Method and system for suspension-based magnetic separation

Abstract

Various embodiments of methods, devices, systems, and kits for magnetic levitation-based separation of mixtures or particle swarms including various types of particles are described. Some embodiments of these methods, devices, systems, and kits can be used to separate mixtures or populations of cells including various cell types based on magnetic levitation. Some other embodiments of the described methods, devices, systems, and kits can be used to isolate a mixture or population of cells or a mixture or population of biomolecules based on magnetic levitation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 362,627 filed on April 7, 2022 and U.S. Patent Application No. 63 / 254,946 filed on October 12, 2021, the entire disclosures of which are incorporated herein by reference. Background Art

[0002] Magnetic levitation has recently become a useful method for separating particles (including cells and biomolecules). During magnetic levitation, particles suspended in a paramagnetic fluid medium are exposed to a magnetic field gradient, which produces a non-uniform pressure equal to the magnetic energy density in the paramagnetic fluid medium. In the magnetic field gradient, the particles (or "objects") subjected to magnetic levitation appear to be repelled by the strong magnetic field region. In fact, the object is replaced by an equal volume of paramagnetic fluid medium. The attractive interaction between the paramagnetic fluid medium and the high magnetic field region causes the object to levitate magnetically. Conditions and systems for suspending living cells have been established, and both eukaryotic and prokaryotic cells have been shown to have unique magnetic levitation properties. See Durmus et al., 2015, "Magnetic levitation of single cells", Proc Natl Acad Sci USA 112 (28): E3661-8 ("Durmus"). Systems for collecting cells at different suspension heights and collecting them for downstream analysis and other applications have been developed. Summary of the invention

[0003] The terms "invention," "said invention," "the invention," and "the present invention" as used herein are intended to refer broadly to all subject matter of this patent application and the claims that follow. Statements containing these terms should not be construed as limiting the subject matter described in this disclosure or limiting the meaning or scope of the following patent claims. The embodiments covered by the present invention are defined by the claims rather than by the Summary of the Invention. The Summary of the Invention is a high-level overview of various aspects of the invention and introduces some of the concepts described and illustrated in this document and the drawings. The Summary of the Invention is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The present subject matter should be understood by reference to appropriate portions of the entire specification, any or all of the drawings, and each claim. Some exemplary embodiments of the present invention are discussed below.

[0004] Included in embodiments of the invention and described in the present disclosure are cell separation methods. Some embodiments of the method include the following steps: combining a first suspension height altering agent with a first type of cell in a cell population comprising a plurality of cell types, wherein the first suspension height altering agent comprises a first paramagnetic or superparamagnetic particle and a first linker that preferentially binds to the first type of cell, thereby forming a first complex, wherein the first complex comprises the first suspension height altering agent bound to a single cell of the first type; forming a suspension in a paramagnetic fluid medium, wherein the suspension comprises a plurality of first complexes and a plurality of cells of a plurality of cell types; introducing the suspension into a processing channel of a flow cell cartridge; and exposing the processing channel to a magnetic field for a first period of time, wherein the first period of time is sufficient to separate at least some of the first complexes from the cells of the plurality of cell types that are not bound to the first suspension height altering agent in the processing channel, thereby forming a first portion of the suspension and a second portion of the suspension, wherein the first portion is enriched in the first complex relative to the suspension, and the second portion is depleted of the first complex relative to the suspension. Some embodiments of the method include the following steps: combining a magnetic agent with a cell population comprising a plurality of cell types, wherein the magnetic agent comprises magnetic particles and a linker that preferentially binds to cells of a target type of the plurality of cell types, thereby forming a magnetic complex, wherein the magnetic complex comprises the magnetic agent bound to a single cell of the target type; forming a suspension in a paramagnetic fluid medium, wherein the suspension comprises a plurality of the magnetic complexes and a plurality of the cells of the plurality of cell types; introducing the suspension into a processing channel of a flow cell cartridge; and exposing the processing channel to a magnetic field for a period of time sufficient to cause at least some of the plurality of magnetic complexes to migrate to one or more sides of the processing channel and to be fixed thereon, thereby forming a suspension depleted of the magnetic complexes.

[0005] Also included in embodiments of the present invention are kits for magnetic levitation, and described in the present disclosure. For example, a magnetic levitation kit may include a paramagnetic fluid medium and one or more suspension height changers, or a separation component of one or more of the suspension height changers, capable of forming a complex with a single cell, wherein each suspension height changer includes paramagnetic or superparamagnetic particles, and a linker that preferentially binds to a target cell type. In another example, a magnetic levitation kit may include a paramagnetic fluid medium and one or more magnetic agents, or a separation component of one or more magnetic agents, capable of forming a complex with a single cell, wherein each magnetic agent includes magnetic particles and a linker that preferentially binds to a target cell type. Also included in embodiments of the present invention are systems for cell separation. An exemplary system may include magnetic particles that are capable, alone or in combination with other agents, of preferentially binding to a first type of cells in a cell population comprising a plurality of cell types and forming a first complex of the particles and the first type of cells; a flow cell cartridge comprising a first outlet channel and a processing channel; a station comprising a retaining block for the flow cell cartridge and one or more magnets, the one or more magnets being positioned to expose the processing channel of the flow cell cartridge located in the retaining block to a magnetic field, wherein exposing the processing channel of the flow cell cartridge containing a suspension of cells of the plurality of cell types in a paramagnetic fluid medium to the magnetic field allows the first complex to be separated from cells of the plurality of cell types that are not bound to the first non-magnetic particles in the processing channel and from cells of other types of the plurality of cell types, and wherein the first complex is suspended in the processing channel of the flow cell cartridge below that of the plurality of cell types that are not complexed in the magnetic particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 is a schematic diagram of an exemplary magnetic levitation system.

[0007] Figure 2 is a schematic diagram of two views of an exemplary flow cell cartridge of a magnetic levitation system.

[0008] Figure 3 is a schematic diagram of a method according to an exemplary embodiment of the present invention.

[0009] Figure 4 is a schematic diagram of a method according to an exemplary embodiment of the present invention.

[0010] Figure 5 are photographic images showing separation of cells complexed with magnetic microbeads according to some embodiments described in the present disclosure.

[0011] Figure 6 is a dot plot showing the effect of antibody surface coverage on cell separation according to an exemplary embodiment of the present invention.

[0012] Figure 7 is a schematic diagram of the microparticle model complexed with the cell surface.

[0013] Figure 8 is a table showing the density calculation of the microparticle-cell complex, where the density of the microparticle is 1.063 g / cm 3 , the diameter of the cell is 11.5μm. DETAILED DESCRIPTION

[0014] Cells have inherent properties that determine their behavior under the action of magnetic levitation. Durmus et al. showed that the height at which cells are suspended in a paramagnetic fluid medium ("suspension curve") corresponds to the cell density, and that different cell types can be distinguished based on their characteristic magnetic levitation curves. See also co-pending, co-owned U.S. Patent Application No. 17 / 449,438, filed on September 29, 2021, which is incorporated herein by reference, describing methods and systems for separating cells by magnetic levitation, including methods for separating dead cells from living cells. Improved methods, devices, systems, and kits for separating particles (including cells) by magnetic levitation are described in the present disclosure.

[0015] The inventor has found a method for improving the separation of particles (such as cells) during magnetic suspension. The inventor has found that by combining cells with paramagnetic or superparamagnetic particles, when cells are suspended in a paramagnetic fluid medium in a flow cell barrel of a magnetic suspension system and exposed to a magnetic field, they can change the suspension height of the cells. The suspension height can be limited by the vertical position of the cells in the flow cell barrel of the magnetic suspension system. In other words, the inventor can control the suspension height of the cells during magnetic suspension by combining cells with paramagnetic or superparamagnetic particles. In one example, cells with cell-specific surface markers are combined with superparamagnetic particles connected with anti-surface marker antibodies. The resulting cell and superparamagnetic particle complex (each complex includes a cell with a cell-specific surface marker combined with one or more superparamagnetic particles) is suspended in a paramagnetic medium and magnetically suspended in the processing channel of the flow cell barrel of the magnetic suspension system.

[0016] In the above examples, the suspension height of the complex of cells and superparamagnetic particles is affected by the number of superparamagnetic particles in each complex. Depending on the number of superparamagnetic particles attached to each cell, the complex "falls" (i.e., is fixed to the bottom of the processing channel, resulting in "depletion" of the paramagnetic medium of the complex) or suspends in the processing channel, which may be because the magnetic force in the processing channel is insufficient to pull the complex to the bottom of the channel. In a series of experiments, the number of superparamagnetic particles per cell in the complex was changed by changing the ratio of particles to cells ("PTC ratio") during complex formation to about 1 to about 100,000. The inventors found that increasing the PTC ratio reduces the suspension height of the complex. When the PTC ratio is in the range of about 10,000 to 50,000, the complex falls to the bottom of the processing channel of the flow cell cartridge, effectively achieving depletion of the paramagnetic medium of the complex. When the PTC ratio is in the range of about 1 to 1,000, the suspension height of the complex in the processing channel is lower than that of the same cell not complexed with superparamagnetic particles, but does not fall to the bottom of the processing channel. In view of the above findings, the inventors have realized that in some cases, it may be beneficial to use smaller paramagnetic or superparamagnetic particles in order to obtain a higher concentration during complex formation, which may lead to an increase in the PTC ratio. The inventors have also found that in addition to the PTC ratio and the applied magnetic field strength, various other parameters can also affect the suspension height. Some of these parameters are the nature of the material contained in the paramagnetic or superparamagnetic particles (which can affect the magnetic susceptibility of the particles), the particle size and the particle density.

[0017] Therefore, by connecting paramagnetic or superparamagnetic particles to the surface marker characteristics of the cell type of interest found in the cell group containing multiple cell types, and selecting one or more parameters that affect the suspension height, the inventor can selectively change the suspension height of the cell belonging to the cell type of interest. As discussed in detail in the present disclosure, paramagnetic or superparamagnetic particles can be used to separate specific cells from a mixed cell group suspended in a paramagnetic medium by magnetic suspension, thereby producing a fraction (fraction) that is rich in or exhausted of the cell type of interest. These fractions can then be taken out from the flow cell of the magnetic suspension system to achieve the separation of the cell type of interest. As described in the present disclosure, paramagnetic or superparamagnetic particles can be used to separate various components of interest from various types of heterogeneous mixtures by magnetic suspension, such as separating organelles, nucleic acids or other molecules. Components of interest (such as cells, organelles, nucleic acids) are sometimes referred to as "analytes".

[0018] As also discussed in detail in the present disclosure, magnetic microparticles (which include nanoparticles, which may be ferromagnetic, ferrimagnetic, paramagnetic, or superparamagnetic) can be used to selectively deplete a mixed population of analytes (such as cells or other particles) suspended in a paramagnetic medium of a specific analyte (such as a cell type or other type of particle) by forming a complex of the magnetic particles and a specific analyte and allowing them to migrate and adhere to one or more sides of a processing channel of a magnetic levitation cartridge under the influence of magnetic forces during magnetic levitation. The paramagnetic fluid medium depleted of the analyte of interest can then be removed from the processing channel.

[0019] Thus, the present disclosure describes various embodiments of methods, devices, systems, and kits for separation of mixtures or particle groups including various types of particles based on magnetic suspension separation. The methods, devices, methods, and kits conceived by the inventors can be used for various applications. Some embodiments of the methods, devices, systems, and kits described in the present disclosure can be used to separate cells or cell subpopulations from heterogeneous mixtures or cell groups including various cell types based on magnetic suspension. Some other embodiments of the methods, devices, systems, and kits described in the present disclosure can be used to separate mixtures or groups of organelles or other cellular components (including intracellular and extracellular components, such as but not limited to endosomes or exosomes) based on magnetic suspension. Some other embodiments of the methods, devices, systems, and kits described in the present disclosure can be used to separate mixtures or groups of biomolecules or molecular complexes based on magnetic suspension, such as separating nucleic acids, such as separating nucleic acid libraries during next generation sequencing (NGS), or separating lipoproteins. Some embodiments of the methods, devices, systems, and kits described in the present disclosure can be used to separate mixtures based on magnetic suspension or to separate groups of cells that have been absorbed by endocytosed magnetic particles.

[0020] The methods, devices, systems and kits described in the present disclosure have various advantages over previously known separation methods based on magnetic levitation. Some of these advantages are improved separation accuracy and speed, improved reproducibility, and the ability to separate complex multi-component mixtures. Other advantages are the ability to suspend molecules that are difficult to suspend to a specific position during magnetic levitation, an example of which is RNA (such as RNA released from lysed cells), and the ability to separate cell types with very similar densities (thus, they cannot be separated by magnetic levitation themselves) by magnetic levitation. Terms and concepts

[0021] Some terms and concepts are discussed below. They are intended to help understand various embodiments of the present invention in conjunction with the rest of this document and the accompanying drawings. These terms and concepts can be further clarified and understood based on the conventions accepted in the field of the present invention and the descriptions provided by the entire present document and / or the accompanying drawings. Some other terms can be defined explicitly or implicitly in other parts of this document and the accompanying drawings, and can be used and understood based on the generally accepted practices in the field of the present invention and the descriptions provided by the entire present document and / or the accompanying drawings. Undefined terms can also be defined and understood based on the generally accepted practices in the field of the present invention and are explained in the context of this document and / or the accompanying drawings.

[0022] Unless the context dictates otherwise, singular terms shall include the plural, and plural terms shall include the singular. Generally, the terms and techniques associated with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry are well-known and commonly used. Unless otherwise indicated, known methods and techniques are generally performed according to well-known conventional methods, as described in various general and more specific references. The terms used in the laboratory procedures and techniques described in this disclosure are well-known and commonly used. other

[0023] As used in this disclosure, the terms "a," "an," and "the" may refer to one or more, unless expressly stated otherwise.

[0024] The use of the term "or" is used to mean "and / or" unless explicitly stated to refer to only alternatives, or the alternatives are mutually exclusive, although the present disclosure supports a definition referring to only alternatives and "and / or." As used in the present disclosure, "another" may mean at least a second or more.

[0025] As used in this disclosure, and unless otherwise indicated, the terms "include" or "including," and in some cases similar terms (such as "have" or "having"), mean "comprising."

[0026] When numerical ranges are provided in this disclosure, unless otherwise stated, the numerical ranges include the range endpoints. Unless otherwise stated, numerical ranges in this disclosure include all values ​​and subranges as if expressly written out.

[0027] As used in this disclosure, the terms "about" and "approximately" generally refer to an acceptable degree of error in a measured quantity for a given measured property or precision. Exemplary degrees of error are within 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a given value or range of values. For example, any reference to "about X" or "approximately X" expressly refers to at least value X, 0.9X, 0.91X, 0.92x, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. In another example, the term "about" or "approximately" associated with a reference value may include a range of values ​​of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Thus, the expression "about X" or "approximately X" is intended to describe a claim limitation such as "0.98X". Unless otherwise stated, the numerical values ​​given in this disclosure are approximate, meaning that the term "about" or "approximately" can be inferred when not explicitly stated. When the terms "about" or "approximately" are applied to the beginning of a numerical range, they apply to both ends of the range. When a series of values ​​begins with the terms "about" or "approximately", these terms are intended to modify each value contained in the series.

[0028] The terms "plurality" or "population", when used in connection with particles, such as but not limited to cells (e.g., as in "plurality of cells" or "population of cells"), refer to groups of particles (i.e., more than one particle) that include various numbers of particles. For example, a plurality of particles or a population of particles (e.g., cells) may include 2 or more, 10 or more, 100 or more, 500 or more, 10 or more, 150 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 100 or more, 1500 or more, 10 ... 3 or more, 10 4 or more, 10 5 or more, 10 6 or more, or 10 7 or more particles.

[0029] The terms "peptide", "polypeptide" or "protein" are used to refer to a polymer of amino acids linked by natural amide bonds and / or non-natural amide bonds. A peptide, polypeptide or protein may include moieties other than amino acids (e.g., lipids or sugars). A peptide, polypeptide or protein may be produced by synthetic or recombinant techniques.

[0030] The terms "oligonucleotide," "polynucleotide," or "nucleic acid" include DNA or RNA molecules, including molecules produced synthetically or by recombinant techniques. An oligonucleotide, polynucleotide, or nucleic acid may be single-stranded or double-stranded.

[0031] The term "small molecule" includes molecules (organic, organometallic or inorganic), organic molecules and inorganic molecules having a molecular weight greater than about 50Da and less than about 2500Da, respectively. Small organic (e.g.) molecules can be less than about 2000Da, between about 100Da to about 1000Da, or between about 100Da to about 600Da, or between about 200Da to about 500Da.

[0032] The term "paramagnetism" and related terms and expressions refer in this disclosure to materials and particles that have paramagnetic properties or paramagnetism. Paramagnetism is a form of magnetic characteristic of a material that has a weak attraction under the influence of an external magnetic field. In the absence of an external magnetic field, paramagnetic materials do not retain any magnetic properties. See, e.g., Britannica, editor of the Encyclopaedia, "Paramagnetism", Encyclopedia Britannica, December 20, 2006. In other words, paramagnetic materials have a low sensitivity ("magnetic susceptibility") to magnetic fields, but do not retain magnetism once the magnetic field is removed.

[0033] The term "superparamagnetism" and related terms and expressions refer to particles that show superparamagnetic properties or superparamagnetism in the present disclosure. Superparamagnetism is a phenomenon observed in small ferromagnetic or ferrimagnetic particles. If the size of these particles is small enough (within the nanoparticle size range), their magnetization direction can be randomly flipped under the influence of temperature. The time between two flips is called the Néel relaxation time. If the time used to measure the magnetization intensity of the particles is much longer than the Néel relaxation time and there is no external magnetic field, their average magnetization intensity appears to be zero, and they are said to be in a superparamagnetic state. See Pedro M. Enriquez-Navas and Maria L. Garcia-Martin "Application of Inorganic Nanoparticles for Diagnosis Based on MRI" Frontiers of Nanoscience 4: 233-245 (2012), doi.org / 10.1016 / B978-0-12-415769-9.00009-1. Superparamagnetic materials exhibit magnetic susceptibility in the presence of an externally applied magnetic field, but not in the absence of an applied magnetic field. Separation, Isolation and Concentration

[0034] As used in this disclosure, the term "concentration" refers to the amount of a first component contained within a second component and can be based on the number of particles per unit volume, the amount of moles per unit volume, the weight per unit volume, or based on the volume of the first component per volume of the combined components.

[0035] As used in the present disclosure, the terms "isolate", "separate", "separate", "purify" and their respective related terms and expressions can be used interchangeably. These terms can be used to refer to a method in which the amount of one or more components of interest present in a sample is increased relative to one or more other components. With respect to a particle or component (which can be a cell), these terms can refer to one or more of the following: separating such a component from other components, increasing the concentration of a component in a solution, or separating a component in a solution from other components. For example, a particle in a solution can be considered to be "separated" if it is separated from other particles in the solution and / or is located within a specified portion of the solution. In another example, if after processing the solution, the concentration of such a particle or component is increased in 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, then the particle or component in the solution is considered to be "separated". A particle of interest in a solution containing multiple types of particles can be considered "separated" if, after processing the solution, the ratio of the concentration of the particle of interest to the concentration of other types of particles increases, or if the ratio of the concentration of the particle of interest to the concentration of other types of particles increases by at least about 10%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, or if the concentration of other components of the solution (including but not limited to particle types other than the particle of interest) decreases to at least about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%. Suspension height changing agent; magnetic agent

[0036] The suspension height changing agent (i.e., an agent capable of changing the suspension height of a particle during magnetic suspension) comprises (i) magnetic (paramagnetic or superparamagnetic) microparticles and (ii) a linker that preferentially binds to a specified (e.g., first) type of particle (e.g., but not limited to a cell). The suspension height changing agent also preferentially binds to a specified (e.g., first) type of particle (e.g., but not limited to a cell) with the aid of the linker. Suspension height changing agents are sometimes referred to as "tags". The process of attaching a suspension height changing agent or magnetic particle to a cell (or other particle) is sometimes referred to as "labeling".

[0037] The inherent characteristic ability of a suspension height altering agent to change the suspension height of particles forming a complex with it under certain conditions may be referred to as the "suspension height altering property", "suspension height altering property" or other related terms and expressions of the suspension height altering agent. The suspension height altering property of the suspension height altering agent may be affected by factors such as the material contained in the magnetic particles of the suspension height altering agent, the particle size and the particle density.

[0038] Magnetic agents (i.e., agents that can adhere particles to one or more sides of a processing channel of a flow cell cartridge during magnetic levitation) include (i) magnetic (ferromagnetic, ferromagnetic, paramagnetic or superparamagnetic) microparticles and (ii) linkers that preferentially bind to a specified (e.g., first) type of particles (e.g., but not limited to cells). The magnetic agent, with the aid of the linker, also preferentially binds to a specified (e.g., first) type of particles (e.g., but not limited to cells). Magnetic agents and / or suspension height altering agents may be referred to as "tags" or "magnetic tags". The process of attaching one or more suspension height altering agents to cells (or other particles) is sometimes referred to as "labeling" or "magnetic labeling". It should be understood that in some cases, the terms "magnetic agent" and "suspension height altering agent" may be used to refer to agents containing the same type of magnetic microparticles, because various parameters discussed elsewhere in this disclosure affect the behavior of cells (or other particles) labeled with a particular agent during a separation process based on magnetic levitation (e.g., whether the complex will suspend lower than unlabeled cells or particles in the mixture, or whether it will "fall" from the mixture to the bottom of the processing channel of the flow cell cartridge). Magnetic particles

[0039] The terms "microparticles", "microspheres" and "microbeads" (which include "nanospheres", "nanospheres", "nanoparticles" and "nanobeads", respectively) in the context of the present disclosure refer to magnetic (including ferrimagnetic, ferromagnetic, paramagnetic and superparamagnetic) particles having one or more dimensions (such as length, width, diameter or circumference) of about 500 μm or less. Microparticles can have a generally spherical or non-spherical shape.

[0040] Microparticles (including ferrimagnetic, ferromagnetic, paramagnetic and superparamagnetic microparticles, including nanoparticles) used in embodiments of the present invention can have a range of sizes. For example, microparticles (which can be nanoparticles), such as superparamagnetic microparticles (which can be superparamagnetic nanoparticles) or paramagnetic microparticles (which can be paramagnetic nanoparticles), can have the following cross-sectional dimensions (e.g., diameter, length, width): about 500 μm or less, about 100 μm or less, about 50 μm or less, about 20 μm or less, about 10 μm or less, about 5 μm or less, about 1 μm (1000 nm) or less, about 0.5 μm (500 nm) or less, about 0.25 μm (250 nm) or less, about 0.1 μm (100 nm) or less, about 0.05 μm (50 nm) or less, or about 0.025 μm (100 nm) or less. 25nm) or less, such as a cross-sectional size range of about 500μm to about 0.0005μm (0.5nm), 500μm to about 0.001μm (1nm), about 500μm to about 0.01μm (10nm), about 500μm to about 0.025μm (25nm), about 500μm to about 0.05μm (50nm), about 500μm to about 0.1μm (100nm), about 500μm to about 0.25μm (250nm), about 500μm to about 0.5μm (500nm), about 500μm to about 1μm (1000nm), about 500μm to about 10μm, about 500μm to about 20 μm, about 500 μm to about 100 μm, about 100 μm to about 0.0005 μm (0.5 nm), about 100 μm to about 0.001 μm (1 nm), about 100 μm to about 0.01 μm (10 nm), about 100 μm to about 0.025 μm (25 nm), about 100 μm to about 0.05 μm (50 nm), about 100 μm to about 0.1 μm (100 nm), about 100 μm to about 0.25 μm (250 nm), about 100 μm to about 0.5 μm (500 nm), about 100 μm to about 1 μm (1000 nm), about 100 μm to about 10 μm, about 100 μm to about 20 μm, about 20 μm to about 0.0005 μm (0.5 nm), about 20 μm to about 0.001 μm (1 nm), about 20 μm to about 0.01 μm (10 nm), about 20 μm to about 0.025 μm (25 nm), about 20 μm to about 0.05 μm (50 nm), about 20 μm to about 0.1 μm (100 nm), about 20 μm to about 0.25 μm (250 nm), about 20 μm to about 0.5 μm (500 nm), about 20 μm to about 1 μm (200 nm), or about 20 μm to about 10 μm, about 10 μm to about 0.0005 μm (0.5 nm), about 10 μm to about 0.001 μm (1 nm), about 10 μm to about 0.01 μm (10 nm), about 10 μm to about 0.025 μm (25 nm), about 10 μm to about 0.05 μm (50 nm), about 10 μm to about 0.1 μm (100 nm), about 10 μm to about 0.25 μm (250 nm), about 10 μm to about 0.5 μm (500 nm), about 10 μm to about 1 μm (200 nm), about 5 μm to about 0.0005 μm (0.5 nm), about 5 μm to about 0.001 μm (1 nm), about 5 μm to about 0.01 μm (10 nm), about 5 μm to about 0.025 μm (25 nm), about 5 μm to about 0.05 μm (50 nm) ), about 5 μm to about 0.1 μm (100 nm), about 5 μm to about 0.25 μm (250 nm), about 5 μm to about 0.5 μm (500 nm), about 5 μm to about 1 μm (200 nm), about 1 μm to about 0.0005 μm (0.5 nm), about 1 μm to about 0.001 μm (1 nm), about 1 μm to about 0.01 μm (10 nm), about 1 μm to about 0.025 μm (25 nm), about 1 μm to about 0.05 μm (50 nm), about 1 μm to about 0.1 μm (100 nm), about 1 μm to about 0.25 μm (250 nm), about 1 μm to about 0.5 μm (500 nm), about 0.5 μm (500 nm) to about 0.0005 μm (0.5 nm), about 0.5 μm (500 nm) to about 0.001 μm (1 nm), about 0.5 μm (500 nm) to about 0.01 μm (10 nm), about 0.5 μm (500 nm) to about 0.025 μm (25 nm), about 0.5 μm (500 nm) to about 0.05 μm (50 nm), about 0.5 μm (500 nm) to about 0.1 μm (100 nm), about 0.5 μm (500 nm) to about 0.25 μm (250 nm), about 0.25 μm (250 nm) to about 0.0005 μm (0.5 nm), 0.25 μm (250 nm) to about 0.001 μm (1 nm), about 0.25 μm (250 nm) to about 0.01 μm (10 nm), about 0.25 μm (250 nm) to about 0.025 μm (25 nm), about 0.25 μm (250 nm) to about 0.05 μm (50 nm), about 0.25 μm (250 nm) to about 0.1 μm (100 nm), about 0.1 μm (100 nm) to about 0.0005 μm (0.5 nm), about 0.1 μm (100 nm) to about 0.001 μm (1 nm), about 0.1 μm (100 nm) to about 0.01 μm (10 nm), about 0.1 μm (100 nm) to about 0.025 μm (25 nm), about 0.1 μm (100 nm) to about 0.05 μm (5 nm), about 0.05 μm (5 nm) to about 0.0005 μm (0.5 nm), about 0.05 μm (50 nm) to about 0.001 μm (1 nm), about 0.05 μm (50 nm) to about 0.01 μm (10 nm), or about 0.05 μm (50 nm) to about 0.025 μm (25 nm).

[0041] Magnetic microparticles (including ferrimagnetic, ferrimagnetic, paramagnetic and superparamagnetic microparticles, including nanoparticles) used in embodiments of the present invention may be composed of any number of ferromagnetic, ferrimagnetic or paramagnetic materials or combinations thereof, or may include any number of ferromagnetic, ferrimagnetic or paramagnetic materials or combinations thereof, including but not limited to: metals, such as but not limited to iron, magnesium or molybdenum, metal salts, or metal oxides (e.g., iron oxide), suitable ceramics, and / or suitable composite materials, such as monodisperse nanoporous silica containing iron oxide particles within a porous silica network. An example of a magnetic microparticle used in embodiments of the present invention is a microparticle having a magnetic core (e.g., but not limited to a Fe2O3 core) and a polymer coating (e.g., the coating may be made of or include one or more of polystyrene, dextran, polyethylene glycol (PEG), polymethyl methacrylate (PMMA) or polyethylene). Some other examples of magnetic microparticles used in embodiments of the present invention are dextran-coated magnetic nanoparticles, gold-coated magnetic nanoparticles or silica-coated magnetic nanoparticles, such as those described in U.S. Pat. No. 7,169,618.

[0042] It will be appreciated that, according to the methods described in the present disclosure, the magnetic microparticles used in a single separation process may include multiple (two or more) types of microparticles having different suspension height changes and / or magnetic properties. In some exemplary embodiments, these different types of microparticles may be coupled to different linkers. For example, one (first) type of magnetic microparticle used in a separation process may be coupled to a linker for marking a first cell type (e.g., CD8+T cells), while a different (second) type of magnetic microparticle may be coupled to a linker for marking a second cell type (e.g., CD4+T cells). In some embodiments according to the present disclosure, suspension height changers comprising the same linker (or comprising linkers having the same specificity) will be combined with microparticles having the same suspension change or magnetic properties. Factors Affecting Microparticle Selection

[0043] The binding of the suspension height modifying agent or magnetic agent to the cell or other object and the behavior of the resulting complex during the magnetic suspension process depends on several interrelated variables, including, but not limited to, the density of the microparticles contained in the density modifier, the ratio of microparticles to cells during complex formation (PTC ratio discussed elsewhere in this disclosure), the size of the microparticles, the size of the cells or other objects being labeled, the ratio of the above sizes, the density of the linker on the surface of the microparticles, and the material of the microparticles. The above and other factors affect the selection of microparticles for a particular separation process and can be estimated using theoretical calculations, some of which will be discussed below.

[0044] In one example, the density of the cell complex is estimated as cells (m 细胞 ) and all bound particles (n*m MS ) divided by the total mass of the cell (V 细胞 ) and all bound beads (n*V MS )’s volume. To estimate the theoretical maximum number of microparticles that can bind to cells, microparticles and cells were modeled as hard spheres forming a monolayer of beads around the cell surface, e.g. Figure 7 The volume that a particle can occupy on the cell surface is calculated by dividing the volume by a diameter equal to the particle diameter (d MP ) plus twice the cell diameter (d 细胞 The diameter of the complex is determined by subtracting the cell volume from the spheroid volume. Figure 7 Indicated as d 复合物 . V 壳 =V 复合物 -V 细胞 In the above calculation, the volume of the shell around the cell was multiplied by the spheroid packing factor (random arrangement of equal spheres) of 0.64 and divided by the volume of a single microsphere to determine the number of microspheres that could bind to the cell. The packing factor assumes random packing of spheres. This is probably a conservative estimate of how many spheres there might be around a larger central sphere.

[0045] By determining as an upper limit the maximum number of microparticles that can be accommodated around a cell, estimates of the density of microparticle-cell complexes can be determined for beads of various sizes. Figure 8 The table shown in Figure 1 describes the theoretical density (g / cm2) of the microparticle (microparticles are of various sizes and have a wide range of particle diameters)-cell complexes. 3) and the number of particles bound to each cell. The density of the particles used in the calculations was assigned to be 1.18 g / cm 3 The density of the cells used in the calculations is 1.063 g / cm 3 , with a diameter of 11.5μm. Figure 8 In the table shown, the table cells highlighted in dark gray represent complexes with approximately 40% to 60% coverage of the cell surface by microparticles. The table cells highlighted in light gray represent complexes with approximately 80% to 100% coverage of the cell surface by microparticles. 3 The complexes are marked with a single or double asterisk, respectively. Figure 8 The particular calculation shown chose 1.13 g / cm 3 This value is used because it represents the cutoff density for the specific conditions of the exemplary magnetic levitation experiment. Complexes with densities below the cutoff value will be collected at the bottom of the processing channel of the flow cell cartridge and thus separated from complexes with densities above the cutoff value, which will float higher at the top of the processing channel of the flow cell cartridge. Other cutoff values ​​can be used depending on the specific experimental conditions and the desired results. Figure 8 The calculations shown in estimate that for larger particles, fewer particles are needed per cell to increase the density of complexes above the chosen cutoff, but there is also less space on the cell surface to accommodate these larger particles. Figure 8 Microparticles with diameters less than 3 μm will not form complexes at densities above the chosen cutoff, as this would require >100% coverage of the cell surface by microparticles, as determined by the calculations shown in . Microparticles with diameters of approximately 4-5 μm can achieve densities above the chosen cutoff and cover approximately 50% of the cell surface, based on the calculations shown.

[0046] Figure 8The calculations shown in estimate the number of particles per cell in the complex to achieve a specific density. Given that the binding kinetics of the suspension-height altering agent or magnetic agent are primarily driven by ligand-receptor binding interactions, a higher ratio of particles to cells (PTC ratios discussed elsewhere in this disclosure) is required during complex formation to achieve a target ratio of particles per cell in the complex. In order to balance binding mechanics, steric hindrance, and diffusion of particles through solution to interact with cells, the effective concentration of the linker contained in the suspension height altering agent or magnetic agent (which contains particles and linkers) needs to be maximized during complex formation, for example by selecting particles of smaller diameter so as to be able to increase the number of particles during complex formation. Another factor to consider is the product of the number of linker molecules bound to the surface of each particle and the total number of particles in the suspension. For example, particles with an average diameter of 150 nm, a streptavidin coverage of 50% per particle, and four binding sites per streptavidin molecule will provide 1×10 -14 μmol concentration of biotinylated antibody conjugated to streptavidin. For 2 × 10 5 For a sample of 10 cells, a 50,000:1 microparticle-to-cell ratio will produce an antibody concentration of approximately 1 μM, which is 10 to 100 times the dissociation constant (KD) of a typical antibody interaction (10-100 nm). In contrast, keeping all other conditions the same, a 40:1 microparticle-to-cell ratio and microparticles with an average diameter of 5 μm will still produce an antibody concentration of approximately 900 nm. However, in this case, the amount of antibody attached to each microparticle is approximately 1000 times greater. This limits the access of antibodies to cells during complex formation because all available antibodies are located in a smaller total number of microparticles. It can be expected that for higher affinity linkers, efficient complex formation requires lower PTC ratios because the interaction between the linker and the cell is more likely to persist. Lower affinity linkers may require higher PTC ratios to effectively form complexes in order to reach and maintain the target number of microparticles / cells in the complex. Production and sources of microparticles

[0047] Some methods for producing magnetic microparticles are described, for example, in Lu et al., "Modular and Integrated Systems for Nanoparticle and Microparticle Synthesis—A Review" Biosensors (2020) 10(11):165. Commercially available microparticles may also be used. Some exemplary sources of magnetic microparticles are: Nanopartz (Loveland, Colorado, USA), Biolegend (San Diego, California; e.g., MojoSort TMnanobeads), BD Biosciences (San Jose, CA; e.g., BD TM IMag nanoparticles), Thermo Fisher Scientific (Waltham, MA; e.g., ), Creative Diagnostics (New York, NY, USA), Spherotech (Lake Forest, IL, USA), Bangs Laboratories (Fischer, IN, USA), Miltenyi Biotec (Bergisch Gladbach, North Rhine-Westphalia, Germany; e.g., MicroBeads), Bio-Techne (Minneapolis, MN), Bioclone (San Diego, CA; e.g., BcMag strains), Polysciences (Warrington, PA, USA), and STEMCELL Technologies (Vancouver, Canada; e.g., RapidSphere TM micro strains). Connectors

[0048] "Linkers" are used to connect magnetic particles to components of interest (e.g., cells, organelles, nucleic acids of interest). Linkers specifically bind to cells or other analytes. Linkers can include one or more specific binding molecules, examples of which are discussed in more detail elsewhere in this disclosure. An example of a linker includes an antibody that specifically binds to a cell surface protein displayed on a cell of interest. Other types of linkers include aptamers, ligands (which bind through cell surface receptors) (including but not limited to small molecule ligands and polypeptide or protein ligands), lipophilic tags, and nucleic acids (at least a portion of which is complementary to the target nucleic acid). For example, mRNA can be removed from a sample by connecting oligo-dT to magnetic particles, which are then bound to the poly-A tail at the end of the mRNA. In another example, total RNA can be removed from a sample by connecting random hexameric oligonucleotides to magnetic particles, which are then bound to random RNA hexanucleotides. Antibody

[0049] The term "antibody" and related terms, in the broadest sense, are used in the present disclosure to refer to any product, composition or molecule comprising at least one epitope binding site, meaning a molecule that is capable of specifically binding to an "epitope" - a region or structure within an antigen. The term "antibody" includes any class of complete immunoglobulins (i.e., complete antibodies), including natural, natural-based, modified and non-natural (engineered) antibodies, and fragments thereof. The term "antibody" includes "polyclonal antibodies" (which react to the same antigen, but may bind to different epitopes within the antigen) and "monoclonal antibodies" ("mAbs") (substantially homogeneous antibody groups or antibodies obtained from substantially homogeneous antibody groups). The antigen binding sites of the individual antibodies that make up the mAb group are composed of polypeptide regions that are similar (although not necessarily identical) in sequence. The term "antibody" also includes fragments, variants, modified and engineered antibodies, such as those artificially produced ("engineered") antibodies by recombinant technology. For example, the term "antibody" includes, but is not limited to, chimeric and hybrid antibodies, antibodies with dual or multiple antigenic or epitope specificities, and fragments thereof, such as F(ab')2, Fab', Fab, hybrid fragments, single-chain variable fragments (scFv), "third generation" (3G) fragments, fusion proteins, single domain and "miniaturized" antibody molecules and "nanobodies". Aptamers

[0050] Nucleic acid aptamers are RNA or single-stranded DNA molecules that can fold into various structures and bind to a variety of targets, including other nucleotides or proteins. For example, aptamers for tumor cell surface markers (including HER-2, a breast cancer cell surface marker) can be used to separate or remove tumor cells from tissue samples. For example, aptamers targeting tumor cell surface protein biomarkers and the selection of these aptamers are discussed in Mercier et al., Cancers (Basel) 9(6):69(2017) doi:10.3390 / cancers9060069. Lipophilic tags

[0051] The linking agent can be a lipophilic tag. Lipophilic tags are lipophilic molecules that can bind to and / or insert into lipid membranes (such as cell membranes and organelle membranes). Examples of lipophilic molecules include sterol lipids (e.g., cholesterol or tocopherol), sterol lipids, lignoceric acid, and palmitic acid. Lipophilic tags themselves cannot achieve specific binding. However, they can be used to specifically target cell membranes in a mixture of cells and other particles. In addition, different samples can be labeled with differentially labeled lipophilic tags. Other specific binding partners

[0052] The expression "specific binding molecule" means a molecule that can specifically or selectively bind to another molecule or a region or structure within another molecule, which may be referred to as a "target", "ligand" or "binding partner". The term "specific binding", "selective binding" or related terms refers to a binding reaction in which a specific binding molecule or a composition containing it binds to its binding partner without binding to any other substance in large amounts under specified conditions. The binding to any other substance other than the binding partner is generally referred to as "non-specific binding" or "background". For example, the absence of a significant amount of binding is considered to be binding less than 1.5 times the background (i.e., non-specific binding level or slightly above the non-specific binding level). Some non-limiting examples of specific binding are antibody-antigen or antibody-epitope binding, oligonucleotide or polynucleotide binding to other oligonucleotides or polynucleotides, oligonucleotide or polynucleotide binding to proteins or polypeptides (and vice versa), protein or polypeptide binding to other proteins or polypeptides, receptor-ligand binding and carbohydrate-lectin binding. Therefore, a specific binding molecule may be or may include a protein, polypeptide, antibody, oligonucleotide or polynucleotide, receptor or ligand. Specific binding molecules can be natural or engineered. For example, in embodiments of the present invention, engineered and naturally occurring nucleic acid or peptide aptamers can be used as specific binding molecules. This list is not restrictive, and other types of specific binding molecules can also be used. The term "target molecule" is used to represent a molecule or a portion thereof, including a biological molecule (such as but not limited to a protein, peptide, lipid, nucleic acid, fatty acid or carbohydrate molecule (such as oligosaccharide)) or a non-biological molecule (including small molecules, such as small molecule drugs or small molecule ligands). Specific binding molecules, such as antibodies, specifically bind to target molecules. Indirect combination

[0053] Specific binding molecules, such as but not limited to antibodies, can be directly attached to magnetic particles, such as by surface conjugation, coating or adsorption. However, specific binding molecules do not have to be directly attached to magnetic particles, and can be used to make magnetic particles and target cells complex through intermediate non-covalent binding interactions. For example, the specific binding molecule is a biotinylated antibody that can specifically bind to target cells, and the density-adjusting particles are coated with avidin, streptavidin, neutravidin or any form of modified avidin, which can be referred to as "avidin-like compounds". The intermediate binding interaction between the avidin-like compound on the magnetic particles and the biotin portion of the antibody allows the formation of a complex between the target cell and the density-adjusting particles. In another example, the specific binding molecule is an antibody ("primary antibody") that can specifically bind to target cells, and the magnetic particles are coated with protein A, protein S or anti-antibody (i.e., an antibody against a primary antibody). The intermediate binding interaction between protein A, protein S or anti-antibody on the magnetic particles and the primary antibody allows the formation of a complex between the target cell and the magnetic particles. Fluidics

[0054] As used in this disclosure, the term "fluidics" refers to a system, device or element for handling, processing, ejecting and / or analyzing a fluid sample that includes at least one "channel" as defined elsewhere in this disclosure. The term "fluidics" includes, but is not limited to, microfluidics and nanofluidics.

[0055] As used in the present disclosure, the terms "channel", "flow channel" and "fluidic channel" are used interchangeably and refer to a path on a fluidic device in which fluidic can flow. The channel includes a path with a maximum height dimension of about 100mM, about 50mM, about 30mM, about 25mM, about 20mM, about 15mM, about 10mM, about 5mM, about 3mM, about 2mM, about 1mM or about 0.5mM. For example, the channel between the magnets may have the following cross-sectional dimensions (width by height): about 10mM×10mM, about 10mM×5mM, about 10mM×3mM, about 10mM×2mM, about 10mM×1mM, or about 10mM×0.5mM, about 5mM×10mM, about 5mM×5mM, about 5mM×3mM, about 5mM×2mM, about 5mM×1mM, about 5mM×0.5mM, about 3mM×10mM, about 3mM×5mM, about 3mM×3mM, about 3mM×2mM, about 3mM×1mM , about 3mM×0.5mM, about 2mM×10mM, about 2mM×5mM, about 2mM×3mM, about 2mM×2mM, about 2mM×1mM, about 2mM×0.5mM, about 1mM×10mM, about 1mM×5mM, about 1mM×3mM, about 1mM×2mM, about 1mM×1mM, about 1mM×0.5mM, about 0.5mM×10mM, about 0.5mM×5mM, about 0.5mM×3mM, about 0.5mM×2mM, about 0.5mM×1mM or about 0.5mM×0.5mM. The internal height of the channel can be inconsistent in its cross section, and the geometry of the cross section can be any shape, including circular, square, oval, rectangular or hexagonal. The cross section can vary along the length of the channel. The term "channel" includes, but is not limited to, microchannels and nanochannels, and with respect to any reference to a channel in this disclosure, such channel may include a microchannel or a nanochannel.

[0056] As used in this disclosure, the term "fluidically coupled" or "fluidically connected" means that a fluid can flow between two components so coupled or connected. magnetic levitation

[0057] The expression "magnetic levitation" in the context of the present disclosure and, for example, as described in U.S. Patent Application No. US14 / 407,736, generally refers to subjecting a diamagnetic, paramagnetic, ferromagnetic, ferrimagnetic or antiferromagnetic material suspended in a paramagnetic fluid medium or an "object" suspended in a paramagnetic fluid medium to a magnetic field, such as a magnetic field gradient formed between two magnets. The magnetic field produces an uneven pressure, which is equivalent to the magnetic energy density in the paramagnetic fluid medium. In the magnetic field gradient, the object appears to be repelled by the high magnetic field area. In fact, the object is replaced by an equal amount of paramagnetic fluid medium. The attractive interaction between the paramagnetic fluid medium and the high magnetic field area can lead to the "levitation" of the object. In a dual magnet setting, the "levitation height" of the object can be defined as desired. For example, in some embodiments, the "levitation height" can be defined as the distance between the center of the suspended object and the top surface of the bottom magnet, but any desired reference point can be used. By applying a magnetic field in such a way that the force on the object is offset by another uniform force (e.g., gravity), a balance of the object is achieved, which is directly related to its density. Paramagnetic fluid medium

[0058] In the context of the present disclosure, "paramagnetic fluid medium" may include paramagnetic materials and solvents. In some embodiments of the methods described in the present disclosure, the paramagnetic fluid medium is biocompatible, i.e., it can be mixed with living cells and does not affect the viability of the cells or affect cell behavior. The paramagnetic material may include one or more of the following: gadolinium, titanium, vanadium, dysprosium, chromium, manganese, iron, nickel, gallium, including their ions. For example, the paramagnetic material may include one or more of the following ions: titanium (III) ions, gadolinium (III) ions, vanadium (I) ions, nickel (II) ions, chromium (III) ions, vanadium (III) ions, dysprosium (III) ions, cobalt (II) ions, and gallium (III) ions. In some embodiments, the paramagnetic material comprises 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 may include one or more of the following: [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][FeCl4]. In an exemplary embodiment, the paramagnetic material is gadobutrol. The paramagnetic material can 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 an exemplary embodiment, the paramagnetic material comprises gadolinium and the paramagnetic material is present in the paramagnetic fluid medium at a concentration of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, about 10 mM to about 50 mM, about 25 mM to about 75 mM, about 50 mM to about 100 mM, about 50 mM to about 200 mM, about 50 mM to about 300 mM, about 50 mM to about 400 mM, about 50 mM to about 500 mM, about 50 mM to about 600 mM, about 50 mM to about 700 mM, about 50 mM to about 800 mM, about 50 mM to about 900 mM, or about 50 mM to about 1 M. It should be understood that in addition to the paramagnetic material and the solvent, the paramagnetic fluid medium may contain other ingredients such as salts or additives, such as, but not limited to, additives for maintaining cell integrity. Magnetic levitation systems and components

[0059] For example, exemplary magnetic levitation systems and components thereof are described in Durmus et al. and U.S. Patent Application No. 17 / 449,438 filed on September 29, 2021. Although the various embodiments of the invention provided in this disclosure are not limited to any particular magnetic levitation system, a brief description is included to facilitate understanding of the methods, kits, and systems according to embodiments of the invention. Figure 1 An exemplary magnetic levitation system is schematically shown. Flow cell cartridge

[0060] At least some methods and kits according to embodiments of the present invention relate to flow cell cartridges for use in magnetic levitation systems. Figure 2 An exemplary flow cell cartridge is schematically shown in . An exemplary flow cell cartridge may include a planar substrate including an upper surface and a lower surface; a first longitudinal side forming an imaging surface; a second longitudinal side forming an illumination surface; and first and second lateral sides; an inlet hole on the upper surface; an inlet channel; a sample processing channel, which is fluidically connected to the inlet channel and positioned substantially parallel to the longitudinal side; a sample separator, which is located in the processing channel; a plurality of outlet channels, which are fluidically connected to the processing channel; and a plurality of collection holes, which are fluidically connected to each of the plurality of outlet channels. The planar configuration allows all required flow cell cartridge functions to be integrated into the flow cell cartridge and improves performance and repeatability in a laboratory or clinical setting. In operation, it is important to make the processing channel and the flow into the outlet channel as turbulent as possible for improved performance. The processing channel can be offset within the plane of the planar substrate to spatially bias the imaging surface.

[0061] The flow cell cartridge can be formed by injection molding, etching, laser ablation, machining or 3D printing. When imaging is required in the flow cell cartridge, the planar substrate includes an optically transparent material. Glass, plastic or polymer materials (including cycloolefin polymers (COP) or cycloolefin copolymers (COC)) are some examples of suitable optically transparent materials. The size of the planar substrate can be at least 50mM in length, 20mM in width, and at least 1.5mM in thickness. The optional range is at least 100mM in length, at least 35mM in width, and at least about 2 to about 6mM in thickness. The longitudinal side of the barrel can be used as a waveguide for illumination and imaging. For this reason, the processing channel is offset in the plane of the substrate, and is parallel and adjacent to the imaging longitudinal side of the substrate. The distance from the imaging side wall can be about 0.5mM to about 10mM, preferably about 0.5mM to about 5mM, and optionally about 1mM to about 3.5mM. In one embodiment, the processing channel is spaced about 2mM from the imaging wall. The volume of the processing channel can be about 10 μL to about 800 μL, about 50 μL to about 600 μL, 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. The total volume of the outlet channel can be greater than the volume of the processing channel. When operating in a system embodiment, the flow distribution between the two outlet channels can be uniform, or can be in the range of about 4:1 to about 1:4, about 3:1 to about 1:3, or about 2:1 to about 1:2, or can differ from 1:1 by about 50% or less, or about 40% or less, or about 30% or less, or about 15% or less.

[0062] The flow cell cartridge optionally includes a collection hole on a planar substrate. The collection hole is characterized by having an inlet that is fluidically connected to the outlet channel. The inlet can be located at the first hole height and is configured to have a step that transitions from the inlet hole to the bottom of the hole. This provides a transition surface for the sample fraction to flow into the hole, and can inhibit the siphoning back of the sample fraction into the outlet channel and the formation of bubbles in the collection hole. The outlet channel in the collection hole can be provided with an opening that is located at a height of the bottom plate leaving the collection hole, which is higher than the opening of the inlet channel. The internal outlet can be connected to a flow regulator. In some cases, the flow regulator is a separate pump to provide a flow through the flow cell cartridge. In operation, the collection hole is sealed with a layer of material or film to provide a closed system, thereby allowing the sample and sample fraction to flow through or be pumped through the flow cell cartridge. When assembling the flow cell cartridge layers, if an adhesive is used, a biocompatible adhesive can be used for biological applications. Correct adhesive selection is necessary to minimize or prevent adhesive components from leaching into solution, adhering to cells or binding molecules in solution, being autofluorescent, having a texture that increases surface area and therefore impact on cells, and being overly hydrophilic or hydrophobic. Examples of suitable adhesives are silicone or silicone-based adhesives. Separation system

[0063] Exemplary magnetic levitation-based separation systems (e.g. Figure 1 ) includes a receiving block for holding a flow cell cartridge; an optical system including an optical sensor, a lens, and an illumination source; and a plurality of flow regulating components. The receiving block removably places the flow cell cartridge in optical alignment with the optical system, removably engages with a magnetic component near a processing channel of the flow cell cartridge, and removably places a plurality of outlet channels of the flow cell cartridge in fluidic communication with a plurality of flow regulating components. The optical system can be configured to provide microscopic imaging of the processing channel of the flow cell cartridge. Optionally, the optical system can be constructed and arranged to provide imaging for fluorescence emission using an optional ultraviolet light exciter module. The optical system may include a visible light illumination source, which is constructed and arranged to provide light transmission through the processing channel within the planar substrate. The receiving block is constructed and arranged to hold the planar flow cell cartridge in an orientation relative to the optical system so that the imaging optics are aligned with the imaging side of the planar cartridge, and the visible light emitter is in a direction that illuminates the illumination side of the planar flow cell cartridge. Optionally, the optical system may further include one or more ultraviolet or visible illumination sources constructed and arranged to direct illumination at an angle to the imaging side of the planar barrel to excite fluorophores within the processing channel of the barrel. For imaging fluorescent entities within the processing channel, the optical system may optionally include a dual bandpass filter that preferably passes emission radiation in a band centered around wavelengths of approximately 524 nm and 628 nm.

[0064] An optional feature of the receiving block is a series of flow regulator adapters connected to the outlet on the top or bottom of the flow cell cartridge. The adapter helps to communicate with the flow regulator (such as the pump in the system) flow control, and is in fluid communication with the outlet channel (such as the collecting hole outlet channel) of the flow cell. Once the flow cell cartridge is inserted into the receiving block, the receiving block is mechanically actuated to support the cartridge, the illumination and imaging side of the planar cartridge are aligned with the optical imaging system, the magnetic components are aligned to position them above and below the flow cartridge processing channel, and when necessary, the flow regulator adapter is placed in communication with the corresponding outlet channel flow control of the flow cell cartridge. The flow regulator of the system provides flow to the sample and sample fraction in the flow cell cartridge. In the separation process, the flow rate range provided by the flow regulator can be increased from as low as 1 μ L per minute to as high as 1 mL per minute. The flow rate may be equal to or at least about 25 μL / min, equal to or at least about 50 μL / min, equal to or at least about 100 μL / min, equal to or at least about 200 μL / min, equal to or at least about 250 μL / min, equal to or at least about 300 μL / min, or about 300 μL / min to about 1 mL / min. The total sample volume flow rate may be about 50 μL / min, about 75 μL / min, about 100 μL / min, about 150 μL / min, about 200 μL / min, or about 300 μL / min. When operating in a system embodiment, the flow distribution between the two outlet channels may be uniform, or may be in the range of about 4:1 to about 1:4, about 3:1 to about 1:3, or about 2:1 to about 1:2, or may differ from 1:1 by about 50% or less, or about 40% or less, or about 30% or less, or about 15% or less.

[0065] The magnetic suspension system enables particles suspended in a paramagnetic fluid medium to be magnetically suspended in a processing channel or an inlet channel of a flow cell cartridge. The paramagnetic interaction of the magnetic field with the particles in the paramagnetic fluid medium can produce a repulsive or attractive effect on the particles to promote their separation or concentration. 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 range from about 1 mega-Gauss oersted to about 1000 mega-Gauss oersted, or from about 10 mega-Gauss oersted to about 100 mega-Gauss oersted. The surface field strength of the magnet can range from about 0.01 Tesla to about 100 Tesla, or from about 1 Tesla to about 10 Tesla. The remanence range of the magnet can range from about 0.5 Tesla to about 5 Tesla, or from about 1 Tesla to about 3 Tesla. The magnet may be made of a material including a neodymium alloy of iron and boron, neodymium, an alloy of aluminum and nickel, an alloy of neodymium and iron, an alloy of aluminum and cobalt and iron, samarium-cobalt, an alloy of other rare earth elements and iron, an alloy of rare earth elements and nickel, ferrites, or a combination thereof. When the magnetic levitation system includes a plurality of magnets, the magnets may be made of the same material or of different materials.

[0066] An asymmetric magnetic field can be obtained by using a stronger magnetic material on one side of the flow channel channel of the flow cell cartridge and a weaker magnetic material on the opposite side of the flow control channel of the flow cell cartridge. An asymmetric magnetic field can be obtained by positioning the magnet closer on one side of the channel than on the other side. An asymmetric magnetic field can be obtained by using a magnetic material on one side of the flow control channel of the flow cell cartridge and using a substantially similar magnetic material on the opposite side of the flow control channel of the flow cell cartridge. The upper magnet and the lower magnet can have substantially the same size. In one example, the upper magnet can include neodymium and the lower magnet can include samarium-cobalt. Alternatively, the upper magnet can include samarium-cobalt and the lower magnet can include neodymium. Alternative magnet configurations can be used. The magnetic suspension system can include a plurality of upper magnets and a plurality of lower magnets, which are positioned around the flow channel channel of the flow cell cartridge. In one example, the upper magnet can include a front upper magnet, a center upper magnet, and a rear upper magnet. The lower magnet can include a front lower magnet, a center lower magnet, and a rear lower magnet. In another example, the magnetic levitation system can include a front upper magnet, a rear upper magnet, a front lower magnet, and a rear lower magnet, wherein the magnets are positioned around the flow channel channel of the flow cell cartridge, and the front upper magnet and the rear lower magnet are positioned in a magnetic repulsion direction. Exemplary NdFeB magnetic component dimensions include: the bottom magnet component is about 50×15×2mM (magnetized by a 15mM axis), and the top magnet component is about 50×5×2mM (magnetized by a 5mM axis). Other exemplary magnet sizes include 60×15×2mM, 60×5×2mM, 75×10×3mM, 75×20×3mM, and 25×15×2mM. An exemplary magnet configuration for a magnetic suspension system includes an upper magnet and a lower magnet having a size of about 75×20×3.2 mM, and the spacing between the upper magnet and the lower magnet is 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. Another exemplary magnet configuration for a magnetic suspension system has an upper magnet and a lower magnet, wherein the lower magnet extends into the inlet channel of the flow cell cartridge. The bottom magnet size can be about (50 mM to about 100 mM)×(about 10 mM to about 30 mM)×(about 2 mM to about 4 mM). sample

[0067] Unless otherwise limited, the term "sample (sample or samples)" and related terms and expressions as used in the present disclosure are not intended to be limiting. These terms refer to any product, composition, cell, tissue or organism. Generally, the term "sample" is not intended to be limited by its source, origin, procurement method, treatment, processing, storage or analysis method or any modification. Some examples of samples are solutions, suspensions, supernatants, precipitates or particles. The sample can contain cells or tissues or be mainly composed of cells or tissues, or can be prepared by cells or tissues. However, the sample does not necessarily contain cells. The sample can be a mixture of biomolecules or contain biomolecules, such as nucleic acids, polypeptides, proteins (including antibodies), lipids, carbohydrates, etc. The sample can be a biological sample. For example, a "sample" can be any cell or tissue sample or extract derived from a cell, tissue or subject, and includes samples of animal cells or tissues and cells of non-animal origin (including plant and bacterial samples). The sample can be obtained directly from an organism, or bred, or cultured. Some exemplary samples are cell extracts (e.g., cell lysates), cell nuclear suspensions, liquid cell cultures, cell suspensions, biological fluids (including but not limited to blood, serum, plasma, saliva, urine, cerebrospinal fluid, amniotic fluid, tears, lavage fluid from the lungs, or interstitial fluid), tissue sections, including needle biopsies, microscopic sections, frozen tissue sections, or fixed cell and tissue samples. Exemplary cell types

[0068] Cells labeled with suspension height changes and / or magnetic agents can be, but are not limited to, human: human cells, non-human animal cells, plant cells, eukaryotic cells, prokaryotic cells, etc. For example, labeled cells can be, but are not limited to, human or non-human immune cells, endothelial cells, and T cells. Labeled cells can be in a heterogeneous population of unlabeled cells, which can be, for example, but not limited to: human cells, non-human animal cells, plant cells, eukaryotic cells, prokaryotic cells, etc. Labeled cells can be from different cell lineages, or can be the same cell lineage, but different in activation state, differentiation state, or some other characteristics. Labeling cells

[0069] One or more (2,3,4 or more than 4) cell types can be processed in a single separation step.When marking two or more cell types, each cell type can be marked with different suspension height changes and / or magnetic agents.Alternately, two or more different cell types can be marked with the same suspension height change and / or magnetic agent.For example, two or more different cell types can have the same surface marker, so the suspension height change and / or magnetic agent comprising the same linking agent can be combined with two cell types.In another example, the suspension height change and / or magnetic agent include two or more different linking agents, which can be combined with corresponding two or more surface markers.In this case, the suspension height change and / or magnetic agent can be combined with two or more different cell types, and these cell types have different surface markers corresponding to two or more different linking agents.In some embodiments, two or more different cell types can be marked with different suspension height change agents with the same suspension height change characteristic. Marking process

[0070] As part of the process of labeling cells, magnetic particles containing a linker specific to the target cell type (which together can be referred to as a "suspension height changing agent" or "magnetic agent") are mixed with the cells. The ratio of magnetic particles to cells in the mixture (PTC ratio, discussed elsewhere in this disclosure) is optimized based on various parameters. One of these parameters can be the target cell type, which determines its suspension curve, but can also affect the number of suspension height changes and / or magnetic agent units that each cell may be complexed with. For example, if the linker is specific to a cell surface marker, the number of markers on a specific target cell type will determine how many units of suspension height change and / or magnetic agent can bind to the cell. Another parameter can be the affinity of the linker (such as an antibody) for a specific cell type. Another parameter is the strength of the magnetic field applied during the magnetic suspension process. Other parameters that can be considered when determining the PTC ratio can be the magnetic susceptibility, particle size, and / or particle density of the particles contained in the suspension height change and / or magnetic agent. It should be understood that other parameters not listed here can also be considered, and the PTC ratio can be experimentally determined and / or optimized for specific labeling and / or separation applications. Non-limiting PTC ratios used in embodiments of the methods described in the present disclosure can be, but are not limited to: about 1 to about 100,000, about 1 to about 50,000, about 1 to about 10,000, about 1 to about 1,000, about 1 to about 100, about 10,000 to about 100, about 10,000 to about 100,000 to about 50,000, or about 50,000 to about 100,000. When multiple cell types are present, magnetic microparticles with different specific linkers for each cell type can be mixed together before adding to the cell mixture, or magnetic microparticles and cells with different linkers can be mixed together in one step. In some cases, the labeling process can be performed continuously on the cell mixture, meaning that microparticles with different linkers specific for each cell type can be applied after each labeling and separation step. Cell separation methods

[0071] Improved methods for separating cells by magnetic levitation are described in the present disclosure and are also included in embodiments of the present invention. These methods may also be referred to as "cell separation methods (cell separation methods or methods of separating cells or methods of cell isolation or cell isolation methods or methods of isolating cells or cell segregation methods or methods of segregating cells)", "cell concentration methods (cell concentration methods or methods of concentrating cells)", and other related terms and expressions, but are not intended to be limiting.

[0072] The method described in the present disclosure can be used to separate one or more types of cells from a cell group including a variety of cell types. Such a variety of cell types can include animal cells, including human cells and non-human animal cells, mixtures of human cells and non-human cells, plant cells, and cells from other sources, including but not limited to bacterial cells, protozoan cells, algae cells, etc. A variety of cell types may include dead cells, living cells, healthy cells, pathological cells, infected cells, transfected cells or genetically modified cells. Cells separated according to the method disclosed herein may include cells in various states (e.g., stem cells, differentiated cells, etc.). Cells separated according to the method disclosed herein may be obtained directly from an organism (or an organism itself), or proliferated or cultured. Before separating cells according to the method described in the present disclosure, cells may be subjected to various treatments, storage or processing procedures. Generally, the terms "cell (cell or cells)", "cell type" (or related terms and expressions) are not intended to be limited by their source, origin, procurement method, treatment, processing, storage or analysis method or any modification. Some non-limiting examples of cell types that may be suitable for separation by the methods described in the present disclosure are macrophages, alveolar type II (ATII) cells, stem cells, adipocytes, cardiomyocytes, embryonic cells, tumor cells, lymphocytes, red blood cells (red blood cell or erythrocyte), epithelial cells, ova (ova or egg cell), sperm cells, T cells, B cells, bone marrow cells, immune cells, hepatocytes, endothelial cells, stromal cells and bacterial cells. Cell populations including multiple cell types can be derived from various types of samples, which will be discussed elsewhere in the present disclosure.

[0073] The cell separation method according to some embodiments of the present invention involves changing one or more suspension heights and / or binding of magnetic agents to specific types of cells (which may be referred to as "target cells" or "target cell types") found in a cell population comprising a variety of cell types. This combination may also be described as "forming a complex", "complex formation" or "compound". The preferential binding of suspension height changes and / or magnetic agents to target cells is achieved by using a linker comprising a binding molecule that can specifically or selectively bind to target cells. This molecule may be referred to as a "specific binding molecule". Examples of specific binding molecules are specific antibodies for cell surface markers or specific molecules for target cell types. Some examples of surface markers are CD45, CD3, CD4, CD8, CD19, CD40, CD56, CD11b, CD14, CD15, EpCAM, ICAM, CD235, HER-2, HER-3, CD66e, integrin, EPL-selectin, EGFR, EGFRVIII, PDGFRβ, c-MET, MUC-1, OX-40, CD28, CD133, CD30TNFRSF8, CTLA4, CD71, CD16αVCAM-1, nucleolin and myelin basic protein. It should be understood that different cells can have different surface markers. For example, human and non-human animal cells, such as mouse cells, can have different surface markers. The embodiment of the cell separation method of the present invention can utilize more than one (one or more, two or more, three or more, four or more, etc.) specific binding molecules. In other words, the embodiment of the cell separation method of the present invention can utilize a variety of specific binding molecules that can form complexes with different target cell types in a cell population containing a variety of cell types.

[0074] In the embodiment of the cell separation method that relates to suspension height change and / or magnetic agent is combined with one or more specific types of cells, combination can be realized by various steps.For example, under the condition of magnetic (for example, paramagnetism or superparamagnetism) microparticle combined with single cell of target cell type to form complex, by contacting, combining or incubating suspension height change agent to realize combination, the suspension height change agent comprises the linker containing specific binding molecule, the specific binding molecule and the cell group containing multiple cell types (may include target cell type), and each complex is combined with one or more microparticles on the cell of target cell type.However, the embodiment of the cell separation method of the present invention does not necessarily include any step related to suspension height change and / or magnetic agent combined with cell.Suspension height change and / or magnetic agent and target cell complex can be formed before the method starts, and can be provided at the beginning of the cell separation method according to the embodiment of the present invention.In other words, the method can start from the step of providing suspension height change and / or magnetic agent and target cell type (or target cell) complex, and the cell is optionally included in the cell group comprising multiple cell types.

[0075] Some embodiments of the cell separation method according to the present invention include one or more steps of forming a suspension of a complex of a plurality of cells of a plurality of cells of a plurality of suspension height changes and / or magnetic agents and target cell types in a paramagnetic fluid medium. In some embodiments, such a suspension can be provided at the beginning of the method. The cell separation method according to an embodiment of the present invention relates to introducing a suspension into a processing channel of a flow cell cartridge of a magnetic levitation system. The flow cell cartridge includes at least one outlet channel and a processing channel with a length and a vertical height. The cell separation method according to an embodiment of the present invention relates to exposing the processing channel to a magnetic field for a period of time, which is sufficient to make at least some complexes (or at least one complex) of one or more suspension height changes and / or magnetic agents and cells of a target cell type, and cells of a plurality of cell types that are not combined with one or more suspension height changes and / or magnetic agents are separated, thereby forming a first part of a suspension rich in complexes relative to the suspension, and a second part of a suspension depleted of complexes relative to the suspension. Exposure to a magnetic field can be carried out in a stop flow mode or a continuous flow mode of a flow cell cartridge.

[0076] In some embodiments of the cell separation method, the vertical position of the flow cell tube in the magnetic field is variable, which may affect the suspension height of the compound (or at least one compound) of one or more suspension height changers and target cell types relative to the magnet of the magnetic suspension system. Changing the vertical position of the flow cell tube can be advantageously used to approach different parts of the suspension. In some embodiments of the cell separation method, the composition of the paramagnetic fluid can be adjusted to improve cell separation. For example, the concentration of paramagnetic compounds can change the physical space occupied by a certain density range, so that the specific density range in the separation channel can be targeted by adjusting the concentration of paramagnetic compounds. Along with the increase of paramagnetic compound concentration in the paramagnetic fluid, the particle density range that can be suspended between the magnets becomes wider. However, if the magnet spacing is not adjusted in this case, the physical separation distance between particles of different densities becomes smaller. On the contrary, along with the reduction of paramagnetic compound concentration in the paramagnetic fluid, the particle density range that can be suspended between the magnets narrows, but the physical separation distance between particles of different densities increases. Therefore, in the cell separation method according to an embodiment of the present invention, the concentration of the paramagnetic compound in the paramagnetic fluid and / or the magnet spacing in the magnetic suspension system can be adjusted to optimize the purity and / or yield of the separation product (particles of interest).

[0077] In an embodiment of a cell separation method utilizing multiple suspension height changes and / or magnetic agents having different suspension change characteristics, the suspension height changes and / or magnetic agents are capable of forming complexes with different target cell types in a cell population containing multiple cell types, and after exposure to a magnetic field, more than two parts of the suspension can be formed. For example, if a method utilizes two different suspension height changers that are capable of binding to two different target cell types in a cell population containing multiple cell types, two different types of complexes can be formed in the suspension when exposed to a magnetic field: a first complex of a first suspension height changer and a single cell of the first target type, and a second complex of a second suspension height changer and a single cell of the second target type. In this case, the first suspension height changer and the second suspension height changer can be selected so that the suspension height of the first complex is different from the suspension height of the second complex, and is also different from the suspension height of other types of cells in the group. In one example, when exposed to a magnetic field, at least three different parts of the suspension will be formed in the processing channel of the flow cell cartridge: a part enriched in the first complex, a part enriched in the second complex, and a part (or parts) depleted of the first complex and the second complex. In this case, the first and second portions (which may be referred to as "fractions") may require the same or different lengths of exposure to the magnetic field to form. In another example, upon exposure to the magnetic field, the first complex may "fall" from the suspension to the bottom of a processing channel of a flow cell cartridge, and two different portions of the suspension may then form in the processing channel - a portion enriched in the second complex, and a portion depleted in the first complex and the second complex.

[0078] The cell separation method according to an embodiment of the present invention may also include taking out different parts of the suspension from the processing channel of the flow cell cartridge or from the flow cell cartridge together. The taking out of different parts or fractions can be carried out through one or more outlet channels of the flow cell cartridge, and may include flowing the suspension along the length of the processing channel.

[0079] exist Figure 3 An exemplary embodiment of a cell separation method is schematically shown in FIG. Figure 3The embodiment shown in is an example of a cell separation method using magnetic particles with suspension-changing properties. The magnetic particles (which may be paramagnetic or superparamagnetic) are contained in a suspension height changer (4), which is capable of forming a complex with a first cell type (1), wherein the suspension height of the complex (3) is lower than that of the uncomplexed first type (1) cells. The suspension height changer allows the separation of a portion of a sample that is rich in the complex (3) or depleted of the complex, thereby allowing the separation of cells of the first type from a cell population containing multiple cell types. The exemplary cell population comprises at least two different cell types, a first type (1) and a second type (2), which have different surface markers (e.g., proteins, carbohydrates, or other biomolecules). When performing the exemplary method, the exemplary cell population is contacted with an exemplary suspension height changer (4), which comprises a paramagnetic or superparamagnetic particle conjugated to an antibody, the antibody being capable of specifically binding to a surface marker of the first type (1) of the two cell types. The particles bind to the surface marker of the first cell type (1) and form a complex with the cells of the first type (1). When subjected to magnetic levitation, the complexes are suspended lower in the processing channel of the flow cell cartridge than cells of the second type (2) that do not form complexes with the microparticles. A complex-enriched fraction (Fraction I) or a complex-depleted fraction (Fraction II) is removed from the flow cell cartridge, resulting in separation of the first type and the second type of cells.

[0080] Figure 4 Another exemplary embodiment of the cell separation method is schematically shown in FIG. Figure 4 The embodiment shown in is an example of a cell separation method using magnetic particles that are immobilized on the side of a processing channel of a flow cell cartridge when subjected to magnetic levitation. Cells that are not complexed with the magnetic particles are suspended in the processing channel. Figure 4The illustrated embodiment uses magnetic particles to deplete a cell population containing at least two cell types, a first type (1) and a second type (2). The magnetic particles (which may be ferromagnetic, ferrimagnetic, paramagnetic or superparamagnetic) are included in a magnetic agent (4) capable of forming a complex with the first cell type (1), and during magnetic suspension, the complex (3) is magnetically pulled to the side of the processing channel of the flow cell cartridge. The magnetic agent allows a portion of the sample depleted of the complex to be separated, thereby allowing the second type of cells to be separated from a cell population containing multiple cell types. The exemplary cell population contains at least two different cell types, a first type (1) and a second type (2), which have different surface markers (e.g., proteins, carbohydrates or other biomolecules). When performing the exemplary method, the exemplary cell population is contacted with an exemplary magnetic agent (4), which contains magnetic particles conjugated to antibodies that are capable of specifically binding to surface markers of the first type (1) of the two cell types. The particles bind to the surface markers of the first cell type (1) and form a complex with the cells of the first type (1). When exposed to a magnetic field during the magnetic levitation process, the complex (3) migrates to the side of the processing channel of the flow cell cartridge. The fraction depleted of the complex is removed from the flow cell cartridge, resulting in the separation of the first type and the second type of cells. In some embodiments, after the portion depleted of the first type of complex is removed from the flow cell cartridge, the exposure of the processing channel to the magnetic field can be stopped, and the first type of complex that migrated to one or more sides of the processing channel is then released into the processing channel to form a suspension rich in the first type of complex. The suspension rich in the first type of complex can then be removed from the flow cell cartridge, thereby separating the first type of cells (which form part of the first complex).

[0081] Magnetic agents according to embodiments of the present invention may but need not include paramagnetic or superparamagnetic particles. Ferromagnetic or ferrimagnetic particles are also suitable for inclusion in magnetic agents according to embodiments of the present invention, and are used in separation methods using such magnetic agents. It should be understood that suspension altering agents comprising particles having superparamagnetic or paramagnetic properties can act as magnetic agents under suitable conditions, and are used in the methods shown in the above exemplary descriptions. For example, as discussed elsewhere in this disclosure, as the number of superparamagnetic or paramagnetic particles connected to each cell in a cell complex with a suspension height altering agent increases, the complex can be fixed to the bottom of a processing channel ("dropping" of the suspension) during magnetic suspension. Systems and kits for particle separation

[0082] Kits and systems for separating particles (such as cells) by magnetic suspension are described in the present disclosure, and these kits and systems are included in embodiments of the present invention. Exemplary kits include one or more types of suspension height changers and / or magnetic agents (as described in detail elsewhere in the present disclosure), or separate components of one or more types of suspension height changers and / or magnetic agents. Each suspension height changer or magnetic agent can form a complex with a separate particle (such as a cell). Each suspension height changer or magnetic agent comprises magnetic particles and a linker that preferentially binds to a target cell type. For suspension height changers, the magnetic particles can be paramagnetic or superparamagnetic. For magnetic agents, the magnetic particles can be ferromagnetic, ferrimagnetic, paramagnetic or superparamagnetic. In embodiments of kits comprising one or more types of magnetic particles and one or more types of linkers as separate components, one or more types of magnetic particles can be surface modified with a portion that can mediate non-covalent interactions with one or more types of linkers. The kit can also include one or more linkers. Embodiments of the kit can include a paramagnetic fluid medium. Embodiments of the kit may also include one or more other components, such as, but not limited to, antibodies, conjugates, buffers (including, but not limited to, buffers formulated to reduce nonspecific binding of particles to non-target cells), flow cell cartridges, or materials designed to optimize depletion or recovery of target particles (e.g., cells) from a mixed particle population.

[0083] An exemplary system for separating particles is a cell separation system, which includes one or more types of suspension height changers and / or magnetic agents (as described in detail elsewhere in the present disclosure). Each suspension height changer and / or magnetic agent can form a complex with a single cell. Each suspension height changer or magnetic agent comprises a magnetic particle and a linker that preferentially binds to a target cell type. For suspension height changers, the magnetic particles can be paramagnetic or superparamagnetic. For magnetic agents, the magnetic particles can be ferromagnetic, ferrimagnetic, paramagnetic or superparamagnetic. In addition to one or more types of suspension height changers and / or magnetic agents, the exemplary system includes a paramagnetic fluid medium. The exemplary system also includes a flow cell cartridge described elsewhere in the present disclosure, a station including a holding block for a flow cell cartridge, and one or more magnets, the magnet being positioned to expose the processing channel of the flow cell cartridge located in the holding block to a magnetic field. Example

[0084] The following examples are offered to illustrate, but not to limit, the claimed invention. Example 1: Isolation of Jurkat cells using magnetic beads

[0085] Jurkat cells ("Jurkat") were isolated using microbeads, such as Figure 5As shown. Streptavidin-coated high-iron superparamagnetic nanobeads (300 nm diameter) were obtained from Spherotech (Forest Lake, IL, USA). CD45 anti-human antibodies were bound to the microbeads. 15 μL of human anti-CD45 antibody (clone 2D1, Biolegend, San Diego, CA) was mixed with 9 μL of 5 μM free biotin. The resulting mixture was then mixed with 35 μL of superparamagnetic nanobeads and incubated at room temperature for 30 minutes. At the end of the incubation period, the reaction was quenched by adding an excess of 1 μM biotin and then incubated for 5 minutes to completely bind all free biotin binding sites on the nanobeads. The nanobeads were then washed 3 times with 1×PBS / 0.5% BSA to remove all unbound antibodies and biotin, thereby obtaining nanobeads bound to anti-human CD45 biotinylated antibodies. Since the nano beads are incubated with a solution containing 90% free biotin and 10% biotinylated antibodies, about 90% of the potential biotin binding sites on the nano beads are bound to biotin, and 10% are bound to the biotinylated antibodies. The Jurkat cells of the human T cell line (ATCC TIB-152, Jurkat clone E6.1) are stained with Calcein AM (Thermo Fisher Scientific Inc.) at a final concentration of 10 μM to provide green fluorescence. H358 cells (NCI-H358, derived from Berkeley Cell Culture Facility, Berkeley, California) are stained with CellTracker Red CMPTX (Thermo Fisher Scientific Inc.) at a final concentration of 10 μM to provide red fluorescence. The two types of stained cells are washed 3 times to remove excess fluorescent staining. The stained Jurkat and H358 cells are mixed to about 50% H358 cells / about 50% Jurkat cells ("H358 / Jurkat mixture"). Nanobeads conjugated with anti-human CD45 biotinylated antibody were added to the H358 / Jurkat mixture, with a total cell input of 10 6 , which means that there are about 1 million cells in total, about 500,000 Jurkat cells and about 500,000 H358 cells. Nanobeads conjugated with anti-human CD45 biotinylated antibodies were added to this cell mixture, and the resulting mixture was incubated at 25°C for 15 minutes. The bead-to-cell ratio was 50,000:1, which means that about 25×10 9Nanobeads conjugated with anti-human CD45 biotinylated antibodies were added to the H358 / Jurkat mixture. Paramagnetic fluid medium containing 1M gadobutrol was then added to the cell / nanobead mixture to a final concentration of 75mM. The resulting suspension was loaded into LeviCell TM The suspension was exposed to a magnetic field ("equilibrated") for 20 minutes in the LeviCell instrument and then flowed through the flow cell. A control experiment was performed (no magnetic beads were added).

[0086] Figure 5 Photographic images (reproduced in grayscale) of a cell mixture at the end of the equilibrium phase in a flow cell of a control experiment (left) and a mixture with magnetic beads (right) are shown. As shown in the images obtained from the control experiment, CD45+Jurkat cells (labeled with green fluorescent Calcein AM dye) and CD45-H358 cells (labeled with red fluorescent CellTracker Red dye) are both suspended in approximately the same position in the flow cell. In the image with a bead to cell ratio of 50,000:1, CD45+Jurkat cells (green fluorescence) are no longer suspended because the magnetic particles bound to the cells have pulled the cells out of the solution (depleted cells), while CD45-H358 cells are still suspended in the same position as they were in the control reaction. Figure 5 The results shown show that Jurkat CD45+ cells were depleted by 99.87% by the Nexcelom cell counter. To determine the depletion level, the output sample was collected from the flow cell cartridge and its volume was measured using a micropipette. 20 μl of the sample was placed in a cell counter chip (Nexcellom Bioscience, Lawrence, Massachusetts) and placed in the Nexcelom Cellometer instrument. The green-labeled Jurkat cells and red-labeled H358 cells were automatically counted and the concentration of each type of cell was calculated. The calculated concentration was multiplied by the sample volume to obtain the total number of cells in each output sample and each input sample set aside before. The depletion level was calculated as the inverse ratio of the total amount of Jurkat cells in the top output portion to the input portion: No depletion was observed in control experiments. Example 2: Comparison of different complex formations

[0087] The method of isolating Jurkat cells using magnetic microbeads was performed essentially as described in Example 1, but with the following modifications. Two different complex formations were tested. Under the first set of conditions, streptavidin-conjugated superparamagnetic nanobeads were mixed with cells that had been labeled with biotinylated antibodies. Under the second set of conditions, superparamagnetic nanobeads were first conjugated with antibodies and then mixed with cells. In the first experimental protocol, 15% CD45 was labeled with a saturating concentration of biotin-conjugated anti-human CD45 antibody. neg / 85% CD45 pos The mixture of cells was added to bind all available CD45 on the cell surface. Excess antibody was washed away and the labeled cells were incubated with streptavidin-conjugated superparamagnetic nanobeads. In the second experimental scheme, streptavidin-conjugated superparamagnetic nanobeads were mixed with biotin-conjugated antibodies, and the remaining free streptavidin was blocked by free biotin. The antibody-linked superparamagnetic nanobeads were then incubated with the cell mixture to bind CD45. Due to the interaction with streptavidin-biotin (KD of about 10 -6 Up to 10 -9 mol / L), the antibody-CD45 interaction (KD is about 10 -6 Up to 10 -9 mol / L) has a lower affinity, so nearly 10 times more superparamagnetic nanobeads are needed to achieve similar CD45 pos Due to the number of beads required, it is not feasible to label all available biotin binding sites with antibodies due to the costs involved, which is why free biotin is used to block the unbound binding sites on the beads.

[0088] A certain amount of linker needs to be bound to each microparticle to achieve a saturated "solution phase" concentration of linker to maximize binding to labeled cells. Complete coverage of the microparticle surface with linker is ideal. However, there may be a large number of potential ligand binding sites on the microparticle surface, which may require an impractically large amount of linker to achieve complete surface coverage. In lieu of complete surface coverage, in some cases, a minimum required amount of linker needs to be bound to the microparticle surface to provide a sufficient "solution phase" concentration of linker to bind a sufficiently high proportion of labeled cells. Since leaving free binding sites on the microparticles can increase non-specific binding of the microparticles to cells, it may be advantageous to add a "filler" or "blocking agent" to cover these binding sites. Example 3: Effect of particle size on cell separation

[0089] It is theoretically predicted and experimentally confirmed that the most effective binding of a suspension height altering agent or a magnetic agent to a cell is performed using particles of the smallest possible size, so that the product of the number of linker molecules per particle and the number of particles per unit volume of solution is as close as possible to the saturation concentration of a particular linker in the solution. For example, two different types of particles made of the same material and having the same density can have different sizes, the first particle having a diameter of 5 μm and the second particle having a diameter of 1 μm. Compared with 1 μm particles, the particle volume of 5 μm particles is 125 times that of the former and the surface area is 25 times that of the former. Assuming that a fixed surface area is required for each antibody binding event, 5 μm particles can bind 25 times the linker molecules of 1 μm particles. However, in the solution, the concentration of 1 μm particles will be 125 times higher at the same particle weight volume concentration. Therefore, at the same weight volume concentration, the effective solution concentration of particle-bound linkers for 1 μm particles is 5 times higher than that of 5 μm particles. Therefore, by using smaller particles and using a higher number of particles per unit volume, it is easier to reach the saturation concentration of particle-bound linkers in the solution. It was determined experimentally that in order to TM To achieve effective cell separation on a magnetic levitation platform, the size of the microparticles must be small enough to reach saturation concentration in solution and large enough to contain enough magnetic material (such as Fe3O4) to move the complexes formed with cells downward toward the fixed magnet and / or effectively change the density of the complexes with cells to achieve their levitation height. The effect of microparticle size on the density of the complexes of cells and density modifiers is described in Figure 8 , which are discussed elsewhere in this disclosure. Example 4: Testing the effect of antibody surface coverage on cell separation

[0090] The method of isolating Jurkat cells using magnetic microbeads was performed essentially as described in Example 1, but with the following modifications. Streptavidin-coated nanobeads were incubated with different amounts of biotinylated anti-CD45 antibodies to cover different percentages of available streptavidin binding sites (binding site data provided by the manufacturer). The remaining binding sites were blocked with biotinylated BSA and / or free biotin. The resulting nanobeads bound to anti-human CD45 biotinylated antibodies were incubated with 15% CD45 neg Cells / 85% CD45 pos The cell mixture was incubated together and subjected to magnetic levitation separation. pos Depletion of cells and CD45 neg The cell yield was used to evaluate the effectiveness of the separation method. Figure 6 The results shown show that 10% antibody coverage of the beads is sufficient to provide >99.9% CD45 posCell depletion while maintaining >50% CD45 neg Cell yield. Reducing antibody coverage directly reduces CD45 pos Depletion of cells and CD45 neg This may be due to an increase in nonspecific binding. Example 5: Testing different particulate materials

[0091] According to the exemplary embodiments described in the present disclosure, a variety of particulate materials were tested in a cell separation process. Some particulate tests showed more nonspecific binding than others. Nonspecific binding of the particulates to iron / polymer, silica, or gold coatings was observed primarily. Microbeads from Creative Diagnostics (New York, NY) showed low nonspecific binding, as expected, as the manufacturer explicitly states that the microbeads are blocked to reduce nonspecific binding. 100nm superparamagnetic high iron beads obtained from Creative Diagnostics showed >99% depletion when cells were labeled with antibodies prior to mixing at a 1000 bead-to-cell ratio. Conjugating the particulates to antibodies prior to mixing with cells resulted in 87% depletion at a 10,000 bead-to-cell ratio. Conjugating the particulates to antibodies prior to mixing with cells reduced the less nonspecific binding observed.

[0092] Initial testing of magnetic beads obtained from Spherotech (Forest Lake, IL, USA) involved 5 cm and 0.5 μm paramagnetic microparticles, which have a polystyrene / iron oxide shell around a polystyrene core and are marketed as having "regular iron content". These paramagnetic microparticles showed significant nonspecific binding to all cells, regardless of cell size. Smaller Spherotech microparticles (0.5 μm and 0.3 μm) marketed as "high iron" were also tested. These high iron microparticles have an iron oxide core and a polystyrene shell. The 0.5 μm microparticles showed little nonspecific binding, but did not achieve >90% depletion. The specific differences between high iron content and low iron content 0.5 μm beads indicate the importance of material selection (such as microparticle coating and iron content) for the performance of depletion beads. 0.3 μm high iron content beads achieved >95% depletion.

[0093] Ferrofluids obtained from Bio-Techne (Minneapolis, MN) contain 100-300 nm superparamagnetic microparticles with a polymer coating. U.S. Patent No. 7,169,618 proposes a variety of coating possibilities for these microparticles, including silanization and carboxydextran or aminodextran coatings. Direct conjugation of antibodies or streptavidin to the surface of the microparticles is also envisioned. Streptavidin conjugation of biotinylated antibodies to microparticles was performed. Since the biotin binding capacity of the microparticles was unknown, the streptavidin-conjugated beads were first titrated with a set amount of biotinylated antibody. >99% CD45 was achieved with 20 μl of ferrofluid labeled with 25 μl of biotinylated antibody. pos Depletion of CD45 cells. When 400 μl of ferrofluid was labeled with 25 μl of biotinylated antibody, a reduction in depletion to about 85% was observed, indicating that insufficient antibody was present to achieve adequate antibody levels on the microparticle surface. A 50 μl sample of ferrofluid was used to titrate the antibody concentration. Almost 100% depletion of CD45 was observed for all antibody volumes tested (ranging from 25 μl to 6.25 μl). pos The cells were depleted. When the cells were first incubated with the antibody, the ferrofluid showed better depletion performance, achieving >99.5% depletion. Minimal non-specific binding was observed when labeled with an isotype control or no antibody. These beads were not used for further testing as it was not available for production. However, the patent recently expired, allowing for the possibility of producing these beads.

[0094] BD IMag TM Microparticles were obtained from Luna Nanotech (San Jose, CA). These microparticles have a superparamagnetic iron oxide core and a polymer shell. Initial testing used streptavidin-conjugated microparticles with biotinylated antibodies to specifically target CD45. pos Since the biotin-binding capacity of the beads was unknown, the cells were first stained with the antibody, washed, and then mixed with streptavidin-conjugated microbeads. This resulted in almost 100% depletion, as well as a good yield of the desired cell population.

[0095] Dextran CLIO magnetic nanoparticles were obtained from Luna Nanotech (Markham, Ontario, Canada). These nanoparticles consist of cross-linked dextran with one to three 7-14 nm iron oxide spheres within each nanoparticle. Using nanobeads conjugated to anti-CD45 antibodies, the average depletion was approximately 92%, and some significant nonspecific binding was observed.

[0096] Gold-coated nanoparticles coated with an iron oxide core were obtained from Nanopartz TM(Loveland, Colorado). CD45 anti-human antibodies were conjugated to 100 nm streptavidin-conjugated nanoparticles and tested in depletion experiments. Approximately 98% depletion was achieved, however, when beads without antibody were used and quenched with free biotin, nonspecific binding to all cells was observed. In subsequent experiments, when the bead-to-cell ratio was >50,000:1, nonspecific binding of anti-CD45 coated beads to CD45 was observed. Neg There was some non-specific binding of the cells to . Further testing with alternative blocking agents is needed to make depletion feasible.

[0097] It should be understood that the embodiments and implementations described in this disclosure are for illustrative purposes only, and those skilled in the art will appreciate that various modifications or changes will be made based on these embodiments and implementations, and these modifications or changes will be included in the spirit and scope of this application and the scope of 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 method for cell separation, comprising: a) combining a magnetic agent with a cell population comprising a plurality of cell types, wherein the magnetic agent comprises magnetic microparticles and a linker that preferentially binds to cells of a target type of the plurality of cell types, thereby forming a magnetic complex, wherein the magnetic complex comprises the magnetic agent bound to a single cell of the target type; b) forming a suspension in a paramagnetic fluid medium, wherein the suspension comprises a plurality of the magnetic complexes and a plurality of cells of the plurality of cell types; c) introducing the suspension into a processing channel of a flow cell cartridge; and d) exposing the processing channel to a magnetic field for a period of time sufficient to cause at least some of the plurality of magnetic complexes to migrate to and be fixed to one or more sides of the processing channel, thereby forming a suspension depleted of the magnetic complexes. 2 . The method of claim 1 , wherein the linking agent comprises an antibody or a domain of the antibody that is capable of specifically binding to a moiety on the surface of a cell of the target type.

3. The method of claim 2, wherein the combining of the magnetic agent comprises: binding the antibody or a domain of the antibody to the moiety on the surface of the cell of the target type; and, The antibody or the domain of the antibody is bound to the magnetic particle.

4. The method according to any one of claims 1 to 3, wherein the linking agent is bound to the magnetic particle by one or more covalent or non-covalent interactions.

5. The method according to any one of claims 1 to 4, further comprising: e) removing at least a portion of the suspension depleted of the magnetic complexes from the flow cell cartridge.

6. The method according to claim 5, after executing step (e), further comprising: f) stopping exposing the processing channel to the magnetic field, thereby releasing the magnetic complexes fixed to one or more sides of the processing channel, thereby forming a suspension rich in the magnetic complexes; and g) removing at least part of the suspension with the magnetic complexes from the flow cell cartridge.

7. The method of claim 5 or 6, wherein step (e) or (g) is performed through the outlet channel of the flow cell cartridge.

8. The method according to any one of claims 1 to 7, wherein step (d) is performed in a stopped flow mode of the flow cell cartridge.

9. A method according to any one of claims 1 to 8, wherein step (e) or (g) comprises flowing the suspension along the length of the processing channel.

10. The method according to any one of claims 1 to 9, wherein the magnetic particles are paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic particles. The method of claim 10 , wherein the magnetic particles comprise a metal, a metal salt or a metal oxide.

12. The method of any one of claims 1 to 11, wherein the target type of cell is selected from the group consisting of macrophages, alveolar type II (ATII) cells, stem cells, adipocytes, cardiomyocytes, embryonic cells, tumor cells, lymphocytes, erythrocytes, epithelial cells, egg cells, sperm cells, T cells, B cells, bone marrow cells, immune cells, hepatocytes, endothelial cells, stromal cells, and bacterial cells.

13. The method of any one of claims 2 to 11, wherein the moiety on the cell surface of the target type is selected from the group consisting of: CD45, CD3, CD4, CD8, CD19, CD40, CD56, CD11b, CD14, CD15, EpCAM, ICAM, CD235, HER-2, HER-3, CD66e, integrin, EPL-selectin, EGFR, EGFRVIII, PDGFRβ, c-MET, MUC-1, OX-40, CD28, CD133, CD30 TNFRSF8, CTLA4, CD71, CD16αVCAM-1, nucleolar protein, and myelin basic protein.

14. A method for cell separation, comprising: a) binding a first suspension height altering agent to a first type of cell in a cell population comprising a plurality of cell types, wherein the first suspension height altering agent comprises a first paramagnetic or superparamagnetic particle and a first linking agent that preferentially binds to the first type of cell, thereby forming a first complex comprising the first suspension height altering agent bound to a single cell of the first type; b) forming a suspension in a paramagnetic fluid medium, wherein the suspension comprises a plurality of the first complexes and a plurality of cells of the plurality of cell types; c) introducing the suspension into a processing channel of a flow cell cartridge; and d) exposing the processing channel to a magnetic field for a first time period, the first time period being sufficient to separate at least some of the first complexes from cells of the plurality of cell types that are not bound to the first suspension height altering agent in the processing channel, thereby forming a first portion of the suspension and a second portion of the suspension, wherein the first portion is enriched in the first complex relative to the suspension and the second portion is depleted in the first complex relative to the suspension.

15. The method of claim 14, wherein the cells of the first type are selected from the group consisting of macrophages, alveolar type II (ATII) cells, stem cells, adipocytes, cardiomyocytes, embryonic cells, tumor cells, lymphocytes, erythrocytes, epithelial cells, egg cells, sperm cells, T cells, B cells, bone marrow cells, immune cells, hepatocytes, endothelial cells, stromal cells, and bacterial cells.

16. The method of claim 14 or 15, wherein the first complex is suspended in the processing channel of the flow cell cartridge below the plurality of cell types not bound to the first suspension height altering agent.

17. The method according to any one of claims 14 to 16, wherein the first linking agent comprises a first antibody or a domain of the first antibody capable of specifically binding to a first moiety on the surface of the cell of the first type.

18. The method of claim 17, wherein the first portion is selected from the group consisting of CD45, CD3, CD4, CD8, CD19, CD40, CD56, CD11b, CD14, CD15, EpCAM, ICAM, CD235, HER-2, HER-3, CD66e, integrin, EPL-selectin, EGFR, EGFRVIII, PDGFRβ, c-MET, MUC-1, OX-40, CD28, CD133, CD30 TNFRSF8, CTLA4, CD71, CD16αVCAM-1, nucleolar protein, and myelin basic protein.

19. The method of claim 17 or 18, wherein the combination of the first suspension height altering agent comprises: binding of said first antibody or a domain of said first antibody capable of specifically binding to said first moiety on the surface of said cell of said first type to said first moiety on the surface of said cell of said first type; and, Binding of said first antibody or said domain of said first antibody to said first paramagnetic or superparamagnetic particle.

20. The method of any one of claims 14 to 19, wherein the first linking agent is bound to the first paramagnetic or superparamagnetic microparticle by one or more covalent or non-covalent interactions.

21. The method according to any one of claims 14 to 20, further comprising: e) removing at least a portion of the first fraction and / or removing at least a portion of the second fraction from the flow cell cartridge.

22. The method according to claim 21, further comprising: f) removing at least a portion of the first fraction and / or removing at least a portion of the second fraction from the flow cell cartridge.

23. The method of claim 21, wherein step (e) is performed through a first outlet channel of the flow cell cartridge.

24. The method according to any one of claims 14 to 23, further comprising: In step (a), a second suspension height altering agent is bound to a second type of cells in the cell population comprising a plurality of cell types, wherein the second suspension height altering agent comprises a second paramagnetic or superparamagnetic particle and a second linking agent that preferentially binds to the second type of cells of the plurality of cell types, wherein the binding of the second suspension height altering agent is performed under conditions where the second suspension height altering agent binds to a single cell of the second type to form a second complex, each second complex comprising the second suspension height altering agent bound to the second type of cell, In step (b), the suspension further comprises a plurality of the second complexes; In step (d), the flow cell cartridge is exposed to the magnetic field for a second time period, and the second time period is sufficient to separate at least some of the second complexes from cells of the multiple cell types that are not bound to the second suspension height altering agent, from the first complexes, and from cells of other types of the multiple cell types in the processing channel, thereby forming a third part of the suspension and a fourth part of the suspension, wherein the third part is enriched in the second complex relative to the suspension and the fourth part is depleted of the second complex relative to the suspension.

25. The method of claim 24, wherein the second linking agent comprises a second antibody or a domain of the second antibody that is capable of specifically binding to a second moiety on the surface of the cell of the second type.

26. The method of claim 25, wherein the incorporation of the second fly height altering agent comprises: binding of said second antibody or a domain of said second antibody capable of specifically binding to said second moiety on the surface of said cell of said second type to said second moiety on the surface of said cell of said second type; and, Binding of the second antibody or a domain of the second antibody to the second microparticle.

27. The method of any one of claims 24 to 26, wherein the second linking agent is bound to the second paramagnetic or superparamagnetic microparticle by one or more covalent or non-covalent interactions.

28. The method of any one of claims 20 to 27, wherein the one or more non-covalent interactions of the first linker or the second linker independently comprise one or more of the following: an interaction of biotin with an avidin-like molecule, an antibody-antigen interaction, or an interaction between an antibody binding protein or a domain of the antibody binding protein and its binding partner.

29. The method of any one of claims 24 to 28, wherein the first time period coincides with the second time period.

30. The method according to any one of claims 24 to 25, further comprising: f) removing at least a portion of the third part or removing at least a portion of the fourth part from the processing channel and / or the flow cell cartridge.

31. The method of claim 30, wherein step (f) is performed through a second outlet channel of the flow cell cartridge.

32. The method of any one of claims 14 to 31, wherein step (d) is performed in a stopped flow mode of the flow cell cartridge.

33. A method according to any one of claims 21 to 32, wherein steps (e) and (f) comprise flowing the suspension along the length of the processing channel.

34. The method of any one of claims 21 to 33, wherein steps (e) and (f) are simultaneous or sequential.

35. The method of any one of claims 14 to 34, wherein the vertical position of the flow cell cartridge in the magnetic field is variable.

36. The method of any one of claims 14 to 35, wherein the first paramagnetic or superparamagnetic microparticle and / or the second paramagnetic or superparamagnetic microparticle is a paramagnetic microparticle.

37. The method of claim 36, wherein the paramagnetic microparticles comprise a paramagnetic metal, a paramagnetic metal salt, or a paramagnetic metal oxide.

38. The method of claim 36, wherein the paramagnetic particles comprise iron oxide.

39. The method according to any one of claims 14 to 38, wherein the first paramagnetic or superparamagnetic particle and / or the second paramagnetic or superparamagnetic particle is a superparamagnetic particle.

40. The method of claim 39, wherein the superparamagnetic particles comprise iron or iron oxide.

41. The method of any one of claims 14 to 40, wherein one or more of the first type of cells and / or one or more of the second type of cells are living cells.

42. The method of any one of claims 14 to 40, wherein one or more of the first type of cells in the first complex and / or one or more of the second type of cells in the second complex are living cells.

43. A magnetic suspension kit comprising a paramagnetic fluid medium and one or more suspension height altering agents, or isolated components of said one or more suspension height altering agents, capable of forming a complex with a single cell, Each suspension height altering agent comprises a paramagnetic or superparamagnetic particle and a linking agent that preferentially binds to a target cell type.

44. The kit of claim 43, wherein the linking agent preferentially binds to a cell surface marker selected from the group consisting of CD45, CD3, CD4, CD8, CD19, CD40, CD56, CD11b, CD14, CD15, EpCAM, ICAM, CD235, HER-2, HER-3, CD66e, integrin, EPL-selectin, EGFR, EGFRVIII, PDGFRβ, c-MET, MUC-1, OX-40, CD28, CD133, CD30 TNFRSF8, CTLA4, CD71, CD16αVCAM-1, nucleolar protein, and myelin basic protein.

45. The kit of claim 43 or 44, wherein the linking agent is covalently bound to the paramagnetic or superparamagnetic microparticle.

46. ​​The kit method according to claim 43 or 44, wherein the kit comprises one or more types of said paramagnetic or superparamagnetic microparticles and one or more types of said linking agents as separate components of said one or more said suspension height altering agents, wherein said one or more types of said paramagnetic or superparamagnetic microparticles are surface modified with moieties capable of mediating non-covalent interactions with said one or more types of said linking agents.

47. The kit of claim 46, wherein the non-covalent interaction comprises one or more of the following: an interaction of biotin with an avidin-like molecule, an antibody-antigen interaction, or an interaction between an antibody binding protein or a domain of the antibody binding protein and its binding partner.

48. A magnetic suspension kit comprising a paramagnetic fluid medium and one or more magnetic agents, or separated components of said one or more magnetic agents, capable of forming a complex with a single cell, Each magnetic agent comprises a paramagnetic particle and a linker that preferentially binds to a target cell type.

49. The kit of claim 48, wherein the linking agent that preferentially binds to the cell surface marker is selected from the group consisting of: CD45, CD3, CD4, CD8, CD19, CD40, CD56, CD11b, CD14, CD15, EpCAM, ICAM, CD235, HER-2, HER-3, CD66e, integrin, EPL-selectin, EGFR, EGFRVIII, PDGFRβ, c-MET, MUC-1, OX-40, CD28, CD133, CD30 TNFRSF8, CTLA4, CD71, CD16αVCAM-1, nucleolar protein, and myelin basic protein.

50. The kit of claim 48 or 49, wherein the magnetic particles are paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic particles.

51. The kit of claim 50, wherein the magnetic particles comprise a metal, a metal oxide, or a metal salt.

52. The kit according to any one of claims 48 to 51, wherein the linking agent is covalently bound to the magnetic particles.

53. The kit according to any one of claims 48 to 51, wherein the kit comprises one or more types of said magnetic particles and one or more types of said linking agents as separate components of said one or more said magnetic agents, wherein said one or more types of said magnetic microparticles are surface modified with moieties capable of mediating non-covalent interactions with said one or more types of said linking agents.

54. The kit of claim 52, wherein the non-covalent interaction comprises one or more of the following: an interaction of biotin with an avidin-like molecule, an antibody-antigen interaction, or an interaction between an antibody binding protein or a domain of the antibody binding protein and its binding partner.

55. The kit of any one of claims 42 to 53, wherein the linking agent is an antibody or a domain of the antibody capable of specifically binding to a moiety on the surface of the target cell type.

56. A system for cell separation, comprising Magnetic microparticles, which are capable, alone or in combination with other agents, of preferentially binding to a first type of cell in a cell population comprising a plurality of cell types and forming a first complex of the microparticle and the first type of cell; a flow cell cartridge including a first outlet channel and a process channel; a station comprising a holding block for the flow cell cartridge and one or more magnets positioned to expose the processing channels of the flow cell cartridge located in the holding block to a magnetic field, wherein exposing the processing channel of the flow cell cartridge containing a suspension of cells of the plurality of cell types in a paramagnetic fluid medium to the magnetic field allows the first complex to be separated in the processing channel from cells of the plurality of cell types that are not bound to the first non-magnetic microparticles and from other types of cells of the plurality of cell types, and wherein the first complex is suspended lower in the processing channel of the flow cell cartridge than the plurality of cell types not complexed in magnetic particles.

57. The kit of claim 50, wherein the other reagents comprise a linking agent that preferentially binds to cells of the first type.

58. The kit of claim 57, wherein the magnetic microparticles are surface modified with moieties capable of mediating non-covalent interactions with the linking agent, or wherein the linking agent is covalently bound to the magnetic microparticles.

59. The kit according to any one of claims 56 to 58, wherein the magnetic particles are paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic particles.

60. The kit of claim 59, wherein the magnetic particles comprise a metal, a metal oxide, or a metal salt.

Images