PARTICLE SEPARATION SYSTEM AND METHOD

Abstract

A magnetic levitation particle separation system and method for use with a multi-channel flow cell. The system can include a core for receiving and holding the flow cell with upper and lower clamps for securing and positioning the flow cell relative to an array of magnets. The system is configured to image particle separation in a processing channel of the flow cell and to regulate a temperature of the flow cell. The core is removable as a single unit to facilitate reconfiguration of the system.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 294,915, filed December 30, 2021, and U.S. Provisional Patent Application No. 63 / 253,448, filed October 7, 2021, the entire contents and disclosures of which are incorporated herein by reference. [Technical field]

[0002] Systems and methods for separating particles, particularly those that utilize magnetic levitation for particle separation. [Background technology]

[0003] Magnetic levitation has recently emerged as a useful method for separating particles, including cells, biomolecules, and other types of particles. By flowing particles through a channel exposed to a magnetic field gradient, particles can be separated according to type or state, with different levitation heights due to the physical properties of the particles. Once separated into distinct bands with different levitation heights, the separated particles can be analyzed and collected. Summary of the Invention

[0004] This patent describes several examples of magnetic levitation systems and methods for improved particle separation, as well as examples of multi-channel flow cell cartridges for use with these systems and methods. The systems, methods and cartridges can be configured to separate a relatively large number of particles in a single processing run.

[0005] In one example, a multi-channel flow cell cartridge comprises: (a) a substrate; and (b) a plurality of fluidic separation channels formed in the substrate, each of the fluidic channels including a magnetic levitation processing channel in fluid communication with an inlet and at least two outputs, each of the processing channels having at least one partition vertically separating a portion of the processing channel into at least an upper portion and a lower portion, the upper portion in fluid communication with one of the outputs and the lower portion in fluid communication with the other of the outputs, each of the processing channels further comprising at least one imaging surface extending along the length of the processing channel, the imaging surface configured to enable imaging of the magnetic levitation of particles within the processing channel.

[0006] In some cases, each of the processing channels can have two opposing imaging surfaces that extend along the processing channel.

[0007] In some cases, the processing channels may be coplanar.

[0008] In some cases, the cartridge can have several channel beams formed in a substrate, the channel beams extending between the inlet and outlet ends of the flow cell cartridge, each of the channel beams having one of the processing channels.

[0009] In some cases, each of the channel beams may be spaced apart from adjacent channel beams.

[0010] In some cases, the cartridge may further comprise several openings extending through the substrate, each of the openings extending between two adjacent channel beams along the length of the processing channel, the openings being configured for optical access to the imaging surface.

[0011] In some cases, each of the channel beams may include at least one alignment tab.

[0012] In some cases, the substrate may further include a sidewall extending downwardly away from the fluid channel.

[0013] In some cases, each of the side walls can include an opening configured for optical access to one of the imaging surfaces.

[0014] In another example, a magnetic levitation particle separation system for use with a flow cell cartridge having multiple processing channels includes: (a) a holder configured to receive and hold a flow cell cartridge; (b) an array of magnets configured to apply a magnetic field gradient to the processing channel when the flow cell cartridge is received and held by the holder, the array of magnets being positioned at least one above and below the processing channel when the flow cell cartridge is received and held by the holder; (c) an imager configured to image the vertical separation of particles in the processing channel while the flow cell cartridge is received and held by the holder and while the array of magnets is positioned at least one of above and below the processing channel; Equipped with.

[0015] In some cases, a magnetic levitation particle separation system may have a maximum particle separation capacity of at least about 100 million particles in a single separation run.

[0016] In some cases, a magnetic levitation particle separation system may have a maximum particle separation capacity of at least about 100 million particles in a single separation run, the particles having a characteristic dimension of 1-5 microns.

[0017] In some cases, the magnetic levitation particle separation system may have a maximum particle separation capacity of at least about 100 million spherical particles in a single separation run, the particles having an average diameter of at least about 3 microns.

[0018] In some cases, a magnetic levitation particle separation system may have a maximum particle separation capacity of at least about 20 million particles per processing channel in a single separation run.

[0019] In some cases, the magnetic levitation particle separation system may have a maximum particle separation capacity of at least about 20 million particles per processing channel in a single separation run, the particles having a characteristic dimension of 1-5 microns.

[0020] In some cases, the magnetic levitation particle separation system may have a maximum particle separation capacity of at least about 20 million spherical particles per processing channel in a single separation run, the particles having an average size of at least about 5 microns.

[0021] In some cases, the imager may be configured to image the vertical separation of particles within the processing channel by a bent optical path.

[0022] In some cases, the curved optical path may include a path portion that extends between a magnet positioned directly above one of the processing channels and a magnet positioned directly below one of the processing channels.

[0023] In some cases, the curved optical path may include a second portion that extends through an opening in the holder between two adjacent magnets of the magnet array.

[0024] In some cases, the imager may include an optical periscope, and the system is configured to move the periscope to a position adjacent to one of the processing channels while the flow cell cartridge is received and held by the holder and while the array of magnets is positioned above and below the multiple processing channels of the flow cell cartridge.

[0025] In some cases, the system may be configured to scan the periscope along the length of the processing channels while the flow cell cartridge is received and held by the holder and while an array of magnets is positioned above and below multiple processing channels of the flow cell cartridge.

[0026] In some cases, the holder may have several openings sized and positioned to allow the periscope to move through the openings and into a position adjacent to the processing channel.

[0027] In some cases, the holder can be a core unit having an upper clamp and a lower clamp, the core configured to receive and clamp the flow cell cartridge between the upper clamp and the lower clamp, and the array of magnets can include a first array of magnets mounted in the upper clamp and a second array of magnets mounted in the lower clamp.

[0028] In some cases, the imager may be configured to image vertical separation of particles in one of the processing channels by an optical path that includes a first portion of the optical path extending between a magnet of a first array positioned directly above the processing channel and a magnet of a second array positioned directly below the processing channel, and a second portion of the optical path extending between a magnet of the first array positioned directly above the processing channel and another magnet of the first array positioned above an adjacent processing channel.

[0029] In some cases, the holder may be a core unit configured to regulate the temperature of a processing channel of a flow cell cartridge.

[0030] In some cases, the core unit may have several temperature transfer surfaces positioned such that the temperature transfer surfaces are in contact with the processing channels when the flow cell cartridge is received and held by the core unit.

[0031] In some cases, the thermally conductive surfaces may be end faces of pillars formed from a thermally conductive material, each of the pillars carrying a magnet of the magnet array.

[0032] In some cases, the system may be configured to cool the processing channel by conduction by cooling the pillars.

[0033] In some cases, the core may have a cooling assembly in contact with the pillars and configured to conductively cool the pillars.

[0034] In some cases, the cooling assembly may include a temperature control plate, a thermoelectric cooler, and a heat sink.

[0035] In some cases, the core unit can include air channels extending along the pillars from the cooling assemblies toward the pillar end surfaces, the air channels configured to cool the pillars and the flow cell cartridges by convective airflow.

[0036] In some cases, the core unit may be a removable unit of the system.

[0037] In some cases, the core unit may include a flow cell cartridge holder, a fluid connector assembly configured to fluidly connect the flow cell cartridge to a pump subsystem, a magnet array, and a cooling assembly.

[0038] In some cases, the system may further include a second core unit having a second flow cell cartridge holder, a second fluid connector assembly, a second magnet array and a second cooling assembly, wherein the second magnet array has a different configuration than the magnet array of the other core unit, and the system is configured to accommodate and operate both core units in an alternating manner.

[0039] In another example, a particle separation method includes: (a) loading a multi-channel flow cell cartridge having at least a first processing channel and a second processing channel into a particle separation system; (b) flowing a first suspension of particles and a paramagnetic fluid into the first processing channel; (c) flowing a second suspension of particles and a paramagnetic fluid into the second processing channel; (d) exposing the first and second suspensions in the first and second processing channels to one or more magnetic field gradients; (e) separating the particles in the first processing channel into one or more bands and separating the particles in the second processing channel into one or more bands; (f) imaging the one or more separated bands in the first processing channel and imaging the one or more separated bands in the second processing channel; and (g) separately collecting at least one of the bands.

[0040] In some cases, imaging the separated bands may include imaging the separated bands using an imaging subsystem of the particle separation system, the imaging subsystem having at least one optical component positioned at least partially within a region between the first processing channel and the second processing channel.

[0041] In some cases, the multi-channel flow cell cartridge can include an opening positioned between the first processing channel and the second processing channel, and an optical component of the imaging subsystem is positioned at least partially within the opening during imaging of at least one of the processing channels.

[0042] In some cases, the optical component may be a reflector.

[0043] In some cases, the imaging subsystem may further include a second optical component positioned across one of the first or second processing channels, the second optical component being on an opposite side of the opening to the at least one optical component during imaging of at least one of the processing channels.

[0044] In some cases, the method may further include using a particle separation system to move at least one optical component into the opening, then imaging at least one of the first and second processing channels, and then moving the at least one optical component out of the opening.

[0045] In some cases, the method may further include using a particle separation system to move at least one optical component from one position within the aperture to another position within the aperture, and imaging at least one of the first and second processing channels at different locations along the length of the channel.

[0046] In some cases, the openings can be elongated openings that extend along the first and second processing channels.

[0047] In some cases, imaging the separated bands may include imaging the separated bands using an imaging subsystem of the particle separation system, the imaging subsystem having at least one bent optical path.

[0048] In some cases, loading the multi-channel flow cell cartridge into the particle separation system may include positioning the multi-channel flow cell cartridge relative to an array of magnets.

[0049] In some cases, the array of magnets can include at least a first magnet and a second magnet, where the first magnet is adjacent to a first separation channel and the second magnet is adjacent to a second separation channel when the multi-channel flow cell cartridge is loaded into the particle separation system.

[0050] In some cases, the first magnet may have a polarity that does not match the polarity of the second magnet.

[0051] In some cases, the array of magnets may include an array of upper magnets and an array of lower magnets, and the multi-channel flow cell cartridge is positioned between the upper magnet array and the lower magnet array when the multi-channel flow cell cartridge is loaded into the particle separation system.

[0052] In some cases, the array of upper magnets may be an array of alternating polarity magnets and the array of lower magnets may be an array of alternating polarity magnets. [Brief description of the drawings]

[0053] [Figure 1] FIG. 1 is a schematic example of particle separation in a fluid channel by magnetic levitation. [Diagram 2] FIG. 1 is a diagram of an example of a particle separation system. [Diagram 3] FIG. 1 is a diagram of an example of a particle separation system comprising several particle separation devices connected by a computer network. [Figure 4A] FIG. 1 shows an example of a multi-channel flow cell cartridge. [Figure 4B] FIG. 1 shows an example of a multi-channel flow cell cartridge. [Figure 4C] FIG. 4B shows the components of the multi-channel flow cell cartridge of FIGS. 4A and B before assembly. [Figure 5A] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5B] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5C] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5D] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5E] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5F] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5G] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5H] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5I] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5J] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5K] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5L] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5M] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 5N] FIG. 4D shows the main body parts of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 6A] FIG. 4D shows the top capping film component of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 6B] FIG. 4D shows the bottom capping film component of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 6C] FIG. 4C shows a capping film component for the collection wells of the multichannel flow cell cartridge of FIGS. 4A and 4C. [Figure 6D] FIG. 4D shows the outlet membrane film component of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 7]FIG. 4D shows the processing channels of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 8A] FIG. 4D shows the processing channels of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 8B] FIG. 4D shows the processing channels of the multi-channel flow cell cartridge of FIGS. 4A and 4C. [Figure 9] FIG. 2 is a schematic example of a core of a particle separation system. [Figure 10A] FIG. 13 illustrates another example of a core of a particle separation system shown in an unclamped configuration. [Figure 10B] FIG. 10B shows the core of FIG. 10A in a clamped configuration, with a multi-channel flow cell cartridge clamped between the upper and lower clamps of the core. [Figure 11] FIG. 10C shows the core of FIGS. 10A and 10B with the upper clamping piece of the core removed. [Figure 12] FIG. 1C shows a portion of the lower clamp of the core of FIGS. 10A and 10B in more detail. [Figure 13] FIG. 1C shows the pillar body and magnets of the lower clamp of the core of FIGS. 10A and 10B. [Figure 14] FIG. 10B shows the upper clamp of the core of FIGS. [Figure 15] FIG. 15 shows the underside of the upper clamp of FIG. [Figure 16] FIG. 2 shows a schematic diagram of an example of a magnet array of a particle separation system relative to a processing channel of a flow cell cartridge. [Figure 17] FIG. 2 shows a schematic diagram of an example of a magnet array of a particle separation system relative to a processing channel of a flow cell cartridge. [Figure 18A] FIG. 10B shows a cross-section of the core of FIGS. [Figure 18B] FIG. 10B shows a cross-section of the core of FIGS. [Figure 19] FIG. 10B shows a cross-section of the core of FIGS. [Figure 20]FIG. 1 illustrates an example of a removable core of a particle separation system. [Figure 21] FIG. 1 illustrates an example of a removable core of a particle separation system. [Figure 22] FIG. 1 illustrates an example of a removable core of a particle separation system. [Figure 21] FIG. 1 illustrates an example of a removable core of a particle separation system. [Figure 22] FIG. 1 illustrates an example of a removable core of a particle separation system. [Figure 23] FIG. 2 illustrates an example imaging subsystem of a particle separation system. [Figure 24] FIG. 2 illustrates an example imaging subsystem of a particle separation system. [Diagram 25] FIG. 2 illustrates another example of an imaging subsystem of a particle separation system. [Figure 26] FIG. 1 illustrates an example of a method of operation of the particle separation system. [Figure 27] FIG. 13 illustrates an example of a display of a particle separation system showing imaging obtained from three particle separation channels. [Figure 28] 27 shows an example of a method of operating the particle separation system in a different operating sequence than that shown in FIG. 26. [Figure 29] 29 shows an example of a method of operating the particle separation system in a different operating sequence than shown in FIGS. 26 and 28. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Cells, biological materials, and other types of particles have properties that dictate how they behave when magnetically levitated. Durmus et al. have shown that the height to which cells levitate in a paramagnetic fluid medium corresponds to cell density, and that different cell types can be distinguished based on their characteristic magnetic levitation profiles. See Durmus et al., 2015, "Magnetic levitation of single cells," Proc Natl Acad Sci USA 112(28):E3661-8. See also co-pending and commonly owned U.S. patent application Ser. No. 17 / 449,438, filed Sep. 29, 2021, the entire contents of which are incorporated herein by reference.

[0055] An example of particle separation using magnetic levitation is shown generally in Figure 1. In Figure 1, a heterogeneous mixture 10 of different types of diamagnetic particles is suspended in a paramagnetic fluid medium within a fluid channel 12. The particle mixture flows into a region of the fluid channel 12 where magnets 14, 16 subject the particles to a magnetic field gradient.

[0056] The magnetic field generates a non-uniform pressure in the paramagnetic fluid medium, which corresponds to the magnetic energy density. Under a magnetic field gradient, the diamagnetic particles appear to be repelled from the region of the strong magnetic field. In fact, the diamagnetic particles are displaced by an equal volume of the paramagnetic fluid medium. The attractive interaction between the paramagnetic fluid medium and the region of the strong magnetic field allows the diamagnetic particles to be "levitated". By applying the magnetic field such that the force exerted on the diamagnetic particles is opposed by another uniform force (e.g., gravity), a balance of the diamagnetic particles is achieved, which is directly related to the density of the diamagnetic particles. In this way, the particles can be separated into separate bands 18, 20, 22 with different levitation heights depending on the density of the particles.

[0057] Once the particles have equilibrated into different bands, these bands can be collected and analyzed separately. In the example of Figure 1, partitions 24, 26 separate the fluidic channel 12 into separate portions, in this example collection subchannels 28, 30, 32. The collection subchannels 28, 30, 32 are set up to collect particle bands 18, 20, 22 that are within a particular flying height range.

[0058] Terms and Concepts The terms and concepts used in this patent disclosure may be further clarified and understood by the descriptions and figures provided throughout this disclosure in accordance with accepted practices in the relevant art.

[0059] Isolate As used in this disclosure, the terms "separate," "isolate," "segregate," "purify," "concentrate," and their respective associated terms and expressions may be used interchangeably. For example, particles in a solution may be considered "separated" if the particles are separated from other types of particles in the solution and / or located within a predefined portion of the solution. In another example, particles in a solution may be considered "separated" if, after processing of the solution, the concentration of said particles is increased in the solution or a portion of the solution by at least about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1.5:1, or 1.1:1. A particle of interest in a solution containing multiple types of particles may be considered to be "isolated" if, after processing of the solution, the ratio of the concentration of the particle of interest to the concentration of other types of particles is increased, or the ratio of the concentration of the particle of interest to the concentration of other types of particles is increased by at least about 10%, about 50%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, or the concentration of other particles (including, but not limited to, types of particles other than the particle of interest) in the solution is decreased by less than about 20%, less than about 15%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.5%.

[0060] Fluidic Devices As used in this disclosure, the term “fluidic device” refers to a system, device, or element for handling, processing, discharging, and / or analyzing a fluid sample that comprises at least one “channel.” The term “fluidic device” includes, but is not limited to, microfluidic devices and nanofluidic devices.

[0061] channel As used in this disclosure, the terms "channel," "flow path," "fluid channel," and "fluidic channel" are used interchangeably and refer to a passageway in a fluidic device through which a fluid can flow. Channels include passageways having a maximum height dimension of about 100 mm, about 50 mm, about 30 mm, about 25 mm, about 20 mm, about 15 mm, about 10 mm, about 5 mm, about 3 mm, about 2 mm, about 1 mm, or about 0.5 mm. For example, the channel 12 in FIG. 1 may be approximately 10 mm×10 mm, approximately 10 mm×5 mm, approximately 10 mm×3 mm, approximately 10 mm×2 mm, approximately 10 mm×1 mm, approximately 10 mm×0.5 mm, approximately 5 mm×10 mm, approximately 5 mm×5 mm, approximately 5 mm×3 mm, approximately 5 mm×2 mm, approximately 5 mm×1 mm, approximately 5 mm×0.5 mm, approximately 3 mm×10 mm, approximately 3 mm×5 mm, approximately 3 mm×3 mm, approximately 3 mm×2 mm, approximately 3 mm×1 mm, approximately 3 mm×0.5 mm, approximately 2 mm The channel may have cross-sectional dimensions (height x width) of about 10 mm, about 2 mm x 5 mm, about 2 mm x 3 mm, about 2 mm x 2 mm, about 2 mm x 1 mm, about 2 mm x 0.5 mm, about 1 mm x 10 mm, about 1 mm x 5 mm, about 1 mm x 3 mm, about 1 mm x 2 mm, about 1 mm x 1 mm, about 1 mm x 0.5 mm, about 0.5 mm x 10 mm, about 0.5 mm x 5 mm, about 0.5 mm x 3 mm, about 0.5 mm x 2 mm, about 0.5 mm x 1 mm, or about 0.5 mm x 0.5 mm. The internal dimensions height and / or width of the channel may be one-lobed or non-uniform across the cross-section of the channel, and geometrically the cross-section may be any shape including round, square, oval, rectangular, or hexagonal. The cross-section may vary along the length of the channel. The term "channel" includes, but is not limited to, microchannels and nanochannels, and for any reference to a channel in this disclosure, the channel may be a microchannel or a nanochannel.

[0062] Fluidly coupled As used in this disclosure, the terms "fluidly coupled" or "fluid communication" mean that a fluid can flow between two components so coupled or in communication.

[0063] Magnetic levitation As used in this disclosure, the term "magnetic levitation" generally includes subjecting a diamagnetic, paramagnetic, ferromagnetic, or antiferromagnetic material suspended in a paramagnetic fluid medium to a magnetic field, such as a magnetic field gradient formed between two magnets. Magnetic levitation can include suspending a material in a magnetic fluid.

[0064] paramagnetic fluid medium As used in this disclosure, a "paramagnetic fluid medium" includes a paramagnetic material and a solvent. The paramagnetic fluid medium, at least in some implementations, may be biocompatible, i.e., capable of being mixed with living cells without significantly affecting the viability or behavior of the living cells. The paramagnetic material may include, but is not limited to, one or more of gadolinium, titanium, vanadium, dysprosium, chromium, manganese, iron, nickel, and gallium ions. For example, the paramagnetic material may include one or more of the following ions: titanium (III), gadolinium (III), vanadium (I), nickel (II), chromium (III), vanadium (III), dysprosium (III), cobalt (II), and gallium (III). In some examples, the paramagnetic material may include a chelated compound, such as, but not limited to, a gadolinium chelate, a dysprosium chelate, or a manganese chelate. In some examples, the paramagnetic material can be one or more of [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], [AlaCl][FeCl4], [AlaCl]2[MnCl4], [AlaCl]3[GdCl6], [AlaCl]3[HoCl6], [AlaCl]3[DyCl6], [GlyC2], or [FeCl4]. The paramagnetic material may be present in the paramagnetic fluid medium at a concentration of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 nM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 150 mM, 200 mM, 250 mM, 300 mM, 500 mM, 1 M, 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.In one example, the paramagnetic material is gadobutrol and is present 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, or 100 mM, about 10 mM to about 50 mM, about 25 mM to about 75 mM, or about 50 mM to about 100 mM, or about 10 mM to 1 M. In some cases, the paramagnetic material is present at a concentration of up to 10 M. It is understood that the paramagnetic fluid medium may include other components, in addition to the paramagnetic material and the solvent, such as salts or additives, including but not limited to additives that function to maintain cellular integrity.

[0065] sample Unless otherwise stipulated, the terms "sample" or "samples" and related terms and phrases used in this disclosure are not intended to be limiting. These terms refer to any product, composition, cell, tissue, organism, or other particle of interest. In general, the terms "sample" or "samples" are not intended to be limited by source, origin, method of procurement, treatment, processing, storage, or analysis, or any modification. Some examples of samples are solutions, suspensions, supernatants, precipitates, or pellets. A sample can contain cells or tissues, or can be composed primarily of cells or tissues, or can be prepared from cells or tissues. However, a sample need not contain cells. A sample can be a mixture of biomolecules, such as nucleic acids, polypeptides, proteins (including antibodies), lipids, carbohydrates, or can contain biomolecules. A sample can be a biological sample. For example, a "sample" can be any cell or tissue sample or extract from a cell, tissue, or subject, and can include animal cells or tissues, as well as samples of cells that are not of animal origin, including plant samples and bacterial samples. Samples can be obtained directly from an organism or can be grown or cultured. Some exemplary samples are cell extracts (e.g., cell lysates), suspensions of cell nuclei, liquid cell cultures, cell suspensions, bodily fluids (including but not limited to blood, serum, plasma, saliva, urine, cerebrospinal fluid, amniotic fluid, tears, pleural fluid, lavage fluid or interstitial fluid from the lungs), tissue sections including needle biopsies, microscope slides, frozen tissue sections or fixed cell and tissue samples. In yet other examples, the sample can be a non-biological sample that includes non-biological particles.

[0066] Exemplary Cell Types and Other Kinds of Particles to be Separated The particle separation systems and methods described herein can be used with a wide variety of samples, including a wide variety of cell types. As used herein, the term "particle" or "particles" can include cells, organisms, organelles, nuclei, organic materials such as organic polymers and proteins, organic or inorganic particulate matter, and particles such as beads, bubbles, and fragments. Exemplary cell types include human cells, non-human animal cells, plant cells, eukaryotic cells (e.g., but not limited to, immune cells, endothelial cells, yeast, and T cells), prokaryotic cells including bacteria, and the like. Cell types can include dead cells, live cells, healthy cells, diseased cells, infected cells, transfected cells, or genetically engineered cells. Cells separated by the methods of the present disclosure can be obtained directly from an organism (or can be the organism itself), or can be grown or cultured. In general, the terms "cell", "cells", and "cell types" (or related terms and phrases) are not intended to be limited by source, origin, method of procurement, treatment, processing, storage, or analysis, or any modification.

[0067] Some non-limiting examples of cell types that may be suitable for separation by the methods described herein are macrophages, alveolar type II (ATII) cells, stem cells (e.g., adipocytes or cardiomyocytes), germ cells, tumor cells, lymphocytes, erythroid cells (red blood cells), epithelial cells, ova (egg cells), sperm cells, T cells, B cells, bone marrow cells, immune cells, stem cells, endothelial cells, stromal cells, and bacterial cells. A population of cells comprising multiple cell types may be derived from various types of samples, which are discussed elsewhere in this disclosure.

[0068] Cells may be subjected to various treatments, storage or processing procedures before being separated using the systems and processes described herein. In other examples, the particle separation systems and processes described herein may also be used with cells or particles that have not been stained, labeled, tagged or otherwise treated. In other examples, cells or particles (or a particular subset of cells or particles) in a sample may be associated with a density modifier.

[0069] Other matters As used in this disclosure, the terms "a," "an," and "said" can refer to one or more, unless expressly stated otherwise. Unless otherwise dictated by context, singular terms shall include the plural terms and plural terms shall include the singular term.

[0070] Use of the term "or" is used to mean "and / or," unless expressly indicated to refer only to alternatives or that the alternatives are mutually exclusive, however, the present disclosure supports the provision to refer only to alternatives and "and / or." As used in this disclosure, "another" can mean at least a second or more.

[0071] As used in this disclosure, unless otherwise indicated, the terms "including," "comprising," and similar terms (such as "having" or "involving") mean inclusion without limitation.

[0072] When numerical ranges are presented in this disclosure, unless otherwise indicated, the numerical ranges include the endpoints of the ranges. Unless otherwise indicated, the numerical ranges in this disclosure include all values ​​and subranges exactly as if they were explicitly written down.

[0073] System Architecture 2 is a schematic example of a particle separation system 200. In this example, the particle separation system 200 includes a multi-channel flow cell 202, a core 204, a pumping subsystem 206, an imaging subsystem 208, a user interface 210, and a controller 212. The core 204 is configured to house and hold the multi-channel flow cell 202, expose the multi-channel flow cell 202 to a magnetic field gradient, regulate the temperature of the multi-channel flow cell 202, and fluidly connect the multi-channel flow cell 202 to the pumping subsystem 206. The pumping subsystem 206 is configured to move fluid through the channels of the flow cell 202. The imaging subsystem 208 is configured to image particle separation within the channels of the flow cell 202. The user interface 210 and controller 212 facilitate monitoring and control of the particle separation system 200. The components of the system 200 are housed in a housing 214 having an access door 216 that allows for insertion and removal of the multi-channel flow cell 202.

[0074] The particle separation system 200 of FIG. 2 is capable of magnetically levitating particles suspended in a paramagnetic fluid medium within the processing channels of a multi-channel flow cell 202. The interaction of the magnetic field with the paramagnetic properties of the particles in the paramagnetic fluid medium can exert a repulsive or attractive effect on the particles to facilitate separation or enrichment of the particles. The magnetic field in the magnetic fluid medium is generated by a magnet, which can be a permanent magnet or an electromagnet. The maximum energy product of the magnet can be in the range of about 1 Megagauss Oersted to about 1000 Megagauss Oersted or in the range of about 10 Megagauss Oersted to about 100 Megagauss Oersted. The surface magnetic field strength of the magnet can be in the range of about 0.01 Tesla to about 100 Tesla or in the range of about 1 Tesla to about 10 Tesla. The remanence of the magnet can be in the range of about 0.1 Tesla to about 5 Tesla or in the range of about 1 Tesla to about 3 Tesla. The magnets can be made from materials including neodymium alloys with iron and boron, neodymium, alloys of aluminum and nickel, neodymium alloys with iron, aluminum and cobalt alloyed with iron, samarium-cobalt, other alloys of rare earth elements and iron, alloys of rare earth alloys and nickel, ferrite, or combinations thereof. All of the magnets in a particle separation system may be made from the same material or from different materials.

[0075] An asymmetric magnetic field can be achieved by using a more ferromagnetic material on one side of the fluid channel of the flow cell and a less ferromagnetic material on the other side of the flow cell. An asymmetric magnetic field in a channel can also be achieved by positioning magnets on one side of the channel closer together compared to magnets on the other side. An asymmetric magnetic field can also be achieved by using a magnetic material on one side of the flow cell and a substantially similar magnetic material on the other side of the flow cell. The upper and lower magnets can be substantially the same size. In one example, the upper magnet can be neodymium and the lower magnet can be samarium-cobalt. Alternatively, the upper magnet can be samarium-cobalt and the lower magnet can be neodymium. Alternatively, alternative magnet configurations can be used. The magnetic levitation system can include multiple upper magnets and multiple lower magnets positioned around each fluid channel of the multi-channel flow cell.

[0076] Exemplary NdFeB magnetic component dimensions include approximately 50x15x2mm for the bottom magnet component (magnetized through a 15mm axis) and 50x5x2mm for the top magnet component (magnetized through a 5mm axis). Other exemplary magnet dimensions include 60x15x2mm, 60x5x2mm, 75x10x3mm, 75x20x3mm, and 25x15x2mm. An exemplary magnet configuration for the separation channel of a multi-channel flow cell includes upper and lower magnets with dimensions of about 75 x 10 x 3.2 mm, with the spacing between the upper and lower magnets being about 2.5 mm, about 3.0 mm, about 3.5 mm, about 2.9 mm, about 3.0 mm, about 3.1 mm, about 3.2 mm, about 3.3 mm, or about 2.72 mm, about 2.88 mm, about 2.98 mm, about 3.18 mm, about 3.20 mm, or about 3.37 mm.

[0077] The pumping subsystem 206 is configured to move fluid through the channels of the flow cell 202. In one particular example, the flow cell 202 includes four separation channels, each of which splits into two subchannels after separation, and the pumping subsystem includes eight pumps, one for each subchannel of the flow cell 202. In the particular example shown in FIG. 2, the pumps are syringe pumps, although other pumps may be used. The flow rate of each separation channel achieved by the pumping subsystem may range from 1 μL / min to 100 mL / min during separation. The flow rate may be about 25 μL / min or more, about 50 μL / min or more, about 100 μL / min or more, about 200 μL / min or more, about 250 μL / min or more, about 300 μL / min or more, or about 300 μL / min to about 1 mL / min. The total sample volume flow rate in each separation channel of the flow cell can be about 50 μL / min, about 75 μL / min, about 100 μL / min, about 150 μL / min, about 200 μL / min, about 300 μL / min, or about 600 μL / min. During operation of embodiments of the system, the flow rate split between the two outlet channels can be an even split, or can range from about 9:1 to 1:9, about 4:1 to about 1:4, about 3:1 to about 1:3, or about 2:1 to about 1:2, or can vary from 1:1 by about 90% or less, 50% or less, or about 40% or less, or about 30% or less, or about 15% or less. The system can be configured to vary the split of flow rate between outlet channels associated with each separation channel.

[0078] The particle separation system 200 may be a stand-alone system or may be centrally monitored and controlled by another computer. Figure 3 shows an example of several particle separation devices 200 centrally controlled by a computer 300 via a network 302.

[0079] Flow Cell 4A and 4B show an example of a multi-channel flow cell (in this example, a flow cell cartridge) from the top and bottom that can be used for particle separation. FIG. 4C shows an exploded view of the flow cell cartridge. In the example shown, the components of the flow cell cartridge 400 include a main body 402, a top capping film 404, a bottom capping film 406, a collection well capping film 408, an outlet membrane 410, an outlet membrane film 412, and a label 414.

[0080] The main body of the flow cell cartridge can be formed by injection molding, etching, laser ablation, machining, or 3D printing. If imaging within the flow cell cartridge is desired, the main body, or at least a portion of the main body, can be formed from an optically transparent or translucent material. Glass, plastic, or polymeric materials, including cyclic olefin polymers (COP) or cyclic olefin copolymers (COC), are some non-limiting examples of suitable materials.

[0081] The flow cell cartridge 400 comprises an inlet well 434 and a collection well 438. The processing channels extend through a channel beam 432 that fluidly connects the inlet well 434 to the collection well 438. As shown in this example, the four processing channels and the channel beam are coplanar with respect to one another. The flow cell cartridge further comprises a collection well outlet 442 that is fluidly connected to the collection well 438. By applying negative pressure (e.g., by the pumping subsystem 206 shown in FIG. 2) to the collection well outlet 442, fluid is drawn from the inlet well 434 through the processing channels in the channel beam 432 and into the collection well 438. The volume of each processing channel can be about 10 μL to about 800 μL, about 50 μL to about 600 μL, about 100 μL to about 400 μL, about 150 μL to about 300 μL, at least about 150 μL, at least about 200 μL, at least about 250 μL, or at least about 300 μL. As discussed in more detail below, in this particular example, each of the processing channels splits into two outlet channels. In some implementations, the total volume of the outlet channels can be greater than the volume of the processing channel. When used in the exemplary particle separation system, the flow rate split between the two outlet channels can be an equal split or can range from about 4:1 to about 1:4, from about 3:1 to about 1:3, or from about 2:1 to about 1:2, or can vary from 1:1 by about 50% or less, or about 40% or less, or about 30% or less, or about 15% or less.

[0082] 4A, an opening 474 extends through the flow cell cartridge 400 on each side of the channel beam 432. As discussed in more detail below, the openings 474 facilitate imaging of particles within the processing channels.

[0083] 5A-5J show the main body 402 of the flow cell cartridge of FIGS. 4A and 4B. In this particular example, the main body 402 is generally rectangular and defines an inlet end 416 and an outlet end 418 at opposite longitudinal ends of the main body 402. The main body 402 has a top surface 420 and a bottom surface 422 opposite the top surface 420, both of which extend between the inlet end 416 and the outlet end 418. The main body 402 further includes a sidewall 424 extending downwardly from the bottom surface 422 to define a cavity in the back of the flow cell cartridge (see, e.g., FIGS. 5C and 5D).

[0084] In this example, the sidewall 424 of the main body 402 extends between an inlet end sidewall 428 and an outlet end sidewall 430 and includes two support beams 426 that are parallel to a channel beam 432 (described further below). The support beams 426 are offset horizontally and vertically from the channel beam 432 to support the main body and prevent twisting and bending. In some implementations, this can be beneficial in maintaining optical alignment during imaging.

[0085] In this example, the inlet end 416 comprises a number of inlet wells 434, four in this particular example. The inlet wells 434 may extend from the top surface 420 of the main body 402. As shown in the top view of FIG. 5K, the inlet wells 434 may comprise cylindrical sidewalls. As shown in the top view of FIG. 5E, the cylindrical sidewalls of the inlet wells 434 may be spaced apart from one another. Additionally, the top surface of the inlet end may comprise an elongated recess 436 between the cartridge edge and the inlet wells 434. The elongated recess 436 may be designed to provide a handle for a user to perform the operations of inserting and removing the flow cell cartridge into and from the particle separation system.

[0086] As also shown in the top view of FIG. 5E, the outlet end can include a number of collection wells 438. In the particular example shown, there are two collection wells 438 for each inlet well 434 and processing channel 440, with the inlet well 434 fluidly coupled to the processing channel 440, and the processing channel 440 fluidly coupled to the two collection wells 438. In other cases, the flow cell cartridge can include other numbers of inlet and collection wells in other ratios, such as, but not limited to, 1:1, 1:2, 1:3, 1:4, or other ratios. For example, FIG. 1 shows a processing channel that splits into three outlet subchannels. Returning to FIG. 5E, the collection wells 438 can extend from the top surface 420 of the main body 402. Each pair of two collection wells 438 can be aligned longitudinally with the corresponding inlet well 434 such that the spacing between the centers of adjacent collection wells 438 is narrower than the spacing between adjacent inlet wells 434. As shown, the collection wells 438 can also have elliptical sidewalls as shown in Figure 5M. Due to the closer spacing of the collection wells 438 relative to the inlet wells 434, the elliptical sidewalls of the collection wells can touch each other.

[0087] 5E, the top surface 420 of the outlet end 418 may also include a collection well outlet 442 between the outer edge of the cartridge and the collection wells 438. As will be discussed further below, each collection well 438 may be fluidly connected to a dedicated collection well outlet 442.

[0088] 5E (top) and 5F (bottom), each inlet well 434 is fluidly connected to two collection wells 438 by a processing channel 440. Each processing channel 440 comprises a processing segment 444 located within the channel beam 432, a segment 446 connecting the inlet well 434 to the processing segment 444, and two segments 448 connecting the processing segment 444 to the two collection wells 438.

[0089] The processing channel 440 is defined by slots in the main body 402, both single- and double-door slots defined in the top and bottom surfaces 420 and 422 of the main body 402, through-holes connecting the single-door slots defined in the top surface 420 to the single-door slots defined in the bottom surface 422, and capping films 404 and 406 coupled to the top and bottom surfaces 420 and 422 of the main body 402 that seal the open sides of the single- and double-door slots. For example, as shown in Figures 5E and 5F, through-holes 450 in the collection well 434 fluidly connect the collection well 434 to the channel segment 446, which is a single-door slot in the bottom surface 422. The processing segment 444 is a double-door slot that extends from the top surface 420 to the bottom surface 422 (although one or more internal partitions may also be included within portions of the processing segment 444, as discussed further below). Segment 448 is also defined by slots in the top and bottom surfaces 420, 422 and through holes extending through the top and bottom surfaces 420, 422. Figures 7, 8A and 8B show the top and bottom capping films 404, 406 sealing the slots to form fluid channels.

[0090] 5A-5H, the channel beams 432 containing the processing segments 444 of the processing channels 440 may be generally rectangular prism shaped and may extend longitudinally parallel to one another. Each channel beam 432 may define a portion of a top surface 420 and a bottom surface 422 such that the top surface 420 and the bottom surface 422 are continuous between the inlet end 416 and the outlet end 418. In some implementations, the continuous top surface 420 and the continuous bottom surface 422 may beneficially enable the top capping film 404 and the bottom capping film 406 to form a continuous seal of the channel 440, at least in the area where the slots for the processing channels 440 are formed in said surfaces.

[0091] 5A, each channel beam 432 may also include an alignment protrusion 460. In other examples, the channel beams may include more than one alignment protrusion on one or both sides of the channel beam. The protrusions 460 may be interlocked with portions of the core to facilitate and / or maintain alignment of the channel beams 432 during use.

[0092] 7 and 8A show a cross-section of one of the channel beams 432. As shown in FIG. 7 and 8A, each of the channel beams 432 comprises a channel beam top surface 452 that is part of the top surface 420, a channel beam bottom surface 454 that is part of the bottom surface 422, a first channel side 456, and a second channel side 458 opposite the first channel side 456. In this example, the main body 402 of the flow cell cartridge 400 is formed from a transparent or semi-transparent material (to at least some wavelengths of light) to facilitate illumination and / or imaging of the processing channel 440 through the surface of the channel beam 432, including the first and second channel sides 456, 458, as will be discussed further below.

[0093] 8B shows a longitudinal cross-section of a portion of one of the channel beams 432 toward the outlet end of the processing portion 444 of the processing channel 440. As shown, a partition 462 can divide the processing segment 444 into upper and lower portions 464 and 466, respectively, whereby fluid flowing along the upper portion 464 enters a channel segment 448 that extends to one of the collection wells 438 and fluid flowing along the lower portion 466 enters a different channel segment 448 that extends to a different collection well 438, as will be discussed further below.

[0094] 5K and 5L show one of the inlet wells 434 of the flow cell cartridge 400 in more detail. As shown, the inlet wells 434 include through-holes 450 for fluidly connecting the inlet wells 434 to the processing channel 440. Although not shown in FIG. 5L, when assembled, the bottom capping film 406 forms a seal around each through-hole 450 and over the slot to define a channel segment 446 connecting the inlet wells 434 to the processing segment 444 of the processing channel 440. To facilitate aspiration of the entire sample and to avoid air bubbles being aspirated along with the sample, the interior shape of the inlet wells 434 can define a large radius corner and may be sloped toward the inlet through-hole 450. Additionally, the ramp provides guidance to the user to pipette the sample to the appropriate location within the inlet well. In some embodiments, each of the inlet channel portions can include a valve, e.g., a capillary valve, to prevent undesired backflow of fluid from the processing channel 440 into the inlet wells 434.

[0095] 5M and 5N show one of the collection wells 438 of the flow cell cartridge 400 in more detail. As shown, the collection well 438 comprises a collection through hole 468 that extends from the bottom surface 422 of the main body 402 into the interior of the collection well 438. As shown in FIG. 5N, the collection through hole 468 enters the interior of the collection well 438 above the bottom of the collection well 438. The interior shape of the collection well 438 comprises a ramp surface 470 configured to direct collected fluid away from the collection through hole 468. The top of the ramp can define a curved edge so that fluid is not pinned down at the ramp and the sample is not retained. These features and the funnel shape of the bottom of the collection well facilitate pipetting for withdrawal of the sample from the collection well.

[0096] As shown in FIG. 4A, the upper edge of the collection well 438 can be sealed by a capping film 408. In this particular example, by sealing the upper edge by the capping film 408, negative pressure can be applied to the interior of the collection well, for example by a pump, to draw fluid through the flow path into the collection well. In this example, negative pressure is applied via a collection well outlet 442, for example, fluidly connected to the collection well at an opening 472, as shown in FIG. 5M and FIG. 5N. As shown in FIG. 5N, the opening 472 connects to the collection well 438 above the height at which the collection through-hole 468 connects to the collection well 438. The collection well 438 is configured to reduce, if not completely prevent, the drawing of fluid from the collection well into the opening 472, thereby reducing, if not completely eliminating, the risk of cross-contamination of samples from one flow cell cartridge with samples in other flow cell cartridges that are later used with the particle separation system. The collection well 438 is configured to retain collected fluid in the well after pumping is completed, so that fluid can be collected from the well at a later time. 5N, the upwardly open, "up and over" profile of the fluid pathway, including the locations of the through-holes 468, 472, facilitates retention of collected fluid within the collection well 438. In other examples, the collection well may be positioned lower within the cartridge body to facilitate retention of fluid within the well.

[0097] 4A, the collection well outlet is covered by an outlet membrane film 412. The outlet membrane film 412 can include a gas permeable material, such as PTFE, to allow escape of gas while retaining liquid, thus further reducing, if not eliminating, the risk of liquid from the flow cell cartridge 400 entering other components of the particle separation system while still allowing application of negative pressure to the processing channels by the pumping subsystem of the particle separation system.

[0098] The flow cell cartridge 400 described above in this example includes four processing channels 440 that define parallel flow paths, each of which fluidically connects an inlet well 434 to two collection wells 438. Each of the parallel flow paths includes a common portion that flows from the inlet well 434 through an inlet through-hole 450, through a channel segment 446, and then into the processing segment 444. From the processing segment 444, the fluid flow path includes two alternative paths, including a first path and a second path, in the example shown. The first path flows above the divider 462 through a collection through-hole 468 into a channel segment and a first collection well 438. The second path flows below the divider 462 into a different channel segment and a different collection well 438. Thus, by vertically separating particles of a sample within the processing channel segment 444 and allowing the sample to flow out of the processing channel segment 444, the separated samples can be collected separately in different collection wells 438.

[0099] core Figure 9 shows the core 204 of the schematic example of Figure 2. In this example, the core comprises a holder configured to receive and hold a flow cell. In particular, the core comprises an upper clamp 502, a lower clamp 504, a clamp plate 506, a temperature control plate and thermoelectric cooler 508, and a fan and heat sink unit 510. Each of these elements is discussed in further detail below.

[0100] The upper and lower clamps 502, 504 are configured to receive and secure a flow cell cartridge (such as flow cell cartridge 400 of FIG. 4A) between the upper clamp 502 and the lower clamp 504, and the clamp plate 506 guides the movement of the upper clamp 502 between the clamped and unclamped positions. Figures 10A and 10B show another example of the core when the upper clamp 502 is in the unclamped (FIG. 10A) and clamped (FIG. 10B) positions. The upper clamp 502 is slidably mounted on four posts 512 (see also FIG. 11, which shows the core without the upper clamp), so that the upper clamp 502 can translate along a vertical axis between the clamped and unclamped positions. The upper clamp 502 further includes a pin 544 (not shown in FIGS. 10A and 10B, see instead FIG. 14) that extends outward from the side of the upper clamp 502 and into the inner cam track of the clamp plate 506. In other implementations, the pin 544 may be replaced by a roller bearing.

[0101] FIG. 11 shows the core of FIGS. 10A and 10B with the upper clamp 502 removed so that the inner cam track 514 in one of the clamp plates can be seen, otherwise the core is partially disassembled. As can be seen in FIG. 11, the cam track 514 slopes from the upper portion to the lower portion. As can also be seen in FIG. 11, the clamp plate 506 is mounted on a slide 516 such that the cam plate 506 can translate along a horizontal axis. A linear actuator 518 controls the horizontal position of the clamp plate 506. By actuating the linear actuator 518 to move the clamp plate 506 rearward along the horizontal axis, the upper clamp 502 can be moved from the unclamped position of FIG. 10A to the clamped position of FIG. 10B. As the clamp plate 506 moves rearward, the ramped cam track 514 cooperates with the pin 544 on the upper clamp 502 to force the upper clamp 502 down the post 512, translating the upper clamp 502 into a clamping position.

[0102] FIG. 12 shows the components of the lower clamp 504 of the core of FIGS. 10 and 11 in more detail. In this example, the lower clamp 504 includes four magnet pillar assemblies 520 sized and positioned to correspond to the four channel beams 432 of the flow cell cartridge 400 shown in FIGS. 4A and 4B. FIG. 13 shows one of the magnet pillar assemblies 520 removed from the rest of the lower clamp 504. Each magnet pillar assembly 520 includes a pillar body 522 with a slot 524 for receiving a magnet 526. The pillar body 522 has a flat upper surface 528 that contacts the underside of the channel beam 432 when the flow cell cartridge 400 is installed and clamped within the core. The pillar body 522 may be formed from a thermally conductive material (such as aluminum, copper, silver, copper alloy, aluminum alloy, or a multi-layer structure with a conductive outer layer) to facilitate temperature regulation, as discussed in more detail below.

[0103] Returning to FIG. 12, the pillar assembly 520 is surrounded by a lower clamp body 530. A portion of the lower clamp body 530 defines a seat 532 that is shaped and sized to fit inside a cavity in the underside of the flow cell cartridge 400 shown in FIGS. 4A and 4B. The top of the pillar assembly 520 extends through an opening 534 in the lower clamp body. In this particular example, the top of the pillar assembly 520 is located slightly above the top surface of the lower clamp body 530 such that when the flow cell cartridge is positioned on the seat 532, the top of the pillar assembly 520 contacts the underside of the channel beam of the flow cell cartridge 400 and the top surface of the lower clamp body 530 does not contact the channel beam. In this particular example, the top 528 of the pillar body is also located slightly above the top surface of the magnet 526, as shown in FIG. In this particular example, when a flow cell cartridge having four processing channels is positioned between the upper clamp 502 and the lower clamps 502, 504 and clamped, each of the magnets 526 is positioned directly below one of the processing channels.

[0104] The opening 534 in the lower clamp body 530 is larger than the pillar body 522 positioned inside the opening 534, defining an air channel around the pillar body 522 to further facilitate temperature regulation, as discussed in more detail below.

[0105] Although not specifically mentioned in the drawings, the pillar body 522 and / or the lower clamp body 530 and / or other parts of the lower clamp may include set screws or other components for fine tuning and adjusting the position of the magnet 526.

[0106] 12, the body 530 of the lower clamp 504 includes slots 536 between each of the portions of the body 530 in which the magnet pillar assemblies 520 are mounted. The slots provide clearance and allow access to portions of the imaging subsystem, as discussed in more detail below.

[0107] FIG. 14 shows the upper clamp 502 of the core of FIGS. 10 and 11 in more detail from above the upper clamp 502. The upper clamp 502 comprises an upper clamp body 538 supported by a carrier 540. In this example, a resilient element 542 (e.g., a coil spring) between the carrier and the upper clamp 502 biases the upper clamp body 538 downwardly away from the carrier to facilitate uniform clamping of the flow cell cartridge even with dimensional variations in the manufactured cartridges. The carrier 540 comprises a plain bearing 542 to accommodate the upper clamp 502 and allow vertical translation along the post 512. FIG. 14 also shows a pin 544 housed in a cam track 514 of the clamp plate 506.

[0108] 14, the upper clamp body 538 includes an opening 546 that extends through the clamp body from the top surface to the bottom surface of the clamp body. As discussed in more detail below, the opening 546 provides clearance and allows access to portions of the imaging subsystem.

[0109] 15 shows a close-up of a portion of the upper clamp 502 from underneath. The lower clamp body includes openings 548 for accommodating magnets 550. In this particular example, when a flow cell cartridge having four processing channels is positioned and clamped between the upper clamp 502 and the lower clamp 504, each of the magnets 550 is positioned directly above one of the processing channels. As with the lower clamp 504, the upper clamp 502 can include set screws or other components for fine-tuning the position of each of the magnets 550.

[0110] The upper clamp body 538 shown in FIG. 15 includes resilient contacts 552 positioned and configured to contact alignment features of the flow cell cartridge (e.g., protrusions 460 shown in FIG. 5A) that clamp the flow cell cartridge flat and otherwise seat the flow cell cartridge precisely between the upper and lower clamps. In the particular example shown, the resilient contacts 552 are spring plungers with downwardly biased contact ends. In the particular example shown, when the flow cell cartridge is clamped between the upper and lower clamps 502, 504, the lower ends of the resilient contacts 552 contact one or more of the protrusions of the flow cell cartridge closest to the processing channel, and the magnet 550 and the surrounding portion of the upper clamp body 538 are positioned just above, but not touching, the top surface of the flow cell cartridge closest to the processing channel of the flow cell cartridge.

[0111] 15, the upper clamp body 538 further comprises a series of fluid connection ports 554 positioned and configured to fluidly connect the flow cell cartridge to the pumping subsystem. In this particular example, each connection includes a port opening 556 surrounded by a raised sealing ring 558.

[0112] 16 and 17 show schematic examples of magnet arrangements (e.g., the upper and lower magnets 526, 550 of the core shown in FIGS. 10A and 10B) relative to a flow cell cartridge when the cartridge is clamped within the core. As shown, opposite magnet poles may be positioned above and below the processing portion 444 of the processing channel of the cartridge. Additionally, as shown in FIG. 16, adjacent processing channels may include opposite magnet polarities. In this example, alternating magnet polarities help minimize magnetic field cancellation between adjacent magnet pairs. In other examples, the magnets may be arranged in a non-alternating configuration. In some applications, the magnets may be arranged to prioritize certain aspects of the magnetic separation process. For example, in one example, the magnets may be arranged in an alternating configuration to optimize the speed and dynamic range of the separation process. In another example, the magnets may be arranged in a non-alternating configuration to optimize the resolution of the separation process. As described below, the separation system may include several removable units, allowing a user to replace one unit (e.g., having a particular magnet arrangement) with another unit (e.g., having a different magnet arrangement).

[0113] As mentioned above, the core can include components for regulating the flow cell temperature. Figures 18A and 18B show a cross section of the core of Figures 10A and 10B. In this example, the cooling assembly of the core includes a thermoelectric (Peltier) module 560, a temperature controlled plate 562 positioned on the top surface of the thermoelectric module 560, a heat sink 564 and a fan 566 below the thermoelectric module 560. In this example, the pillar body 522 and lower clamp body 530 mentioned above are also responsible for regulating the flow cell temperature.

[0114] In the example of Figures 18A and 18B, passing an electric current through the thermoelectric module 560 (which may be one or more individual modules) creates a temperature difference between two of the surfaces of the thermoelectric module 560, cooling one of these surfaces (in this example, the top surface of the module 560) and heating the other surface (in this example, the bottom surface of the module 560). On the heated side of the module 560, the generated heat is dissipated by a heat sink 564, facilitated by a fan 566. On the cooled side of the module 560, in the example shown in Figure 18 (and in the enlarged view of Figure 19), the bottom surface of the pillar body 522 is in direct contact with a temperature-controlled plate 562, which is in direct contact with the cooled side of the module 560, and as a result, when the thermoelectric module 560 is operated, the temperature-controlled plate 562 is cooled by conduction. This cools the pillar body 522 by conduction, which in turn cools the flow cell cartridge in direct contact with the top surface of the pillar body 522 by conduction. As can also be seen in Figures 18 and 19, the cavity in the lower clamp body 530 defines air channels 570, so that the air cooled by the temperature control plate 562 flows along the sides of the pillar body 522, further cooling the pillar body 522 by convection, and rises around the channel beam 432 of the flow cell cartridge, further cooling the cartridge by convection. Although not shown in full detail in the drawings, a further fan unit (separate from the fan used to cool the heat sink) can also move air through the channels in the temperature control plate 562, and said cooled air moves upwards around the pillar body 522 through the air channels 570. In short, the core in this example is configured to cool the flow cell cartridge by both conductive and convective heat transfer. A thermistor or other feedback mechanism (not specifically mentioned in the drawings) can be used to sense the temperature and adjust the operation of module 560 or other cooling modules to thereby regulate the temperature of the flow cell cartridge to a desired level or range.Alternatively, the same thermoelectric module and associated components can be used to warm the cartridge above ambient temperature. Alternatively, the same thermoelectric module and associated components can be used to maintain a fixed temperature at or near ambient temperature, thereby reducing fluctuations. In other words, the temperature regulating components of the particle separation system can be configured to cool, warm, or alternatively cool and warm the flow cell cartridge.

[0115] In some implementations, the particle separation system may be configured such that an end user or technician may relatively easily remove the core as a single unit and replace it with another core. For example, with reference to Figures 10, 11, and 18, the removable unit may include an upper clamp 502, a lower clamp 504, a clamp plate 506, a linear actuator 518, a temperature control plate and thermoelectric module 508, a heat sink 564, and a fan 566, all of which may be interconnected to allow removal from and installation into the particle separation system as a single unit. In some cases, the removable / replaceable core unit may allow reconfiguration of the particle separation system by changing the number, type (e.g., field strength) and arrangement of magnets in the clamping block, changing the number of particle separation channels the system can process simultaneously, changing aspects of the temperature control system, etc.

[0116] 20-22 show an example of a particle separation system with a removable / replaceable core. As shown in FIG. 20, the particle separation system includes a tray 600 that can be slid in and out to access a removable core 602 after removing a portion of the cover of the system. The alignment pins 604 of the removable core 602 can fit into slots in the tray 600 to facilitate proper alignment and installation of the removable core 602 within the tray 600. FIG. 21 shows the alignment pins of the removable core seated within the slots in more detail. FIG. 21 also shows a thumbscrew 608 on the tray 600 for securing the tray 600 within the system after it is slid in.

[0117] FIG. 22 shows diagrammatically one of two runners 610 to which a tray (not shown) is slidably mounted to allow the tray to slide in and out. As shown in FIG. 22, the runner 610 includes a guide that is wider at one end to facilitate insertion of the tray into the runner. As also shown in FIG. 22, the guide narrows or pinches at the opposite end to provide precise vertical alignment of the removable core when the tray is slid fully in. Spring features at the end of the guide bias the tray and removable core upwards, resulting in little or no vertical clearance between the top of the guide and the parts of the tray that interface with the guide when the tray is slid fully in.

[0118] In one exemplary method, a removable core unit can be removed and replaced with another core unit by first removing all or part of the particle separation system housing. A tray holding the core unit can then be slid out of the housing, and the fluidic, electrical and other connections between the core unit and the rest of the system can be disconnected. The removable core unit (including upper and lower clamps, clamp plate, linear actuator, temperature control plate and thermoelectric module, heat sink and fan) can then be lifted out as a single unit and removed from the system. Another core unit (having the same or different configuration as the removed core unit) can then be lowered into the tray as a single unit, and the alignment pins of the removable core fit into the slots in the tray to effect proper alignment and installation of the core unit within the tray. The tray holding the new core unit can then be slid back into the system, and the fluidic, electrical and other connections between the core and the rest of the system are connected. Finally, the thumbscrews are tightened to secure the tray into the system, and the housing is replaced. The thumb screws may be replaced by clamps, including but not limited to over-center clamps or quarter-turn clamps, or fasteners that require a tool such as a hex key.

[0119] Imaging The particle separation system 200 shown in FIG. 2 includes an imaging subsystem 208 for imaging the processing channel and / or particles within the processing channel of the flow cell cartridge 202, including before, during and / or after particle separation by magnetic levitation.

[0120] The imaging subsystem may be constructed to provide microscopic imaging of the processing channel of the flow cell cartridge. Optionally, the imaging subsystem may be constructed and arranged to provide imaging of fluorescent emission using an optional ultraviolet light excitation module. The imaging subsystem may include a visible optical illumination source constructed and arranged to perform optical transmission through the processing channel of the flow cell. The imaging system may employ optics to enable bright field illumination, dark field illumination and / or fluorescent detection of sample components. To image fluorescent elements in the processing channel, the imaging subsystem may also optionally include a dual bandpass filter that transmits radiation emitted in bands preferably centered around wavelengths of about 524 nm and 628 nm.

[0121] For the multi-channel flow cell cartridge 400 shown in FIGS. 4-8, imaging may be performed through the sides 456, 458 (see, e.g., FIG. 7) of the channel beam 432. As shown in FIG. 4A, an opening 474 in the flow cell cartridge 400 (including openings extending through the top and bottom surfaces of the cartridge and / or through the side walls of the cartridge) exposes the channel sides 456, 458 of the channel beam 432. The opening (which in this example is generally rectangular) allows optical access to the sides 456, 458 so that the contents of the processing portion of the processing channel 444 can be imaged. The imaging subsystem may include any device that allows or enhances the ability to view and / or record particles in real time as they pass through the processing channel, thereby allowing observation and / or measurement of particle isolation, including the degree and / or rate of particle separation. Visualization may also include analysis of collected images.

[0122] 23 and 24 show an example of an imaging subsystem that can be used in a particle separation system. In this example, the imaging subsystem includes an illumination source 652 configured to illuminate particles in a processing channel (schematically shown at 660), collection optics 654, and reflector optics 656 and 658. As shown in FIGS. 25 and 26, the imaging subsystem further includes a reflector optics 662 configured to image the illuminated particles in the processing channel 660, an objective lens 664, and one or more sensors 666. The imaging subsystem can also be configured for use with a wide variety of sensors, such as CCD, CMOS, or other sensors. Although not specifically mentioned in FIGS. 25 and 26, the imaging subsystem can include one or more precision translation stages for adjusting the focus of the optical system of the system. Although also not specifically mentioned in FIGS. 25 and 26, the imaging subsystem can include one or more adjustable apertures or other components for adjusting or changing the depth of field for imaging the processing channel 660.

[0123] The imaging subsystem, or at least components of the imaging subsystem, can be mounted to a multi-degree-of-freedom device for positioning the imaging subsystem to image the processing channels of the multi-channel flow cell cartridge. In one example, the imaging subsystem is mounted to a multi-stage translation device configured to move the imaging subsystem along the x-, y-, and z-axes.

[0124] 23 and 24 , the reflector optical features 658 and 662 are attached to the distal end of a periscope arm 668. The periscope arm is configured to allow the reflector optical features 658 and 662 to be lowered through the opening 546 in the upper clamp body and into the opening 474 of the flow cell cartridge 400 and the slot 536 in the lower clamp body 530. As a result, the reflector optical features 658 and 662 can be positioned adjacent to the sides 456, 458 of the channel beam 432 of the multichannel flow cell cartridge 400 while leaving most of the components of the imaging subsystem positioned higher than the multichannel flow cell cartridge 400.

[0125] 25 shows a schematic example in cross section of how the components of the imaging subsystem can be positioned relative to the parts of the upper clamp, flow cell cartridge, and lower clamp during imaging. As shown in FIG. 25, the periscope arm 668 extends through an opening between parts of the upper clamp 502, downwardly from between the channel beams 432 of the flow cell cartridge, into a slot 536 formed in the lower clamp body 530. In this way, even if the channel beams 432 are arranged in a planar array of several parallel channels, both sides of each channel beam 432 can be imaged, even if the top surface of the channel beam is obstructed by the magnet 550 and other parts of the upper clamp body, and the bottom surface of the channel beam 432 is covered by the top surface of the pillar body 522 and the magnet 526 of the lower clamp. In other words, the imaging subsystem is configured to image particle separation within each processing channel while still allowing positioning of magnets above and below each processing channel, as discussed earlier in this specification, and positioning of pillar body 522 and other components of lower clamp 504 that facilitate cooling of the flow cell cartridge by conductive and convective heat transfer.

[0126] 25, the imaging subsystem is configured to image particle separation in the processing channel with a curved optical path, with most of the imaging subsystem components and structures located above the various components of the core unit. In this particular example, the optical path starts from the illumination source 652 and collection optics 654, rotates 90° at a reflector optical mechanism 656, travels downward through an opening in the upper clamp body, rotates 90° at a reflector optical mechanism 658 attached to the distal end of a periscope arm 668, travels through the processing channel of the channel beam 432 (below magnet 550 and other parts of the upper clamp, and above magnet 526, pillar body 522, and other parts of the lower clamp body 530), rotates 90° at a reflector optical mechanism 662, travels upward through another opening in the upper clamp body, and then travels to the objective lens 664 and sensor 666.

[0127] As previously mentioned, the imaging subsystem may be mounted on a multi-axis translation device for positioning and moving the imaging subsystem along the x-axis, y-axis, and z-axis. In some implementations, this facilitates scanning the periscope arm and the remainder of the imaging subsystem along the length of each channel beam. In an exemplary mode of operation, the multi-axis translation device may position the imaging subsystem with the periscope arm straddling one of the channel beams of the flow cell, and then scan along the length of the processing channel to image the entire length or at least a substantial length of the processing channel in the channel beam. The translation device may then uncouple the periscope arm from around the first channel beam, move it to an adjacent channel beam, and scan to image the processing channel along its length. The above process may be repeated until all of the channel beams have been scanned along the length of the processing channel, and may be repeated continuously or periodically during operation of the particle separation system.

[0128] In the particular example shown, the imaging subsystem utilizes a periscope with a distally mounted reflector for optically accessing the imaging surface of the multi-channel flow cell. In other examples, the flow cell itself may have built-in reflectors on one or both sides of the reflector to facilitate imaging, or the system and / or flow cell may otherwise be configured to not require a periscope for optical access.

[0129] Example of how it works FIG. 26 illustrates an example of a method of operating a particle separation system.

[0130] In step 1002, a multi-channel flow cell cartridge is selected and one or more inlet wells of the cartridge are loaded with a sample for analysis suspended in a paramagnetic fluid medium. An operator can enter identifying information about the cartridge into the system, such as by scanning a unique barcode affixed to the cartridge.

[0131] In step 1004, the operator inputs operational parameters for the particle separation system. The operational parameters may include the type of particle for analysis (e.g., size or type of cells, cellular components (e.g., nuclei) or other particles for analysis), the presence or absence of debris in the fluid sample and / or the type of debris, the concentration of the agent for flotation, which fraction is most desired to be collected, and the temperature at which the process is run. Different operational parameters may result in different run (flotation) times and / or pump parameters, may capture particles of interest from one fraction (e.g., bottom fraction) rather than another fraction (e.g., top fraction), or may cause other changes to the operation of the components of the system. The operational parameters may also include selecting an image analysis mode (e.g., continuous scan vs. single scan for each processing channel), selecting which channels of the flow cell cartridge are run, and (for networked systems) which particular devices are used. In some implementations, the particle separation system may be configured to receive and apply different run parameters to different channels of the flow cell cartridge. An operator can enter the parameters using a user interface on the device itself, or in the case of a networked system, the parameters can be entered at a central computer.

[0132] In step 1006, the flow cell cartridge is loaded into the particle separation system. The operator positions the flow cell cartridge between the upper and lower clamps of the core of the device, which clamps the flow cell cartridge into place. Clamping the flow cell cartridge fluidly connects the collection well outlet of the cartridge to the syringe pump of the pumping subsystem.

[0133] In step 1008, the syringe pump applies negative pressure to the fluidic channel of the cartridge via the collection well outlet, drawing the sample suspension from the inlet well into the processing portion of the fluidic channel of the cartridge.

[0134] In step 1010, the particles are separated by magnetic levitation in the processing channels. Some or all of the separation can be performed during residence time in the processing channels, and the pumping subsystem does not induce fluid flow in the processing channels. Before, during, and / or after particle separation, the system can image the processing channels, allowing visualization of the separation and other analysis of particles in the processing channels. Figure 27 shows an example of an image of three processing channels of a cartridge along the length of the processing channels. The images show that the particles in each processing channel have been magnetically levitated within each channel and separated into two distinct bands of particles.

[0135] In step 1012, the operator can identify and input the split line locations into the system and delineate a vertical split line for each processing channel below which one of the bands of particles is generally collected and above which the other band of particles is generally collected. Figure 27 shows an example of split lines identified for each channel along with graphical buttons for adjusting each split line up or down within the processing channel.

[0136] In step 1014, the pumping subsystem is operated to draw off the bands of separated particles into separate collection wells. In some implementations, the system can use the data associated with the indicated split line position to adjust the operation of the pumping subsystem during fluid extraction. For example, if a particular channel split line is set relatively high or low within a processing channel, the system can adjust the operation of two syringe pumps associated with the processing channel such that one of the syringe pumps applies a greater negative pressure to the particular collection well outlet connected to it compared to the other syringe pump connected to the other collection well outlet associated with the processing channel. Such adjustments can change the vertical level or split line at which separated particles flowing from the processing channel during extraction are collected into two different outlet channels.

[0137] The above-described systems and methods are not limited to the particular sequence of operations illustrated in Figure 26. Figures 28 and 29 show further examples of sequences of operations that may be utilized.

[0138] The system and method may allow for the separation of samples with a relatively large number of cells or other particles. For example, the system and method may be capable of handling samples with a total of 100 million or more particles to be separated (e.g., particles having an average size of 3 microns) in one processing run. As another example, the system and method may be capable of handling 20 million or more particles to be separated (e.g., particles having an average size of 5 microns) per processing channel in one processing run. Separation of a large number of cells or particles is achieved rapidly without exposing the particles to stresses associated with other separation techniques such as FACS or centrifugation. Rapid separation of a large number of particles is performed by a method that includes loading a sample containing particles and a sample medium containing a paramagnetic compound or a magnetic fluid into multiple separation channels, applying a magnetic force to the sample using at least one magnet to affect separation, optionally collecting at least two separated sample fractions, and optionally imaging the particles in the sample before, during and / or after separation. In this method, the sample may contain about 1,000,000 to about 100,000,000 particles. The total time for separation may range from about 1 minute to about 20 minutes. In the rapid separation method, the concentration of the particle of interest is increased in the solution or in a portion of the solution by a ratio of at least about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1.5:1, or 1.1:1. Particles and / or cells of interest in a solution containing multiple types of particles may be considered "isolated" if, after processing of the solution, the ratio of the concentration of the particle of interest to the concentration of other types of particles is increased, or if the ratio of the concentration of the particle of interest to the concentration of other types of particles is increased by at least about 10%, about 50%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, or if the concentration of other particles (including, but not limited to, types of particles other than the particle of interest) in the solution is decreased by less than about 20%, less than about 15%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.5%.In a preferred embodiment of the rapid separation method, the viability of the separated cells or organisms in the concentrated collected portion of the sample is at least 70% relative to the viability of the cells or organisms in the starting sample before separation. Preferably, the viability of the separated cells or organisms in the concentrated collected portion of the sample is at least 75%, 80%, 85%, 90%, 95%, 97% or 99% relative to the viability of the cells or organisms in the starting sample before separation. The starting sample may be about 1,000,000 to about 100,000,000 particles, about 5,000,000 to about 100,000,000 particles, about 10,000,000 to about 100,000,000 particles, about 20,000,000 to about 100,000,000 particles, about 30,000,000 to about 100,000,000 particles, about 40,000,000 to about 100,000,000 particles, or about 50,000,000 to about 100,000,000 particles. The particle size may include about 00,000,000 particles, about 50,000,000 to about 100,000,000 particles, about 60,000,000 to about 100,000,000 particles, about 70,000,000 to about 100,000,000 particles, about 80,000,000 to about 100,000,000 particles, or about 90,000,000 to about 100,000,000 particles. The time taken to separate and collect the separated particle fraction may be 20 minutes or less, 18 minutes or less, 15 minutes or less, 12 minutes or less, 10 minutes or less, 8 minutes or less, 5 minutes or less, 3 minutes or less, or less than 1 minute. The particle size may range in dimensions, such as diameter, from about 100 nm to 100 μm or more. For prokaryotic cells, a typical lower limit may be about 400 nm, while eukaryotic cells may range from about 10 μm to about 100 μm, with human female egg cells being at the higher end of the aforementioned range. Other particles or organisms may be larger. In one embodiment of the rapid large volume separation method described above, the separated particles are cells. Cell types separated by the method described above may include prokaryotic cells, including human cells, non-human animal cells, plant cells, eukaryotic cells (e.g., but not limited to, immune cells, endothelial cells, yeast and T cells), bacteria, etc., cells, organisms, organelles, nuclei, organic materials such as organic polymers and proteins, particulate matter of organic or inorganic compounds, and particles such as beads, bubbles and fragments.The multiple cell types may include dead cells, live cells, healthy cells, diseased cells, infected cells, transfected cells, or genetically engineered cells. The cells separated by the methods of the present disclosure may be obtained directly from an organism (or may be the organism itself) or may be grown or cultured. In one particular embodiment of the rapid large-volume separation method, live cells are separated from dead cells. In another embodiment of the separation method, live transfected cells and / or CRISPR modified or gene edited cells are separated from dead transfected cells and / or CRISPR modified or gene edited cells and / or untransfected cells and / or unCRISPR modified or gene edited cells. In another embodiment of the method, healthy cells are separated from diseased cells. In a further embodiment of the method, cell nuclei are separated from cells, cell fragments, and / or sample debris. In various embodiments, the methods are performed utilizing the cartridges and systems described herein.

[0139] It is understood that the examples and embodiments described in this disclosure are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art in light of this, and these modifications or changes should be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited in this disclosure are incorporated herein by reference in their entirety for all purposes.

Claims

1. (a) a substrate; (b) a plurality of fluid separation channels formed in the substrate, (i) each of the fluid channels includes a processing channel in fluid communication with an inlet and at least two outputs; (ii) each of said processing channels comprises at least one partition that, in use, vertically separates a portion of said processing channel into at least an upper portion and a lower portion, said upper portion being in fluid communication with one of said outputs and said lower portion being in fluid communication with the other of said outputs; (iii) each of the processing channels further comprises at least one imaging surface extending along the length of the processing channel, the imaging surface configured to enable imaging of particles within the processing channel; a plurality of fluidic separation channels; A multi-channel flow cell device further comprising a plurality of channel beams formed in the substrate, the channel beams extending between an inlet end and an outlet end of the flow cell device, each of the channel beams comprising one of the processing channels, and the channel beams spaced apart from adjacent channel beams.

2. The multi-channel flow cell device of claim 1, each of the processing channels comprising two opposing imaging surfaces extending along the processing channel; Multichannel flow cell device.

3. The multi-channel flow cell device according to claim 1, the substrate further comprising sidewalls extending downwardly away from the fluid channels, each of the sidewalls comprising an opening configured for optical access to one of the imaging surfaces; Multichannel flow cell device.

4. A magnetic levitation particle separation system for use with the multi-channel flow cell device of claim 1 or 2, comprising: (a) a holder configured to receive and hold the flow cell device; (b) an array of magnets configured to apply a magnetic field gradient to the processing channel when the flow cell device is received and held by the holder, the array of magnets being positioned at least one above and below the processing channel when the flow cell device is received and held by the holder; (c) an imager configured to image vertical separation of particles within the processing channel while the flow cell device is received and held by the holder and while the array of magnets is positioned at least one of above and below the processing channel; and 1. A magnetic levitation particle separation system comprising:

5. A magnetic levitation particle separation system as described in claim 4, The system, in one isolated run, i) has a minimum particle separation capacity of at least about 1 million particles or at least about 5 million particles; or ii) a minimum particle separation capacity of at least about 5 million particles, said particles having a characteristic dimension of 1 to 5 microns; or iii) a minimum particle separation capacity of at least about 5 million spherical particles, said particles having an average diameter of at least about 3 microns; or iv) a minimum particle separation capacity of at least about 20 million particles per processing channel; or v) a minimum particle separation capacity of at least about 20 million particles per said processing channel, said particles having a characteristic dimension of 1 to 5 microns; or vi) A magnetic levitation particle separation system having a minimum particle separation capacity of at least about 20 million spherical particles per said processing channel, said particles having an average size of at least about 5 microns.

6. The magnetic levitation particle separation system of claim 4, the imager is configured to image vertical separation of particles in the processing channel by a bent optical path; and optionally the curved optical path includes a path portion extending between a magnet positioned directly above one of the processing channels and a magnet positioned directly below one of the processing channels, and further optionally: The magnetic levitation particle separation system, wherein the curved optical path further includes a second portion extending through an opening in the holder between two adjacent magnets in the array of magnets.

7. The magnetic levitation particle separation system of claim 4, the imager comprises an optical periscope, and the system is configured to move the periscope to a position adjacent one of the processing channels while the flow cell device is received and held by the holder and while the array of magnets is positioned above and below the plurality of processing channels of the flow cell device; and optionally i) the system is configured to scan the periscope along the length of the processing channels while the flow cell device is received and held by the holder and while the array of magnets is positioned above and below the plurality of processing channels of the flow cell device; and / or ii) A magnetic levitation particle separation system, wherein said holder comprises a plurality of openings sized and positioned to allow said periscope to move through the openings into a position adjacent said processing channel.

8. The magnetic levitation particle separation system of claim 4, the holder comprises a core unit having an upper clamp and a lower clamp, the core configured to receive and clamp the flow cell device between the upper clamp and the lower clamp, the array of magnets including a first array of magnets mounted in the upper clamp and a second array of magnets mounted in the lower clamp, and optionally 1. A magnetic levitation particle separation system, wherein the imager is configured to image vertical separation of particles within one of the processing channels using an optical path comprising: a first portion of the optical path extending between a magnet of the first array positioned directly above the processing channel and a magnet of the second array positioned directly below the processing channel; and a second portion of the optical path extending between the magnet of the first array positioned directly above the processing channel and another magnet of the first array positioned above an adjacent processing channel.

9. The magnetic levitation particle separation system of claim 4, the holder comprises a core unit configured to regulate the temperature of the processing channel of the flow cell device; and optionally the core unit comprising a plurality of temperature transfer surfaces configured to contact the processing channels when the flow cell device is received and held by the core unit, and further optionally the temperature conducting surface comprises end faces of pillars formed from a temperature conducting material, each of the pillars holding a magnet of the magnet array; and optionally configured to cool the pillars thereby cooling the processing channel by conduction, and further optionally the core comprises a cooling assembly in contact with the pillar and configured to conductively cool the pillar; and further optionally: (i) the cooling assembly comprises a temperature control plate, a thermoelectric cooler, and a heat sink; or (ii) the core unit further comprises an air channel extending along the pillar from the cooling assembly toward the pillar end surface, the air channel configured to cool the pillar and the flow cell device by convective airflow.

10. The magnetic levitation particle separation system of claim 9, The core unit comprises a removable unit of the system, optionally comprising: the core unit comprising a flow cell device holder, a fluid connector assembly configured to fluidly connect the flow cell device to a pump subsystem, the magnet array and a cooling assembly, and further optionally A magnetic levitation particle separation system, wherein the system further comprises a second core unit having a second flow cell device holder, a second fluid connector assembly, a second magnet array, and a second cooling assembly, the second magnet array having a different configuration than the magnet array of the other core unit, and the system is configured to accommodate and operate both core units in an alternating manner.

11. A method for separating particles, comprising: (a) loading a multi-channel flow cell device having at least a first processing channel and a second processing channel into a particle separation system; (b) flowing a suspension of first particles and a paramagnetic fluid into the first processing channel; (c) flowing a second particle suspension and a paramagnetic fluid into the second processing channel; (d) exposing the first and second suspensions in the first and second processing channels to one or more magnetic field gradients; (e) separating the particles in the first processing channel into one or more bands and separating the particles in the second processing channel into one or more bands; (f) imaging the one or more separated bands in the first processing channel and imaging the one or more separated bands in the second processing channel; (g) separately collecting at least one of said bands; A particle separation method comprising:

12. The particle separation method according to claim 11, comprising: imaging the separated bands includes imaging the separated bands using an imaging subsystem of the particle separation system, the imaging subsystem comprising at least one optical component positioned at least partially within a region between the first processing channel and the second processing channel; and optionally the multi-channel flow cell device comprises an opening positioned between the first processing channel and the second processing channel, the optical component of the imaging subsystem being at least partially positioned within the opening during imaging of at least one of the processing channels, and further optionally: The method of particle separation, wherein the optical component comprises a reflector.

13. The particle separation method according to claim 12, comprising: A particle separation method, wherein the imaging subsystem further comprises a second optical component positioned across one of the first or second processing channels, the second optical component being on the opposite side of the opening from the at least one optical component during imaging of at least one of the processing channels.

14. The particle separation method of claim 12, comprising: using the particle separation system to move the at least one optical component into the opening, then imaging at least one of the first and second processing channels, and then moving the at least one optical component out of the opening. and optionally further comprising: i) using the particle separation system to move the at least one optical component from one position within the aperture to another position within the aperture and imaging at least one of the first and second processing channels at different locations along the length of the channel; and / or ii) the openings comprise elongated openings extending along the first and second processing channels; 15. The particle separation method of claim 11, comprising: i) imaging the separated bands includes imaging the separated bands using an imaging subsystem of the particle separation system, the imaging subsystem having at least one bent optical path; or ii) loading the multi-channel flow cell device into the particle separation system includes positioning the multi-channel flow cell device relative to an array of magnets, and further optionally: a) the array of magnets comprises at least a first magnet and a second magnet, wherein the first magnet is adjacent to a first separation channel and the second magnet is adjacent to a second separation channel when the multi-channel flow cell device is loaded into the particle separation system; and optionally the first magnet has a polarity that does not match the polarity of the second magnet; or b) the array of magnets comprises an array of upper magnets and an array of lower magnets, and the multi-channel flow cell device is placed between the array of upper magnets and the array of lower magnets when the multi-channel flow cell device is loaded into the particle separation system; and optionally, A method of particle separation, wherein the upper array of magnets comprises an array of alternating polarity magnets and the lower array of magnets comprises an array of alternating polarity magnets.

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