Methods for isolating target cells

Abstract

The present invention provides a method for isolating viable target cells from a sample, the method comprising: subjecting a sample comprising a suspension of viable target cells displaying molecules on their cell surface to a concentration of about 1.45 g / cm 3 the target cell / microparticle complexes are separated from the unbound material in the sample by washing the unbound material through a filter while retaining the target cell / microparticle complexes; mechanically dissociating the target cell / microparticle complexes and eluting the viable target cells through the filter while retaining the microparticles having the capture ligand covalently immobilized on their surface; and a cartridge, kit of parts, device configured for use in the method, as well as a medicament comprising the viable target cells obtained by the method.

Description

[Technical field]

[0001] 1.Technical Field The present invention is within the field of cell separation technology and, in particular, relates to methods, cartridges and devices for targeted cell capture, separation and isolation. [Background technology]

[0002] 2. Background of the invention Cell separation and target cell isolation from complex biological matrices such as tissues, blood, and other samples is important in several industrial and research processes. At its core, cell separation requires the selective enrichment of target cells based on their physical, chemical, or biological properties. One of the main goals of target cell isolation is to produce viable cells that retain the original biological properties found in the original tissue or source. This is particularly important in cell and gene therapy, where cell health and metabolism are critical for the success of such treatments.

[0003] Examples of such cell and gene therapy include hematopoietic stem cell transplantation for the treatment of leukemia or solid cancer. The cells effective in the treatment (e.g., nucleated cells, including hematopoietic stem cells) are separated from bone marrow or peripheral blood by removing red blood cells, and then administered to the patient. Cord blood blood banks require cells to be cryopreserved before use, and red blood cells are removed to prevent hemolysis that may occur during cryopreservation. Furthermore, transplantation of bone marrow, umbilical cord blood, or peripheral blood cell fractions enriched in stem cells, e.g., mesenchymal stem cells, hematopoietic stem cells, and endothelial progenitor cells, has been used to promote angiogenesis or nerve regeneration in the treatment of ischemic diseases, e.g., cerebral infarction, myocardial infarction, and ischemia. Furthermore, granulocytes may cause undesirable side reactions, e.g., inflammation that may reduce the therapeutic effect, and it may be desirable to remove them from cell therapy products.

[0004] Cell isolation is also important in the manufacture of cell therapies, such as CAR-T cells, autologous and heterologous stem cell treatments, and other novel therapeutic approaches. Sometimes, further processing of isolated cells is necessary in both industrial and research areas. The success and efficacy of these processes is directly affected by the quality and purity of the isolated cell suspension. Residual components, such as nanoparticles, antibodies, and buffers, can interfere with downstream processes (i.e. cell growth, gene editing) and should be excluded from the final product if possible.

[0005] The same is true for the clinical use of isolated cells in autologous and allogeneic transplantation procedures. Isolated cells contaminated with residual buffers and antibodies can negatively affect the outcome of the procedure. Moreover, the clinical use of these contaminated cells is not permitted in some countries due to regulatory constraints. This limits the access of cell therapy.

[0006] Current technologies available on the market utilize different means of cell separation and enrichment and fall mainly into the following categories: 1) Magnetic particle separation: This method uses magnetic nanoparticles that are functionalized with cell-specific antibodies and capture target cells as they pass through a magnetic field source. If cells bind to the magnetic particles, the magnetic field will hold the magnetic particles and non-target cells will be washed away. If the magnetic field is removed, the cells will be released. The magnetic particles are typically nanoparticles of iron oxide, e.g. magnetite (Fe3O4), with dimensions ranging from 1 to 100 nanometers, giving them superparamagnetic properties. Superparamagnetic particles differ from more common ferromagnetic materials in that they only exhibit magnetic behavior in the presence of an external magnetic field. This property relies on the small dimensions of the particles, which allows them to be separated in suspension together with whatever they are bound to. Since nanoparticles often have diameters smaller than or similar to the diameter of the cells, it is not possible to separate the cells and particles by size, and the cells bound to the nanoparticles are separated by a magnet. The cells can be eluted from the nanoparticles by the use of buffers, which can greatly affect the viability of the cells, and the magnetic nanoparticles are then separated from the cell solution by the use of a magnetic field. One drawback of this method is that some residual magnetic nanoparticles may remain in the final cell solution. This may occur due to loss of magnetic properties of the particles, making it difficult to ensure that the removal of the magnetic particles from the solution of target cells has been completely performed. Furthermore, this method requires large and expensive equipment to ensure magnetic separation and requires significant operator execution time (see, for example, EP3037171A1, US10119970B2, CA2854240A1, EP0819250A1, EP0760102A1, US5385707A, EP2444158A2, US2002146848A1, WO2019103103A1, and CN107278270A). 2) Non-magnetic bead separation: This method uses porous or non-porous microbeads of various sizes coated with antibody receptors (e.g., protein A, protein G, avidin, and others). The antibody receptors are used to capture antibodies that can capture the target cells of interest. Non-target cells are washed away in a column or strainer. The target cells are eluted by 1) a competitive binding compound (e.g., biotin in the case of avidin) that preferentially binds to the coated receptor and releases the secondary antibody and cells from the beads, 2) an elution buffer that may contain high salt content, 3) low pH, 4) enzymes, or 5) other chemicals and animal-derived compounds that facilitate the release of the antibodies from the coated receptor. The resulting cell suspension therefore contains an elution buffer that may negatively affect cell viability or cell behavior, thereby interfering with downstream processing. Furthermore, column separation by gravity is unreliable because the flow is controlled only by the density of the beads in the solution and the gravity, resulting in non-uniform separation times and efficiency. These methods are also limited in the scale of input volumes due to the physical limitations of column separation (see, e.g., US10196631B2, JP2018138913A, EP2734538A2, WO2015166049A1, US2014315297A1, and US2017299585A1). 3) Separation by centrifugation and density: This is the most widely used standard form of cell separation based on the physical properties of cells, such as size and density. These methods use centrifugation and density gradients to capture the cells of interest. The main drawback of these methods is that cells are purified based only on their physical properties and not on their biological properties, such as surface antigens. In cell therapy, specific cell populations need to be isolated based on their surface antigens (called CD antigens) and cannot be differentiated based on the physical properties of cells alone. Extended centrifugation and the use of density reagents can have a negative effect on cell behavior and viability (see, for example, JP2012143256A and WO2018194061A1).

[0007] As a result of these and other shortcomings of cell separation methods and systems in the art, there is a need for methods, devices, and systems that provide efficient separation of target cells based on surface antigens without the use of magnetic particles and without the use of non-magnetic particles coated with antibody receptors that can be used directly in therapy. Summary of the Invention

[0008] 3. Overview of the Invention To address this need, the present invention provides methods, cartridges, and devices that provide efficient and specific separation of viable target cells based on molecules, e.g., antigens, present on the surface of the target cells without the use of magnetic particles or antibody receptor coated beads. The present invention provides high purity and high cell viability of isolated target cells in a selected buffer or medium.

[0009] These objects are solved by a method for isolating viable target cells from a sample, a cartridge configured for use in such a method, a kit of parts and / or a device configured for use in the above method according to the claims. Specific embodiments are derivable from the dependent claims and the description.

[0010] Thus, in one aspect, the present invention provides a method for isolating viable target cells from a sample, comprising the steps of: a. A sample containing a suspension of viable target cells displaying molecules on their cell surface is immersed in a 25 mL aliquot of about 1.45 g / cm 3 contacting said molecule with non-porous microparticles having a density equal to or greater than 10 μm and a diameter of about 10 μm to 200 μm, said microparticles having a capture ligand covalently immobilized on the surface of said microparticles capable of specifically binding said molecule; b. incubating the sample substantially without agitation to allow target cell / microparticle complexes to form; c. separating the unbound material in the sample from the target cell / microparticle complexes by washing the unbound material through a filter while retaining the target cell / microparticle complexes; d. Mechanically dissociating the target cell / microparticle complexes and eluting the viable target cells through the filter while retaining the microparticles having the capture ligand covalently immobilized on their surface. The present invention relates to a method comprising the steps of:

[0011] In a further aspect, the present invention relates to a cartridge adapted to be used for separating viable target cells from a sample using the method according to the present invention.

[0012] In a further aspect, the present invention relates to an apparatus configured to separate viable target cells from a sample using the method according to the present invention.

[0013] In a further aspect, the present invention provides a cartridge and a method for producing a cartridge having a coating composition comprising: 3 and a container containing a suspension of non-porous microparticles having a density equal to or greater than 10 μm and a diameter of about 10 μm to 200 μm, wherein a capture ligand capable of specifically binding to a molecule on a cell surface is covalently immobilized on the surface of the microparticles.

[0014] The proposed invention eliminates the shortcomings of current technology by allowing for automated or manual isolation of target cells from a sample using non-porous microparticles with a specified density and a specified diameter range, free of other antibody binding receptors and with capture ligands, e.g. antibodies, covalently attached to their surface. At the same time, the invention ensures the purity and high viability of the isolated target cell suspension, thereby eliminating the need for downstream processing.

[0015] The methods, cartridges, and devices according to the invention allow for the capture of specific target cells from a sample by directly binding cell surface molecules on the target cells to capture ligands that recognize these cell surface molecules, e.g., by binding cell surface antigens to capture antibodies covalently bound to non-porous microparticles. After washing the sample free of unbound cells and other undesirable material, the target cells are eluted by mechanical dissociation or disruption of cell surface molecule-capture ligand interactions, e.g., cell surface antigen-antibody interactions, by adjusting the flow rate or flow speed of the solutions, by mixing, pipetting, and / or sonication. By using the methods, cartridges, and devices according to the invention, there is no need for the addition of special chemical elution buffers or elution additives, e.g., enzymes, biotin-streptavidin, and other compounds. The methods, cartridges, and devices according to the invention also eliminate the need for unnecessary and / or biologically incompatible materials, e.g., bacterial proteins (protein A, protein G) and animal-derived products, e.g., BSA, casein, and others. The invention does not rely on elution of antibodies into the final cell suspension, as do other approaches. The method, cartridge and device according to the invention do not rely on the magnetic properties of magnetic particles that are eluted and present in the final cell suspension in other systems. The method, cartridge and device according to the invention allow a rapid and controlled cell separation procedure that can last less than one hour, thereby reducing cell death and changes in cell metabolism as well as unwanted activation of cells. Furthermore, the method, cartridge and device according to the invention can provide a closed and sterile environment and can be adapted for small (e.g. 10 ml sample) or large (multiple liter sample) applications. The method, cartridge and device according to the invention result in separated target cells eluted in a selected medium or physiological buffer, without unwanted additives or impurities, with limited processing time and high cell viability and health.

[0016] Thus, in further aspects, the present invention also relates to a medicament comprising a target cell suspension obtained according to the method of the present invention, a target cell suspension obtained according to the method of the present invention for use as a medicament, as well as a method of treating a subject in need of a target cell suspension obtained according to the method of the present invention for the treatment of a disease. [Brief description of the drawings]

[0017] 4. Brief description of the drawings [Figure 1] Results of Bradford supernatant analysis calculated as percentage of protein remaining in the supernatant solution from the starting protein concentration. [Diagram 2] Calculated surface saturation of 40 μm non-porous silica microparticles (Glantreo Ltd., Ireland) coated with anti-CD90 antibodies (BIOTEM, France) based on experimental results obtained by Bradford spectrophotometry of the supernatant. [Diagram 3] FIG. 1 illustrates the components connected to one system used for the manual cell separation method of target cells according to the invention: filter, incubation tube, and syringe. The system is sterile and meant for one-time use. Syringe 1 is used for incubation of viable target cells and non-porous microparticles, as well as for elution buffer. Filter 3 is used to separate viable target cells in the sample from non-porous microparticles. Long tube 4 and short tube 5 are used to facilitate connection to filter 3 and manipulation of first syringe 1 and second syringe 2. Second syringe 2 is used as a receiving syringe for collection of viable target cells. First syringe 1 and second syringe 2 can be replaced with clean syringes if necessary to carry out the method. [Figure 4a]1 is a microscopic image of non-porous microparticles after carrying out steps a and b of the cell separation method according to the present invention using positive control cells. Silica non-porous microparticles (purchased from Glantreo Ltd., Cork, Ireland) with a diameter of 40 μm and with immobilized anti-CD90 antibodies on their surface bind to CD90 positive cells (Jurkat cells) and form target cell / microparticle complexes. CD90 positive cells are identified as small round shiny structures located on the surface of the large spherical non-porous microparticles. [Figure 4b] 1 shows a microscopic image of non-porous microparticles after carrying out steps a and b of the cell separation method according to the present invention using negative control cells. Silica non-porous microparticles (purchased from Glantreo Ltd., Cork, Ireland) with a diameter of 40 μm and immobilized anti-CD90 antibodies (BIOTEM, France) on their surface do not bind to CD90 negative cells (Kasumi cells) and do not form target cell / microparticle complexes. [Diagram 5] FIG. 1 is a front view of a cartridge for the separation of target cells according to the invention, used in a device. In the front view, a valve 13 can be seen at the end of each luer lock 1-10, to which a syringe or other fluid container can be attached. The front side of the cartridge is connected to the device, whereby the device has access to the valve to control the open / closed position. The cartridge is made of female luer locks 1-10, to which a male luer lock can be connected, a valve 13 allowing control of the fluid flow, a filter 11, used to prevent non-porous particulates from contaminating the separated viable target cells, and a hollow tube 12 connecting all the syringes or other fluid containers into one closed system. [Figure 6]FIG. 1 is a front view of a rotated version of a cartridge for the separation of target cells according to the invention used in a device. The orientation of the cartridge shown allows for better control of the flow of liquids and solids through the system. Female luer locks 1-10 are in position to which syringes or other fluid containers with male luer locks are connected. Filter 11 prevents non-porous particulates from contaminating the separated viable target cells. Tubing 12 is used as an incubation space and also connects all syringes or other fluid containers into one closed system. [Figure 7] Rear view of a cartridge according to the invention with female luer locks 1-10 and valves located on only one side of the cartridge. The illustrated method of orienting the luer locks and valves allows for easy attachment of syringes and other fluid containers. Furthermore, the illustrated system allows for attachment of more ports and possibly more filters and tubes to the incubation tube. The female luer locks 1-10 are in the position where a syringe or other fluid container with a male luer lock would be connected. Filter 11 prevents non-porous particulates from contaminating the isolated viable target cells. Tube 12 is used as an incubation space, which also connects all the syringes or other fluid containers into one closed system. A schematic diagram of the front view is also provided in this figure. [Figure 8] 2 is a flow chart of a method according to the present invention; [Figure 9]1 is an exemplary process flow diagram illustrating a method used in various embodiments of the present invention. The diagram illustrates the basic process of cell separation using a device according to the present invention. An incubation space 1 is connected to an inlet stream 2 and an outlet stream 3. Element 4 represents the entry point of all streams into one inlet stream 2, and element 16 represents the splitter of the outlet stream 3. A sample containing a suspension of viable target cells is pushed by pump 23 with stream 15 through valve 14. When the cells pass valve 14, they enter stream 10 and mixing point 8. Non-porous microparticles are pushed with stream 13 by a second pump 27, and the non-porous microparticles pass valve 12. When the non-porous microparticles pass valve 12, they enter stream 9 and mixing point 8, where the non-porous microparticles are mixed with the sample containing a suspension of viable target cells. The stream then enters element 4, which is the entry point of all streams into inlet stream 2 and incubation space 1, where viable target cell / microparticle complexes are formed. After the incubation is completed and the cell / microparticle complexes are formed, the washing of unbound and non-target cells starts. The flow of the washing buffer is controlled by pump 29 and valve 5. The washing buffer passes through stream 6 and valve 5. Once the washing buffer has passed the valve, it flows to stream 7 and the entry point of all inlet streams 4. The washing buffer passes through inlet stream 2 and fills the incubation space 1. The incubation space 1 is equipped with a filter that is smaller than the non-porous microparticles and larger than the target cells (cells that have formed cell / microparticle complexes), and acts as a barrier for the non-porous microparticles as well as the larger non-target cells and unbound materials. During the first wash (washing of unbound materials and non-target cells), the liquid flows into the outlet stream 3 and then through the flow divider 16, and the unbound materials and non-target cells pass through stream 18 and valve 20 and finally into stream 22, where they are collected in a suitable fluid container. After the washing is completed, the mechanical disruption and elution of viable target cells starts. In the basic process, the mechanical disruption is performed with the same buffer. The flow of the buffer solution is controlled by pump 29 and passes through valve 5 into stream 6 and stream 7 .From here, buffer enters the entry points of all inlet streams 4 and inlet stream 2, filling the incubation space. The mechanical forces of the fluid in the incubation space cause disruption of the cell / microparticle complexes and allow the target cells to be eluted. The microparticles remain on one side of the filter in the incubation space, while the viable target cells pass through the filter and flow splitter 16 in stream 3. Stream 19 passes through valve 21 into stream 24, where the viable target cells are collected in a suitable fluid container. The outlet stream may optionally be equipped with a pump and there may optionally be two or more streams of wash buffer. [Figure 10a] FIG. 1 shows enrichment in the target cell fraction before and after separation of a leukopheresis sample (healthy donor) using microparticles containing anti-CD4 antibodies and the manual method according to the invention. [Figure 10b] The viability of the samples before and after separation is shown. Cells retained 100% of their starting viability in the target cell fraction. [Figure 11a] 1 shows enrichment in the target cell fraction before and after isolation of a stromal vascular fraction sample (healthy donor) using microparticles containing anti-CD34 antibodies and the manual method according to the present invention. [Figure 11b] The viability of the samples before and after separation is shown. Cells retained 74% of their starting viability in the target cell fraction. [Figure 12a] 1 shows enrichment in the target cell fraction before and after isolation of a stromal vascular fraction sample (healthy donor) using microparticles containing anti-CD90 antibodies and the manual method according to the invention. [Figure 12b] The viability of the samples before and after separation is shown, with cells retaining 75% of their starting viability in the target cell fraction. [Figure 13a] FIG. 1 shows the enrichment in the target cell fraction before and after separation of a leukopheresis sample (healthy donor) using microparticles containing anti-CD4 antibodies and an automated method according to the invention. [Figure 13b] The viability of the samples before and after separation is shown. Cells retained 98% of their starting viability in the target cell fraction. [Figure 14a]FIG. 1 shows the enrichment in the target cell fraction before and after separation of a PBMC sample (healthy donor) using microparticles containing anti-CD19 antibodies and the manual method according to the invention. [Figure 14b] The viability of the samples before and after separation is shown. Cells retained 70% of their starting viability in the target cell fraction. [Figure 15a] FIG. 1 shows the enrichment in the target cell fraction before and after isolation of human DPS (dental pulp stem) cells mixed with a leukopheresis sample (healthy donor) using microparticles containing anti-CD90 antibodies and the manual method according to the invention. [Figure 15b] The viability of the samples before and after separation is shown. Cells retained 77% of their starting viability in the target cell fraction. [Figure 16a] FIG. 1 shows enrichment in the target cell fraction before and after separation of human MS (mesenchymal stem) cells mixed with a leukopheresis sample (healthy donor) using microparticles containing anti-CD90 antibodies and the manual method according to the invention. [Figure 16b] The viability of the samples before and after separation is shown. Cells retained 89% of their starting viability in the target cell fraction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] 5. Detailed Description of the Invention As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. As used herein, "and" is used interchangeably with "or" unless expressly specified otherwise. All embodiments according to any aspect of the invention may be used in combination unless the context clearly dictates otherwise. Unless the context clearly requires otherwise, the word "comprise," "comprising," and the like, throughout this specification and claims, should be construed in an inclusive sense, i.e., "including but not limited to," rather than an exclusive or exhaustive sense. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "on," and "under," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application. The entire disclosures of all publications cited herein are incorporated by reference. The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. The following description of the invention is made primarily with reference to the method of the invention, however, references herein to the method of the invention equally apply to and relate to the cartridges and devices of the invention, unless otherwise indicated.

[0019] In one aspect, the invention provides a method for isolating viable target cells from a sample, comprising the steps of: a. A sample containing a suspension of viable target cells displaying molecules on their cell surface is immersed in a 25 mL aliquot of about 1.45 g / cm 3 contacting said molecule with non-porous microparticles having a density equal to or greater than 10 μm and a diameter of about 10 μm to 200 μm, said microparticles having a capture ligand covalently immobilized on the surface of said microparticles capable of specifically binding said molecule; b. incubating the sample substantially without agitation to allow target cell / microparticle complexes to form; c. separating the unbound material in the sample from the target cell / microparticle complexes by washing the unbound material through a filter while retaining the target cell / microparticle complexes; d. Mechanically dissociating the target cell / microparticle complexes and eluting the viable target cells through the filter while retaining the microparticles having the capture ligand covalently immobilized on their surface. The present invention relates to a method comprising the steps of:

[0020] target cell The present invention provides a method for separating viable cells of interest, referred to herein as "viable target cells" or "target cells", from other cells and materials present in a sample. As used herein, the term "viable" in reference to target cells refers to the cells being able to survive after being cultured in a culture medium that supports the growth of the target cells, or being able to survive after transplantation or implantation, both before and after carrying out the method according to the invention. Suitable culture media for the assessment of target cell growth and cell viability are known to those skilled in the art. Cell viability assays use various markers as indicators of metabolically active (living) cells. Examples of commonly used markers include staining with vital dyes, e.g., propidium iodide, measuring ATP levels, measuring the ability to reduce substrates, and detecting enzyme / protease activity specific to living cells, or measuring cell proliferation, e.g., by measuring DNA content or DNA synthesis in replicating cells. Cell proliferation assays are performed using standard methods, including enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence, and high content imaging.

[0021] The target cell may be any cell of interest that can be used in cell therapy, so long as it displays a molecule on its cell surface that can bind to the capture ligand covalently attached to the non-porous microparticles used in the methods of the present invention, and is capable of passing through the pores of a filter selected to retain the non-porous microparticles.

[0022] Target cells may be from any mammal, for example primates, including monkeys and humans, bovines, including cattle, ovines, including sheep, equines, including horses, canines, including wild and domestic dogs, swine, including wild and domestic pigs, murines, including mice and rats, or cells derived from these animals, which may be genetically engineered.

[0023] Target mammalian cells, preferably human cells, include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), fibroblasts, muscle cells, pancreatic cells, cardiac cells derived from any tissue or organ, including, but not limited to, heart, liver, kidney, colon, intestine, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymph glands, adenoids, tonsils, bone marrow, and blood), and spleen, as well as cell lines established from these primary cells.

[0024] Target cells may be healthy naturally occurring cells or cells derived from these cells (i.e. cancer cells, cells derived from diseased tissues or organs, cells infected with any pathogen including viruses, transfected cells, transformed cells, or established cell lines), and are preferably human cells or cells derived from human cells.

[0025] Typically, human cells have a diameter of about 5-100 μm.

[0026] In a preferred embodiment, the target cell is a bone marrow or hematopoietic cell or cell line. Examples of hematopoietic cells include granulocytes, T lymphocytes, monocytes, T regulatory cells, T helper cells, cytotoxic T cells, B lymphocytes, platelets, natural killer cells, hematopoietic stem cells and hematopoietic progenitor cells, which are preferably human.

[0027] Among human hematopoietic cells, neutrophils have a mean diameter of 8.3 μm, lymphocytes have a mean diameter of 6.05 μm, and monocytes have a mean diameter of 8.13 μm (see Downey et al., J Appl. Physiol. 69(5): pp. 1767-78, 1990). Other large cell types include megakaryocytes and myeloid cells, which are involved in the production of blood platelets. These cells can reach a diameter of about 100 μm. Red blood cells, also known as erythrocytes, are one of the smallest and most abundant human cells. These cells have a characteristic biconcave disc shape and a depression in which the nucleus has been lost during maturation, with corresponding diameters of about 7-8 μm and a length of about 100 μm. 3 It has a volume of

[0028] CAR-T cells (chimeric antigen receptor cells) are T cells genetically engineered to produce an artificial chimeric antigen receptor on their surface. CAR-T cell immunotherapy is one of the newest approaches for the treatment of hematopoietic malignancies, including B-cell lymphoma, T-cell lymphoma, acute myeloblastic leukemia, Hodgkin's lymphoma, and multiple myeloma. This is particularly important for patients who have failed other therapeutic strategies. In principle, the therapy is performed by harvesting the white blood cell fraction of the patient's blood by leukopheresis. T cells are separated from other cells in the blood by their size and density, and the remaining blood is returned to the patient's bloodstream. The obtained T cells are then washed, thereby removing the anticoagulant. The T cells are then further separated into specific subtypes. Separation of T cell subtypes is typically performed using specific antibodies bound to magnetic particles. This is problematic because these particles may lose their magnetic ability and remain bound on the cells, and even if the magnetic particles are successfully removed, residual antibodies may remain bound to the antigens on the cell surface. These are then transplanted into the patient together with the isolated cells. Now the method and device according to the invention can offer a solution, since the final cell eluate is essentially free of any antibodies or microparticles. After separation of the cells into specific subtypes, the T cells are activated in vitro by using artificial antigen presenting cells, antibody-coated beads, or special drugs. After activation, a sequence encoding the CAR (chimeric antigen receptor) is integrated into the genome of the T cells. Integration of the sequence can be performed by various input methods, for example by electroporation of the DNA molecule itself, via transposon / transposase systems, viral vectors (e.g. retroviral and lentiviral vectors). Successfully transformed T cells express the chimeric antigen receptor on their surface. The structure of the CAR can be adjusted so that the action of the CAR-T cells is directed to a specific target cell. After T cell expansion, the engineered T lymphocytes are returned to the patient via blood transfusion, where lymphocytes equipped with specific cell recognition receptors perform their function. One reason this form of treatment is not widely available is the high cost of production.The device according to the invention can be designed to significantly reduce the cost of the cell separation process, while the resulting viable target cell population is free of magnetic particles, thus increasing safety, and the cell suspension is free of residual antibodies or animal-derived products, thus not requiring further purification. Thus, the device according to the invention can be used for the isolation of CAR-T cells for CAR-T cell therapy, which can treat a wide range of cancers, autoimmune diseases, chronic inflammatory diseases, post-transplant graft rejection, and other disease conditions. The isolated T cells can, of course, be used without subsequent genetic modification.

[0029] Tumor infiltrating lymphocytes (TILs) consist of all lymphoid cell populations that have invaded tumor tissue. They are found in several solid tumors and have emerged as important biomarkers in predicting treatment efficacy and outcome. In breast cancer, TILs consist mainly of cytotoxic (CD8+) and helper (CD4+) T cells, as well as a small percentage of B cells and natural killer cells. These tumor infiltrating lymphocytes can be isolated from samples using the device according to the invention and later expanded and returned to the patient as an add-on therapy for the treatment of various cancers. Markers by which TILs can be selected are CD3 for T cells, CD8 for cytotoxic T lymphocytes, CD4 for helper T cells, and FOXP3 for regulatory T cells.

[0030] In another preferred embodiment, the target cells are stem cells, preferably human stem cells. Examples of stem cells include hair follicle stem cells, cardiac stem cells, neural stem cells, multipotent muscle cells, hepatic stem cells, hematopoietic stem cells, mesenchymal / stromal stem cells, dental pulp cells, periodontal ligament cells, adipose tissue derived stem and progenitor cells, embryonic stem cells and induced pluripotent cells, which are preferably human cells, as well as any other cell type derived from these pluripotent cells.

[0031] Bone marrow is composed of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and supportive stromal cells. All types of hematopoietic cells, including myeloid and lymphatic lineages, are generated in the bone marrow. HSCs give rise to white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes). Mesenchymal stem cells are multipotent stem cells that can differentiate into various cell types, such as osteoblasts, chondrocytes, muscle cells, and bone marrow adipocytes. Whole bone marrow transplants can be used to treat certain types of cancer, such as leukemia, myeloma, lymphoma, and other blood and immune system diseases that affect the bone marrow. Using the device of the present invention, specific bone marrow cells can be isolated. Mesenchymal stem cells can also be isolated from adipose tissue and / or obtained as waste during other surgeries not intended solely for cell isolation, such as liposuction or abdominoplasty.

[0032] Cell markers that can be used for the specific isolation of hematopoietic stem cells: BMI-1, CD31, CD33, CD34, CD38, CD41, CD44, CD45, CD48, CD90(Thy1), CD105, CD106, CD117, CD127, CD150, EPCR, Ly6A / E(sca-1), MYB, Mcl-1, PTEN, SCF, STAT5a, STAT5b, VEGFR2.

[0033] Cell markers that can be used for the specific isolation of mesenchymal stem cells: The presence of CD73, CD90, and CD105, and the absence of CD34, CD45, and HLA-DR antigens.

[0034] Microvascular endothelial cells (MVECs) are located in the smallest blood vessels of the circulatory system, e.g., capillaries. They have an important role in vascular homeostasis and endothelial dysfunction that can lead to a number of diseases, including atherosclerosis.

[0035] Cell markers that can be used for specific isolation of MVECs: CD31 (endothelial cells), CD34 (progenitor cells, endothelial cells of blood vessels), ICAM-1 / CD54 (vascular endothelium), LYVE-1 (lymphatic endothelial cells), Tie-2 / Tek (vascular endothelial cells), VCAM-1 / CD106 (quiescent endothelial cells, inducible following injury), VE-cadherin (at junctions between endothelial cells and on HSCs and MSCs), VEGF-R2 (endothelial cells, endothelial cell precursors), von Willebrand factor-vWF (endothelial megakaryocytes).

[0036] Other examples of target cells of interest include retinal pigment epithelial cells, which express, for example, CD140b, CD56, GD2, and CD184, among others, as the most important antigens on their cell surface. The retinal pigment epithelium (RPE) is a specialized epithelium that exists at the interface between the neural retina and the choriocapillaris, forming the outer blood-retinal barrier. The retinal pigment epithelium is a fundamental component of the retina and plays an essential role in visual function. Damage to the structure and function of the retinal pigment epithelium leads to various retinopathies, such as age-related macular degeneration (AMD), which is one of the major causes of blindness worldwide.

[0037] Retinal pigment epithelial cells can be isolated using the methods and devices according to the invention based on their cell surface markers, namely CD140b, CD56, GD2, CD184, Mitf, ZO-1, RPE65, CRALBP, CD104, CD164, CD220, EGER, CD10, CD30, CD49a, CD49b, CD50, CD171, TRA-1-60, and CD326.

[0038] In another embodiment, the target cells are tumor cells. Isolation and detection of tumor cells, including circulating tumor cells (CTCs), have an important role in early cancer diagnosis and prognosis, i.e., facilitating access to cells and studying the molecular and genetic profiles of these metastatic cells before clinically detectable metastasis occurs. CTC methods are currently used mainly for research purposes, and only a few methods have been accepted for clinical application due to the difficulties caused by the heterogeneity of CTCs, isolation of CTCs from blood, and lack of complete clinical validation. Using the cell isolation method according to the present invention, highly pure and highly viable cancer cells can be obtained for characterization and study of such cells and for research purposes by covalently attaching capture ligands to non-porous microparticles that specifically bind to cancer cells expressing cell surface markers that are not normally expressed on non-cancerous cells of the same type.

[0039] Furthermore, the method according to the invention can be used to generate CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, Citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN205 cells, McCoy cells, mouse L cells, line 2071 (mouse L) cells, line LM (mouse L) cells, L-MTK-(mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, C11 cells, and Janssen cells, or their derivatives, mouse myeloma NSO or SP2 / 0 or rat myeloma YB2 / 0 cells, or their derivatives, and other established cell lines and lines thereof (e.g. 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells, and PER-C6 retinal cells), MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, I-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RCAny mammalian or human cell line can be isolated, including 256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof that have been modified, e.g., by genetic manipulation or infection, to contain and / or express genes that are not normally expressed in these cells.

[0040] Cell surface molecules As used herein, a "cell surface molecule" or "cell surface molecule" may be any molecule that is expressed or displayed on the cell surface of a target cell and thereby capable of being recognized and bound by a capture ligand covalently attached to a non-porous microparticle. Cell surface molecules may be proteinaceous or non-proteinaceous.

[0041] In a preferred embodiment of the invention, the molecule on the cell surface is an antigen capable of being recognized by an antibody or derivative thereof that is covalently attached to the non-porous microparticle.

[0042] Examples of human cell lines and antigens found on cell surfaces that can be recognized by capture ligands covalently attached to non-porous microparticles according to the invention include: Hematopoietic stem cells: Sca-1, CD27, CD34, CD38, CD43, CD48, CD117, and CD150 Mesenchymal / stromal stem cells: STRO-1, CD105, CD73, CD90 Granulocytes: CD45, CD11b, CD15, CD24, CD114, CD182 T-lymphocytes: CD45, CD3 Monocytes: CD4, CD45, CD14, CD114, CD11a, CD11b, CD91, CD16 T regulatory cells: CD4, CD25 T helper cells: CD45, CD3, CD4 Cytotoxic T cells: CD45, CD3, CD8 B lymphocytes: CD45, CD19, CD20, CD24, CD38, CD22 Platelets: CD45, CD61 Natural killer cells: CD16, CD56, CD31, CD30, CD38 Retinal pigment epithelial cells: CD140b, CD56, GD2, and CD184 Hair follicle stem cells: CD90 Cardiac stem cells: CD34, CD117, GPR4 Pluripotent myocytes: CD73, CD105 Liver stem cells: CDCP1, CD90 There is.

[0043] Other examples of proteinaceous molecules on the cell surface of various mammalian and human cells that can be recognized by capture ligands covalently attached to non-porous microparticles according to the invention include CD2, CD5, CD21 / CD35 (CR2 / CR1), CD23, CD40, CD45R / B220, CD69, CD70, CD79a (Igα), CD79b (Igβ), CD80, CD86, CD93 (C1Rqp), CD137 (4-1BB), CD138 (Syndecan-1), CD252 (OX40L), CD26 7, CD268 (BAFF-R), CD279 (PD1), IgD, IgM, CD9, CD49f, CD324, CD338, SSEA-3, SSEA-4, SSEA-5, TRA-1-60, TRA-1-81, TRA-2-49, TRA-2-54, CD54, CD62E (E-selectin), CD106 (VCAM-1), CD144 (VE-cadherin), CD146 (MUC18, Mel-CAM), CD201 (EPCR), CD202b (Tie2 / Tek), CD309 (VEGFR2 - Flk-1), podoplanin, VEGFR3, CD110, CD111, CD133, CD135 (Flk-2), CD243 (MDR-1), CD271 (NGFR), CD11c, CD16 / 32, CD33, CD64, CD68, CD85k (ILT3), CD107b, CD115, CD163, CD195 (CCR5), CD282, CD284, F4 / 80, GITRL, HLA-DR, Mac-2 (galectin-3), MHC class II, CD203c, FcεRIα, CD44, CD349 (Frizzled-9), TNAP, CD45RA , CD45RB, CD62L, CD6, CD47, CD90.1, CD90.2, CD100, CD112 (Nectin-2), CD166 (ALCAM), CD172a / b (SIRPα / β), CD200 (OX2), CD231 (TALLA), CD304 (Neuropil CD325 (N-cadherin), CX3CR1, CXCR7, CD10, CD13, CD64, CD66b, CD88, CXCR1, CXCR2, GR-1, JAML, TLR2, CD49b, CD57, CD122, CD158(Kir), CD161(NK-1.1), CD244 (2B4), CD314 (NKG2D), CD319 (CRACC), CD328 (Siglec-7), CD335 (NKp46), Ly49, Ly108, Vα24-Jα18 TCR (iNKT), CD1a, CD1b, CD1c, CD83, CD85g / ILT7, CD123, CD197 (CCR7), CD273 (B7-DC, PD-L2), CD303 (BDCA-2), DC marker (33D1), F4 / 80, HLA-DR, MHC class II, Siglec H, CD29, CD49d, CD50 (ICAM-3), CD51, CD10 2 (ICAM-2), CD106 (VCAM-1), CD140a (PDGFRα), lymphotoxin beta receptor (LTβR), Madcam-1, neuroganglioside, TLR1, TLR2, TLR4, CD84, CD126 (IL-6Rα), CD154 (CD40L), CD185 (CXCR5), CD252 (OX40L), CD278 (ICOS), TCR α / β, CD26, CD94, CD119, CD183, CD191(CCR1), CD254(TRANCE, RANKL), CD366(Tim-3), IL-18R, TNF-α, TNF-β, CCR8, CD193(CCR3), CD194(CCR4), CD294(CRTH2), Includes CD365 (Tim-1), IL-1R, TGF-β, CCR10, CD196 (CCR6), CD39, CD103, CD134, CD152 (CTLA-4), CD223, FR4, GARP, GITR, STRO-1, and STRO-3, NCAM, CD133, and SSEA-1. .

[0044] Examples of non-proteinaceous molecules on the cell surface of mammalian cells that can be recognized by capture ligands covalently attached to non-porous microparticles according to the invention include glycans (e.g., Galp1-3GlcNAc1-R, Galp1-4GlcNAcp1-R, Galp1-3GalNAca1-R, Galp1-3GalNAcp1-R), lipids and phospholipids (e.g., sphingomyelin, phosphatidylserine, phosphatidylcholine), and non-proteinaceous aptamers (e.g., tenascin-C (TN-C) and synthetic DNA / RNA / XNA molecules).

[0045] sample As used herein, the term "sample" may be any sample that contains a suspension of viable target cells. Samples can be prepared from tissues or organs taken from an individual, or specimens that have been treated to release the target cells.

[0046] The samples may be solutions containing viable target cells derived from mammalian, preferably human, tissue or organ extracts, as well as cell lines established from primary cells derived from tissues or organs including, but not limited to, heart, liver, kidney, colon, intestine, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymph glands, adenoids, tonsils, bone marrow, and peripheral blood cells, placenta / umbilical cord blood, blood including menstrual blood), spleen, and fibroblasts, or solutions obtained by roughly isolating cells from such solutions, as well as solutions diluted with water, any of the buffers and / or culture media described herein, such as the physiological buffers described herein, physiological saline, Ringer's solution containing divalent cations, such as calcium ions or magnesium ions, cell culture media, such as RPMI, MEM, IMEM, or DMEM, and phosphate buffers, such as PBS, HBSS, TBS, DPBS, EBSS, etc.

[0047] Preferred samples containing target cells to be separated according to the methods of the present invention are mammalian, preferably human, whole blood, apheresis samples, bone marrow aspirates, biopsy samples, liquefied tissue samples (e.g., enzymatically digested lipoaspirate samples), cell culture samples, bioreactor cultures, single cell suspensions, and the like.

[0048] In one embodiment, the sample is preferably derived from cells cultured in suspension, or the sample is a suspension of cells cultured on a substrate, such as a glass or plastic flask or plate, and dissociated from the substrate by enzymatic means, such as accutase or trypsin, or other means, such as washing with EDTA, washing the cells with a balanced salt solution or calcium- and magnesium-free medium, or by mechanical agitation.

[0049] Preferably, before a sample containing viable target cells is contacted with the non-porous microparticles, at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 99% or more of the cells in the sample are individual cells.

[0050] In carrying out the methods of the invention, if the sample contains or is suspected of containing debris, such as tissue, bone, organ, cell aggregates, or other fragments, that would otherwise interfere with performance or clog filters, in a preferred embodiment the sample can be pretreated to partially or completely remove this debris. This can be accomplished by filtering and / or centrifuging the sample and resuspending the cells in the sample under conditions that essentially do not remove single viable target cells from the sample.

[0051] non-porous microparticles The method of the present invention uses a suspension of non-porous microparticles. As used herein, the term "non-porous microparticles" refers to a suspension of non-porous microparticles having a density of about 1.45 g / cm 3 The term refers to any spherical or non-spherical, non-porous particle having a (dry weight) density of 10 μm or more and a diameter of approximately 10 μm to 200 μm, and having a capture ligand that can specifically recognize a molecule on the surface of a target cell to form a target cell / microparticle complex covalently immobilized on the surface of the microparticle.

[0052] The term "non-porous," when referring to a "microparticle," means that the "non-porous microparticle" has a smooth surface that is essentially free of depressions or indentations on the surface that can capture target cells and prevent them from mechanically dissociating from the non-porous microparticle when performing the methods of the invention. A "non-porous microparticle," as used herein, has one or more capture ligands covalently attached to its surface, such that at least about 90%, preferably at least about 95%, and more preferably at least about 99% of the capture ligands are available to bind to molecules on the surface of a target cell when using the methods of the invention.

[0053] In a preferred embodiment, the non-porous microparticles have a density of about 1.45 g / cm 3 More preferably, about 1.65 g / cm 3 More preferably, about 1.85 g / cm 3 More preferably, about 1.95 g / cm 3 It has a density (dry weight) of or greater.

[0054] In a further preferred embodiment, the non-porous microparticles have a diameter of about 10 μm to 200 μm, preferably 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0055] In certain embodiments, the non-porous microparticles have a density of about 1.45 g / cm 3 It has a density (dry weight) of 10 μm or more and a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0056] In certain embodiments, the non-porous microparticles have a density of about 1.65 g / cm 3 It has a density (dry weight) of 10 μm or more and a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0057] In certain embodiments, the non-porous microparticles have a density of about 1.85 g / cm 3 It has a density (dry weight) of 10 μm or more and a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0058] In certain embodiments, the non-porous microparticles have a density of about 1.95 g / cm 3 It has a density (dry weight) of 10 μm or more and a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0059] The non-porous microparticles used in the method according to the invention are made from at least one inorganic material, preferably but not limited to SiO2 (silicon dioxide), silicone, gold, silver, or platinum, or a composite material made from an inorganic shell of SiO2, silicone, gold, or silver and an organic polymer or copolymer material, such as but not limited to PMMA (poly(methyl methacrylate)), PLA (polylactic acid), ABS (acrylonitrile butadiene styrene), and nylon.

[0060] In a preferred embodiment, the non-porous microparticles are made of SiO2 glass. Preferably, the SiO2 glass non-porous microparticles have a density of about 2 g / cm 3 and a diameter of about 40 μm. An example of a silica non-porous microparticle having a diameter of 40 μm is a silica non-porous microparticle available from Glantreo Ltd. (Cork, Ireland). An example of a non-porous microparticle having a diameter of 40 μm (PNPP40.0NAR, Glantreo Ltd., Cork, Ireland) is used according to the invention in some of the examples and is shown in Figures 4a and 4b.

[0061] Other examples of commercially available non-porous microparticles include: Glantreo, Ltd. (Cork, Ireland): Non-porous Silica particles: 10 μm (PNPP10.0NAR), 20 μm (PNPP20.0NAR), 40 μm (PNPP40.0NAR), 50 μm (PNPP50.0NAR), 70 μm (PNPP70.0NAR); CD Bioparticles (New York, NY, USA): Non-porous Silica particles: 20 μm (DNG-E009), 30 μm (DNG-E010), 40 μm (DNG-E011); EPRUI (Wujiang District, Suzhou, China): Non-porous Silica particles: 20μm (EPRUI-Si-20), 30μm (EPRUI-Si-30), 40μm (EPRUI-Si-40), 50μm (EPRUI-Si-50); Abvigen (Newark, NJ, USA): Non-porous Silica particles: 20μm (ABM-3-362), 30μm (ABM-3000), 40μm (ABM-4000), 50μm (ABM-5000), 60μm (ABM-6000), 7 0μm (ABM-7000), 80μm (ABM-8000), 90μm (ABM-9000), 100μm (ABM-10000), 200μm (ABM-20000); VWR Chemicals (Radnor, PA, USA: Non-porous Silica gel particles: 40~63μm (27623.323), 40~63μm (7631-86-9), 60~200μm (84893.290), 63~200μm (27647.325), and MilliporeSigma Supelco, Merck (Darmstadt, Germany): Non-porous Silica gel particles: 40~60μm (1.09385), 40~75μm (80442), 40~75μm (53698), 75~200μm (78991) There is.

[0062] In a preferred embodiment, the non-porous microparticles have a density of about 1.45 g / cm 3 More preferably, about 1.65 g / cm 3 More preferably, about 1.85 g / cm 3 More preferably, about 1.95 g / cm 3 The non-porous SiO2 glass particles have a density (dry weight) of at least 1000 g / cm2.

[0063] In a further preferred embodiment, the non-porous microparticles are non-porous SiO2 glass microparticles having a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0064] In certain embodiments, the non-porous microparticles have a density of about 1.45 g / cm 3 The non-porous SiO2 glass particles have the above (dry weight) density and a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0065] In certain embodiments, the non-porous microparticles have a density of about 1.65 g / cm 3 The non-porous SiO2 glass particles have the above (dry weight) density and a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0066] In certain embodiments, the non-porous microparticles have a density of about 1.85 g / cm 3 The non-porous SiO2 glass particles have the above (dry weight) density and a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0067] In certain embodiments, the non-porous microparticles have a density of about 1.95 g / cm 3 The non-porous SiO2 glass particles have the above (dry weight) density and a diameter of about 10 μm to 200 μm, preferably about 25 μm to 150 μm, more preferably about 30 μm to 100 μm, and most preferably about 35 μm to 50 μm.

[0068] In certain preferred embodiments, the SiO2 glass non-porous particulates have a density of about 2 g / cm 3 and a diameter of about 40 μm.

[0069] The non-porous microparticles may have conductive properties in some embodiments of the present invention. Surprisingly, it has been determined that by adjusting the density, diameter, and surface properties of the microparticles, complexes can be formed between target cells and non-porous microparticles through interactions between capture ligands covalently attached to the surface of the microparticles and molecules on the surface of the target cells that are strong enough to withstand mechanical forces that arise when the target cell / non-porous microparticle complexes are washed with a filter that is permeable to the target cells but not the non-porous microparticles, and weak enough to be broken only by mechanical forces that allow the target cells to pass through the filter in a viable state. Elution of cells is achieved exclusively by the use of mechanical forces. Thus, while it is possible to carry out the methods of the present invention using magnetic non-porous microparticles, the present invention does not rely on the magnetic properties of such particles to separate cells. Thus, in a preferred embodiment of all aspects of the methods of the present invention, the non-porous microparticles are non-magnetic.

[0070] The non-porous microparticles may be in the form of a dry powder, for example a dried powder containing a non-toxic preservative, such as trehalose or saccharose, and in the form of a suspension. The solution used for the non-porous microparticle suspension may be any solution that maintains the viability of the target cells after addition to the sample. For example, the solution may be a buffer solution as described herein, a cell culture medium, or a medium used for transfection, transplantation or embedding, preservation, or cryopreservation of the cells as described herein, and / or a solution used to perform the assays described herein.

[0071] Functional groups on the surface of the non-porous microparticles and on the linkers that can be used to form covalent bonds between the surface and the capture ligand The non-porous microparticles used in the method according to the invention have one or more capture ligands covalently immobilized on the surface of the microparticles and capable of recognizing one or more molecules presented on the cell surface of the target cells. The capture ligands can be covalently attached to the non-porous microparticles by using coupling agents, such as common bifunctional agents, or by binding via a spacer. Depending on the reactive groups on the carrier surface, a suitable coupling agent is used. In the case where the surface has -OH groups, such as materials made of SiO2, a silane surface modifier is used to introduce functional groups to the surface of the carrier. The functional groups can be, for example, amino, amide, imide, hydrazide, carboxyl, thiol, hydroxyl, azide, alkyl, phenyl, epoxy, ester, halide, and acyl halide. Preferably, amino, carboxyl, thiol, and epoxy groups are used. Most preferably, carboxyl groups are used. In the case where the carrier material is a metal, such as gold, copper, silver, and platinum, the functionalization of the material can be carried out by using disulfide bridges formed on the material surface. Typically, linkers with a thiol group (-SH) are used for this type of functionalization, such as various alkanethiols with a thiol group at one end of the molecule and amino, amide, imide, hydrazide, carboxyl, thiol, hydroxyl, azide, alkyl, phenyl, epoxy, ester, halide, and acyl halide at the other end.

[0072] Conjugating, joining, bonding, connecting, immobilizing, coupling of a first unit to a second unit The covalent bond formed between the capture ligand and the surface can be formed directly between the functionalized surface and the capture ligand or through a bifunctional linker that can react with functional groups on both the surface carrier and the ligand molecule. The linker may or may not include a PEG (polyethylene glycol) spacer. Linkers that can be used for the formation of a covalent bond between the non-porous microparticle and the capture ligand can be, but are not limited to, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), DCC (N',N'-dicyclohexylcarbodiimide), NHS (N-hydroxysuccinimide), sulfo-NHS (N-hydroxysulfosuccinimide), DMA (dimethyladipimidate), DMP (dimethylpimelimidate), DMS (dimethylsuberimidate), glutaraldehyde, glutaraldehyde polymers, cyanogen bromide, cyanuric chloride, SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate), sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), or any other bifunctional linker or spacer that can activate the surface of the particle and then react with functional groups found in the structure of the capture ligand. The size of the PEG spacer that can be used in the present invention can vary from 0.4 kDa to more than 20 kDa. The non-porous microparticle and the capture ligand are bound to each other directly or through a linker. The capture ligand is prevented from leaking out of the non-porous microparticle by using only the method of covalent binding between the surface and the capture ligand without using any affinity ligand. Preferably, a linker that does not contain PEG (polyethylene glycol) is used in the present invention. Any other linker that can ensure the formation of a covalent bond between the functional group on the surface of the non-porous microparticle and the group that can be found in the structure of the capture ligand, as well as other functional groups that can be found on the surface of the non-porous microparticle that can react with the group of the capture ligand, with or without a linker molecule, is not a limiting factor in the implementation of the present invention.

[0073] Capture Ligand The non-porous microparticles used in the methods according to the invention have at least one capture ligand covalently attached to their surface and capable of specifically binding to a molecule displayed on the cell surface of a target cell in a sample to form a complex between the target cell and the non-porous microparticle, i.e. a "target cell / microparticle complex".

[0074] As used herein, the term "specifically binds" or "specifically binds to" when referring to any of the capture ligands described herein means that when performing the method according to the invention, the capture ligand on the non-porous microparticle binds to a molecule on the surface of a target cell, thereby forming a target cell / microparticle complex, without any substantial binding of the capture ligand to non-target cells in the sample. The capture ligand has an affinity for the molecule on the surface of the target cell, thereby maintaining the target cell / microparticle complex when unbound material is washed away from the sample. When the capture ligand is multivalent, i.e., when a particular capture ligand has more than one binding site for a molecule on the surface of the target cell, the capture ligand has an affinity for the molecule on the surface of the target cell, thereby maintaining the target cell / microparticle complex when unbound material is washed away from the sample.

[0075] In one embodiment, the term "capture ligand" refers to a proteinaceous receptor that can recognize a molecule on the surface of a target cell of interest. In a preferred embodiment, the proteinaceous receptor capture ligand is about 10 -5 That's about 10 -6 That's about 10 -7 That's about 10 -8 That's about 10 -9 That's about 10 -10 That's about 10 -11 More than or about 10 -12 The capture ligand specifically binds to a molecule on the surface of a target cell with a dissociation constant of 10 or greater. -5 ~10 -12 , more preferably 10 -9 ~10 -11 , or more preferably 10 -7 ~10 -10In a preferred embodiment, the affinity constant of the capture ligand is on the nanomolar order (about 10 -9 Binding affinity can be measured using a variety of known analytical methods, such as radioligand binding assays, surface plasmon resonance, fluorescence energy resonance transfer, and affinity chromatography.

[0076] In a preferred embodiment, the capture ligand is an antibody or derivative thereof that specifically binds to an antigen on the surface of a target cell. An antibody is a molecule having one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. It is known that the basic immunoglobulin structural unit consists of a tetramer. Each tetramer consists of a pair of two identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. The capture ligand may exist as an original immunoglobulin, i.e., an antibody, or a derivative thereof, in various forms, including, for example, FabFc2, Fab, Fv, Fd, F(ab')2, Fv fragments containing only the variable regions of the light and heavy chains, Fab or F(ab')2 fragments containing the variable regions and parts of the constant regions, single chain antibodies, e.g., scFv, CDR-grafted antibodies, dAbs, nanobodies, etc. The heavy and light chains of the Fv may be derived from the same or different antibodies, thereby producing a chimeric Fv region. The antibody may be an engineered antibody (e.g., oligomeric, reduced, oxidized, and labeled antibodies). The antibody may be of animal (particularly mouse, rabbit, or rat) or human origin, and may be chimeric or humanized. As used herein, the term "antibody" encompasses these various forms.

[0077] In a preferred embodiment, the capture ligand is about 10 -5 That's about 10 -6 That's about 10 -7 That's about 10 -8That's about 10 -9 That's about 10 -10 That's about 10 -11 More than or about 10 -12 Antibodies that recognize antigens on the surface of target cells with dissociation constants equal to or greater than 10, such as FabFc2, Fab, Fv, Fd, F(ab')2, Fv fragments containing only the variable regions of the light and heavy chains, Fab or F(ab')2 fragments containing the variable and constant regions, single chain antibodies such as scFv, CDR-grafted antibodies, dAbs, nanobodies, etc. In a preferred embodiment, the capture ligand is 10 -5 ~10 -12 , more preferably 10 -9 ~10 -11 , or more preferably 10 -7 ~10 -10 In a preferred embodiment, the affinity constant of the antibody is on the nanomolar order (about 10 -9 ).

[0078] Examples of antibodies on the surface of target cells: anti-Sca-1, anti-CD27 , anti-CD34, anti-CD38, anti-CD43, anti-CD48, anti-CD117, anti-CD150, anti-STRO-1 , anti-CD105, anti-CD73, anti-CD90, anti-CD45, anti-CD15, anti-CD24, anti-CD114, anti-CD182, anti-CD3, anti-CD4, anti-CD14, anti-CD11a, anti-CD11b, anti-CD91, anti-CD2 5. Anti-CD8, anti-CD19, anti-CD20, anti-CD24, anti-CD22, anti-CD61, anti-CD16, anti-CD56, anti-CD31, anti-CD30, anti-CD38, anti-CD140b, anti-CD56, anti-GD2, anti-CD184, anti-CD90, anti-CD34, anti-CD117, anti-GPR4, anti-CD73, anti-CD105, anti-CDCP1, anti-CD2, anti-CD5, anti-CD21 / CD35 (CR2 / CR1), anti-CD23, anti-CD40, anti-CD4 5R / B220, anti-CD69, anti-CD70, anti-CD79a (Igα), anti-CD79b (Igβ), anti-CD80, anti-CD86, anti-CD93 (C1Rqp), anti-CD137 (4-1BB), anti-CD138 (Shindekan-1), anti-CD252 (OX40L), anti-CD267, anti-CD268 (BAFF-R), anti-CD279 (PD1), anti-IgD, anti-IgM, anti-CD9, anti-CD49f, anti-CD324, anti-CD338, Anti-SSEA-3, Anti-SSEA-4, Anti-SSEA-5, Anti-TRA-1-60, Anti-TRA-1-81, Anti-TRA-2-49, Anti-TRA-2-54, Anti-CD54, Anti-CD62E (E-Selectin), Anti-CD106 (VCAM-1), Anti-CD144 (VE-Cardiol), Anti-CD146 (MUC18, Mel-CAM), Anti-CD201 (EPCR), Anti-CD202b (Tie2 / Tek), Anti-CD309 (VEGFR2 -Flk-1), anti-potropranin, anti-VEGFR3, anti-CD110, anti-CD111, anti-CD133, anti-CD135 (Flk-2), anti-CD243 (MDR-1), anti-CD271 (NGFR), anti-CD11c, anti-CD16 / 32, anti-CD33, anti-CD64, anti-CD68, anti-CD85k (ILT3), anti-CD107b, anti-CD115, anti-CD163, anti-CD195 (CCR 5), anti-CD282, anti-CD284, anti-F4 / 80, anti-GITRL, anti-HLA-DR, anti-Mac-2 (Galexin-3), anti-MHC Class II, anti-CD203c, anti-FcεRIα, anti-CD44, anti-CD349 (Freezul-9), anti-TNAP, anti-CD45RA, anti-CD45RB, anti-CD62L, anti-CD6, anti-CD47, anti-CD90.1, anti-CD90.2, anti-CD10 0, anti-CD112 (Nekuchin-2), anti-CD166 (ALCAM), anti-CD172a / b (SIRPα / β), anti-CD200 (OX2), anti-CD231 (TALLA), anti-CD304 (Nylopilin-1), anti-CD325 (N-cadherin), anti-CX3CR1, anti-CXCR7, anti-CD10, anti-CD13, anti-CD64, anti-CD66b, anti-CD88, anti-CXCR1, anti CXCR2, anti-GR-1, anti-JAML, anti-TLR2, anti-CD49b, anti-CD57, anti-CD122, anti-CD158 (Kir), anti-CD161 (NK-1.1), anti-CD244 (2B4), anti-CD314 (NKG2D), anti-CD319 (CRACC), anti-CD328 (シグレック-7), anti-CD335 (NKp46), anti-Ly49, anti-Ly108, anti-Vα24-Jα18TCR (iNKT), anti-CD1a, anti-CD1b, anti-CD1c, anti-CD83, anti-CD85g / ILT7, anti-CD123, anti-CD197 (CCR7), anti-CD273 (B7-DC, PD-L2), anti-CD30 3 (BDCA-2), anti-DC marker (33D1), F4 / 80, anti-HLA-DR, anti-MHC class II, anti-Siglec H, anti-CD29, anti-CD49d, anti-CD50 (ICAM-3), anti-CD51, anti-C D102 (ICAM-2), anti-CD106 (VCAM-1), anti-CD140a (PDGFRα), anti-lymphotoxin beta receptor (LTβR), anti-Madcam-1, anti-neural ganglioside, anti-TLR1, anti-TLR4, anti-CD84, anti-CD126 (IL-6Rα), anti-CD154 (CD40L), anti-CD185 (CXCR5), anti-CD252 (OX40L), anti-CD278 (ICOS), anti-TCR α / β, anti-CD26, anti-CD94, anti-CD119, anti-CD183, anti-CD191 (CCR1), anti-CD254 (TRANCE, RANKL), anti-CD366 (Tim-3), anti-IL-18R , anti-TNF-α, anti-TNF-β, anti-CCR8, anti-CD193 (CCR3), anti-CD194 (CCR4), anti-CD294 (CRTH2), anti-CD365 (Tim-1), anti-IL-1R, anti-TCR α / β, anti-TGF-β, anti-CCR10, anti-CD196 (CCR6), anti-CD39, anti-CD103, anti-CD134, anti-CD152 (CTLA-4), anti-CD223, anti-FR4, anti-GARP, anti-GITR, anti-STRO-1 and anti-STRO-3, anti-NCAM, anti-CD133, anti-SSEA-1, anti-SSEA-3, anti-SSEA-4 antibodies, or fragments thereof. Protein receptors having specific binding regions capable of recognizing certain proteins listed above are also considered capture ligands (e.g., Sca-1, CD27, CD34, CD38, and other receptors).

[0079] The non-porous microparticles may have one or more different capture ligands covalently bound to their surface that can specifically bind to a particular molecule on the surface of a target cell. The non-porous microparticles may also have one or more different capture ligands covalently bound to their surface that can specifically bind to different molecules on the surface of the same target cell.

[0080] The method according to the present invention can also be used to simultaneously separate different target cell populations from the same sample.When two or more different types of target cells in a sample are simultaneously separated from other unbound substances in the sample, the same non-porous microparticles used in the method can have different capture ligands covalently bound to their surface, which can specifically bind to different molecules on the surface of each target cell.Alternatively, the method can be carried out using a mixture of two or more different populations of non-porous microparticles, each of which can recognize and bind to a different target cell.

[0081] unbound substance Unbound material may include cells and materials that are not specifically recognized by the capture ligands on the surface of the non-porous microparticles, such as materials that are not specifically recognized by the antibodies present on the non-porous microparticles, such as components of the buffer and / or medium, such as ions, cations, sugars, lipids, residual chemicals, plasma, cell debris, cell waste and other metabolites, viruses, virus-like particles, exosomes, proteins, lipids, DNA, and / or non-target cells (cells that do not express the target molecule on their surface). The type and number of non-target cells that may be present in a sample depends on the type of sample. Non-target cells are cells present in a sample that do not express on their cell surface the specific molecule of interest present on viable target cells of interest, or that express the specific molecule of interest at a level that does not essentially result in the formation of a stable cell / microparticle complex upon washing of the target cell / microparticle complex according to the present invention. If the non-target cells in the sample are smaller than the size of the pores or mesh of the filter used to separate the target cells, then these non-target cells will pass through the filter and be washed away, while the target cell / microparticle complexes will be retained by the filter and separated from the viable target cells in the sample along with other unbound material. As used herein, the phrase "(and optionally non-target cells)" is used to describe this situation. On the other hand, if the non-target cells are larger than the size of the pores or mesh of the filter, then the non-target cells will be retained by the filter along with the target cell / microparticle complexes, but will not pass through the filter when the target cell / microparticle complexes are mechanically dissociated, remaining on the same side of the filter as the non-porous microparticles.

[0082] Filters The filters used in the method according to the invention for removing unbound material (and optionally non-target cells) from target cells are biocompatible, do not substantially affect cell viability and can be made of the following materials: spider silk, metals and alloys, such as gold, silver, platinum, copper, carbon, graphene, enamel, ceramics, glass, cellulose, cellulose acetate, collagen, chitosan, lignin, hydrogels, arginine, cotton, nanofibrous cellulose, polyamides, such as nylon, polyimides, polyesters, polyethylene, polypropylene, polyvinyl chloride, polyvinyl chloride-acrylic acid copolymers, polyvinyl alcohol, vinylidene chloride, polytetrafluoroethylene ... The porous substrate may be made from any porous matrix material that comprises, consists essentially of, or consists of any of the following: polyfluoroethylene, polychlorotrifluoroethylene, tetrafluoroethylene-hexafluoropropylene copolymers, polydimethylsiloxane, polyethylene terephthalate, polyether urethane, polyether urethane urea, polystyrene, polycarbonate, polyether ether ketone, polysulfone, acrylic polymers such as polymethyl methacrylate, polyacrylonitrile, etc., polycaprolactam, polyhexamethylene adipamide, polymethylpentene, and poly 2-hydroxyethyl methacrylate.

[0083] Preferred filter materials are resistant to sterilization (sterilizable) and include polyamides, such as nylon, polypropylene, polyvinyl chloride, polyethylene, polyimides, polycarbonate, polysulfone, and polymethylpentene.

[0084] In a preferred embodiment, the filter is a flexible membrane (or film-like sheet) of a material such as polyamine, e.g., nylon, having a thickness in the range of about 0.01 mm to about 0.5 mm, with about 0.05 mm to about 0.2 mm being more common. Other thicknesses can also be used. The filter may be a single layer membrane or may include two or more layers sealed together or separated to form a bilayer membrane.

[0085] Those skilled in the art will appreciate that there is an interactive relationship between the size of the filter pores or mesh, the diameter of the non-porous microparticles, and the diameter of the target cells. In carrying out the methods according to the invention, the size of the filter pores or mesh and the diameter of the non-porous microparticles are selected such that the non-porous microparticles do not pass through the pores or mesh of the filter, while unbound material (and optionally non-target cells) and the target cells pass through the pores or mesh of the filter.

[0086] The size of the pores or mesh of the filter is preferably about 9 μm to 100 μm, more preferably about 20 μm to 80 μm, and most preferably about 25 μm to 45 μm. In one particular preferred embodiment, the size of the pores or mesh is about 31 μm. This is applicable to cell separation when the target cells are smaller than 30 μm. When the cells of interest for separation (target cells) are larger than 30 μm, the size of the pores or mesh should be adjusted to the specific cell type. Furthermore, when the size of the target cells exceeds the preferred diameter of the non-porous microparticles, the size of the non-porous microparticles is selected to have a larger diameter than the cells of interest. In that case, the size of the pores or mesh of the filter is selected according to these parameters.

[0087] In a preferred embodiment, the filter is a nylon membrane with a pore or mesh size of about 30 μm.

[0088] Examples of commercially available filter membranes for use in the practice of the present invention are: MERCK MILLIPORE (Darmstadt、Germany): Omnipore Membranes: 10μm (JCWP); Polypropylene Prefilters: 10μm (AN1H)、30μm (AN3H); Polypropylene Net Filters: 25μm (PP25)、45μm (PP45)、80μm (PP80); Nylon Net Filters: 10μm (NY10)、11μm (NY11)、20μm (NY20)、30μm (NY30)、40μm (NY41)、60μm (NY60)、80μm (NY80)、100μm (NY1H); Spectrum Chemical Mfg. Corp. (New Brunswick、NJ、USA): Polyester Mesh Filters: 10μm (888-13699-PK)、15μm (888-13702-PK)、21μm (888-13705-PK)、43μm (888-13714-PK); Nylon Mesh Filters: 10μm (888-13687-PK)、20μm (888-13684-PK)、30μm (888-13681-PK)、41μm (888-13675-PK)、53μm (888-13669-PK)、60μm (888-13666-PK)、70μm (888-13660-PK)、100μm (888-13654-PK); Stainless Steel Mesh Filters: 30μm (888-14221-PK)、51μm (888-14224-PK); PEEK Mesh Filters (ポリエステル): 35μm (158-27388-PK); Tisch scientific (Cleves、OH、USA): Nylon Mesh Filters: 31μm (ME17232)、18μm (ME17233)、80μm (ME17225)、38μm (ME17231)、10μm (ME17399)、85μm (ME17224)、70μm (ME17226)、64μm (ME17227)、60μm (ME17228)、52μm (ME17229)、44μm (ME17230); Membrane Solutions (Auburn、WA、USA): Nylon Mesh Filter: 20μm (MENY025020)、30μm (MENY025030)、41μm (MENY025041)、60μm (MENY025060)、80μm (MENY025080)、100μm (MENY025100); pluriSelect Life Science (Leipzig, Germany): Re-Strainer: 10µm (43-75010-40), 20µm (43-75020-40), 40µm (43-75040-40), 70µm (43-75070-40). (43-75100-40); Syringe Strainer: 10µm (43-71010-50), 15µm (43-71015-50), 20µm (43-71020-50), 30µm (43-71030-50), 40µm (43-71040-50), 50µm (43-71050-50) 60μm (43-71060-50) 70μm (43-71070-50) 85μm (43-71085-50) 100μm (43-71005-50); Uberstrainer: 10µm (43-70010-03), 15µm (43-70015-03), 20µm (43-70020-03), 30µm (43-70030-03), 40µm (43-70040-03), 60µm (43-70060-03) 70μm (43-70070-03) 85μm (43-70085-03) 100μm (43-70100-03); pluriStrainer (Free Strainer): 10µm (43-50010-03), 15µm (43-50015-03), 20µm (43-50020-03), 30µm (43-50030-03), 40µm (43-50040-51)、50µm(43-50050-03)、60µm(43-50060-03)、70µm(43-50070-51); Genesee Scientific (San Diego, CA, USA): Olympus Advanced Cell Strainers: 40µm (25-375), 70µm (25-376), 100µm (25-377), and so on Bio-Rad, Inc. (Hercules, CA, USA): ProFlow Cell Filters: 30µm (12012576), 50µm (12012574), 70µm (12012573).

[0089] Implementation of the method according to the invention A variety of solutions can be used during the practice of the methods of the invention, depending on the source of the sample, optional dilution of the sample, the solution used to suspend the non-porous microparticles, the desired properties of the wash solutions, and the desired properties of the elution solution used to obtain viable target cells, as well as other factors. Such solutions include, but are not limited to, buffer solutions or cell culture media, solutions or media used for transfection, transplantation, embedding, and / or storage or cryopreservation of cells, as described herein. Examples of such buffer solutions include PBS (phosphate buffered saline), DPBS (Dulbecco's phosphate buffered saline), HBSS (Hanks' Balanced Salt Solution), EBSS (Earle's Balanced Salt Solution), HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid), MOPS (3-(N-morpholino)-propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), BES (bis(2-hydroxyethyl)amine), MOPSO (β-hydroxy-4-morpholinepropanesulfonic acid), ACES (N--2-aminoethanesulfonic acid), TAPS (tris(hydroxymethyl)methylamino)propanesulfonic acid), Bicine (diethanolglycine), and Tricine (N-(tri(hydroxymethyl)methyl)glycine) (Sigma / ThermoFisher). The buffer may optionally be supplemented with a protein, such as albumin or other proteinaceous material, as well as surfactants, such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and foam reducing agents, such as Pluronic® F68, glycols, glycerol, or glycerol esters. When water is used to dilute the solutions or suspensions described herein, the water may be distilled, double-distilled, or ultrapure water.

[0090] The cell culture media that may be used in the various steps of the method and / or after cell separation depends on the separated cell sample. Different cells or cell types may have very specific requirements for media, serum, and complements. Information regarding the medium used for transfection of a particular cell line can be obtained from the cell bank from which the cells were purchased. The skilled artisan will be familiar with which medium to use for further cell growth, differentiation, transfection, embedding, transplantation, or freezing. Exemplary cell culture media include MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), RPMI-1640 (Roswell Park Memorial Institute Medium 1640), and IMDM (Iscove's Modified Dulbecco's Media) (Sigma / ThermoFisher). Exemplary media used for cell transfection include, for example, Opti-MEM (Minimum Essential Medium with reduced serum content, ThermoFisher), which is recommended for transfection with RNA. Exemplary media used for embedding cells include ECM (Early Cleavage Medium), Endothelial Cell Medium, Cook IVF Cell Culture Media (Cook, Australia), P-1 Medium (Irvine Scientific, CA, USA), HTF Medium (Irvine Scientific, CA, USA), and MultiBlast Medium (Irvine Scientific, CA, USA). Examples of media for storing or cryopreserving cells include 5%-10% DMSO or glycerol, 90% FBS with 5%-10% DMSO or glycerol, and any of the cell growth media listed above supplemented with 5%-10% DMSO, 20% FBS, and 0.1 M trehalose. The choice of medium for storage or freezing depends on the cell line used. DMSO is not suitable for all cell lines, especially when serum-free.Methylcellulose or polyvinylpyrrolidone can be used for serum-free cell lines.

[0091] The temperature of some of the steps for carrying out the methods of the invention, including the steps of (optionally) pretreating the sample, (optionally) diluting the sample, contacting the sample with non-porous microparticles, incubating the sample containing the target cells with non-porous microparticles, separating unbound material from the target cell / microparticle complexes, and / or mechanically dissociating and eluting the viable target cells as described above, is not critical so long as the target cells remain viable. Typically, the temperature is at or below the temperature at which the target cells grow in their native environment or culture conditions, and may be the same or different temperature as the temperature at which other steps in the methods of the invention are carried out.

[0092] To reduce or eliminate clogging of the filter, when carrying out the method of the present invention, the filter is preferably positioned so that non-porous particulates do not accumulate over a large percentage of the filter's surface. For example, in one embodiment, the filter is oriented horizontally, the sample is placed under the filter, and the upflow of the solution for washing the sample and / or eluting and obtaining the target cells is oriented vertically, so that the target cells are first collected on top of the filter and the non-porous particulates fall off the filter. In another embodiment, the filter is oriented vertically, the sample is placed on one side of the filter, and the flow of the solution for washing the sample and / or eluting and obtaining the target cells is oriented horizontally, so that the target cells are collected on the other side of the filter and the non-porous particulates remain on the other side of the filter.

[0093] Contacting a sample containing viable target cells with non-porous microparticles If the sample contains or is likely to contain debris, such as tissue, bone, organ, cell aggregates, or other fragments, that may otherwise interfere with performance or clog filters when carrying out the method according to the invention, in a preferred embodiment the sample is pretreated to partially or completely remove this debris, which can be achieved by filtering the sample under conditions that do not essentially remove viable target cells from the sample.

[0094] According to the method of the present invention, 1.45 g / cm 3 Non-porous microparticles having a density equal to or greater than 10 μm and a diameter between 10 μm and 200 μm, and having a capture ligand capable of specifically binding to a molecule covalently immobilized on the surface of the microparticles, are contacted with a sample containing a suspension of viable target cells displaying the molecule on their cell surface, under conditions such that the target cells in the sample remain viable and become mixed with the non-porous microparticles, e.g., by mixing the sample stream with the suspension of non-porous microparticles and / or by shaking, stirring and / or agitation.

[0095] In one embodiment according to the invention, the method is initiated by directly contacting the sample with a suspension of non-porous microparticles in a solution. In another embodiment, the sample is diluted with a liquid after the addition of the non-porous microparticles as a suspension. The sample can be diluted with any liquid before, during or after the addition of the non-porous microparticles that maintains the viability of the target cells, allows the non-porous microparticles to form complexes with the target cells during the incubation of the sample, and can be selected based on the type of cells of interest, i.e., target cells, present in the sample. Such liquids include, but are not limited to, water, buffer solutions or cell culture media, solutions or media used for transfection, transplantation, embedding, and / or storage or freezing of cells as described herein.

[0096] Incubation of a sample containing target cells with non-porous microparticles According to the methods of the present invention, incubation of the non-porous microparticles with a sample containing target cells is carried out substantially without mechanical agitation, preferably without mechanical agitation, whereby the target cells specifically bind to the capture ligand via the molecule to form a stable target cell / microparticle complex.

[0097] The period of incubation is adjusted so that the percentage of target cells in the sample that form stable complexes with the non-porous microparticles is at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably about 99% or 100%.

[0098] Separation of unbound material from target cell / microparticle complexes According to the method of the invention, the step of separating the target cell / microparticle complexes from unbound material in the sample is carried out by washing the unbound material through a filter while retaining the target cell / microparticle complexes. The washing step is carried out by passing a solution through the sample at a flow rate or velocity that does not substantially dissociate the target cell / microparticle complexes but allows for the removal of undesired unbound material.

[0099] The solution used to wash the target cell / microparticle complexes and remove unbound material can be any solution that maintains the integrity of the target cell / microparticle complexes and the viability of the target cells, and can be selected based on the type of cells of interest, i.e., target cells, present in the sample. The solution can be the same solution used to contact and / or incubate the non-porous microparticles with the sample as described herein, or a different solution, so long as the solution maintains the viability of the target cells.

[0100] The flow rate or speed for washing unbound material (and optionally non-target cells) from the sample can be adjusted to match the fluid properties of the starting sample. Washing unbound material is performed at a low flow rate, preferably within the laminar or transitional flow rate range, such that no vortexes are formed. In a preferred embodiment, the solution passes through the filter in a laminar flow. The flow rate for washing unbound material from the sample while retaining the target cell / microparticle complexes is preferably 0.2 mm / s to 0.5 mm / s, more preferably 0.3 mm / s to 0.5 mm / s, and most preferably 0.35 mm / s to 0.45 mm / s.

[0101] The sample, containing a suspension of viable target cells and non-porous microparticles, is washed through a filter to separate the target cell / microparticle complexes from unbound material. Unbound material larger than the pores of the filter does not pass through the filter and does not contaminate the final eluted cell suspension. The flow rate or speed and wash time are adjusted such that at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 99% or more of the unbound material in the original sample is removed, and / or preferably more than 50%, more preferably more than 75%, and most preferably more than 99% of the target cells in the target cell / microparticle complexes are not eluted during the wash.

[0102] Mechanical dissociation and elution of viable target cells According to the method of the invention, viable target cells are obtained by mechanically dissociating the target cell / microparticle complex and eluting the viable target cells through a filter while retaining the non-porous microparticles to which the capture ligand is covalently immobilized. The viable target cells are preferably eluted into a sterile container that can be closed aseptically.

[0103] The solution used to disrupt the target cell / microparticle complexes and / or elute and filter the target cells can be any solution that maintains the viability of the target cells and can be selected based on the type of cells of interest, i.e., target cells, present in the sample. The solution can be the same solution used to contact and / or incubate the non-porous microparticles with the sample and / or wash the sample as described herein, or a different solution so long as the solution maintains the viability of the target cells.

[0104] Mechanical dissociation of the target cell / microparticle complexes can be performed by any means that breaks the capture ligand-surface molecule bond and results in the release of viable target cells from the non-porous microparticles on which the capture ligand is covalently immobilized, for example by passing the sample through a flow of solution. The flow rate or flow rate for the mechanical disruption of the target cell / microparticle complexes can be adjusted to match the fluid properties of the starting sample. The flow rate or flow rate for the mechanical dissociation of the target cell / microparticle complexes depends on the strength of the target cell-microparticle interaction and can be adjusted accordingly. In a preferred embodiment, the viable target cells are eluted after the mechanical dissociation or disruption of the target cell / microparticle complexes, i.e., the dissociation or disruption of the cell surface molecule-capture ligand interaction, e.g., the cell surface antigen-antibody interaction, by, for example, adjusting the flow rate or flow rate of the solution to a flow rate greater than that used to wash the sample to separate unbound material (and optionally non-target cells).

[0105] In a preferred embodiment, the flow velocity is greater than 0.45 mm / s, more preferably greater than 0.55 mm / s, and most preferably greater than 0.65 mm / s.

[0106] In a preferred embodiment, the flow of the solution containing the target cell / microparticle complexes and the elution solution is bidirectional, whereby the fluid flow initially flows in one direction through the filter and then the flow direction is reversed through the filter ("back and forth"). Flow can be initiated from the target cell / microparticle complex side of the filter or from the opposite side of the filter, and can be performed one or more times, provided that the final flow direction is towards a vessel used to obtain the separated target cells.

[0107] In one preferred embodiment of the method of the present invention, the target cells are eluted from the non-porous microparticles while retaining about 95% or more of the capture ligand on the surface of the non-porous microparticles, preferably about 96% or more, preferably about 97% or more, preferably about 98% or more, preferably about 99% or more, more preferably about 99.9% or more, and most preferably about 99.99% or more. In all most preferred embodiments of the method of the present invention, the target cell suspension is essentially free of cell-bound soluble capture ligands that specifically bind to molecules displayed on the target cell surface. Preferably, the eluted viable target cell suspension is essentially free of cell-bound soluble capture ligand antibodies that specifically bind to molecules displayed on the target cell surface. Preferably, the eluted viable target cell suspension contains less than 0.01%, more preferably less than 0.001% of the capture ligand already bound to the non-porous microparticles.

[0108] For certain applications where the number of target cells in a sample is limited, the viability of target cells is of primary concern. Examples of such applications include cell therapy or research where target cells are further genetically modified and / or expanded before use. Some examples of cell therapy that fall into this case are CAR-T therapy, isolation of T cells that are meant to be used without further modification, isolation of pluripotent stem cells, including tumor infiltrating lymphocytes, hair follicle stem cells, cardiac stem cells, neural stem cells, multipotent muscle cells, liver stem cells, hematopoietic stem cells, mesenchymal / stromal stem cells, dental pulp cells, periodontal ligament cells, adipose tissue-derived stem cells and progenitor cells, embryonic stem cells and induced pluripotent stem cells. These cells are preferably human cells, as well as other cell types and applications where cell viability is of primary concern.

[0109] Preferably, at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells obtained according to the methods of the present invention are viable when cultured under conditions that support growth of the target cells and / or compared to the viability of the target cells in the original sample.

[0110] For certain applications, the purity of target cells is of paramount concern when the use of the target cells requires a particular cell type to be separated from other cells that are normally associated with a particular sample. Examples of such applications / target cells include cells used for specific research, tumor cells including circulating tumor cells (CTCs) used for disease detection and prognosis, as well as specific subtypes of T cells (tumor infiltrating T cells, cytotoxic T cells, helper cells, etc.) that can be genetically modified and / or expanded, stem cells such as hair follicle stem cells, cardiac stem cells, multipotent muscle stem cells, neural stem cells, hepatic stem cells, hematopoietic stem cells, mesenchymal / stromal stem cells, dental pulp cells, periodontal ligament cells, adipose tissue derived stem and progenitor cells, pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells, and any other type of human or animal cell for which high specificity is desired.

[0111] In one preferred embodiment, the population of viable target cells obtained according to the methods of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0112] For certain applications, the amount of target cells recovered is a major concern, as the number of target cells may limit the application or use. Examples of such applications / target cells include cell therapy, such as stem cell therapy, T cell therapy, stem cells, such as multipotent muscle cells, neural stem cells, hepatic stem cells, hematopoietic stem cells, mesenchymal / stromal stem cells, dental pulp cells, periodontal ligament cells, adipose tissue derived stem cells and progenitor cells, pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, and the isolation of any other cell type derived from such pluripotent stem cells, endothelial cells, and any other cell type requiring large amounts of cells for therapeutic or research purposes.

[0113] In one preferred embodiment of the present invention, the number of target cells eluted from the non-porous microparticles accounts for at least about 10%, preferably at least about 30%, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, and most preferably at least about 90% of the target cells originally contained in the sample.

[0114] In preferred embodiments, the target cells are eluted from the non-porous microparticles retaining about 95% or more of said capture ligand on the surface of the non-porous microparticles, and at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells obtained according to the methods of the invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample.

[0115] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while at least about 95% of the capture ligands on the surface of the non-porous microparticles are retained, and the population of viable target cells obtained according to the methods of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0116] In a preferred embodiment, while the target cells are eluted from the non-porous microparticles, about 96% or more of the capture ligands on the surface of the non-porous microparticles are retained, and at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells obtained according to the methods of the present invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample.

[0117] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while at least about 96% of the capture ligands on the surface of the non-porous microparticles are retained, and the population of viable target cells obtained according to the methods of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0118] In a preferred embodiment, while the target cells are eluted from the non-porous microparticles, about 97% or more of the capture ligands on the surface of the non-porous microparticles are retained, and at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells obtained according to the methods of the present invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample.

[0119] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while at least about 97% of the capture ligands on the surface of the non-porous microparticles are retained, and the population of viable target cells obtained according to the methods of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0120] In a preferred embodiment, while the target cells are eluted from the non-porous microparticles, about 98% or more of the capture ligands on the surface of the non-porous microparticles are retained, and at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells obtained according to the methods of the present invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample.

[0121] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while about 98% or more of the capture ligands on the surface of the non-porous microparticles are retained, and the population of viable target cells obtained according to the methods of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0122] In a preferred embodiment, while the target cells are eluted from the non-porous microparticles, about 99% or more of the capture ligands on the surface of the non-porous microparticles are retained, and at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells obtained according to the methods of the present invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample.

[0123] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while about 99% or more of the capture ligands on the surface of the non-porous microparticles are retained, and the population of viable target cells obtained according to the methods of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0124] In a preferred embodiment, while the target cells are eluted from the non-porous microparticles, about 99.9% or more of the capture ligands on the surface of the non-porous microparticles are retained, and at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells obtained according to the methods of the present invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample.

[0125] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while at least about 99.9% of the capture ligand on the surface of the non-porous microparticles is retained, and the population of viable target cells obtained according to the methods of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0126] In a preferred embodiment, while the target cells are eluted from the non-porous microparticles, about 99.99% or more of the capture ligands on the surface of the non-porous microparticles are retained, and at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells obtained according to the methods of the present invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample.

[0127] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while about 99.99% or more of the capture ligand on the surface of the non-porous microparticles is retained, and the population of viable target cells obtained according to the methods of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0128] In one preferred embodiment, at least about 50% of the target cells obtained according to the methods of the invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample, and the population of viable target cells obtained according to the methods of the invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0129] In one preferred embodiment, at least about 60% of the target cells obtained according to the methods of the invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample, and the population of viable target cells obtained according to the methods of the invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0130] In one preferred embodiment, at least about 70% of the target cells obtained according to the methods of the invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample, and the population of viable target cells obtained according to the methods of the invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0131] In one preferred embodiment, at least about 80% of the target cells obtained according to the methods of the invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample, and the population of viable target cells obtained according to the methods of the invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0132] In one preferred embodiment, at least about 90% of the target cells obtained according to the methods of the invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample, and the population of viable target cells obtained according to the methods of the invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0133] In one preferred embodiment, at least about 95% of the target cells obtained according to the methods of the invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample, and the population of viable target cells obtained according to the methods of the invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0134] In one preferred embodiment, about 99% or more of the target cells obtained according to the methods of the invention are viable when cultured under conditions that support target cell growth and / or compared to the viability of the target cells in the original sample, and the population of viable target cells obtained according to the methods of the invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0135] Medicament comprising a viable target cell suspension obtained by the method of the present invention In a further aspect, the present invention relates to a viable target cell suspension obtainable according to the method of the present invention, a medicament comprising a viable target cell suspension obtainable according to the method of the present invention for use as a medicament for the prevention and / or treatment of a disease, and / or a method of treating a subject in need of a viable target cell suspension obtainable according to the method of the present invention, comprising the step of administering said viable target cell suspension to a subject for the prevention and / or treatment of a disease, wherein the viable target cell suspension is selected from the group consisting of granulocytes, T lymphocytes, monocytes, T regulatory cells, T helper cells, cytotoxic T cells, B lymphocytes, tumor infiltrating lymphocytes, platelets, natural killer cells, hematopoietic stem and progenitor cells, mesenchymal / stromal stem cells, hair follicle stem cells, cardiac stem cells, multipotent muscle cells, neural stem cells, hepatic stem cells, dental pulp cells, periodontal ligament cells, retinal pigment epithelial cells, adipose tissue a suspension of mammalian cells, preferably human cells, comprising cells selected from the group of mammalian derived stem and progenitor cells, pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, Car-T cells, microvascular endothelial cells (MVEC), primary epithelial cells, such as keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells and retinal epithelial cells, neural tissue from the heart, liver, kidney, colon, intestine, esophagus, stomach, brain and spinal cord, vascular tissue from the lungs, arteries, veins and capillaries, fibroblasts from any tissue or organ, including lymphoid tissue from lymph glands, adenoids, tonsils, bone marrow and blood, spleen, muscle cells, pancreatic cells, cardiac cells, and any cells established from these primary cells, wherein the eluted viable target cell suspension is essentially free of soluble capture ligands bound to the cells that specifically bind to molecules presented on the target cell surface. Preferably, the eluted viable target cell suspension is essentially free of soluble capture ligand antibodies bound to the cells that specifically bind to molecules presented on the target cell surface. Preferably, the eluted suspension of viable target cells contains less than 0.01%, more preferably less than 0.001%, of the capture ligand previously bound to the non-porous microparticles.

[0136] Preferably, at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably about 99% or more of the target cells in the medicament obtained by the method of the present invention are viable when cultured under conditions that support growth of the target cells and / or compared to the viability of the target cells in the original sample.

[0137] Preferably, the population of viable target cells in the medicament obtained by the method of the present invention is enriched by at least about 1.25-fold, preferably at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 3-fold, preferably at least about 4-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 30-fold, preferably at least about 40-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 250-fold, preferably at least about 500-fold, preferably at least about 1000-fold or more relative to the population of non-target cells in the original sample.

[0138] In one embodiment of the present invention, the method according to the present invention is carried out under sterile conditions using the device and / or cartridge according to the present invention as a module in GMP manufacturing. The device can be connected aseptically to an inlet cell source and has an outlet connected to, but not limited to, an IV bag, other type of collection container, or downstream processing machine, such as a bioreactor or other cell culture or processing container. This embodiment is particularly preferred when viable target cells are used as a medicine.

[0139] The viable target cell suspension obtained by the method according to the invention may be diluted, for example with a physiologically acceptable buffer or medium, or concentrated, for example by sterile filtering the viable target cell suspension to partially remove fluid, prior to administration. Furthermore, the medicament according to the invention may comprise one or more other medicaments, which may be added to the viable target cell suspension at any time prior to administration to the patient.

[0140] cartridge In a further aspect, the present invention relates to a cartridge configured to be used to separate viable target cells from a sample using the method according to the present invention described herein, the use of the cartridge in such a method has not yet been developed and is particularly advantageous as it allows for the separation of target cells in a simple, inexpensive, sterile and flexible manner, providing viable target cells with high purity and viability in a selected medium.

[0141] In one embodiment, the cartridge comprises: - a filter enclosed in a filter unit having an inlet port and an outlet port; - a fluid conduit having one or more valves connected to the inlet port of the filter and capable of controlling the flow of a source of a sample containing the target cells of the invention, a source of a suspension of non-porous microparticles of the invention, a source of a wash solution, and a source of an elution solution; and - a fluid conduit connected to the outlet port of the filter that includes one or more valves, one valve capable of diverting the target cell solution through the fluid conduit to a target cell collection container and one valve capable of diverting flow through the fluid conduit to a different container; A closed housing cartridge comprising:

[0142] As used herein, a "closed housing cartridge" is a cartridge that allows for sterile contact, mixing, and incubation of a sample containing viable target cells with a suspension of non-porous microparticles in a closed housing, as well as sterile transfer, washing, and elution with one or more solutions. The elements of a cartridge according to the invention, such as filter units, fluid conduits, and valves, are joined in a circuit, such that they can be transiently connected to each other via valves when performing the various steps of a method according to the invention.

[0143] The cartridge housing may be rigid and / or flexible. The cartridge fluid conduits connected to the inlet and / or outlet of the filter may be a composite of flexible and rigid materials, embedded or molded in the cartridge rigid housing, or may be present as tubes. The cartridge fluid conduits may have any shape of cross section, but are preferably rectangular with rounded corners, oval, or circular. These cross sections allow the target cells to pass through the conduit with little mechanical stress. In some embodiments, the cartridge fluid conduits serve as incubation spaces for incubating the sample to form target cell / microparticle complexes to which a liquid-holding container can be attached. In a preferred embodiment, the incubation space is made of a flexible plastic material, such as PVC or silicone.

[0144] The filter of the cartridge is enclosed within a filter unit such that the filter acts as a porous barrier between the inlet and outlet ports, allowing any fluid medium flowing through the inlet port to pass through the filter, and any filtrate that passes through the filter, i.e., a suspension of viable target cells or a washing solution with or without unbound material (and optionally non-target cells), to pass through the outlet port.

[0145] The valves in the cartridge according to the invention can access, prevent and / or redirect fluid flow. The valves can be any type of mechanical valve. When used in the device according to the invention, the valves can also be operated by electronic means. In a preferred embodiment, the parts of the cartridge containing the valves are made from a hard plastic material, for example polysulfone.

[0146] In a preferred embodiment, the cartridge is first washed with culture medium, a buffer solution, or water to remove air from the cartridge. In some embodiments of the invention, the target cells are washed and eluted with the same solution. In other embodiments, the solution for eluting the target cells is different from the solution for washing the sample containing the target cell / microparticle complexes. In this latter and other embodiments, the cartridge according to the invention may include one or more valves connected to a source of a solution for eluting the target cells that is different from the solution for washing the target cell / microparticle complexes. For example, the cartridge can be connected to a source of liquid for diluting any of the solutions and / or suspensions used to carry out the methods of the invention.

[0147] The cartridge and its elements, such as filter units, fluid conduits, valves, etc., can be made from any biocompatible plastic material, including PVC (polyvinyl chloride), PA (polyamide), PE (polyethylene), PS (polystyrene), epoxy resins, silicone rubber, natural rubber, polyurethane, PP (polypropylene), polyester, PEEK (polyetheretherketone), polyphenylsulfone, polysulfone, nylon, PMMA (poly(methyl methacrylate)), polysulfones, polyphosphazene, thermoplastic elastomers, polydimethylsiloxane, PTFE (polytetrafluoroethylene), etc. In a most preferred embodiment, the cartridge according to the invention is configured to separate viable target cells from a sample using the method according to the invention, whereby the resulting viable target cell suspension can be sterilely grown in culture, stored, frozen, or introduced or reintroduced into the body of a mammal, preferably a human, for use in therapy, for example. In such an embodiment, the cartridge and its components, i.e., the filter unit, fluid conduits, and valves, are made from biocompatible plastic materials that are resistant to sterilization, including PVC (polyvinyl chloride), PA (polyamide), PE (polyethylene), PS (polystyrene), epoxy resins, silicone rubber, natural rubber, polyurethane, PP (polypropylene), polyester, PEEK (polyetheretherketone), polyphenylsulfone, polysulfone, nylon, PMMA (poly(methyl methacrylate)), polysulfones, polyphosphazene, thermoplastic elastomers, polydimethylsiloxane, PTFE (polytetrafluoroethylene), and others.

[0148] As used herein, "sterilization" refers to the process of destroying or eliminating all forms of microbial life by physical or chemical methods, such as autoclaving, pressurized steam, dry heat, ethylene oxide (EtO) gas, nitrogen dioxide gas, ozone, hydrogen peroxide gas plasma, electron beam sterilization, and liquid chemicals. In one embodiment, the cartridge is contained within a sterilizable package. In a most preferred embodiment, the cartridge according to the present invention is contained within a sealed sterile package.

[0149] In one embodiment of the present invention, the method is performed manually, whereby the fluid flow can be controlled by hand. One example for performing the method manually is shown in Figure 3. The method uses the following elements: a first syringe 1, a second syringe 2, a filter 3, a long tube 4, and a short tube 5. The method can also be performed using a commercially available cell strainer (see list of commercially available filters and strainers) and a centrifuge tube.

[0150] However, in a preferred embodiment, the method according to the invention is carried out using a device and / or a cartridge according to the invention. In one preferred embodiment of the invention, the method is automated and uses a device and / or a cartridge according to the invention.

[0151] In a preferred embodiment, the cartridge is a single-use replaceable part and is designed to be installed in the device according to the invention. In one preferred embodiment of the invention, the cartridge is designed to be modular, whereby the cartridge is installed and connected to the device of the invention such that the valves of the cartridge are operated by the control system of the device and the flow of solutions is measured, set and / or regulated through the valves of the cartridge.

[0152] In one embodiment of the invention, the cartridge is designed to separate one or more specific types of target cells from one or more specific samples. In such cases, the cartridge can be designed with filters and / or fluid conduits to match a particular sample and used to separate a particular target cell type.

[0153] In certain embodiments, the total cell treatment time employing the methods of the invention is less than 1 hour, less than 45 minutes, less than 30 minutes, less than 25 minutes, or less than 20 minutes.

[0154] Parts Kit In a further aspect, the present invention relates to a cartridge according to the present invention for separating viable target cells from a sample using a method according to the present invention, and a method for separating viable target cells from a sample using a cartridge having a flow rate of about 1.45 g / cm 3 and a container containing a suspension of non-porous microparticles having a density of 10 μm or more and a diameter of about 10 μm to 200 μm, wherein a capture ligand capable of specifically binding to a molecule on the surface of a cell is covalently immobilized to said surface of the microparticle. In a further embodiment, the kit of parts comprises one or more containers containing solutions for carrying out the method of the invention, such as solutions for washing the target cell / microparticle complexes and / or solutions for eluting viable target cells or liquids for diluting these solutions. In a preferred embodiment, the components of the kit of parts are sterilizable, and most preferably the components of the kit of parts are contained within a sealed sterile package.

[0155] Device In one aspect, the present invention relates to an apparatus configured to separate viable target cells from a sample using the method according to the present invention. The apparatus according to the present invention is capable of controlling fluid flow and fluid flow rate, and the process of cell separation according to the present invention can be partially or fully automated.

[0156] "Automated" means that the device can be programmed to perform the processing steps of the method of the invention without substantial operator involvement. Of course, it will be understood that the automated system of the invention may involve operator activity, including loading cartridges and / or containers and inputting processing parameters to perform the method of the invention. Additional manual steps may also be required. However, in a preferred embodiment, once the containers for the sample, the suspension of non-porous microparticles, the solutions for washing and elution, and the containers for collecting the viable target cell suspension are connected to the cartridge, the device is capable of processing a sample containing viable target cells without substantial operator intervention.

[0157] According to this aspect of the invention, the apparatus comprises means for securely mounting a cartridge according to the invention such that the valves of said cartridge are mechanically operated by an electronic control system, means for connecting said cartridge to one or more containers containing one or more solutions and / or suspensions, means for transferring the solutions and / or suspensions, and a control system for setting, measuring and / or regulating the flow of solutions and / or suspensions through the valves of the cartridge while performing the method according to the invention.

[0158] The device of the present invention may include one or more of the following: a keyboard, a touch screen, a barcode reading device, communication elements for WIFI, NFC (near field communication), or Bluetooth, a pre-treatment filter, a motor, a vibration isolation unit for any or all pumps or motors, sensors or electrodes for measuring pressure, flow, impedance, conductivity, resistance, current, pH, temperature, humidity, gas percentage, radiation, movement, volume, weight, magnetic flux, time, turbidity, optical density, an alarm, a cell counting device, a heating unit, a cooling unit, a graphic display unit, data storage devices such as CD and USB flash drive sticks, a port for NFC, etc. The device can be directly connected to a fixed power source or can be mobile operated by battery power.

[0159] Control System An electronic control system controls the flow of fluids into, through, and out of the cartridge. The control system of the device of the invention is any suitable human-machine interface controller capable of accepting inputs, transmitting outputs, and performing operations to carry out the methods of the invention. According to certain embodiments, the controller may include a programmable microprocessor, which can be programmed to cause the contacting, incubation, washing, and elution steps of the methods of the invention, as well as other additional steps, preferably in an automated manner. The controller can be programmed to have unidirectional or bidirectional flow in one or more of these steps, whereby fluid flow initially flows in one direction and then the direction of flow is reversed ("back and forth").

[0160] The controller may receive user input through a device, such as a keyboard, a touch screen, a handheld device, such as a smartphone or tablet, and / or a barcode reader or scanner, or may receive user input by connecting directly to the device, such as via a radio frequency identification (RFID) reader, or by connecting to other processing systems through a local network, or by connecting to other computing devices (e.g., servers) through a local network, wide area network, or the Internet. According to such an embodiment, the device may include internal transceiver devices for wireless communication technologies, such as WLAN, WIFI, Bluetooth, etc. The controller may be connected to one or more sensors in the device that measure one or more variables before, during, or after performing a method according to the invention, and may receive process data input from these sensors. Variables measured by these sensors include whether the cartridge or one or more containers are properly connected to the device, the level of any solution or suspension in the container used to carry out the method of the invention, pressure, flow rate, flow rate, impedance, conductivity, resistance, current, pH, temperature, humidity, gas percentage, radiation, motion, volume, weight, magnetic flux, time, etc. at any point in the system. The controller can be connected to and output data to a graphic display unit or screen, a video display unit or screen, a data storage device such as a CD, USB stick, or can transmit data to a device such as a computer, a handheld device such as a smartphone or electronic tablet, etc., through a local network or through other computing devices (e.g., a server) via a local network, a wide area network, or the Internet. According to such an embodiment, the device may include an internal transceiver device for wireless communication technologies such as WLAN, WIFI, Bluetooth, NFC, etc.

[0161] Fluid container In a preferred embodiment, the device of the present invention has one or more means enclosed within the device for connecting the cartridge to one or more containers, preferably made from any one or more of the biocompatible materials mentioned herein.

[0162] The containers can be made of any rigid or flexible biocompatible material mentioned herein, including glass, rigid or flexible plastics, and can be in the form of a bottle, a tube, e.g., a sample tube, a centrifuge tube, a syringe, a flask, e.g., a tissue culture flask, a bag, e.g., a collapsible bag or an IV bag, which is preferably closable, optionally sealed and aseptically re-openable, etc. Preferably, the containers for receiving the sample, the container for receiving the suspension of non-porous microparticles, the container for receiving the washing and / or elution solution, and the container into which the target cells are eluted are sterile syringes, bottles, or IV bags.

[0163] In a preferred embodiment, the device of the present invention comprises one or more spaces enclosed within the device for securely storing and / or holding one or more containers connected to a cartridge according to the present invention.

[0164] In a preferred embodiment, the device is sterilely connected to a container containing a source of sample containing viable target cells, a container containing a source of non-porous microparticles, a container containing a source of wash solution, elution solution, and / or diluent, and a cartridge of the invention which is sterilely connected to a sterile container for collecting the separated viable target cells. The containers used to collect unbound material and / or non-target cells may also be sterile.

[0165] Solution / Suspension Transfer System The device comprises at least one means for moving, stopping and / or reversing the solutions and suspensions used in the method according to the invention. Preferably, the device comprises at least one pump, including positive displacement pumps, such as syringe pumps, syringe plungers, infusion pumps, peristaltic pumps, diaphragm pumps, gear pumps, screw pumps, hollow disk pumps, vibration pumps, water hammer pumps, pulsar pumps, air lift pumps, centrifugal pumps, propeller pumps, axial pumps, mixed flow pumps, siphon pumps, piston pumps, pulse meter steam pumps, rotary lobe pumps, rotary vane pumps, ejection jet pumps and electromagnetic pumps. In one embodiment of the invention, the fluid containers are connected to the main cartridge and there may be valves positioned after any container, and the valves may be in different positions to control the flow through the system. In one embodiment, the movement of the valves and syringe plungers is controlled by the use of a fluid control system. In another embodiment of the invention, the flow can be controlled by a pump that can automatically control the flow rate or flow rate of the fluids entering or leaving the system. In one preferred embodiment, the one or more pumps are capable of pumping bidirectionally, whereby fluid flow initially flows in one direction and then the direction of flow is reversed ("back and forth"). EXAMPLES

[0166] 6. Working Example The present invention provides a unique method, cartridge, and device for cell separation that allows for a high yield of viable target cells in suspension essentially free of capture ligands on the surface of the cells or in the suspension fluid, and allows for timely separation of viable target cells from a sample with cell viability maintained, while eliminating the need for downstream processing and purification of the sample, and allowing for aseptic processing for subsequent culturing of the target cells or use in subsequent treatments. The present invention is described in detail in the following examples, which may represent two or more embodiments of the invention. The present invention is not limited in scope by the exemplified embodiments, which are intended to be illustrative of individual aspects of the invention. Indeed, from the above description and the accompanying drawings, various modifications to the present invention, in addition to the embodiments shown and described herein, will become apparent to those skilled in the art. Such modifications are intended to be within the scope of the appended claims. The following examples are offered by way of illustration and not by way of limitation.

[0167] [Example 1] Preparation of anti-CD90 antibodies covalently immobilized on carboxyl silica microparticles 20 mg of 40 μm diameter carboxyl-functionalized silica nonporous microparticles (Glantreo Ltd., Ireland), 1 μL of EDC (1-ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride) (concentration 100 mg / mL), and 1 μL of NHS (N-hydroxysuccinimide) or sulfo-NHS (N-hydroxysuccinimide) (concentration 60 mg / mL) are mixed in an Eppendorf tube at room temperature in MES buffer, pH=6. The activated non-porous microparticles are centrifuged and the supernatant is discarded to remove excess reagent. 15 μg of anti-CD90 antibody (purchased from BIOTEM, France) is dissolved in 1 mL of PBS buffer and added to the activated silica microparticles. After mixing for 2.5 hours at room temperature, the remaining active sites on the surface of the microparticles are blocked by the addition of 100 μL of 0.1 M ethanolamine solution. After blocking of the remaining groups, the microparticles are centrifuged and the supernatant is saved for the determination of the protein concentration. After washing with PBS, the microparticles are used for cell separation.

[0168] [Example 2] Preparation of anti-CD90 antibodies covalently immobilized on epoxy-functionalized silica microparticles Non-functionalized silica non-porous microparticles with a diameter of 40 μm were purchased from Glantreo Ltd, Ireland. To prepare silica non-porous microparticles functionalized with epoxy groups, non-functionalized silica microparticles are silanized with GLYMO. 20 mg of epoxy-functionalized silica non-porous microparticles are washed in 1 M phosphate buffer, pH=7, at room temperature in an Eppendorf tube. Then, 15 μg of anti-CD90 antibody (purchased from BIOTEM, France) is added to the Eppendorf tube. The reaction is carried out overnight. The remaining active sites on the non-porous microparticle surface are blocked with 100 μL of 0.1 M ethanolamine. After blocking the remaining groups, the non-porous microparticles are centrifuged and the supernatant is saved for the determination of protein concentration as described below. After washing with PBS, the non-porous microparticles are used for cell separation.

[0169] To determine whether the immobilization was successful, an indirect method known to those skilled in the art for measuring the protein concentration in the sample can be performed. The protein concentration was measured by using the Bradford method for low concentration samples. This method can be performed by mixing 1 mL of the supernatant sample with 1 mL of Bradford reagent in a cuvette. The absorbance value of the sample is measured after 10 minutes. The measurement is performed in the cuvette with a NanoDrop™ One / OneC Microvolume UV-Vis Spectrophotometer. The results for the microparticles prepared in Example 1 are shown in Figure 1. The starting anti-CD90 antibody solution is expressed as 100% protein. The protein concentration in the supernatant that was saved during the immobilization procedure was measured and the ratio of the amount of protein in micrograms in the starting sample and the supernatant was calculated. The percentage of protein remaining in the supernatant is shown in Figure 1. The difference in the mass of protein in the starting antibody solution and the supernatant can be calculated, which is considered to be the amount of antibody bound to the non-porous microparticles. FIG. 1 shows that approximately 60% of the added anti-CD90 antibody was covalently bound to the silica microparticles.

[0170] The specific surface area available for antibody binding on 40 μm non-porous silica microparticles can be calculated based on the following formula:

[0171]

number

[0172] The surface saturation by proteins bound to non-porous silica particles can be calculated based on the following formula:

[0173]

number

[0174] Diameter 40μm, density 2g / cm 3 For non-porous silica particles, the theoretical maximum amount of antibody per gram of silica particles is 0.2 mg (Ab) / g (MP).

[0175] The experimental results in Figure 2 show agreement with the values ​​of surface saturation by anti-CD90 antibodies, which are consistent with the calculated theoretical maximum. In two different experiments, the experimental values ​​of surface saturation of 40 μm non-porous silica microparticles have the same results, and therefore it can be concluded that all of the antibodies bound to the microparticles are bound to the surface of the microparticles.

[0176] The average surface area of ​​the 40 μm non-porous silica particles was measured using a Micromeritics TriStar II analyzer. 2 / g particles.

[0177] [Example 2a] Preparation of anti-CD4 antibodies covalently immobilized on carboxyl silica microparticles. 1 g of 40 μm diameter carboxyl-functionalized silica nonporous microparticles (Glantreo Ltd., Ireland), 28 μL of EDC (1-ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride) (concentration 100 mg / mL), and 28 μL of NHS (N-hydroxysuccinimide) or sulfo-NHS (N-hydroxysuccinimide) (concentration 60 mg / mL) are mixed in an Eppendorf tube at room temperature in MES buffer, pH=6. The activated non-porous microparticles are centrifuged and the supernatant is discarded to remove excess reagent. 110 μg of anti-CD4 antibody (purchased from Proteintech, USA) is dissolved in 3 mL of PBS buffer and added to the activated silica microparticles. After mixing for 2.5 hours at room temperature, the remaining active sites on the surface of the microparticles are blocked by the addition of 500 μL of 0.1 M ethanolamine solution. After blocking of the remaining groups, the microparticles are centrifuged and the supernatant is saved for the determination of the protein concentration. After washing with PBS, the microparticles are used for cell separation.

[0178] [Example 2b] Preparation of anti-CD34 antibody covalently immobilized on carboxyl silica microparticles 1 g of 40 μm diameter carboxyl-functionalized silica nonporous microparticles (Glantreo Ltd., Ireland), 28 μL of EDC (1-ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride) (concentration 100 mg / mL), and 28 μL of NHS (N-hydroxysuccinimide) or sulfo-NHS (N-hydroxysuccinimide) (concentration 60 mg / mL) are mixed in an Eppendorf tube at room temperature in MES buffer, pH=6. The activated non-porous microparticles are centrifuged and the supernatant is discarded to remove excess reagent. 110 μg of anti-CD34 antibody (purchased from BIOTEM, France) is dissolved in 3 mL of PBS buffer and added to the activated silica microparticles. After mixing for 2.5 hours at room temperature, the remaining active sites on the surface of the microparticles are blocked by the addition of 500 μL of 0.1 M ethanolamine solution. After blocking of the remaining groups, the microparticles are centrifuged and the supernatant is saved for the determination of the protein concentration. After washing with PBS, the microparticles are used for cell separation.

[0179] Modes for carrying out the invention [Example 3] Manual cell isolation The elements for carrying out the manual cell separation method are shown in Figure 3. A hollow tube (element number 4, Figure 3) with an inner diameter of 4 mm and a length of 50 cm is connected to two 30 mL syringes (first syringe 1 and second syringe 2, Figure 3) with a filter (element 3, Figure 3) attached to one of them. In the first syringe 1, cells and non-porous microparticles are mixed in a volume of 6 ml and the syringe is attached to tube 4 in Figure 3. The second syringe 2 is a clean syringe with no fluid in it. The plunger of the first syringe 1 is pressed and the liquid containing the non-porous microparticles is transferred to tube 4. The tube is now in a horizontal position and the system is in the incubation phase. After 15 minutes, a new syringe containing 25 mL of elution / wash buffer is placed in the position of the first syringe 1. The elution / wash buffer is pushed through the tube. The tube is now in a vertical position and all the liquid is transferred to the second syringe 2 once it has passed through tube 4. Remove the second syringe and replace it with a new clean syringe containing 10 mL of elution buffer in place of the second syringe 2. Press the two syringes 1, 2 one after the other back and forth through tube 5 10 times until all the liquid has returned to the syringe in position 2. Wash tube 5 with a further 10 mL of elution buffer and collect the elution buffer in the syringe in position 2.

[0180] [Example 4] Cell Separation in a Device According to the Invention One of the orientations of the cartridge and the positions of the connection valves according to the invention is shown in FIG. 5. The cells are placed in a syringe connected to a port at position 10 in FIG. 5, and the non-porous microparticles suspended in a buffer at physiological pH are placed in a syringe connected to a port at position 9 in FIG. 5. A syringe filled with air is placed in position 3 in FIG. 5 and is used to purge the system. Positions 1, 2 and 3 below the filter 11 in FIG. 5 are empty and therefore can be used as receiving syringes for non-target and target cells. The ports at positions 4 and 5 are connected to tubing that can be connected to a waste bottle. The first step is to wash the system with an elution / wash buffer and to purge it with air. The fluids from the syringes connected to positions 9 and 10 (FIG. 5) are then mixed by pushing the liquid in and out of the syringes. Once the contents of the two syringes are well mixed, the incubation of the cells with the non-porous microparticles begins. After the time programmed in the device (15 min), washing of cells that did not bind to the non-porous microparticles is performed by employing a laminar flow in the syringe at position 9 and washing three times with 10 mL of elution / wash buffer. After washing off the cells that did not bind to the non-porous microparticles, the system is rinsed again with elution / wash buffer and air to remove all cells from the cartridge. After thorough washing, elution of cells bound to the microparticles is performed. Elution is performed mechanically using a high flow rate or flow rate through the syringe at position 9 (Figure 5) and the tubing of the cartridge (element number 12, Figure 5). Once the process is completed, the receiving syringe at position 1 (Figure 5) contains the target cells eluted from the non-porous microparticles. The sample is free of animal-derived products and free of antibodies (or fragments thereof) that may contaminate the sample. A filter (item no. 11, FIG. 5) placed above the syringe at position 1 (FIG. 5) prevents non-porous microparticles from entering the target cell sample, so that at the end of the process, all non-porous microparticles remain in the cartridge. The eluted target cells show a very high viability (>90%), allowing the cells to be used for their further application without further purification steps.

[0181] [Example 5] Cell Separation in a Device According to the Invention One of the orientations of the cartridge according to the invention and the positions of the connection valves is shown in Figure 6. Cells are placed in the syringe placed in position 1 (Figure 6) and non-porous microparticles suspended in a buffer at physiological pH are placed in the syringe placed in position 2 (Figure 6). A syringe filled with air is placed in position 5 (Figure 6) and is used to purge the system. Positions 3 and 4 in Figure 6 are connected to syringes filled with buffer for elution / washing of cells. The syringes in positions 8 and 9 placed after the filter 11 in Figure 6 are empty and therefore can be used as receiving syringes for non-target material. The syringe placed in position 10 contains the elution buffer and serves as a receiving syringe for target cells. The ports in positions 6 and 7 are connected to tubes that can be connected to a waste bottle.

[0182] The first step in using the device is to wash the system with elution / wash buffer and to purge the system with air. The fluids from the syringes at positions 1 and 2 (Figure 6) are then mixed by pushing the liquids in and out of the syringes. Once the fluids in the two syringes are well mixed, the mixture is pushed out of the syringes, filling the tube connected to the filter (item number 12, Figure 6). Incubation of the cells with the non-porous microparticles is carried out in tube 12 of Figure 6. After the time programmed in the device (15 minutes), washing of the cells that did not bind to the non-porous microparticles is carried out by adopting a slow laminar flow through tube 12 of Figure 6 and washing with 40 mL of elution / wash buffer. After thorough washing, elution of the cells bound to the microparticles is carried out. Elution is carried out mechanically with a high flow rate or flow velocity in the cartridge, back and forth from the syringes at position number 10 (Figure 6) through tube 12 of Figure 6 in the cartridge. Once the process is complete, the receiving syringe at position 10 (Figure 6) contains the target cells eluted from the non-porous microparticles. The sample is free of animal-derived products and free of antibodies or fragments thereof that may contaminate the sample. A filter (element number 11, Figure 6) in front of the syringe at position number 10 (Figure 6) prevents the non-porous microparticles from entering the target cell sample, so that at the end of the process, all non-porous microparticles remain on top of the filter 11 in Figure 6. The eluted target cells show a very high viability (>90%), allowing the cells to be used for their further application without further purification steps.

[0183] [Example 6] Cell Separation in a Device According to the Invention The cartridge shown in Figure 7 is more user friendly and can ensure that the positions of ports 1-10 allow the flow through the cartridge to be adopted in both directions. Moreover, this allows the system to be a completely closed and sterile environment. As shown in Figure 7, the syringe is positioned facing downwards. The cells placed in the syringe located at port number 9 in Figure 7 and the non-porous microparticles placed in the syringe located at port number 10 in Figure 7 are mixed in a first step and pushed through the system. The incubation is carried out in a tube (element number 12, Figure 7). Positions 1-8 may be connected to syringes that may be empty or filled with elution / wash buffer, one location is provided with an air filter. After an incubation time of 15 minutes, the system is flushed with 30 mL of elution buffer to remove unbound cells. After the unbound cells are removed from the system, elution of the target cells bound to the non-porous microparticles is carried out with a flow back and forth through tube 12 in Figure 7. Filters (item no. 11, FIG. 7) are placed in front of the target and non-target receiving syringes to prevent non-porous particulates from contaminating the target cell sample.

[0184] [Example 7] Cell Separation in a Device According to the Invention The non-porous microparticles are pre-loaded into the incubation space, a tube with two filters at each end. The cells are transferred from an IV bag attached to a pump that controls the flow rate or speed. The incubation is performed by using a steady flow of cell suspension. After a 15 minute incubation period, the cell flow is stopped and 40 mL of elution / wash buffer is run through the tube to remove unbound cells. After the unbound cells are removed from the system, elution of the target cells bound to the non-porous microparticles is performed by back and forth flow through the tube controlled by the pump.

[0185] [Example 8] Manual method for cell separation according to the present invention A sample containing a suspension of viable target cells and wash buffer is prepared. The buffer is prepared by warming a commercial cell buffer stock buffer (Stock buffer catalogue number 60-00080-10 purchased from pluriSelect Life Science, Leipzig, Germany) to room temperature and diluting 10-fold. Human Kasumi (CD90-) cells (DSMZ accession number ACC 220) and human Jurkat (CD90+) cells (ATCC accession number TIB-152) are centrifuged at 300 rpm for 10 min. The cell pellet is resuspended in 3 mL of buffer. Cells are counted and diluted to a concentration of 5 × 10 5 Prepare 3 mL of cell suspension at 1000 x 1000 cells / mL. Place 1 mL of cells in a sterile, commercially available centrifuge tube. Add 20 mg of non-porous silica microparticles activated with anti-CD90 antibodies prepared by the procedure provided in Example 1. Mix gently 5 times to contact the cells and non-porous microparticles, and leave the mixture to incubate at room temperature for 30 minutes. The formation of target cell / microparticle complexes using Jurkat (CD90+) cells is shown in Figure 4a. On the other hand, Kasumi (CD90-) cells do not form target cell / microparticle complexes, as shown in Figure 4b. Place a 30 μm pore size filter on top of a sterile, commercially available centrifuge tube and pour the solution containing the target cell / microparticle complexes on top of the filter. Wash the target cell / microparticle complexes with 16 mL of wash buffer by pouring the buffer over the target cell / microparticle complexes to remove all unbound and non-targeted materials. Close the openings on the filter to prevent liquid from passing through, and place everything on a clean, sterile, commercially available centrifuge tube. Add 2 mL of wash buffer and pipette up and down 20 times to mechanically disrupt the target cell / microparticle complexes and eluate the target cells. Wash the filter with an additional 10 mL of wash buffer. Count both cell lines in the target cell eluate. The results are shown in Table 1 along with those of Example 9 and are expressed as a percentage of the starting cell count.

[0186] [Example 9] Automated cell separation method according to the present invention A sample containing a suspension of viable target cells and wash buffer is prepared. The buffer is prepared by warming a commercial cell buffer (Stock buffer catalogue no. 60-00080-10 purchased from pluriSelect Life Science, Leipzig, Germany) to room temperature and diluting 10-fold. Kasumi (CD90-) cells (DSMZ ACC 220) and Jurkat (CD90+) cells (ATCC-TIB-152) are centrifuged at 300 rpm for 10 min. The cell pellet is resuspended in 4 mL of buffer. Cells are counted and diluted to a concentration of 3.5 x 10 5 Prepare 4 mL of cell suspension at 100 cells / mL.

[0187] Prepare two 10 mL syringes: in the first syringe, transfer 3 mL of cells suspended in buffer, and in the second syringe, transfer 40 mg of non-porous silica microparticles activated with anti-CD90 antibody (prepared by the procedure provided in Example 1) and suspended in 3 mL of buffer.

[0188] Syringes are attached to the cartridge (Figure 7), along with either a wash buffer or an empty syringe to receive non-target and target samples. 1 - Empty 30mL syringe, 3 - empty 50mL syringes, 5 - empty 50mL syringes, 7 - 50mL syringe containing 50mL of wash buffer, 9 and 10 - 10 mL syringes containing microparticles and cells, respectively.

[0189] Select the program for the specific cell type on the device, place the cartridge with the syringe firmly attached to the device and run the program. The device according to the invention was programmed so that in a first step the cells and microparticles are mixed and transferred to a tube (element 12, FIG. 7) for incubation at room temperature. After 15 minutes of incubation, washing of unbound material and non-target cells is performed by washing the tube with 30 mL of washing buffer. This washing buffer is the same as the buffer used to prepare the cell suspension. After this step, elution of the cells by mechanical disruption of the cell / microparticle complexes is performed by adding 7 mL of washing buffer and pushing the liquid "back and forth" through the tube in 5 steps. The final cell suspension (of viable target cells) is collected in syringe 1.

[0190] After the instrument completes the program, syringe numbers 1, 3, and 5 are collected and the target cells in the target elution syringe are counted. The results are shown in Table 1 together with the results of Example 8 and are expressed as a percentage of the starting cell number. The results provided below are obtained under the conditions described above in Examples 8 and 9 and carried out with a fixed amount of non-porous microparticles (20 and 40 mg of activated non-porous microparticles). The yield is improved if a larger amount of microparticles is used in the same experimental setting.

[0191] [Table 1]

[0192] To determine whether anti-CD90 antibodies were present in the eluted target and non-target cells separated in the above examples, cells contained in the target eluate (i.e., the final cell suspension containing viable target cells) and the non-target eluate were subjected to flow analysis.

[0193] Specifically, Kasumi (CD90-) and Jurkat (CD90+) cells contained in the target and non-target eluates were stained with goat anti-human Fc FITC affinity antibody (Invitrogen). As negative controls, Kasumi (CD90-) and Jurkat (CD90+) cells from the starting cell suspension were used (as these cells were not contacted with anti-CD90 antibody). Unstained Kasumi (CD90-) and Jurkat (CD90+) cells were used as additional negative controls.

[0194] The presence of anti-CD90 antibodies on the cell surface of eluted cells is expected to result in a signal above background in the FITC channel during flow cytometry analysis. The percentages of FITC-negative and FITC-positive cells detected are shown in Table 2.

[0195] In the negative control groups (i.e., unstained Kasumi (CD90-) cells, unstained Jurkat (CD90+) cells, Kasumi (CD90-) cells and Jurkat (CD90+) cells stained with secondary antibodies), extremely low positive FITC signals representing background noise were detected.

[0196] Importantly, the staining of Kasumi (CD90-) and Jurkat (CD90+) cells from the target and non-target eluates (eluates subjected to the cell separation method using the device according to the present invention) was below the background noise value detected in the negative control group. In fact, no FITC-positive cells were detected in either Kasumi (CD90-) or Jurkat (CD90+) cells contained in the target and non-target eluates, demonstrating that no antibodies from the non-porous microparticles were present in the cells eluted after the cell separation method according to the present invention, i.e., the cells were essentially free of anti-CD90 antibodies.

[0197] [Table 2]

[0198] To determine whether anti-CD90 antibodies were present in the final target eluate (i.e., the final cell suspension containing viable target cells) and non-target eluates collected in the above examples, Kasumi (CD90-) and Jurkat (CD90+) cells were stained with the target and non-target eluates, followed by staining with a secondary goat anti-human Fc FITC affinity antibody (Invitrogen). The stained cells were then analyzed using flow cytometry as described above.

[0199] Kasumi (CD90-) cells were used as negative controls. In addition, unstained Jurkat (CD90+) cells and Jurkat (CD90+) cells stained with secondary antibody alone served as additional negative controls. CD90+ Jurkat cells stained with anti-CD90 antibody served as positive controls.

[0200] If anti-CD90 antibodies are present in the target and / or non-target eluates, a signal in the FITC channel is expected during flow cytometry analysis. The percentages of FITC-negative and FITC-positive cells detected are shown in Table 3.

[0201] As shown in Table 3, there is no anti-CD90 antibody in the target and non-target eluates. As shown above, very low FITC signals were detected in the negative control group, representing background noise. Samples stained with target and non-target eluates gave signals equivalent to the negative control group. That is, the eluates are essentially free of anti-CD90 antibody.

[0202] [Table 3]

[0203] [Example 10] Manual cell separation method according to the present invention for isolation of CD19+ B lymphocytes from peripheral blood mononuclear cell (PBMC) samples Isolation of PBMCs from whole blood samples Prepare 50 ml of PBMC culture medium: 10% FBS in RPMI. Place 10 ml of Histopaque-1077 in a 50 ml centrifuge tube and carefully layer 10 ml of whole blood sample on top of the histopaque. Centrifuge for exactly 30 min at 400 g at room temperature. After centrifugation, carefully aspirate the top layer within 0.5 cm of the opaque interface containing the mononuclear cells with a Pasteur pipette. Discard the top layer and transfer the opaque interface with a Pasteur pipette to a clean 50 ml tube. Wash the cells by adding HBSS (up to the top 50 ml), mix by gently pipetting up and down with a Pasteur pipette and centrifuge the cell suspension at 250 g for 10 min.

[0204] Aspirate and discard the supernatant. Resuspend the cell pellet in 1 ml of Red Blood Cell Lysis Buffer (Roche, Cat. No. 11814389001) and incubate the sample for 5 min at room temperature. Add 30 mL of HBSS, mix by gently pipetting up and down with a Pasteur pipette, and centrifuge the cell suspension at 250 g for 10 min. Repeat these steps twice. Resuspend the cell pellet in PBMC culture medium and freeze or use the isolated PBMC cell sample immediately.

[0205] Isolation of CD19+ B lymphocytes from PBMC samples Prepare cell and wash buffer. Prepare cell and wash buffer by warming 10x buffer stock solution (catalog number 60-00080-10, purchased from pluriSelect Life Science, Leipzig, Germany) to room temperature and diluting the buffer stock solution 10-fold. Resuspend the PBMC pellet in 3 mL of diluted buffer. Count cells and dilute to a concentration of 5x10 5Prepare 3 mL of cell suspension at 1000 x 1000 cells / mL. Place 1 mL of cells in a sterile, commercially available centrifuge tube. Add 20 mg of non-porous silica microparticles activated with anti-CD19 antibodies. Mix gently 5 times to bring the cells into contact with the non-porous microparticles and leave the mixture to incubate at room temperature for 30 minutes. Place a 30 μm pore size filter on top of the sterile, commercially available centrifuge tube and pour the cell and microparticle mixture onto the filter. Wash the complexes with 16 mL of buffer by pouring the wash buffer over the cell / microparticle complexes to remove all unbound material and non-target cells that are small enough to pass through the filter. Close the opening on the filter so that no liquid can pass through and place everything on top of a clean, sterile, commercially available centrifuge tube. Add 2 mL of wash buffer and mechanically disrupt the target cell / microparticle complexes by pipetting up and down 20 times. Elute the target cells through the filter into a collection tube and wash the filter with an additional 10 mL of wash buffer. Collection tubes containing non-target and target cells are prepared for analysis on a MACS Quant Flow Cytometer.

[0206] In a human PBMC sample with a starting content of CD19+ B lymphocytes in the sample of 2%, the cell separation method according to the invention using non-porous microparticles with anti-CD19+ antibodies resulted in an approximately 20-fold enrichment of CD19+ B lymphocytes (purity 40%) with a viability of 78%. Furthermore, the results of another cell separation using a PBMC sample from a different (healthy) donor are shown in FIG. A schematic diagram of the method according to the invention is shown in FIGS.

[0207] [Example 11] Manual method for cell separation from human leukopheresis samples according to the present invention Preparation and freezing of human leukopheresis samples: After leukopheresis collection (the process of running donor blood through a machine to remove white blood cells and return all other blood cells and plasma to the bloodstream) is complete, store the collection bag containing the cell suspension in a 4°C refrigerator. For a 100mL apheresis, prepare 50mL apheresis medium (RPMI + 10% FBS), 400mL DPBS with 2% FBS for washing, and RPMI freezing medium (RPMI + 20% FBS + 20% DMSO). Sterilize scissors with ethanol and cut one of the tubes coming out of the collection bag. Pour the cell suspension into a sterile container. Prepare 10mL aliquots of apheresis cells in 50mL tubes and add chilled DPBS + 2% FBS on top. Centrifuge at 300×g, 10°C for 10 minutes. Discard the supernatant and resuspend the pellet in 5mL chilled apheresis medium. If necessary, filter the cells through a 70 µm strainer into a new 50 mL tube, where they can be frozen at -80 °C.

[0208] Thawing leukopheresis samples: Thaw the sample, e.g., at 37°C in a water bath or by warming by hand. Add 10 mL of DPBS while ice crystals are still present in the tube. Transfer the cell suspension to a 50 mL tube and top up with DPBS. Centrifuge at 300 x g for 10 min and resuspend the pellet in 10 mL of wash buffer. Pass the cell suspension through a 70 μm strainer to remove any clumped cells.

[0209] Cell isolation procedure: Prepare a sample containing a suspension of viable target cells and an animal protein-free, isotonic, physiological pH buffer, such as DPBS supplemented with 0.1% human albumin. Thaw the human leukopheresis sample and centrifuge at 300 rpm for 10 min. Count the cells and determine a concentration of 5×10 5Prepare 3 mL of cell suspension at 1000 x 1000 cells / mL. Place 1 mL of cells in a sterile, commercially available centrifuge tube. Add 20 mg of non-porous silica microparticles activated with anti-CD4 antibodies prepared by the procedure provided in Example 2a. Mix gently 5 times to bring the cells and non-porous microparticles into contact, and leave the mixture to incubate at room temperature for 30 minutes. Place a 30 μm pore size filter on top of the sterile, commercially available centrifuge tube and pour the solution containing the target cell / microparticle complexes onto the filter. Wash the target cell / microparticle complexes with 16 mL of wash buffer by pouring the buffer over the target cell / microparticle complexes to remove all unbound and non-targeted material. Close the opening on the filter to prevent liquid from passing through, and place everything on a clean, sterile, commercially available centrifuge tube. Add 2 mL of wash buffer and pipette up and down 20 times to mechanically disrupt the target cell / microparticle complexes and elute the target cells. Wash the filter with an additional 10 mL of wash buffer. Measure the purity and viability of the target and non-target fractions. The results are shown in Figures 10a and 10b as an increase in the purity of target cells in the samples after separation and in the viability of the samples. The purity of the cells was determined using a Miltenyi MACSQuant® VYB Flow Cytometer. The cells were stained with Miltenyi VioGreen CD4 dye (cat. no. 130-113-223) at a concentration of 1:100 and PI at a concentration of 1:1000. They were incubated for 10 minutes at room temperature in the dark and washed with a buffer containing 1x PBS, 0.5% BSA, and 2mM EDTA according to the manufacturer's protocol. Each sample was read in 450 μL with gate P4 (viable single cells) for 20'000 events. The viability of the cells was determined using a Miltenyi MACSQuant® VYB Flow Cytometer. Cells were stained with Propidium Iodide Solution (Miltenyi, Cat. No. 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. Cells were incubated at room temperature in the dark for 10 min and washed with MACS buffer. Each sample was read for 20'000 events in 450 μL.Data analysis was performed using MACSQuantify™ Software.

[0210] [Example 12] Manual method for cell isolation from human stromal vascular fraction samples according to the present invention Preparation of human stromal vascular fraction (SVF) samples The starting material is a lipoaspirate sample of adipose tissue collected in a bag during the liposuction procedure. Cut the sample bag and pour the lipoaspirate into a plastic or glass container. Wash the bag with 50 mL of PBS and add it to the remaining lipoaspirate. Wait 2-3 minutes for the fat to separate from the remaining liquid. Remove and discard the lower liquid phase and dispense 45 mL of lipoaspirate into a 50 mL tube. Centrifuge the lipoaspirate for 10 minutes at 430 x g. Remove the upper PBS and oil layer by pushing the tip of a 10 mL stripper through the fat layer. Add PBS to a total volume of 50 mL and centrifuge again. Repeat three times. Remove the PBS after the final centrifugation. Add the same volume of collagenase solution in digestion buffer as the adipose tissue, warmed to 37°C. Mix well and incubate for 30 minutes on an inclined roller mixer in a 37°C incubator. Centrifuge for 10 minutes at 600 x g and discard the supernatant. Remove as much of the fat layer as possible. Resuspend the SVF pellet in each tube in 5 mL of DPBS. Transfer the pellet in one 50 mL tube through a 100 μm strainer. Centrifuge at 600 x g for 10 minutes, remove the supernatant, and resuspend the pellet in each tube in 10 mL of 1 x Red Blood Cell Lysis Buffer. Incubate at room temperature in the dark for 10 minutes. Centrifuge at 600 x g for 10 minutes, and aspirate the supernatant. Resuspend the pellet in 10 mL of culture medium or buffer. The cells are now ready for the isolation procedure.

[0211] Cell isolation procedure: Prepare a sample containing a suspension of viable target cells from a human stromal vascular fraction sample and an animal protein-free, isotonic, physiological pH buffer, such as DPBS supplemented with 0.5% human albumin. Count the cells and culture at a concentration of 5×10 5Prepare 3 mL of cell suspension at 1000 x 1000 cells / mL. Place 1 mL of cells in a sterile, commercially available centrifuge tube. Add 20 mg of non-porous silica microparticles activated with anti-CD90 (or anti-CD34) antibodies prepared by the procedures provided in Example 1 and Example 2b, respectively. Mix gently 5 times to bring the cells and non-porous microparticles into contact, and leave the mixture to incubate at room temperature for 30 minutes. Place a 30 μm pore size filter on top of the sterile, commercially available centrifuge tube and pour the solution containing the target cell / microparticle complexes onto the filter. Wash the target cell / microparticle complexes with 16 mL of washing buffer by pouring the buffer over the target cell / microparticle complexes to remove all unbound and non-targeted material. Close the opening on the filter to prevent liquid from passing through, and place everything on a clean, sterile, commercially available centrifuge tube. Add 2 mL of washing buffer and pipette up and down 20 times to mechanically disrupt the target cell / microparticle complexes and elute the target cells. Wash the filter with an additional 10 mL of washing buffer. The purity and viability of the target and non-target fractions are measured. The results are shown in Figures 11a and 11b for the use of anti-CD90 antibody and in Figures 12a and 12b for the use of anti-CD34 antibody as an increase in the purity of target cells in the samples after separation and in the viability of the samples. The purity of the cells was determined using a Miltenyi MACSQuant® VYB Flow Cytometer. The cells were stained with Miltenyi VioGreen CD90 dye (cat. no. 130-112-683) at a concentration of 1:50 (or with Miltenyi anti-human CD34 antibody (cat. no. 130-113-179) at a concentration of 1:50 when anti-CD34 antibody was used). In both cases, they were incubated for 10 minutes at room temperature in the dark and washed with a buffer containing 1x PBS, 0.5% BSA, and 2mM EDTA according to the manufacturer's protocol. Each sample was read in 400 μL with gate P4 (viable single cells) for 20'000 events. Cell viability was determined using a Miltenyi MACSQuant® VYB Flow Cytometer.Cells were stained with Propidium Iodide Solution (Miltenyi, Cat. No. 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. Cells were incubated at room temperature in the dark for 10 min and washed with MACS buffer. Each sample was read for 20'000 events in 450 μL. Data analysis was performed using MACSQuantify™ Software.

[0212] [Example 13] Automated method for cell separation from human leukopheresis samples according to the present invention A sample is prepared containing a suspension of viable target cells and an animal protein-free, isotonic, physiological pH buffer, such as DPBS supplemented with 0.1% human albumin. A human leukopheresis sample is thawed and centrifuged at 300 rpm for 10 minutes according to the method presented in Example 11. Cells are counted and a concentration of 1×10 6 Prepare 4 mL of cell suspension at 100 cells / mL.

[0213] Prepare two 10 mL syringes: in the first syringe, transfer 3 mL of cells suspended in buffer, and in the second syringe, transfer 160 mg of non-porous silica microparticles activated with anti-CD4 antibody (prepared by the procedure provided in Example 2a) and suspended in 3 mL of buffer.

[0214] Syringes are attached to the cartridge (Figure 7), along with either a wash buffer or an empty syringe to receive non-target and target samples. 1 - Empty 30mL syringe, 3 - empty 50mL syringes, 5 - empty 50mL syringes, 7 - 50mL syringe containing 50mL of wash buffer, 9 and 10 - 10 mL syringes containing microparticles and cells, respectively.

[0215] Select the program for the specific cell type on the device, place the cartridge with the syringe firmly attached to the device and run the program. The device according to the invention was programmed so that in a first step the cells and microparticles are mixed and transferred to a tube (element 12, FIG. 7) for incubation at room temperature. After 15 minutes of incubation, washing of unbound material and non-target cells is performed by washing the tube with 30 mL of washing buffer. This washing buffer is the same as the buffer used to prepare the cell suspension. After this step, elution of the cells by mechanical disruption of the cell / microparticle complexes is performed by adding 7 mL of washing buffer and pushing the liquid "back and forth" through the tube in 5 steps. The final cell suspension (of viable target cells) is collected in syringe 1.

[0216] After the instrument has completed the program, syringes no. 1, 3, and 5 are collected and the purity and viability in the target and non-target fractions are measured. The results are shown in Fig. 13a and 13b, expressed as the purity and viability of target cells in the samples before and after separation. The purity of the cells was determined using a Miltenyi MACSQuant® VYB Flow Cytometer. The cells were stained with Miltenyi anti-human CD4 antibody (cat. no. 130-113-230) at a concentration of 1:300. They were incubated for 10 min at room temperature in the dark and washed with a buffer containing 1x PBS, 0.5% BSA, and 2 mM EDTA according to the manufacturer's protocol. Each sample was read in 450 μL with gate P4 (viable single cells) for 20'000 events. The viability of the cells was determined using a Miltenyi MACSQuant® VYB Flow Cytometer. Cells were stained with Propidium Iodide Solution (Miltenyi, Cat. No. 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. Cells were incubated at room temperature in the dark for 5 min and washed with MACS buffer. Each sample was read for 20'000 events in 400 μL. Data analysis was performed using MACSQuantify™ Software.

[0217] [Example 14] Manual method for cell separation of DPS cells mixed with human leukopheresis samples according to the present invention Sample preparation: Human leukopheresis samples are thawed using the procedure described in Example 11. DPS (dental pulp stem) cells (CD90+) are added to the human leukopheresis samples to obtain a ratio of 50% DPS cells to 50% total leukopheresis cells.

[0218] Cell isolation procedure: A sample is prepared containing a suspension of viable target cells of human leukopheresis cells and DPS cells and an animal protein-free, isotonic, physiological pH buffer, such as DPBS supplemented with 0.1% human albumin. 1 mL of cells is placed in a sterile, commercially available centrifuge tube. 20 mg of non-porous silica microparticles activated with anti-CD90 antibodies prepared by the procedure provided in Example 1 are added. Gently mix 5 times to contact the cells and non-porous microparticles, and the mixture is left to incubate at room temperature for 30 minutes. A filter with a pore size of 30 μm is placed on top of the sterile, commercially available centrifuge tube, and the solution containing the target cell / microparticle complexes is poured on top of the filter. The target cell / microparticle complexes are washed with 16 mL of wash buffer by pouring the buffer on top of the target cell / microparticle complexes to remove all unbound and non-targeted materials. The opening on the filter is closed to prevent liquid from passing through, and everything is placed on a clean, sterile, commercially available centrifuge tube. Add 2 mL of wash buffer and pipette up and down 20 times to mechanically disrupt the target cell / microparticle complex and elute the target cells. Wash the filter with an additional 10 mL of wash buffer. Measure the purity and viability of the target and non-target fractions. The results are shown in Figures 15a and 15b as an increase in the purity of target cells in the samples after separation and in the viability of the samples. The purity of the cells was determined using a Miltenyi MACSQuant® VYB Flow Cytometer. The cells were stained with Miltenyi VioGreen CD90 dye (catalog number 130-112-683) at a concentration of 1:50. They were incubated for 10 minutes at room temperature in the dark and washed with a buffer containing 1x PBS, 0.5% BSA, and 2 mM EDTA according to the manufacturer's protocol. Each sample was read in 400 μL for 20'000 events on gate P4 (viable single cells). Data analysis was performed using MACSQuantify™ Software. Cells were stained with Propidium Iodide Solution (Miltenyi, Cat. No. 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. They were incubated at room temperature in the dark for 5 minutes and read directly.Each sample was read for 20'000 events in 450 μL. Data analysis was performed using MACSQuantify™ Software.

[0219] [Example 15] Automated method for cell separation of MS (mesenchymal stem) cells mixed with human leukopheresis samples according to the present invention Sample preparation: Human leukopheresis samples are thawed using the procedure described in Example 11. Human leukopheresis samples are spiked with MS (mesenchymal stem) cells (CD90+) to obtain a ratio of 33% MS cells to 66% total leukopheresis cells.

[0220] Cell isolation procedure: A sample is prepared containing a suspension of viable target cells of human leukopheresis cells and MS cells and an animal protein-free, isotonic, physiological pH buffer, such as DPBS supplemented with 0.1% human albumin. The human leukopheresis sample is thawed and centrifuged at 300 rpm for 10 minutes according to the method presented in Example 11. The cells are counted and centrifuged at a concentration of 1×10 6 Prepare 4 mL of cell suspension at 100 cells / mL.

[0221] Prepare two 10 mL syringes: in the first syringe, transfer 3 mL of cells suspended in buffer, and in the second syringe, transfer 160 mg of non-porous silica microparticles activated with anti-CD4 antibody (prepared by the procedure provided in Example 2a) and suspended in 3 mL of buffer.

[0222] Syringes are attached to the cartridge (Figure 7), along with either a wash buffer or an empty syringe to receive non-target and target samples. 1 - Empty 30mL syringe, 3 - empty 50mL syringes, 5 - empty 50mL syringes, 7 - 50mL syringe containing 50mL of wash buffer, 9 and 10 - 10 mL syringes containing microparticles and cells, respectively.

[0223] Select the program for the specific cell type on the device, place the cartridge with the syringe firmly attached to the device and run the program. The device according to the invention was programmed so that in a first step the cells and microparticles are mixed and transferred to a tube (element 12, FIG. 7) for incubation at room temperature. After 15 minutes of incubation, washing of unbound material and non-target cells is performed by washing the tube with 30 mL of washing buffer. This washing buffer is the same as the buffer used to prepare the cell suspension. After this step, elution of the cells by mechanical disruption of the cell / microparticle complexes is performed by adding 7 mL of washing buffer and pushing the liquid "back and forth" through the tube in 5 steps. The final cell suspension (of viable target cells) is collected in syringe 1.

[0224] After the instrument has completed the program, syringes no. 1, 3, and 5 are collected to measure the purity and viability in the target and non-target fractions. The results are shown in Fig. 16a and Fig. 16b, expressed as the purity and viability of target cells in the samples before and after separation. The purity of the cells was determined using a Miltenyi MACSQuant® VYB Flow Cytometer. The cells were stained with Miltenyi anti-human CD90 PE-Vio® 770 antibody (cat. no. 130-114-904) at a concentration of 1:400. They were incubated for 10 min at room temperature in the dark and washed with a buffer containing 1x PBS, 0.5% BSA, and 2 mM EDTA according to the manufacturer's protocol. Each sample was read for 20'000 events in 450 μL on gate P4 (viable single cells). Cell viability was determined using a Miltenyi MACSQuant® VYB Flow Cytometer. Cells were stained with Propidium Iodide Solution (Miltenyi, Cat. No. 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. Cells were incubated at room temperature in the dark for 5 min and washed with MACS buffer. Each sample was read for 20'000 events in 400 μL. Data analysis was performed using MACSQuantify™ Software.

Claims

1. A method for isolating viable target cells from a sample, a. A sample containing a suspension of viable target cells that present molecules on their cell surface, at approximately 1.45 g / cm³. 3 A nonporous microparticle having the above density and a diameter of approximately 10 μm to 200 μm, wherein a capture ligand capable of specifically binding to the molecule is covalently immobilized on the surface of the microparticle, and the step of bringing the nonporous microparticle into contact with the microparticle. b. A step of incubating the sample without substantially stirring it to form a target cell / microparticle complex, c. A step of separating the unbound substance in the sample from the target cell / microparticle complex by washing the unbound substance through a filter while retaining the target cell / microparticle complex. d. A step of mechanically dissociating the target cell / microparticle complex and eluting the viable target cells through the filter while retaining the microparticles to which the capture ligand is covalently immobilized on the surface of the microparticles. A method that includes this.

2. The method according to claim 1, wherein the target cells are suitable for cell therapy.

3. The capture ligand is 10 -5 ~10 -12 , more 10 -9 ~10 -11 , or comfort 10 -7 ~10 -10 , comfortably about 10 -9 The method according to claim 1, wherein the molecule specifically binds to the surface of the target cell with a dissociation constant of the order of .

4. The capture ligand is an antibody, FabFc 2 , Fab, Fv, Fd, F(ab') 2 , an Fv fragment containing only the variable regions of the light and heavy chains, a Fab or F(ab') containing a part of the variable region and a constant region 2 fragment, single-chain antibody, scFv, CDR-grafted antibody, dAb, and / or nanobody, the method according to claim 1

5. The method according to claim 1, wherein the cell surface molecule is selected from the group consisting of human CD2, CD3, CD4, CD8, CD11a, CD11b, CD14, CD15, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD31, CD34, CD38, CD43, CD45, CD48, CD56, CD61, CD73, CD90, CD91, CD105, CD114, CD117, CD140b, CD150, CD182, CD184, CD271, CDCP1, GD2, GPR4, Sca-1, and STRO-1.

6. The method according to claim 1, further comprising the step of obtaining a pharmaceutical product comprising a viable target cell suspension of eluted viable target cells, wherein the eluted viable target cell suspension essentially does not contain a capture ligand.

7. Target cells include human granulocytes, T lymphocytes, monocytes, T regulatory cells, T helper cells, cytotoxic T cells, B lymphocytes, tumor-infiltrating lymphocytes, platelets, natural killer cells, hematopoietic stem cells and progenitor cells, mesenchymal / stromal stem cells, hair follicle stem cells, cardiac stem cells, pluripotent muscle cells, neural stem cells, hepatic stem cells, dental pulp cells, periodontal ligament cells, retinal pigment epithelial cells, adipose tissue-derived stem cells and progenitor cells, pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells, Car-T cells, and microvascular endothelial cells (MVECs). The method according to claim 1, comprising, selected from a group of cells established from these primary cells, including primary epithelial cells, such as keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells and retinal epithelial cells, nerve tissue derived from the heart, liver, kidney, colon, intestine, esophagus, stomach, brain and spinal cord, vascular tissue derived from the lungs, arteries, veins and capillaries, lymph glands, adenoids, tonsils, bone marrow and blood, fibroblasts, spleen, muscle cells, pancreatic cells, cardiac cells, and lymphoid tissue derived from any tissue or organ.

8. The method according to claim 1, wherein the target cells are selected from the group consisting of monocytes, T lymphocytes, B lymphocytes, CAR-T cells, and stem cells.

9. The method according to claim 1, wherein the sample is selected from the group consisting of human whole blood, apheresis, bone marrow aspirate, biopsy, liquefied tissue, such as enzymatically hydrolyzed liposuction, cell culture, bioreactor culture, tumor cells, and single-cell suspension.

10. A closed housing cartridge configured for use in separating viable target cells from a sample according to the method described in any one of claims 1 to 9.

11. - A filter enclosed within a unit having an inlet port and an outlet port. - A fluid conduit connected to the inlet of a filter, having one or more valves capable of controlling the flow of a source of sample containing target cells, a source of suspension of nonporous microparticles, a source of washing solution, and a source of elution solution, and - A fluid conduit connected to the outlet of a filter, comprising one or more valves, wherein one valve can redirect the fluid conduit to a target cell solution or to a different container for washing solution and unbound material. A closed housing cartridge according to claim 10, including the following:

12. A closed housing cartridge according to claim 10, and approximately 1.45 g / cm³ 3 A kit of components comprising a container containing a suspension of nonporous microparticles having the above density and a diameter of approximately 10 μm to 200 μm, wherein a capture ligand capable of specifically binding to molecules on the cell surface is covalently immobilized on the surface of the microparticles.

13. A closed housing cartridge according to claim 10, wherein the closed housing cartridge is contained within a sealed sterile package.

14. The parts kit according to claim 12, wherein the components of the parts kit are contained in sealed sterile packaging.

15. The aforementioned nonporous fine particles are approximately 1.95 g / cm³ 3 The method according to any one of claims 1 to 9, having the above density.

16. The kit of components according to claim 12, wherein the nonporous fine particles have a density of about 1.95 g / cm³ or more.

17. The method according to any one of claims 1 to 9, wherein the nonporous fine particles have a diameter of about 35 μm to 50 μm.

18. The kit of components according to claim 12, wherein the nonporous fine particles have a diameter of about 35 μm to 50 μm.

19. The nonporous fine particles are silicon dioxide (SiO 2 The method according to any one of claims 1 to 9, which is made from ).

20. The kit of components according to claim 12, wherein the non-porous fine particles are made from silicon dioxide (SiO₂).

21. The method according to any one of claims 1 to 9, wherein the eluted viable target cell suspension essentially does not contain a capture ligand antibody that specifically binds to a molecule presented on the surface of the target cells.

22. A device configured to separate viable target cells from a sample according to the method described in any one of claims 1 to 9, - Means for securely mounting a closed housing cartridge such that a valve of the cartridge is mechanically operated by an electronically controlled system, configured for use in separating viable target cells from a sample according to the method described in any one of claims 1 to 9. - Means for connecting the cartridge to one or more containers containing one or more solutions and / or suspensions, - Means for moving solutions and / or suspensions, - A control system configured to set, measure, and / or regulate the flow of solution and / or suspension via a valve in the cartridge while carrying out the method according to any one of claims 1 to 9. A device that includes this.

23. The apparatus according to claim 22, comprising one or more pre-processing filters, a keyboard, a touchscreen, a barcode reader device, a communication element for Wi-Fi, Near Field Communication (NFC) or Bluetooth®, a motor, a vibration isolation unit for any or all of the pumps or motors, a sensor or electrode for measuring pressure, flow rate, impedance, conductivity, resistance, current, pH, temperature, humidity, gas percentage, radiation, motion, volume, weight, magnetic flux, time, turbidity, optical density, an alarm, a cell counting device, a heating unit, a cooling unit, a graphic display unit, a data storage device such as a CD, a USB flash drive stick, or a port for NFC.

24. A pharmaceutical product comprising a viable target cell suspension obtained according to the method of claim 6, wherein the eluted viable target cell suspension is essentially free of the capture ligand.

25. A viable target cell suspension obtained according to the method of claim 6 for use as a pharmaceutical, wherein the eluted viable target cell suspension is essentially free of capture ligands.

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