field.
  • This antibody binds to an epitope on the fetal NRBCs (made from repeating N-acetyl lactosamine units of a given structure), thereby attaching at least a portion ofthe fetal NRBCs to the substrate. Non- adherent cells are then washed from the substrate.
  • a pulsed, variable strength magnetic field as utilized in the current invention.
  • U.S. Patent No. 5,639,669 to Ledley discloses a device and method for separating maternal blood cells and fetal blood cells from a mixture comprising the two.
  • the device and method disclosed by Ledley uses ultrasonic mixing to enhance the separation.
  • Ledley's method applies an electromagnetic field to a treated sample containing maternal and fetal blood cells
  • Ledley's method and apparatus differ from those ofthe current invention.
  • the Ledley method and apparatus do not utilize a pulsed, variable strength magnetic field as utilized in the current invention.
  • Ledley's device and method require manipulation of an extensively pre-treated solution containing the maternal and fetal blood cells to achieve appropriate conditions of 0 2 concentration, pH, CI " ion concentration, CO 2 concentration and temperature ofthe solution. Once the optimal conditions are finally reached, the solution is subjected to a magnetic field.
  • Ledley utilizes ultrasonic vibrations to aid in the separation process.
  • the invention comprises a pulsed, laminar magnetic field cell transport apparatus and a pulsed, laminar magnetic field separation method capable of isolating highly purified cell sub-populations from small amounts of heterogeneous cell populations.
  • One method according to the current invention uses magnetic particles which incorporate biological ligands, in particular monoclonal antibodies, which bind to the specific antigenic cell markers on the cells desired to be removed from the heterogenous population. More than one type ofthe magnetic particle, each type having a different ligand, may be used to remove one or more desired cell type.
  • the heterogenous population containing the cells is then placed on a substrate medium.
  • the heterogenous population is exposed to laminar magnetic fields, whose direction, strength and duration can be controlled.
  • the desired cells then travel through or across the medium and are separated from cells not attached to magnetic particles in the heterogenous population.
  • each magnetic particle may bind to more than one cell, transporting more than one cell with the movement of each magnetic particle.
  • subsets of magnetic particle-carrying cells can also be separated from the solution. This further level of separation may be a function of cell isotypes having a different number of antigenic surface markers on their surface.
  • This difference in the number of antigenic surface markers causes a different amount of particles to bind to the surface of the cell isotypes, thereby increasing the effect ofthe magnetic fields generated by the apparatus ofthe current invention on the cells having the particles bound thereto.
  • the current invention has the potential to increase quite significantly the specificity and sensitivity ranges now available in currently utilized magnetic bead separating devices.
  • It is a further obj ect ofthe invention to provide a method for separating biological materials and other substances from a mixture containing desired and undesired components comprising placing a sample of said mixture onto a substrate material; exposing said substrate coated with said sample to a magnetic field of sufficient strength to cause the desired components to migrate across said substrate; and repeatedly activating and deactivating said magnetic field in a pulsing manner with a frequency sufficient to cause said desired magnetic components to separate spatially from the undesired components.
  • Figure 1 is a block diagram of the components ofthe pulsed, laminar magnetic field transport system ofthe invention.
  • Figure 2 is a perspective view of a cabinet for the capacitor bank ofthe device of the invention
  • Figure 3 is a schematic showing the wi ⁇ ng of the power source, capacitor bank, switching/junction box and coil chamber of a first embodiment of the device of the invention
  • Figure 4 is a perspective view of a first embodiment of the winding core of the coil chamber ofthe device ofthe invention
  • Figure 5 A is a front view of the coil chamber ofthe invention
  • Figure 5B is a side view of the coil chamber of the invention
  • Figure 5C is a top view of the platform, showing the substrate base in place
  • Figure 6A is a top view of the holder for the substrate base of the invention
  • Figure 6B is a sectional view of the substrate base holder taken along line 6B in
  • Figure 7A is a graph showing the lines of force in kilo-gausses across a ho ⁇ zontal plane in the empty cell separation chamber using continuous DC current
  • the left most values are measurements at the center of the chamber s field
  • the successive values show measurements every 2 centimeters away from the center along a central ho ⁇ zontal plane
  • Figure 7B is a graph showing the lines of the force in kilo-gausses at the same sites as seen in Figure 7A and indicating the increase generated by a one second pulse through the core following the discharge of the capacitors in se ⁇ es Note that this pulse results in a greater than three fold increase in magnetic force at any given site in the chamber
  • Figure 8 A is a schematic showing the wi ⁇ ng ofthe power source, capacitor bank, switching/junction box and coil chamber of a second embodiment of the device of the invention
  • Figure 8B is a front elevational view of the control panel of the device of the invention.
  • Figure 9 is a perspective view of a second embodiment of the winding core of the device ofthe invention
  • Figure 10 is a perspective view of the winding core of Figure 9 with the coils in place
  • Figure 1 1 includes an end elevational view (Fig 11 A) and a side elevational view
  • FIG. 12 is an illustration of a timing diagram for generating a se ⁇ es of pulses for delivery to the winding coil
  • Figure 13 is an illustration of a timing diagram for generating a se ⁇ es of overlapping pulses for delivery to the winding coil
  • the apparatus 10 As illustrated in Figure 1, the apparatus 10 according to the current invention generates a pulsed magnetic field by the discharge of capacitors 31 which transmit a transient elect ⁇ c current through coil 57
  • the apparatus compnses a power source 20, a bank of capacitors 30, a switching/junction box 40, and a coil chamber 50
  • the power source 20, capacitor bank 30, switching/junction box 40, and coil chamber 50 may all be contained in a single housing unit (not pictured)
  • the vanable output voltage of the power source 20 should convert standard alternating elect ⁇ cal current (AC) to a direct current source (DC) for charging the capacitor bank 30 and should be compatible with the voltage rating of the capacitor bank 30
  • the vanable output voltage of the power source 20 should further have a sufficient rating to maintain the charging time of the capacitor bank 30 to less than 10 seconds
  • the power, current, and voltage output levels of the power source 20 can be controlled by the user of the apparatus Preferred power sources deliver an electromotive force of between about 5 and about 50 Volts to the capacitor bank 30 (as used herein, the term “about' " means within a margin of commonlv acceptable error for the determination being made, using standard methods)
  • the current and voltage output levels of power source 20 should be vanably controllable by the user of the apparatus of the invention, either manually or through the operation of automated circuits 35
  • the automated circuits 35 can be programmed as known in the art to operate without constant user supervision. Typical power supplies are readily available from a commercial supplier. An example of a suitable power supply is the Sola Copper Line series.
  • the power from power source 20 is fed to capacitor bank 30 through power cable 15.
  • the power cable 15 should be rated to carry a current of 20 amps over a length of 20 feet.
  • a preferred power cable 15 is an AWG #12 cable.
  • the capacitor bank 30 comprises a plurality of capacitors 31. The means used for placement ofthe capacitors 31 are unimportant, as long a proper cooling is allowed about the capacitors. In one embodiment, the capacitors could be stored in a substantially upright position, with the major axes of the generally cylindrical bodies of the capacitors 31 in a substantially vertical position.
  • FIG. 2 illustrates an embodiment wherein capacitors 31 are stored in a cabinet 32.
  • Cabinet 32 stores capacitors 31 in a position wherein the major axes ofthe generally cylindrical bodies of the capacitors 31 are in a substantially horizontal position. Regardless of their orientation, the capacitors 31 is preferably stored in a manner that allows ease of access to the anode 28 and cathodes 29 ofthe capacitors 31 and prevents movement ofthe capacitors 31, although movement ofthe capacitors 31 should not affect their performance.
  • Cabinet 32 comprises a front panel 33 and rear panel 34 with support means 35 attaching the front panel 33 to rear panel 34.
  • the support means 35 are side panels 36 and top and bottom panels 37, 38.
  • the support means 35 could also comprise a plurality of spacers of equal length placed between front panel 33 and rear panel 34, as long as such spacers provide necessary support for cabinet 32 to allow cabinet 32 to stably support the capacitors 31 when placed in cabinet 32.
  • Front panel 33, rear panel 34, and support means 35 could be prepared from any material known in the art that would support the cabinet 32 and the capacitors 31 stored therein. Non-limiting examples include, wood, sheet metal, ABS plastics, and the like.
  • Front panel 33 has placed therein a plurality of openings 39 of sufficient size to allow passage of at least individual capacitors 31 there through. Front panel 33 should have sufficient opening spaces 39 to allow storage in cabinet 32 of as many capacitors 31 as are desired. Capacitors 31 are placed in cabinet 32 in a position allowing easy access to the anode 28 and cathode 29 of capacitors 31. In embodiments wherein rear panel 34 has no openings placed therein to allow the opposite end of the capacitors 31 (the end not having the anode 28 or cathode 29 thereon) to rest thereagainst when capacitors 31 are placed in cabinet 32, capacitors 31 are maintained in position (wherein the major axes ofthe generally cylindrical bodies of the capacitors 31 are substantially horizontal) by cradles 27.
  • Cradles 27 may also be used in those embodiments wherein the rear panel 34 has openings through which the opposite ends of capacitors 31 pass.
  • Cabinet 32 may be structured to have the anodes 28 and cathodes 29 exposed, or cabinet can have a cover or be placed inside an external enclosure.
  • capacitors 31 comprise a bank of at least six individual capacitors, more preferably twelve individual capacitors.
  • the individual capacitors have a voltage of about 40 volts each and a capacitance of about 420,000 ⁇ F each for use with a winding coil 57 having at least about 20,000 ampere-turns.
  • Junction-switching box 40 comprises a circuit of resistors, switches and diodes, as pictured in Figure 3.
  • junction- switching box 40 allows the capacitors 31 to be charged in parallel (to facilitate rapid charging) and discharged in series (to facilitate maximum acceleration of current flow through the winding coil 57).
  • junction-switching box 40 comprises resistors having values of about 5, 3.5, 1.0, 0.5, and 0.1 ohm.
  • the control panel of junction-switching box 40 has four control switches for operator control, Figures 2 and 3. The switches can be controlled manually or via automated or programmed operation. The switches are spring loaded and can be mounted on the front panel of the junction-switching box 40 for easy access to the operator ofthe device, when manually operated. Each switch has three positions. In the middle position all switches are open.
  • switch SI When switch SI is placed in the down position, the DC voltage from the power source 20 is connected to the surge resistor Rl which is in parallel with the resistors of the time charge control circuits. The output of surge resistor Rl is connected to switch S2.
  • switch S2 When switch S2 is in the down position, the DC voltage is connected to the capacitors in parallel.
  • Switch S3 in the down position lights lamp L2.
  • Test Terminal TP2 measures the voltage on the capacitors 31 in parallel or in series.
  • Test Terminal TPI is system ground. With switches SI, S2, and S3 in the down position the capacitors are charged by the power source 20. With switches SI, S2, and S3 in the up position, the capacitors are in series configuration (240 V), and are connected to the output relay RLY4.
  • RLY5 connects power source 20 to coil 57 providing a DC bias voltage to coil 57.
  • coil 57 remains energized via power source 20 when the capacitors 31 are not energizing the coil 57. More preferably, power source 20 constantly energizes coil 57.
  • the 120 V, 60 Hz power supply operates the relays.
  • FIG 4 illustrates a first embodiment of a winding core 51 of the current invention.
  • Winding core 51 comprises a central tube 52 and end braces 53.
  • Central tube 52 is an elongate structure with a port 54 on either end thereof.
  • Central tube 52 provides the chamber wherein the support base 60 (not shown) is placed.
  • Central tube 52 can be of any shape that permits generation of a laminar magnetic field of uniform density and will allow insertion of support base 60 into the interior thereof and placement of winding coil 57 (not shown) on the exterior thereof.
  • Preferably central tube 52 has a cross- sectional shape that is rectangular. As illustrated, central tube 52 comprises generally rectangular upper and lower panels 55 continuously joined to side panels 56.
  • Preferably upper and lower panels have dimensions of about 10 inches by about 8.25 inches, and side panels have dimensions of about 10 inches by abut 4.5 inches. These dimensions create ports of about 4.5 by about 8.25 inches.
  • Winding core 51 can be manufactured from any non-magnetic material, strong enough to support the weight ofthe winding coil 57, and which does not interfere with the concentration ofthe magnetic field generated inside the central tube 52 by winding coil 57. Examples of such materials are polyethylene, glass, ABS plastic and wood.
  • a first preferred construction of coil chamber 50 is illustrated in Figures 5 A and
  • Winding core 51 has winding coil 57 placed on the exterior thereof.
  • Winding coil 57 comprises conductive material, preferably copper wire, wound about the winding core 51.
  • the copper wire of winding coil 57 is placed on the exterior surface of winding core 51 , and wound thereabout in a helical manner. Each rotation ofthe copper wire is known as a "turn.”
  • Each portion of copper wire placed during a turn abuts against the prior laid turn, thereby covering the entire exterior portion ofthe central tube 52 of winding core 51.
  • the winding of copper wire of winding coil 57 is continued in the opposite direction along the layer of copper wire already wound onto central tube 52.
  • winding core 51 is determined by the number of turns and the capacitance of capacitor bank 30 (not shown). The number of turns is calculated to maximize the amount of current passing through winding coil 57 when capacitor bank 30 is discharged.
  • winding coil 57 comprises about 1000 turns.
  • the foregoing circuit configuration allows a field strength inside the coil chamber 50 of about 2.5 Tesla.
  • coil chamber 50 has magnetic elements 58 placed in the interior portion thereof. Magnetic elements 58 are prepared of a ferromagnetic material and help to increase the strength of the magnetic field generated by current passing through winding coil 57.
  • the strength ofthe magnetic field inside the coil chamber 50 is related to the number of turns and the current flowing through the winding coil 57 by the following:
  • i 0 is the current in the loop
  • n is the number of turns in winding coil 57
  • r is the radius ofthe winding coil 57
  • 1 is the length of winding coil 57.
  • FIG. 8A A second preferred embodiment for wiring the connections ofthe device ofthe current invention, including power sources 82, 84, capacitors 81 Junction-switching box 80, and winding coils 87, 88 is illustrated in Figure 8A.
  • Junction-switching box 80 comprises a circuit of switches and diodes, as pictured in Figure 8 A.
  • Capacitor switches A, B, C, and D selectively electrically connect capacitors 81 with variable output power source 82 and coils 87, 88.
  • Each capacitor switch A - D has two positions. When capacitor switches A - D are placed in the up position, the variable power source 82 is connected to the capacitors 81 through surge control element 86 and diodes 83.
  • capacitor switches A - D With capacitor switches A - D in the up position the capacitors 81 are charged by the variable output power source 82. With switches A - D in the down position, the capacitors 81 are connected to coils 87, 88 through diodes 89. Timing switch 85 controls the timing ofthe switching of capacitor switches A -
  • Timing switch 85 is preferably a rotating cam or more preferably an electronic timing switch.
  • the circuit of junction-switching box 80 allows the capacitors 81 to be charged individually or as sub-groups wired in parallel (to facilitate rapid charging) and discharged sequentially to create a series of pulses in coils 87, 88 Fig. 12.
  • the timing of timing switch 85 is preferably selected, with respect to the discharge rate of capacitors 81, to cause sequential pulses from capacitors 81 to overlap in time, Figure 13.
  • These pulses are delivered to primary coil 87 and secondary coil 88. These coils are constructed with the primary coil 87 containing more turns than the secondary coil 88.
  • the primary coil 87 contains six times more turns than the secondary coil 88.
  • the sample chamber 96 is located within secondary coil 88, Figure 11. Having fewer turns in the secondary coil 88, while maintaining a larger number of turns in this primary coil 87, minimizes heating of the sample contained within secondary coil 88 while maintaining a strong magnetic field.
  • Fig. 8B illustrates a front view ofthe front panel associated with the circuit of Fig. 8 A.
  • FIGS 9 - 11 illustrate a second preferred embodiment of a winding core 91 for use with the second preferred embodiment of the wiring the connections, discussed above.
  • Winding core 91 comprises a vertical post 92, secondary coil core 93, horizontal post 95, horizontal bar 98 and reduced field intensity return pole 94 which are operably connected.
  • vertical post 92, horizontal post 95, and secondary coil core 93 form a monolithic structure.
  • Winding core 91 can be manufactured from any ferromagnetic material.
  • winding core 91 is made from transformer laminate.
  • Rectangular horizontal post 95 extends at 90 ° from rectangular vertical post 92. The end of horizontal post 95 distal to vertical post 92, secondary coil core 93, is formed to receive secondary coil 88.
  • secondary coil core 93 has a decreased cross sectional area compared to that of horizontal post 95.
  • Vertical post 92 can be of any shape that will allow placement of winding coils 87 on the exterior thereof.
  • Secondary coil core 93 can be of any shape that permits generation of a laminar magnetic field of uniform density.
  • Preferably secondary coil core 93 has a cross sectional shape that is rectangular.
  • Preferably secondary coil core 93 has dimensions of about 3 inches by about 0.5 inches in cross section and length of about 6 inches, Figure 11.
  • Preferably vertical post 92 has dimensions of about 3 inches by about 3 inches in cross section, and horizontal post 95 has dimensions of about 3 inches in width by about 3 inches in height.
  • Rectangular horizontal bar 98 extends at 90° from rectangular vertical post 92 and lies parallel to horizontal post 95. The end of horizontal bar 98 distal to vertical post 92 is operably connected to reduced field intensity return pole 94.
  • Return pole upper face 97 is designed to minimize the strength ofthe magnetic field in the vicinity of reduced field intensity return pole 94 while maximizing magnetic field strength in chamber 96.
  • the surface area of return pole upper face 97 relative to that of secondary coil core 93 and its position relative to secondary coil core 93 are designed to achieve this effect.
  • the surface area of return pole upper face 97 is 100 times the surface area of secondary coil core 93.
  • return pole upper face 97 lies below a horizontal plane containing secondary coil core 93 and lies more distal to primary coil 87 than the distal-most extremity of secondary coil 88.
  • the shape of reduced field intensity return pole 94 may be any shape that maintains the above mentioned relationships between the relative areas and locations of return pole upper face 97 and secondary coil core 93.
  • the shape of reduced field intensity return pole 94 is chosen to additionally reduce its weight.
  • a preferred shape is trapezoidal in cross- section, having a reduced area at the surface opposite to return pole upper face 97 as seen in Fig. 9.
  • Winding coils 87, 88 comprise conductive material, preferably copper wire.
  • the copper wire of winding coils 87, 88 is placed on the exterior surface of winding core 91, and wound thereabout in a helical manner. More preferably, the winding coils 87, 88 comprise flat conductive bands whose flat shape permit decreased weight ofthe winding coils 87, 88.
  • the winding begins with primary coil 87. Each portion of copper wire placed during a turn abuts against the prior laid turn, thereby covering the entire exterior portion ofthe vertical post 92 of winding core 91.
  • coil 87 is composed of several modular coils operably connected and stacked on top of one another around vertical post 92. Preferably, 8 modular coils are used.
  • Secondary coil 88 proceeds in a continuous manner after winding ofthe primary coil 87 by running the copper wire (not shown) from the last turn of primary coil 87 along horizontal post 95, where the winding of secondary coil 88 begins.
  • Secondary coil 88 is wound in an analogous manner to that of primary coil 87 with secondary coil 88 having fewer turns than primary coil 87. The number of turns is calculated to maximize the amount of current passing through coils 87, 88 when capacitors 81 are discharged.
  • primary coil 87 comprises about 1200 turns and secondary coil 88 comprises about 200 turns.
  • the winding of secondary coil 88 further differs from that of primary core 87 in that the winding extends beyond the end of secondary coil core 93 to from sample chamber 96 where the support base (not shown) is placed.
  • This allows a field strength inside the coil chamber 50 of about 3 Tesla. and 0.3 Tesla at the return pole upper face 97.
  • Such a reduced field at the return pole upper face 97 has the benefit of protecting an operator from the stronger magnetic field when the return pole upper face 97 is positioned between the operator and chamber 96.
  • chamber 96 has magnetic elements (not shown) placed in the interior portion thereof.
  • Magnetic elements are prepared of a ferromagnetic material and help to increase the strength ofthe magnetic field generated by current passing through winding coils 87, 88.
  • Figure 7 A shows the lines of force in kilo-gausses across a horizontal plane in the empty coil chamber 50 using continuous DC current.
  • Table 1 The numbers in the column to the left are measurements at the center of the chamber's field. The successive columns show measurements every 2 centimeters away from the center along a central horizontal plane.
  • Figure 7B shows the lines ofthe force in kilo-gausses at the same sites as seen in Figure 7A and indicates the increase generated by a one second pulse through the core following the discharge ofthe capacitors in series. The values plotted are shown in Table 2. This pulse results in a greater than three fold increase in magnetic force at any given site in the coil chamber 50. Table 1
  • Support base 60 is illustrated in Figures 6 A and 6B.
  • Support base 60 comprises a slide base 61, preferably prepared from a standard microscope slide.
  • Slide base 60 has walls 62 placed on the upper surface thereof in a pattern creating transport area 63.
  • Walls 62 preferably comprise glass bars.
  • Transport area 63 is coated with a separation medium, also referred to herein as a "substrate material".
  • the separation medium is a substance which allows differential migration of components in the solution under the effect ofthe magnetic field generated by current passing through winding coil 57. Separation media can be any such substance as known in the art for magnetophoretic substrates.
  • the separation medium is a solution comprising a polymeric material or any other material the viscosity of which can be manipulated (e.g., high concentrations of short carbohydrates).
  • the viscosity of the solution can be varied depending on the size and shape of the components to be magnetically separated, as would be apparent to one of skill in the art of magnetophoretic or electrophoretic separation. In general, the viscosity is adjusted such that the component to be separated is substantially unable to diffuse through the material unless the requisite magnetic field is applied.
  • the substrate material comprises a colloidal solution of a polymer, which is of sufficient viscosity to prevent spontaneous diffusion ofthe cells.
  • a particularly preferred substrate material is methylcellulose , as described in greater detail in the sections below. Methylcellulose is advantageous because the viscosity of methylcellulose solutions can be adjusted, but also because it can be combined with various cellular growth media.
  • methyl cellulose does not autofluoresce like other polymeric solutions (e..g., agar), thereby enabling its advantageous use with fluoresence detection methods.
  • Other substrate materials that could be used to separate various cells or subcellular components include, but are not limited to, agarose, agar and polyacrylamide.
  • a sample containing a component of interest (sometimes referred to herein as a "target substance") is exposed to a solution containing magnetic particles.
  • the magnetic particles have ligands capable of directly or indirectly binding to the target substance.
  • the magnetic particles bind with the target substance to form a complex, referred to herein as a "magnetic transport complex.”
  • a sample of the solution containing the magnetic transport complex is placed along one edge ofthe transport area 63 on the support base 60, in the separation medium.
  • the support base 60 is placed inside the coil chamber 50.
  • the power source 20 is activated and the capacitor bank 30 is charged.
  • Capacitors 31 in capacitor bank 30 are charged in a parallel wired configuration. This allows the maximum charge to be placed on the capacitors in a minimum amount of time. The capacitors 31 of capacitor bank 30 are then discharged in series, releasing current into winding coil 57. This maximizes the charge released into winding coil 57. The capacitors 31 are discharged in series in a pulsed manner. The discharge pulses last about 0.86 seconds per discharge and repeats about once every two minutes. Each pulse generates a magnetic field of at least about 0.7 Tesla, more preferably about
  • the magnetic beads may be removed.
  • a sample of blood taken from a pregnant woman was co-incubated withboth a fluorescein labeled monoclonal antibody to CD 71 and with 0.1 micro meter diameter magnetic beads having a monoclonal antibody to the same antigen.
  • the sample was placed at a starting point in the transport area 63.
  • phase contrast microscopy revealed the separation of cells. Virtually every cell that moved through the magnetic field was seen to be positive for CD71 , as seen at 190X using a Zeiss photo-microscope with epi-fluorescence illumination and appropriate FITC filters for detection of fluorescein.
  • the pulsed magnetic separation device and system described herein can be used to improve many separation applications in which magnetic separation is currently utilized.
  • Such applications include separation of biological or non-biological substances.
  • biological substances that can be separated these include eucaryotic and procaryotic cells, subcellular organelles, viruses, proteins, nucleic acids, carbohydrates, ligands or complex molecules comprising nucleic acids, proteins, lipids and/or carbohydrates.
  • non-biological applications these include removal of toxic compounds from industrial waste streams or other environmentally hazardous sites, or the detection of contaminants in sewage treatment processes and the like.
  • a material is separable by the methods described herein if the material possesses at least one characteristic determinant, which is capable of being recognized by and bound to a ligand which is attachable to a magnetic particle.
  • Materials having such characteristic determinants are referred to herein as "target substances” or “desired components”. If the target substance is a cell, it is referred to herein as a “target cell.”
  • target substances or “desired components”. If the target substance is a cell, it is referred to herein as a “target cell.”
  • characteristic determinant is used herein to refer to substances such as antigens, haptens, and other complex molecules (e.g., carbohydrates, glycoproteins, etc.), which are capable ofthe above-described specific binding to a ligand.
  • Ligand is used herein to refer to any substances or group of substances having a specific binding affinity for a given characteristic determinant, to the substantial exclusion of other substances.
  • Monoclonal antibodies are preferred for use as the ligand.
  • polyclonal antibodies or non-antibody receptors including antigens for antibody-producing cells or antigen processing cells, lectins, such as concanavalin A and various agglutinins, biotin-labelled reagents or hapten-labelled reagents, may be used, if desired.
  • the methods of the invention may be structured as "direct” or “indirect” protocols, or some combination thereof.
  • the ligand is attached directly to the magnetic particles, and magnetic complexes are obtained by incubating test samples containing the target substance with the ligand-coated particles.
  • the target substance is incubated with a free ligand and the magnetic particles comprise a capture agent capable of recognizing and binding specifically to the ligand, so as to form a complex comprising target substance, ligand, capture agent and magnetic particle.
  • suitable capture agents include Protein A or Protein G, where immunoglobulin is used as the ligand; avidin, where a biotin-labelled reagent is used as the ligand; and anti-hapten, where a hapten-labelled reagent is used as the ligand.
  • biotin or a hapten may be used to facilitate capture of lectin ligands, e.g., concanavalin A and various agglutinins, which bind selectively to membrane-containing target substances whose characteristic determinants comprise carbohydrate or glycoprotein components.
  • Hapten/anti-hapten pairs suitable for this purpose include dinitrophenol (DNP)/anti-DNP, fluorescein/anti-fluorescein or arsanilic acid/anti-arsanilic acid.
  • the magnetic separation methods and devices ofthe invention may be used to carry out cell separation for isolation and or analysis of specific cell populations. Because high levels of recovery and purity are achievable by the methods ofthe invention, these methods are particularly suitable for removal or isolation of rare cells from a mixed population of cells.
  • Such separations include, but are not limited to, enrichment of stem cells from bone marrow or peripheral blood, isolation of fetal cells from maternal blood, isolation of transfected cells, and removal or isolation of tumor cells from various mixed cell populations. Such separations may be accomplished by positive selection or negative depletion, or both, in accordance with the present invention.
  • target cell a cell subset enriched by negative depletion is actually a non-target cell, since it is not bound to an antibody or other ligand. Instead, cells to be depleted from the population are target cells, within the definition.
  • Cells recovered by such separation methods may be utilized for numerous purposes, including further analysis (e.g., by flow cytometry or other methods) or for therapeutic purposes (e.g., re-introduction of enriched populations of stem cells to patients).
  • the methods and devices of the present invention may be used to particular advantage in a combined strategy for isolating a small population of rare cells (e.g., stem cells, fetal cells) from a mixed cell population, while simultaneously purging the population of unwanted cells (e.g., tumor cells).
  • This may be accomplished simply by incorporating a receptor for the unwanted cell type in the negative depletion separation systems described hereinabove.
  • the unwanted population comprises a subpopulation of cells already targeted for depletion in such a system, no additional antibodies need be added.
  • the unwanted target cell populations are B-cells, already targeted for removal in the hematopoietic stem cell enrichment process.
  • additional monoclonal antibodies directed to various tumor cells or other unwanted cells are added to the negative depletion antibody mix.
  • Panning methods to remove CD 45+ cells from the granulocyte layer was done by first coating apetri-dish with 11 ml of goat anti-mouse IgG ( 10 ⁇ g/ml in 0.05 M Tris Buffer -Capel-INC Pharm. Inc.) at room temperature. The petri-dish was washed three times with PBS and once with 1% fetal calf serum in PBS. To 2 — 3 million cells/ml of the granulocyte layer, was added 10 ⁇ l of CD45 monoclonal antibody (10 ⁇ g/ml in PBS - Immunotech, Inc.) at room temperature for 20 minutes.
  • Micro Bead and Fluorescent Dye Attachment Two types of CD 71 monoclonal antibody-coated magnetic beads were used, which differ in their size. The larger beads were super-magnetic, mono dispersed polystyrene micro-spheres averaging 4.5 microns in diameter (Dynal. Inc. N.Y. Cat # M- 450). The smaller super paramagnetic micro beads were extremely small with a diameter range of between 50 and 100 nanometers ( Miltenyi Bioec Co. Ca. Cat# 426-01). Paramagnetic, as used herein, refers to a material which is magnetizable when placed within a magnetic field. Attachment was accomplished by taking 20 ⁇ l of the bead solution, adding it to 1 ml of cell suspension and incubating at a temperature of between 0° and 4°C for 1 hour.
  • the desired cells were labeled with fluorescent compounds to locate them in the methylcellulose media after magnetic separation, while maintaining their viability for tissue culturing.
  • Direct immuno-fluorescence staining ofthe cells possessing the CD 71 surface antigen by monoclonal antibody conjugated with fluorescein (FITC, Becton- Dickson Co. Cat # 347513) was done by adding 20 ⁇ l of FITC-labeled-CD 71 to about 1 ml of cell suspension and incubating at a temperature of between 0° and 4°C for 30 minutes.
  • the beads were added first for a 1 hour incubation followed by the FITC 5 antibodies for a 30 minute incubation.
  • Methylcellulose solutions of various viscosities were used. Aqueous solutions were prepared using methylcellulose powder (Sigma Chemical Co. Mo. Cat #
  • G' less modular-fluid nature
  • G" storage modular- solid nature
  • ETA* complex viscosity
  • Magnetic Separation A glass slide was made with methyl cellulose transport medium having the desired viscosity layered onto its surface. An aliquot of the solution containing the magnetic bead-labeled cells was placed at one edge of the methylcellulose to form a starting line. When the magnetic field was activated, the cells were drawn towards the center ofthe chamber in a linear manner. This produced a line of cell movement away from the starting point, creating a separation from the undesired component. The separated cells can be seen to create a band, that can be taken off the slide using a stereoscope to locate the separated cells, and a pipette to aspirate off these cells. Cell Aspiration of CD 71+ Cells by Micro-manipulation
  • the cells with magnetic beads have been separated from the non-magnetic cells, they form a line that can be seen under a stereo microscope.
  • the tip of a pipette that is on a micro manipulator can be placed at this cell line and the cells can be aspirated into the pipette using negative pressure.
  • the cells that have been withdrawn can then be placed onto a microscope slide for multicolor FISH and fluorescent antibody staining techniques to analyze for chromosome abnormalities. Individual cells can also be placed into wells for PCR studies.
  • FISH Fluorescent In-Situ Hybridization

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Abstract

La présente invention concerne une méthode visant à séparer des matières biologiques et autres substances. Un mélange, contenant des composants ciblés et des composants non ciblés, est exposé à des particules magnétiques ayant des ligands capables de se fixer sur les composants ciblés et/ou non ciblés pour former un mélange magnétique. Ledit mélange magnétique est placé sur un substrat, et ce dernier, recouvert par le mélange magnétique, est ensuite exposé à un champ magnétique suffisamment puissant pour faire migrer les composants magnétiques d'un côté à l'autre du substrat. Le champ magnétique est augmenté ou réduit à plusieurs reprises par impulsion, avec une fréquence suffisante pour permettre la séparation spatiale des composants magnétiques ciblés des composants magnétiques non ciblés. L'invention concerne également un dispositif permettant de séparer les matières biologiques et pouvant s'utiliser pour augmenter et réduire le champ magnétique par impulsion.
PCT/US2000/000274 1999-01-06 2000-01-06 Methode et dispositif permettant de separer des matieres biologiques et autres substances Ceased WO2000040947A1 (fr)

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AU26016/00A AU2601600A (en) 1999-01-06 2000-01-06 Method and apparatus for separating biological materials and other substances
US09/869,741 US7364921B1 (en) 1999-01-06 2000-01-06 Method and apparatus for separating biological materials and other substances
CA002358069A CA2358069A1 (fr) 1999-01-06 2000-01-06 Methode et dispositif permettant de separer des matieres biologiques et autres substances
EP00904229A EP1151271A4 (fr) 1999-01-06 2000-01-06 Methode et dispositif permettant de separer des matieres biologiques et autres substances

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WO2008156688A2 (fr) * 2007-06-15 2008-12-24 Purdue Research Foundation Séparation magnétophorétique non-linéaire de substances biologiques
EP3106229B1 (fr) * 2015-06-17 2020-07-29 IMEC vzw Tri cellulaire magnétique dynamique
CN113125707A (zh) * 2020-01-10 2021-07-16 陈琪 一种分离磁微粒的方法、装置及设备
CN113218737A (zh) * 2020-01-21 2021-08-06 陈琪 一种通过使磁微粒往复运动来分离磁微粒的方法和装置

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