Methods and systems for cell separation

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 362,630, filed April 7, 2022, and U.S. Provisional Application No. 63 / 254,944, filed October 12, 2021. The entire contents and disclosures of these applications are incorporated herein by reference. Background of the Invention

[0004] Magnetic levitation is a recently emerged 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, generating a non-uniform pressure equivalent 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 high magnetic field region. In fact, the object is displaced 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 profiles. See Durmus et al., 2015, "Magneticlevitation of single cells," Proc Natl Acad Sci USA 112 (28): E3661-8 ("Durmus"). Systems have been developed for collecting cells at different suspension heights and collecting them for downstream analysis and other applications. SUMMARY OF THE INVENTION

[0006] The terms "invention," "described invention," and "present invention" as used in this document are intended to refer broadly to all subject matter of this patent application and the claims that follow. Statements containing these terms should be understood not to limit the subject matter described in this disclosure or to limit the meaning or scope of the appended patent claims. The embodiments covered by the present invention are defined by the claims rather than this summary. This summary 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. This summary 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 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.

[0007] Included in embodiments of the present invention and described in the present disclosure is a method for cell separation, comprising the steps of: combining a first density modifying agent with a first type of cell in a cell population comprising a plurality of cell types, wherein the first density modifying agent comprises a first non-magnetic 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 density modifying agent bound to a single cell of the first type; forming a suspension in a paramagnetic fluid medium, the suspension comprising a plurality of first complexes and a plurality of 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 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 by the first density modifying 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. Also included in embodiments of the invention and described in the present disclosure is a magnetic levitation kit comprising a paramagnetic fluid medium and one or more density modifiers or separate components of one or more density modifiers that are capable of forming complexes with single cells, wherein the density of each complex is different from the density of the single cell, and wherein each density modifier comprises non-magnetic microparticles and a linker that preferentially binds to a target cell type. Also included in embodiments of the present invention and described in the present disclosure is a system for cell separation, comprising: a first non-magnetic particle that is capable, alone or in combination with a first other reagent, of preferentially binding to a first type of cells of a cell population comprising a plurality of cell types and forming a first complex of the particle and the first type of cells, the density of the first complex being different from the density of the first type of cells and other types of cells of the plurality of cell types; a flow cell box comprising a first outlet channel and a processing channel; a station comprising a holding block for the flow cell box and one or more magnets positioned to expose the processing channel of the flow cell box located in the holding block to a magnetic field, wherein exposing the processing channel of the flow cell box 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 by the first non-magnetic particle and from cells of the other types of the plurality of cell types, wherein the processing channel of the flow cell box contains a suspension of cells of the plurality of cell types in a paramagnetic fluid medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is a schematic diagram of an exemplary magnetic levitation system.

[0010] Figure 2 is a schematic diagram of two views of a flow cell cassette of an exemplary magnetic levitation system.

[0011] Figure 3 is a schematic diagram of a method according to an exemplary embodiment of the present invention.

[0012] Figure 4 is a schematic diagram of a method according to an exemplary embodiment of the present invention.

[0013] Figure 5 is a schematic diagram of a cell complexed with a microvesicle according to some embodiments described in the present disclosure.

[0014] Figure 6 are photographic images showing the isolation of cells complexed with microvesicles according to some embodiments described in the present disclosure.

[0015] Figure 7 is a box plot showing the isolation of cells complexed with microvesicles according to some embodiments described in the present disclosure.

[0016] Figure 8 is a schematic diagram of cells complexed with microbeads according to some embodiments of the present disclosure.

[0017] Fig. 9 are photographic images showing separation of cells complexed with polymer microbeads according to some embodiments described in the present disclosure.

[0018] Fig.10 are photographic images showing the separation of cells complexed with gold microbeads according to some embodiments described in the present disclosure.

[0019] Fig.11 is a dot plot showing the suspension height of cells in the absence and presence of antibodies conjugated to gold nanoparticles.

[0020] Fig.12 is a schematic diagram of the microparticle model complexed with the cell surface.

[0021] Fig.13 is a table showing the density calculation of microparticle-cell complexes, where the density of microparticles is 1.063 g / cm 3 , the diameter of the cell is 11.5μm. DETAILED DESCRIPTION OF THE INVENTION

[0023] Cells have inherent properties that determine their behavior when subjected to magnetic levitation. Durmus et al. showed that the height at which cells are suspended in a paramagnetic fluid medium ("suspension profile") corresponds to the cell density, and that different cell types can be distinguished based on their unique magnetic levitation profiles. 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, which describes methods and systems for separating cells by magnetic levitation, including methods for separating dead cells from living cells. The present disclosure describes improved processes (methods), devices, systems, and kits for separating particles (including cells) by magnetic levitation.

[0024] The inventor has found a method for improving particle (e.g., cell) separation during a magnetic suspension process. The inventor has found that when cells are suspended in a paramagnetic fluid medium in a flow cell box of a magnetic suspension system and exposed to a magnetic field, by combining cells with non-magnetic particles, they can change the suspension height of cells. The suspension height can be limited by the vertical position of the cells in the flow cell box of a magnetic suspension system. In other words, the inventor can control the suspension height of cells in a magnetic suspension process by compounding cells with non-magnetic particles that change in density. In one example, cells with cell-specific surface markers are connected to low-density non-magnetic particles coupled with anti-surface marker antibodies. The resulting complex is suspended in a paramagnetic medium and magnetically suspended in a processing channel of a flow cell box of a magnetic suspension system. If the density of cells (cell-particle complexes) combined with non-magnetic particles is lower than the density of uncombined (uncompounded) cells, the cell complexes with low-density non-magnetic particles are suspended higher in the processing channel than the same cells that are not compounded with low-density non-magnetic particles. In another example, cells with cell-specific surface markers are complexed with high-density non-magnetic microparticles coupled to anti-surface marker antibodies, the resulting complexes are suspended in a paramagnetic medium, and suspended in a processing channel of a flow cell box of a magnetic suspension system. If the density of cells bound to non-magnetic microparticles (cell-microparticle complexes) is higher than the density of unbound (uncomplexed) cells, the cell complexes with high-density non-magnetic microparticles are suspended lower in the processing channel than the same cells not complexed with high-density microparticles. By coupling non-magnetic microparticles of varying density with antibodies specific to surface markers of a cell type of interest found in a cell population containing multiple cell types, the inventors were able to selectively change the suspension height of cells belonging to the cell type of interest.

[0025] In the above examples, the suspension height of the complex of cells and microparticles is affected by the number of microparticles in each complex. The number of microparticles per cell in the complex can be changed by changing the ratio of particles to cells ("PTC ratio") from about 1 to about 100,000 during the complex formation process. The PTC ratio may also be referred to as a particle-to-cell ratio, a microparticle-to-cell ratio, a bead-to-cell ratio or other related terms and expressions. The inventors have found that increasing the PTC ratio reduces the suspension height of the complex. In some cases, in order to obtain a higher concentration during the complex formation process, it may be beneficial to use smaller microparticles, which may result in 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 also affect the suspension height of the complex of cells and microparticles. Some of these parameters are the nature of the material contained in the microparticles, the size of the microparticles, and the density of the microparticles.

[0026] The suspension height of the cell or particle in the flow cell box depends on the position of the flow cell box relative to the magnet of the magnetic suspension system. Therefore, the flow cell box can be moved relative to the magnet to capture the cells suspended in various positions due to being marked with a density modifier. As discussed in detail in the present disclosure, the non-magnetic particles that change in density can be used to separate specific cells from a mixed cell group suspended in a paramagnetic medium by magnetic suspension, thereby producing fractions enriched or exhausted for a cell type of interest. These fractions can then be recovered from the flow cell of the magnetic suspension system, thereby achieving the separation of a cell type of interest. Also as described in the present disclosure, the non-magnetic particles that change in density can be used to separate various components of interest, such as organelles, nucleic acids, or other molecules, from various types of heterogeneous mixtures by magnetic suspension. Components of interest (e.g., cells, organelles, nucleic acids) are sometimes referred to as "analytes".

[0027] Therefore, the present disclosure describes various embodiments of methods, devices, systems and kits for separation based on magnetic suspension of mixtures or groups of particles including various types of particles. The processes, devices, methods and kits conceived by the inventors can be used for a variety of 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 for separation based on magnetic suspension of mixtures or groups of organelles or other cellular components (including intracellular and extracellular components, such as but not limited to endosomes or exosomes). Still other embodiments of the methods, devices, systems and kits described in the present disclosure can be used for separation based on magnetic suspension of mixtures or groups of biomolecules or molecular complexes, such as separation of nucleic acid libraries in the next generation sequencing (NGS) process, or separation of lipoproteins.

[0028] 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 include 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 would otherwise be difficult to suspend to a specific location during magnetic levitation, an example being RNA (e.g., RNA released from lysed cells), and the ability to separate cell types with very similar densities by magnetic levitation (and therefore cannot be separated using magnetic levitation without the aid of density-changing non-magnetic particles described in the present disclosure).

[0029] Terms and concepts

[0030] Many terms and concepts are discussed below. They are intended to help understand various embodiments of the present invention in conjunction with the remainder 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 throughout this document and / or the accompanying drawings. Some other terms may be defined explicitly or implicitly in other parts of this document and the accompanying drawings, and may be used and understood based on the conventions accepted in the field of the present invention, the descriptions provided throughout this document and / or the accompanying drawings. Undefined terms may also be defined and understood based on the conventions accepted in the field of the present invention and are explained in the context of this document and / or the accompanying drawings.

[0031] Unless the context dictates otherwise, singular terms shall include the plural, and plural terms shall include the singular. In general, 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. Known methods and techniques are generally performed according to well-known conventional methods and as described in various general and more specific references, unless otherwise indicated. The terms used in conjunction with the laboratory procedures and techniques described in this disclosure are well known and commonly used.

[0032] Miscellaneous

[0033] As used in this disclosure, the terms "a," "an," and "the" may refer to one or more, unless specifically stated otherwise.

[0034] The use of the term "or" is used to mean "and / or" unless explicitly indicated to refer to only alternatives, or the alternatives are mutually exclusive, although the present disclosure supports definitions and "and / or" referring only to alternatives. As used in the present disclosure, "another" may mean at least a second or more.

[0035] As used in this disclosure, the term "including," and in some cases similar terms (eg, "having"), means "comprising" unless otherwise stated.

[0036] When numerical ranges are provided in this disclosure, the numerical ranges are inclusive of the range endpoints unless otherwise stated. Unless otherwise stated, numerical ranges in this disclosure include all values ​​and subranges as if expressly written out.

[0037] As used in the present disclosure, the terms "about" and "approximately" generally refer to an acceptable degree of error of an amount measured in the case of a given measured property or precision. An exemplary degree of error is 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" specifically represents 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 can include a numerical range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Thus, the expression "about X" or "approximately X" is intended to describe a claim limitation of, for example, "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 also 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 included in the series.

[0038] The term "plurality" or "population" when used with particles such as, but not limited to, cells (e.g., as "plurality of cells" or "population of cells"), refers to a population of particles (i.e., more than one particle) that includes a varying number of particles. For example, a plurality 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 ...000 or more, 1000 or 3 or more, 10 4 or more, 10 5 or more, 10 6 or more, or 10 7 or more particles.

[0039] The terms "peptide", "polypeptide" or "protein" are used to refer to polymers 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 synthesized or produced by recombinant technology.

[0040] The terms "oligonucleotide," "polynucleotide," or "nucleic acid" encompass DNA or RNA molecules, including molecules produced synthetically or by recombinant technology. An oligonucleotide, polynucleotide, or nucleic acid may be single-stranded or double-stranded.

[0041] The term "small molecule" includes molecules (organic molecules, organometallic molecules or inorganic molecules), 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 and about 1000Da, or between about 100Da and about 600Da, or between about 200Da and about 500Da.

[0042] Separation, isolation and concentration

[0043] As used in this disclosure, the term "concentration" refers to the amount of a first component contained in a second component and can be based on the number of particles per unit volume, moles per unit volume, weight per unit volume, or based on the volume of the first component per volume of the combined components.

[0044] As used in the present disclosure, the terms "isolate, separate, segregate", "purify" and their respective related terms and expressions can be used interchangeably. These terms can be used to refer to a procedure for enriching the amount of one or more components of interest relative to one or more other components present in a sample. With respect to particles or components (which can be cells), such terms can mean one or more of the following: separating such components from other components, increasing the concentration of components in a solution, or separating components in a solution from other components. For example, if a particle in a solution is separated from other particles in the solution and / or is located in a defined portion of the solution, the particle can be considered to be "separated". In another example, if after processing the solution, the concentration of the particle or component in the solution is increased at 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, the particle or component is considered to be "separated". A particle of interest within a solution containing multiple types of particles may 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 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%.

[0045] Density regulator

[0046] The density modifier comprises (i) non-magnetic particles and (ii) a linker that preferentially binds to cells of a specific cell (e.g., a first) type. The density modifier, by virtue of the linker, similarly preferentially binds to cells of a specific cell (e.g., a first) type. The density modifier is sometimes referred to as a "tag." The process of connecting a density modifier to a cell (or other object) is sometimes referred to as "tagging."

[0047] Non-magnetic particles

[0048] The terms "microparticles", "microspheres", "microbeads" and "microbubbles" (which encompass "nanospheres", "nanospheres", "nanoparticles", "nanobeads" and "nanobubbles", respectively) refer to non-magnetic particles (as described below) having one or more dimensions (e.g., length, width, diameter or circumference) of about 500 μm or less. Microparticles can be described as having a characteristic density, as described below. Microparticles can have a generally spherical shape or a non-spherical shape. "Microbubbles" and "microbeads" are special subtypes of microparticles.

[0049] The term "microbubble" generally refers to a particle with an empty space or cavity (or multiple empty spaces or cavities) inside, the total volume of which accounts for a considerable (at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%) proportion of the particle's internal volume. The empty space or cavity (or multiple empty spaces or cavities) can be filled with a gas. Microbubbles can be described as "hollow".

[0050] In the context of the present disclosure, the term "microbead" generally refers to a microparticle that does not have a significantly large empty space or cavity (or multiple empty spaces or cavities) inside, the total volume of the empty spaces or cavities occupying no more than 50%, 40%, 30%, 20%, 10% or 5% of the internal volume of the microbead. Microbeads may be described as "solid" or "substantially solid" (in the sense of not having a significantly large internal cavity). In the context of the present disclosure, microbeads may also be referred to as "anchors." "

[0051] Microparticles used in embodiments of the present invention may have a range of sizes and densities. For example, the cross-sectional dimensions (e.g., diameter, length, width) of the microparticles may be 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 (2 5nm) or less, for example, having a cross-sectional size in the range of about 500 μm to about 0.01 μm (10 nm), about 500 μm to about 0.025 μm (25 nm), about 500 μm to about 0.05 μm (50 nm), about 500 μm to about 0.1 μm (100 nm), about 500 μm to about 0.25 μm (250 nm), about 500 μm to about 0.5 μm (500 nm), about 500 μm to about 1 μm (1000 nm), about 500 μm to about 10 μm, about 500 μm to about 20 μm, about 500 μm to about 100 μm, 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 (100 nm), about 100 μm to about 1 μm (10 ...1 μm (100 nm), about 100 μm to about 0.25 μm (250 nm), about 100 μm to about 0.5 μm (100 nm), about 100 μm to about 0.1 μm (100 nm), about 100 μm to about 0.1 μm (100 nm), about 100 μm to about 0.1 μm (100 nm), about 100 μm to about 0.1 μm (100 nm), about 100 μm to about 0.1 μm (100 nm), about 100 μm to about 0.1 μm (100 nm), about 100 μm to about 0.1 μm (100 nm), about 100 μm to about ), about 100 μm to about 10 μm, about 100 μm to about 20 μm, 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 (20 nm), about 20 μm to about 1 μm (200 nm), or about 20 μm to about 10 μm.

[0052] The microparticles used in embodiments of the present invention can be composed of any number of materials or combinations thereof or can include any number of materials or combinations thereof, including but not limited to glass, silica, ceramics, non-magnetic metals (e.g., gold, silver, or platinum) or lipids. In some embodiments, microparticles are polymer microparticles. Polymer microparticles can include or be composed of various types of polymers, such as but not limited to polymethyl methacrylate (PMMA), polystyrene, polypropylene, polyethylene, polyacrylamide, alginic acid and its salts or agarose. Some non-limiting examples of microparticles comprising material combinations are gold-coated silica particles and silica-coated gold particles. It should be understood that microparticles can include various other combinations of two or more materials.

[0053] The microparticles can have a range of densities. More specifically, the density modifier can have a range of densities, but it is expected that the linker moiety has a relatively small effect on the density of the density modifier, so that the microparticle density serves as a reasonable approximation of the density of the density modifier. It should be understood that all references to microparticle density will refer equally to the density of the density modifier. Microparticles having a density within the following ranges can be used in the separation methods described herein: about 0 g / cm 3 To about 21.5g / cm 3 , about 0g / cm 3 To about 19.5g / cm 3 , about 0.001g / cm 3 To about 21.5 / cm 3 , about 0.001g / cm 3 To about 19.5g / cm 3 , about 0.01g / cm 3 To about 21.5g / cm 3 , about 0.01g / cm 3 To about 19.5g / cm 3 , about 0.1g / cm 3 To about 21.5g / cm 3 , about 0.1g / cm 3 To about 19.5g / cm 3 , about 0.1g / cm 3 To about 21.5g / cm 3 , about 0.1g / cm 3 To about 19.5g / cm 3 , about 0.5g / cm 3 To about 21.5g / cm 3 , about 0.5g / cm 3 To about 19.5g / cm 3 , about 0.7g / cm 3 To about 19.5g / cm 3, about 0.7g / cm 3 To about 21.5g / cm 3 , about 0.9g / cm 3 To about 21.5g / cm 3 , about 0.9g / cm 3 To about 19.5g / cm 3 , about 0g / cm 3 To about 0.9g / cm 3 , about 0.001g / cm 3 To about 0.9g / cm 3 , about 0.01g / cm 3 To about 0.9g / cm 3 , about 0.1g / cm 3 To about 0.9g / cm 3 , about 0.5g / cm 3 To about 0.9g / cm 3 , about 0.7g / cm 3 To about 0.9g / cm 3 , about 1.2g / cm 3 To about 21.5g / cm 3 , about 2g / cm 3 To about 21.5g / cm 3 , about 3g / cm 3 To about 21.5g / cm 3 , about 4g / cm 3 To about 21.5g / cm 3 , about 5g / cm 3 To about 21.5g / cm 3 , about 6g / cm 3 To about 21.5g / cm 3 , about 7g / cm 3 To about 21.5g / cm 3 , about 8g / cm 3 To about 21.5g / cm 3 , about 9g / cm 3 To about 21.5g / cm 3 , about 10g / cm 3 To about 21.5g / cm 3 , about 11g / cm 3 To about 21.5g / cm 3 , about 12g / cm 3 To about 21.5g / cm 3 , about 13g / cm 3 To about 21.5g / cm 3 , about 14g / cm 3 To about 21.5g / cm3 , about 15g / cm 3 To about 21.5g / cm 3 , about 16g / cm 3 To about 21.5g / cm 3 , about 17g / cm 3 To about 21.5g / cm 3 , about 18g / cm 3 To about 21.5g / cm 3 , about 19g / cm 3 To about 21.5g / cm 3 or about 20g / cm 3 To about 21.5g / cm 3 .

[0054] It should be appreciated that the microparticles used in a single separation process may include multiple (two or more) microparticles having different densities. For example, one density microparticle may be coupled to a first cell type (e.g., CD8+T cells), and a different density microparticle may be coupled to a second cell type (e.g., CD4+T cells). In general, in a single cell separation process, density modifiers comprising the same linking agent (or comprising a linking agent having the same specificity) will associate with microparticles having the same density.

[0055] Factors Affecting Particle Selection

[0056] The binding of density modifiers to cells or other objects and the behavior of the resulting complexes during magnetic levitation depend 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 ratios 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 microparticle surface, and the microparticle material. 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.

[0057] In one example, the density of the cell complex is estimated as the mass of the cell (m 细胞 ) and the mass of all bound particles (n*m MS ) divided by the volume of the cell (V 细胞 ) and the sum of the volumes of all bound beads (n*VMS).

[0058]

[0059] To estimate the maximum number of microparticles that could theoretically bind to cells, microparticles and cells were modeled as hard spheres, with a monolayer of beads forming around the cell surface, e.g. Fig.12The available volume of the cell surface occupied by the particle is obtained 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 volume of the cell from the volume of the sphere. Fig.12 Indicated as d 复合物 .

[0060] V 壳 =V 复合物 -V 细胞

[0061]

[0062]

[0063]

[0064] In the above calculation, the volume of the shell around the cell is multiplied by the spherical packing factor of 0.64 (random packing of equal spheres) and divided by the volume of a single microsphere to determine the number of microspheres that can bind to the cell. The packing factor assumes random packing of spheres. This is probably a conservative estimate of how many spheres can be packed around a larger central sphere.

[0065] By determining as an upper limit the maximum number of microparticles that can fit around a cell, estimates of the density of microparticle-cell complexes can be determined for beads of various sizes. Fig.13 The table shown in describes the theoretical density (g / cm) of particle-cell complexes with various sized particles over a range of particle diameters and numbers of particles bound per cell. 3 The density of the particles used in the calculation is 1.18 g / cm 3 The cell density used in the calculations was 1.063 g / cm 3 , with a diameter of 11.5μm. Fig.13 In the table shown in , the table cells highlighted in dark gray represent complexes with approximately 40-60% coverage of the cell surface by microparticles. The table cells highlighted in light gray represent complexes with approximately 80-100% coverage of the cell surface by microparticles. 3 The composites are marked with a single or double asterisk. 3 The value used for Fig.13The specific calculation is shown because it represents the cutoff density for the specific conditions of an exemplary magnetic levitation experiment. Complexes with densities below the cutoff value will be collected in the bottom portion of the processing channel of the flow cell cassette, thereby being separated from complexes with densities above the cutoff value, which will be suspended higher in the top of the processing channel of the flow cell cassette. Other cutoff values ​​may be used depending on the specific experimental conditions and desired results. Fig.13 The calculations shown in estimate that for larger particles, fewer particles are needed per cell to increase the density of the complexes above the chosen cutoff, but there is also less space available on the cell surface to accommodate these larger particles. Fig.13 The calculations shown determine that microparticles with diameters less than 3 μm will not form complexes at densities above the chosen cutoff because it would require >100% coverage of the cell surface by microparticles. Based on the calculations shown, microparticles with diameters of approximately 4-5 μm can achieve densities above the chosen cutoff with approximately 50% coverage of the cell surface.

[0066] Fig.13 The calculations shown in estimate the number of microparticles per cell in the complex to achieve a specific density. Given that the binding kinetics of the density modifier are primarily driven by ligand-receptor binding interactions, a higher microparticle-to-cell ratio (PTC ratio discussed elsewhere in this disclosure) is required during complex formation to achieve a target ratio of microparticles per cell in the microparticle complex. In order to balance binding mechanics, steric hindrance, and diffusion of microparticles through solution to interact with cells, it is necessary to maximize the effective concentration of linker contained in the density modifier (which comprises microparticles and linker) during complex formation, for example, by selecting microparticles of smaller diameter so that the number of microparticles can be increased during complex formation. Another factor to consider is the product of the number of linker molecules bound to the surface of each microparticle and the total number of microparticles in the suspension. For example, microparticles with an average diameter of 150 nm, a 50% streptavidin coverage per particle, and four binding sites per streptavidin molecule will provide 1x10 -14 μmol concentration of biotinylated antibody conjugated to streptavidin. For 2x10 5 For a sample of 10 cells, a microparticle-to-cell ratio of 50,000:1 would yield an antibody concentration of approximately 1 μM, which is the dissociation constant (K) for a typical antibody interaction. D) (10-100 nm). In contrast, microparticles with an average diameter of 5 μm and a microparticle-to-cell ratio of 40:1 will still produce an antibody concentration of about 900 nm, with all other conditions remaining constant. However, in this case, the amount of antibody attached to each microparticle is approximately 1000 times greater. This limits the entry of antibodies into the cell during complex formation because all available antibodies are concentrated on a smaller total number of microparticles. It can be expected that for higher affinity linkers, lower PTC ratios will be required for efficient complex formation because the interaction between the linker and the cell is more likely to be sustained. Lower affinity linkers may require higher PTC ratios for efficient complex formation to achieve and maintain the target number of microparticles per cell in the complex.

[0067] Manufacturing and sourcing of microparticles

[0068] For example, Lu et al. "Modular and Integrated Systems for Nanoparticle and Microparticle Synthesis—A Review" Biosensors (2020) 10(11): 165. https: / / doi.org / 10.3390 / bios10110165 describes some methods for producing microparticles. Commercially available microparticles can also be used. Some exemplary sources of microparticles are Nanopartz (Loveland, Colorado, USA), Cospheric (Santa Barbara, California, USA), Creative Diagnostics (New York, New York, USA), Spherotech (Lake Forest, Illinois, USA), Bangs Laboratories (Fishers, Indiana, USA), PolyAn (Berlin, Germany), Polysciences (Warrington, Pennsylvania, USA), and Lab261 (Palo Alto, California, USA).

[0069] Connectors

[0070] "Connectors" are used to couple non-magnetic particles to components of interest (e.g., cells of interest, organelles, nucleic acids). Connectors specifically bind to cells or other analytes. An example of a connector is an antibody that specifically binds to a cell surface protein displayed on a cell of interest. Other types of connectors include aptamers, ligands (bound by 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 coupling Oligo-dT to microparticles, and then the microparticles are bound to the Poly-A tail at the end of the mRNA. In another example, total RNA can be removed from a sample by coupling random hexamer oligonucleotides to microparticles, and then the microparticles are bound to random RNA hexanucleotides.

[0071] Antibody

[0072] The term "antibody" and related terms are used in the broadest sense in the present disclosure to represent any product, composition or molecule containing at least one epitope binding site, meaning a molecule that can specifically bind to an "epitope" - a region or structure within an antigen. The term "antibody" encompasses whole immunoglobulins (i.e., complete antibodies) of any class, including natural, natural-based, modified and non-natural (engineered) antibodies and fragments thereof. The term "antibody" encompasses "polyclonal antibodies", which react to the same antigen, but can bind to different epitopes within the antigen, and "monoclonal antibodies" ("mAb"), meaning a substantially homogeneous antibody population or an antibody obtained from a substantially homogeneous antibody population. The antigen binding sites of the individual antibodies constituting the mAb group are composed of polypeptide regions with similar (although not necessarily identical) sequences. The term "antibody" also encompasses fragments, variants, modified and engineered antibodies, such as those artificially produced ("engineered") by recombinant technology. For example, the term "antibody" encompasses, but is not limited to, chimeric and hybrid antibodies, antibodies with dual or multiple antigenic or epitope specificities, and fragments 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".

[0073] Aptamers

[0074] 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 breast cancer cell surface marker HER-2) can be used to separate or remove tumor cells from tissue samples. Aptamers targeting tumor cell surface protein biomarkers and the selection of such aptamers are discussed in, for example, Mercier et al., Cancers (Basel) 9 (6): 69 (2017) doi: 10.3390 / cancers9060069.

[0075] Lipophilic tags

[0076] The linking agent can be a lipophilic tag. A lipophilic tag is a lipophilic molecule that can associate with lipid membranes such as cell membranes and organelle membranes and / or insert therein. Examples of lipophilic molecules include sterol lipids (e.g., cholesterol or tocopherol), steryl 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 with other particles. In addition, different samples can be labeled with differentially labeled lipophilic tags.

[0077] Other specific binding partners

[0078] 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, under specified conditions, a specific binding molecule or a composition containing it binds to its binding partner and does not bind to any other substance in a significant amount. The binding to any other substance other than the binding partner is generally referred to as "non-specific binding" or "background". The lack of significant amount binding is considered to be, for example, 2 / 3 of 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 oligonucleotide or polynucleotide, oligonucleotide or polynucleotide binding to protein or polypeptide (vice versa), protein to polypeptide, other protein or polypeptide binding, 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, engineered and naturally occurring nucleic acid or peptide aptamers can serve as specific binding molecules in embodiments of the present invention. This list is not intended to be limiting, and other types of specific binding molecules can be used. The term "target molecule" is used to represent a molecule or part thereof, including biomolecules (such as but not limited to proteins, peptides, lipids, nucleic acids, fatty acids or carbohydrate molecules such as oligosaccharides), or non-biological molecules (including small molecules, such as small molecule drugs or small molecule ligands). Specific binding molecules, such as antibodies, specifically bind to target molecules.

[0079] Indirect combination

[0080] Specific binding molecules, such as but not limited to antibodies, can be directly attached to density adjustment microparticles, such as by surface conjugation, coating or adsorption. However, the specific binding molecule does not need to be directly attached to the density adjustment microparticles, and can be used to complex the density adjustment molecule with the target cell through an intermediate non-covalent binding interaction. For example, the specific binding molecule is a biotinylated antibody capable of specifically binding to the target cell, and the density adjustment microparticles are coated with avidin, streptavidin, neutravidin or any form of modified avidin, which can be referred to as an "avidin-like compound". The intermediate binding interaction between the avidin-like compound on the density adjustment microparticle and the biotin portion of the antibody allows the target cell and the density adjustment microparticle to form a complex. In another example, the specific binding molecule is an antibody capable of specifically binding to the target cell ("primary antibody"), and the density adjustment microparticles are coated with protein A, protein S or anti-antibody (i.e., an antibody against the primary antibody). The intermediate binding interaction between the protein A, protein S or anti-antibody on the density-changing microparticles and the primary antibody allows the target cell and the density adjustment microparticles to form a complex.

[0081] Fluidics

[0082] As used in this disclosure, the term "fluidics" refers to a system, device or element for manipulating, processing, ejecting and / or analyzing a fluid sample, which includes at least one "channel" as defined elsewhere in this disclosure. The term "fluidics" includes, but is not limited to, microfluidics and nanofluidics.

[0083] As used in this disclosure, the terms "channel," "flow channel," "fluidic channel," and "fluidic channel" are used interchangeably and refer to a path on a fluidic device through which a fluid can flow. A channel includes a path having a maximum height dimension of about 100 mM, about 50 mM, about 30 mM, about 25 mM, about 20 mM, about 15 mM, about 10 mM, about 5 mM, about 3 mM, about 2 mM, about 1 mM, or about 0.5 mM. For example, the channel between the magnets may have the following cross-sectional dimensions (height times width): about 10mM×10mM, about 10mM×5mM, about 10mM×3mM, about 10mM×2mM, about 10mM×1mM, or about, 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×1 In some embodiments, the channel can be shaped like a channel of about 100 mm, about 200 mm, about 300 mm, about 150 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about 200 mm, about The term "channel" includes, but is not limited to, microchannels and nanochannels, and for any reference to a channel in this disclosure, such a channel may include a microchannel or a nanochannel.

[0084] As used in this disclosure, the term "fluidly coupled" or "fluidically connected" means that a fluid can flow between two components so coupled or connected.

[0085] magnetic levitation

[0086] The expression "magnetic levitation" in the context of the present disclosure and as described, for example, in U.S. Patent Application No. US14 / 407,736 generally relates to suspending a diamagnetic, paramagnetic, ferromagnetic, ferrimagnetic or antiferromagnetic material or "object" in a paramagnetic fluid medium to a magnetic field, such as a magnetic field gradient formed between two magnets. The magnetic field produces a non-uniform pressure 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 region. In fact, the object is displaced by an equal volume of the paramagnetic fluid medium. The attractive interaction between the paramagnetic fluid medium and the high magnetic field region can result in the "levitation" of the object. In a dual magnet setup, 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 levitated 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 opposite to another uniform force (e.g., gravity), the equilibrium reached by the object is directly related to its density.

[0087] Paramagnetic fluid medium

[0088] In the context of the present disclosure, a "paramagnetic fluid medium" can be a paramagnetic material and a solvent. In some embodiments of the methods described in the present disclosure, the paramagnetic fluid medium is biocompatible, i.e., can be mixed with living cells without affecting the viability of the cells or affecting 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 [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 an amount 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 The paramagnetic fluid medium may be present in a concentration of 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 also include other components, such as salts or additives, such as, but not limited to, additives that function to maintain cell integrity.

[0089] Magnetic levitation systems and components

[0090] For example, exemplary magnetic levitation systems and components thereof are described in Durmus et al. and in 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 methods, kits, and systems according to embodiments of the invention. Figure 1 An exemplary magnetic levitation system is schematically shown.

[0091] Flow cell box

[0092] 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 box is schematically shown. The exemplary flow cell box can include a planar substrate, which includes 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 that is fluidically connected to the inlet channel and positioned substantially parallel to the longitudinal side, a sample flow divider in the processing channel, a plurality of outlet channels that are fluidically connected to the processing channel, and a plurality of collecting holes that are fluidically connected to each of the plurality of outlet channels. The planar configuration allows all necessary flow cell box functions to be integrated into the flow cell box, and improves performance and reproducibility in a laboratory or clinical environment. In operation, in order to enhance performance, it is important that the processing channel and the flow entering the outlet channel are as free of turbulence as possible. The processing channel can be offset in the plane of the planar substrate to be spatially biased to the imaging surface.

[0093] The flow cell box can be formed by injection molding, etching, laser ablation, machining or 3D printing. When imaging is required in the flow cell box, the planar substrate includes an optically transparent material. Glass, plastic or a polymer material including cycloolefin polymer (COP) or cycloolefin copolymer (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 1.5mM in thickness. The optional range is at least 100mM in length, 35mM in width, and about 2 to about 6mM in thickness. The longitudinal side of the box can serve as a waveguide for illumination and imaging. For this reason, the processing channel is offset in the plane of the substrate and 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 150 μL, at least about 200 μL, at least about 250 μL, or at least about 300 μL. The combined volume of the outlet channels can be greater than the volume of the processing channel. The flow rate distributed between the two outlet channels can be evenly distributed or can vary 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 when operating in a system embodiment, can vary from 1:1 by about 50% or less, or about 40% or less, or about 30% or less, or about 15% or less.

[0094] The flow cell box optionally includes a collection hole on a planar substrate. The collection hole has an inlet that is in fluid communication with the outlet channel. The inlet can be at a first hole height and is configured with 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 sample fraction from flowing back into the outlet channel and forming bubbles in the collection hole. The outlet channel in the collection hole can be provided with an opening, the height of the opening from the bottom plate of the collection hole is higher than the opening of the inlet channel. The internal outlet can be arranged to be connected to a flow regulator. In some cases, the flow regulator is a separate pump to provide a flow through the flow cell box. In operation, the collection hole is sealed with a layer of material or film to provide a closed system to allow the sample and sample fraction to flow or pump through the flow cell box. When using an adhesive when assembling the flow cell box layer, a biocompatible adhesive can be used for biological applications. Correct adhesive selection is necessary to minimize or prevent the adhesive component from leaching into the solution, adhering to the binding molecules in the cell or solution, having autofluorescence, having a texture that increases the surface area and thus increases the effect on the cell, and excessive hydrophilicity or hydrophobicity. Examples of suitable adhesives are silicone or silicone-based adhesives.

[0095] Separation system

[0096] Exemplary magnetic levitation-based separation system ( Figure 1 ) includes a receiving block for holding a flow cell box, 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 box in optical alignment with the optical system, removably engages a magnetic component of a processing channel adjacent to the flow cell box, and removably places a plurality of outlet channels of the flow cell box in fluid communication with a plurality of flow regulating components. The optical system may be configured to provide microscopic imaging of the processing channel of the flow cell box. Optionally, the optical system may be constructed and arranged to provide imaging of fluorescence emission with 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 box in an orientation relative to the optical system so that the imaging optical device is aligned with the imaging side of the planar box, and the visible light emitter is in a direction that illuminates the illumination side of the planar flow cell box. Optionally, the optical system may further include one or more UV or visible illumination sources constructed and arranged to direct UV illumination at an angle to the imaging side of the planar box to excite fluorophores within the processing channel of the box. To image fluorescent entities within the processing channel, the optical system optionally includes a dual bandpass filter that preferably passes emitted radiation in a band centered around wavelengths of about 524 nm and 628 nm.

[0097] An optional feature of the receiving block is a series of flow regulator adapters that engage outlets on the top or bottom of the flow cell box. The adapters facilitate fluid communication with a flow regulator (e.g., a pump in the system) and with an outlet channel of the flow cell (e.g., a collection hole outlet channel). After the flow cell box is inserted into the receiving block, the receiving block is mechanically driven to support the box, align the illumination side and imaging side of the planar box with the optical imaging system, align the magnetic components to position them above and below the flow cell processing channels, and place the flow regulator adapters to be in fluid communication with the corresponding outlet channels of the flow cell box when necessary. The system's flow regulators provide flow for samples and sample fractions within the flow cell box. During the separation process, the flow rate provided by the flow regulator can range from as low as 1 μL per minute to as high as 1 mL per minute. The flow rate may be at least about 25 μL per minute, at least about 50 μL per minute, at least about 100 μL per minute, at least about 200 μL per minute, at least about 200 μL per minute, at least about 250 μL per minute, at least about 300 μL per minute, or about 300 μL per minute to about 1 mL per minute. 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. The flow rate distributed between the two outlet channels can be evenly distributed or can vary 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 when operating in a system embodiment, can vary from 1:1 to about 50% or less, or about 40% or less, or about 30% or less, or about 15% or less.

[0098] The magnetic suspension system can make the particles in the paramagnetic fluid medium suspended in the processing channel or the inlet channel of the flow cell box magnetically levitate. The interaction of the paramagnetic properties of the particles in the magnetic field and the paramagnetic fluid medium can provide repulsion or attraction to the particles to promote their separation or concentration. The magnetic field in the magnetic fluid medium is produced by a magnet, which can be a permanent magnet or an electromagnet. The maximum magnetic 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 be in the range of about 0.01 Tesla to about 100 Tesla, or about 1 Tesla to about 10 Tesla. The remanence of the magnet can be in the range of about 0.5 Tesla to about 5 Tesla, or about 1 Tesla to about 3 Tesla. The magnets may be made of materials including neodymium alloys of iron and boron, neodymium, alloys of aluminum and nickel, alloys of neodymium and iron, alloys of aluminum and cobalt and iron, samarium cobalt, alloys of other rare earth elements and iron, alloys of rare earth elements and nickel, ferrite, or combinations thereof. When the magnetic levitation system includes multiple magnets, the magnets may be made of the same material, or of different materials.

[0099] An asymmetric magnetic field can be achieved by using a stronger magnetic material on one side of the fluid channel of the flow cell box and a weaker magnetic material on the opposite side of the fluid channel of the flow cell box. An asymmetric magnetic field can be achieved by placing a magnet closer to one side of the channel than the other side. An asymmetric magnetic field can be achieved by using a magnetic material on one side of the fluid channel of the flow cell box and using a substantially similar magnetic material on the opposite side of the fluid channel of the flow cell box. 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 multiple upper magnets and multiple lower magnets positioned around the fluid channel of the flow cell box. In one example, the upper magnet can include a front upper magnet, a central upper magnet, and a rear upper magnet. The lower magnet can include a front lower magnet, a central lower magnet, and a rear lower magnet. In another example, the magnetic levitation system may 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 fluid channel of the flow cell box, and the front upper magnet and the rear lower magnet are located in the direction of magnetic repulsion. Exemplary NdFeB magnetic component dimensions include, for the bottom magnet component, about 50×15×2mM (magnetized by a 15mM axis); for the top magnet component, about 50×5×2mM (magnetized by a 5mM axis). Other exemplary magnet dimensions include 60×15×2mM, 60×5×2mM, 75×10×3mM, 75×20×3mM, and 25×15×2mM. An exemplary magnet configuration for a magnetic levitation system includes an upper magnet and a lower magnet having dimensions of about 75×20×3.2 mM, and a spacing between the upper magnet and the lower magnet of 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 levitation system has an upper magnet and a lower magnet, wherein the lower magnet extends into the inlet channel of the flow cell box. The bottom magnet dimensions can be about 50 mM to about 100 mM×about 10 mM to about 30 mM×about 2 mM to about 4 mM.

[0100] sample

[0101] Unless otherwise limited, the term "sample" 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. In general, the term "sample" is not intended to be limited by its source, origin, procurement method, treatment, processing, storage or analysis or any modification. Some examples of samples are solutions, suspensions, supernatants, sediments or agglomerates. Samples can contain cells or tissues or be mainly composed of cells or tissues, or can be prepared from cells or tissues. However, samples do not need to include cells. Samples can be mixtures of biomolecules or contain biomolecules, such as nucleic acids, polypeptides, proteins (including antibodies), lipids, carbohydrates, etc. Samples can be biological samples. For example, "sample" can be any cell or tissue sample or an 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. Samples can be obtained directly from organisms, 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, lung lavage fluid, or interstitial fluid), tissue sections including needle biopsies, microscope slides, frozen tissue sections, or fixed cell and tissue samples.

[0102] Exemplary cell types

[0103] For the purpose of illustration and not limitation, the cells labeled with the density modifier can be human cells, non-human animal cells, plant cells, eukaryotic cells, etc. For example, the labeled cells can be, but are not limited to, human or non-human immune cells, endothelial cells, and T cells. The labeled cells can be in a heterogeneous population of unlabeled cells, which can be human cells, non-human animal cells, plant cells, eukaryotic cells, prokaryotic cells, etc. for the purpose of illustration and not limitation. The labeled cells can be from different lineages, or can be the same lineage, but with different activation states, differentiation states, or some other characteristics.

[0104] Labeling cells

[0105] One or more than one (2,3,4 or more than 4) cell types can be processed in a single separation step. When marking two or more cell types, different density modifiers can be used to mark each cell type. Alternatively, the same density modifier can be used to mark two or more different cell types. For example, two or more different cell types can have the same surface marker, and the density modifier comprising the same linking agent can therefore be combined with the two cell types. In another example, the density modifier comprises two or more different linking agents, which can be combined with corresponding two or more surface markers. In this case, the density modifier can be combined with two or more different cell types with 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 density modifiers with the same density.

[0106] Marking process

[0107] As part of the labeling process, microparticles containing a linking agent specific to the target cell type can be mixed with cells at a ratio optimized for the target cell type. The ratio of microparticles 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 characteristics, but can also affect the number of microparticles containing the linking agent that each cell can complex with. For example, if the linking agent is specific to a cell surface marker, the number of markers on a specific target cell type will determine how many units of microparticles containing the linking agent can bind to the cell. Another parameter can be the affinity of the linking agent (e.g., an antibody) for a specific cell type. Another parameter is the strength of the magnetic field applied during magnetic levitation. Other parameters that can be considered when determining the PTC ratio can be microparticle size and / or microparticle density. It should be understood that other parameters not listed here can also be considered, and for specific labeling and / or separation applications, the PTC ratio can be experimentally determined and / or optimized. 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 100,000, about 10,000 to about 50,000, or about 50,000 to about 100,000. When multiple cell types are present, microparticles with different linkers specific for each cell type can be mixed together before being added to the cell mixture, or microparticles with different linkers and cells 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.

[0108] Design and selection of density regulator

[0109] The design and selection of one or more density regulators and amount thereof used in embodiments of the present invention have considered various situations.For example, some cells of the same type can be marked with the same density regulator of different numbers.Therefore, the cells of the same type marked with the same density regulator may have different densities and suspension heights.In some cases, this problem can be solved at least in part by adjusting the ratio of the estimated number of target cells in the density regulator and the sample, because the difference in the number of density regulators of the target cell type can be more obvious than other ratios under certain ratios.In another example, the target cells with the same number of surface markers per surface area can be marked with the density regulator unit of different numbers.In some cases, this problem can be solved at least in part, for example, by increasing the ratio of the estimated number of target cells in the density regulator and the sample in an attempt to saturate all available surface markers.In other examples, this problem can be solved at least in part by increasing the incubation time of density regulator and target cells to reach saturation, increase the amount of the antibody combined with the density regulator to increase local concentration or use the binding molecule with increased affinity to the cell surface target.In another example, some cells can show multiple cell surface markers and can be marked (intentionally or unintentionally) with lower and higher density modifiers. This may produce multiple complex populations containing different cells and beads at different ratios. In some cases, this may make the separation between different cell populations difficult. If this problem occurs, it can be at least partially solved, for example, by increasing the incubation time of the density modifier with the target cells to reach saturation, increasing the amount of the antibody bound to the density modifier to increase the local concentration, or by using a binding molecule with increased affinity for the cell surface target. In another exemplary method, a subset of cells with multiple surface markers can be labeled with two different density modifiers so that the density change is regulated between no labeling and all-one labeling. It should be understood that the method discussed above can be used in various combinations of two or more methods.

[0110] Cell separation methods

[0111] Described in the present disclosure and included in embodiments of the present invention is an improved method (process) for cell separation by magnetic levitation. Such methods may also be referred to as "cell separation methods", "methods for separating cells", "methods for cell separation", "cell separation methods", "methods for separating cells", "cell concentration methods", "methods for concentrating cells", "cell separation methods (cell segregation methods)", "methods for separating cells" and other related terms and expressions, which are not intended to be limiting.

[0112] 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. This variety of cell types can include animal cells, including human cells and non-human animal cells, a mixture of human and non-human cells, plant cells and cells from other sources, including but not limited to bacterial cells, protozoan cells, algae cells, etc. Various cell types can 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 can include cells in various states (for example, stem cells, differentiated cells, etc.). Cells separated according to the method disclosed herein can be obtained directly from an organism (or the organism itself), or propagated or cultured. Before separating cells according to the method described in the present disclosure, cells can be subjected to various treatments, storage or processing procedures. In general, the terms "cell", "cell type" (or related terms and expressions) are not intended to be limited by their source, origin, acquisition method, treatment, processing, storage or analysis 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, erythrocytes, epithelial cells, egg cells, sperm cells, T cells, B cells, bone marrow cells, immune cells, hepatocytes, endothelial cells, stromal cells, and bacterial cells. Cell populations comprising a variety of cell types can be derived from various types of samples, which are discussed elsewhere in the present disclosure.

[0113] The cell separation methods according to some embodiments of the present invention involve performing density-modified microparticles and binding of a specific type or types of cells (which may be referred to as "target cells" or "target cell types") found in a cell population comprising a plurality of cell types. Such binding may also be described as "forming a complex," "complex formation," or "complexing." Preferential binding of density-modified microparticles to target cells is achieved by using binding molecules that are capable of specifically or selectively binding to target cells. Such molecules may be referred to as "specific binding molecules." An example of a specific binding molecule is an antibody specific for a cell surface marker or a molecule specific for a target cell type. 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, nucleolar protein 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, may 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.

[0114] In embodiments of cell separation methods involving density-changing microparticles combined with specific types or multiple types of cells, the combination can be accomplished by various steps. For example, the combination can be achieved by contacting, combining or incubating a cell group containing multiple cell types (possibly including a target cell type) with a density-changing microparticle containing a specific binding molecule under certain conditions, wherein the density changes the microparticles in combination with a single cell of a target cell type to form a complex, and each complex, one or more microparticles are bound to the cell of the target cell type. In another example, the combination can be achieved by contacting, combining or incubating a cell group containing multiple cell types (possibly including a target cell type) with a specific binding molecule. However, the embodiment of the cell separation method according to the present invention does not need to include any steps related to the combination of microparticles with cells with density changes. The density changes the complex of microparticles and target cells can be formed before the method starts, and is provided at the beginning of the cell separation method according to an embodiment of the present invention. In other words, the method can start with providing one or more densities to change the step of a complex of cells (or target cells) of microparticles and target cell types, optionally included in a cell group containing multiple cell types.

[0115] Some embodiments of the cell separation method according to the present invention include one or more steps, which involve forming a suspension of a complex of one or more density-changing microparticles and cells of target cell types and multiple cells of multiple 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 includes introducing the suspension into the processing channel of the flow cell box of the magnetic suspension system. The flow cell box includes at least one outlet channel and a processing channel with a certain length and vertical height. The cell separation method according to an embodiment of the present invention includes exposing the processing channel to a magnetic field for a period of time, which is sufficient to separate at least some (or at least one complex) of the complex of one or more density-changing microparticles and cells of target cell types from cells of multiple cell types that are not bound to the density-changing microparticles, 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 the magnetic field can be performed in a stop flow mode or a continuous flow mode of the flow cell box.

[0116] In some embodiments of the cell separation method, the vertical position of the flow cell box in the magnetic field is variable, which can affect the suspension height of the complex (or at least one complex) of the cell of one or more density-changing microparticles and target cell types relative to the magnet of the magnetic suspension system. Changing the vertical position of the flow cell box 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 the paramagnetic compound changes the physical space occupied by a certain range of densities, so that the specific density range in the separation channel can be targeted by adjusting the concentration of the paramagnetic compound. With the increase of the concentration of the paramagnetic compound 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 the particles of different densities will become smaller. On the contrary, with the reduction of the concentration of the paramagnetic compound in the paramagnetic fluid, the particle density range that can be suspended between the magnets narrows, but the physical separation distance between the 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).

[0117] In an embodiment of a cell separation method utilizing a plurality of specific binding molecules capable of forming complexes with different target cell types in a cell population containing a plurality of cell types, more than two suspension portions may be formed after exposure to a magnetic field. For example, if a method utilizes two different specific binding molecules capable of binding to two different target cell types in a cell population containing a plurality of cell types, two different types of complexes may be formed in the suspension when exposed to a magnetic field: a first complex of a first type of microparticle and a single cell of a first target type, and a second complex of a second type of microparticle and a single cell of a second target type. In this case, the first type of microparticle and the second type of microparticle are selected so that the density of the first complex is different from the density of the second complex, and is also different from the density of other types of cells in the group. In one example, when the flow cell box is exposed to a magnetic field, at least three different portions of the suspension will subsequently be formed in the processing channel of the flow cell box: a portion enriched by the first complex, a portion enriched by the second complex, and a portion (or portions) depleted of the first complex and the second complex. In this case, different portions of the suspension (which may be referred to as "fractions") may require the same or different exposure times to the magnetic field to form.

[0118] The cell separation method according to an embodiment of the present invention may also include withdrawing different parts of the suspension from the processing channel of the flow cell box or from the flow cell box together. The withdrawal of different parts or fractions can be carried out through one or more outlet channels of the flow cell box and can involve flowing the suspension along the length of the processing channel.

[0119] An exemplary embodiment of a cell separation method is schematically shown in Figure 3 middle. Figure 3 The embodiment shown in is an example of a cell separation method that uses microparticles having a buoyant density lower than a target cell type (first type) in a cell population containing at least two cell types (first type and second type) and also including some dead cells. The living cells of the two different cell types ((1) - living cells of the first type; ((2) - living cells of the second type) have different surface markers (e.g., proteins, carbohydrates, or other biomolecules). The cell population is contacted with non-magnetic microparticles coupled to antibodies that are capable of specifically binding to surface markers of the first type of the two cell types. The non-magnetic microparticles bind to the surface markers of the first cell type and form a complex with the cells of the first type. The density of the complex is lower than the density of non-complexed cells of the first or second type. When subjected to magnetic suspension, the complex is suspended in the processing channel of the flow cell box higher than the cells of the second type that have not formed a complex with the microparticles. The dead cells of the first type (3) are also suspended lower than the complex. The dead cells of the second type may not have as many exposed surface markers, or may have a lower density than the living cells of the first type. A fraction enriched in complexes formed by the living cells of the first type is recovered from the flow cell box, resulting in the separation of the living cells of the first type.

[0120] Another exemplary embodiment of the cell separation method is schematically shown in Figure 4 middle. Figure 4 The embodiment shown in is to use microparticles with a buoyant density higher than that of the target cell type (second type) to separate the target cell type from a cell population containing at least two cell types (a first type (1) and a second type (2)) and also including some dead cells of the first type (3). The living cells of the two different cell types have different surface markers (e.g., proteins, carbohydrates, or other biomolecules). The cell population is contacted with microparticles coupled to antibodies that are capable of specifically binding to surface markers of the second type of the two cell types. The microparticles bind to the surface markers of the second cell type and form a complex with the cells of the second type. The density of the complex is higher than the density of non-complexed cells of the first type or the second type. When subjected to magnetic suspension, the complex is suspended in the processing channel of the flow cell box lower than the cells of the first type that have not formed a complex with the microparticles. Dead cells are also suspended lower than non-complexed cells. The fraction depleted of the complex is recovered from the flow cell box, resulting in the separation of the living cells of the first type.

[0121] Particle Separation Systems and Kits

[0122] Described in the present disclosure and included in embodiments of the present invention are kits and systems that can be used to separate particles (e.g., cells) by magnetic levitation. Exemplary kits include one or more types of density-modified non-magnetic microparticles that are capable of forming complexes with individual cells. Such non-magnetic microparticles are described elsewhere in the present disclosure. In addition to one or more types of density-modified non-magnetic microparticles, the kit may also include a paramagnetic fluid medium. In addition to one or more types of density-modified non-magnetic microparticles, the kit may also include one or more linkers. 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 boxes, or materials designed to optimize the consumption or recovery of target cells in a mixed cell population.

[0123] An exemplary system for separating particles is a cell separation system that includes one or more types of density-modified microparticles that are capable, alone or in combination with other agents, of preferentially binding to a target cell type in a cell population comprising a plurality of cell types and forming a complex of the microparticle and cells of the target cell type. The system also includes a flow cell cassette as described elsewhere in the present disclosure, a station including a holding block for the flow cell cassette, and one or more magnets positioned to expose a processing channel of the flow cell cassette located in the holding block to a magnetic field. Example

[0124] The following examples are offered to illustrate, but not to limit the claimed invention.

[0125] Example 1: Isolation of Jurkat cells using microvesicles

[0126] The isolation of Jurkat cells ("Jurkat") using microvesicles was completed and Figure 5-Figure 7. SIMB3-4 microbubbles were purchased from Advanced Microbubbles Inc. (Newark, California). Jurkat cells of the human T cell line (ATCC TIB-152, Jurkat clone E6.1) were stained with calcein AM (Thermo Fisher Scientific Inc.) at a final concentration of 10 μM to provide green fluorescence, and washed 3 times to remove excess stain. The stained Jurkat cells were mixed with H358 cells of the human lung cell carcinoma cell line that do not express CD45 (NCI-H358 obtained from the Berkeley Cell Culture Facility in Berkeley, California) to obtain a sample containing about 50% H358 cells and about 50% Jurkat cells ("H358 / Jurkat mixture"). The H358 / Jurkat mixture was incubated on ice for 1 hour with 1.25 μg / ml of biotinylated anti-CD45 antibody (clone HI30, Biolegend, San Diego, California), and then washed 4 times to remove unbound antibodies. The stained cells were mixed with streptavidin-conjugated microvesicles at a microvesicle:cell ratio of approximately 20:1 for 15 min at room temperature and (Eppendorf, Enfield, CT, USA) at 500 rpm. A suspension containing 1 M gadobutrol was then added to the cell / microbubble mixture to a final concentration of 75 mM, and the resulting suspension was loaded into the LeviCell TM The cells were placed in a flow cell box on a magnetic levitation platform (Levitas Bio, Menlo Park, CA, USA). Figure 5 Schematic representation of a complex of Jurkat cells stained with biotinylated antibodies and streptavidin-conjugated vesicles. The suspension was exposed to a magnetic field ("equilibrium") for 20 minutes within the LeviCell instrument and then flowed through the flow cell. In a series of suspension experiments, the cassette was either not raised from the bottom magnet (standard configuration) or raised from the bottom of the magnet with a 0.012" or 0.015" brass shim. The presence of the shim improves the position of the flow cell cassette between the two magnets, allowing the cassette to be located higher in the magnetic field, such as Figure 6 As shown. The above experiment illustrates the following embodiments of the present invention. Cells are suspended in a processing channel of a flow cell box at a fixed position in a magnetic field (i.e., between two magnets). The inherent position of the cell in the magnetic field, which is affected by cell density and magnetic susceptibility, can be called a "suspension profile." By moving the flow cell up or down, the cell can be located at a lower or higher position in the flow cell, respectively, while maintaining the suspension profile. The ability to adjust the position ("suspension height") of the suspended cell in the processing channel of the flow cell box can be used to improve the collection of separated fractions.

[0127] Figure 6 Shown is a photo image of a cell mixture using different gaskets at the end of the equilibrium phase in a flow cell, as shown in the image. The darker cell band near the middle of the flow cell corresponds to the H358 cell without microvesicles, because their surface does not bind CD45 antibodies. The Jurkat cells combined with microvesicles are suspended above the cell band, but do not form a single band in this case. According to cell size and the number of microvesicles combined with each cell, the Jurkat-microvesicle complex is suspended at a distance above its original position, and some of these Jurkat cell suspensions are up to the top of the flow cell. When using 0.015 " gasket to raise the flow cell, the complex that was in an invisible height (i.e., fixed on the channel top) before raising the flow cell does become visible. This proves that the Jurkat / bubble complex can raise the suspension height of Jurkat cells higher than the cells that are not combined with bubbles, and some of these complexes are raised to the top of the flow cell. Figure 7 The bar graph shown summarizes the percentage of Jurkat cells present in the sample at the input part, top part and bottom part (as shown) of the flow cell box.Echo fluorescence microscope is used to count cells on a disposable hemocytometer chip.Then the total cell count from 4 corner grids is divided by 4, then divided by 10,000 (conversion factor of hemocytometer chip) to obtain cell number / ml.Green fluorescence Jurkat cells are counted (due to calcein fluorescence staining) and compared with the total number of cells to obtain %Jurkat value.The top part is recovered from the flow cell, the unbound cells of the centrifugal precipitation, and the supernatant is counted separately from the precipitation to determine how many Jurkat cells are compounded with microvesicles and therefore float in the supernatant.The results show that, compared with the suspended sample input, the supernatant of the top fraction is enriched by Jurkat cells, and when the flow cell is raised using a gasket, the purity of Jurkat cells increases.As the flow cell rises with the gasket, the purity of the Jurkat cells present in the precipitation cells from the top part also increases. The bottom fraction was depleted of Jurkat cells compared to the input suspension sample.

[0128] Example 2: Isolation of Jurkat cells using polymer microbeads

[0129] The isolation of Jurkat cells using polymer microbeads was completed and Figure 8 and Fig. 9 12μmDiagPoly TMStreptavidin PMMA beads ("microbeads") were obtained from Creative Diagnostics (Alpharetta, Georgia). Jurkat cells (human T cell line ATCC TIB-152, Jurkat clone E6.1) were stained with calcein AM to provide green fluorescence as described in Example 1. H358 cells (NCI-H358 obtained from the Berkeley Cell Culture Facility in Berkeley, California) were stained with CellTracker Red CMPTX (Thermo Fisher Scientific Inc.) at a final concentration of 10 μM to provide red fluorescence. Both types of stained cells were washed three times to remove excess fluorescent dye. The stained Jurkat and H358 cells were mixed as described in Example 1. ～ 15% H358 cells / ～ 85% Jurkat cells ("H358 / Jurkat mixture"). As described in Example 1, the H358 / Jurkat mixture was incubated with biotinylated anti-CD45 antibody on ice for 1 hour, followed by 4 washes to remove unbound antibody. The antibody-stained cells were then mixed with microbeads at a 1:1 bead to cell ratio for 5 minutes at room temperature and (Eppendorf, Enfield, Connecticut, USA) and mixed at 500 rpm. Then, a suspension containing 1 M gadobutrol was added to the cell / microbubble mixture to a final concentration of 75 mM. The resulting suspension was loaded into the LeviCell TM The sample was placed in a flow cell box on a magnetic levitation platform (Levitas Bio, Menlo Park, CA, USA) and then flowed through the flow cell. Figure 8 Schematic representation of Jurkat cells ("Jurkat") complexed with streptavidin-conjugated microbeads using a biotinylated antibody linker.

[0130] Fig. 9 is a composite photographic image of bright field, red and green fluorescence of the frame at the end of the 20 minute equilibration step (reproduced in grayscale). Unbound H358 cells remain in the top band of the flow cell along with some unlabeled Jurkat cells (unlabeled live cells have the lowest density and lowest suspension profile distribution in the mixture). Below them are Jurkat cells complexed with PMMA microbeads. Their suspension profile distribution (LP) depends on how many beads are attached to each cell, with more beads causing the cells to suspend lower in the flow cell. At the bottom are the unbound PMMA microbeads, which have the highest density and highest suspension profile distribution. In Fig. 9 In the magnetic levitation experiment shown, the fraction containing H358 cells recovered from the flow cell was enriched to 47%, corresponding to a 92% depletion of Jurkat cells.

[0131] Example 3: Isolation of Jurkat cells using gold microbeads

[0132] The isolation of Jurkat cells using gold microbeads was completed and Fig.10 and 11 As shown in . Jurkat cells (described in Example 1) were labeled with 50 μg / ml of biotinylated anti-CD45 antibody (clone HI30, Biolegend, San Diego, CA) on ice for 1 hour and then washed 4 times to remove unbound antibody. Therefore, the labeled Jurkat cells were incubated with gold nanoparticles for 150 minutes ( Fig.10 "Jurkat + Gd 150min incubation" and Fig.11 Unlabeled Jurkat cells not incubated with gold nanoparticles ( Fig.10 "Single Jurkat" and Fig.11 The “without Au NPs” in the example was used as a control. Magnetic levitation was performed essentially as described in the previous example. Fig.11 The LP in the flow cell is calculated based on the position of the cell band in the middle of the suspension. Fig.10 The images captured during the experiments shown were used to calculate the average levitation value. Fig.11 The bars in the middle represent the thickness of the cell band. When the suspension reaches equilibrium, the approximate ～ After 10 minutes, cells incubated with gold nanoparticles for 150 minutes were suspended with a higher LP, translating to being suspended lower in the flow cell and therefore at a higher density compared to cells without any gold nanoparticles.

[0133] Example 4: Effect of particle size on cell separation

[0134] It may be advantageous to use a lower amount of density modifier for cell separation. However, it is necessary to ensure that at least one particle is bound to each cell, which requires a higher ratio of particles to cells to reach a saturation equilibrium binding concentration. Larger particles are provided at the same weight / volume concentration (mass concentration) as smaller particles, so a given volume of 12 μm beads in suspension contains about 8 times fewer particles than the same volume of 5 μm particles. In addition, as the size of the particles increases, their surface area also increases, so more linkers are required for each particle. Since the linker is fixed to the surface of the beads, the increase in the amount of linker per particle as the particle size increases does not directly translate into an increase in the "solution phase" linker concentration. Given any type of particle with a certain density but different sizes and at the same mass concentration, and assuming that the linker covers a fixed proportion of the particle surface area, regardless of particle size, the linker molecules are better distributed in a larger number of smaller particles (with fewer linker molecules per particle) than in fewer larger particles. Larger particles also take up a lot of space in the processing channel of the flow cell box, effectively setting an upper limit on the number of particles that can be used. The following experiments were conducted to test the effect of particle size on cell separation.

[0135] Isolation of Jurkat cells using polymer microbeads was performed essentially as described in Example 2, with the following modifications. Separate experiments were performed using 12 μm and 5 μm streptavidin-conjugated PMMA beads ("microbeads"). The microbeads were coated with biotinylated anti-CD45 antibodies to bind to all available biotin binding sites. The biotin binding capacity of the 5 μm microbeads was estimated by minimizing the biotin binding capacity of the 12 μm microbeads. The antibody-conjugated microbeads were conjugated to 15% CD45 阴性 / 85% CD45 阳性 The cell mixture was incubated with 12 μm and 5 μm beads at a bead to cell ratio of 1:1 and 60:1, respectively. The 12 μm beads at a 1:1 ratio depleted approximately 55% of the CD45 阳性 Cells. 阳性 The cell-bound 12 μm beads substantially reduced the resulting complex suspension height, but this fraction diffused in the processing channel and many beads were not bound to cells. The 5 μm beads depleted approximately 85-87% of CD45 阳性 cells, but requires a 60:1 bead-to-cell ratio. A higher bead-to-cell ratio may improve the performance of 12 μm beads, but a 60:1 ratio would require almost 12 times as much antibody as 5 μm beads when using the same concentration of antibody-coated beads. Furthermore, such a large number of large beads would far exceed the capacity of the flow cell processing channels.

[0136] It is theoretically predicted and experimentally confirmed that the binding of the suspension height changing agent or magnetic agent to the cell is most effective when the smallest possible size of particles is used, 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 the specific 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 and the surface area is 25 times. Assuming that a fixed surface area is required for each antibody binding event, 5 μm particles can bind 25 times more linker molecules than 1 μm particles. However, in solution, at the same particle weight / volume concentration, the concentration of 1 μm particles will increase 125 times. Therefore, at the same weight / volume concentration, the effective solution concentration of particle-bound linkers for 1 μm particles is 5 times that of 5 μm particles. Therefore, by using smaller particles and using a larger number of particles per unit volume, it is easier to reach the saturation concentration of particle-bound linkers in solution. Experiments have determined 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 the solution and large enough to effectively change the density of the complex of cells and density modifiers. The effect of microparticle size on the density of the complex of cells and density modifiers is described in Fig.13 , which is discussed elsewhere in this disclosure.

[0137] It should be understood that the examples and embodiments described in the present disclosure are for illustrative purposes only, and that those skilled in the art will suggest various modifications or changes thereto, and that these modifications or changes will be included within the spirit and scope of the present application and within the scope of the appended claims. All publications, patents, and patent applications cited in the present disclosure are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for cell separation, comprising: a) binding a first density modifier to a first type of cell in a cell population comprising a plurality of cell types, wherein the first density modifier comprises a first non-magnetic 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 density modifier bound to a single cell of the first type; b) forming a suspension in a paramagnetic fluid medium, the suspension comprising a plurality of first complexes and a plurality of cells of a 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 by the first density modifier 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. 2 . The method of claim 1 , wherein the first linking agent comprises a first antibody or a domain of the first antibody that is capable of specifically binding to a first moiety on the surface of the first type of cell.

3. The method of claim 2, wherein the combining of the first density modifier comprises: binding of a first antibody or a domain of said first antibody that is capable of specifically binding to a first moiety on the surface of said first type of cell; and, The first antibody or the domain of the first antibody is bound to the first non-magnetic microparticle. 4 . The method according to claim 1 , wherein the first linking agent is bound to the first non-magnetic microparticle via a first one or more covalent or non-covalent interactions.

5. The method according to any one of claims 1 to 4, further comprising: e) withdrawing at least a portion of the first part or withdrawing at least a portion of the second part from the processing channel.

6. The method according to claim 5, further comprising: f) withdrawing at least a portion of the first portion or withdrawing at least a portion of the second portion from the flow cell cartridge.

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

8. The method according to any one of claims 1 to 7, further comprising: In step (a), a second density modifier is bound to a second type of cells in a cell population comprising a plurality of cell types, wherein the second density modifier comprises a second non-magnetic particle and a second linker that preferentially binds to the second type of cells of the plurality of cell types, wherein the binding of the second density modifier is performed under the condition that the second density modifier binds to a second type of single cell to form a second complex, each second complex comprising the second density modifier bound to the second type of cell, wherein the density of the second complex is different from the density of the second type of cells, the density of the first complex, and the density of other types of cells of the plurality of cell types; In step (b), the suspension further comprises a plurality of the second complexes; In step (d), the flow cell box 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 by the second density modifier, from the first complexes, and from other types of cells 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 in the second complex relative to the suspension.

9. The method of claim 8, wherein the first portion and the third portion are identical, and / or the second portion and the fourth portion are identical.

10. The method of claim 8 or 9, 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 second type of cell.

11. The method of claim 10, wherein the combining of the second density modifier comprises: The second antibody or the domain of the second antibody that is capable of specifically binding to the second part on the surface of the second type of cell binds to the second part on the surface of the second type of cell; and, Binding of the second antibody or a domain of the second antibody to the second microparticle.

12. The method of any one of claims 8 to 11, wherein the second linking agent is bound to the second non-magnetic microparticle via one or more non-covalent interactions.

13. The method of any one of claims 4 to 12, wherein the one or more non-covalent interactions of the first linker or the second linker independently comprise one or more of an interaction between biotin and an avidin-like molecule, an antibody-antigen interaction, or an interaction between an antibody binding protein or a domain of an antibody binding protein and its binding partner.

14. The method according to any one of claims 8 to 13, wherein the first time period and the second time period coincide.

15. The method according to any one of claims 8 to 10, further comprising: f) Retracting at least a portion of the third part or retracting at least a portion of the fourth part from the processing channel and / or from the flow cell cartridge.

16. The method of claim 15, wherein step (f) is performed through a second outlet channel of the flow cell cartridge.

17. The method according to any one of claims 1 to 16, wherein step (d) is performed in a stopped flow mode or a continuous flow mode of the flow cell cartridge.

18. A method according to any one of claims 5 to 17, wherein steps (e) and (f) comprise flowing the suspension along the length of the processing channel.

19. The method according to any one of claims 5 to 18, wherein steps (e) and (f) are simultaneous or sequential.

20. The method of any one of claims 1 to 19, wherein the vertical position of the flow cell cassette in the magnetic field is changeable.

21. The method of any one of claims 1 to 20, wherein the first microparticles and / or the second microparticles are polymeric microparticles.

22. The method of claim 21, wherein the polymer microparticles comprise one or more of polymethyl methacrylate (PMMA), polystyrene, polypropylene, or polyethylene.

23. The method of claim 21 or 22, wherein the polymer microparticles are polymer microbeads or polymer microbubbles.

24. The method according to any one of claims 1 to 20, wherein the first particles and / or the second particles are glass particles.

25. The method of claim 24, wherein the glass particles are glass microbeads or glass microbubbles.

26. The method according to any one of claims 1 to 19, wherein the first particles and / or the second particles are non-magnetic metal particles.

27. The method of claim 26, wherein the non-magnetic metal particles are microbeads or microbubbles.

28. The method of claim 26 or 27, wherein the non-magnetic metal particles comprise gold, silver or platinum.

29. The method of any one of claims 1 to 20, wherein the first microparticles and / or the second microparticles are microbubbles.

30. The method of claim 29, wherein the microvesicles are lipid microvesicles.

31. The method of any one of claims 1 to 30, wherein the one or more cells of the first type and / or the one or more cells of the second type are living cells.

32. The method of any one of claims 1 to 31, wherein one or more cells of the first type in the first complex and / or one or more cells of the second type in the second complex are living cells.

33. A magnetic levitation kit comprising a paramagnetic fluid medium and one or more density modifiers or separate components of said one or more density modifiers, which are capable of forming complexes with single cells, wherein the density of each complex is different from the density of a single cell, and wherein each density modifier comprises non-magnetic microparticles and a linker that preferentially binds to a target cell type.

34. The kit of claim 33, wherein the non-magnetic microparticles are microbeads or microbubbles.

35. The kit of claim 34, wherein the non-magnetic particles are metal particles or polymer particles.

36. The kit of claim 35, wherein the metal particles comprise gold, silver or platinum.

37. The kit of claim 35, wherein the polymer microparticles comprise one or more of polymethyl methacrylate (PMMA), polystyrene, polypropylene or polyethylene.

38. The kit of claim 34, wherein the non-magnetic microparticles are lipid microbubbles.

39. The kit of claim 34, wherein the non-magnetic particles are glass particles.

40. The kit of any one of claims 33 to 39, wherein the linking agent is an antibody or a domain of the second antibody capable of specifically binding to a second moiety on the surface of the target cell type.

41. The kit according to any one of claims 33 to 40, wherein the linking agent is covalently bound to the non-magnetic microparticles.

42. A kit according to any one of claims 33 to 41, wherein the kit comprises one or more types of non-magnetic microparticles and one or more types of linking agents as separate components of the one or more density modifiers, wherein the one or more types of non-magnetic microparticles are surface modified with moieties capable of mediating non-covalent interactions with the one or more types of linking agents.

43. The kit of claim 42, wherein the non-covalent interaction comprises one or more of an interaction between an aptamer binding protein and its aptamer, an interaction between biotin and 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.

44. A system for cell separation, comprising a first non-magnetic microparticle capable of, alone or in combination with a first other agent, 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 microparticle and the first type of cells, the first complex having a density different from that of the first type of cells and of cells of other types different from the plurality of cell types; a flow cell cartridge comprising a first outlet channel and a processing channel; a station comprising a holding block for a flow cell cartridge and one or more magnets positioned to expose 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 box to the magnetic field allows the first complex to be separated from cells of multiple cell types that are not bound by the first non-magnetic particles and from other types of cells of the multiple cell types in the processing channel, and the processing channel of the flow cell box contains a suspension of cells of the multiple cell types in a paramagnetic fluid medium.

45. The system of claim 44, further comprising: second non-magnetic microparticles, capable of, alone or in combination with a second agent, preferentially binding to a second type of cells in the cell population and forming a second complex of the second microparticles and the second type of cells, the density of the second complex being different from the density of the second type of cells, different from the density of the first complex, and different from the density of other types of cells in the plurality of cell types, Wherein exposing the processing channel of the flow cell box to the magnetic field allows the second complex to be separated from cells of multiple cell types that are not bound by the second non-magnetic particles, from the first complex, and from other types of cells of the multiple cell types in the processing channel, and the processing channel of the flow cell box contains a suspension of cells of the multiple cell types in the paramagnetic fluid medium.

46. ​​The system of claim 44 or 45, wherein the first microparticles and / or the second microparticles are polymer microparticles.

47. The system of claim 46, wherein the polymer microparticles comprise one or more of polymethylmethacrylate (PMMA), polystyrene, polypropylene, or polyethylene.

48. The system of claim 46 or 47, wherein the polymer microparticles are polymer microbeads or polymer microbubbles.

49. The system of any one of claims 44 to 48, wherein the first particles and / or the second particles are glass particles.

50. The system of claim 49, wherein the glass particles are glass microbeads or glass microbubbles.

51. The system of any one of claims 44 to 50, wherein the first particles and / or the second particles are non-magnetic metal particles.

52. The system of claim 51, wherein the non-magnetic metal particles are microbeads or microbubbles.

53. The system of claim 51 or 52, wherein the non-magnetic metal particles comprise gold, silver, or platinum.

54. The system of any one of claims 44 to 53, wherein the first particles and / or the second particles are microbubbles.

55. The method of claim 54, wherein the microvesicles are lipid microvesicles.

Images