Particle separator system, materials, and methods of use

Abstract

The present invention concerns systems, materials, and methods of cell, cell organelle and / or particle separation and / or concentration utilizing magnetic levitation to affect separation. In some embodiments, the separation and / or concentration is performed in the wells of a multi-well plate.

Description

Attorney Docket LEV-06-PCT PARTICLE SEPARATOR SYSTEM, MATERIALS, AND METHODS OF USE CROSS REFERENCE TO RELATED APPLICATIONS [0000.1] This application claims priority benefit of U.S. Provisional Application Ser. No. 63 / 664,699, filed June 26, 2024, and U.S. Provisional Application Ser. No. 63 / 763,195, filed February 25, 2025, the contents of which are incorporated herein their entireties. TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates generally to the concentration of particulate containing samples, such as cells or biomolecules, in order to isolate such particles within a medium and to isolate particle depleted medium. In some embodiments, the present invention relates generally to the separation and / or concentration of cells and / or cell organelles, from unwanted species such as small molecules, e.g. dyes, cellular debris, small particles and dead cells. BACKGROUND OF THE INVENTION

[0002] Isolation of particles contained within a medium is an important step in many chemical and biological processes. In some processes there may be a need to simply isolate a particle to facilitate the use or manipulation of the particle, whereas in other processes there may be a need to separate the particle from other particles that are also present in the medium. Various devices have been developed to facilitate such particle isolation and separation. In addition, there have been attempts to develop devices that rely on the magnetic properties of the particles and their surrounding medium in order to separate out particles of interest from heterogenous populations of particles.

[0003] A common need when working with cells is to concentrate the cells by reducing the volume that the cells are suspended in. The most common procedure for cell concentration is to centrifuge the cells to form a pellet and removing a large portion of the media. Centrifugation involves the application of centrifugal force to separate particles from a solution according to their size, shape, density, viscosity of the medium,Attorney Docket LEV-06-PCT and rotor speed. However, there are instances in which centrifugation is undesirable, where centrifugation can create damage to the cells or activate the cells. For example, centrifugation with T cells can lead to activation of the cells. Additionally, when working with rare or low volume samples, bulk separation techniques such as centrifugation can be extraordinarily wasteful or laborious and do not easily allow for fractionation of the sample. Also, when the particles to be separated are fragile or labile, such as when working with biological entities, precise conditions to enhance particle stability can be challenging.

[0004] Magnetic levitation is a technique that uses competing gravitational (buoyant) and magnetic forces to form an effectively continuous density gradient in an aqueous paramagnetic medium in a magnetic field, and allows separations of particles suspended in the medium on the basis of their densities. However, present methods for separating particles, including magnetic levitation are not amenable to high throughput screening techniques. The high-throughput devices and methods described herein address these issues by providing alternative methods for concentrating particles, e.g. cells and cell organelles of interest, and producing contaminant-depleted and / or particle- depleted medium that does not depend on the high mechanical forces that are required during centrifugation. SUMMARY OF THE INVENTION

[0005] The inventive embodiments provided in this Summary of the Invention are meant to be illustrative only and to provide an overview of selected embodiments disclosed herein. The Summary of the Invention, being illustrative and selective, does not limit the scope of any claim, does not provide the entire scope of inventive embodiments disclosed or contemplated herein, and should not be construed as limiting or constraining the scope of this disclosure or any claimed inventive embodiment.

[0006] In one aspect, provided herein is a fluidic concentrator device or system (System 1) for conducting magnetic levitation separation of samples in a multi-well plate, the system comprising: a levitation sample fixture comprising:Attorney Docket LEV-06-PCT a multi-well plate comprising a top surface and a plurality of wells, wherein said wells are optionally transparent; a magnet array comprising a plurality of magnets disposed in between and below the wells, configured to provide a magnetic field in each of the wells; a magnet holder configured to receive and hold the magnets of the magnet array; optionally, a mirror assembly comprising: mirrors positioned to project, substantially parallel to the top surface of the plate, images of the wells along their vertical axes; and optionally a mirror holder; optionally, a plurality of metal pins configured to attenuate and / or adjust the magnetic fields in the wells; and optionally, a imaging array disposed beneath the multi-well plate and the magnet array.

[0007] Embodiments of System 1

[0008] The present disclosure further provides the following embodiments of System 1: 1.1 System 1, further comprising a fluid transfer system. 1.2 System 1.1, wherein the fluid transfer system comprises means to dispense or remove all or part of a sample or other fluid into or out of the wells; e.g., a pipette or pipetting robot. 1.3 System 1, or 1.1-1.2, further comprising means to move the multi-well plate in a vertical direction relative to the magnet array. 1.4 Any System 1 or 1.1 et seq., wherein the magnet array comprises permanent magnets. 1.5 System 1.4, wherein for each well: a first magnet and a second magnet are disposed adjacent to the well to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the first magnet faces the first pole of the second magnet; andAttorney Docket LEV-06-PCT a third magnet is stacked under the first magnet, and a fourth magnet is stacked under the second magnet, wherein the third and fourth magnets each comprise a first pole and a second pole, and wherein the second pole of the third magnet faces the second pole of the fourth magnet. System 1.5, wherein the magnets are rectangular magnets; optionally configured substantially in accordance with FIG. 1. System 1.4, wherein the magnets are rectangular magnets arranged in a linear Halbach array. System 1.4, wherein the magnets are ring magnets that surround the wells. System 1.8, wherein the magnets are radially magnetized. System 1.9, comprising for each well two ring magnets stacked one upon the other, wherein the poles of the stacked magnets are radially opposed. System of claim 1.10, wherein the stacked ring magnets are configured to create a strong gradient at the interface with a low field zone in the height of the upper magnet. System 1.8, wherein the magnets are configured to be axially magnetized. Any System 1 or 1.1 et seq., wherein the magnets provide a magnetic field in each of the wells of between about 0.1 Tesla and about 2.0 Tesla and optionally between about 0.3 Tesla and about 1.0 Tesla at the surfaces of the magnets, varying to zero Tesla in certain locations due to superposition of fields from the plurality of magnets. Any System 1 or 1.1 et seq., wherein the imaging array is present and comprises: a) a microscope, e.g., a USB microscope and means for moving the microscope underneath the multi-well plate from well to well; or b) a camera array comprising a plurality of cameras; for example a motorized camera array. System 1.14, wherein the plurality of cameras comprise one or more of built-in lenses, motorized focus, and zoom-in capability; and wherein each camera isAttorney Docket LEV-06-PCT configured to capture images from one to twenty-four of the wells; for example from four wells or from six wells. System 1.14 or 1.15, further comprising a graphics processing unit (GPU) comprising graphics software that integrates two or more of the images from the cameras. Any System 1 or 1.1 et seq., wherein the mirror assembly is present and comprises a mirror holder and mirrors. System 1.17, wherein where the mirrors are either: a) a separate layer with reflective surfaces; or b) a mirror-coating disposed on the mirror holder. Any System 1 or 1.1 et seq., wherein the mirror assembly is present and the mirrors are positioned at about 40 ° to about 50 ° with respect to the vertical axis substantially perpendicular to the main surface of the multi-well plate. Any System 1 or 1.1 et seq., wherein the mirror assembly is present and the mirrors are configured substantially in accordance with FIG. 2. Any System 1 or 1.1 et seq., wherein the wells are conical. Any System 1 or 1.1 et seq., wherein the wells have four vertical sides and a square bottom. Any System 1 or 1.1 et seq., wherein the levitation sample fixture comprises a plurality of metal pins configured to attenuate and / or adjust the shape of the magnetic fields in the wells. System 1.23, wherein the metal pins are steel rods disposed in between the wells and are configured to increase the magnetic field gradients within the wells. Any System 1 or 1.1 et seq., wherein the multi-well plate is configured to fit in the footprint of a standard 96-well PCR plate; or is in accordance with ANSI SLAS microplate standards; e.g., with a footprint of 127.76 mm × 85.48 mm ± 0.5 mm. Any System 1 or 1.1 et seq., wherein compatibility with standards or common equipment is achieved by setting the spacing between wells to 9 mm. System 1.4, wherein for each well:Attorney Docket LEV-06-PCT a first magnet and a second magnet are disposed adjacent to, and on opposite sides of, the well to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the first magnet faces the first pole of the second magnet; a third magnet is stacked under the first magnet, and a fourth magnet is stacked under the second magnet, wherein the third and fourth magnets each comprise a first pole and a second pole, and wherein the second pole of the third magnet faces the second pole of the fourth magnet; a fifth magnet and a sixth magnet are disposed adjacent to, and on opposite sides of, the well, to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the fifth magnet faces the first pole of the sixth magnet, and wherein the fifth and sixth magnets are oriented at approximately 90 degrees in the horizontal plane relative to the first and second magnets; and a seventh magnet is stacked under the fifth magnet, and an eighth magnet is stacked under the sixth magnet, wherein the seventh and eighth magnets each comprise a first pole and a second pole, and wherein the second pole of the seventh magnet faces the second pole of the eighth magnet. System 1.27, wherein the magnets are rectangular magnets; optionally configured substantially in accordance with FIG. 26B. Any System 1.27 et seq., wherein the magnets provide a magnetic field in each of the wells of between about 0.1 Tesla and about 2.0 Tesla and optionally between about 0.3 Tesla and about 1.0 Tesla at the surfaces of the magnets, varying to zero Tesla in certain locations due to superposition of fields from the plurality of magnets. Any System 1.27 et seq., wherein the imaging array is present and comprises: a) a microscope, e.g., a USB microscope and means for moving the microscope underneath the multi-well plate from well to well; or b) a camera array comprising a plurality of cameras; for example a motorized camera array.Attorney Docket LEV-06-PCT System 1.30 wherein the plurality of cameras comprise one or more of built-in lenses, motorized focus, and zoom-in capability; and wherein each camera is configured to capture images from one to twenty-four of the wells; for example from four wells or from six wells. System 1.30 or 1.31, further comprising a graphics processing unit (GPU) comprising graphics software that integrates two or more of the images from the cameras. Any System 1.27 et seq., wherein the mirror assembly is present and comprises a mirror holder and mirrors. System 1.33, wherein where the mirrors are either: a) a separate layer with reflective surfaces; or b) a mirror-coating disposed on the mirror holder. Any System 1.27 et seq., wherein the mirror assembly is present and the mirrors are positioned at about 40 ° to about 50 ° with respect to the vertical axis substantially perpendicular to the main surface of the multi-well plate. Any System 1.27 et seq., wherein the mirror assembly is present and the mirrors are configured substantially in accordance with Figure. 2. Any System 1.27 et seq., wherein the wells are conical. Any System 1.27 et seq., wherein the wells have four vertical sides and a square bottom. Any System 1.27 et seq., wherein the levitation sample fixture comprises a plurality of metal pins configured to attenuate and / or adjust the shape of the magnetic fields in the wells. System 1.39, wherein the metal pins are disposed in between the wells, for example diagonally in between the wells, and are configured to increase the magnetic field gradients at selected area(s) within the wells. System 1.39 or 1.40, wherein the metal pins are disposed substantially as shown in FIG. 26B. Any System 1.39-1.41, wherein the metal pins comprise or are made of a material with high magnetic susceptibility such as steel, iron, cobalt, nickel, andAttorney Docket LEV-06-PCT alloys such as manganese alloy, magnesium alloy, nickel alloy, and chromium alloy, for example steel, manganese alloy, nickel alloy or chromium alloy rods. 1.43 Any System 1.27 et seq., wherein the multi-well plate is configured to fit in the footprint of a standard 96-well PCR plate; or is in accordance with ANSI SLAS microplate standards; e.g., with a footprint of 127.76 mm × 85.48 mm ± 0.5 mm. 1.44 Any System 1.27 et seq., wherein compatibility with standards or common equipment is achieved by setting the spacing between wells to 9 mm, or any other standard or non-standard microplate or well plate layout. 1.45 Any System 1.1 et seq., wherein the fluid transfer system comprises a pipette or pipetting robot. 1.46 System 1.45, wherein the pipette or pipetting robot is equipped with gripper or manipulator arm. 1.47 System 1.45 et seq., wherein the pipettor tips are aligned with the centers of the sample wells. 1.48 System 1.47, wherein the pipetting robot comprises an optical alignment system. 1.49 System 1.48, wherein the optical alignment system is used to align or verify alignment of the pipettor tip(s) with the center of the sample well(s).

[0009] In a further aspect, provided herein is a method ("Method 1") for separating live cells from a sample comprising said live cells and one or more contaminating species, the method comprising: loading a) the sample; and b) a paramagnetic medium comprising a paramagnetic compound or ferrofluid, into a well of a system according to System 1 and 1.1 et seq. to form a sample fluid or sample suspension in said well; and subjecting the sample fluid or sample suspension to a magnetic force from at least one magnet from the magnet array, to effect separation of the live cells from the contaminating species; and optionally imaging the sample fluid or sample suspension prior to, during, and / or after the separation.Attorney Docket LEV-06-PCT

[0010] Embodiments of Method 1

[0011] The present disclosure further provides the following embodiments of Method 1: 1.1 Method 1, wherein the contaminating species is selected from one or more of a dissolved or suspended compound (e.g., a dye or dyes, antibodies, etc.); cellular debris; small particles (e.g., micro- or nano-beads); and dead cells. 1.2 Method 1.1, wherein the contaminating species is a dye or dyes. 1.3 Method 1.1, wherein the contaminating species is cellular debris. 1.4 Method 1.1, wherein the contaminating species is dead cells. 1.5 Any Method 1 or 1.1 et seq., wherein the paramagnetic medium comprises one or more of a paramagnetic salt, a paramagnetic metal chelate, or a paramagnetic ionic liquid; for example a water soluble paramagnetic metal chelate. 1.6 Any Method 1 or 1.1 et seq., further comprising collecting the separated live cells. 1.7 Method 1.6, wherein the separated live cells are collected from the well in a pipette tip. 1.8 Method 1.7, wherein the separated live cells are collected by the steps of: a) moving the multi-well plate vertically upward relative to the magnet array while maintaining the separated live cells in place with the magnetic field, thereby causing the separated live cells to migrate to the bottom of the well; and b) either: i) removing the liquid above the separated live cells with the pipette tip, leaving the separated live cells in a reduced volume of fluid at the bottom of the well; or ii) removing the separated live cells from the bottom of the well with the pipette tip. 1.9 Method 1.7, wherein the separated live cells are collected by the steps of: a1) moving the pipette tip a vertical direction while maintaining the separated live cells in place with the magnetic field, to bring the cells to the top of the pipette tip; and a2) dispensing the liquid below the cells while retaining the separated live cells in the pipette tip;Attorney Docket LEV-06-PCT or b1) moving the pipette tip a vertical direction while maintaining the separated live cells in place with the magnetic field, to bring the separated live cells to the bottom of the pipette tip; and b2) selectively dispensing the separated live cells, leaving unwanted liquid in the pipette tip. Method 1.7, wherein the separated live cells are collected by the steps of: a) inserting the pipette tip into the sample past the levitation position of the separated live cells; b) drawing the liquid up into the pipette tip while maintaining the separated live cells in place with the magnetic field; and c) dispensing just the separated live cells. Method 1.10, wherein step (a) is performed early in the levitation process. Method 1.7, wherein the separated live cells are collected by the steps of: a) after achieving levitation equilibrium, aspirating the liquid into the pipette tip until the separated live cells are aspirated; b) withdrawing the pipette tip from the well and immersing it into a second well in the plate or a separate plate that contains a washing liquid; c) aspirating the separated live cells into the washing liquid while retaining the liquid in the pipette; i.e., slowly removing the pipette tip and the liquid therein, while the field maintains the position of the levitated cells, thus separating them from the original liquid; and d) optionally repeating steps (a)-(c). Method 1.7, wherein the live cells are levitated by radial magnets, and collected by the steps of: a) aspirating the entire sample into the pipette tip; b) moving the pipette tip and sample vertically upwards until they are clear of, and above, the magnets; c) slowly lowering the tip into the magnetic field, and allowing a spheroid of particles to begin to coalesce in the region of minimal field;Attorney Docket LEV-06-PCT d) after the tip completes passage through the region of minimal field, ejecting most of the tip's contents while the tip is moved slowly upwards, leaving a concentrated sample at the bottom of the tip; and e) optionally transferring the concentrated sample. Method 1.7, wherein the live cells are levitated by radial magnets, and collected by the steps of: a) slowly aspirating the entire sample into the pipette tip creating a spheroid of particles at the region of minimal field; b) moving the pipette tip vertically upwards through the magnetic field while the particle spheroid remains stationary relative to the field; c) rapidly moving the sample to a destination, e.g., an output well; and d) depositing a small volume containing the spheroid in the destination. Any Method 1 or 1.1 et seq., wherein the magnetic field strength at a surface of at least one of the magnets adjacent to the wells is between about 0.1 Tesla and about 2.0 Tesla and optionally between about 0.3 Tesla and about 1.0 Tesla Any Method 1 or 1.1 et seq., wherein the paramagnetic compound is present in the sample solution at a concentration of from about 20 mM to about 500 mM, optionally from about 50 mM to about 175 mM, and further optionally from about 70 mM to about 150 mM. Any Method 1 or 1.1 et seq., wherein the enriched recovered sample comprises at least about 60%, at least about 70%, at least about 80% or at least about 90% live cells. Any Method 1 or 1.1 et seq., wherein the yield of live cells in the enriched recovered sample fraction is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total live cell composition of the sample. Any Method 1 or 1.1 et seq., wherein the sample is loaded into the well or wells, and / or transferred out of the well or wells, using a pipetting robot. Any Method 1 or 1.1 et seq., wherein the magnetic levitation is monitored and / or recorded in real time by the visualization system.Attorney Docket LEV-06-PCT 1.21 Any Method 1 or 1.1 et seq., further comprising removing the multi-well plate and replacing it with another multi-well plate. 1.22 Any Method 1 or 1.1 et seq., wherein the contaminating species is a dye or dyes. 1.23 Any Method 1 or 1.1 et seq., wherein the visualization system is used to adjust the parameters for transferring the sample into or out of the well.

[0012] In a further aspect, provided herein is a method ("Method 2") for enriching a population of interest, said population of interest comprising one or more of live cells, a cell organelle or organelles, e.g. nuclei or chloroplasts, organoids, and sub-mm sized organisms, the method comprising: loading into a well of a system according to any System 1 and 1.1 et seq., individually or as a pre-mix, a) a sample comprising the population of interest; and b) a paramagnetic medium comprising a paramagnetic compound or ferrofluid, to form a sample fluid or sample suspension in said well; and subjecting the sample fluid or sample suspension to a magnetic force from at least one magnet from the magnet array, to effect enrichment of the population of interest; and optionally imaging the sample fluid or sample suspension prior to, during, and / or after the enrichment.

[0013] Embodiments of Method 2

[0014] The present disclosure further provides the following embodiments of Method 2: 2.1 Method 2, wherein the paramagnetic medium comprises one or more of a paramagnetic salt, a paramagnetic hydrophobic metal chelate, a paramagnetic metal chelate, or a paramagnetic ionic liquid. 2.2 Method 2 or 2.1, further comprising collecting the population of interest. 2.3 Method 2.2, wherein the separated population of interest is collected from the well in a pipette tip. 2.4 Method 2.3, wherein the separated population of interest is collected by the steps of: a) moving the multi-well plate vertically upward relative to the magnet array while maintaining the separated population of interest in place with theAttorney Docket LEV-06-PCT magnetic field, thereby causing the cells to migrate to the bottom of the well; and b) either: i) removing the liquid above the separated population of interest with the pipette tip, leaving the separated population of interest in a reduced volume of fluid at the bottom of the well; or ii) removing the separated population of interest from the bottom of the well with the pipette tip. Method 2.3, wherein the separated population of interest is collected by the steps of: a1) moving the pipette tip a vertical direction while maintaining the separated population of interest in place with the magnetic field, to bring the separated population of interest to the top of the pipette tip; and a2) dispensing the liquid below the separated population of interest while retaining the separated cells in the pipette tip; or: b1) moving the pipette tip a vertical direction while maintaining the separated population of interest in place with the magnetic field, to bring the cells to the bottom of the pipette tip; and b2) selectively dispensing the separated population of interest, leaving unwanted liquid in the pipette tip. Method 2.3, wherein the separated population of interest is collected by the steps of: a) inserting the pipette tip into the sample past the levitation position of the separated population of interest; b) drawing the liquid up into the pipette tip while maintaining the separated population of interest in place with the magnetic field; and c) dispensing just the separated population of interest. Method 2.6, wherein step (a) is performed early in the levitation process. Method 2.3, wherein the separated population of interest is collected by the steps of:Attorney Docket LEV-06-PCT a) after achieving levitation equilibrium, aspirating the liquid into the pipette tip until the separated population of interest is aspirated; b) withdrawing the pipette tip from the well and immersing it into a second well in the plate or a separate plate that contains a washing liquid; c) aspirating the separated population of interest into the washing liquid while retaining the liquid in the pipette; and d) optionally repeating steps (a)-(c). Method 2.3, wherein the live cells are levitated by radial magnets, and collected by the steps of: a) aspirating the entire sample into the pipette tip; b) moving the pipette tip and sample vertically upwards until they are clear of, and above, the magnets; c) slowly lowering the tip into the magnetic field, and allowing a spheroid of particles to begin to coalesce in the region of minimal field; d) after the tip completes passage through the region of minimal field, ejecting most of the tip's contents while the tip is moved slowly upwards, leaving a concentrated sample at the bottom of the tip; and e) optionally transferring the concentrated sample. Method 2.3, wherein the live cells are levitated by radial magnets, and collected by the steps of: a) slowly aspirating the entire sample into the pipette tip creating a spheroid of particles at the region of minimal field; b) moving the pipette tip vertically upwards through the magnetic field while the particle spheroid remains stationary relative to the field; c) rapidly moving the sample to a destination, e.g., an output well; and d) depositing a small volume containing the spheroid in the destination. Any Method 2 or 2.1 et seq., wherein the magnetic field strength at each of the wells is between about 0.8 Tesla and about 2.0 Tesla and optionally between about 0.9 Tesla and about 1.4 Tesla. Any Method 2 or 2.1 et seq., wherein the paramagnetic compound is present in the sample solution at a concentration of from about 50 mM to about 200 mM,Attorney Docket LEV-06-PCT optionally from about 65 mM to about 175 mM, and further optionally from about 70 mM to about 150 mM. 2.13 Any Method 2 or 2.1 et seq., wherein the enriched recovered sample comprises at least about 60%, at least about 70%, at least about 80% or at least about 90% live cells. 2.14 Any Method 2 or 2.1 et seq., wherein the yield of live cells in the enriched recovered sample fraction is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total live cell composition of the sample. 2.15 Any Method 2 or 2.1 et seq., wherein the sample is loaded into the well or wells using a pipetting robot. 2.16 Any Method 2 or 2.1 et seq., wherein the magnetic levitation is monitored and / or recorded in real time by the visualization system. 2.17 Any Method 2 or 2.1 et seq., further comprising removing the multi-well plate and replacing it with another multi-well plate. 2.18 Any Method 2 or 2.1 et seq., wherein the contaminating species is a dye or dyes. 2.19 Any Method 2 or 2.1 et seq., the visualization system is used to adjust the parameters for transferring the sample into or out of the well.

[0015] In a further aspect, provided herein is a kit ("Kit 1") for separating live cells from a sample comprising said live cells and one or more contaminating species, comprising one or more components selected from buffers, solvents, proteins, particles, enzymes, stabilizers and the like.

[0016] The numbered embodiments herein are non-limiting of the invention and may incorporate further elements and alternatives described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a cross-sectional view of wells of a multi-well plate of a levitation sample fixture of the invention showing one configuration of the magnets as described herein.Attorney Docket LEV-06-PCT

[0018] FIG. 2 is a cross-sectional view of a well of a multi-well plate of a levitation sample fixture of the invention showing one configuration of the mirror as described herein.

[0019] FIG. 3 is an exploded view of a levitation sample fixture according to the present disclosure comprising mirrors..

[0020] FIG. 4 is an exploded view of a levitation sample fixture according to the present disclosure comprising metal pins.

[0021] FIG. 5 depicts the magnetic gradient of a Linear Halbach Array according to the present disclosure.

[0022] FIG. 6 depicts a comparison of the magnetic fields imposed by a linear array and a Halbach array within a well of a multi-well plate in accordance with an embodiment as described herein.

[0023] FIG. 7 depicts one configuration of radially magnetized ring magnets and their resulting magnetic field in accordance with an embodiment as described herein.

[0024] FIG. 8 is a depiction of a System in accordance with an embodiment as described herein.

[0025] FIG. 9 shows cross-sectional views of placement of magnets and mirrors in accordance with an embodiment as described herein.

[0026] FIG. 10 shows the effect of metal pins on the levitation of fluorescent polystyrene microparticles in accordance with an embodiment as described herein.

[0027] FIG. 11 shows cross-sectional views of placement of magnets and metal pins in accordance with an embodiment as described herein.

[0028] FIG. 12 depicts an exemplary workflow using the device of the present disclosure.

[0029] FIG. 13 shows the separation of beads by a System according to the present disclosure.

[0030] FIG. 14 depicts retrieval of levitated cells in accordance with an embodiment as described herein.

[0031] FIG. 15 depicts retrieval of levitated cells in accordance with a further embodiment as described herein.Attorney Docket LEV-06-PCT

[0032] FIG. 16 depicts retrieval of levitated cells in accordance with a further embodiment as described herein.

[0033] FIG. 17 depicts retrieval of levitated cells in accordance with a further embodiment as described herein.

[0034] FIG. 18 depicts retrieval of cells levitated with radial magnets in accordance with a further embodiment as described herein.

[0035] FIG. 19 depicts retrieval of cells levitated with radial magnets in accordance with a further embodiment as described herein.

[0036] FIG. 20 is a detailed view of a well plate design in accordance with an embodiment as described herein.

[0037] FIG. 21 is a detailed view of a well plate design containing mirrors in accordance with an embodiment as described herein.

[0038] FIG. 22 shows separation of live Jurkat cells in accordance with an embodiment as described herein.

[0039] FIG. 23 shows UMAPs of clustered cell-drug populations separated in accordance with an embodiment as described herein.

[0040] FIG. 24 shows a comparison of the clustering among cell-drug populations separated in accordance with an embodiment as described herein

[0041] FIG. 25 shows a magnetic array wherein four sets of magnets are disposed around each well, and the resulting magnetic levitation forces created thereby.

[0042] FIG. 26A shows the polarity of the magnets in magnetic arrays of the invention having two sets of magnets disposed around each well, and a magnetic array of the invention (designated "LV") having four sets of magnets disposed around each well.

[0043] FIG. 26B shows an overhead view of wells of a multi-well plate of a levitation sample fixture of the invention wherein four sets of magnets are disposed around each well (designated "LV"), and the placement of optional metal pins.

[0044] FIG. 27 shows the washing of a cell sample by an immersion method of the present disclosure in accordance with Method 1.12.

[0045] FIG. 28 shows the magnetic field lines of an exemplary Dual Stacked Magnetic Array of the present disclosure and a LV Array of the present disclosure.Attorney Docket LEV-06-PCT

[0046] FIG. 29 shows a comparison of the particle height and response time for an exemplary Dual Stacked Magnetic Array of the present disclosure and a LV Array of the present disclosure.

[0047] FIG. 30 shows the results of debris and dead cell removal evaluated with FACS.

[0048] FIGs. 31A and 31B show the results of dead cell removal evaluated with FACS after manual wash and levitation wash, respectively.

[0049] FIG. 32 shows an exploded view of a LV Array of the present disclosure. DETAILED DESCRIPTION I. Definitions / Nomenclature

[0050] The following definitions are provided to aid in understanding the invention. Unless otherwise defined, all terms of art, notations and other scientific or engineering terms or terminology used herein are intended to have the meanings commonly understood by those of skill. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be assumed to represent a substantial difference over what is generally understood in the art but is intended to compliment such general understandings. To the extent a definition herein is inconsistent with what is generally understood in the art, unless expressly stated otherwise, both the definition provided herein and what is generally understood in the art shall be deemed to be within the scope of the present invention as alternative embodiments.

[0051] As used herein unless otherwise indicated, open terms such as “contain,” “containing,” “include,” “including,” and the like mean comprising.

[0052] Some embodiments herein contemplate numerical ranges. When a numerical range is provided, the range includes the range endpoints unless otherwise indicated. Unless otherwise indicated, numerical ranges include all values and subranges therein as if explicitly written out.

[0053] As used herein, the article “a” means one or more unless explicitly stated otherwise.Attorney Docket LEV-06-PCT

[0054] Some values herein are modified by the term “about.” In some instances, the term “about” in relation to a reference numerical value can include a range of values plus or minus 10% from that value. For example, the amount “about 10” can include amounts from 9 to 11. In other embodiments, the term “about” in relation to a reference numerical value can include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Where a series of values is prefaced with the term “about,” the term is intended to modify each value included in the series.

[0055] As used herein, the term “asymmetric” about a magnetic field means that the magnetic field in the region of an associated fluidic channel or well of a multi-well plate is not symmetric about one or more planes passing through the center of the fluidic channel, or the center of the well of the multi-well plate.

[0056] As used herein, the terms “capillary” or “capillary tube,” refer to a tube having a channel as defined hereinbelow.

[0057] As used herein, the term “concentration” means the amount of a first component contained within a second component, and may be based on the number of particles per unit volume, a molar amount per unit volume, weight per unit volume, or based on the volume of the first component per volume of the combined components.

[0058] As used herein, the term “fluidically coupled” or “fluidic communication” means that a fluid can flow between two components that are so coupled or in communication.

[0059] As used herein, the terms “isolate” or “isolating” or “separate” or “separating” or “segregate” or “segregating” are used interchangeably, and they are in reference to a component means separating such component from other components, and includes increasing the concentration of a component within a solution, or separating a component from other components in a solution, or a combination of both increasing the concentration of a component within a solution while separating such component from other components in the solution. A particle within a solution is deemed “isolated” if it is segregated from other particles within the solution and / or positioned within a defined portion of the solution. A particle or component within a solution is also deemed “isolated” if after processing the solution the concentration of such particle or component is increased by a ratio of at least about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1,Attorney Docket LEV-06-PCT 20:1, 10:1, 5:1, 3:1 or 2:1. Particles of interest within a solution containing other particles are deemed “isolated” if after processing such solution the ratio of the concentration of such particles of interest to the concentration of such other particles is increased, or if the ratio of the concentration of such particles of interest to the concentration of such other particles is increased by at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, or if the concentration of such other components is decreased to less than about 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%.

[0060] As used herein, the term “fluidic” refers to a system, device or element for handling, processing, ejecting and / or analyzing a fluid sample including at least one “channel” as defined hereinabove. The term “fluidic” includes, but is not limited to, microfluidic and nanofluidic.

[0061] As used herein, the term “fluidic function” refers to any operation, function or process performed or expressed on a fluid or sample in a fluidic system, including, but not limited to filtration, pumping, fluid flow regulation, controlling fluid flow and the like.

[0062] As used herein, the term “particle” refers to any matter, including, but not limited to atoms, chemical elements, molecules, compounds, biomolecules, cells, necrotic cells, live cells, apoptotic cells, cancer cells, cancer or tumor circulating cells, cell nuclei, blood, plasma, proteins, lipids, bodily fluid, nucleic acids, nucleotides, amino acids, peptides, antibodies, antigens, carbohydrates, microorganisms, bacteria, viruses, fungi, sperms, gametes, eggs, embryos, or any physical substance with its largest dimension in any direction being less than about 3 mm, 2 mm, 1 mm, 0.5 mm, 0.25 mm, 100 microns, 75 microns, 50 microns, 40 microns, 30 microns, 20 microns, 10 microns, 5 microns, 2 microns, 1 micron, or 0.1 micron. The particle may have the largest dimension in any direction being about 0.001 micron to about 3 mm, about 0.1 micron to about 2 mm, about 0.5 microns to about 1.5 mm, about 10 microns to about 1 mm, or about 20 microns to about 100 microns.

[0063] As used herein, the term “concentrate” or “concentrating” refers to making a substance in a medium with increased population density or purer by removing water, aqueous or non-aqueous medium or other substances. The substance is a type of particle or a mixture of particles as described herein. Typically, concentrating as described hereinAttorney Docket LEV-06-PCT involves facilitated sedimentation of the particles or a mixture of particles in a medium, thereby bringing the particles or a mixture of to a particular area. Alternatively, concentrating may involve separating a particular type of particle from a mixture of particles and collecting the particular type of particle in a collecting channel, typically with pre-determined volume of a liquid medium. The concentrating need not involve spinning or rotating the bulk sample in order to concentrate the particles. Concentration performed by the present invention allows of separation of particles without significant damage, lysis, or shearing of the particles. Additionally, under certain conditions of operation, the present invention provides for flocculation or crystallization within a sample during operation and isolation of the flocculated or crystallized particles of the sample.

[0064] As used herein, the term “multi-well plate” refers to a removable flat plate with multiple "wells" (i.e., depressions) that are used as small test tubes, wherein at least the wells of the multi-well plate may be optically transparent. In some embodiments, the multi-well plate can have 6, 12, 24, 48, 96, 384 or 1536 sample wells, arranged in a 2:3 rectangular matrix. In some embodiments, the multi-well plate is in accordance with ANSI SLAS microplate standards; e.g., with a footprint of 127.76 mm × 85.48 mm ± 0.5 mm. In some embodiments, the well positions of the multi-well plate is in accordance with ANSI SLAS microplate standards; e.g., wherein 96-well plates have a 9 mm well-to-well spacing, 384-wells a 4.5 mm spacing, and 1536-wells a 2.25 mm spacing. In some embodiments, the multi-well plate has the footprint and / or wall thickness of a standard 96-well PCR plate. The wells of the multi-well plate can be round or square, and have various geometries at the bottom of the well, including F- Bottom, V-Bottom, U-Bottom and C-Bottom geometries. In some embodiments, the diameter of the wells is between about 0.25 mm to about 20 mm.

[0065] As used herein, the term "well" refers to a well of a multi-well plate of the present disclosure.

[0066] Where methods and steps described herein indicate certain events occurring in certain order, those of ordinary skill in the art will recognize that the ordering of certain steps may be modified and that such modifications are in accordanceAttorney Docket LEV-06-PCT with the variations of the invention. Additionally, certain steps may be performed concurrently in a parallel process when possible, as well as performed sequentially. II. System Embodiments

[0067] In one embodiment, the System includes a levitation sample fixture that includes a) a removable multi-well plate comprising a top surface and a plurality of optically transparent wells; b) a magnet array comprising a plurality of magnets disposed in between and below the wells, configured to provide a magnetic field in each of the wells; and c) a magnet holder configured to receive and hold the magnets of the magnet array. In further embodiments, the levitation sample fixture can include a mirror assembly for projecting cross-section images of the wells vertically downwards to an image-capturing device. In further embodiments, the levitation sample fixture can include a plurality of metal pins configured to attenuate and / or adjust the magnetic fields in the wells. In further embodiments, the System can include an imaging array for capturing images from the mirrors. In further embodiments, the System can include a fluid transfer system, i.e., means to dispense or remove all or part of a sample or other fluid into or out of the wells; e.g., a pipetting robot.

[0068] In further embodiments, the System can further include a deck or stage holding the levitation sample fixture, the imaging array and the fluid transfer system. FIG. 8 depicts one exemplary System (23) comprising a deck or stage (24) adapted to engage and hold levitation sample fixture (25). The System further includes fluid transport system (26) and control panel (27). In further embodiments, the System can include means to move the multi-well plate in a vertical direction relative to the fluid transfer system; and / or means to move the levitation sample fixture and / or the multi-well plate in a vertical direction relative to the magnet array. In some embodiments, the movement of the levitation sample fixture and / or multi-well plate in a vertical direction is accomplished by mounting the levitation plate (levitation sample fixture and / or the multi- well plate) on a single- or multi-axis mechanism, e.g. a motion stage. The mechanism can be driven, for example, mechanically, e.g. via a motor and lead screw, or a belt drive, or a via way to store energy such as a loaded spring; manually; by piezoelectric drive by direct or indirect means; pneumatically; hydraulically; or using magnetic forces.Attorney Docket LEV-06-PCT III. Multi-well Plate

[0069] The multi-well plate is a removable plate having a top surface and is configured to contain a plurality of optically transparent wells. Such plates are well known and are used in numerous applications such as PCR. In some embodiments, the dimensions and well configuration of the multi-well plate are in accordance with ANSI SLAS microplate standards; e.g., with a footprint of 127.76 mm × 85.48 mm ± 0.5 mm. The multi-well plate can have any standard number of sample wells - e.g., 6, 12, 24, 48, 96, 384 or 1536 sample wells, preferably arranged in a 2:3 rectangular matrix. In one embodiment, the multi-well plate has 96 sample wells arranged in a 2:3 rectangular matrix.

[0070] In some embodiments, the well positions of the multi-well plate is in accordance with ANSI SLAS microplate standards; e.g., wherein 96-well plates have a 9 mm well-to-well spacing, 384-wells a 4.5 mm spacing, and 1536-wells a 2.25 mm spacing. In some embodiments, the multi-well plate has the footprint and / or wall thickness of a standard 96-well PCR plate. The wells of the multi-well plate can be round or square, and have various standard geometries at the bottom of the well, including F-Bottom, V-Bottom, U-Bottom and C-Bottom geometries.

[0071] FIG. 20 shows an example of a multi-well plate having wells with increased volumes. In this example, the plate would comprise 96 wells, with volume between 200 and 10,000 µL, or more preferably between 500 and 2,000 µL

[0072] In some embodiments, the mirrors can be incorporated into the multi-well plate, obviating the need for inclusion of a mirror holder. FIG. 21 shows an example of such a design.

[0073] FIG. 32 shows an exploded view of one embodiments of a LV Array according to the present disclosure. The stacked Magnet Array, e.g. as shown in Figures 25 and 26B, fits into mating spaces in the Magnet Holder. The Magnet Array is covered by a Top Plate, which contains holes for accommodating the wells, for example the wells of a 96-well plate as described above.Attorney Docket LEV-06-PCT IV. Magnet Array / Magnetic Field

[0074] The present disclosure provides for methods and devices for concentrating using a magnetic field within a well of a multi-well plate. The interaction of the magnetic field with the paramagnetic medium provides separation of components in the sample according to density of the species.

[0075] In accordance with one preferred embodiment, the magnets are permanent magnets. In accordance with one embodiment, the maximum energy product of magnets range from about 1 Mega-Gauss Oersted to about 1000 Mega-Gauss Oersted, and more preferably ranges from about 10 Mega-Gauss Oersted to about 100 Mega-Gauss Oersted. In accordance with an embodiment, the surface field strength of magnets range from about 0.1 Tesla to about 100 Tesla, and more preferably ranges from about 1 Tesla to about 10 Tesla. In accordance with an embodiment, the remanence of magnets range from about 0.5 Tesla to about 5 Tesla, and more preferably ranges from about 1 Tesla to about 3 Tesla.

[0076] In accordance with a preferred embodiment, magnets are made from a material comprising neodymium alloys with iron and boron, neodymium, alloys of aluminum with nickel, neodymium alloys with iron, aluminum and cobalt alloyed with iron, samarium-cobalt, other alloys of rare earth elements with iron, alloys of rare earth alloys with nickel, ferrite, or combinations thereof. In accordance with an embodiment comprising a plurality of magnets, magnets are made from the same material or are made from different materials. In some embodiments, suitable magnets include N50-N53 magnets, for example N52 magnets. In some embodiments, the magnet fixtures are 83 mm x 6.4 mm x 4.6 mm.

[0077] Exemplary Magnet Configurations

[0078] Stacked linear array: In accordance with a preferred embodiment, for each well, a first magnet and a second magnet are disposed adjacent to the well to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the first magnet faces the first pole of the second magnet; and a third magnet is stacked under the first magnet, and a fourth magnet is stacked under the second magnet, wherein the third and fourth magnets each comprise a first pole and a second pole, and wherein the second pole of the third magnet faces theAttorney Docket LEV-06-PCT second pole of the fourth magnet. In some such embodiments, the magnets are rectangular. In one exemplary embodiment, the dimensions of the magnets are 83 mm x 6.4 mm x 4.6 mm. An exemplary depiction of this configuration is shown in FIG. 1, where magnets 1, 2, 3 and 4 correspond respectively to the first, second, third and fourth magnets described above. As shown in the upper panel, well (60) is positioned such that the bottom of well (60) is at the interface of upper magnets (1) / (2) and lower magnets (3) / (4), with the equilibrium levitation position of the levitated sample (61) occurring at a positive distance z above the bottom of the well. The lower panel shows the magnetic field strength relative to the magnets.

[0079] Linear Halbach array: In accordance with a further embodiment, the magnets can be arranged in a linear Halbach array, which has a spatially rotating pattern of magnetization and results in augmentation the magnetic field on one side of the array while cancelling the field to near zero on the other side. The Halbach configuration adds a magnetic gradient component in the XY plane (parallel to the bar magnets) and the YZ plane, focusing the sample in two planes. An exemplary depiction of this configuration is shown in FIG. 5, and a simulation comparison of the magnetic flux density in the stacked linear array and the Halbach array is shown in FIG. 6. As can be seen in FIG. 6, the Halbach array provides a stronger magnetic field near the bottom and the walls of the well.

[0080] Four-Stacked Magnet ("LV") Array: In one preferred embodiment, the stacked linear array contains four sets of stacked magnets surrounding each well. In one such embodiment, a first magnet and a second magnet are disposed adjacent to, and on opposite sides of, the well to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the first magnet faces the first pole of the second magnet; a third magnet is stacked under the first magnet, and a fourth magnet is stacked under the second magnet, wherein the third and fourth magnets each comprise a first pole and a second pole, and wherein the second pole of the third magnet faces the second pole of the fourth magnet; a fifth magnet and a sixth magnet are disposed adjacent to, and on opposite sides of, the well, to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the fifth magnet faces the first pole of the sixth magnet, andAttorney Docket LEV-06-PCT wherein the fifth and sixth magnets are oriented at approximately 90 degrees in the horizontal plane relative to the first and second magnets; and a seventh magnet is stacked under the fifth magnet, and an eighth magnet is stacked under the sixth magnet, wherein the seventh and eighth magnets each comprise a first pole and a second pole, and wherein the second pole of the seventh magnet faces the second pole of the eighth magnet. FIG. 25 in the right-hand panel shows an exemplary configuration of the magnet array. FIG. 26B shows an overhead view of the array, and a depiction of the magnetic forces generated around the wells.

[0081] Figures 25 and 26A shows the effect of magnetic forces from the system containing two stacked magnets surrounding each well, and the system containing four stacked magnets surrounding each well (the latter being designated the "LV" system). As can be seen, the presence of the additional two stacked magnets results in force being applied from four direction, with the result that the sample is levitated into a shape that is more spherical and less ellipsoid relative to the 2-stacked magnet configuration. This can also be seen in FIG. 28, which shows the magnetic field lines of dual stacked magnet and LV arrays. It can be seen that the LV configuration results in greater focusing into the central portion of the well, including at the corners, where the field of the 2-stacked magnet configuration appears to be less strong. FIG. 29 shows a comparison of the particle height and response time for an exemplary dual stacked magnetic array of the present disclosure, and a LV Array of the present disclosure. It can be seen that the LV array results in a comparable focused particle height in the wells, and also has a similar response time.

[0082] In some embodiments, the LV array contains a plurality of blocks that each contain four wells, and their associated stacked magnets. Such a repeating block is shown in FIG. 26B.

[0083] Ring magnet array: In accordance with a further embodiment, the magnets of the array can be ring magnets that surround the wells. The ring magnets can be axially magnetized or radially magnetized. In one embodiment, two ring magnets are stacked one upon the other and are disposed around one or more wells, for example all the wells, of the multi-well plate. Preferably, the poles of the stacked magnets are radially opposed. As shown in FIG. 7, the radially opposed magnets are configured toAttorney Docket LEV-06-PCT create a strong gradient at the interface with a low field zone in the height of the upper magnet, which creates a trap that confines particles within the lowest magnetic field.

[0084] In some embodiments, the magnets of the array that are adjacent to the wells (i.e., in a linear array), or are surrounding the wells (i.e., in a ring magnet array), comprise two stacked magnets. As shown in Figures 1 and 7, in some embodiments, the bottom of the wells (60) (z = 0) is positioned at the interface of the stacked magnets for the levitation procedure, and the levitated species, e.g., live cells, levitate to a point at a positive distance z above the bottom of the well, where the magnetic field is lowest.

[0085] In accordance with an embodiment, an asymmetric magnetic field is achieved by using a stronger magnetic material on one side of a multi-well plate well and a weaker magnetic material on the opposite side of the multi-well plate well. In accordance with a preferred embodiment, an asymmetric magnetic field is achieved by using a magnetic material on one side of a multi-well plate well and a substantially similar magnetic material on the opposite side of the multi-well plate well. In accordance with such embodiment, the upper magnet and lower magnets may be substantially the same size. In accordance with such embodiment, upper magnet may comprise neodymium, lower magnet may comprise samarium-cobalt, and wherein both magnets are substantially the same size. Alternatively, upper magnet may comprise samarium- cobalt, lower magnet may comprise neodymium, and wherein both magnets are substantially the same size. V. Magnet Holder

[0086] The magnet holder forms the base of the levitation sample fixture and is configured to receive and hold the magnets of the magnet array, for example rectangular magnets and ring magnets, and provides the support for the other components of the levitation sample fixture. In some embodiments, the magnet holder is made of non- ferrous metals such as aluminum or titanium, structural plastics such as polyoxymethylene or PEEK, and hardwoods. In some embodiments, the magnet holder has same footprint dimensions as a standard multi-well plate, i.e., of 127.76 mm x 85.48 mm; ±0.5 mm. Exemplary depictions of the magnet holder (22) are shown in Figures 3 and 4.Attorney Docket LEV-06-PCT VI. Mirror Assembly / Mirrors

[0087] In some embodiments, the levitation sample fixture further includes a mirror assembly containing mirrors that are positioned to project, substantially parallel to the top surface of the plate, cross-sectional images of the wells along their vertical axes; and optionally, a mirror holder. In some preferred embodiments, the mirrors are positioned at about 40 ° to about 50 ° with respect to the vertical axis substantially perpendicular to the main surface of the multi-well plate. Exemplary exploded views of such embodiments are shown in Figures 2 and 3, and cross-sectionals view of wells showing exemplary placement of the mirrors and magnets is shown in Figures 2 and 9.

[0088] FIG. 3 depicts an exemplary levitation sample fixture (24) that comprises a mirror array. In this embodiment, shown in FIG. 3, the mirror holder is a single plate (8) having a top surface (9) and a bottom surface (10), with holes (11) configured to accommodate the wells (60) of the multi-well plate. The mirror holder (8) has protrusions (12) that extend down from the bottom surface (10) of the mirror holder that include a planar surface (13) upon which a mirror (14) or a reflective coating is disposed. The protrusions are configured to provide placement of mirrors adjacent to the wells (60), and to provide that the angle of the mirror or reflective coating with respect to the vertical axis substantially perpendicular to the main surface of the multi-well plate is about 40 ° to about 50 °. The protrusions (12) and mirrors (14) of the mirror holder (8) extend downward into the magnet array (21). The magnets (71) and (72) of the magnet array (21) are held by magnet holder 22. In one embodiment, the mirrors are configured substantially in accordance with FIG. 2, which shows the placement of mirror holder (6) and mirrors (7) relative to wells (5) and magnets (1)-(4), as well as exemplary dimensions for one embodiment of the wells and magnets. In further embodiments, mirrors or reflective coatings are present only at selected wells, individually, or in rows and / or columns. In some such embodiments, mirrors or reflective coatings are present in one row of wells and / or one column of wells, or more than one row and / or column of wells, up to all of the rows and / or columns of wells.

[0089] In one embodiment, the mirrors are contained in a separate layer having a reflective coating. For example, the separate layer provides a support surface forAttorney Docket LEV-06-PCT reflective material, e.g. reflective adhesive-backed foil, to be added. The support surface is configured such that the desired view of the wells is achieved, by adding foil as individual pieces or long strips, providing for advantages during manufacturing. VII. Metal Pin Containing Embodiments

[0090] In some embodiments, the levitation sample fixture further includes a plurality of metal pins configured to attenuate and / or adjust the magnetic fields in the wells. Preferably, the pins comprise or are made of a material with high magnetic susceptibility such as steel, iron, cobalt, nickel, and / or alloys such as magnesium alloy, nickel alloy, and chromium alloy. In one embodiment, the pins are steel rods. In some embodiments, the pins are cylindrical. In further embodiments, the pins are in the shape of posts with square cross-section. In a preferred embodiment, the pins are disposed in between the wells and are configured to increase the magnetic field gradients within the wells. FIG. 4 is an exploded view of a levitation sample fixture (15) according to the present disclosure comprising metal pins. In this embodiment, the metal pins (16) are held by a pin holder (17) that has posts (18) to accept pins (16), and holes (19) configured to accept the wells (60) of well plate (20). The metal pins are disposed in between the magnets (71) and (72) of magnet array (21), which in turn are held by magnet holder (22). A cross-sectional view of a well, showing the placement of magnetic pins (16) relative to the well (60) and the magnets (1)-(4) is shown in FIG. 11.

[0091] FIG. 10 shows one exemplary placement of the magnetic pins in between the sample wells (60). As a result of the perturbation of the magnetic field by the pins on the equilibrium position of levitated species, the embodiment with metal pins provides levitation of cells and / or other particles that is more centered in the well, and does not extend to the walls of the cells. In the embodiment without pins, lacking the magnetic field perturbation, the equilibrium position of the isolated species is less centered in the well, and extends from the walls of the wells, which makes collection more difficult.

[0092] FIG. 26B shows an exemplary placement of the pins in a LV array, with the pins placed diagonally in between the sample wells. While not wishing to be bound by a particular theory, it is believed that the presence of the pins results in increased field focusing in the corners, resulting in more rapid and / or stronger focusing.Attorney Docket LEV-06-PCT

[0093] In one embodiment, two of the stacked magnets surrounding each well of the LV array can be replaced by metal pins as described above. While not wishing to be bound by a particular theory, it is believed that in some embodiments, the pins will strengthen the magnetic fields in the wells in a similar fashion to the stacked magnets. For example, referring to FIG. 26B, the stacked magnets in Rows A, B, C and D could be replaced with pins, with or without the presence of the optional metal pins diagonal to the wells; or the stacked magnets in Rows 1, 2, 3, 4, 5 and 6 could be replaced with pins, with or without the presence of the optional metal pins diagonal to the wells. VIII. Imaging Array

[0094] In some embodiments, the System further includes a visualization component or imaging array. The visualization component may comprise any device which enables or enhances the ability to view in real time and / or to record the levitation of the desired species in the wells as the levitation proceeds, thereby enabling observation and / or measurement of the isolation of the desired species, including the extent of isolation and / or the rate of isolation. Visualization may also include analysis of the size, shape, or other characteristics of the particles and / or other components of the sample. The visualization system may employ optics to allow bright-field illumination, dark-field illumination, and / or fluorescent detection of sample components.

[0095] In one embodiment, the visualization component is an imaging array disposed beneath the multi-well plate and magnet array. In one embodiment, the visualization component comprises a microscope, e.g., a USB microscope, and means for moving the microscope underneath the multi-well plate from well to well, for example a motorized stage, manual placement, or pneumatic positioners. In a further embodiment, the imaging array comprises a camera array comprising a plurality of cameras, and means for moving the cameras underneath the multi-well plate from well to well, for example a motorized stage. In one embodiment, the camera array is a motorized camera array. In some such embodiments, the plurality of cameras comprises one or more of built-in lenses, motorized focus, and zoom-in capability. In one embodiment, each camera is configured to capture images from a plurality of the wells. In a preferred embodiment,Attorney Docket LEV-06-PCT each camera is configured to capture images from one to twenty-four of the wells; for example from four wells or from six wells.

[0096] In one embodiment, the visualization component further comprises a graphics processing unit (GPU) comprising graphics software that can integrate two or more of the images from the cameras. In some embodiments, the graphics software can integrate up to all of the images from the cameras, providing an array of up to all the images from up to all of the wells, in a single graphic. Integrating multiple images can also consist of one or more of: merging images into a single larger image, combining images from multiple times in a single data structure to allow for analysis of differences over time, or collating a subset of images for the purposes of a report. IX. Fluid Transfer System

[0097] In some embodiments, the System further includes a fluid transfer system for transferring fluids into and out of the wells. In one embodiment, the fluid transfer system includes means to dispense or remove all or part of a sample or other fluid into or out of the wells. One example of such means is a pipette or pipetting robot. Such pipetting robots are widely employed in conjunction with devices such as plate readers, and other high-throughput applications. Pipettors

[0098] Pipetting technique affects washing and recovery performance. Use of a wide bore pipette tip allows for rapid solution introduction and removal and for enhanced recovery of levitated cells. Preferably, introduction of the pipette tip into a levitated sample should displace the volume of solution substantially equivalent to the displacement volume of the pipette tip. The volume withdrawn by the pipettor is preferred to be equivalent to the volume displaced by the insertion of the pipette tip. In an embodiment, the volume is continuously withdrawn as the pipette is inserted into the sample so that the total volume of sample well remains substantially constant during introduction of the pipette tip into the sample solution.

[0099] The pipetting techniques disclosed herein are applicable to single and multichannel pipettors including 4, 6, 8, 12, 16, 24, 48, 96 and 384 channel pipettors. Examples of manual multichannel pipettors include Eppendorf Research Plus pipettors,Attorney Docket LEV-06-PCT Gilson PIPETMAN L or G Multichannel pipettors, Rainin Pipet-Lite XLS+ pipettors, Thermo Fisher Finnpipette F2 / F3 pipettors, Integra Viaflow / Voyager pippetors, and Sartorius Tacta Multichannel pipettors. Examples of electronic multichannel pipettors include Eppendorf Xplorer / Xplorer plus pipettors, Gilson PIPETMAN M pipettors, Rainin E4 XLS+ pipettors, and Thermo Fisher E1-ClipTip pipettors. For high-throughput applications, the pipettors are preferably incorporated in to automated pipetting robots such as or similar to Hamilton Microlab STAR / STARlet / VANTAGE robots, Analytik Jena CyBio FeliX robots, Integra Assist / Assist Plus, Tecan Freedom EVO / Fluent robots, Opentrs OT-2 / OT-3 robots, Agilent Bravo Liquid Handling Platforms, Eppendorf epMotion robots, Formulatrix MANTIS + TEMPEST + F.A.S.T. robots, and Beckman Coulter Biomek Series robots. The pipettor robots can optionally be equipped with a gripper or manipulator arm to facilitate high-throughput performance of particle isolation and / or washing, and for incorporation of the recovered and / or washed samples into downstream applications such as sequencing library preparation, PCR preparation, ELISA workflows, proteomic screening, flow sorting, transfection, and cell culture. Use of multichannel pipettors in high-throughput applications with multiwell plates is preferred. The pipettor, and thus, the tips should preferably be substantially horizontally level with the plane of the multiwell plate in the levitation Array or other configurations of the stacked Magnet Array in the Magnet Holder, which should level on the deck of the pipettor robot. Alignment of the pipettor tips is preferred to be with the center of the sample wells. In an embodiment, the pipettor robot is equipped with an optical alignment system to align or verify alignment of the pipettor tip(s) with the center of the sample well(s). In applications utilizing a stacked linear array or linear Halbach array, horizontal movement of the pipette tip(s) along the line of levitated particles during removal of particles from the sample solution can enhance recovery of the levitated particles of interest. Sample Volumes

[0100] Sample volumes processed by the magnetic arrays of the present invention can range from about 10 uL to about 400 mL with additional operable ranges in high- throughput applications from about 10 uL to about 1 mL, about 15 uL to about 900 uL,Attorney Docket LEV-06-PCT about 20 uL to about 800 uL, about 25 uL to about 500 uL, and about 25 uL to about 100 uL. Examples of high-throughput sample volumes include about 25 uL, about 30, 40, 50, 60, 70, 80, 90, and 100uL. Large volume applications of about 1 mL to about 30 mL can be singularly processed (or in a large array fashion) using high magnetic field strength permanent magnets such as NdFeB (N52) magnets, rare-earth-free magnets (e.g. Fe-Ni or Fe-Co alloys) and electromagnets in an LV array as configured in in Figures 25 and 26A. Volumes of about 30 mL to about 400 mL, including volumes of about 50 mL to about 500 mL, such as the volume of a Leukopak, can be processed in the methods of the present invention using REBCO high-temperature superconducting magnets with a maximum field strength of about 32 T, an Nb₃Sn low-temperature superconducting magnets with a maximum field strength of about 23 T, a NbTi low-temperature superconducting magnet with a maximum field strength of about 12 T, or a higher field strength cooled resistive Bitter magnets in circular configurations with maximum field strengths of about 45 T. X. Magnetic Beads

[0101] Magnetic beads are widely used for the depletion of specific cells or organelles or types of debris, such as myelin, from complex biological samples through magnetic separation. These beads can be coated with antibodies, streptavidin / avidin, oligonucleotides, lectins, small molecules including drugs, protein ligands such as those acting as receptor or substrate mimics, affinity tags such as recombinant tagged proteins, and aptamers. When mixed with a particle sample, the beads attach to the intended targets. When the sample well containing the sample and magnetic beads are placed into the magnetic field of the LV Array or other configurations of the stacked Magnet Array in the Magnet Holder, a magnetic field is applied drawing the bead-bound targets to the side of the well, allowing unbound components to be removed. This method enables the efficient, rapid, and gentle removal of specific populations and debris—such as T cells, red blood cells, mitochondria, exosomes, and myelin—without affecting the remaining sample. Following removal of the levitated particles and removal of the well from the magnet array, the magnetic particles and their bound substrate can be recovered from the well, either manually or by robot.Attorney Docket LEV-06-PCT XI. Paramagnetic Medium

[0102] Samples processed by magnetic facilitated concentration by the present invention will typically have an added paramagnetic component or an added diamagnetic component. In accordance with the method of the present invention, a substance containing particles of interest is combined with a paramagnetic medium to create a sample fluid or sample suspension. The paramagnetic medium comprises a paramagnetic material and a solvent. In accordance with a preferred embodiment, the paramagnetic medium is biocompatible, i.e. capable of being mixed with live cells and without impacting the viability of the cells or impacting cellular behavior, e.g. impacting gene expression. The paramagnetic material may be selected from the group comprising gadolinium, titanium, vanadium, dysprosium, chromium, manganese, iron, nickel, gallium, including ions thereof and combinations thereof. In accordance with an embodiment the paramagnetic material is selected from the group comprising titanium (III) ion, gadolinium (III) ion, vanadium (I) ion, nickel (II) ion, chromium (III) ion, vanadium (III) ion, dysprosium (III) ion, cobalt (II) ion, and gallium (III) ion. In accordance with a preferred embodiment, the paramagnetic material comprises a chelated compound. In accordance with a preferred embodiment, the paramagnetic material comprises a gadolinium chelate, e.g., gadobutrol, a dysprosium chelate, or a manganese chelate. In accordance with an embodiment, the paramagnetic medium comprises a paramagnetic material, salts, and other additives that function to maintain cellular integrity. In an embodiment of the invention the paramagnetic material may be [Aliq]2 [MnCl4], [Aliq]3 [GdCl6], [Aliq]3 [HoCl6], [Aliq]3 [HoBr6], [BMIM]3 [HoCl6], [BMIM] [FeCl4], [BMIM]2[MnCl4], [BMIM]3[DyCl6], BDMIM]3[DyCl6], [AlaC1] [FeCl4], [AlaC1]2 [MnCl4], [AlaC1]3 [GdCl6], [AlaC1]3 [HoCl6], [AlaC1]3 [DyCl6], [GlyC2] [FeCl4] as described in U.S. Patent Application Serial Number 14 / 407,736 which is incorporated herein by reference.

[0103] In accordance with an embodiment, the paramagnetic material may be present in the paramagnetic medium at a concentration of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 150 mM, 200 mM, 250 mM, 300 mM, 500 mM, or 1 M. In accordance with an embodiment, theAttorney Docket LEV-06-PCT paramagnetic material may be present in the paramagnetic medium at a concentration of about 10 mM to about 50 mM, about 25 mM to about 75 mM, about 50 mM to about 100 mM, about 100 mM to about 150 mM, about 150 mM to about 200 mM, about 200 mM to about 250 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, or about 500 mM to about 1 M.

[0104] In accordance with an embodiment, the paramagnetic material comprises gadolinium, e.g. gadobutrol, and is present in the paramagnetic medium at a concentration of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or100 mM. In accordance with an embodiment, the paramagnetic material comprises gadolinium, e.g. gadobutrol, and is present in the paramagnetic medium at a concentration of about 10 mM to about 50 mM, about 25 mM to about 75 mM, or about 50 mM to about 100 mM. XII. Isolation and Concentration Methods

[0105] A population of interest, for example organic or inorganic particles, may be concentrated by the methods of the present invention. The particles may be biological entities such as cells, cell fragments, organelles (e.g., cell nuclei and chloroplasts), clusters, tissue, tissue components, microorganisms including bacteria, fungi (yeasts and molds), viruses, protozoa, and algae and fragments, organelles, clusters, and other components thereof. Particles can be macromolecules, complexes, chelates, conjugates, crystals, amorphous solids, gels, coagulates, and the like. DNA, RNA, proteins, are concentratable under methods of the present invention. Beads, shells, nanoparticles, laminates, and precipitates and coprecipitates may likewise be concentrated. Numerous applications require the isolation of particles, including applications requiring the separation of like particles from other particles, identification of particles, and the treatment or otherwise manipulation of particles. Such applications include, but are not limited to, separating live and dead cells, separating cell nuclei from live and dead cells and nuclear debris, isolation and / or treatment of circulating tumor cells, emulsion PCR enrichment, production of plasma such as platelet rich plasma, isolating sperm for specific traits such as gender selection, bacterial load testing, antibiotic resistance testing, identification of sepsis or blood contamination, immune cell isolation, compoundAttorney Docket LEV-06-PCT screening, exosome separation, or extracellular vesicles separation. The particle isolation methods of the present invention may be utilized in any of these applications.

[0106] Particles present in a sample medium are concentrated in a particle concentrating device under conditions that substantially enrich particle concentration and substantially deplete a layer of sample medium. Sample medium with heterogeneous particle populations may be selectively enriched based on size, density, and paramagnetic heterogeneity and selective orientation of magnetic forces and processing channel flow rates. The heterogeneous population of particles may be derived from biological samples. In some cases, the biological samples are, as illustrating examples, a bodily fluid including blood, saliva, urine, sperm, plasma, serum, and stool; swabs including skin, anal, nasal and vaginal swabs or environmental swabs from a door handle; and proximal fluids including tears, lavage fluid from lungs, or interstitial tissue fluids from a breast. In some cases, the biological samples are, as illustrating examples, live and dead cells, lysed cells, circulating tumor cells, nucleic acids, nucleotides, amino acids, peptides, proteins, antigens, antibodies, or immune cells (e.g., white blood cells, T cells, phagocytic cells). In some cases, the biological samples are, as illustrating examples, a biomolecule, cell, protein, lipid, carbohydrate, microorganism, virus, virion, or bacteria.

[0107] Level of concentration of the particle enriched fraction over particle concentration in the sample medium is at least 30%, preferably 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In an embodiment, the particle depleted fraction is substantially free of particles.

[0108] FIG. 12 shows a typical workflow for a device of the present invention. Samples are dispensed into wells of the multi-well plate (about 2 minutes), and levitation is commenced. Levitation is allowed to proceed until the components reach their levitation equilibrium positions, typically 5-30 minutes, during which time the wells are imaged and recorded in real time. The fraction. of interest (e.g., a small volume containing live cells) is then collected as described below in a process that typically takes about 5 minutes or less.

[0109] In some embodiments, a population of interest, e.g., particles of interest, is separated from one or more contaminating species by the methods described herein. In some embodiments, the contaminating species is selected from one or more of aAttorney Docket LEV-06-PCT dissolved or suspended compound (e.g., a dye or dyes, antibodies, etc.); cellular debris; small particles (e.g., micro- or nano-beads); and dead cells.

[0110] In one embodiment, live cells can be isolated from a sample comprising said live cells and one or more contaminating species. In one such embodiment, the sample and a paramagnetic medium comprising a paramagnetic compound or ferrofluid are loaded into a well of a system as described herein (e.g., System 1) to form a sample fluid or sample suspension in the well. The sample fluid or sample suspension is then subjected to the magnetic force from at least one magnet from the magnet array, and the components of the fluid or suspension are allowed to levitate to their equilibrium positions. FIG. 13 illustrates the concentration of particles (e.g., live and dead cells) utilizing a device of the present invention. The lower left panel shows a representation of the starting position and equilibrium levitation positions of live and dead cells in a well of the multi-well plate. The image on the lower right is a composite of images taken of all 96 cells used to separate green and red colored beads of different density. The top left image (28) is first in time and shows a diffuse pattern, which over time sharpens to show clear separation of the beads , as shown in bottom left image (29). These images are shown in detail in Figures 13a and 13b, respectively.

[0111] Collection of a particles can be achieved by any of several modalities. FIG. 14 depicts the separation of levitated cells (31) from unwanted components (30) using a pipette tip (32), for example a pipette tip of a pipetting robot. Retrieval of levitated cells (31) can also capture additional liquid that may contain unwanted components (30). Accordingly, it is advantageous to minimize the volume of liquid collected with the cells. FIG. 15 depicts the retrieval of cells by a) moving the multi- well plate (20) vertically upward relative to the magnet array (21) while maintaining the separated live cells (31) in place with the magnetic field, thereby causing the separated live cells (31) to migrate to the bottom of the well; and then either: i) removing the liquid above the separated live cells with the pipette tip (32), leaving the separated live cells (31) in a reduced volume of fluid at the bottom of the well; or ii) removing the separated live cells (31) from the bottom of the well with the pipette tip (32). The arrow inside the pipette tip (32) in FIG. 15 indicates movement of liquid, and the dashed arrow outside the pipette tip (32) in FIG. 15 indicates movement of the multi-well plate (20).Attorney Docket LEV-06-PCT

[0112] FIG. 16 depicts the retrieval of cells by either 1) moving the pipette tip (32) a vertical direction while maintaining the separated live cells (31) in place with the magnetic field, to bring the cells to the top of the pipette tip (32); and 2) dispensing the unwanted liquid below the cells (33) while retaining the separated live cells (31) in the pipette tip (32); or 1) moving the pipette tip (32) in a vertical direction while maintaining the separated live cells (33) in place with the magnetic field, to bring the separated live cells (33) to the bottom of the pipette tip (32); and 2) selectively dispensing the separated live cells (33), leaving unwanted liquid (33) in the pipette tip. In a further embodiment, retrieval of cells can be accomplished by a) early in the levitation, inserting the pipette tip (32) into the sample past the levitation position of the separated live cells (31); b) drawing the liquid up into the pipette tip (32) while maintaining the separated live cells (31) in place with the magnetic field; and c) dispensing just the separated live cells (31).

[0113] Figures 17 and 27 depict the retrieval of cells by an immersion method according to the following steps: a) levitating the cells, optionally in a staining mix; b) after achieving levitation equilibrium, aspirating the liquid into the pipette tip (32) until the separated live cells (31) are aspirated; b) withdrawing the pipette tip (32) from the well (60) and immersing it into a second well (34) in the plate or a separate plate that contains a washing liquid (35); c) aspirating the separated live cells (31) into the washing liquid (35) while retaining the liquid in the pipette tip (32); i.e., slowly removing the pipette tip and the liquid therein, while the field maintains the position of the levitated cells, thus separating them from the original liquid; and d) optionally repeating steps (a)- (c).

[0114] FIG. 18 depicts one mode of retrieval of cells (31) levitated by radial magnets (36) and (37). The entire sample is aspirated into the pipette tip (32). The pipette tip (32) and sample (38) are then moved vertically upwards until they are clear of, and above, radial magnets (36) and (37). The tip (32) is then slowly lowered into the magnetic field, allowing a spheroid of particles (31) to begin to coalesce in the region of minimal field. As the tip (32) continues to move downward, the volume in the tip (32) completes passage through the region of minimal field and the spheroid (31) grows. Then most of the contents of tip (32) are ejected while the tip (32) is moved slowlyAttorney Docket LEV-06-PCT upwards, leaving a concentrated sample (31) at the bottom of the tip (32) that can be transferred.

[0115] FIG. 19 depicts another mode of retrieval of cells levitated by radial magnets (36). and (37). The entire sample is slowly aspirated into the pipette tip (32) creating a spheroid of particles, e.g., levitated live cells, (31) at the region of minimal field. The pipette tip (32) is then moved vertically upwards through the magnetic field while the particle spheroid (31) remains stationary relative to the field. The sample is then rapidly moved to an output well (39) and a small volume containing the spheroid (31) is deposited in the output well (39). The methods described herein produce exceptional recovery and purity of live cell fractions. The enriched recovered sample fraction comprises at least about 60%, at least about 70%, at least about 80% or at least about 90% live cells and the yield of live cells in the enriched recovered sample fraction is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total live cell composition of the sample.

[0116] In some embodiments wherein stained cells are levitated, the cells can first be stained and after a period of time, for example one hour, the cells can then be levitated and separated. In some other embodiments wherein stained cells are levitated, the cells can be mixed with the stain(s) and immediately placed into the levitation well and levitated - i.e., staining and levitating the cells at the same time. In some further embodiments, one or more of the staining and levitating steps can be performed at room temperature, or at reduced temperature, for example at 4º C.Attorney Docket LEV-06-PCT

[0117] Isolation of particles, e.g., live cells, as described herein is rapidly accomplished without subjecting the particles to the stresses associated with other washing and / or separation techniques such as FACS or wash / centrifugation. The total time for separation can range from about 1 minute to 2 hours, for example from about 12 minutes to about 40 minutes. Cells within a solution containing multiple types of particles may be deemed “separated” if, after processing the solution, the ratio of the cells of interest to the concentration of other types of particles is increased, or if the ratio of the concentration of the cells to the concentration of other types particles is increased by at least about 10%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, or if the concentration of non-live cell particles in the solution is decreased by at least about 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%. In a preferred embodiment, the integrity of isolated cells enriched collected portion of the sample is greater than 30% of the integrity of similar cells isolated by a method comprising centrifugation. Preferably the integrity of isolated cells in the enriched collected portion of the sample is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the integrity of similar cells isolated by a method comprising centrifugation. The cell types separated by the above method can include human cells, non-human animal cells, plant cells, eukaryotic cells (for example, but not limited to, immune cells, endothelial cells, yeasts and T-cells). Cells isolated according to the methods of this disclosure can be directly obtained from an organism, or from propagated or cultured cells. In a particular embodiment of the rapid, high capacity separation method, live cells are separated from dead cells. In another embodiment of the method, cells are separated from cell fragments, and / or sample debris.

[0118] The methods described herein provide information not easily obtainable by other means. For example, the methods of the present disclosure can be used to obtain information from within viable cell populations. In one embodiment, the cells can be levitated in the presence of a drug in accordance with the present disclosure, and information can be obtained regarding the interaction of the drug and the cell population. In further embodiments, information can be obtained regarding interactions between cells, e.g. immune cells and / or macrophages attacking tumor cells, without the concern of whether cells adhered to the plate and in solution exhibit differences behavior.Attorney Docket LEV-06-PCT

[0119] As shown in the Examples below, the methods described herein provide a superior alternative to current particle washing and / or separation techniques such as FACS, flow cytometry, and flow sorting. Examples Example 1 Isolation of beads

[0120] A mixture of green and red beads (fluorescent polystyrene microparticles) having different density was subjected to magnetic levitation in a 96-well plate according to the present disclosure. As shown in FIG. 13, the image on the lower right is a composite of images taken of all 96 cells used to separate the green and red colored beads. The top left image (28) is first in time and shows a diffuse pattern, which over time sharpens to show clear separation of the beads, as shown in bottom left image (29). These images are shown in detail in Figures 13a and 13b, respectively. Example 2 - Cell Enrichment

[0121] Samples containing 80,000 Jurkat cells having a cell viability of 51% were placed in wells of a multi-well plate as described herein and subjected to magnetic levitation as described herein. As shown in FIG. 22, the levitated fraction at the top of the well had a viability of over 80%, and a yield of >50%. These data show that the levitation of cells on a System according to the present disclosure enables enrichment of live cells. Example 3 - Cell-Drug interactions

[0122] Samples were prepared containing 50,000 H358 cells in 25ul and containing 75mM Gd levitation agent were placed in wells of a 96-well plate. The samples were levitated in the presence of 30 nM Paclitaxel and 66 µM cisplatin in accordance with the methods of the present disclosure. FIG. 23 shows UMAPs of the clustered populations that were separated by the technique. FIG. 24 shows a comparison of the clustering due to the drug treatment conditions.Attorney Docket LEV-06-PCT Example 4: HTS Washing – Debris and Dead Cell Removal Evaluated with FACS (Fluorescence-activated Cell Sorting)

[0123] A FACS staining workflow was performed on a compromised sample of PBMCs. The FACS plots in FIG. 30 show forward and side scatter plots comparing 3 cycles of standard manual washing (with a centrifuge) to 3 cycles of Levitation washing. The results show that levitation washing effectively removes debris from the sample compared to manual washing methods, and that levitation washing can produce a high- quality sample from a low-quality input. Example 5: HTS Washing –Dead Cell Removal Evaluated with FACS

[0124] A FACS staining workflow was performed on a compromised sample of PBMCs. The FACS plots in Figures 31A and 31B show forward and side scatter plots that were used to create a gate to identify lymphocytes in the sample. The viable fraction of cells in the lymphocyte population was identified by the presence of PI signal indicating dead cells. The results show that levitation washing effectively removed dead cells from the input population while manual washing did not. Example 6: Wash Factor Measurement

[0125] An experiment was performed to measure the wash factor across 3 cycles of Levitation washing. The input condition was of a known concentration of a fluorophore. The fluorophore concentration was measured at each step of the wash cycle using a fluorimeter. The measured wash factor after on wash was 17, and after two washes was 462. The fluorophore concentration at the third step (after the third wash) was below the fluorimeter’s detection range. However, the wash factor is assumed to be 4600, i.e., a 10x dilution due to the final wash step being a 1:10 dilution. Example 7: HTS Viable Cell Enrichment – Single Step Workflow

[0126] All wells of a 96-well plate were loaded with Jurkat cells with a starting viability of 50% in levitation medium containing 150 mm gadobutrol. The samples were enriched using a single-step Levitation-based viable cell enrichment protocol wherein theAttorney Docket LEV-06-PCT plate was placed in the levitation array in a Levicell system, and the samples levitated to their equilibrium positions. Live cells levitate at a different height than dead cells. A pipette was used to retrieve the live cells from the well and transfer them to an output well. The mean viability of the transferred sample was measured to be 89%. This indicates the successful enrichment of viable cells using the technology. Example 8: CD45 Depletion

[0127] All wells of The LeviSelect CD45 depletion kit was used to remove CD45+ cells from a mixture of 85% Jurkat (CD45 pos) and 15% H358 (CD45 neg) cells and levitated on the HTS platform.. Jurkat (CD45+) cells were stained with Calcein and H358 (CD45-) cells were stained with celltracker red (CTR) and then mixed: 85% Jurkat and 15% H358. A standard depletion reaction on 1e6 total cells / 270ul, and 50ul was aliquoted into wells of the LV array as described herein. A “concentrated” depletion on 5e5 cells / 50ul was also conducted similarly. Measurements were made of 1) the depletion rate - i.e., the number of Jurkat cells in the output / the number of Jurkat cells in the input; 2) the yield - i.e., the number of H358 cells in the output / the number of H358 cells in the input; and 3) the total viability in the input and output. Both the conventional and “concentrated” methods worked well to deplete CD45+ cells on the Ruby system, with a yield of > than 24%. The results showed that the CD45 positive cells were effectively removed leaving a pure sample of CD45 negative cells. XIII. Disclosed Embodiments are Non-Limiting

[0128] While various embodiments of the present invention have been shown and described herein, it is emphasized that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may be made without departing from the invention herein in its various embodiments. Specifically, when any range is described herein, unless clearly stated otherwise, that range includes all values therein and all sub-ranges therein.

[0129] Also, and more generally, in accordance with disclosures, discussions, examples and embodiments herein, there may be employed conventional fluidics, molecular biology, cellular biology, microbiology, and recombinant DNA techniquesAttorney Docket LEV-06-PCT within the skill of the art. Resources incorporated by reference herein are for their respective content and teachings found therein. Such incorporation, at a minimum, is for the specific teaching and / or other purpose that may be noted when citing the reference herein. If a specific teaching and / or other purpose is not so noted, then the published resource is specifically incorporated for the teaching(s) indicated by one or more of the title, abstract, and / or summary of the reference. If no such specifically identified teaching and / or other purpose may be so relevant, then the published resource is incorporated in order to more fully describe the state of the art to which the present invention pertains, and / or to provide such teachings as are generally known to those skilled in the art, as may be applicable. However, it is specifically stated that a citation of a published resource herein shall not be construed as an admission that such is prior art to the present invention. Also, in the event that one or more of the incorporated published resources differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls as a preferred embodiment, and any contradiction may be viewed as an alternative embodiment.

[0130] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

Claims

Attorney Docket LEV-06-PCT What is claimed is:

1. A system for conducting magnetic levitation separation of samples in a multi-well plate, the system comprising: a levitation sample fixture comprising: a multi-well plate comprising a top surface and a plurality of wells, wherein said wells are optionally optically transparent; a magnet array comprising a plurality of magnets disposed in between and below the wells, configured to provide a magnetic field in each of the wells; a magnet holder configured to receive and hold the magnets of the magnet array; optionally, a mirror assembly comprising: mirrors positioned to project, substantially parallel to the top surface of the plate, images of the wells along their vertical axes; and optionally a mirror holder; optionally, a plurality of metal pins configured to attenuate and / or adjust the magnetic fields in the wells; and optionally, an imaging array disposed beneath the multi-well plate and magnet array.

2. The system of claim 1, further comprising a fluid transfer system (e.g., means to dispense or remove all or part of a sample or other fluid into or out of the wells; e.g., a pipetting robot).

3. The system of claim 2, further comprising means to move the multi-well plate in a vertical direction relative to the magnet array.

4. The system of any preceding claim, wherein the magnet array comprises permanent magnets.

5. The system of claim 4, wherein, for each well:Attorney Docket LEV-06-PCT a first magnet and a second magnet are disposed adjacent to the well to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the first magnet faces the first pole of the second magnet; and a third magnet is stacked under the first magnet, and a fourth magnet is stacked under the second magnet, wherein the third and fourth magnets each comprise a first pole and a second pole, and wherein the second pole of the third magnet faces the second pole of the fourth magnet.; or a first magnet and a second magnet are disposed adjacent to, and on opposite sides of, the well to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the first magnet faces the first pole of the second magnet; a third magnet is stacked under the first magnet, and a fourth magnet is stacked under the second magnet, wherein the third and fourth magnets each comprise a first pole and a second pole, and wherein the second pole of the third magnet faces the second pole of the fourth magnet; a fifth magnet and a sixth magnet are disposed adjacent to, and on opposite sides of, the well, to impart a magnetic field that is inside the well; wherein each magnet comprises a first pole and a second pole, and the first pole of the fifth magnet faces the first pole of the sixth magnet, and wherein the fifth and sixth magnets are oriented at approximately 90 degrees in the horizontal plane relative to the first and second magnets; and a seventh magnet is stacked under the fifth magnet, and an eighth magnet is stacked under the sixth magnet, wherein the seventh and eighth magnets each comprise a first pole and a second pole, and wherein the second pole of the seventh magnet faces the second pole of the eighth magnet.

6. The system of claim 5, wherein the magnets are rectangular magnets configured substantially in accordance with FIG. 1 or FIG. 26B.Attorney Docket LEV-06-PCT 7. The system of claim 4, wherein the magnets are arranged in a linear Halbach array.

8. The system of claim 4, wherein the magnets are ring magnets that surround the wells.

9. The system of claim 8, wherein the magnets are radially magnetized.

10. The system of claim 9, comprising for each well two ring magnets stacked one upon the other, wherein the poles of the stacked magnets are radially opposed.

11. The system of claim 10, wherein the stacked ring magnets are configured to create a strong gradient at the interface with a low field zone in the height of the upper magnet.

12. The system of claim 8, wherein the magnets are configured to be axially magnetized.

13. The system of any preceding claim, wherein the magnets provide a magnetic field in each of the wells of between about 0.1 Tesla and about 2.0 Tesla and optionally between about 0.3 Tesla and about 1.0 Tesla, at the surfaces of the magnets, varying to zero Tesla in certain locations due to superposition of fields from the plurality of magnets.

14. The system of any preceding claim, wherein the imaging array is present and comprises: a) a microscope, e.g., a USB microscope and means for moving the microscope underneath the multi-well plate from well to well; or b) a camera array comprising a plurality of cameras; for example a motorized camera array.Attorney Docket LEV-06-PCT 15. The system of claim 14, wherein the plurality of cameras comprise one or more of built-in lenses, motorized focus, and zoom-in capability; and wherein each camera is configured to capture images from one to four of the wells; for example from two to three of the wells.

16. The system of claim 14 or claim 15, further comprising a graphics processing unit (GPU) comprising graphics software that integrates two or more of the images from the cameras.

17. The system of any preceding claim, wherein the mirror assembly is present and comprises a mirror holder and mirrors.

18. The system of claim 17, wherein where the mirrors are either: a) a separate layer with reflective surfaces; or b) a mirror-coating disposed on the mirror holder.

19. The system of any preceding claim, wherein the mirror assembly is present and the mirrors are positioned at about 40 ° to about 50 ° with respect to the vertical axis substantially perpendicular to the main surface of the multi-well plate.

20. The system of any preceding claim, wherein the mirror assembly is present and the mirrors are configured substantially in accordance with FIG.

2.

21. The system of any preceding claim, wherein the wells are conical.

22. The system of any preceding claim, wherein the wells have four vertical sides and a square bottom.

23. The system of any preceding claim, wherein the levitation sample fixture comprises a plurality of metal pins configured to attenuate and / or adjust the shape of the magnetic fields in the wells.Attorney Docket LEV-06-PCT 24. The system of claim 23, wherein the metal pins are steel rods disposed in between the wells, either linearly or diagonally between the wells, and are configured to increase the magnetic field gradients within the wells, e.g., within the walls of the wells, or at preselected area(s) within the wells.

25. The system of any preceding claim, wherein the multi-well plate is configured to fit in the footprint of a standard 96-well PCR plate, or is in accordance with ANSI SLAS microplate standards; e.g., with a footprint of 127.76 mm × 85.48 mm ± 0.5 mm.

26. A method for separating live cells from a sample comprising said live cells and one or more contaminating species, the method comprising: loading a) the sample; and b) a paramagnetic medium comprising a paramagnetic compound or ferrofluid, into a well of a system according to any of claims 1-25 to form a sample fluid or sample suspension in said well; and subjecting the sample fluid or sample suspension to a magnetic force from at least one magnet from the magnet array, to effect separation of the live cells from the contaminating species; and optionally imaging the sample fluid or sample suspension prior to, during, and / or after the separation.

27. The method of claim 26, wherein the contaminating species is selected from one or more of a dissolved or suspended compound (e.g., a dye or dyes, antibodies, etc.); cellular debris; small particles (e.g., micro- or nano-beads); and dead cells.

28. The method of claim 26, wherein the contaminating species is a dye or dyes.

29. The method of claim 26, wherein the contaminating species is cellular debris.Attorney Docket LEV-06-PCT 30. The method of claim 26, wherein the contaminating species is dead cells.

31. The method of any of claims 26-30, wherein the paramagnetic medium comprises one or more of a paramagnetic salt, a paramagnetic hydrophobic metal chelate, a paramagnetic metal chelate, or a paramagnetic ionic liquid.

32. The method of any of claims 26-30, further comprising collecting the separated live cells.

33. The method of claim 32, wherein the separated live cells are collected from the well in a pipette tip.

34. The method of claim 33, wherein the separated live cells are collected by the steps of: a) moving the multi-well plate vertically upward relative to the magnet array while maintaining the separated live cells in place with the magnetic field, thereby causing the separated live cells to migrate to the bottom of the well; and b) either: i) removing the liquid above the separated live cells with the pipette tip, leaving the separated live cells in a reduced volume of fluid at the bottom of the well; or ii) removing the separated live cells from the bottom of the well with the pipette tip.

35. The method of claim 33 wherein the separated live cells are collected by the steps of: a1) moving the pipette tip a vertical direction while maintaining the separated live cells in place with the magnetic field, to bring the cells towards the top of the fluid in the pipette tip; and a2) dispensing the liquid below the cells while retaining the separated live cells in the pipette tip; orAttorney Docket LEV-06-PCT b1) moving the pipette tip a vertical direction while maintaining the separated live cells in place with the magnetic field, to bring the separated live cells to the bottom of the pipette tip; and b2) selectively dispensing the separated live cells, leaving unwanted liquid in the pipette tip.

36. The method of claim 33, wherein the separated live cells are collected by the steps of: a) inserting the pipette tip into the sample past the levitation position of the separated live cells; b) drawing the liquid up into the pipette tip while maintaining the separated live cells in place with the magnetic field; and c) dispensing just the separated live cells.

37. The method of claim 36, wherein step (a) is performed early in the levitation process.

38. The method of claim 33, wherein the separated live cells are collected by the steps of: a) after achieving levitation equilibrium, aspirating the liquid into the pipette tip until the separated live cells are aspirated; b) withdrawing the pipette tip from the well and immersing it into a second well in the plate or a separate plate that contains a washing liquid; c) aspirating the separated live cells into the washing liquid while retaining the liquid in the pipette; i.e., slowly removing the pipette tip and the liquid therein, while the field maintains the position of the levitated cells, thus separating them from the original liquid; and optionally repeating steps (a)-(c).

39. The method of claim 33, wherein the live cells are levitated by radial magnets, and collected by the steps of:Attorney Docket LEV-06-PCT a) aspirating the entire sample into the pipette tip; b) moving the pipette tip and sample vertically upwards until they are clear of, and above, the magnets; c) slowly lowering the tip into the magnetic field, and allowing a spheroid of particles to begin to coalesce in the region of minimal field; d) after the tip completes passage through the region of minimal field, ejecting most of the tip's contents while the tip is moved slowly upwards, leaving a concentrated sample at the bottom of the tip; and e) optionally transferring the concentrated sample.

40. The method of claim 33, wherein the live cells are levitated by radial magnets, and collected by the steps of: a) slowly aspirating the entire sample into the pipette tip creating a spheroid of particles at the region of minimal field; b) moving the pipette tip vertically upwards through the magnetic field while the particle spheroid remains stationary relative to the field; c) rapidly moving the sample to a destination, e.g., an output well; and d) depositing a small volume containing the spheroid in the destination.

41. A method for enriching a population of interest, said population of interest comprising one or more of live cells, a cell organelle or organelles e.g. nuclei or chloroplasts, the method comprising: loading, into a well of a system according to according to any of claims 1-25, individually or as a pre-mix, a) a sample comprising the population of interest; and b) a paramagnetic medium comprising a paramagnetic compound or ferrofluid, to form a sample fluid or sample suspension in said well; and subjecting the sample fluid or sample suspension to a magnetic force from at least one magnet from the magnet array, to effect enrichment of the population of interest; and optionally imaging the sample fluid or sample suspension prior to, during, and / or after the enrichment.Attorney Docket LEV-06-PCT 42. The method of claim 41, wherein the paramagnetic medium comprises one or more of a paramagnetic salt, a paramagnetic metal chelate, or a paramagnetic ionic liquid; for example a water soluble paramagnetic metal chelate.

43. The method of claim 41 or claim 42, further comprising collecting the population of interest.

44. The method of claim 41, wherein the separated population of interest is collected from the well in a pipette tip.

45. The method of claim 42, wherein the separated population of interest is collected by the steps of: a) moving the multi-well plate vertically upward while maintaining the separated population of interest in place with the magnetic field, thereby causing the cells to migrate to the bottom of the well; and b) either: i) removing the liquid above the separated population of interest with the pipette tip, leaving the separated population of interest in a reduced volume of fluid at the bottom of the well; or ii) removing the separated population of interest from the bottom of the well with the pipette tip.

46. The method of claim 42, wherein the separated population of interest is collected by the steps of: a1) moving the pipette tip a vertical direction while maintaining the separated population of interest in place with the magnetic field, to bring the separated population of interest to the top of the pipette tip; and a2) dispensing the liquid below the separated population of interest while retaining the separated cells in the pipette tip; or:Attorney Docket LEV-06-PCT b1) moving the pipette tip a vertical direction while maintaining the separated population of interest in place with the magnetic field, to bring the cells to the bottom of the pipette tip; and b2) selectively dispensing the separated population of interest, leaving unwanted liquid in the pipette tip.

47. The method of claim 42, wherein the separated population of interest is collected by the steps of: a) inserting the pipette tip into the sample past the levitation position of the separated population of interest; b) drawing the liquid up into the pipette tip while maintaining the separated population of interest in place with the magnetic field; and c) dispensing just the separated population of interest.

48. The method of claim 47, wherein step (a) is performed early in the levitation process.

49. The method of claim 42, wherein the separated population of interest is collected by the steps of: a) after achieving levitation equilibrium, aspirating the liquid into the pipette tip until the separated population of interest is aspirated; b) withdrawing the pipette tip from the well and immersing it into a second well in the plate or a separate plate that contains a washing liquid; c) aspirating the separated population of interest into the washing liquid while retaining the liquid in the pipette; and optionally repeating steps (a)-(c).

50. The method of any of claims 26-49, wherein the magnetic field strength at a surface of at least one of the magnets adjacent to the wells is between about 0.1 Tesla and about 2.0 Tesla and optionally between about 0.3 Tesla and about 1.0 Tesla.Attorney Docket LEV-06-PCT 51. The method of any of claims 26-50, wherein the paramagnetic compound is present in the sample solution at a concentration of from about 20 mM to about 500 mM, optionally from about 50 mM to about 175 mM, and further optionally from about 70 mM to about 150 mM.

52. The method of any of claims 26-51, wherein the enriched recovered sample comprises at least about 60%, at least about 70%, at least about 80% or at least about 90% live cells.

53. The method of any of claims 26-51, wherein the yield of live cells in the enriched recovered sample fraction is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total live cell composition of the sample.

54. The method of any of claims 26-53, wherein the sample is loaded into the well or wells, and / or transferred out of the well or wells, using a pipetting robot.

55. The method of any of claims 26-54, wherein the magnetic levitation is monitored and / or recorded in real time by the visualization system.

56. The method of any of claims 26-55, further comprising removing the multi-well plate and replacing it with another multi-well plate.

57. The method of any of claims 26-56, wherein the contaminating species is a dye or dyes.

58. The method of any of claims 26-57, wherein the visualization system is used to adjust the parameters for transferring the sample into or out of the well.

59. The system of claim 23 or claim 24, wherein the metal pins are disposed substantially as shown in FIG. 26B.Attorney Docket LEV-06-PCT 60. The system of any of claim 23-25 and 59, wherein the metal pins are steel, manganese alloy, nickel alloy or chromium alloy rods.

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