Magnetic separation

Abstract

To provide a method for collecting a target biological population from a biological sample in an automated cell culture system.SOLUTION: The method is provided repeating the following steps: a. binding the target biological population to the magnetic particles; b. circulating the biological sample through one or more fluid pathways of the automated cell culture system; c. exposing a target biological population bound to magnetic particles to a magnetic field gradient to capture the target biological population bound to the magnetic particles; d. circulating the unbound components of the biological sample through one or more fluidic pathways of the automated cell culture system; e. inserting a magnetic field shield / barrier between the target biological population bound to magnetic particles and the magnetic field to release the target biological population bound to magnetic particles; and f. circulating the target biological population bound to the magnetic particles through one or more fluid pathways of the automated cell culture system.SELECTED DRAWING: Figure 21

Description

Technical Field

[0001] The present invention relates to a device, method, and system for automatically magnetically separating a target from a biological sample. The device, method, and system have been found to be useful in a variety of clinical and experimental settings.

Background Art

[0002] Magnetic separation has been utilized as a method for separating magnetic impurities from fluids through the application of various different processes (U.S. Pat. Nos. 3,985,646, 4,054,513, and 5,137,629). Magnetic separation technology has also been applied to the separation of populations of biological materials using magnetic beads coated with antibodies or polymers that bind to various biological targets including viruses, bacteria, and cells (U.S. Pat. Nos. 3,970,518, 4,219,411, 4,795,698, and 5,385,707). Subsequently, the biological targets can be extracted from the fluid suspension using, for example, one of the previously developed magnetic separation devices described in U.S. Pat. Nos. 4,710,472, 5,691,208, 6,193,892, and Zborowski et al. (Journal of Magnetism and Magnetic Materials, vol. 194, pp. 224 - 230, 1999). The magnetic field generated by the separation device exerts a force on the magnetic beads suspended within the fluid suspension, and the beads can be withdrawn from the fluid suspension as described by Shevkoplyas et al. (Lab on a Chip, vol. 7, pp. 1294 - 1302, 2007) and Warnke (IEEE Transactions on Magnetics, vol. 39, issue 3, pp. 1771 - 1777, 2003), and any biological material bound to the magnetic beads can also be withdrawn. This allows for the isolation of the desired population either by removing it from the fluid suspension (also known as positive selection) or by removing all other populations from the fluid suspension and leaving only the non - magnetically bound target population (also known as negative selection). Isolating cells such as T cells or stem cells from heterogeneous cell populations is essential for the development of cell therapies used in the treatment of various diseases.

[0003] One system utilizes a suspension that is stationary within surrounding magnets (EasySep (trademark) by STEMCELL Technologies (registered trademark)). Other systems are also known that are automated and use magnetic beads to isolate a target population (AutoMACS (registered trademark) by Milytenyi Biotec, and RoboSep (trademark) by STEMCELL (trademark) Technologies).

[0004] However, there remains an unmet need for rapid and reliable magnetic separation of a selected target within a biological sample, where the application of a magnetic field can be automated, customized, and controlled to achieve a desired high yield and high purity of target separation. SUMMARY OF THE INVENTION

[0005] The present invention provides a method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising: a. binding the target biological population to magnetic particles; b. circulating the biological sample through one or more fluidic pathways of the automated cell culture system; c. exposing the target biological population bound to the magnetic particles to a magnetic field gradient; d. repeating steps b - c one or more times; and e. collecting the target biological population bound to the magnetic particles.

[0006] Also provided herein is a method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising: a. binding the target biological population to magnetic particles; b. circulating the biological sample through one or more fluid pathways of the automated cell culture system; c. exposing the target biological population bound to the magnetic particles to a magnetic field gradient to capture the target biological population bound to the magnetic particles; d. circulating the unbound components of the biological sample through one or more fluid pathways of the automated cell culture system; e. inserting a magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field to release the target biological population bound to the magnetic particles; f. circulating the target biological population bound to the magnetic particles through one or more fluid pathways of the automated cell culture system; g. repeating steps b-f one or more times; and h. collecting the target biological population bound to the magnetic particles.

[0007] In additional embodiments herein, a method for collecting a target biological population from a biological sample in an automated cell culture system is provided, the method comprising: a. binding a non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluid pathways of the automated cell culture system; c. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient; d. repeating steps b-c one or more times; and e. collecting the target biological population.

[0008] In a further embodiment of the present specification, a method for collecting a target biological population from a biological sample in an automated cell culture system is also provided, the method comprising: a. binding a non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluid pathways of the automated cell culture system; c. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient to capture the non-target biological population bound to the magnetic particles; d. circulating the target of the biological sample through one or more fluid pathways of the automated cell culture system; e. inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field to release the non-target biological population bound to the magnetic particles; f. circulating the non-target biological population bound to the magnetic particles through one or more fluid pathways of the automated cell culture system; g. repeating steps b-f one or more times; and h. collecting the target biological population.

Brief Description of the Drawings

[0009] The following description of typical embodiments described herein will be better understood when read in conjunction with the accompanying drawings. For purposes of explaining the invention, embodiments that are presently typical are shown in the drawings. It should be understood, however, that the invention is not limited to the exact arrangements and instrumentalities of the embodiments shown in the drawings. Note that like reference numerals refer to like elements throughout the various embodiments shown in the drawings and referred to herein.

[0010] The description herein will be more fully understood in consideration of the following drawings.

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DETAILED DESCRIPTION OF THE INVENTION

[0011] All published documents, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The published documents and patent applications considered herein present only the disclosures prior to the filing date of this application. No statement in this specification should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Further, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. In case of conflict, the specification including the definitions shall govern. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. The following definitions are provided to facilitate the understanding of the present invention as used herein.

[0012] As used herein, the articles “a” and “an” preceding an element or component are intended to be non - limiting with respect to the number of instances (i.e., occurrences) of that element or component. Thus, “a” or “an” should be interpreted to mean one or at least one, and the singular form of the element or component shall also include the plural unless the number is clearly intended to be singular.

[0013] As used herein, the terms “invention” or “the invention” are non - limiting terms and are not intended to refer to any one particular aspect of the invention, but rather to encompass all possible aspects described in this specification and the claims.

[0014] As used herein, the terms “comprising,” “comprises,” “containing,” “has,” and their inflected forms and cognates mean “including, but not limited to.”

[0015] As used herein, the term “about” modifying the amount of a component, element or reactant employed refers to variations that may occur, for example, through typical measuring and liquid - handling procedures used in making concentrates or solutions. Further variations may also occur from inadvertent errors in the measuring procedures, differences in the manufacture, source, or purity of the components employed to make the compositions or to carry out the methods. In one aspect, the term “about” means within 10% of the reported numerical value. In another aspect, the term “about” means within 5% of the reported numerical value. In yet another aspect, the term “about” means within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reported numerical value.

[0016] When a range of values is recited, it is merely for the purpose of convenience or brevity and includes all the possible sub-ranges as well as the individual numerical values within and surrounding the recited range. All numerical values, unless otherwise specified, include values that are physically near, and integer values do not exclude fractional values. Values of sub-ranges and physically near values shall be considered as specifically disclosed values.

[0017] It should be understood that any component defined in this specification may, in some cases, be expressly excluded from the claimed invention for purposes of disclaimer or negative limitation.

[0018] As used herein, the terms "near", "about", and "substantially" mean each relevant relationship, measurement, quantity, amount, or degree that does not have a detrimental result or effect as compared to the recited term, embodiment, operation, or range of the present invention.

[0019] As used herein, for example, the terms "vertical", "horizontal", "parallel", "opposite", "straight", "lateral", "parallel", "right angle", and any other terms referring to angular relationships also mean approximately yet functional and / or practical respective relationships.

[0020] As used herein, the terms "preferred", "preferably", "exemplary", "typically", or "optionally" do not limit the scope of the present invention or its embodiments.

[0021] As used herein, the terms "substantial", "substantially" (or their synonyms) mean a measurement, range, quantity, or degree that encompasses the majority or a large portion of the entity being referred to in relation to the situation, or means at least a moderate, or further high, or large, or more efficient, or more significant degree as compared to the reference entity or with respect to the reference subject matter.

[0022] As used herein, the terms "negligible" and "slight" (or synonyms thereof) mean each respective relevance, measurement result, amount, quantity, or degree that is sufficiently small as to have no practical result as compared to the term being referenced or with respect to the scope of the invention.

[0023] As used herein, the term "may" means an option or effect that is not included, and / or not used, and / or not implemented, and / or does not occur, which option, without limiting the scope of the invention, constitutes at least a part of some embodiments of the invention or their results.

[0024] As used herein, a "sample" may be any sample, or may be a "biological sample" that can be derived from a plant, human, animal or microbial source. A sample is typically a heterogeneous sample from which a target is selected, isolated and collected. The target can be a cell, DNA, RNA, protein, peptide, microbe, virus, etc. A biological sample contains a target population.

[0025] Biological samples can include body fluid samples, somatic cell samples, or biological tissue samples. Examples of biological fluid samples or body fluid samples include urine, lymph, blood, plasma, serum, saliva, cervical mucus, cervical-vaginal fluid, vaginal fluid, milk, breast milk, synovial fluid, semen (semen and seminal fluid), feces, sputum, cerebrospinal fluid, tears, mucus, interstitial fluid, follicular fluid, amniotic fluid, aqueous humor, vitreous humor, ascites (peritoneal fluid and ascites), sweat, lymphatic fluid, lung sputum and lung lavage fluid, or samples derived therefrom. A biological tissue sample is a sample that contains cells, usually an aggregate of specific types of cells and intercellular substances, and forms one of the structural substances of the structure of humans, animals, plants, bacteria, fungi, or viruses, including connective tissue, epithelial tissue, muscle tissue, and nerve tissue. Examples of biological tissue samples also include organs, tumors, lymph nodes, arteries, and individual cells. For example, the sample may be a tissue sample suspected of being cancerous. Biological tissue samples can be processed to first separate the cell aggregates.

[0026] In a plurality of embodiments, the biological sample is a blood cell, a white blood cell, or a platelet. White blood cells include neutrophils, lymphocytes (T helper cells, cytotoxic T cells, T-killer cells, T cells including natural killer cells, and B lymphocytes), monocytes, eosinophils, basophils, macrophages, and dendritic cells.

[0027] As used herein, a "target cell" is typically a cell that is intended to be separated or concentrated from other cells (e.g., for testing or diagnosis) that has unique characteristics such as a specific type of cell or a selective affinity, such as binding to a specific antibody or other compound or other particle, compared to other cells. In certain embodiments, the unique characteristic is a selective affinity that binds or associates with magnetic beads to form magnetic target cells.

[0028] As used herein, the term "patient sample" is defined as a biological sample taken from any animal from which diagnosis, screening, monitoring, or treatment is anticipated. Animals include mammals. A patient refers to a subject such as a mammal, primate, human, or domestic animal that has a disease condition or in which a disease condition is to be determined or treated. A patient sample can be a source of the biological population from which it originated.

[0029] As used herein, the term "antibody" is intended to include monoclonal and polyclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), or antigen-binding portions thereof, and includes, but is not limited to, F(ab), Fv fragments such as scFv, single-chain antibodies, chimeric antibodies, humanized antibodies, recombinantly engineered antibodies, and Fab expression libraries. Bispecific antibodies can also be immobilized on magnetic particles.

[0030] As used herein, a "labeling moiety" is directly or indirectly detectable. The labeling moiety may be a labelable label and may be used in conjunction with magnetic particles. Direct labeling moieties include radioisotopes; enzymes whose products are detectable (e.g., luciferase, β-galactosidase, etc.); fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin, cyanine dyes, cascade blue, PerCP, Cy5, Cy7, allophycocyanin (APC), PECy5, or tandem conjugates of other different fluorescent dyes, Texas red, etc.); fluorescent emitting metals, e.g., 152 Euor other substances of the lanthanide series added to the protein via a metal chelate group such as, for example, EDTA; chemiluminescent compounds such as luminol, isoluminol, acridinium salts, etc.; bioluminescent compounds such as luciferin, aequorin (green fluorescent protein), etc.; and metal compounds. Examples of the indirect labeling moiety include labeling molecules that bind to the polypeptide, such as, for example, antibodies specific for the polypeptide. In this case, the binding molecule to be labeled is labeled as described above and is labeled as one of a specific binding pair such as, for example, biotin (one of the biotin-avidin specific binding pair), digoxigenin (one of the digoxigenin - antibody against digoxigenin specific binding pair), etc. Alternatively, the labeling moiety may be any suitable label including, but not limited to, those described herein.

[0031] For example, magnetic particles labeled with a binding partner such as an antibody, protein or nucleic acid molecule are commercially available from Miltenyi Biotec GmbH (Friedrich Ebert Str.68, D - 51429, Bergisch Gladbach, Germany). Methods for magnetically labeling biomolecules are known in the art and any known method can be used. For example, U.S. Patent No. 6,020,210 describes methods for preparing magnetic particles and for attaching biomolecules to the magnetic particles. The first member of a specific binding pair may be associated with the magnetic particle, in which case the biomolecule to be modified includes a moiety that binds to a member of the specific binding pair. Alternatively, the magnetic particle is linked to an antibody or an immunoreactive fragment thereof via, for example, a linker or spacer (such as, for example, a nucleic acid linker). By adding a spacer or linker, the biomolecule can be presented more flexibly and, by careful chemical manipulation, ligands can be added in a specific orientation. There are many chemical manipulation methods used for these linkages and many companies have published protocols to assist those skilled in the chemical arts.

[0032] Examples of members of specific binding pairs that can be added to magnetic particles include, but are not limited to, oligo dT (for binding to nucleic acid molecules containing, for example, a poly A portion at the 3' end); oligonucleotides having a specific nucleotide sequence (for binding to nucleic acid molecules containing a complementary nucleotide sequence); avidin (e.g., streptavidin) (for binding to biotinylated biomolecules); antigen-binding polypeptides, such as immunoglobulins (Ig) or epitope-binding fragments thereof (for binding to biomolecules containing an epitope recognized by the Ig); polynucleotide-binding proteins (for binding to polynucleotides), such as transcription factors, translation factors, etc.; Ni chelate or Co chelate (for immobilizing polyhistidine-tagged proteins); receptor-ligand systems, or other specific protein-protein interaction pairs; aptamers (e.g., nucleic acid ligands for three-dimensional molecular targets); lectins (for binding to glycoproteins); lipids and phospholipids (for binding to lipid-binding proteins), such as phosphatidylserine and annexin V. Those skilled in the art will recognize other members of specific binding pairs that can be bound to magnetic particles.

[0033] Biomolecules may be linked (covalently or non-covalently) to magnetic particles by direct chemical bonds or by physical association. Such methods are known in the art. Biochemical conjugation is described, for example, in “Bioconjugate Techniques” by Greg T. Hermanson, Academic Press. Non-covalent interactions such as, for example, ionic bonds, hydrophobic interactions, hydrogen bonds, and / or van der Waals forces may be used to link biomolecules to magnetic particles. For example, standard non-covalent interactions used to bind biomolecules to chromatography matrices may be used. One non-limiting example of such non-covalent interactions that can be used to bind biomolecules to magnetic particles is DNA binding to silica in the presence of chaotropic salts. Those skilled in the art are aware of other such non-covalent bonds and conditions for achieving such binding. See, for example, Molecular Cloning, Sambrook and Russell, Cold Spring Harbor Laboratory Press.

[0034] As used herein, “magnetic particles” are used, for example but not limited to, as labels for biomolecular targets in biological samples such as antibodies, DNA, polypeptides, and cells, and assist in the separation from complex mixtures of samples. Magnetic particles may be classified according to size as follows: microbeads less than about 50 nm; nanobeads from about 100 to about 200 nm; and dynabeads from about 1 to 5 μm. Furthermore, magnetic particles may be adapted for selective affinity (functionalized) for linkage or binding to desired biomolecular targets such as, for example, fluorescent labels, antibodies, nucleic acids.

[0035] For example, various magnetic particles are available from many sources including Dynal (Norway), Advanced Magnetics (Cambridge, Mass., USA), Immuncon (Philadelphia, USA), Immunotec (Marseille, France) and Miltenyi Biotec GmbH (Germany). Preferred magnetic labeling methods include colloidal superparamagnetic particles in the size range of 5 to 200 nm, preferably 10 to 100 nm in size. These magnetic particles enable quantitative magnetic labeling of cells, and accordingly, the amount of the linked magnetic label will be proportional to the amount of the bound product. Colloidal particles with various specificities are available, for example, via Miltenyi Biotec GmbH.

[0036] As used herein, "separation" includes isolating or collecting and accumulating target cells from the surrounding fluid bulk, where the bulk is, for example, a fluid mixture or suspension or emulsion of cells or a combination thereof, and also includes the meaning of concentration or enrichment of target cells compared to the surrounding bulk or the provided cell sample (obtaining a precipitate as in precipitation or centrifugation).

[0037] As used herein, "depletion" related to separation is the removal of target cells from the bulk (obtaining the supernatant as in precipitation or centrifugation).

[0038] As used herein, "highly qualitative" (separation, depletion) means separation of highly pure target cells from which substantially other cells are excluded or which contain only a small amount of other cells, for example, about 10% to about 1% or less of the separated cells, and in the case of depletion, vice versa.

[0039] As used herein, "highly quantitative" (separation, depletion) means a high degree of recovery and separation from a sample of substantially all target cells or a very large amount of target cells, such as about 80% to about 99% or more of the separated cells, and in the case of depletion, vice versa.

[0040] Whenever the attachment, adhesion, or sticking of cells to the wall of a tube, or similar terms related to such an action, are mentioned in this specification, it should always be noted that it does not necessarily mean that the cells directly attach to the wall. Rather, for example, it means that they are indirectly connected, joined, or attracted to the wall by, for example, chains of cells or groups of cells.

[0041] As used in this specification, "magnetic shielding" reduces and / or blocks the magnetic field in space by blocking the magnetic field with a "magnetic field shield" (also referred to in this specification as a magnetic field shield / barrier, and these terms are interchangeable).

[0042] As used in this specification, "HMPSM" means a substance with high permeability and magnetic saturation, which generates a highly concentrated magnetic field within itself and efficiently reduces and / or eliminates the influence of the magnetic field.

[0043] As used in this specification, a "magnetic field shield / barrier" is a controllable structure with respect to the use of a magnetic field.

[0044] As used in this specification, an "electromagnet" is a type of magnet in which the magnetic field is generated by an electric current. The magnetic field disappears when the current disappears. An electromagnet usually consists of a wire wound around a coil. The electric current passing through the wire generates a magnetic field, which is concentrated in the hole at the center of the coil. The wire turns are often wound around a magnetic core made of a ferromagnetic or ferrimagnetic material, such as iron. The magnetic core concentrates the magnetic flux and creates a more powerful magnet.

[0045] As used in this specification, a "permanent magnet" is a permanent magnet as opposed to an electromagnet, which only acts like a magnet when an electric current flows through it. Permanent magnets are made of substances such as magnetite (Fe 3 O 4 )), which is the most magnetic natural mineral, or neodymium, a powerful magnetic composite material.

[0046] As used herein, "magnet array" is one or more magnets. The one or more magnets may be permanent magnets or electromagnets. The one or more permanent magnets may be in a linear array, may have different sizes and different strengths, may be configured in opposite pole directions perpendicular to the axis of the linear array, or may be configured by rotating 90° to each other in a plane perpendicular to the axis of the linear array. Any number of magnets in the array may be physically held together or may be adhered to hold each other. The permanent magnet may be a magnet of a material selected from iron, neodymium, samarium-cobalt, or alnico.

[0047] The overall non-limiting summary of the present invention and the implementation of the present invention are presented below. The summary describes exemplary implementations of embodiments / aspects of the present invention and presents a constitutive basis for various and / or alternative and / or diverse aspects / embodiments, some of which are described later.

[0048] The present disclosure relates to devices, methods, and systems for magnetically separating and collecting a desired biomolecular target in a biological sample via positive or negative selection. As presented herein, a magnetic field is generated substantially adjacent to a biological sample containing a desired magnetized biomolecular target. The magnetic field can be automatically switched "on" and "off", and as a result, magnetic fields of desired intensities, continuous durations, intermittent durations, pulsed durations, and combinations thereof are generated. This is achieved by introducing a magnetic field shield (also referred to herein as a magnetic field shield / barrier) to functionally control the application of the magnetic field facing / added to the biological sample. The magnetic field shield / barrier is disposed between the magnetic field source and the biological sample, and as a function of a material with high magnetic permeability and saturation (HMPSM: high magnetic permeability and saturation material), it generates a highly concentrated magnetic field within itself, efficiently reducing and / or eliminating the influence of the magnetic field on the biological sample containing the magnetized biomolecular target.

[0049] In an aspect of the invention, the cell biological material is cultured in a bioreactor tube, and the desired cells are the biomolecular targets for magnetic separation and collection.

[0050] The devices, methods, and systems described herein generally involve a biological sample (a heterogeneous biological population), typically but not limited to cells, having magnetic beads that bind to a specific biomolecular target (a specific cell type) in the sample, resulting in "magnetized cells". Typical binding methods can include: i) directly binding magnetic beads conjugated to an antibody of the biological target, and ii) using a multi-step process in which the biological target is bound to an antibody or binding pair conjugated to another antigen. Subsequently, this antigen / binding pair is bound to magnetic beads conjugated to each antibody / binding pair. During magnetic separation, the magnetized cells (expressing the antigen; positively selected), which are the target cells bound to the magnetic beads, are attracted to a location near the magnet, while the cell population not bound to the beads (negatively selected) remains in the medium of the biological sample and is easily removed from the bound population. An alternative process for magnetic cell selection is to use antigen-presenting magnetic microbeads to stimulate some types of biological processes on the target cells (e.g., T cell activation using anti-CD3 and anti-CD28 conjugated to magnetic activation beads). After stimulation, the magnetic beads are removed, and downstream processing must then be performed. This requires treating the cell suspension with an effective magnet.

[0051] During separation, magnetically inclined particles receive a force vector F from the applied magnetic field B that acts on the paramagnetic particles as defined by Equation 1 below (Pamme, 2006).

Number

[0052] where V is the volume of the particle, and Δ X is the difference in magnetic susceptibility (ability to be magnetized) between the particle and the surrounding medium, and μ 0is the magnetic permeability of vacuum, and B·∇ is the dot product between the magnetic field and the gradient operator. From this equation, it is clear that the success of the magnetic separation system depends on many parameters. First is the particle size. If the particles are large, they will experience a strong magnetic force. There are three typical size classifications for magnetic particles. i) Less than 50 nm (e.g., MACS® microbeads from Miltenyi Biotec), ii) 100 - 200 nm (e.g., nanobeads from BioLegend®), or iii) 1 - 5 μm (e.g., Dynabeads® from Invitrogen), and the larger the size, the easier the separation. Next is to increase the magnetic susceptibility of the beads compared to the surrounding medium. Most beads often consist of an iron core, and since the surrounding medium is not actually magnetized, this value is typically already relatively large. Finally, by increasing the magnetic field gradient, the force applied to the magnetic beads can be dramatically increased. This is because the magnetic field gradient produces a non-uniform force on the N and S poles of the magnetic particles due to the sparse nature of the space of the high-gradient magnetic field (Figure 1A). This non-uniform force on the particles causes particle movement. In a completely uniform magnetic field, equal and opposite forces are generated on the two poles of the magnetic particle, resulting in a net zero force on the particle and no net movement (Figure 1B). Furthermore, increasing the size of the magnetic particle increases the difference in the magnetic force applied from the gradient between the two poles compared to smaller beads (Figures 2 and 3).

[0053] There exists means that can "switchably" induce (able to be turned on and off) a magnetic field that can generate a gradient for attracting magnetic beads for the purpose of automated separation, isolation, and collection. For example, an electromagnet is formed by winding a current-carrying wire around a magnetically sensitive substance (e.g., iron) (Figure 4A). The electromagnet can be switched on or off by respectively applying or removing an electric current passing through the wire. Another method is an electro-permanent magnet, which consists of a hard magnet with a high magnetic coercivity (a high magnetic field for switching magnetic poles) and a soft magnet with a low coercivity (Figure 4B). Both magnets are connected to each other along with a paramagnetic substance (e.g., iron) to complete the magnetic circuit. The soft magnet is wound with a current-carrying wire, and by pulsing a strong electric current in the wire, the magnetic poles of the soft magnet can be switched. When the poles are not aligned, the "current" of the magnet flows through the paramagnetic substance, and no external magnetic field is observed. However, when the poles are aligned, the magnetic flux moves through the air, and an external magnetic field is generated. The last method is to use a permanent magnet to generate a magnetic field. By using a substance with a high magnetic saturation, it is possible to block the magnetic field on one side of the magnet (Figure 4C).

[0054] In an aspect of the present invention, an array of strong permanent magnets with alternating switching directions is used (Figure 4C). This generates a strong magnetic gradient that spreads radially from the array, as well as a gradient that linearly follows the axis of the array. A modified version of this design is the Halbach array, where the magnets in the array are rotated 90° relative to each other (Figure 4D). This induces a significant increase in the magnetic field on one side of the magnet, while the magnetic field on the other side is attenuated (Kang et al.).

[0055] By designing this controllable magnetic field, it becomes possible to perform a continuous activity of controlling the isolation (either through positive or negative selection) of a biological fraction of a target containing a biomolecular target, while at the same time the non-target biological fraction can be removed and discarded.

[0056] The ability to switch the magnetic field on and off during the processing of biological samples using magnetic separation to enable the automation of both positive and negative cell selection is a major operational requirement. The devices and methods described herein enable the automated collection of target biological populations, thereby reducing the overall process complexity and operational costs.

[0057] The switchable magnetic field described herein, between "on" and "off", controls the magnetic field faced by biological samples labeled with magnetic particles, such as magnetic beads, by introducing a magnetic field shield / barrier. Through this controllable magnetic field, a subsequent continuous process of target biological retention, secondary release, and capture enables the creation of a very compact and energy-efficient magnetic separation system.

[0058] The magnetic field shield / barrier functions due to the inherent properties of HMPSM. This high permeability and magnetic saturation result in a highly concentrated magnetic field within this material. By placing HMPSM as a magnetic field shield / barrier between the magnetic field source and the biological sample, it is possible to substantially eliminate the influence of the magnetic field on the biological sample. Through the activation of this controllable magnetic barrier, it becomes possible to magnetically separate the target population from biological samples with high reproducibility and relatively low cost.

[0059] Figure 1 presents the theory of how magnetic particles / beads (e.g., those used for cell separation and activation) respond to a magnetic field gradient. This is mathematically demonstrated in Equation 1. Each magnetic bead has an N pole and an S pole. When exposed to a magnetic field gradient, the magnetic forces acting on each pole of the particle (one attracting and one repelling) are different, resulting in a net force that can move the particle (Figure 1A). By comparison, in the absence of a magnetic field gradient, the forces acting on each pole are equal and opposite (Figure 1B). Therefore, no net force is applied to the magnetic particle and no movement is induced. These necessary magnetic field gradients can be generated using an array of magnets arranged such that the poles alternate between N and S. Further, as shown in Figure 1C, high magnetic field gradients can be generated by using an array of multiple strong and small magnets arranged such that the poles alternate between the N direction and the S direction in the array.

[0060] Figure 2 presents the sizes of magnetic beads that are widely used in the magnetic separation of cells. The current range of bead sizes is from about 50 nm MACS® microbeads from Miltenyi to about 5 μm Dynabeads® from Invitrogen. By increasing the bead size, the difference in the forces acting on each pole of the bead also increases, and thus the sensitivity of the bead to respond to a magnetic field often increases.

[0061] Figure 3 describes an overview of how a fluid can be more efficiently pushed out in response to an induced magnetic field by large-sized magnetic beads. In some embodiments, the beads are simultaneously exposed to an attractive and a repulsive force from the magnetic field on each of the poles of the bead. As the distance from the magnetic field source increases, the magnitude and gradient of the magnetic field decrease. Therefore, the net force that the bead experiences depends on the distance from the magnetic field source and the diameter of the bead. Larger beads have a greater distance between both poles, and as a result, the difference in the magnetic forces acting on the poles also increases. Similarly, as the gradient increases (i.e., as the proximity to the magnet increases), the difference in the forces acting on each pole of the bead also increases. This increase in the net force causes the attraction of the beads towards the magnet and out of the fluid to occur earlier.

[0062] Figure 4 presents a method of generating a magnetic field that can rotate on / off for application to biological samples using an automated control system. In Figure 4A, an electromagnet is presented, with the wire wound around a ferromagnetic material such as iron, for example. By applying a current to the wire, the magnetic field can quickly and easily be turned on and conversely, turned off. However, the magnetic field generated thereby is low. Furthermore, the electromagnet produces a significant level of heat, which is a major problem for the local cell culture environment. Figure 4B presents an electro-permanent magnet consisting of both a non-switchable rare earth magnet and a pole-switchable alnico permanent magnet, both of which are incorporated into a ferromagnetic material forming a magnetic circuit. When the poles of the permanent magnets are aligned, the carbon steel selects the same direction and generates a net magnetic flux in the air around the magnet (this can be used to draw magnetic beads out of a fluid suspension). However, when the poles of the two permanent magnets are opposite, the magnetic flux is confined to the ferromagnetic material and the extraction of magnetic beads is inhibited. The directionality of the alnico magnet is switched by applying a very large-scale and short-term current pulse through the coil of wire around the alnico magnet, which is accompanied by a transient large-scale magnetic field. However, the magnetic gradient resulting from such a setup is also very low compared to the rare earth magnet, and this method also generates electromagnetic interference that can have unknown harmful effects on any peripheral electronics. Figure 4C shows an array of permanent magnets with opposite pole directions, with HMPSMs such as iron, cobalt iron, and Hiperco50 on one side, for example. By utilizing this HMPSM, the magnetic flux of the magnetic field is amplified on the opposite side while decreasing to a slight level on the HMPSM side. By operating the HMPSM as if it were between the permanent magnet and the magnetic beads, the force acting on the beads can be reduced to virtually zero. Conversely, by moving the HMPSM to the opposite side of the permanent magnet, a very strong magnetic force can be induced on the beads. Finally, in Figure 4D, a Halbach linear array is presented, where a 90° rotation of the magnets generates an amplified magnetic field on one side of the array while on the opposite side of the array, the magnetic field is significantly reduced or eliminated.

[0063] Figure 5 presents computer modeling showing the function of the HMPSM (x - marked) on the permanent magnet. The magnetic flux is mainly transmitted through the air around the magnet. However, the magnetic flux is not strong enough to be transmitted through the HMPSM. Therefore, a large magnetic flux density does not occur in the HMPSM on the opposite side of the magnet, but the magnetic flux density is mainly amplified at the two poles of the permanent magnet.

[0064] Figure 6 shows the layout of an example cassette that is widely used in an automated cell culture system with a separation tube for magnetic separation using magnetic beads, which extends along the full length of the cassette face. This tube is aligned with an array of permanent magnets placed within the automated cell culture system. The separation tube is connected to various tubes and bags within the cassette, and the cassette is used for positive or negative selection of target cells. By using a tube as long as possible, the volume that can flow through the separation tube increases, and accordingly, the processing time for generating magnetic separation is shortened.

[0065] Figure 7 presents an embodiment of the separation process presented in Figure 6. Magnetic - bound beads, either bound or unbound to cells, can flow into the separation tube 701 (Figure 7A). In some embodiments, the separation tube 701 is aligned with a permanent - magnet array 704 as shown in Figure 7B. In other embodiments, the separation tube 701 is aligned with an electromagnet replacing the permanent - magnet array 704. The permanent - magnet array 704 consists of permanent magnets with N - poles (702) and S - poles (703) alternating, generating the highest possible magnetic - flux gradient. The magnetic beads in the separation tube 701 are attracted to the magnet array 704, thereby successfully separating the magnetic beads from the fluid suspension.

[0066] FIG. 8 shows an embodiment of a magnetic field shield / barrier with an “on” magnetic field. The separation tube 701 extends along the entire length of the permanent magnet array 704. A magnetic field shield 801 generated from HMSPM surrounds a portion of the permanent magnet array 704 (shown as a paramagnetic sheath 801). In some embodiments, the magnetic field shield 801 is made of pure iron. In some embodiments, the magnetic field shield 801 is made of a soft magnetic iron alloy such as, for example, ferrite steel, silicon iron, nickel iron, or cobalt iron. In some embodiments, the magnetic field shield 801 is made of a soft magnetic alloy of cobalt, vanadium, and iron such as, for example, an alloy of about 49% cobalt, about 2% vanadium, and the balance iron. In some embodiments, the sheath 801 can be made of Hiperco50 or Hiperco 50A. In some embodiments, the paramagnetic properties of the magnetic field shield 801 amplify the magnetic field gradient applied to the separation tube 701 by the magnet array 704. In fact, this paramagnetism increases the magnetic force acting on the magnetic beads, so that the magnetic beads are removed from the fluid suspension more efficiently, while the non-magnetic substances are not affected and can pass through the separation tube smoothly. The permanent magnet array 704 and the magnetic field shield 801 are further connected to a servo 802 and a gear train 803, so that the entire assembly can be rotated completely and turned off and on as needed.

[0067] FIG. 9 shows the same assembly as in FIG. 8, but with the magnets in the “off” position. The separation tube 701 still extends along the permanent magnet array 704. However, in the off position, the magnet array 704 and the magnetic field shield 801 are rotated using a rotary servo 802 and a gear train 803, and accordingly, the magnetic field shield 801 is between the separation tube 701 and the permanent magnet array 704. As shown in FIGS. 4 and 5, the HMPSM material used for the magnetic field shield 801 impedes the passage of magnetic flux. Therefore, the magnetic flux gradient to which the separation tube 701 is exposed is greatly reduced, and the magnetic field cannot be maintained on the magnetic beads. By applying a high-speed liquid or gas flow to the separation tube 701, the magnetic beads and the cells magnetically coupled thereto can be flushed out.

[0068] Figure 10 is a cross-sectional view of the interface between the separation tube 701, the magnetic field shield 801, and the permanent magnet array 704. When in the "on" position, as shown in Figure 10A, a large magnetic field acts on the separation tube 701 to attract magnetic particles. When in the "off" position, as shown in Figure 10B, the magnetic field acting on the separation tube 701 is slight, and the cells and beads that were previously bound can be efficiently removed.

[0069] Figure 11 is a side view of the interface between the cassette 1101 and the automated cell culture device 1102. The separation tube 701 is attached to the cassette 1101, and accordingly, the magnetic fraction or the non-magnetic fraction is moved from different regions within the cassette 1101 and to different regions. Conversely, the magnetic separation assembly (consisting of 704 and 801) is included within the automated cell culture device 1102. The control system associated with the device 1102 can control the servo 802 and the gear train 803 to rotate the magnet array 704 and the magnetic field shield 801, such that when associated with the separation tube 701, the magnetic field is "on" or "off". When the cassette 1101 and the device 1102 are connected, the separation tube 701 and the separation assembly (collectively 704 and 801) are aligned, such that efficient magnetic separation can be performed. Further, by using a peristaltic pump associated with the device 1102, fluid (with or without magnetic beads and cells) can be transferred from or removed from the separation tube 701.

[0070] FIG. 12 is composed of a side view of a separation tube 701 installed between the outer wall of the cassette 1101 and the outer wall of the cell culture device 1102, and shows various means for increasing the number and scale of the magnetic field gradient in the separation tube 701. FIG. 12A shows a paramagnetic / diamagnetic spacer 1201 extending along the entire length of the separation tube 701 on the outer surface of the cassette 1101. This spacer 1201 acts to compress the tube 701 so that the distance between the magnetization element flowing through the tube 701 and the magnet array 704 is minimized. Moreover, due to the magnetization (when it is paramagnetic) of the spacer 1201 from the magnet array 704, another magnetic field gradient is formed in the separation tube 701 on the side closest to the cassette 1101. Further, by cutting the spacer 1201 into even smaller pieces along the entire length of the spacer 1201 (not shown), a high magnetic field gradient can be generated at the end portions of each small piece 1201 of the spacer. FIG. 12B shows a paramagnetic wire mesh 1202 in the separation tube 701, indicating that a high magnetic field gradient is generated around the strands of the mesh 1202 when exposed to the magnetic field generated by the permanent magnet array 704. FIG. 12C shows paramagnetic particles 1203 installed in the separation tube 701, which generate a localized magnetic field gradient when exposed to the magnetic field derived from the permanent magnet array 704. FIG. 12D shows a paramagnetic rod 1204 (not shown) extending along the entire length of the separation tube 701 and crushed into small longitudinal pieces, which can generate a high magnetic field gradient at the end of each rod 1204 when exposed to the magnet array 704. FIG. 12E shows a series of paramagnetic rods 1205 extending along the entire length of the separation tube 701, and a high magnetic field gradient is formed between the rods 1205 when exposed to the magnetic field derived from the array 704. FIG. 12F shows a paramagnetic sleeve 1206 extending along the entire length of the separation tube 701, and a high magnetic field gradient is formed in the holes when exposed to the magnetic field derived from the array 704. FIG. 12G shows a paramagnetic coating 1207 of the separation tube 701 consisting of small aberrations, and a high magnetic field gradient is generated between them when exposed to the magnetic field derived from the array 704.FIG. 12H shows a paramagnetic filter 1208 placed within the separation tube 701, which generates a high magnetic field gradient within the filter pores when exposed to the magnetic field derived from the array 704.

[0071] Optionally, or in some embodiments of the present invention, various parameters can be adjusted, such as, for example, magnetic field strength, spatial distribution (concentration) of the biomolecular target, and / or other parameters such as, for example, temperature. For example, it is possible to adjust the flow rate and / or viscosity and / or elasticity of the biological sample fluid to separate the target cells while at least substantially preventing the aggregation of non-target cells. In some embodiments, a fluid may be used to wash away the separated target cells. Optionally adjusting the flow pattern and the velocity of the washing fluid facilitates the removal of the target cells from the tube wall (i.e., facilitates removal or release). By changing the flow so abruptly, turbulence or impact is induced, which aids in the removal or destabilization of the target cells on the tube wall.

[0072] In certain embodiments, the release of the separated cells from the separation tube by various methods (e.g., demagnetization, bubbling, vibration, enzymes, sound waves, and combinations thereof) and combinations of methods may be performed before and / or simultaneously with the washing of the cells from the tube. This peripheral treatment may be performed to improve the separation and characteristics of the desired target population with respect to quality and / or quantity. For example, RBC lysis may be used to assist in the removal of adherent RBCs and improve purity, which in turn facilitates the separation of T cells from the overall PBMC population.

[0073] Enzymes such as, for example, DNase may be used to assist in cell release.

[0074] Furthermore, when the goal is high-quality or high-purity depletion (rather than collection of target cells), a stronger, sufficiently strong magnetic field than that used for collection may be applied, sacrificing non-target cells adhering to and / or aggregating on the wall.

[0075] Devices, systems, and methods may be embodied in a kit that includes one or more reagents, one or more magnetic particles, one or more binding partners, a magnet array, a magnetic field shield, and / or instructions for use, and in their use for one or more implementations of the present invention.

[0076] Even without further explanation, those skilled in the art will be able to make and use the present invention and carry out the claimed methods using the foregoing description and the following exemplary embodiments. Therefore, the following practical embodiments specifically illustrate typical aspects of the present invention and are in no way considered to limit the remainder of the present disclosure. Therefore, the embodiments are for illustrative purposes only and are not used in any way to limit the scope of the present invention.

Example

[0077] Example 1 - Separation of beads from fluid T cells (derived from Lonza PBMC) activated and expanded using CD3 / CD28 Dynabeads® (Invitrogen) were processed using magnetic separation systems (704 and 801). The goal was to separate the T cells from the beads. 31 ml of bead-cell (1.6x10 6A fluid suspension of 100 ng / ml of cells (T cell medium, 94% X-Vivo 15, 5% HS, 1% P / S, 10 ng / ml IL-2) was flowed through the separation tube 701 at different flow rates (5, 10, and 20 ml / min). The magnetic separation assemblies (704 and 801) were set to the "on" position. The fluid collected from the separation tube was named the "cell fraction" since it was unlikely that cells (not magnetically bound) were removed from the fluid suspension. By manually rotating the magnet assemblies (704 and 801) to the "off" position (without using 802 and 803), the paramagnetic material 801 was between the separation tube 701 and the magnet array 704. Three flush cycles (each consisting of 4 ml alternating air and fluid rinses at 40 ml / min) in the separation tube 701 were performed to wash away and collect the Dynabeads® and all cells attached to the walls of the tube 701. This was named the "magnetic fraction" since it consisted of all magnetically attracted cells and beads.

[0078] Cell counts from both fractions were used to calculate the percentage of cells successfully separated from Dynabeads® (i.e., percentage of total cells obtained in the "cell fraction"). At all applied flow rates, the percentage of cells successfully separated from Dynabeads® was approximately 95% (Figure 13A). Both fluid fractions were also counted using a hemacytometer to determine the number of Dynabeads® in each fraction. At all flow rates, the percentage of Dynabeads® removed from cells was at least 95% (Figure 13B).

[0079] Example 2 - Continuous flow of bead-bound cells through the separation tube Streptavidin nanobeads (BioLegend®) were bound to passaged Jurkats on the outside of cassette 1101 and positively selected using magnetic separation assemblies (704 and 801). Cells (10 7 cells / ml) were first treated with a blocking agent (5 μl / 10 7Cells, Human TruStain FcX (trademark), Biolegend (registered trademark) were added, and the cells and the agent were incubated together at room temperature for 10 minutes to block non-specific binding. Biotin-conjugated primary antibody cocktail (10 μl / 10 7 Cells, human CD14+ monocyte isolation, Biolegend (registered trademark) binds to the target cells, which were added and the mixture was incubated at 2 - 8°C for 15 minutes. Streptavidin-coated nanobeads (10 μl / 10 7 cells) were similarly added to the cell suspension at 2 - 8°C for an additional 15 minutes. Streptavidin on the nanobeads binds to the biotin on the antibody of the target cells, thereby magnetically binding the cells. To ensure a pure population of magnetically bound cells, the bound cells were pre-sorted with an EasySep (trademark) (STEMCELL Technologies (registered trademark) magnet for 5 minutes, and then the supernatant filled with unbound cells was discarded.

[0080] To separate Jurkats using the magnet array 704, Jurkats (1.5 - 2 ml / min, 3 ml) were passed through the separation tube 701 at different flow rates (1 - 5 ml / min), and at the same time the magnetic separation assembly (704 and 801) was turned on. The magnetically attracted cells exited the fluid suspension and adhered to the wall of the tube 701 closest to the magnet array 704. All cells that were not removed from the fluid by the activated magnet were captured as the "negative fraction" (collected in a volume of 9 - 12 ml). The magnetic separation assembly (704 and 801) was manually rotated to the "off" position and three wash cycles (described in Example 1) were performed to capture the "positive fraction". All of the above steps were performed using isolation buffer (98% DPBS, 2% FBS).

[0081] By using cell counts of both the negative fraction and the positive fraction, it becomes possible to determine the percentage of cells that were captured but missed (all cells in the "negative fraction" relative to the number of cells that flowed through), as well as the release efficiency of the positively captured cells (cells that were captured and then successfully entered the "positive fraction"). Increasing the capture flow rate in separation tube 701 increases the number of cells that the system misses capturing (Figures 14A and 14C). This is because the time that the bound cells are exposed to the magnetic field is shortened. However, by increasing the capture flow rate to 5 ml / min, the release rate of the bound cells was significantly improved, reaching almost 100% (Figures 14B and 14D). This is likely due to fewer cells leaving the suspension at a high flow rate and being trapped at various connections in the tube circuit. Modifying the inner diameter of the separation tube (thick tube - 1 / 8" ID (inner diameter) (about 0.32 cm), thin tube - 3 / 32" ID (about 0.24 cm)) slightly increases the capture failure rate (Figures 14A and 14C). However, cell release is also slightly improved (Figures 14B and 14D). These results are likely due to the increased fluid velocity and the shear stress on the walls of the thin tube.

[0082] Example 3 - Passing bead-bound cells through the separation tube multiple times Jurkats were magnetically bound and pre - selected as described in Example 2. Similar to Example 2, Jurkats (2x10 6 cells / ml, 3 ml) were passed through separation tube 701 in isolation buffer at a flow rate of 5 ml / min while the magnetic separation assembly (704 and 801) was turned on. Cells not captured by the magnet array 704 were collected as the first - pass negative fraction in isolation buffer (collection volume was 12 ml). Again, the cells were flowed through separation tube 701 at a capture flow rate of 5 ml / min, and cells that were still not captured were collected as the second - pass negative fraction (collected again as 12 ml). After the second pass, the separation assembly (704 and 801) was rotated to the "off" position, and the cells captured in separation tube 701 were subjected to 3 wash - through cycles (described in Example 1) to obtain the positive fraction. All of the above steps were performed using isolation buffer.

[0083] Similar to Example 2, the cells in each fraction were counted and quantified for capture failure and release efficiency (as the percentage of total cells flowed through Tube 701 for both passes). By further passing the cells that passed through the separation tube 701, the proportion of cells that were captured and lost was successfully reduced (Figure 15A), and there was no negative decrease in release compared to a single pass (Figure 15B).

[0084] Example 4 - "Waiting time" in the separation tube Jurkats were magnetically bound and pre-selected as described in Example 2. To efficiently increase the period during which the bound cells were exposed to the magnetic field, after being flowed through the separation tube 701, the magnetic assemblies (704 and 801) were rotated to the "on" position while maintaining Jurkats (1.5x10 6 , 3 ml) in a stationary state for various periods (1 - 5 minutes). The tube 701 was gently rinsed at 12 ml, 5 ml / min, and the effluent was collected as the negative fraction. After the waiting time, the separation assemblies (704 and 801) were rotated to the "off" position, and three wash - through cycles (described in Example 1) were performed to collect the positive fraction. All of the above steps were carried out using the isolation buffer.

[0085] The collected negative and positive fractions were counted again to quantify capture failure and release rate. A significant trend was observed that capture failure decreased as the waiting time increased (Figure 16A). This is likely due to the additional time for the magnetic particles to respond to the magnetic field. Furthermore, despite the increase in waiting time, the release rate for each waiting time was close to 100% (Figure 16B). This suggests that the improvement in capture rate is not simply due to an increase in cell loss at the connection of the tube circuit.

[0086] Adding a waiting time to the process increases the likelihood that negative cells will be captured by the process (hereinafter referred to as "false positives"). To quantify the proportion of false positives, Jurkats that had not undergone binding to magnetic beads were flowed through the separation tube 701 at various capture flow rates (5 ml / min or 10 ml / min) (3x106 cells / ml, 3 ml). Similarly, the cells were allowed to wait for various waiting times (1 minute or 3 minutes), and various "negative fraction" washes were performed. This wash consists of a high-flow rate air or fluid slash with the magnetic assemblies (704 and 801) left in the "on" position. Since the cells that were flowed through did not bind, all cells were predicted to appear as the "negative fraction", and the cells that did not appear were considered "false positives". Despite using a more standard capture sequence (a capture flow rate of 5 ml / min and a waiting time of 3 minutes), the false positive rate was poor (30%), and by increasing the capture flow rate and decreasing the waiting time, it was reduced to approximately 20% (Figure 17). Furthermore, by combining both a sufficient reduction in the waiting time and an acceleration of the capture flow rate (Condition 5), and by adding an air wash of 40 ml / min (Condition 7), the false positive rate could be reduced to less than 3% (Figure 17).

[0087] Example 5 - Separation of a mixed cell population and Purification Dissolved human peripheral blood mononuclear cells (PBMCs) were bound to Dynabeads® and CD3+ cells were selected from the heterogeneous cell population by positive selection (from ThermoFisher Scientific). PBMCs (10 7 cells / ml) were first incubated with CD3 antibody (5 μl / 10 7 cells, FlowComp™ human CD3 antibody, Invitrogen) at 2-8°C for 10 minutes. Then the PBMCs were bound to FlowComp™ Dynabeads (15 μl / 10 7 cells, Invitrogen) at room temperature with tilting and shaking for 15 minutes. The bead-bound cells (5-10x10 6Cells / ml, 1.5 ml) were flowed through separation tube 701 through magnet array 704 using various process parameters (flow rate, waiting time, number of passes, as described in FIG. 18). All cells not captured by the magnet (CD3-negative cells) were sent to the waste liquid and not characterized. The magnet array 704 was rotated to the "off" position, and the tube 701 was subjected to three wash cycles to obtain a "positive fraction". This fraction consisted of bead-bound CD3+ cells. All of the above steps were carried out in isolation buffer supplemented with 2 mM EDTA.

[0088] Viability (0.031 μl / 100 μl, Live / Dead™ Green, Invitrogen), CD3 (5 μl / 100 μl, PE mouse anti-human CD3, BD Biosciences), and in some experiments, CD14 (0.625 μl / 100 μl, CD14 monoclonal antibody-Pacific Blue, Invitrogen) were fluorescently stained for the pre-separation fraction and the post-separation fraction (300,000 cells per well), and analyzed using flow-assisted cell sorting (FACS) to evaluate the phenotype of the obtained fractions. The initially flowed population was found to be heterogeneous but mainly CD3+ (FIG. 18 - black line). As a comparison, various process parameters were verified using the magnet array 704. It was observed that changing the waiting time from 0 minutes to 2 minutes had only a limited effect on the purity of the output cells (FIGS. 18A, B and FIGS. 18F, G). However, when the waiting time was further increased to 5 minutes, the cell purity decreased significantly (FIGS. 18C, D). The flow rate also had only a limited effect on cell purity between 3 ml / min and 10 ml / min (FIGS. 18G, H). The greatest improvement in cell purity was achieved by adding a second pass to the process, with the purity improved to 97.5% CD3+ cells (FIG. 18E).

[0089] Example 6 - Magnets of various sizes for magnetic bead capture Permanent magnets (702 and 703) of various sizes (1 / 8 inch and 1 inch in length) were used to assemble the magnet array 704. The magnitude of the magnetic field was measured at various specific distances from the magnets using a gaussmeter (AlphaLab Inc.) (Figure 19A). From the magnitude measurements, an estimate of the magnetic field gradient was calculated (Figure 19B). It was found that it is desirable to shorten the length of the magnet to produce the strongest gradient. However, as the gradient increases, the effective range of the magnet decreases more rapidly than when using a long magnet. This indicates the possibility of using various magnet sizes in the magnet array 704 to achieve various separation purposes, such as short-distance separation of weakly bound targets and long-distance separation of strongly bound targets.

[0090] To verify the effect of the size of magnets 702 and 703 on cell separation, Jurkat cells were conjugated to BioLegend® nanobeads as described in Example 2. Cells conjugated to the beads (2.5x10 6 cells / ml, 1.5 ml) were flowed through the magnet array 704 in the separation tube 701 at a flow rate of 5 ml / min and a waiting time of 5 minutes. Unbound cells were flushed out of the tube 701 at 5 ml / min using 12 ml of isolation buffer (98% PBS, 2% FBS, 2 mM EDTA). The magnetic field generated by the array 704 was removed from the tube 701, and three wash cycles (described in Example 1) were performed to remove the positive fraction from the tube. The results were compared with the results obtained from a typical magnet assembly (704 and 801). It was observed that the magnets 702 and 703 with a length of 1 / 8 inch reduced capture failure compared to the magnets 702 and 703 with a length of 1 inch (Figure 19C). All of the above steps were performed using isolation buffer.

[0091] Example 7 - Additives to facilitate capture of weakly binding biological targets One way to vary or improve the ease of capture of the weakly bound target / small beads is to reduce the distance between the magnet array 704 and the separation tube 701 and increase the magnitude and gradient of the average magnetic field received within the tube 701, as described in Example 6. To do this, a spacer 1201 with a thickness of 3 / 32 inches was placed between the separation tube 701 and the cassette 1101. Jurkat cells (2.5 - 5x10 6 cells / ml, 1.5 ml) bound to BioLegend® nanobeads as described in Example 2 were flowed into the separation tube 701 at a flow rate of 5 ml / min and allowed a waiting time of 3 or 5 minutes. Thereafter, the non-captured cells were flushed out at 5 ml / min (11 - 13 ml), and three wash cycles (described in Example 1) were performed to collect the captured cell fraction. From the results obtained by adding the spacer 1201 to this setup, it was shown that using the spacer improved the level obtained from a 3-minute waiting time to a 5-minute waiting time with respect to capture failure (Figure 20A). All of the above steps were performed using an isolation buffer.

[0092] Another way to reduce the distance between the biological target and the magnetic field is to include magnetized objects (1202 - 1208) in the separation tube 701. The magnetic field generated by the magnet array 704 can be amplified by these objects (1202 - 1208). The magnetic field gradient is inversely proportional to the size of the objects (1202 - 1208), and the effective range is proportional to the size of the objects (1202 - 1208). To demonstrate this, a paramagnetic mesh 1202 was added to the separation tube 701. Jurkat cells (1.25x10 7 cells / ml) and MACS® microbeads (20 μl / 10 7 cells, CD3 microbeads - human, Miltenyi Biotec) were combined by incubating the cells and the pre-bound beads together for 15 minutes at 4 - 8°C. After binding, the cells were flowed into the separation tube 701 containing the paramagnetic mesh 1202 at 5 ml / min (9x10 6Cells / ml, 1.5 ml), with a waiting time of 5 minutes. Non-captured cells were flushed out from tube 701 at 5 ml / min using 12 ml of isolation buffer. The magnet array 704 was rotated to the off position, and three flushing cycles (described in Example 1) were performed to remove the cells positively captured from tube 701. To explain non-specific capture by mesh 1202, the results were normalized against the results obtained from a bead-free control (Jurkat not bound to microbeads), where all capture was due to physical restraint from mesh 1202. By using a paramagnetic mesh, a relative increase in cell capture of 10% was observed with beads compared to without beads (Figure 20B). The above steps were performed using isolation buffer supplemented with 2 mM EDTA.

[0093] Method Utilizing Recirculation and Magnetic Fields As described herein, in exemplary embodiments, a method for magnetically separating a target within a biological sample appropriately utilizes recirculation of the sample through a plurality of (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) magnetic separation cycles. Such recirculation dramatically increases the yield of the desired target.

[0094] The methods described herein are appropriately implemented in an automated cell culture system and, in some embodiments, can be executed in a cassette within the automated cell culture system. Figure 6 shows an example of a cassette, and Figure 21 shows the placement of this cassette in the flow diagram of an automated cell culture system. As shown in Figure 21, the automated cell culture system appropriately includes a cell growth chamber 2102, several fluid paths 2104, a magnetic field source 2106, and a reagent input location 2108.

[0095] Figure 21 shows an example of an embodiment in which a recirculation path / line is illustrated. In this case, a biological sample containing a target biological population circulates through the recirculation path / line (thick line) a plurality of times. Between each pass, the sample is appropriately exposed to a magnetic field gradient by a magnetic field source 2106 (e.g., a permanent magnet or an electromagnet).

[0096] The methods described herein utilize the recirculation of a biological sample to extract a target biological population and may rely on positive selection methods, negative selection methods, or combinations thereof.

[0097] Positive selection method In embodiments that utilize a positive selection method to isolate and capture a target biological population, the present disclosure provides a method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising binding the target biological population to magnetic particles, circulating the biological sample through one or more fluidic pathways of the automated cell culture system, exposing the target biological population bound to the magnetic particles to a magnetic field gradient, repeating the circulation step and the exposure step one or more times, and collecting the target biological population bound to the magnetic particles. In additional embodiments, the method may further comprise removing the target biological population from the bound magnetic particles.

[0098] Such positive selection methods rely on the direct extraction of a target biological population from a biological sample that utilizes a magnetic field to positively select a desired target population from the sample.

[0099] As described herein, the target biological population is suitably bound to magnetic particles. Methods for binding magnetic particles to a target biological population are described herein and suitably use an antibody, protein, or nucleic acid. As described herein, the target biological population suitably comprises one or more cells, viruses, bacteria, proteins, DNA, and / or RNA. In an exemplary embodiment, the target biological population is a population of T cells and suitably is a population of T cells produced to comprise a desired receptor. The biological sample from which the target population is extracted may also include other cells, viruses, bacteria, proteins, DNA, RNA, etc. that are not desired (i.e., non-target populations).

[0100] Additional steps that may be included in the positive selection method described herein include washing the biological sample (e.g., cell population), washing the magnetic particles, transferring the target biological population to a cell culture zone (e.g., growth chamber), and transferring the non-target biological population to a waste chamber and ultimately removing it from the automated cell culture system.

[0101] The biological sample is circulated, for example, through one or more fluid pathways of the automated cell culture system described in FIG. 21. In a plurality of embodiments, the biological sample may begin as a cell culture sample at the input location 2108 of the system, or in a plurality of embodiments, may begin as a cell culture sample in the cell growth chamber 2102, and then is transferred to an area where magnetic particles containing an antibody or other agent are provided, and the magnetic particles bind to the desired target population (e.g., cells). This binding to the magnetic particles may occur within the growth chamber 2102 within the system or at any input location 2108.

[0102] Thereafter, the biological sample passes through a section of the automated cell culture system that includes a source of magnetic field 2106, and as a result, the target biological population bound to the magnetic particles is exposed to a magnetic field gradient. As a result of this exposure, the target biological population (e.g., the desired cell population) becomes bound to the source of the magnetic field (e.g., gathers on the side of the separation tube 701 or other similar device), i.e., adjacent to the magnetic field source that generates the magnetic field. This separation withdraws the target biological population (or at least a portion of the target biological population) from the sample. The target biological population bound to the magnetic particles is then appropriately collected. Exemplary methods of collecting the target biological population include removing and washing the target biological population after exposure to the magnetic field. In a plurality of embodiments, the target biological population is collected by circulating a gaseous phase fluid and then circulating a liquid phase fluid one or more times through the system. Suitably, the gaseous phase fluid includes one or more of air, nitrogen, oxygen, and carbon dioxide. In further embodiments, the liquid phase includes one or more of water, buffered saline, culture medium, animal serum, chelating agents, and enzymes.

[0103] As described herein, it has been found that by recirculating the sample through multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) magnetic separation cycles, the amount of the target population removed from the sample increases. Thus, in suitable embodiments, the steps of the positive selection method of circulating a biological sample through one or more fluid pathways of an automated cell culture system and exposing the target biological population bound to magnetic particles to a magnetic field gradient are suitably repeated two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times). As shown in FIG. 21, this circulation suitably occurs through a recirculation cycle where the sample passes adjacent to the source of the magnetic field 2106 that binds to the target population, and then the sample recirculates and passes adjacent to the source of the magnetic field again to further remove the target population that could not be captured in the previous pass, and then finally the final target sample is collected. This cycle can be repeated a desired number of times until the goal of the target population is achieved or until it is determined through statistical or other means that additional cycles will no longer dramatically increase the yield and / or purity of the target population. After collection of the target biological population, the method suitably includes removing the target biological population from the bound magnetic particles, whereby the target population can further be processed or utilized by, for example, various means described herein.

[0104] In additional embodiments, the recirculation method described herein can include rinsing the target population bound to a magnetic source (e.g., the separation tube used in a magnetically based method), and then transferring the rinsed target population to a growth chamber for further processing and / or expansion. These elements of capture, rinse, and transfer can then be performed on another biological sample containing the magnetically bound target population.

[0105] In multiple embodiments, the magnetic field gradient to which the target population is exposed is provided by one or more permanent magnets. Exemplary materials that can be utilized for the permanent magnets are described herein and suitably include magnetite, neodymium, samarium-cobalt, and / or alnico. As described herein, in multiple embodiments, the permanent magnets are suitably configured in a linear array, such as the magnet array 704 of FIG. 7B.

[0106] In additional embodiments, the magnetic field gradient is provided by one or more electromagnets as described herein.

[0107] In further embodiments, provided herein is a positive selection method that includes collecting a target biological population from a biological sample in an automated cell culture system, the method comprising binding the target biological population to magnetic particles, circulating the biological sample through one or more fluidic paths of the automated cell culture system, exposing the target biological population bound to the magnetic particles to a magnetic field gradient to capture the target biological population bound to the magnetic particles, circulating unbound components of the biological sample through one or more fluidic paths of the automated cell culture system, inserting a magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field to release the target biological population bound to the magnetic particles, circulating the target biological population bound to the magnetic particles through one or more fluidic paths of the automated cell culture system, repeating the step of circulating the biological sample to the step of circulating the target biological population one or more times, and collecting the target biological population bound to the magnetic particles. In additional embodiments, the positive selection method may further include removing the target biological population from the bound magnetic particles.

[0108] As described herein, the positive selection method utilizes a design in which a biological sample passes, for example, through a separation tube 701 (as shown, for example, in FIG. 7A). Within the biological sample, the target biological population is bound to magnetic particles. The method suitably includes circulating the biological sample through one or more fluidic paths in front of or including the separation tube 701 and a magnetic source. The target biological population bound to the magnetic particles is suitably exposed to a magnetic field gradient that captures the target biological population bound to the magnetic particles (and suitably recirculated through this magnetic field one or more times). For example, as shown in FIG. 8, the biological sample passes through the separation tube 701, and the bound magnetic particles and target sample are captured by the magnetic field against the side of the tube (see also FIGS. 24D-24E).

[0109] Thereafter, unbound components in the biological sample (i.e., unwanted cells, proteins, DNA, or other structures) circulate through one or more fluidic paths and are removed from the separation tube 701.

[0110] Thereafter, a magnetic field shield / barrier is suitably inserted between the target biological population bound to the magnetic particles and the magnetic field, and the target biological population bound to the magnetic particles is released from the magnet. Thereafter, the target biological population bound to the magnetic particles circulates through one or more fluidic paths of an automated cell culture system and is collected, for example, in a separation area of an automated cell manipulation system. Various methods for collecting the target biological population are described herein.

[0111] Suitably, the steps of circulating the biological sample, exposing the sample (and the target biological population bound to the magnetic particles), inserting a magnetic field shield / barrier between the target population and the magnetic field, and collecting the target biological population are repeated one or more times (suitably two or more times, three or more times, four or more times, five or more times, etc.). With each pass through this cycle, the yield of the target biological preparation increases. As described herein, the target biological population is then suitably removed from the bound magnetic particles.

[0112] As described herein, a target biological population suitably includes one or more of cells, viruses, bacteria, proteins, DNA and / or RNA, and in multiple embodiments, includes T cells. Methods and compounds for binding magnetic particles to a target biological population are described herein, and they suitably include the use of antibodies, proteins or nucleic acids.

[0113] Exemplary magnetic fields are described herein, and the magnetic fields are suitably generated by permanent magnets or electromagnets. Materials used in the fabrication of permanent magnets are described herein, and include, for example, magnetite, neodymium, samarium-cobalt, and / or alnico. In multiple embodiments, the permanent magnets are configured in a linear array. In embodiments where an electromagnet is utilized, the insertion of a magnetic field shield / barrier can be replaced by turning off the electromagnet, for example, simply removing the current from the electromagnet and stopping the magnetic field.

[0114] As described throughout this specification, in multiple embodiments, the magnetic field shield / barrier suitably includes materials with high permeability and magnetic saturation. As described herein, in multiple embodiments, the magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field. Such embodiments are illustrated in FIGS. 8, 9, and 10A-10B and described in detail herein.

[0115] Negative selection method In an embodiment using a negative selection method for a target biological population, the present specification provides a method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising binding a non-target biological population to magnetic particles, circulating the biological sample through one or more fluidic paths of the automated cell culture system, exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient, repeating the circulation step to the exposure step one or more times, and collecting the target biological population. The method may further include collecting the non-target biological population for proper removal as waste.

[0116] As used herein, the negative selection method utilizes the binding of magnetic particles to a "non-target biological sample", which refers to one or more cells, proteins, DNA, RNA not included in the "target biological population", and thus is to be removed from the biological sample in order to leave behind the target biological population. In such negative selection methods, magnetic separation is used to separate the non-target biological population and the remaining target biological population is collected from the sample.

[0117] As described herein, the non-target biological population is suitably bound to magnetic particles. Methods for binding magnetic particles to the non-target biological population are described herein and the methods suitably use antibodies, proteins or nucleic acids. As described herein, the non-target biological population suitably includes one or more cells, viruses, bacteria, proteins, DNA and / or RNA. In an exemplary embodiment, the target biological population is a population of T cells, suitably T cells produced to contain a desired receptor, while the non-target biological population includes any other cells, proteins, etc. in the sample that are removed in order to leave behind the target population. The biological sample from which the target population is removed may also contain other unwanted cells, viruses, bacteria, proteins, DNA, RNA, etc.

[0118] Biological samples are circulated through one or more fluid pathways of an automated cell culture system as described, for example, in FIG. 21. In multiple embodiments, a biological sample may begin as a cell culture sample at an input location 2108, or in a cell growth chamber 2102, or at other locations within the system, and is then transferred to an area where magnetic particles containing an antibody or other agent are provided, and the magnetic particles bind to an undesired non-target population (e.g., undesired cells, proteins, DNA, etc.). This binding to the magnetic particles may occur within the growth chamber, at the input location 2108, or within other chambers within the system.

[0119] Thereafter, the biological sample passes through a section of the automated cell culture system that includes a source of magnetic field 2106, and as a result, the non-target biological population bound to the magnetic particles is exposed to a magnetic field gradient (the target biological population is also exposed but does not react with the magnetic field). As a result of this exposure, the non-target biological population (e.g., a population of undesired cells, proteins, etc.) becomes bound to the source of the magnetic field (e.g., gathers on the side of a separation tube 701 or other similar device), i.e., adjacent to the magnetic field. This separation draws out the non-target biological population (or at least a portion of the non-target biological population) from the sample. The target biological population that is not bound to the magnetic particles is then appropriately collected. Exemplary methods of collecting the target biological population include filtering, removing, and washing the target biological population from the sample after exposure to the magnetic field (and thus removing the non-target biological population).

[0120] In multiple embodiments, the target biological population is collected by circulating a gas-phase fluid and then circulating a liquid-phase fluid one or more times through the system. Suitably, the gas-phase fluid includes one or more of air, nitrogen, oxygen, and carbon dioxide. In further embodiments, the liquid phase includes one or more of water, buffered saline, culture medium, animal serum, chelating agents, and enzymes.

[0121] As described herein, it has been found that by recirculating the sample through multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) magnetic separation cycles, the amount and / or purity of the target population removed from the sample is increased. Thus, in suitable embodiments, the step of circulating a biological sample through one or more fluid pathways of an automated cell culture system, exposing the non-target biological population bound to magnetic particles to a magnetic field gradient, and collecting the target biological population in a negative selection method is preferably repeated two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times). As shown in FIG. 21, this circulation preferably occurs through a recirculation cycle where the sample passes adjacent to the source of the magnetic field 2106 that binds to the non-target population and the target population is collected, and then the sample is recirculated and passed adjacent to the source of the magnetic field again to further remove non-target populations that could not be captured in the previous pass. This cycle can be repeated the desired number of times until the goal of the target population is achieved or until it is determined via statistical or other means that additional cycles will no longer dramatically increase the yield and / or purity of the target population. After collection of the target biological population, the method preferably further comprises processing, filtering, or utilizing the target biological population by various means described herein.

[0122] In multiple embodiments, the magnetic field gradient to which the non-target population is exposed is provided by one or more permanent magnets. Exemplary materials that can be utilized for permanent magnets are described herein and preferably include magnetite, neodymium, samarium-cobalt, and / or alnico. As described herein, in multiple embodiments, the permanent magnets are preferably configured in a linear array, such as the magnet array 704 of FIG. 7B.

[0123] In additional embodiments, the magnetic field gradient is provided by one or more electromagnets described herein.

[0124] In a further embodiment, provided herein is a negative selection method comprising collecting a target biological population from a biological sample in an automated cell culture system, the method comprising binding a non-target biological population to magnetic particles, circulating the biological sample through one or more fluidic pathways of the automated cell culture system, exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient to capture the non-target biological population bound to the magnetic particles, circulating the target biological population of the biological sample through one or more fluidic pathways of the automated cell culture system, inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field to release the non-target biological population bound to the magnetic particles, circulating the non-target biological population bound to the magnetic particles through one or more fluidic pathways of the automated cell culture system, repeating the collection step of the target biological population one or more times from the biological sample collection step, and collecting the target biological population.

[0125] As described herein, the negative selection method involves collection of the target biological population; the biological sample utilizes a design that passes, for example, through separation tube 701 (e.g., as shown in FIG. 7A). Within the biological sample, the non-target biological population is bound to magnetic particles. The method suitably comprises circulating the biological sample through one or more fluidic pathways before or including separation tube 701. The non-target biological population bound to the magnetic particles is suitably exposed to a magnetic field gradient that captures the non-target biological population bound to the magnetic particles. For example, as shown in FIG. 8, the biological sample passes through separation tube 701 and the bound magnetic particles and non-target sample are captured by the magnetic field against the side of the tube (see also FIGS. 24D - 24E).

[0126] Unbound components in a biological sample (i.e., a target biological population including desired cells, proteins, DNA, or other structures including T cells) then circulate through one or more fluid pathways and are appropriately filtered or collected by other mechanisms in, for example, the separation area of an automated cell manipulation system.

[0127] Subsequently, a magnetic field shield / barrier is appropriately inserted between the non-target biological population bound to magnetic particles and the magnetic field, and the non-target biological population bound to the magnetic particles is released from the magnet. The non-target biological population bound to the magnetic particles then circulates through one or more fluid pathways of an automated cell culture system.

[0128] Appropriately, the steps of circulating the biological sample, exposing the sample (and the non-target biological population bound to the magnetic particles), inserting a magnetic field shield / barrier between the non-target population and the magnetic field, and collecting the target biological population are repeated one or more times (appropriately two or more times, three or more times, four or more times, five or more times, etc.). Through this recycling, the yield and / or purity of the target biological preparation increases each time, thereby enabling the removal of an increasing amount of non-target biological population and the separation and collection of a greater amount of the desired target biological preparation.

[0129] Additional steps that may be included in the negative selection method described herein include washing the biological sample (e.g., cell population), washing the magnetic particles, transferring the target biological population to a cell culture zone (e.g., growth chamber), and transferring the non-target biological population to a waste chamber and ultimately removing it from the automated cell culture system.

[0130] As described herein, suitably, the non-target biological population includes one or more of cells, viruses, bacteria, proteins, DNA, and / or RNA, and in multiple embodiments, the target biological population includes one or more of cells, viruses, bacteria, proteins, DNA, and / or RNA, suitably including T cells. Methods and compounds for binding magnetic particles to non-target biological populations are described herein, and they suitably include the use of antibodies, proteins, or nucleic acids.

[0131] Exemplary magnetic fields are described herein, and the magnetic fields are suitably generated by permanent magnets or electromagnets. Materials used in the production of permanent magnets are described herein, for example, including magnetite, neodymium, samarium-cobalt, and / or alnico. In multiple embodiments, the permanent magnet is composed of a linear array. In embodiments where an electromagnet is utilized, the insertion of a magnetic field shield / barrier can be replaced by turning off the electromagnet, for example, simply removing the current from the electromagnet and stopping the magnetic field.

[0132] As described throughout this specification, in multiple embodiments, the magnetic field shield / barrier suitably includes materials with high permeability and magnetic saturation. As described herein, in multiple embodiments, the magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field. Such embodiments are illustrated in FIGS. 8, 9, and 10A - 10B and are described in detail herein.

[0133] In further embodiments, methods for washing and recovering magnetic particles in an automated cell culture system are provided herein.

[0134] Suitably, the method includes: a. circulating magnetic particles through one or more fluidic paths of an automated cell culture system; b. exposing the magnetic particles to a magnetic field gradient to capture the magnetic particles; c. collecting the magnetic particles by applying a gaseous fluid phase and then a liquid fluid phase; d. circulating magnetic particles through one or more fluidic paths of the automated cell culture system; and e. repeating steps c to d one or more times (e.g., two or more times, three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, ten or more times).

[0135] As described herein, in a plurality of embodiments, the magnetic particles are bound to a target biological population, and the binding suitably occurs via an antibody, protein, or nucleic acid.

[0136] In additional embodiments, the magnetic particles are bound to a non-target biological population and include binding via an antibody, protein, or nucleic acid.

[0137] Exemplary non-target and target biological populations are described herein, and suitably the target population is any one or more of cells, viruses, bacteria, proteins, DNA, and RNA, including T cells.

[0138] In a plurality of embodiments, the magnetic field gradient is provided by one or more permanent magnets, including permanent magnets containing magnetite, neodymium, samarium-cobalt, or alnico. The permanent magnets may be configured in a linear array, as described herein. In additional embodiments, the magnetic field gradient is provided by an electromagnet.

[0139] Suitably, as described herein, the magnetic field shield / barrier is inserted between the magnetic particles and the magnetic field, and by blocking the magnetic field, the collection of magnetic particles becomes possible, and the magnetic particles bound to the magnetic source are released. Exemplary substances for use in the magnetic field shield / barrier include substances with high permeability and magnetic saturation. In embodiments where electromagnets are utilized, the current may be removed from one or more electromagnets, and simply by blocking the source of the electromagnets, the collection of magnetic particles becomes possible.

[0140] Examples of gaseous phase fluids that can be utilized in the method include one or more of air, nitrogen, oxygen, and carbon dioxide. Examples of liquid phase fluids that can be used include one or more of water, buffered saline, culture medium, animal serum, chelating agents, and enzymes.

[0141] Bead recovery method Negative selection methods and positive selection methods, including multiple magnetic separations, and data on the recovery of magnetic particles Figures 22A - 22D show the binding of magnetic particles to both target cells (purified cells) and waste cells (non - target population). As shown, the "improvement process" utilizing reagent dose optimization shows high binding between magnetic particles and waste cells (non - target population for negative selection) and low "false negatives".

[0142] Figures 23A - 23B show the release of magnetic particles from cells. As shown, in Figure 23A, the binding mechanism utilized in the automated cell culture system (COCOON) shows bead - cell release similar to the control, demonstrating the ability to recover cells (or other target biological populations) by the positive selection method. Figure 23B shows the percentage of beads remaining in the target cells after release and bead removal using the automated cell culture system.

[0143] To determine the benefits of performing multiple magnetic separations (i.e., recirculating the sample through an automated cell culture system and exposing it to the magnetic field 2, 3, 4, 5 times), experiments were conducted that involved increasing the exposure time to the magnet and decreasing the flow rate. As shown in Figure 24A, increasing the magnet exposure time (from left to right) increased the percentage of bound cells captured from approximately 65% to at least 90%, which was equivalent to the control. Similarly, in Figure 24B, decreasing the flow rate (from right to left) increased the bound cells captured from approximately 60% to approximately 85%, which was similar to the control.

[0144] Table 1 below shows that by performing multiple passes using the magnetic separation method described herein, an increase was obtained in both the bound cells secured and the overall cell yield.

Table 1

[0145] Figure 24C shows the effect of the number of cycles of the wash flow of the fluid that releases the captured biological sample from the separation line, and it is illustrated that a large amount is recovered after the first two flushes.

[0146] Figures 24D - 24E show the capture of magnetic particle - bound cells using a magnetic field (top) and the release of cells (bottom) after performing multiple cycles of fluid wash - out with the magnetic field turned off (as shown in Figure 24C) in a separation tube, demonstrating the effectiveness of the method described herein.

[0147] In FIGS. 25A - 25F, high - level purification of the target biological population (cells) in the positive and negative selection methods is shown, indicating that for both the positive and negative selection methods, there was a similar recovery between the automated cell culture system (COCOON) described herein and the control. FIGS. 25G - 25I show the purification of cells using negative selection, indicating an improvement in purity by performing multiple passes using a recirculation line. The CD3 + CD14 + population is an undesired bead - bound monocyte - T cell aggregate. FIGS. 25J - 25L show population purification using the negative selection process. By performing multiple passes, successful removal of unwanted bead - bound cells was achieved (highlighted by white circles).

[0148] FIGS. 26A - 26C show the advantages of magnetic separation in the automated cell culture system described herein. FIG. 26A shows a significant shortening of the process duration, FIG. 26B shows a reduction in cell loss, and FIG. 26C shows a reduction in volume loss compared to bag - and culture - tube - based controls. FIG. 26D shows population purification using the positive selection process. By multiple passes through the recirculation line, the cell yield is improved compared to a single pass.

[0149] FIGS. 27A - 27B show the ability to recover magnetic particles after washing in the automated cell culture system described herein. Compared to a typically hand - performed process, FIG. 27A shows the effect of consecutive rinses of the separation tube 701 on the absolute recovery amount, and FIG. 27B shows the effect of consecutive rinses of the separation tube 701 on the cumulative particle recovery rate.

[0150] The description of various embodiments and / or examples of the present invention is presented for illustrative purposes and is not intended to be comprehensive or limited to the disclosed embodiments and / or examples. Many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the described embodiments. The terminology used herein is selected to best explain the spirit of the embodiments, the practical application, or the technical improvements in the art, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0151] Preferred embodiments of the present invention are as follows. [1] A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding the target biological population to magnetic particles; b. circulating the biological sample through one or more fluid pathways of the automated cell culture system; c. exposing the target biological population bound to the magnetic particles to a magnetic field gradient; d. repeating steps b - c one or more times; and e. collecting the target biological population bound to the magnetic particles. [2] The method according to [1], wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA. [3] The method according to [1] or [2], wherein the target biological population comprises T cells. [4] The method according to any one of [1] - [3], wherein the magnetic particles are bound to the target biological population via an antibody, protein, or nucleic acid. [5] The method according to any one of [1] - [4], wherein the magnetic field gradient is provided by one or more permanent magnets. [6] The above permanent magnet is the method according to [5], including magnetite, neodymium, samarium-cobalt, or alnico. [7] The above permanent magnet is the method according to any one of [5] to [6], which is composed of a linear array. [8] The above magnetic field gradient is the method according to any one of [1] to [4], which is provided by one or more electromagnets. [9] Steps b to c are repeated at least twice, which is the method according to any one of [1] to [8].

[10] The above target biological population bound to the above magnetic particles is collected by circulating a gas-phase fluid and then a liquid-phase fluid one or more times, which is the method according to any one of [1] to [9].

[11] The above gas-phase fluid contains one or more of air, nitrogen, oxygen, and carbon dioxide, which is the method according to

[10] .

[12] The above liquid phase contains one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, which is the method according to

[10] .

[13] The method according to any one of [1] to

[12] , further including removing the above target biological population from the above bound magnetic particles.

[14] A method for collecting a target biological population from a biological sample in an automated cell culture system, a. Binding the above target biological population to magnetic particles; b. Circulating the above biological sample through one or more fluid paths of the above automated cell culture system; c. Exposing the above target biological population bound to the above magnetic particles to a magnetic field gradient to capture the above target biological population bound to the above magnetic particles; d. Circulating the unbound components of the above biological sample through one or more fluid paths of the above automated cell culture system; e. Inserting a magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field to release the target biological population bound to the magnetic particles; f. Circulating the target biological population bound to the magnetic particles through one or more fluid pathways of the automated cell culture system; g. Repeating steps b - f one or more times; and h. Collecting the target biological population bound to the magnetic particles, a method comprising the same. 〔15〕 The method according to 〔14〕, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA. 〔16〕 The method according to 〔14〕 or 〔15〕, wherein the target biological population comprises T cells. 〔17〕 The method according to any one of 〔14〕 - 〔16〕, wherein the magnetic particles are bound to the target biological population via an antibody, protein, or nucleic acid. 〔18〕 The method according to any one of 〔14〕 - 〔17〕, wherein the magnetic field gradient is provided by one or more permanent magnets. 〔19〕 The method according to 〔18〕, wherein the permanent magnet comprises magnetite, neodymium, samarium - cobalt, or alnico. 〔20〕 The method according to any one of 〔17〕 - 〔19〕, wherein the permanent magnet is composed of a linear array. 〔21〕 The method according to any one of 〔14〕 - 〔16〕, wherein the magnetic field gradient is provided by one or more electromagnets, and optionally, the inserting step in step e is replaced by a step of removing current from the one or more electromagnets. 〔22〕 The method according to any one of 〔14〕 - 〔21〕, wherein steps b - f are repeated at least twice. 〔23〕 The method according to any one of 〔14〕 - 〔22〕, wherein the magnetic field shield / barrier comprises a material with high permeability and magnetic saturation.

[24] The magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field, according to the method described in any one of

[14] to

[23] .

[25] The target biological population is collected by circulating a gaseous phase fluid followed by a liquid phase fluid one or more times, according to the method described in any one of

[14] to

[24] .

[26] The gaseous phase fluid contains one or more of air, nitrogen, oxygen, and carbon dioxide, according to the method described in

[25] .

[27] The liquid phase contains one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, according to the method described in

[25] .

[28] The method further includes removing the target biological population from the bound magnetic particles, according to the method described in any one of

[14] to

[27] .

[29] A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding a non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluid pathways of the automated cell culture system; c. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient; d. repeating steps b to c one or more times; and e. collecting the target biological population.

[30] The target biological population contains one or more of cells, viruses, bacteria, proteins, DNA, and RNA, according to the method described in

[29] .

[31] The target biological population contains T cells, according to the method described in

[29] or

[30] .

[32] The method according to any one of

[29] to

[31] , wherein the non-target biological population is collected. 〔33〕 The magnetic particles are bound to the non-target biological population via an antibody, protein, or nucleic acid, by the method according to any one of 〔29〕 to 〔32〕. 〔34〕 The magnetic field gradient is provided by one or more permanent magnets, by the method according to any one of 〔29〕 to 〔33〕. 〔35〕 The permanent magnet includes magnetite, neodymium, samarium-cobalt, or alnico, by the method according to 〔34〕. 〔36〕 The permanent magnet is composed of a linear array, by the method according to any one of 〔34〕 to 〔35〕. 〔37〕 The magnetic field gradient is provided by one or more electromagnets, by the method according to any one of 〔29〕 to 〔33〕. 〔38〕 Steps b to c are repeated at least twice, by the method according to any one of 〔29〕 to 〔37〕. 〔39〕 The target biological population bound to the magnetic particles is collected by circulating a gas-phase fluid, followed by a liquid-phase fluid one or more times, by the method according to any one of 〔29〕 to 〔38〕. 〔40〕 The gas-phase fluid includes one or more of air, nitrogen, oxygen, and carbon dioxide, by the method according to 〔39〕. 〔41〕 The liquid phase includes one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, by the method according to 〔39〕. 〔42〕 A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding the non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluid paths of the automated cell culture system; c. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient to capture the non-target biological population bound to the magnetic particles. d. circulating the target of the biological sample through one or more fluidic paths of the automated cell culture system; e. inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field to release the non-target biological population bound to the magnetic particles; f. circulating the non-target biological population bound to the magnetic particles through one or more fluidic paths of the automated cell culture system; and g. repeating steps b - f one or more times; and h. collecting the target biological population, a method comprising. 〔43〕 The method according to 〔42〕, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA. 〔44〕 The method according to 〔42〕 or 〔43〕, wherein the target biological population comprises T cells. 〔45〕 The method according to any one of 〔42〕 - 〔44〕, wherein the non-target biological population is collected. 〔46〕 The method according to any one of 〔42〕 - 〔45〕, wherein the magnetic particles are bound to the target biological population via an antibody, protein, or nucleic acid. 〔47〕 The method according to any one of 〔42〕 - 〔46〕, wherein the magnetic field gradient is provided by one or more permanent magnets. 〔48〕 The method according to 〔47〕, wherein the permanent magnet comprises magnetite, neodymium, samarium - cobalt, or alnico. 〔49〕 The method according to any one of 〔47〕 - 〔48〕, wherein the permanent magnet is composed of a linear array. 〔50〕 The method according to any one of 〔42〕 - 〔46〕, wherein the magnetic field gradient is provided by one or more electromagnets, and optionally, the step of inserting e is replaced by the step of removing current from the one or more electromagnets. 〔51〕 Steps b to f are methods described in any one of

[42] to

[50] , which are repeated at least twice.

[52] The magnetic field shield / barrier is a method described in any one of

[42] to

[51] , which contains substances with high permeability and magnetic saturation.

[53] The magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field, which is a method described in any one of

[42] to

[51] .

[54] The target biological population is collected by circulating the liquid phase fluid one or more times after the gas phase fluid, which is a method described in any one of

[42] to

[53] .

[55] The gas phase fluid contains one or more of air, nitrogen, oxygen, and carbon dioxide, which is a method described in

[54] .

[56] The liquid phase contains one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, which is a method described in

[54] .

[57] A method for washing and recovering magnetic particles in an automated cell culture system, comprising: a. circulating the magnetic particles through one or more fluid paths of the automated cell culture system; b. exposing the magnetic particles to a magnetic field gradient to capture the magnetic particles; c. collecting the magnetic particles by applying a gas fluid phase and then a liquid fluid phase; d. circulating the magnetic particles through one or more fluid paths of the automated cell culture system; and e. repeating steps c to d one or more times.

[58] The method according to

[57] , wherein the magnetic particles are bound to a target biological population.

[59] The magnetic particles are bound to the target biological population via an antibody, protein, or nucleic acid, which is a method described in

[58] . The method according to

[57] , wherein the magnetic particles are bound to a non-target biological population. The method according to

[60] , wherein the magnetic particles are bound to the non-target biological population via an antibody, a protein or a nucleic acid. The method according to any one of

[58] to

[59] , wherein the target biological population is any one or more of cells, viruses, bacteria, proteins, DNA and RNA. The method according to

[62] , wherein the target biological population is a T cell. The method according to any one of

[57] to

[63] , wherein the magnetic field gradient is provided by one or more permanent magnets. The method according to

[64] , wherein the permanent magnet contains magnetite, neodymium, samarium-cobalt, or alnico. The method according to any one of

[64] to

[65] , wherein the permanent magnet is composed of a linear array. The method according to any one of

[57] to

[67] , wherein a magnetic field shield / barrier is inserted between the magnetic particles and the magnetic field to enable collection of the magnetic particles. The method according to

[67] , wherein the magnetic field shield / barrier contains a material with high permeability and magnetic saturation. The method according to

[57] , wherein the magnetic field gradient is provided by one or more electromagnets. The method according to

[69] , wherein current is removed from the one or more electromagnets to enable collection of the magnetic particles. The method according to any one of

[57] to

[70] , wherein the gaseous phase fluid contains one or more of air, nitrogen, oxygen and carbon dioxide. ​​​​​​​​​​​​​The liquid-phase fluid contains one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, and is the method according to any one of

[57] to

[71] .

[73] Steps c to d are repeated at least twice, and are the method according to any one of

[57] to

[72] .

[74] A system for magnetically separating and collecting a target biological population from a biological sample, a. A magnetic field source; b. A separation tube for flowing the biological sample, aligned with the magnetic field source; c. A magnetic field shield / barrier configured to be disposed between the magnetic field source and the separation tube; and d. A device for inserting the magnetic field shield / barrier between the magnetic field source and the separation tube, a system comprising the same.

[75] The system is part of an automated cell culture system, and is the system according to

[74] .

[76] The magnetic field shield / barrier is a substance with high permeability and magnetic saturation, and is the system according to

[74] or

[75] .

[77] The substance with high permeability and magnetic saturation is selected from the group consisting of iron, iron alloy, ferrite steel, and Hiperco-50, and is the system according to

[76] .

[78] The magnetic field source includes one or more permanent magnets, and is the system according to any one of

[74] to

[77] .

[79] The permanent magnet includes neodymium, samarium-cobalt, or alnico, and is the system according to

[78] .

[80] The magnetic field source includes a plurality of permanent magnets configured in a linear array, and is the system according to any one of

[74] to

[78] .

[81] The plurality of permanent magnets in the linear array are arranged in opposite pole directions perpendicular to the axis of the linear array, and is the system according to

[80] . 〔82〕 The apparatus for insertion is the system according to any one of 〔74〕 to 〔80〕, which is an apparatus for rotating the magnetic field shield / barrier between the magnetic field source and the separation tube. 〔83〕 The apparatus for rotation is the system according to 〔82〕, which is an electro-mechanical drive assembly controlled by software. 〔84〕 A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding the target biological population to magnetic particles; b. exposing the target biological population bound to the magnetic particles to a magnetic field gradient to capture the target biological population bound to the magnetic particles; c. removing the unbound population of the biological sample; d. inserting a magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field to release the target biological population bound to the magnetic particles; and e. collecting the target biological population bound to the magnetic particles. 〔85〕 The target biological population includes one or more of cells, viruses, bacteria, proteins, DNA, and RNA, according to the method of 〔84〕. 〔86〕 The target biological population includes T cells, according to the method of 〔84〕 or 〔85〕. 〔87〕 The magnetic particles are bound to the target biological population via an antibody, protein, or nucleic acid, according to any one of 〔84〕 to 〔86〕. 〔88〕 The magnetic field gradient is provided by one or more permanent magnets, according to any one of 〔84〕 to 〔87〕. 〔89〕 The permanent magnet includes magnetite, neodymium, samarium-cobalt, or alnico, according to the method of 〔88〕. 〔90〕 The permanent magnet is configured in a linear array, by the method described in any one of

[84] to

[89] .

[91] The magnetic field gradient is provided by one or more electromagnets, and optionally the step of inserting in step d is replaced by the step of removing current from the one or more electromagnets, by the method described in any one of

[84] to

[90] .

[92] The magnetic field shield / barrier contains a material with high permeability and magnetic saturation, by the method described in any one of

[84] to

[91] .

[93] The magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field, by the method described in any one of

[84] to

[92] .

[94] The target biological population is collected by circulating a gas-phase fluid and subsequently a liquid-phase fluid one or more times, by the method described in any one of

[84] to

[93] .

[95] The gas-phase fluid contains one or more of air, nitrogen, oxygen, and carbon dioxide, by the method described in

[94] .

[96] The liquid phase contains one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, by the method described in

[94] .

[97] The method further includes removing the target biological population from the bound magnetic particles, by the method described in any one of

[84] to

[96] .

[98] A method for collecting a target biological population from a biological sample in an automated cell culture system, a. binding a non-target biological population to magnetic particles; b. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient to capture the non-target biological population bound to the magnetic particles; c. collecting the target biological population; and d. Inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field to release the non-target biological population bound to the magnetic particles, a method comprising this. 〔99〕 The method according to

[98] , wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA. 〔100〕 The method according to

[98] or

[99] , wherein the target biological population comprises T cells. 〔101〕 The method according to any one of

[98] to

[100] , wherein the magnetic particles are bound to the non-target biological population via an antibody, protein, or nucleic acid. 〔102〕 The method according to any one of

[98] to

[101] , wherein the magnetic field gradient is provided by one or more permanent magnets. 〔103〕 The method according to

[102] , wherein the permanent magnet comprises magnetite, neodymium, samarium-cobalt, or alnico. 〔104〕 The method according to any one of

[102] to

[103] , wherein the permanent magnet is composed of a linear array. 〔105〕 The method according to any one of

[98] to

[101] , wherein the magnetic field gradient is provided by one or more electromagnets, and optionally the step of inserting d is replaced by the step of removing current from the one or more electromagnets. 〔106〕 The method according to any one of

[98] to

[105] , wherein the magnetic field shield / barrier comprises a material with high permeability and magnetic saturation. 〔107〕 The method according to any one of

[98] to

[106] , wherein the magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field. 〔108〕 The target biological population is collected by circulating the gas-phase fluid and subsequently the liquid-phase fluid one or more times, by the method described in any one of

[98] to

[107] .

[109] The gas-phase fluid contains one or more of air, nitrogen, oxygen, and carbon dioxide, by the method described in

[108] .

[110] The liquid phase contains one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, by the method described in

[108] .

Claims

1. A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding the target biological population to magnetic particles; b. circulating the biological sample through one or more fluid pathways of the automated cell culture system; c. exposing the target biological population bound to the magnetic particles to a magnetic field gradient to capture the target biological population bound to the magnetic particles; d. circulating unbound components of the biological sample through the one or more fluid pathways of the automated cell culture system; e. inserting a magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field gradient to release the target biological population bound to the magnetic particles; f. circulating the target biological population bound to the magnetic particles through the one or more fluid pathways of the automated cell culture system; g. repeating steps b to f one or more times; and h. collecting the target biological population bound to the magnetic particles.

2. The method according to claim 1, wherein the target biological population comprises one or more of T cells, viruses, bacteria, proteins, DNA, and RNA.

3. The method according to claim 1 or 2, wherein the magnetic particles are bound to the target biological population via an antibody, protein, or nucleic acid.

4. The method according to any one of claims 1 to 3, wherein the magnetic field gradient is provided by one or more permanent magnets comprising magnetite, neodymium, samarium-cobalt, or alnico.

5. The method according to claim 4, wherein the one or more permanent magnets are configured in a linear array.

6. The method according to claim 1 or 2, wherein the magnetic field gradient is provided by one or more electromagnets.

7. The method according to any one of claims 1 to 6, wherein steps b to f are repeated at least twice.

8. The method according to any one of claims 1 to 6, wherein the magnetic field shield / barrier comprises a material with high permeability and magnetic saturation.

9. The method according to any one of claims 1 to 8, wherein the magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field gradient.

10. The target biological population is collected by circulating the gas-phase fluid and then the liquid-phase fluid one or more times, wherein the gas-phase fluid contains one or more of air, nitrogen, oxygen, and carbon dioxide, and the liquid-phase fluid contains one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, The method according to any one of claims 1 to 9.

11. The method according to any one of claims 1 to 10, further comprising removing the target biological population from the bound magnetic particles.

12. A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding a non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluid pathways of the automated cell culture system; c. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient to capture the non-target biological population bound to the magnetic particles; d. circulating the target biological population through the one or more fluid pathways of the automated cell culture system; e. inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field to release the non-target biological population bound to the magnetic particles; f. circulating the non-target biological population bound to the magnetic particles through one or more fluid pathways of the automated cell culture system; and g. repeating steps b to f one or more times; and h. collecting the target biological population.

13. The method according to claim 12, wherein the target biological population contains one or more of T cells, viruses, bacteria, proteins, DNA, and RNA.

14. The method according to claim 12 or 13, wherein the non-target biological population is collected.

15. The method according to any one of claims 12 to 14, wherein the magnetic particles are bound to the target biological population via an antibody, protein, or nucleic acid.

16. The method according to any one of claims 12 to 15, wherein the magnetic field gradient is provided by one or more permanent magnets including magnetite, neodymium, samarium-cobalt, or alnico.

17. The method according to claim 16, wherein the one or more permanent magnets are configured in a linear array.

18. The magnetic field gradient is provided by one or more electromagnets, the method according to any one of claims 12 to 15.

19. Steps b to f are repeated at least twice, the method according to any one of claims 12 to 18.

20. The magnetic field shield / barrier comprises a material with high permeability and magnetic saturation, the method according to any one of claims 12 to 19.

21. The magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field gradient, the method according to any one of claims 12 to 19.

22. The target biological population is collected by circulating the liquid phase fluid one or more times after the gas phase fluid, The gas phase fluid contains one or more of air, nitrogen, oxygen and carbon dioxide, The liquid phase fluid is among water, buffered saline, culture medium, animal serum, chelating agent and enzyme containing one or more of them, the method according to any one of claims 12 to 21.

23. A method for collecting a target biological population from a biological sample in an automated cell culture system, a. Binding a non-target biological population to magnetic particles; b. Exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient to capture the non-target biological population bound to the magnetic particles; c. Collecting the target biological population; and d. Inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field gradient to release the non-target biological population bound to the magnetic particles, a method comprising.

24. The target biological population contains one or more of T cells, viruses, bacteria, proteins, DNA and RNA, the method according to claim 23.

25. The magnetic particles are bound to the non-target biological population via an antibody, protein or nucleic acid, the method according to claim 23 or 24.

26. The magnetic field gradient is provided by one or more permanent magnets including magnetite, neodymium, samarium-cobalt, or alnico, the method according to any one of claims 23 to 25.

27. The one or more permanent magnets are configured in a linear array, the method according to claim 26.

28. The magnetic field gradient is provided by one or more electromagnets, the method according to any one of claims 23 to 25. **Claim 29** The method according to any one of claims 23 to 25, wherein the magnetic field shield / barrier comprises a material having high permeability and magnetic saturation. **Claim 30** The method according to any one of claims 23 to 29, wherein the magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field gradient. **Claim 31** The target biological population is collected by circulating a gas-phase fluid and then a liquid-phase fluid one or more times, wherein the gas-phase fluid comprises one or more of air, nitrogen, oxygen, and carbon dioxide, and the liquid-phase fluid comprises one or more of water, buffered saline, culture medium, animal serum, chelating agent, and enzyme, The method according to any one of claims 23 to 30.

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