Method for isolating target cells

Abstract

The present invention relates to a method for isolating viable target cells from a sample as well as a cartridge, a kit, a device and a drug comprising viable target cells obtainable by said method configured for use in said method, the method comprises the step of contacting a sample comprising a suspension of viable target cells, the viable target cells exhibiting molecules on the cell surface, with non-porous microparticles having a density of about 1.45 g / cm3 or greater, a diameter of about 10 [mu] m to 200 [mu] m, and capture ligands covalently immobilized to the surface of the microparticles, the capture ligand is capable of specifically binding to the molecule; incubating the sample without substantially stirring to form a target cell / microparticle complex; separating the target cell / particle complex from the unbound species in the sample by washing the unbound species through a filter while retaining the target cell / particle complex; the target cell / microparticle complex is mechanically dissociated and the viable target cells are eluted through the filter while retaining the microparticles with the capture ligands covalently immobilized to the surface of the microparticles.

Description

1. Technical Field

[0001] The present invention belongs to the field of cell separation technology, and particularly relates to methods, cartridges and devices for capturing, separating and isolating targeted cells. 2. Background technology

[0002] In several industries and research processes, it is crucial to carry out cell separation and targeted cell separation from complex biological matrices (such as tissues, blood and other samples). The core of cell separation is that it needs to carry out the selective enrichment of target cells based on the physical, chemical or biological properties of target cells. One of the main goals of targeted cell separation is to produce viable cells that retain their initial biological properties as seen in the original tissue or source. This is especially important in cell and gene therapy, where cell health and metabolism are crucial to the success of such treatment.

[0003] Some examples of such cell and gene therapy include hematopoietic stem cell transplantation for the treatment of leukemia or solid cancer. By removing red blood cells, cells (such as nucleated cells, including hematopoietic stem cells) that are effective in treatment are separated from bone marrow or peripheral blood, and then applied to the patient. Cord blood banks require cells to be cryopreserved before use, and red blood cells are removed to prevent possible hemolysis during cryopreservation. In addition, the transplantation of cell fractions of bone marrow, cord blood or peripheral blood rich in stem cells (such as mesenchymal stem cells, hematopoietic stem cells and endothelial progenitor cells) has been used for the treatment of ischemic diseases (such as cerebral infarction, myocardial infarction and local ischemia), to promote angiogenesis or neural regeneration. In addition, granulocytes can cause undesirable side effects (such as inflammation), which can reduce the therapeutic effect, and it is expected that they will be removed from cell therapy products.

[0004] Cell separation is also crucial in the preparation of cell therapeutic agents such as CAR-T cells, autologous and allogeneic stem cell therapy and other new treatment methods. Sometimes, the separated cells need to be further processed in both industrial and research fields. The success and effectiveness of these processes are directly affected by the quality and purity of the separated cell suspension. Residual components (such as nanoparticles, antibodies and buffers) can interfere with downstream processes (i.e., cell amplification, gene editing), and if possible, should be excluded from the final product.

[0005] The same is true for the clinical use of isolated cells in autologous and xenotransplantation therapies: isolated cells contaminated by residual buffers and antibodies can negatively affect the outcome of the therapy. Furthermore, due to regulatory restrictions, the clinical use of these contaminated cells is not permitted in several countries. This limits the accessibility of cell therapies.

[0006] The technologies currently available on the market utilize different means of cell separation and enrichment and are mainly divided into the following categories:

[0007] 1) Magnetic particle separation: This method uses magnetic nanoparticles functionalized with cell-specific antibodies that capture target cells as they pass through a magnetic source. Once cells bind to the magnetic particles, the magnetic field retains the magnetic particles and non-target cells are washed away. Once the magnetic field is released, the cells are also released. The magnetic particles are typically nanoparticles of iron oxide (e.g., magnetite (Fe3O4)) ranging in size from 1 to 100 nanometers, which gives them superparamagnetic properties. Superparamagnetic particles are different from more common ferromagnets in that they only exhibit magnetic behavior in the presence of an external magnetic field. This property depends on the small size of the particles and enables them to be separated in suspension along with any substance they are bound to. Since nanoparticles are typically small or have a diameter similar to that of cells, it is not feasible to separate cells and particles by size, so cells bound to nanoparticles are separated by magnets. Cells can be eluted from nanoparticles using a buffer that can highly affect cell viability, and the magnetic nanoparticles can then be separated from the cell solution using a magnetic field. One disadvantage of this method is that some residual magnetic nanoparticles may remain in the final cell solution. This may be due to the loss of magnetic properties of the particles, making it difficult to ensure thorough removal of the magnetic particles from the solution of target cells. In addition, the method requires large and expensive equipment to ensure magnetic separation and requires a lot of manual operation time of the operator (see, for example, EP3037171A1, US10119970B2, CA2854240A1, EP0819250A1, EP0760102A1, US5385707A, EP2444158A2, US2002146848A1, WO2019103103A1, and CN107278270A).

[0008] 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, etc.). Antibody receptors are used to capture antibodies that can capture target cells of interest. Non-target cells are washed away in a column or strainer. Target cells are eluted with an elution buffer, which may contain 1) a competitive binding compound that preferably binds to the coated receptor (e.g., biotin in the case of avidin) and releases secondary antibodies and cells from the beads, 2) high salt content, 3) low pH, 4) enzymes or 5) other chemicals and animal-derived compounds that promote the release of antibodies from the coated receptor. Therefore, the resulting cell suspension contains an elution buffer that can damage the viability or cell behavior of the cells, and thus can hinder downstream processing. In addition, weight column separation is unreliable because the flow rate is controlled only by the density and gravity of the beads in the solution, resulting in uneven separation duration and efficiency. Due to the physical limitations of column separation, these methods are also limited in the scale of incoming volume (see, e.g.,

[0009] US10196631B2, JP2018138913A, EP2734538A2, WO2015166049A1, US2014315297A1 and US2017299585A1).

[0010] 3) Centrifugation and density separation: This is the most widely used standard mode of cell separation based on the physical properties of cells (such as size and density). These methods use centrifuges and density gradients to capture target cells. The main disadvantage of these methods is that cells are purified based only on the physical properties of cells rather than the biological properties of cells (such as surface antigens). Cell therapy requires the separation of specific cell populations based on the surface antigens of specific cell populations (called CD antigens), and specific cell populations cannot be distinguished based solely on the physical properties of cells. Extended centrifugation and the use of density reagents can also negatively affect cell behavior and viability (see, for example, JP2012143256A and WO2018194061A1).

[0011] Due to these and other shortcomings in the cell separation methods and systems in the art, there is a need for methods, devices and systems that can provide effective target cell separation based on the surface antigens of the target cells without the use of magnetic particles and without the use of non-magnetic beads coated with antibody receptors that can be directly used for treatment. 3. Summary of the invention

[0012] To meet this need, the present invention provides methods, cartridges and devices that result in 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 beads coated with antibody receptors. The present invention results in isolated target cells having high purity and high cell viability in a selected buffer or culture medium.

[0013] 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 aforementioned method according to the claims. Different embodiments can be derived from the dependent claims and the description.

[0014] Thus, in one aspect, the present invention relates to a method for isolating viable target cells from a sample, comprising the following steps:

[0015] a. contacting a sample comprising a suspension of viable target cells displaying molecules on the cell surface with nonporous microparticles and a capture ligand covalently immobilized to the surface of the microparticles, wherein the nonporous microparticles have a density of about 1.45 g / cm 3 or larger, with a diameter of about 10 μm to 200 μm, and the capture ligand is capable of specifically binding to the molecule;

[0016] b. incubating the sample substantially without agitation to form a target cell / particle complex;

[0017] c. separating the target cell / microparticle complex from the unbound material in the sample by washing the unbound material through a filter while retaining the target cell / microparticle complex;

[0018] d. Mechanically dissociating the target cell / microparticle complexes and eluting the viable target cells through the filter while retaining the microparticles with the capture ligands covalently immobilized to the microparticle surface.

[0019] In another aspect, the invention relates to a cartridge configured for isolating viable target cells from a sample using a method according to the invention.

[0020] In another aspect, the invention relates to a device configured to separate viable target cells from a sample using a method according to the invention.

[0021] In another aspect, the invention relates to a kit comprising a cartridge and a container, wherein the container comprises a suspension of non-porous microparticles and a capture ligand covalently immobilized to the surface of the microparticles, wherein the non-porous microparticles have a density of about 1.45 g / cm 3or larger, with a diameter of about 10 μm to 200 μm, and the capture ligand is capable of specifically binding to molecules on the cell surface.

[0022] The proposed invention eliminates the disadvantages of the prior art by enabling automatic or manual separation of target cells from a sample using non-porous microparticles having capture ligands (e.g. antibodies) covalently bound on their surface, having a specific density and a specific diameter range, and without any other antibody binding receptors bound thereto. At the same time, the invention ensures the purity and high viability of the isolated target cell suspension and thus eliminates the need for downstream processing.

[0023] The method, cartridge and device according to the present invention allow specific target cells to be captured 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 nonporous microparticles). After washing unbound cells and other undesirable substances from the sample, the target cells are eluted by mechanically dissociating or destroying cell surface molecules-capture ligand interactions such as cell surface antigen-antibody interactions (e.g., by regulating the flow rate or flow rate of the solution, mixing, pipetting and / or ultrasonic treatment). Using the method, cartridge and device according to the present invention, no special chemical elution buffer or addition of any elution additives, such as enzymes, biotin streptavidin and other compounds, is required. In the method, cartridge and device according to the present invention, the need for unnecessary and / or bioincompatible substances (e.g., bacterial proteins (protein A, protein G)) and products of animal origin (e.g., BSA, casein, etc.) is also eliminated. The present invention does not rely on eluting antibodies into the final cell suspension as other methods do. Method according to the present invention, tube and device do not rely on the magnetic properties of magnetic particles that are eluted in other systems and are present in the final cell suspension. Method according to the present invention, tube and device allow a rapid and controlled cell separation operation that can be less than one hour, thereby reducing the change of cell death and cell metabolism and undesirable cell activation. In addition, method according to the present invention, tube and device can provide a closed, sterile environment, and can be adapted to low-capacity (e.g., 10ml sample) or high-capacity (several liters of sample) applications. Method according to the present invention, tube and device produce the separated target cells eluted in a selected culture medium or physiological buffer solution that does not contain undesirable additives or impurities under limited processing time and high cell viability and health.

[0024] Therefore, in another aspect, the present invention also relates to a medicament comprising a target cell suspension obtainable according to the method of the present invention, a target cell suspension obtainable according to the method of the present invention for use as a medicament, and a method of treating a subject in need of a target cell suspension obtainable according to the method of the present invention for the treatment of a disease. 4. Description of the drawings

[0025] Figure 1 Results of the Bradford supernatant assay calculated as the percentage of protein remaining in the supernatant solution based on the starting protein concentration.

[0026] Figure 2 : Surface saturation of 40 μm nonporous silica microparticles (Glantreo Ltd., Ireland) coated with anti-CD90 antibodies (BIOTEM, France) calculated based on experimental results obtained with Bradford spectrophotometry of the supernatant.

[0027] Figure 3 : shows the components (part) that can be used for the manual cell separation method of target cells according to the present invention, namely filters, incubation tubes and syringes connected in one system. The system is sterile and intended for single use. Syringe 1 is used for incubation of viable target cells and non-porous microparticles and for elution buffer. Filter 3 is used to separate viable target cells from non-porous microparticles in a sample. Long tube 4 and short tube 5 are used to more easily connect to filter 3 and operate first syringe 1 and second syringe 2. Second syringe 2 is used as a receiving syringe for collecting viable target cells. When it is necessary to perform the method, the first syringe 1 and the second syringe 2 can be replaced by clean syringes.

[0028] Figure 4a : Microscope images of nonporous microparticles after steps a and b of the cell separation method according to the invention were performed using positive control cells. Silica nonporous microparticles with a diameter of 40 μm (purchased from Glantreo Ltd., Cork, Ireland) and having anti-CD90 antibodies immobilized on their surface bind CD90 positive cells (Jurkat cells) to form target cell / microparticle complexes. CD90 positive cells are identified as small round luminous structures located on the surface of larger spherical nonporous microparticles.

[0029] Figure 4b : Microscope images of nonporous microparticles after performing steps a and b of the cell separation method according to the invention using negative control cells. Silica nonporous microparticles with a diameter of 40 μm (purchased from Glantreo Ltd., Cork, Ireland) and having anti-CD90 antibodies (BIOTEM, France) immobilized on their surface do not bind to CD90 negative cells (Kasumi cells) and do not form any target cell / microparticle complexes.

[0030] Figure 5: Cartridge for isolating target cells according to the invention used in a device: front view. In the front view, a valve 13 can be seen at the end of each Luer lock 1 to 10, to which a syringe or other fluid container can be connected. The front side of the cartridge is connected to the device so that it contacts the valve, thereby controlling the open / closed position. The cartridge is made of: female Luer locks 1 to 10, to which male Luer locks can be connected; valves 13, which allow controlling the flow of fluid; filters 11, which are used to prevent non-porous particles from contaminating the isolated viable target cells; and hollow tubes 12, which connect all syringes or other fluid containers into a closed system.

[0031] Figure 6 : Rotated version of the cartridge for isolating target cells according to the invention used in the device: front view. The shown orientation of the cartridge allows better control of the flow of liquids and solids through the system. Female Luer locks 1 to 10 are located in positions where syringes or other fluid containers with male Luer locks are connected. Filter 11 prevents non-porous particles from contaminating the isolated viable target cells. Tube 12 serves as an incubation space and also connects all syringes or other fluid containers into a closed system.

[0032] Figure 7 : A cartridge according to the invention, wherein female Luer locks 1 to 10 and valves are located only on one side of the cartridge: rear view. The orientation of the Luer locks and valves shown allows for easier connection of syringes or other fluid containers. In addition, the system shown allows for more ports to be connected to incubation tubes and optionally more filters and tubes. Female Luer locks 1 to 10 are located in positions where syringes or other fluid containers with male Luer locks are connected. Filter 11 prevents non-porous particles from contaminating isolated viable target cells. Tube 12 serves as an incubation space and also connects all syringes or other fluid containers into a closed system. A schematic diagram of a front view is also provided in the figure.

[0033] Figure 8 : Flowchart of the method according to the invention.

[0034] Fig. 9: An exemplary process flow diagram of the method used in various embodiments of the present invention is shown. The figure shows the basic process of cell separation using the device according to the present invention. The incubation space 1 is connected to the inlet flow 2 and the outlet flow 3. Element 4 represents the entry point for all flows into one inlet flow 2, and element 16 represents the splitter of the outlet flow 3. A sample of a suspension containing viable target cells is pushed through valve 14 with pump 23 and flow 15. When the cells pass through valve 14, the cells enter flow 10 and enter mixing point 8. Non-porous microparticles are pushed with a second pump 27 and flow 13, and the non-porous microparticles pass through valve 12. When the non-porous microparticles pass through valve 12, the microparticles enter flow 9 and enter mixing point 8, where the non-porous microparticles are mixed with the sample of the suspension containing viable target cells. Then, the flow enters element 4, which is the entry point for all flows into the inlet flow 2 and into the incubation space 1, and a viable target cell / microparticle complex is formed in the incubation space 1. After the incubation is completed and the cell / particle complex is formed, washing of unbound cells and non-target cells begins. The flow of the wash buffer is controlled by pump 29 and valve 5. The wash buffer passes through stream 6 and valve 5. When the wash buffer passes through the valve, the wash buffer enters stream 7 and enters the entry point of all inlet streams 4. The wash buffer passes through the inlet stream 2 and fills the incubation space 1. The incubation space 1 is equipped with a filter that is smaller than non-porous particles and larger than target cells (cells that have formed cell / particle complexes), and it acts as a barrier for non-porous particles, as well as larger non-target cells and unbound substances. During the first wash period - washing unbound substances and non-target cells - the liquid enters the outlet stream 3 and then passes through the flow diverter 16, and the unbound substances and non-target cells pass through stream 18 and valve 20 and finally enter stream 22, where they are collected in a suitable fluid container. After the washing is completed, mechanical disruption and elution of viable target cells begins. In the basic process, mechanical disruption is performed with the same buffer. The flow of buffer is controlled by pump 29, the flow passes through valve 5 into stream 6 and into stream 7. From there, the buffer enters into all the entry points of inlet stream 4 and into inlet stream 2, so that it fills the incubation space. With the mechanical force of the fluid in the incubation space, the destruction of the cell / particle complex is carried out and the target cells can be eluted. The particles are retained on one side of the filter in the incubation space, and the viable target cells pass through the filter in stream 3 and pass through the flow splitter 16. Stream 19 passes through valve 21 and into stream 24, where the viable target cells are collected in a suitable fluid container. The outlet stream can optionally be equipped with a pump and can optionally be more than one wash buffer stream.

[0035] Fig.10a: shows the enrichment in the target cell fraction before and after separation of a leukapheresis sample (healthy donor) using microparticles with anti-CD4 antibodies and the manual method according to the invention.

[0036] Fig.10 b: Viability of samples before and after separation is shown. Cells in the target cell fraction retained 100% of their initial viability.

[0037] Fig.11 a: shows the enrichment in the target cell fraction before and after separation of a stromal vascular fraction sample (healthy donor) using microparticles with anti-CD34 antibodies and the manual method according to the invention.

[0038] Fig.11 b: Viability of samples before and after separation is shown. Cells retained 74% of their initial viability in the target cell fraction.

[0039] Fig.12 a: shows the enrichment in the target cell fraction before and after separation of a stromal vascular fraction sample (healthy donor) using microparticles with anti-CD90 antibodies and the manual method according to the invention.

[0040] Fig.12 b: Viability of samples before and after separation is shown. Cells in the target cell fraction retained 75% of their initial viability.

[0041] Fig.13 a: shows the enrichment in the target cell fraction before and after separation of a leukapheresis sample (healthy donor) using microparticles with anti-CD4 antibodies and the automated method according to the invention.

[0042] Fig.13 b: Viability of samples before and after separation is shown. Cells retained 98% of their initial viability in the target cell fraction.

[0043] Fig.14 a: shows the enrichment in the target cell fraction before and after isolation of a PBMC sample (healthy donor) using microparticles with anti-CD19 antibodies and the manual method according to the invention.

[0044] Fig.14 b: Viability of samples before and after separation is shown. Cells in the target cell fraction retained 70% of their initial viability.

[0045] Fig.15a: shows the enrichment in the target cell fraction before and after separation of human DPS (dental pulp stem) cells mixed with leukapheresis samples (healthy donors) using microparticles with anti-CD90 antibodies and the manual method according to the present invention.

[0046] Fig.15 b: Viability of samples before and after separation is shown. Cells retained 77% of their initial viability in the target cell fraction.

[0047] Fig.16 a: shows the enrichment in the target cell fraction before and after separation of human MS (mesenchymal stem) cells mixed with leukapheresis samples (healthy donors) using microparticles with anti-CD90 antibodies and the manual method according to the present invention.

[0048] Fig.16 b: Viability of samples before and after separation is shown. Cells retained 89% of their initial viability in the target cell fraction. 5. Specific implementation methods

[0049] Unless the context clearly states otherwise, nouns used herein without quantifier modification include plural referents. Unless otherwise clearly stated, "and" used herein can be used interchangeably with "or / or". Unless the context clearly states otherwise, all embodiments of any aspect of the present invention can be used in combination. Unless the context clearly requires otherwise, throughout this specification and claims, the words "include / include" and its variations should be interpreted as inclusive meanings rather than exclusive or exhaustive meanings; that is, interpreted as the meaning of "including but not limited to". Words using singular or plural numbers also include plural and singular numbers, respectively. In addition, when used in this application, the words "herein", "above" and "below" and words of similar meanings should refer to the application as a whole, rather than to any particular part of the application. The entire disclosure of all publications cited herein is incorporated by reference. The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. The following description of the present invention is mainly carried out with reference to the method of the present invention. However, unless otherwise stated, references in this specification to the method of the invention also apply and refer equally to the cartridge and device of the invention.

[0050] In one aspect, the present invention relates to a method for isolating viable target cells from a sample, comprising the following steps:

[0051] a. contacting a sample comprising a suspension of viable target cells displaying molecules on the cell surface with nonporous microparticles and a capture ligand covalently immobilized to the surface of the microparticles, wherein the density of the nonporous microparticles is about 1.45 g / cm 3 or larger, with a diameter of about 10 μm to 200 μm, and the capture ligand is capable of specifically binding to the molecule;

[0052] b. incubating the sample substantially without agitation to form a target cell / particle complex;

[0053] c. separating the target cell / microparticle complex from the unbound material in the sample by washing the unbound material through a filter while retaining the target cell / microparticle complex;

[0054] d. Mechanically dissociating the target cell / microparticle complexes and eluting the viable target cells through the filter while retaining the microparticles with the capture ligands covalently immobilized to the microparticle surface.

[0055] Target cells

[0056] The present invention provides methods for separating viable cells of interest (referred to herein as "viable target cells" or "target cells") from other cells and substances present in a sample. The term "viable" as used herein, when it refers to target cells, means that the cells are able to survive after being cultured in a medium that supports the growth of target cells, or after being transplanted or implanted, before and after the implementation of the method according to the present invention. Suitable culture media for growing target cells and assessing cell viability are known to the skilled person. Cell viability assays use a variety of markers as indicators of metabolically active (living) cells. Some 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 by measuring, for example, DNA content or DNA synthesis in replicating cells. Cell proliferation assays are performed using standard methods, including enzyme-linked immunosorbent assays (ELISA), flow cytometry, immunofluorescence, and high-content imaging.

[0057] The target cell can be any cell of interest that can be used for cell therapy, as long as the target cell displays molecules on its cell surface that can bind to the capture ligand covalently linked to the non-porous microparticle used in the method according to the present invention and can pass through the pores of the filter that selectively retains the non-porous microparticle.

[0058] The target cell can be from any mammal, such as primates (including monkeys and humans), bovines (including cattle), ovines (including sheep), equines (including horses), canines (including wild dogs and domestic dogs), porcines (including wild boars and domestic pigs), murines (including mice and rats), or cells derived from such animals, which may also be genetically engineered.

[0059] 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 from any tissue or organ, including but not limited to heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymph glands, adenoids, tonsils, bone marrow, and blood), and spleen; muscle cells; pancreatic cells; cardiac cells, and cell lines established from such primary cells.

[0060] The target cell can be a healthy, naturally occurring cell or a cell derived from such a cell (i.e., a cancer cell, a cell derived from a diseased tissue or organ, a cell infected by any pathogen (including a virus), a transfected cell, a transformed cell, or an established cell line), and is preferably a human cell or a cell derived from a human cell.

[0061] Generally speaking, human cells are about 5 to 100 μm in diameter.

[0062] In a preferred embodiment, the target cell is a bone marrow or hematopoietic cell or cell line. Some examples of hematopoietic cells include granulocytes, T lymphocytes, monocytes, T regulatory cells, T helper cells, cytotoxic T cells, B lymphocytes, thrombocytes, natural killer cells, hematopoietic stem cells and progenitor cells, which are preferably human.

[0063] Among human hematopoietic cells, the average diameter of neutrophils is 8.3 μm; the average diameter of lymphocytes is 6.05 μm, and the average diameter of monocytes is 8.13 μm (see Downey et al., J Appl. Physiol. 69(5):1767-78, 1990). Other larger cell types include megakaryocytes and myeloid cells, which are responsible for the production of platelets. These cells can be up to about 100 μm in diameter. Red blood cells, also known as erythrocytes, are some of the smallest and most abundant cells in the human body. These cells have a characteristic biconcave disc shape with a depression where the nucleus was lost during maturation, and a corresponding diameter of about 7 to 8 μm and a volume of ≈100 μm. 3 .

[0064] CAR-T cells (chimeric antigen receptor cells) are T cells that are genetically modified to produce artificial chimeric antigen receptors on their surfaces. Immunotherapy with CAR-T cells is one of the latest methods for treating hematological malignancies, including B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's lymphoma and multiple myeloma. This is particularly important for patients who are ineffective with other treatment strategies. In principle, treatment is performed by obtaining the white blood cell fraction in the patient's blood via leukocyte removal. T cells and other cells in the blood are separated according to the size and density of T cells, and the remaining blood is returned to the patient's bloodstream. The obtained T cells are then rinsed to remove the anticoagulant. The T cells are then further separated into specific subtypes. The separation of T cell subtypes is usually performed using specific antibodies bound to magnetic particles. This creates a problem because these particles may lose their magnetic ability and remain bound to the cells, and even if the magnetic particles are successfully removed, the residual antibodies will still be attached to the cell surface antigens. Then they can be transplanted into the patient together with the separated cells. This is where the method and apparatus according to the present invention can provide a solution, because the final cell eluate is substantially free of any antibodies or microparticles. After the cells are separated into specific subtypes, T cells are then activated in vitro by using artificial antigen presenting cells, beads coated with antibodies or special reagents. After activation, the sequence encoding CAR (chimeric antigen receptor) is incorporated into the T cell genome. The integration of the sequence can be completed with different input methods, such as electroporation of the DNA molecule itself, by transposon / transposase system, viral vectors (such as retrovirus and lentiviral vectors). Successfully transformed T cells express chimeric antigen receptors on their surface. The CAR structure can be adjusted so that the effect of CAR-T cells is directed to specific target cells. After T cell amplification, the modified T lymphocytes are returned to the patient by blood transfusion, wherein those equipped with specific cell recognition receptors perform their functions. One of the reasons why this treatment has not been widely used is the high production cost. The device according to the present invention can be designed to greatly reduce the cost of the cell separation process while also improving safety, because the resulting viable target cell population does not contain magnetic particles, and because the cell suspension does not contain residual antibodies or animal-derived products, no further purification is required. Therefore, the device according to the present invention can be used to separate CAR-T cells for CAR-T cell therapy, which can treat a wide range of cancers, autoimmune diseases, chronic inflammatory diseases, transplant rejection after transplantation, and other disease states. Of course, the separated T cells can be used without subsequent genetic modification.

[0065] Tumor infiltrating lymphocytes (TIL) consist of all lymphocyte groups that have invaded tumor tissue. They can be present in many solid tumors and are becoming an important biomarker for predicting treatment efficacy and outcome. In breast cancer, TILs mainly contain cytotoxic (CD8+) and helper (CD4+) T cells and a smaller proportion of B cells and natural killer cells. Those tumor infiltrating lymphocytes can be separated from the sample using a device according to the present invention, and then amplified and returned to the patient's body as an additional treatment for treating a variety of cancers. The markers that can be used to select TILs are: CD3 for T cells, CD8 for cytotoxic T lymphocytes, CD4 for helper T cells, and FOXP3 for regulatory T cells.

[0066] In another preferred embodiment, the target cell is a stem cell, preferably a human stem cell. Some examples of stem cells include hair follicle stem cells, cardiac stem cells, neural stem cells, pluripotent muscle cells, liver stem cells, hematopoietic stem cells, mesenchymal / stromal stem cells, dental pulp cells, periodontal ligament cells, adipose-derived stem cells and progenitor cells, pluripotent stem cells (including embryonic stem cells and induced pluripotent cells), which are preferably human, and any other cell type derived from such pluripotent cells.

[0067] Bone marrow is composed of hematopoietic stem cells (HSC), mesenchymal stem cells (MSC) and supporting stromal cells. All types of hematopoietic cells, including both myeloid and lymphoid systems, are produced in the bone marrow. HSC produces leukocytes (white blood cells), erythrocytes (erythrocytes) and platelets (thrombocytes). Mesenchymal stem cells are multipotent stem cells that can be differentiated into a variety of cell types, such as osteoblasts, chondrocytes, muscle cells and bone marrow adipocytes. Whole bone marrow transplantation can be used to treat certain types of cancer, such as leukemia, myeloma, lymphoma and other blood and immune system diseases that affect bone marrow. Specific bone marrow cells can be separated using the device of the present invention. Mesenchymal stem cells can also be separated from adipose tissue and / or can be obtained as waste materials during other operations, and the other operations are not only intended to be used for cell separation, such as liposuction or abdominoplasty.

[0068] Cell markers that can be used to specifically isolate hematopoietic stem cells:

[0069] 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.

[0070] Cell markers that can be used to specifically isolate mesenchymal stem cells:

[0071] CD73, CD90 and CD105 are present, and CD34, CD45 and HLA-DR antigens are absent.

[0072] Microvascular endothelial cells (MVECs) are located in the smallest blood vessels of the circulatory system, such as capillaries. They play an important role in vascular homeostasis and endothelial dysfunction that can lead to a variety of diseases, including atherosclerosis.

[0073] Cell markers that can be used to specifically isolate MVECs:

[0074] CD31 (endothelial cells), CD34 (progenitor cells, vascular endothelial cells), ICAM-1 / CD54

[0075] (endothelial cells), LYVE-1 (lymphatic endothelial cells, Tie-2 / Tek (endothelial cells), VCAM-1 / CD106 (quiescent endothelial cells - inducible after injury), VE-cadherin (connector between endothelial cells and on HSCs and MSCs), VEGF-R2 (endothelial cells, endothelial cell progenitor cells), von Willebrand factor

[0076] -vWF (endothelial megakaryocytes).

[0077] Some examples of other target cells include retinal pigment epithelial cells, which express, for example, CD140b, CD56, GD2 and CD184 on their cell surfaces as the most important antigens, etc. Retinal pigment epithelium (RPE) is a specialized epithelium, located in the interface between neural retina and choroidal capillaries, where it forms an external blood-retina barrier. Retinal pigment epithelium is the basic component of retina, plays an important role in visual function. The damage of the structure and function of retinal pigment epithelium causes multiple retinal diseases, such as age-related macular degeneration (AMD), which is one of the main causes of global blindness.

[0078] Retinal pigment epithelial cells can be isolated based on their cell surface markers CD140b, CD56, GD2, CD184, Mitf, ZO-1, RPE65, CRALBP, CD104, CD164, CD220, EGER, CD10, CD30, CD49a, CD49b, CD50, CD171, TRA-1-60 and CD326 using the methods and devices according to the present invention.

[0079] In another embodiment, the target cell is a tumor cell. The separation and detection of tumor cells (including circulating tumor cells (circulating tumour cell, CTC)) have an important role in early cancer diagnosis and prognosis, that is, before clinically detectable metastasis occurs, cells that are easily accessible are provided, and the molecules and genetic profiles of these metastatic cells are studied. The CTC method is currently mainly used for research purposes, and due to the difficulties caused by CTC heterogeneity, separation of CTC from blood and lack of comprehensive clinical validation, only a few methods are accepted for clinical application. The cell separation method according to the present invention can be used to obtain highly purified and highly viable cancer cells, for characterizing and studying such cells, and by capturing ligands and non-porous microparticles covalently linked for research purposes, the non-porous microparticles are specifically combined with the cancer cell expressing cell surface markers, and the cell surface markers are not usually expressed on non-cancerous cells of the same type.

[0080] In addition, the method according to the present invention can be used to isolate any mammalian or human cell line, including 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, HEL 299 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, strain 2071 (mouse L) cells, LM strain (mouse L) cells, L-MTK (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells (Indian muntjac cell), SIRC cells, C11 cells, and Jensen cells, or derivatives thereof), mouse myeloma NSO or SP2 / 0 or rat myeloma YB2 / 0 cells and their derivatives, and other established cell lines and strains 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, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28VA13, 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-RC 256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, and TH-1, B1 cells, or derivatives thereof, e.g., altered by genetic engineering or by infection,and contain and / or express genes that are not normally expressed in these cells.

[0081] Cell surface molecules

[0082] As used herein, a "molecule on the cell surface" or "cell surface molecule" can be any molecule expressed or displayed on the cell surface of a target cell such that the molecule can be recognized and bound by a capture ligand covalently attached to a nonporous microparticle. The molecule on the cell surface can be proteinaceous or non-proteinaceous.

[0083] In a preferred embodiment of the present invention, the molecule on the cell surface is an antigen that can be recognized by the antibody or its derivative covalently attached to the non-porous microparticle.

[0084] Some examples of human cell lines and antigens present on their cell surfaces that can be recognized by capture ligands covalently bound to non-porous microparticles according to the present invention are:

[0085] Hematopoietic stem cells: Sca-1, CD27, CD34, CD38, CD43, CD48, CD117, and CD150

[0086] Mesenchymal / stromal stem cells: STRO-1, CD105, CD73, CD90

[0087] Granulocytes: CD45, CD11b, CD15, CD24, CD114, CD182

[0088] T lymphocytes: CD45, CD3

[0089] Monocytes: CD4, CD45, CD14, CD114, CD11a, CD11b, CD91, CD16

[0090] T regulatory cells: CD4, CD25

[0091] T helper cells: CD45, CD3, CD4

[0092] Cytotoxic T cells: CD45, CD3, CD8

[0093] B lymphocytes: CD45, CD19, CD20, CD24, CD38, CD22

[0094] Thrombocytes: CD45, CD61

[0095] Natural killer cells: CD16, CD56, CD31, CD30, CD38

[0096] Retinal pigment epithelial cells: CD140b, CD56, GD2, and CD184

[0097] Hair follicle stem cells: CD90

[0098] Cardiac stem cells: CD34, CD117, GPR4

[0099] Multipotent myocytes: CD73, CD105

[0100] Hepatic stem cells: CDCP1, CD90.

[0101] Other examples of protein molecules on the cell surface of various mammalian and human cells that can be recognized by the capture ligands covalently bound to the non-porous microparticles according to the present 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), CD267, 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, TR A-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, CD 135 (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 (Connexin-2), CD166 (ALCAM), CD172a / b (SIRPα / β), CD200 (OX2), CD231 (TALLA), CD304 (Neuropilin-1), 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, CD102 (ICAM-2), CD106 (VCAM-1), CD140a (PDGFRα), lymphotoxin beta receptor (LTβR), Madcam-1, neurogangliosides, TLR1, TLR2, TLR4, CD84, CD1 26 (IL-6Rα), CD154 (CD40L), CD185 (CXCR5), CD252 (OX40L), CD278 (ICOS), TCRα / β, CD26, CD94, CD119, CD183, CD191 (CCR1), CD254 (TRANCE, RANKL), CD366 (Tim-3), IL-18R, TNF-α, TNF-β, CCR8, C D193(CCR3), CD194(CCR4), CD294(CRTH2), CD365(Tim-1), IL-1R, TGF-β, CCR10, CD196(CCR6), CD 39, CD103, CD134, CD152 (CTLA-4), CD223, FR4, GARP, GITR, STRO-1 and STRO-3, NCAM, CD133, SSEA-1.

[0102] Some examples of non-protein molecules on the cell surface of mammalian cells that can be recognized by the capture ligands covalently bound to the non-porous particles according to the present invention are polysaccharides (e.g., Galp1-3GlcNAc1-R, Galp1-4GlcNAcp1-R, Galp1-3GalNAca1-R, Galp1-3GalNAcp1-R), lipids and phospholipids (e.g., sphingomyelin, phosphatidylserine, phosphatidylcholine) and non-protein aptamers (e.g., tenascin-C (TN-C) and synthetic DNA / RNA / XNA molecules).

[0103] sample

[0104] As used herein, the term "sample" may be any sample that contains a suspension of viable target cells. The sample may be prepared from a tissue or organ taken from an individual or a specimen that has been treated to release target cells.

[0105] The sample may be a solution containing viable target cells from a mammal (preferably a human), a tissue extract or an organ extract, and a cell line that has been established from primary cells from such tissues or organs: the tissues or organs include but are not limited to heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoids, tonsil, bone marrow and blood, the blood including peripheral blood cells, placenta / umbilical cord blood, menstrual blood), spleen and fibroblasts; or a solution obtained by roughly separating cells from such a solution, and such a solution diluted with water, any buffer and / or culture medium described herein, such as the physiological buffer described herein, physiological saline, Ringer's solution containing divalent cations (such as calcium ions or magnesium ions), cell culture medium (such as RPMI, MEM, IMEM or DMEM), and phosphate buffer (such as PBS, HBSS, TBS, DPBS, EBSS, etc.).

[0106] Preferred samples containing target cells to be separated according to the method of the present invention are mammals (preferably humans), whole blood, single blood component samples, bone marrow aspirates, biopsy samples, liquefied tissue samples (such as enzymatically degraded liposuction samples), cell culture samples, bioreactor cultures, single cell suspensions, etc.

[0107] In one embodiment, the sample is preferably derived from cells that have been cultured in suspension, or the sample is a suspension of cells that have been cultured on a matrix (e.g., a glass or plastic flask or plate) and have been dissociated from the matrix by enzymatic methods (e.g., using accutase or trypsin) or other methods (e.g., washing with EDTA, washing the cells with a balanced salt solution or culture medium without calcium and magnesium, or mechanical agitation).

[0108] Preferably, prior to contacting the sample containing viable target cells 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 single cells.

[0109] If the sample contains or is suspected of containing debris that would otherwise hamper performance or clog the filter when performing the method according to the invention, such as fragments of tissue, bone, organs, cell aggregates, etc., then in a preferred embodiment the sample may be pre-treated to partially or completely remove the debris. This may be achieved by filtering the sample and / or by centrifuging and resuspending the cells in the sample without substantially removing individual viable target cells from the sample.

[0110] Non-porous particles

[0111] The method of the present invention uses a suspension of nonporous microparticles. As used herein, the term "nonporous microparticles" refers to any spherical or non-spherical nonporous particles having a (dry weight) density of about 1.45 g / cm 3 or larger, with a diameter of about 10 μm to 200 μm, and a capture ligand covalently fixed to the surface of the microparticle, which can specifically recognize molecules on the surface of target cells to form a target cell / microparticle complex.

[0112] The term "non-porous" when it refers to "microparticles" means that the "non-porous microparticles" have a smooth surface with essentially no depressions or indentations on the surface that can capture target cells and prevent them from mechanically dissociating from the non-porous microparticles when performing the methods according to the present invention. "Non-porous microparticles" as used herein also have one or more capture ligands covalently attached to the 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 for binding to molecules on the surface of target cells when using the methods according to the present invention.

[0113] In a preferred embodiment, the nonporous microparticles have a (dry weight) density of about 1.45 g / cm 3 or greater, more preferably about 1.65 g / cm 3 or greater, more preferably about 1.85 g / cm 3 or greater, and most preferably about 1.95 g / cm 3 or larger.

[0114] In another preferred embodiment, the diameter of the non-porous microparticles is 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.

[0115] In some embodiments, the (dry weight) density of the non-porous microparticles is about 1.45 g / cm 3 or larger, and have 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.

[0116] In some embodiments, the (dry weight) density of the non-porous microparticles is about 1.65 g / cm 3 or larger, and have 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.

[0117] In some embodiments, the (dry weight) density of the non-porous microparticles is about 1.85 g / cm 3 or larger, and have 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.

[0118] In some embodiments, the (dry weight) density of the non-porous microparticles is about 1.95 g / cm 3 or larger, and have 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.

[0119] The nonporous microparticles used in the method according to the present invention are made of at least one inorganic material, preferably, but not limited to, nonporous microparticles made of: SiO2 (silicon dioxide), silicone, gold, silver or platinum, or a composite material made of an inorganic shell SiO2, silicone, gold or silver, and a particle core made of 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.

[0120] In a preferred embodiment, the non-porous particles are made of SiO2 glass. Preferably, the (dry weight) density of the SiO2 glass non-porous particles is about 2 g / cm 3 and about 40 μm in diameter. An example of silica nonporous microparticles having a diameter of 40 μm are those available from Glantreo Ltd. (Cork, Ireland). Some examples of nonporous microparticles having a diameter of 40 μm (PNPP40.0NAR, Glantreo Ltd., Cork, Ireland) have been used in some examples according to the present invention and are Figure 4a and Figure 4b Shown in.

[0121] Other examples of commercially available non-porous microparticles are:

[0122] Glantreo, Ltd. (Cork, Ireland): nonporous silica particles: 10 μm (PNPP10.0 NAR), 20 μm (PNPP20.0 NAR), 40 μm (PNPP40.0 NAR), 50 μm (PNPP50.0 NAR), 70 μm (PNPP70.0 NAR);

[0123] CD Bioparticles (New York, NY, USA): nonporous silica particles: 20 μm (DNG-E009), 30 μm (DNG-E010), 40 μm (DNG-E011);

[0124] EPRUI (Wujiang Disctrict, Suzhou, China): nonporous silica particles: 20 μm (EPRUI-Si-20), 30 μm (EPRUI-Si-30), 40 μm (EPRUI-Si-40), 50 μm (EPRUI-Si-50);

[0125] Abvigen (Newark, NJ, USA): nonporous silica particles: 20 μm (ABM-3-362), 30 μm (ABM-3000), 40 μm (ABM-4000), 50 μm (ABM-5000), 60 μm (ABM-6000), 70 μm (ABM-7000), 80 μm (ABM-8000), 90 μm (ABM-9000), 100 μm (ABM-10000), 200 μm (ABM-20000);

[0126] VWR Chemicals (Radnor, PA, USA: non-porous silica particles: 40-63 μm (27623.323), 40-63 μm (7631-86-9), 60-200 μm (84893.290), 63-200 μm (27647.325), and

[0127] MilliporeSigma Supelco, Merck (Darmstadt, Germany): Non-porous silica particles: 40-60 μm (1.09385), 40-75 μm (80442), 40-75 μm (53698), 75-200 μm (78991).

[0128] In a preferred embodiment, the nonporous particles are SiO2 glass nonporous particles having a (dry weight) density of about 1.45 g / cm 3 or greater, more preferably about 1.65 g / cm3 or greater, more preferably about 1.85 g / cm 3 or greater, and most preferably about 1.95 g / cm 3 or larger.

[0129] In another preferred embodiment, the nonporous particles are SiO2 glass nonporous particles 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.

[0130] In some embodiments, the nonporous particles are SiO2 glass nonporous particles having a (dry weight) density of about 1.45 g / cm 3 or larger, and have 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.

[0131] In some embodiments, the nonporous particles are SiO2 glass nonporous particles having a (dry weight) density of about 1.65 g / cm 3 or larger, and have 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.

[0132] In some embodiments, the nonporous particles are SiO2 glass nonporous particles having a (dry weight) density of about 1.85 g / cm 3 or larger, and have 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.

[0133] In some embodiments, the nonporous particles are SiO2 glass nonporous particles having a (dry weight) density of about 1.95 g / cm 3 or larger, and have 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.

[0134] In some embodiments, the (dry weight) density of the SiO2 glass non-porous particles is about 2 g / cm 3 And the diameter is about 40 μm.

[0135] In some embodiments of the present invention, non-porous microparticles may have conductive properties. It has been unexpectedly determined that, by adjusting the density, diameter and surface properties of microparticles, a complex can be formed between target cells and non-porous microparticles via the interaction of captured ligands covalently linked to the microparticle surface and molecules on the target cell surface, the complex is strong enough on the one hand to withstand the mechanical force generated when using a filter that can permeate target cells but does not permeate non-porous microparticles to wash the target cell / non-porous microparticle complex, and on the other hand the complex is weak enough to be destroyed only by mechanical force, so that the target cell passes through the filter in a viable state. The elution of cells is realized using only mechanical force. Therefore, although magnetic non-porous microparticles can be used to practice the method of the present invention, the present invention does not rely on the magnetic properties of such microparticles to separate cells. Therefore, in a preferred embodiment of all aspects of the inventive method, non-porous microparticles are non-magnetic.

[0136] The non-porous microparticles may also be in the form of dry powders, such as dry powders containing non-toxic preservatives such as trehalose or sucrose, and may also be in the form of suspensions. The solution for the non-porous microparticle suspension may be any solution that maintains the viability of the target cells after being added to the sample. For example, the solution may be a buffer solution as described herein, a cell culture medium as described herein, or a culture medium for cell transfection, transplantation or implantation, storage or cryopreservation, and / or a solution for measuring as described herein.

[0137] Functional groups on the surface of nonporous microparticles and linkers that can be used to form covalent bonds between the surface and the capture ligand

[0138] The nonporous microparticles used in the method according to the present invention have one or more covalently fixed to the microparticle surface and can identify the capture ligands that display one or more molecules on the cell surface of the target cell. As a method for capturing the covalent bonding of the ligand to the nonporous microparticle, a coupling agent can be used, such as a common bifunctional agent, or a method connected by a spacer can also be used. According to the reactive group on the carrier surface, a suitable coupling agent is used. When the surface has an-OH group (such as made of SiO2), a silane surface modifier is used to introduce the functional group into the carrier surface. The functional group can be, for example, amino, acylamino, imino, hydrazide, carboxyl, thiol, hydroxyl, azido, alkyl, phenyl, epoxy, ester, halide and acyl halide. Preferably, amino, carboxyl, thiol and epoxy are used. Most preferably, a carboxyl is used. When the carrier material is a metal such as gold, copper, silver and platinum, the functionalization of the material can be carried out using the disulfide bridge formed on the material surface. Typically linkers with a thiol group (-SH) at one end will be used for this type of functionalization, such as a variety of alkanethiols having a thiol group at one end of the molecule and an amino, acylamino, imino, hydrazide, carboxyl, thiol, hydroxyl, azido, alkyl, phenyl, epoxy, ester, halide, and acyl halide group at the other end.

[0139] conjugate, join, bind, connect, fix, couple a first unit to a second unit

[0140] 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 two functional groups on the surface support and the ligand molecule. The linker can have or not have a PEG (polyethylene glycol) spacer. Linkers that can be used to form a covalent bond between the non-porous microparticles 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 (dimethyl adipimidate), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), 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, which can then react with the functional groups present in the capture ligand structure. The size of the PEG spacer that can be used in the present invention can vary from 0.4kDa to greater than 20kDa. The non-porous microparticles and the capture ligands are directly bound to each other or bound to each other through a linker. Only a covalent bonding method between the surface and the capture ligand is used without any affinity ligand to prevent the capture ligand from leaking from the non-porous microparticles. Preferably, a linker that does not contain PEG (polyethylene glycol) is used in the present invention. Any other linker that can ensure that a covalent bond is formed between a functional group on the surface of the non-porous microparticle and a group that may be present in the capture ligand structure, as well as other functional groups that may be present on the surface of the non-porous microparticles that can react with the group of the capture ligand with or without a linker molecule, are not limiting factors for practicing the present invention.

[0141] Capture ligand

[0142] The non-porous microparticles used in the method according to the present invention have at least one capture ligand, which is covalently linked to the surface of the non-porous microparticles and is capable of specifically binding to molecules displayed on the cell surface of target cells in the sample to form a complex between the target cells and the non-porous microparticles, i.e., a "target cell / microparticle complex".

[0143] As used herein, the term "specifically binds" or "specifically binds to" when referring to any capture ligand described herein means that the capture ligand on the nonporous microparticle binds to a molecule on the surface of a target cell such that a target cell / microparticle complex is formed when the method according to the invention is performed without any substantial binding of the capture ligand to non-target cells in the sample. The capture ligand will have an affinity for the molecule at the surface of the target cell such that the target cell / microparticle complex is maintained when unbound material is washed from the sample. If the capture ligand is multivalent, i.e., a particular capture ligand has more than one binding site for a molecule at the surface of the target cell, the capture ligand will have an affinity for the molecule at the surface of the target cell such that the target cell / microparticle complex is maintained when unbound material is washed from the sample.

[0144] In one embodiment, the term "capture ligand" refers to a protein receptor that can recognize a molecule on the surface of a target cell of interest. In a preferred embodiment, the protein receptor capture ligand binds to the molecule on the surface of the target cell at a rate of about 10 -5 or larger, about 10 -6 or larger, about 10 -7 or larger, about 10 -8 or larger, about 10 -9 or larger, about 10 -10 or larger, about 10 -11 or greater, or about 10 -12 In a preferred embodiment, the capture ligand specifically binds with a dissociation constant of 10 -5 Up to 10 -12 , more preferably 10 -9 Up to 10 -11 , or more preferably 10 -7 Up to 10 -10 The dissociation constant of the capture ligand is nanomolar (about 10 -9 ). Binding affinity can be measured using a variety of known analytical methods, such as radioligand binding assays, surface plasmon resonance, fluorescence resonance energy transfer, and affinity chromatography.

[0145] 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.Antibodies are molecules having one or more polypeptides that are substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as a large number of immunoglobulin variable region genes.Light chains are classified as κ or λ.Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively.It is known that the basic immunoglobulin structural unit comprises a tetramer.Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kD) and a "heavy" chain (about 50 to 70 kD).The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light chains and heavy chains, respectively. The capture ligand can exist in various forms as a complete immunoglobulin (i.e., antibody) or as a derivative thereof, including, for example, FabFc2, Fab, Fv, Fd, F(ab')2, Fv fragments containing only light and heavy chain variable regions, Fab or F(ab')2 fragments containing variable regions and part of constant regions, single-chain antibodies (e.g., scFv), CDR-grafted antibodies, dAbs, nanobodies, etc. The heavy and light chains of Fv can be derived from the same antibody or different antibodies, thereby producing a chimeric Fv region. Antibodies can also be modified antibodies (e.g., oligomers, reduced, oxidized, and labeled antibodies). Antibodies can be of animal (especially mouse, rabbit, or rat) or human origin, or can be chimeric or humanized. As used herein, the term "antibody" includes these various forms.

[0146] In a preferred embodiment, the capture ligand is an antibody, such as FabFc2, Fab, Fv, Fd, F(ab')2, an Fv fragment containing only the light chain and heavy chain variable regions, a Fab or F(ab')2 fragment containing the variable region and part of the constant region, a single chain antibody (such as scFv), a CDR-grafted antibody, a dAb, a nanobody, etc., which is about 10 -5 or larger, about 10 -6 or larger, about 10 -7 or larger, about 10 -8 or larger, about 10 -9 or larger, about 10 -10 or larger, about 10 -11 or greater, or about 10 -12 or greater dissociation constant recognizes antigens on the surface of target cells. In a preferred embodiment, the capture ligand is a 10 -5Up to 10 -12 , more preferably 10 -9 Up to 10 -11 , or more preferably 10 -7 Up to 10 -10 In a preferred embodiment, the affinity constant of the antibody is in the nanomolar order (about 10 -9 ).

[0147] Some examples of antibodies that can recognize ligands on the surface of target cells are: 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-CD25, anti-CD8, anti-CD19, anti-CD20, anti-CD24, anti-CD22, anti-CD61, anti-CD16, anti-CD56, anti-CD31, anti-CD30, and anti-CD40. D38, 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-CD45R / B220, anti-CD69, anti-CD70, anti-CD79a (Igα), anti-CD79b (Igβ), anti-CD80, anti-CD86, anti-CD93 (C1Rqp), anti-CD137 (4-1BB), anti-CD138 (syndecan-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-cadherin), anti-CD146 (MUC18, Mel-CAM), anti-CD201 (EPCR), anti-CD202b (Tie2 / Tek ), anti-CD309 (VEGFR2-Flk-1), anti-podoplanin, 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 (CCR5), anti-CD282, anti-CD284, anti-F4 / 80, anti-GITRL, anti-HLA-DR, anti-Mac-2 (Galectin-3), anti-MHCClass II, anti-CD203c, anti-FcεRIα, anti-CD44, anti-CD349 (Frizzled-9), anti-TNAP, anti-CD45RA, anti-CD45RB, anti-CD62L, anti-CD6, anti-CD47, anti-CD90.1, anti-CD90.2, anti-CD100, anti-CD112 (Connexin-2), anti-CD166 (ALCAM), anti-CD172a / b (SIRPα / β), anti-CD200 (OX2), anti-CD231 (TALLA), anti-CD304 (Neuropilin-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 (Siglec-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-CD303 (BDCA-2), anti-DC marker (33D1), F4 / 80, anti-HLA-DR, anti-MHC class II, anti-SiglecH, anti-CD29, anti-CD49d, anti-CD50 (ICAM-3), anti-CD51, anti-CD102 (ICAM-2), anti-CD106 (VCAM-1), anti-CD140a (PDGFRα), anti-lymphotoxin β receptor (LTβR), anti-Madcam-1, anti-neuroganglioside, anti-TLR1, anti-TLR4, anti-CD84, anti-CD126 (IL-6Rα), anti-CD154 (CD40L), anti-CD185 (CXCR5), anti-CD252 (OX40L), anti-CD278 (ICOS), anti-TCRα / β, anti-CD26, anti-CD94, anti-CD119, anti-CD183, anti-CD191 (CCR1), anti-CD254 (TRANCE, R 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-TCRα / 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 with specific binding regions that recognize the specific proteins listed above are also considered capture ligands (eg, receptors for Sca-1, CD27, CD34, CD38, etc.).

[0148] The non-porous microparticles may comprise one or more different capture ligands covalently bound to their surfaces, which can specifically bind to specific molecules on the surface of target cells. The non-porous microparticles may also comprise one or more different capture ligands covalently bound to their surfaces, which can specifically bind to different molecules on the surface of the same target cell.

[0149] The method according to the present invention can also be used to simultaneously separate different target cell populations from the same sample. If two or more different types of target cells in a sample are to be separated from other unbound substances in the sample at the same time, the same non-porous microparticle used in the method may contain different capture ligands covalently bound to its surface, and the capture ligands can specifically bind to different molecules on the surface of the corresponding target cells. Alternatively, the method can be performed using a mixture of two or more different non-porous microparticle populations, each of which is capable of recognizing and binding to different target cells.

[0150] Unbound substances

[0151] Unbound material may include cells and materials that are not specifically recognized by the capture ligand 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 solution and / or culture medium, such as ions, cations, sugars, lipids, residual chemicals, plasma, cell debris, cell waste products 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 the sample depends on the type of sample. Non-target cells are cells present in the sample that do not express a specific molecule of interest that is present on the target viable target cells on their cell surface, or that express a specific molecule of interest at a level that does not substantially result in the formation of stable cell / microparticle complexes when the target cell / microparticle complexes according to the present invention are washed. If the non-target cells in the sample are smaller than the pore or mesh size of the filter used to separate the target cells, they will be washed through the filter, while the target cell / microparticle complexes are retained by the filter and will be separated from the viable target cells and other unbound materials in the sample. 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 pore or mesh size of the filter, the non-target cells will be retained by the filter along with the target cell / particle complexes, but when the target cell / particle complexes are mechanically dissociated, the non-target cells will not pass through the filter and will be retained on the same side of the filter as the non-porous particles.

[0152] Filters

[0153] The filter used in the method according to the present invention to remove unbound material (and optionally non-target cells) from target cells can be made of any porous matrix material that is biocompatible, does not substantially affect cell viability, and can include, consist essentially of, or consist of any of the following materials: spider silk, metals and alloys such as gold, silver, platinum, copper, carbon, graphene, enamel, ceramic, glass, cellulose, cellulose acetate, collagen, chitosan, lignin, hydrogels, alginate, cotton, nanofibers Cellulose, polyamide (such as nylon), polyimide, polyester, polyethylene, polypropylene, polyvinyl chloride, polyvinyl chloride-acrylic acid copolymer, polyvinyl alcohol, vinylidene chloride, polytetrafluoroethylene, polychlorotrifluoroethylene, tetrafluoroethylene-hexafluoropropylene copolymer, polydimethylsiloxane, polyethylene terephthalate, polyether urethane, polyether urethane urea, polystyrene, polycarbonate, polyether ether ketone, polysulfone, acrylic polymer (such as polymethyl methacrylate, polyacrylonitrile, etc.), polycaprolactam, polyhexamethylene adipamide, polymethylpentene and poly-2-hydroxyethyl methacrylate.

[0154] Preferred filter materials are resistant to sterilization (sterilizable) and include polyamides (eg, nylon), polypropylene, polyvinyl chloride, polyethylene, polyimides, polycarbonates, polysulfones, and polymethylpentene.

[0155] In a preferred embodiment, the filter is a flexible membrane (or thin film sheet) composed of a material such as a polyamine (e.g., nylon) having a thickness 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 may also be used. The filter may be a single layer membrane, or may include two or more layers that are sealed together or separated to form a double layer membrane.

[0156] The skilled person will understand that there is an interactive relationship between the pore or mesh size of the filter, the diameter of the non-porous microparticles and the diameter of the target cells. When practicing the method according to the present invention, the pore or mesh size of the filter and the diameter of the non-porous microparticles are selected so that the non-porous microparticles do not pass through the pores or mesh of the filter, while the unbound substances (and optionally non-target cells) and the target cells pass through the pores or mesh of the filter.

[0157] The size of the pores or meshes 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 a particularly preferred embodiment, the size of the pores or meshes is about 31 μm. When the target cell is less than 30 μm, this is suitable for the separation of cells. If the target cell (target cell) used for separation is greater than 30 μm, the size of the pores or meshes should be adjusted for a specific cell type. In addition, if the size of the target cell exceeds the preferred diameter of the non-porous microparticles, the size of the non-porous microparticles will be selected to have a larger diameter than the target cell. In this case, the size of the pores or meshes in the filter will be selected according to these parameters.

[0158] In a preferred embodiment, the filter is a nylon membrane having a pore or mesh size of about 30 μm.

[0159] Some examples of commercially available filtration membranes useful in practicing the present invention are:

[0160] MERCK MILLIPORE (Darmstadt, Germany): Omnipore membrane: 10 μm (JCWP); polypropylene prefilter: 10 μm (AN1H), 30 μm (AN3H); polypropylene mesh filter: 25 μm (PP25), 45 μm (PP45), 80 μm (PP80); nylon mesh filter: 10 μm (NY10), 11 μm (NY11), 20 μm (NY20), 30 μm (NY30), 40 μm (NY41), 60 μm (NY60), 80 μm (NY80), 100 μm (NY1H);

[0161] Spectrum Chemical Mfg. Corp. (New Brunswick, NJ, USA): Polyester mesh filters: 10 μm (888-13699-PK), 15 μm (888-13702-PK), 21 μm (888-13705-PK), 43 μm (888-13714-PK); Nylon mesh filters: 10 μm (888-13687-PK), 20 μm (888-13684-PK), 30 μm (888-13681-PK), 41 μm (888-13675-PK), 53μm (888-13669-PK), 60μm (888-13666-PK), 70μm (888-13660-PK), 100μm (888-13654-PK); Stainless steel mesh filter: 30μm (888-14221-PK), 51μm (888-14224-PK); PEEK mesh filter (polyester): 35μm (158-27388-PK);

[0162] 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); MembraneSolutions (Auburn, WA, USA): Nylon mesh filters: 20μm (MENY025020), 30μm (MENY025030), 41μm (MENY025041), 60μm (MENY025060), 80μm (MENY025080), 100μm (MENY025100); pluriSelectLife Science (Leipzig, Germany): Re-filters: 10 μm (43-75010-40), 20 μm (43-75020-40), 40 μm (43-75040-40), 70 μm (43-75070-40), 100 μm (43-75100-40); syringe filters: 10 μm (43-71010-50), 15 μm (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); 10μm (43-70010-03), 15μm (43-70015-03), 20μm (43-70020-03), 30μm (43-70030-03), 40μm (43-7 0040-03), 60μm (43-70060-03), 70μm (43-70070-03), 85μm (43-70085-03), 100μm (43-70100-03); 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);

[0163] Genesee Scientific (San Diego, CA, USA): Olympus Advanced Cell Filter: 40 μm (25-375), 70 μm (25-376), 100 μm (25-377), and Bio-Rad, Inc. (Hercules, CA, USA): ProFlow Cell Filter: 30 μm (12012576), 50 μm (12012574), 70 μm (12012573).

[0164] Carrying out the method according to the invention

[0165] In carrying out the method according to the invention, a variety of solutions can be used, depending on the source of the sample, the optional dilution of the sample, the solution used to suspend the non-porous microparticles, the desired properties of the washing solution for obtaining viable target cells and the desired properties of the elution solution and other factors. Such solutions include, but are not limited to, buffered solutions or cell culture media, solutions or culture media for cell transfection, transplantation, implantation and / or storage or cryopreservation as described herein. Some examples of such buffered solutions include PBS (Phosphate-buffered saline), DPBS (Dulbecco's phosphate-buffered saline), HBSS (Hanks'Balanced Salt Solution), EBSS (Earle's Balanced Salt Solution), and PBS (Dulbecco's phosphate-buffered saline). 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-morpholinopropanesulfonic acid), ACES (N-2-aminoethanesulfonic acid), TAPS (tris(hydroxymethyl)methylamino)propanesulfonic acid), Bicine (diethanolglycine), and Tricine (N-(tris(hydroxymethyl)methyl)glycine) (Sigma / ThermoFisher). The buffer may optionally be supplemented with a protein (e.g., albumin or other proteinaceous material), and a surfactant (e.g., polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and an antifoaming agent (e.g., pluronic F68, ethylene glycol, glycerol or glycerides). If water is used to dilute a solution or suspension described herein, it may be distilled, double distilled or ultrapure.

[0166] The cell culture medium after the multiple steps and / or cell separation that can be used for the method depends on the separated cell sample.Different cells or cell types can have very specific culture medium, serum and supplement requirements.Information about the culture medium for transfecting a specific cell line can be obtained from the cell bank that purchases the cell.Those skilled in the art will know what culture medium to use for further cell growth, differentiation, transfection, implantation, transplantation or freezing.Exemplary cell culture medium includes 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 Medium) (Sigma / ThermoFisher).Exemplary culture medium for cell transfection includes, for example, Opti-MEM (with the minimum essential medium that reduces serum content, ThermoFisher), which is recommended for transfecting RNA. Exemplary culture media for cell implantation include ECM (Early Cleavage Medium), endothelial cell culture media, Cook IVF cell culture media (Cook, Australia), P-1 culture media (Irvine Scientific, CA, USA), HTF culture media (Irvine Scientific, CA, USA) and MultiBlast culture media (Irvine Scientific, CA, USA). Some examples of cell storage or cryopreservation culture media include any cell growth medium listed above supplemented with the following: 5% to 10% DMSO or glycerol, 90% FBS containing 5% to 10% DMSO or glycerol, and 5% to 10% DMSO, 20% FBS and 0.1M trehalose. The selection of storage or freezing culture media depends on the cell line used. DMSO is not suitable for all cell lines, particularly if it is serum-free. Methylcellulose or polyvinyl pyrrolidone can be used for serum-free cell lines.

[0167] The temperature at which any step of the method of the invention occurs, including the (optional) step of pretreating the sample, the (optional) step of diluting the sample, contacting the sample with nonporous microparticles, incubating the sample containing the target cells with nonporous microparticles, separating unbound material from the target cell / microparticle complexes, and / or the step of mechanically dissociating and eluting viable target cells, as described above, is not critical, as long as the target cells remain viable. Generally, the temperature will be equal to or lower than the temperature at which the target cells grow in their natural environment or culture conditions, and may be the same or different temperature than the temperature at which the other steps of the method according to the invention are performed.

[0168] In order to reduce or eliminate filter clogging, the filter is preferably placed so that non-porous particles do not accumulate on a large portion of the filter surface when implementing the method of the present invention. For example, in one embodiment, the filter is horizontally oriented, the sample is placed below the filter, and the upward flow of the solution used to wash the sample and / or elute and obtain the target cells is vertically oriented, so that the target cells are initially collected above the filter, and the non-porous particles fall back from the filter. In another embodiment, the filter is vertically oriented, the sample is placed on one side of the filter, and the flow of the solution used to wash the sample and / or elute and obtain the target cells is horizontally oriented, so that the target cells are collected on the other side of the filter, and the non-porous particles are retained on the other side of the filter.

[0169] Contacting a sample containing viable target cells with nonporous microparticles

[0170] If the sample contains or is suspected of containing debris that would otherwise impede performance or clog the filter when performing the method according to the invention, such as fragments of tissue, bone, organs, cell aggregates, etc., then in a preferred embodiment, the sample is pre-treated to partially or completely remove the debris. This can be achieved by filtering the sample under conditions where viable target cells are not substantially removed from the sample.

[0171] According to the method of the present invention, nonporous microparticles and capture ligands covalently fixed to the surface of the microparticles are contacted with a sample containing a suspension of viable target cells and mixed with the nonporous microparticles (e.g., by mixing the sample flow and the suspension of nonporous microparticles and / or by shaking, stirring and / or agitating) under certain conditions, wherein the target cells in the sample remain viable, and the density of the nonporous microparticles is 1.45 g / cm 3 or larger, with a diameter of 10 μm to 200 μm, the capture ligand is capable of specifically binding to the molecule, and the viable target cell displays the molecule on the cell surface.

[0172] In one embodiment according to the present invention, the method starts by making a suspension of non-porous microparticles in a solution directly contact the sample. In another embodiment, after adding non-porous microparticles as a suspension, the sample is diluted with a liquid. Before, during or after adding non-porous microparticles, the sample can be diluted with any liquid, the liquid maintains the viability of the target cell and allows the non-porous microparticles to form a complex with the target cell during the incubation of the sample, and can be selected based on the type of the target cell (i.e., target cell) present in the sample. Such liquids include, but are not limited to, water, buffer solutions or cell culture media, for cell transfection, transplantation, implantation and / or storage or frozen solutions or culture media as described herein.

[0173] Incubate the sample containing target cells with non-porous microparticles

[0174] According to the method of the present invention, incubation of non-porous microparticles with a sample containing target cells is carried out in the absence of substantially mechanical agitation, preferably in the absence of any mechanical agitation, so that the target cells specifically bind to the capture ligand through the molecule and form a stable target cell / microparticle complex.

[0175] The time 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 99% or 100%.

[0176] Separation of unbound material from target cell / particle complexes

[0177] According to the method of the invention, the target cell / particle complex is separated from unbound material in the sample by washing the unbound material through a filter while retaining the target cell / particle complex. The washing step is performed by passing a solution through the sample at a flow rate or flow rate that does not substantially dissociate the target cell / particle complex but allows the removal of unwanted unbound material.

[0178] 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 or a different solution as used to contact and / or incubate the nonporous microparticles with the sample as described herein, as long as the solution maintains the viability of the target cells.

[0179] The flow rate or flow velocity used to wash unbound material (and optionally non-target cells) from the sample can be adjusted to match the fluid properties of the starting sample. Washing of unbound material is performed using a low flow rate, preferably within the range of laminar flow or transitional flow without forming eddies. In a preferred embodiment, the solution is passed through the filter by laminar flow. The flow rate used to wash unbound material from the sample while retaining the target cell / particle complex is preferably 0.2 mm / sec to 0.5 mm / sec, more preferably 0.3 mm / sec to 0.5 mm / sec, and most preferably 0.35 mm / sec to 0.45 mm / sec.

[0180] A sample containing a suspension of viable target cells and non-porous microparticles is washed through a filter to separate target cell / microparticle complexes from unbound material. Unbound material that is larger than the pores of the filter will not pass through the filter and will not contaminate the final eluted cell suspension. The flow rate or flow velocity and washing time are adjusted so 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 washing.

[0181] Mechanical dissociation and elution of viable target cells

[0182] According to the method of the present 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 with the capture ligand covalently fixed to the microparticle surface. Preferably, the viable target cells are eluted into a sterile container that can be aseptically closed.

[0183] 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 or a different solution as used for contacting the nonporous microparticles with the sample and / or incubating the nonporous microparticles with the sample and / or washing the sample as described herein, as long as the solution maintains the viability of the target cells.

[0184] Mechanical dissociation of target cell / microparticle complexes can be performed by any means that disrupts the capture ligand-surface molecule bonds and results in the release of viable target cells from nonporous microparticles having the capture ligand covalently fixed to the microparticle surface, for example, by flowing a solution through the sample. The flow rate or flow rate used to mechanically disrupt the target cell / microparticle complexes can be adjusted to match the fluid properties of the starting sample. The flow rate or flow rate used to mechanically dissociate the target cell / microparticle complexes depends on the strength of the target cell-microparticle interaction and should be adjusted accordingly. In a preferred embodiment, after mechanically dissociating or disrupting the target cell / microparticle complexes, i.e., after dissociating or disrupting the cell surface molecule-capture ligand interaction, such as the cell surface antigen-antibody interaction, the viable target cells are eluted, for example, by adjusting the flow rate or flow rate of the solution to a higher flow rate than the flow rate used to wash the sample to separate unbound material (and optional non-target cells).

[0185] In a preferred embodiment, the flow rate is higher than 0.45 mm / sec, more preferably higher than 0.55 mm / sec, most preferably higher than 0.65 mm / sec.

[0186] In a preferred embodiment, the flow of the solution containing the target cell / particle complexes and the elution solution is bidirectional, such that the fluid flow is first through the filter in one direction and then through the filter in the reverse direction ("back-and-forth"). The flow may start from the target cell / particle side of the filter, or from the other side of the filter, and may be performed one or more times, provided that the final direction of the flow is toward the container for obtaining isolated viable target cells.

[0187] In a preferred embodiment of the method of the present invention, the target cells are eluted from the nonporous microparticles while retaining about 95% or more, 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 of the capture ligand on the surface of the nonporous microparticles. In all the most preferred embodiments of the method of the present invention, the target cell suspension is substantially free of cell-bound and soluble capture ligands that specifically bind to molecules displayed on the surface of the target cells. Preferably, the eluted viable target cell suspension is substantially free of cell-bound and soluble capture ligands that specifically bind to molecules displayed on the surface of the target cells. Preferably, the eluted viable target cell suspension contains less than 0.01%, more preferably less than 0.001% of capture ligands previously bound to the nonporous microparticles.

[0188] For some applications, when the number of target cells in the sample is limited, the viability of the target cells is a primary concern. Some examples of such applications are cell therapy, or the research on further genetic modification and / or amplification of target cells before use. Some examples of cell therapy in this case are CAR-T treatments, separation is intended to be used without further modification of the T cells, separation of 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-derived stem cells and progenitor cells, including embryonic stem cells and induced pluripotent cells (preferably people) and any other cell type, in this case and application, the viability of cells is a primary concern.

[0189] 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 the growth of the target cells and / or compared to the viability of the target cells in the original sample.

[0190] For certain applications, when the use of target cells requires a specific cell type separated from other cells that are usually associated with a specific sample, the purity of the target cells is a primary concern. Some examples of such applications / target cells are cells used for specific studies, tumor cells including circulating tumor cells (CTCs) for detection and prognosis of diseases, specific subtypes of T cells (tumor-infiltrating T cells, cytotoxic T cells, helper T cells, etc.) that can be further genetically modified and / or expanded, stem cells such as hair follicle stem cells, cardiac stem cells, multipotent muscle cells, neural stem cells, liver stem cells, hematopoietic stem cells, mesenchymal / stromal stem cells, dental pulp cells, periodontal ligament cells, adipose-derived stem cells and progenitor cells, pluripotent stem cells including embryonic stem cells and induced pluripotent cells, and any other type of human or animal cells where high specificity is desired.

[0191] In a preferred embodiment, the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0192] For certain applications, the amount of target cells recovered is of primary concern, since the number of target cells may limit the application or use. Some examples of such applications / target cells are cell therapy, such as stem cell therapy, T cell therapy, separation of stem cells such as pluripotent muscle cells, neural stem cells, liver stem cells, hematopoietic stem cells, mesenchymal / stromal stem cells, dental pulp cells, periodontal ligament cells, adipose-derived stem cells and progenitor cells, pluripotent stem cells including embryonic stem cells and induced pluripotent cells, and any other cell type derived from such pluripotent cells, separation of endothelial cells, and any other cell type where a large number of cells are needed for treatment or research purposes.

[0193] In a preferred embodiment of the present invention, the number of target cells eluted from the non-porous microparticles represents about 10% or more, preferably about 30% or more, preferably about 50% or more, preferably about 60% or more, preferably about 70% or more, more preferably about 80% or more, and most preferably about 90% or more of the target cells originally contained in the sample.

[0194] In a preferred embodiment, 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, while 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 method 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.

[0195] In a preferred embodiment, 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, and the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0196] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 96% or more of the capture ligand on the surface of the non-porous microparticles, while 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 method 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.

[0197] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 96% or more of the capture ligand on the surface of the non-porous microparticles, and the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0198] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 97% or more of the capture ligand on the surface of the non-porous microparticles, while 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 method 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.

[0199] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 97% or more of the capture ligand on the surface of the non-porous microparticles, and the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0200] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 98% or more of the capture ligand on the surface of the non-porous microparticles, while 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 method 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.

[0201] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 98% or more of the capture ligand on the surface of the non-porous microparticles, and the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0202] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 99% or more of the capture ligand on the surface of the non-porous microparticles, while 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 method 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.

[0203] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 99% or more of the capture ligand on the surface of the non-porous microparticles, and the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0204] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 99.9% or more of the capture ligand on the surface of the non-porous microparticles, while 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 method 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.

[0205] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 99.9% or more of the capture ligand on the surface of the non-porous microparticles, and the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0206] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 99.99% or more of the capture ligand on the surface of the non-porous microparticles, while 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 method 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.

[0207] In a preferred embodiment, the target cells are eluted from the non-porous microparticles while retaining about 99.99% or more of the capture ligand on the surface of the non-porous microparticles, and the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0208] In a preferred embodiment, at least about 50% of the target cells obtained according to the method of the present invention are viable when cultured under conditions that support the growth of the target cells and / or compared to the viability of the target cells in the original sample, and the viable target cell population obtained according to the method of the present invention is enriched by the following factors relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0209] In a preferred embodiment, at least about 60% of the target cells obtained according to the method of the present invention are viable when cultured under conditions that support the growth of the target cells and / or compared to the viability of the target cells in the original sample, and the viable target cell population obtained according to the method of the present invention is enriched by the following factors relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0210] In a preferred embodiment, at least about 70% of the target cells obtained according to the method of the present invention are viable when cultured under conditions that support the growth of the target cells and / or compared to the viability of the target cells in the original sample, and the viable target cell population obtained according to the method of the present invention is enriched by the following factors relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0211] In a preferred embodiment, at least about 80% of the target cells obtained according to the method of the present invention are viable when cultured under conditions that support the growth of the target cells and / or compared to the viability of the target cells in the original sample, and the viable target cell population obtained according to the method of the present invention is enriched by the following factors relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0212] In a preferred embodiment, at least about 90% of the target cells obtained according to the method of the present invention are viable when cultured under conditions that support the growth of the target cells and / or compared to the viability of the target cells in the original sample, and the viable target cell population obtained according to the method of the present invention is enriched by the following factors relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0213] In a preferred embodiment, at least about 95% of the target cells obtained according to the method of the present invention are viable when cultured under conditions that support the growth of the target cells and / or compared to the viability of the target cells in the original sample, and the viable target cell population obtained according to the method of the present invention is enriched by the following factors relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0214] In a preferred embodiment, about 99% or more of the target cells obtained according to the method 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, and the viable target cell population obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0215] Medicament comprising a viable target cell suspension obtainable according to the method of the invention

[0216] In another aspect, the present invention relates to a medicament comprising a viable target cell suspension obtainable by the method of the present invention, a viable target cell suspension obtainable by the method of the present invention for use as a medicament for preventing and / or treating a disease, and / or a method for treating a subject in need of a viable target cell suspension obtainable by the method of the present invention, the method comprising applying the viable target cell suspension to the subject for preventing and / or treating a disease, wherein the viable target cell suspension is a mammalian cell suspension, preferably a human cell suspension, comprising cells selected from the group consisting of granulocytes, T lymphocytes, monocytes, T regulatory cells, T helper cells, cytotoxic T cells, B lymphocytes, tumor infiltrating lymphocytes, coagulocytes, natural killer cells, hematopoietic stem cells and progenitor cells, mesenchymal / stromal stem cells, hair follicle stem cells, cardiac stem cells, multipotent muscle cells, neural stem cells, liver stem cells, dental pulp cells, Periodontal ligament cells, retinal pigment epithelial cells, adipose-derived stem cells and progenitor cells, pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells, Car-T cells, microvascular endothelial cells (MVECs), primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells and retinal epithelial cells), fibroblasts from any tissue or organ (the any tissue or organ includes heart, liver, kidney, colon, intestine, esophagus, stomach, neural tissue derived from brain and spinal cord, lung, vascular tissue derived from arteries, veins and capillaries, lymphatic tissue derived from lymph glands, adenoids, tonsils, bone marrow and blood, spleen), muscle cells, pancreatic cells, cardiac cells and any cells established by such primary cells, and wherein the eluted viable target cell suspension is substantially free of cell-bound and soluble capture ligands that specifically bind to molecules displayed on the surface of the target cells. Preferably, the eluted viable target cell suspension is substantially free of cell-bound and soluble capture ligands that specifically bind to molecules displayed on the surface of the target cells. Preferably, the eluted viable target cell suspension contains less than 0.01%, more preferably less than 0.001% of capture ligands previously bound to the non-porous microparticles.

[0217] 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 according to the method of the present invention are viable when cultured under conditions that support the growth of the target cells and / or compared to the viability of the target cells in the original sample.

[0218] Preferably, the viable target cell population in the drug obtained according to the method of the present invention is enriched by the following multiples relative to the non-target cell population in the original sample: at least about 1.25 times, preferably at least about 1.5 times, preferably at least about 2 times, preferably at least about 3 times, preferably at least about 4 times, preferably at least about 5 times, preferably at least about 10 times, preferably at least about 20 times, preferably at least about 30 times, preferably at least about 40 times, preferably at least about 50 times, preferably at least about 100 times, preferably at least about 250 times, preferably at least about 500 times, preferably at least about 1000 times or more.

[0219] In one embodiment of the invention, the method according to the invention is carried out under sterile conditions using the device and / or cartridge according to the invention as a module in GMP manufacturing. The device can be connected to an inlet cell source in a sterile manner and have an outlet connected to (but not limited to) an IV bag, other types of collection containers, or downstream processing machines, such as bioreactors or other cell culture or processing vessels. This embodiment is particularly preferred when viable target cells are to be used as a drug.

[0220] The viable target cell suspension obtained by the method according to the invention may be diluted (e.g. by means of a physiologically acceptable buffer or culture medium) or concentrated (e.g. by sterile filtering the viable target cell suspension to partially remove fluid prior to administration). In addition, the medicament according to the invention may contain one or more additional drugs that may be added to the viable target cell suspension at any time prior to administration to a patient.

[0221] cylinder

[0222] In another aspect, the present invention relates to a cartridge configured for isolating viable target cells from a sample using the methods according to the invention described herein. This is particularly beneficial because the use of cartridges in such methods has not yet been developed, and the cartridges enable the isolation of target cells in a simple, inexpensive, sterile and flexible manner, providing viable target cells of high purity and high viability in a selected culture medium.

[0223] In one embodiment, the cartridge is a closed housing cartridge comprising:

[0224] - a filter enclosed in a filter unit having an inlet end and an outlet end;

[0225] a fluid conduit connected to the filter inlet end and having one or more valves capable of regulating the flow of a source of a sample comprising the target cells of the invention, a source of a suspension of nonporous microparticles of the invention, a source of a washing solution and a source of an elution solution, and

[0226] - A fluid conduit connected to the outlet end of the filter, comprising one or more valves, wherein one valve is capable of transferring the target cell solution to a target cell collection container through the fluid conduit and is capable of transferring fluid to a different container through the fluid conduit.

[0227] As used herein, a "closed housing cartridge" is a cartridge that allows aseptic contact, mixing and incubation of a sample containing a suspension of viable target cells and non-porous microparticles within a closed housing, as well as aseptic transport, washing and elution with one or more solutions. The elements of the cartridge according to the invention, such as the filter unit, fluid conduit and valve are connected in an electrical circuit so that each can be instantaneously connected through the valve when performing the different steps of the method according to the invention.

[0228] The tube housing can be rigid and / or flexible.The fluid conduit of the tube connected with the filter inlet and / or outlet can be a composite of flexible material and rigid material, and can be embedded in or molded into the rigid housing of tube or can exist as a tube.The fluid conduit of tube can have a cross section of any shape, but preferably a rectangle, oval or circle with rounded corners.These cross sections allow target cells to pass through conduit with less mechanical stress.In some embodiments, the fluid conduit of tube serves as an incubation space, is used to incubate sample to form a target cell / microparticle complex that a liquid holding container can attach thereto.In a preferred embodiment, the incubation space is made of soft plastic material such as PVC or silicone.

[0229] The cartridge filter is enclosed within a filter unit such that the filter acts as a porous barrier between an inlet end and an outlet end, and any fluid culture medium flowing through the inlet end can pass through the filter, and any filtrate passing through the filter, i.e., a suspension of viable target cells or a wash solution with or without unbound material (and preferably non-target cells), can flow through the outlet end.

[0230] The valve in the tube of the present invention can approach, stop and / or transfer fluid flow. The valve can be any type of mechanical valve. The valve also can be controlled by electronic means when used for device according to the present invention. In a preferred embodiment, the part of the tube that comprises the valve is made of hard plastic material such as polysulfone.

[0231] In a preferred embodiment, the cartridge is first washed with culture medium, 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 used to elute the target cells is different from the solution used to wash the sample containing the target cell / microparticle complex. In these subsequent and other embodiments, the cartridge according to the invention may also include one or more valves connected to a source of solution for eluting the target cells, the solution for eluting the target cells being different from the solution used to wash the target cell / microparticle complex. For example, the cartridge may be connected to a source of liquid for diluting any solution and / or suspension for carrying out the method of the invention.

[0232] The cartridge and its components, such as filter units, fluid conduits, valves, etc., may be made of 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)), polysulphone, polyphosphazene, thermoplastic elastomer, polydimethylsiloxane, PTFE (polytetrafluoroethylene), etc. In a most preferred embodiment, the cartridge according to the present invention is configured to separate viable target cells from a sample using a method according to the present invention, such that the resulting viable target cell suspension is sterile and can be, for example, grown in culture, stored, frozen, or introduced or reintroduced into a mammal (preferably a human) for treatment. In some such embodiments, the cartridge and its elements, i.e., the filter unit, fluid conduits, and valves, are made of biocompatible plastic materials resistant to sterilization including: PVC (polyvinyl chloride), PA (polyamide), PE (polyethylene), PS (polystyrene), epoxy resin, silicone rubber, natural rubber, polyurethane, PP (polypropylene), polyester, PEEK (polyetheretherketone), polyphenylsulfone, polysulfones, nylon, PMMA (poly(methyl methacrylate)), polysulfone, polyphosphazene, thermoplastic elastomer, polydimethylsiloxane, PTFE (polytetrafluoroethylene), and the like.

[0233] As used herein, "sterilization" describes a process of destroying or eliminating all forms of microbial life by physical or chemical methods such as autoclaving, steam under pressure, 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 in a package that can be sterilized. In a most preferred embodiment, the cartridge according to the present invention is contained in a sealed sterile package.

[0234] In one embodiment of the present invention, the method is performed manually so that the fluid flow can be controlled by hand. One example of manually performing the method is 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 commercially available cell strainers (see the list of commercially available filters and strainers) and centrifuge tubes.

[0235] However, in a preferred embodiment, the method according to the invention is performed using a device and / or a cartridge according to the invention.In a preferred embodiment of the invention, the method is automated and uses a device and / or a cartridge according to the invention.

[0236] In a preferred embodiment, the cartridge is a replaceable part for single use and is designed to be fixed in a device according to the present invention. In a preferred embodiment of the present invention, the cartridge is designed to be modular so that the cartridge can be fixed and connected to a device according to the present invention so that the valve of the cartridge can be manipulated by the control system of the device to measure, set, and / or adjust the solution flow rate through the valve of the cartridge.

[0237] In one embodiment of the present invention, cartridge is designed to separate one or more specific types of target cells from one or more specific samples. In such case, cartridge can be designed together with filter and / or fluid conduit to hold specific sample and be used to separate one or more specific target cell types.

[0238] In certain embodiments, the total cell treatment time using the methods of the invention is less than one hour, less than 45 minutes, less than 30 minutes, less than 25 minutes, or less than 20 minutes.

[0239] Reagent test kit

[0240] In another aspect, the present invention relates to a kit comprising a cartridge according to the present invention for isolating viable target cells from a sample using a method according to the present invention; and a container containing a suspension of non-porous microparticles having a molecular weight of about 1.45 g / cm 3 or greater density, a diameter of about 10 μm to 200 μm, the capture ligand is capable of specifically binding to a molecule on the cell surface. In other embodiments, the kit comprises one or more containers with solutions for performing the methods of the invention (e.g., solutions for washing 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 are sterilizable, and most preferably, the components of the kit are contained in sealed sterile packaging.

[0241] Device

[0242] In one aspect, the invention relates to a device configured to separate viable target cells from a sample using a method according to the invention.The device according to the invention can control fluid flow and fluid flow rate and partially or fully automate the cell separation process according to the invention.

[0243] "Automation" means that the device can be programmed to perform processing steps according to the method of the present invention without substantial operator involvement. Of course, even in the automated system of the present invention, it will be understood that operator activities may be involved, including loading the barrel and / or container for performing the method of the present invention and inputting processing parameters. Additional manual steps may also be required. However, in a preferred embodiment, once the container for the sample, the suspension of non-porous particles, the container for the solution of washing and elution, and the container for collecting the viable target cell suspension are connected to the barrel, the device can process samples containing viable target cells without substantial operator intervention.

[0244] According to this aspect of the invention, the device comprises means for securely fixing the cartridge according to the invention so that the valves of the cartridge can be mechanically manipulated by an electronic control system, means for connecting the cartridge to one or more containers having one or more solutions and / or suspensions, means for moving 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 carrying out the method according to the invention.

[0245] The device of the invention may be equipped with one or more keyboards, touch screens, barcode reading devices, communication elements for WIFI, NFC (Near Field Communication) or Bluetooth, pre-treatment filters, motors, vibration isolation units for any or all pumps or motors, sensors or electrodes for measuring pressure, flow, impedance, conductivity, resistivity, current, pH, temperature, humidity, gas percentage, radiation, motion, volume, weight, magnetic flux, time, turbidity, optical density, alarms, cell counting devices, heating units, cooling units, graphic display units, ports for data storage devices (such as CD and USB flash drive sticks), NFC, etc. The device may be operated in a mobile manner with direct connection to a fixed power source or with battery power.

[0246] Control System

[0247] Electronic control system control fluid enters in the cylinder, in the cylinder and from the flow in the cylinder.The control system of device of the present invention is any suitable man-machine interface controller that can receive input, send output and perform the operation for carrying out method of the present invention.According to certain embodiments, this controller can comprise programmable microprocessor, it can be programmed to cause contact according to the method of the present invention, incubation, washing and elution step and other other steps, preferably in an automated manner.This controller can be programmed to make the flow at one or more step places in these steps be unidirectional or bidirectional, make fluid flow first carry out with one direction, and reverse the direction of flow (" back and forth ") subsequently.

[0248] The controller may receive user input via a device such as a keyboard, a touch screen, a handheld device (e.g., a smartphone or tablet) and / or a barcode reader or scanner, or via a radio frequency identification (RFID) reader, etc., by directly connecting to the device or by connecting to other processing systems via a local network or other computer devices (e.g., servers) via a local network, a wide area network or the Internet. According to such an embodiment, the device may include an internal transmitter / receiver device for wireless communication technologies such as WLAN, WIFI, Bluetooth, etc. The controller may also be connected to and receive process data input from one or more sensors within the device, the sensors measuring one or more variables before, during or after performing the method according to the present invention. The variables measured by such 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 for performing the method of the present invention, pressure, flow, flow rate, impedance, conductivity, resistivity, current, pH, temperature, humidity, gas percentage, radiation, motion, volume, weight, magnetic flux, time, etc. at any point in the system. The controller may be connected to and output data to a graphic display unit or screen, a video display unit or screen, a data storage device (e.g., a CD, a USB stick), or to other computer devices (e.g., a server) via a local network or via a local network, a wide area network, or the Internet to send data to devices such as computers, handheld devices (e.g., a smartphone or an electronic tablet), etc. According to such an embodiment, the device may include an internal transmitter / receiver device for wireless communication technologies such as WLAN, WIFI, Bluetooth, NFC, etc.

[0249] fluid container

[0250] In a preferred embodiment, the device of the present invention has one or more components enclosed within the device for connecting the cartridge to one or more containers, preferably made of any one or more of the biocompatible materials mentioned herein.

[0251] The container may be made of any rigid or flexible biocompatible material mentioned herein, including glass, hard or soft plastics, and may be in the form of a bottle, tube such as a sample tube, centrifuge tube, syringe, flask (e.g., tissue culture bottle), bag (e.g., collapsible bag or IV bag), etc., which is preferably capable of being closed, and optionally sealed, and reopened in a sterile manner. Preferably, the container with the sample, the container with the suspension of non-porous microparticles, the container with the washing and / or elution solution, and the container into which the target cells are eluted are sterile syringes, bottles or IV bags.

[0252] In a preferred embodiment, the device of the invention has 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 invention.

[0253] In a preferred embodiment, the device is aseptically connected to a cartridge of the invention, which is aseptically connected to a container having a sample source containing viable target cells, a container having a source of non-porous microparticles, a container having a source of washing solution, an elution solution and / or a dilution liquid, and a sterile container for collecting separated viable target cells. The container for collecting unbound material and / or non-target cells may also be sterile.

[0254] Solution / suspension mobile system

[0255] The device comprises at least one for moving, stopping and / or reversing the parts of the solution and suspension used in the method according to the present invention.Preferably, the device comprises at least one pump, including positive displacement pump such as syringe pump, syringe plunger, infusion pump, peristaltic pump, diaphragm pump, gear pump, screw pump, hollow disk pump, vibration pump, hydraulic piston pump, pulse pump, airlift pump, centrifugal pump, propeller pump, axial flow pump, mixed flow pump, siphon pump, piston pump, pulsation meter steam pump, rotary cam pump, rotary vane pump, jet pump and electromagnetic pump.In one embodiment of the invention, fluid container is connected with main barrel and can be positioned with valve after each container, and the valve can be located at different positions to control the flow through the system.In one embodiment, the movement of fluid control system control valve and syringe plunger is used.In another embodiment of the present invention, flow can be controlled by the pump of the flow rate or flow velocity that can automatically control fluid in and out of system.In a preferred embodiment, one or more pumps can bidirectional pumping, so that fluid first flows in one direction, and reverses the direction of flow (" back and forth ") subsequently.

[0256] 6. Examples

[0257] The present invention provides unique methods, cartridges and devices for cell separation, enabling the separation of viable target cells from a sample in a timely manner, with a high yield of viable target cells in a suspension substantially free of capture ligands on the cell surface or in a fluid of the suspension, and maintaining cell viability, while eliminating the need for downstream processing and purification of the sample, which can be treated in an aseptic manner for subsequent culture of the target cells or subsequently for treatment. The present invention is described in detail in the following examples, which may represent more than one embodiment of the present invention. The present invention is not limited by the scope of the exemplary embodiments, which are intended to illustrate a single aspect of the present invention. In fact, various modifications of the present invention (except those shown and described herein) will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. The following examples are provided by way of example rather than by way of limitation.

[0258] Example 1: Preparation of anti-CD90 antibodies covalently fixed to carboxylated silica microparticles

[0259] 20 mg of carboxyl-functionalized silica nonporous microparticles with a diameter of 40 μm (purchased from Glantreo Ltd., Ireland), 1 μL of EDC ((1-ethyl-3-(3-dimethylamino)propylcarbodiimide, hydrochloride)) (concentration of 100 mg / mL) and 1 μL of NHS (N-hydroxysuccinimide) or sulfo-NHS (N-hydroxysulfosuccinimide) (concentration of 60 mg / mL) were mixed in an Eppendorf tube in MES buffer at pH = 6 at room temperature. . The activated non-porous microparticles were centrifuged and the supernatant was discarded to remove excess reagents. 15 μg of anti-CD90 antibody (purchased from BIOTEM, France) was 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 were blocked by adding 100 μL of 0.1 M ethanolamine solution. After blocking the remaining groups, the microparticles were centrifuged and the supernatant was saved for protein concentration determination. After washing in PBS, the microparticles can be used for cell isolation.

[0260] Example 2: Preparation of anti-CD90 antibodies covalently immobilized to epoxy-functionalized silica microparticles

[0261] Non-functionalized silica non-porous microparticles with a diameter of 40 μm were purchased from Glantreo Ltd, Ireland. The non-functionalized silica microparticles were silanized with GLYMO to prepare silica non-porous microparticles functionalized with epoxy groups. 20 mg of epoxy-functionalized silica non-porous microparticles were washed in 1 M phosphate buffer at pH = 7 in an Eppendorf tube at room temperature. 15 μg of anti-CD90 antibody (purchased from BIOTEM, France) was then added to the Eppendorf tube. The reaction was allowed to proceed overnight. The remaining active sites on the surface of the non-porous microparticles were blocked with 100 μL of 0.1 M ethanolamine. After blocking the remaining groups, the non-porous microparticles were centrifuged as described below and the supernatant was saved for protein concentration determination. After washing in PBS, the non-porous microparticles can be used for cell separation.

[0262] To determine whether the fixation was successful, an indirect method known to those skilled in the art for measuring protein concentration in a sample can be performed. Protein concentration is 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. After 10 minutes, the sample absorbance value is measured. The measurement is performed using a NanoDrop TM One / OneC Microvolume UV-Vis spectrophotometer was performed in a cuvette. The results of the microparticles prepared in Example 1 are Figure 1 The starting anti-CD90 antibody solution is presented as 100% protein. The protein concentration in the supernatant saved during the fixation process was measured and the protein microgram ratio between the starting sample and the supernatant was calculated. The percentage of protein remaining in the supernatant is Figure 1 The difference in protein mass in the starting antibody solution and the supernatant can be calculated and this mass is considered to be the amount of antibody bound to the nonporous microparticles. Figure 1 In the figure, it was shown that about 60% of the added anti-CD90 antibody was covalently bound to the silica particles.

[0263] The specific surface area available for antibody binding on 40 μm nonporous silica particles can be calculated based on the following equation:

[0264]

[0265] AG = surface area / grams of dry powder (μm 2 / g)

[0266] ρS = density of a solid sphere (g / cm 3 )

[0267] d=average diameter (μm)

[0268] AG=7.5*10 10 μm 2 / g.

[0269] The surface saturation of proteins bound to nonporous silica particles can be calculated based on the following equation:

[0270]

[0271] S = amount of representative protein required to achieve surface saturation (mg protein / g microspheres)

[0272] ρS = density of a solid sphere (g / cm 3 )

[0273] d=average diameter (μm)

[0274] C = capacity of the microsphere surface for a given protein (mg protein / m 2 Ball surface)

[0275] C(IgG)=2.5mg / m 2 .

[0276] For a sample with a diameter of 40 μm and a g / cm 3 For non-porous silica particles with a density of 0.1, the theoretical maximum value of milligrams of antibody per gram of silica particles is 0.2 mg (Ab) / g (MP).

[0277] Figure 2 The experimental results in show the consistency of the surface saturation values ​​of anti-CD90 antibodies and are consistent with the calculated theoretical maximum values. The experimental values ​​of the surface saturation of 40 μm non-porous silica particles have the same results in two different experiments, so it can be concluded that all antibodies bound to the particles are bound on the surface of the particles.

[0278] The average surface area of ​​40 μm non-porous silica particles measured with a Micromeritics TriStar II analyzer is 1 m 2 / g particles.

[0279] Example 2a: Preparation of anti-CD4 antibodies covalently immobilized to carboxylated silica microparticles

[0280] At room temperature, 1 g of carboxyl-functionalized silica nonporous microparticles with a diameter of 40 μm (purchased from Glantreo Ltd., Ireland), 28 μL of EDC ((1-ethyl-3-(3-dimethylamino)propylcarbodiimide, hydrochloride)) (concentration of 100 mg / mL) and 28 μL of NHS (N-hydroxysuccinimide) or sulfo-NHS (N-hydroxysulfosuccinimide) (concentration of 60 mg / mL) were mixed in MES buffer at pH = 6 in an Eppendorf tube. 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 microparticle surface are blocked by adding 500 μL of 0.1M ethanolamine solution. After blocking the remaining groups, the microparticles are centrifuged and the supernatant is stored for protein concentration determination. After washing in PBS, the microparticles can be used for cell separation.

[0281] Example 2b: Preparation of anti-CD34 antibody covalently fixed to carboxylated silica microparticles

[0282] At room temperature, 1 g of carboxyl-functionalized silica nonporous microparticles with a diameter of 40 μm (purchased from Glantreo Ltd., Ireland), 28 μL of EDC ((1-ethyl-3-(3-dimethylamino)propylcarbodiimide, hydrochloride)) (concentration of 100 mg / mL) and 28 μL of NHS (N-hydroxysuccinimide) or sulfo-NHS (N-hydroxysulfosuccinimide) (concentration of 60 mg / mL) were mixed in MES buffer at pH = 6 in an Eppendorf tube. 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 microparticle surface are blocked by adding 500 μL of 0.1M ethanolamine solution. After blocking the remaining groups, the microparticles are centrifuged and the supernatant is stored for protein concentration determination. After washing in PBS, the microparticles can be used for cell separation.

[0283] Mode for Carrying Out the Invention

[0284] Example 3: Manual cell separation

[0285] Figure 3 Components for performing a manual cell separation method are depicted in FIG. A hollow tube (component number 4, Figure 3) and two 30mL syringes (first syringe 1 and second syringe 2, Figure 3 ) connection, with a filter (element 3, Figure 3 In the first syringe 1, cells and non-porous microparticles were mixed in a volume of 6 mL and the syringe was attached to tube 4 ( Figure 3 ). The second syringe 2 is a clean syringe with no fluid inside. Pushing the plunger on the first syringe 1 causes the liquid containing the non-porous particles to be transferred to tube 4 while the tube is in a horizontal position, so the system is now in the incubation phase. After 15 minutes, a new syringe containing 25 mL of elution / washing buffer is placed in the position of the first syringe 1. Push the elution / washing buffer through the tube while the tube is placed in a vertical position, and now transfer all the liquid to the second syringe 2 as it passes through tube 4. Unscrew the second syringe 2 and place a new, clean syringe with 10 mL of elution buffer in place of the second syringe 2. Push the two syringes 1 and 2 one after another so that the flow back and forth passes through tube 5, and do this 10 times until all the liquid returns to the syringe in position 2. Wash tube 5 with the 10 mL of elution buffer collected in the syringe in position 2 in addition.

[0286] Example 4: Cell separation on the device according to the invention

[0287] Figure 5 One of the cartridge orientations and positions of the connection valve according to the present invention is depicted in FIG. Figure 5 The non-porous microparticles suspended in a physiological pH buffer are placed in a syringe connected to the port at position 10 in the Figure 5 Place the syringe filled with air in the Figure 5 Position 3 in the filter and used to purge the system. Figure 5 ) below are empty so they can be used as receiving syringes for non-target and target cells. The ports at positions 4 and 5 are connected to tubes that can be connected to waste bottles. The first step is to wash the system with elution / wash buffer and purge with air. Afterwards, the elution buffer from positions 9 and 10 ( Figure 5 ) connected syringes are mixed by pushing the liquid into and out of the syringes. When the contents of the two syringes are fully mixed, the cells are started to be incubated with the non-porous microparticles. After the time programmed on the device (15 minutes) has passed, the cells that are not bound to the non-porous microparticles are washed by washing 3 times with 10mL of elution / wash buffer using laminar flow in the syringe at position 9. After washing away the cells that are not bound to the non-porous microparticles, the system is flushed again with elution / wash buffer and air to remove all cells from the cartridge. After sufficient washing, elution of the cells bound to the microparticles is performed. Use a higher flow rate or flow rate through position 9 ( Figure 5 ) at the syringe and barrel tube (element number 12, Figure 5 ) is eluted mechanically. When the process is completed, position 1 ( Figure 5 ) contains the target cells eluted from the non-porous microparticles. The sample does not contain any animal-derived products and does not contain antibodies (or fragments thereof) that could contaminate the sample. Placed in position 1 ( Figure 5 ) on the syringe filter (component number 11, Figure 5 ) prevents the non-porous microparticles from entering the target cell sample, so at the end of the process, all non-porous microparticles remain in the cartridge. The eluted target cells show very high viability (>90%) and the cells can be used for their further use without undergoing any additional purification steps.

[0288] Example 5: Cell separation on the device according to the invention

[0289] Figure 6 One of the barrel orientations and positions of the connection valve according to the present invention is depicted in FIG. The cells are placed in position 1 ( Figure 6 ) and place the nonporous microparticles suspended in physiological pH buffer in the syringe at position 2 ( Figure 6 ) in the syringe at position 5 ( Figure 6 ) and used to purge the system. Figure 6 Positions 3 and 4 are connected to syringes filled with buffer for eluting / washing cells. Place filter 11 ( Figure 6 ) are empty, so they can be used as receiving syringes for non-target materials. The syringe placed on position 10 contains elution buffer and acts as a receiving syringe for target cells. The ports at positions 6 and 7 are connected to tubes that can be connected to a waste bottle.

[0290] The first step in using the device is to wash the system with elution / wash buffer and purge the system with air. Then, the liquids from positions 1 and 2 ( Figure 6 ) of the syringe. When the fluids in the two syringes are fully mixed, the mixture is pushed out of the syringes to fill the tube connected to the filter (element number 12, Figure 6 ). The cells were incubated with nonporous microparticles in tube 12 ( Figure 6 After the time programmed on the device (15 minutes) has passed, the flow is slowed down by using a laminar flow through tube 12 ( Figure 6), washing of cells not bound to the nonporous microparticles is performed by washing tube 12 with 40 mL of elution / wash buffer. After sufficient washing, elution of cells bound to the microparticles is performed. Elution is performed using a higher flow rate or flow rate from position 10 ( Figure 6 ) in the syringe back and forth through the tube 12 in the barrel ( Figure 6 ) is done mechanically. When the process is finished, position 10 ( Figure 6 ) contains the target cells eluted from the non-porous microparticles. The sample does not contain any animal-derived products and does not contain antibodies or fragments thereof that may contaminate the sample. Figure 6 ) before the syringe (component number 11, Figure 6 ) prevents non-porous particles from entering the target cell sample, so that at the end of the process, all non-porous particles remain on the filter 11 ( Figure 6 ). The eluted target cells showed very high viability (>90%) and the cells could be used for their further use without any additional purification steps.

[0291] Example 6: Cell separation on the device according to the invention

[0292] Figure 7 The cartridge depicted in FIG. 1 ensures that the cartridge is more user friendly and the location of ports 1 to 10 allows flow through the cartridge to be adopted in both directions. In addition, it allows the system to be a completely closed sterile environment. Figure 7 As depicted in FIG. 1 , the syringe is placed facing downward. In the first step, the syringe is placed in the 9th port ( Figure 7 ) and the cells contained in the syringe located at port 10 ( Figure 7 ) are mixed and pushed through the system. Figure 7 ) for incubation. Positions 1 to 8 can be connected to syringes that can be empty or filled with elution / wash buffer, and an air filter is placed in one position. After the 15-minute incubation period, 30 mL of elution buffer is flushed through the system to remove unbound cells. After removing unbound cells from the system, elution of target cells bound to nonporous microparticles is performed using a back-and-forth flow through tube 12 ( Figure 7 ) is performed. The filter (element number 11, Figure 7 ) is placed before the target receiving syringe and the non-target receiving syringe to prevent non-porous particles from contaminating the target cell sample.

[0293] Example 7: Cell separation on the device according to the invention

[0294] The non-porous microparticles are pre-packaged in an incubation space - a tube with two filters at both ends. The cells are transferred from an IV bag attached to a pump that controls the flow or flow rate. The incubation is performed by using a continuous flow of the cell suspension. After the 15-minute incubation period, the cell flow is turned off and 40 mL of elution / wash buffer is flowed through the tube to remove unbound cells. After removing unbound cells from the system, elution of target cells bound to the non-porous microparticles is performed with a back and forth flow through the tube controlled by a pump.

[0295] Example 8: Manual method for cell separation according to the present invention

[0296] Prepare a sample and wash buffer containing a suspension of viable target cells. Prepare the buffer by warming the stock buffer of a commercial cell buffer (stock buffer catalog number 60-00080-10 purchased from pluriSelect Life Science (Leipzig, Germany)) to room temperature and diluting it 10 times. Centrifuge human Kasumi (CD90-) cells (DSMZ accession No.: ACC 220) and human Jurkat (CD90+) cells (ATCC accession No.: TIB-152) at 300 rpm for 10 minutes. Resuspend the cell pellet in 3 mL of buffer. Count the cells and prepare 3 mL of a 5×10 5 1 mL of cells was added to a sterile commercial centrifuge tube. 20 mg of nonporous silica microparticles activated with anti-CD90 antibodies prepared using the procedure provided in Example 1 were added. Gently mixed five times to allow the cells to contact the nonporous microparticles and the mixture was left at room temperature for 30 minutes for incubation. The formation of target cell / microparticle complexes using Jurkat (CD90+) cells was performed in Figure 4a , whereas Kasumi (CD90-) cells did not form any target cell / particle complexes, as shown in Figure 4b As shown in . A filter with 30 μm pores is placed on a sterile commercial centrifuge tube and the solution containing the target cell / particle complexes is poured onto the filter. The target cell / particle complexes are washed with 16 mL of wash buffer by pouring the buffer over the target cell / particle complexes to remove all unbound material and non-target material. The opening on the filter is closed so that liquid cannot pass through and all material is placed in a clean, sterile commercial centrifuge tube. 2 mL of wash buffer is added and pipetted up and down 20 times to mechanically disrupt the target cell / particle complexes and elute the target cells. The filter is washed with an additional 10 mL of wash buffer. The two cell lines in the target cell eluate are counted. The results are shown in Table 1 together with the results of Example 9 and are expressed as a percentage of the starting cell number of the cells.

[0297] Example 9: Automated method for cell separation according to the present invention

[0298] Prepare a sample and wash buffer containing a suspension of viable target cells. Prepare the buffer by warming the stock buffer of a commercial cell buffer (stock buffer catalog number 60-00080-10 purchased from pluriSelect Life Science (Leipzig, Germany)) to room temperature and diluting it 10-fold. Centrifuge Kasumi (CD90-) cells (DSMZ ACC 220) and Jurkat (CD90+) cells (ATCC-TIB-152) at 300 rpm for 10 minutes. Resuspend the cell pellet in 4 mL of buffer. Count the cells and prepare 4 mL of a 3.5×10 5 cells / mL of cell suspension.

[0299] Two 10 mL syringes were prepared: 3 mL of cells suspended in buffer were transferred in the first syringe and 40 mg of nonporous silica microparticles activated with anti-CD90 antibody (prepared by the procedure provided in Example 1) suspended in 3 mL of buffer were transferred in the second syringe.

[0300] Attach the syringe to the cartridge ( Figure 7 ):

[0301] 1 – empty 30 mL syringe;

[0302] 3 – empty 50 mL syringe;

[0303] 5 – empty 50 mL syringe;

[0304] 7 – 50 mL syringe containing 50 mL of wash buffer;

[0305] 9 and 10 – 10 mL syringes containing microparticles and cells, respectively.

[0306] The program for the specific cell type is selected on the device and the cartridge with the syringe is firmly attached to the device and the program is run. The device according to the invention is programmed so that in a first step the cells are mixed with the microparticles and transferred to the tube (element 12, Figure 7) for incubation at room temperature. After 15 minutes of incubation, washing of unbound material and non-target cells was performed by washing the tube with 30 mL of wash buffer, the same buffer used to prepare the cell suspension. After this step, elution of cells by mechanical disruption of cell / particle complexes was performed by adding 7 mL of wash buffer and pushing the liquid through the tube in 5 "back and forth" steps. The final cell suspension (of viable target cells) was collected in syringe 1.

[0307] After the apparatus has finished the procedure, syringes No. 1, 3 and 5 are collected and the number of target cells is counted in the target eluent syringe. The results are shown in Table 1 together with the results of Example 8 and are expressed as a percentage of the starting number of cells. The results presented below were obtained under the conditions described in Examples 8 and 9 above and were performed with a fixed amount of nonporous microparticles (20 and 40 mg of activated nonporous microparticles). The yield increases if a larger amount of microparticles is used under the same experimental setup.

[0308] Table 1: Target cell viability and productivity from Examples 8 and 9.

[0309] Cell lines CD+ or - Antibody Yield Viability model Jurkat CD90+ Anti-CD90 40％ 99％ Manual Kasumi CD90- Anti-CD90 1％ 100％ Manual Jurkat CD90+ Anti-CD90 9％ 97％ automatic Kasumi CD90- Anti-CD90 2％ 93％ automatic Jurkat CD3+ Anti-CD3 21％ 99％ Manual Kasumi CD3- Anti-CD3 0％ 100％ Manual Jurkat CD3+ Anti-CD3 16％ 97％ automatic Kasumi CD3- Anti-CD3 8％ 97％ automatic

[0310] To determine whether any anti-CD90 antibodies were present on the washed target cells and non-target cells isolated in the above examples, flow analysis was performed on the cells contained in the target eluate (ie, the final cell suspension containing viable target cells) and the non-target eluate.

[0311] Specifically, Kasumi (CD90-) cells and Jurkat (CD90+) cells contained in the target eluate and the non-target eluate were stained with goat anti-human Fc FITC affinity antibody (Invitrogen). Kasumi (CD90-) cells and Jurkat (CD90+) cells from the starting cell suspension were used as negative controls (because these cells were not contacted with anti-CD90 antibodies). Unstained Kasumi (CD90-) cells and Jurkat (CD90+) cells were used as additional negative controls.

[0312] If any anti-CD90 antibody is present on the cell surface of the washed cells, a signal in the FITC channel exceeding the background is expected during flow cytometry analysis. The percentages of FITC-negative and FITC-positive cells detected are shown in Table 2.

[0313] In the negative control groups (i.e., unstained Kasumi (CD90-) cells, unstained Jurkat (CD90+) cells, Kasumi (CD90-) cells stained with secondary antibody, and Jurkat (CD90+) cells), very low positive FITC signals were detected, which represented background noise.

[0314] Importantly, the stained Kasumi (CD90-) cells and Jurkat (CD90+) cells from the target eluate and the non-target eluate, which were subjected to the cell separation method using the device according to the present invention, fell below the background noise value detected in the negative control group. In fact, no FITC-positive cells were detected in any of the Kasumi (CD90-) cells and Jurkat (CD90+) cells contained in the target eluate and the non-target eluate, indicating that after the cell separation method according to the present invention, there was no antibody from the non-porous microparticles on the washed cells, i.e., the cells were essentially free of anti-CD90 antibodies. Table 2: Flow cytometric analysis of Kasumi (CD90-) cells and Jurkat (CD90+) cells from non-target eluates and target eluates stained with anti-human Fc FITC affinity secondary antibody (Invitrogen), the percentage of FITC positive and negative cells.

[0315]

[0316]

[0317] To determine whether there is any anti-CD90 antibody in the final target eluate (i.e., the final cell suspension containing viable target cells) and non-target eluate collected in the above example, the target eluate and non-target eluate were used to stain Kasumi (CD90-) cells and Jurkat (CD90+) cells, followed by staining with goat anti-human Fc FITC affinity secondary antibody (Invitrogen). The stained cells were then analyzed using flow cytometry as described above.

[0318] Kasumi (CD90-) cells were used as negative controls. In addition, unstained Jurkat (CD90+) cells and Jurkat (CD90+) cells stained with secondary antibodies alone served as additional negative controls. CD90+Jurkat cells stained with anti-CD90 antibodies served as positive controls.

[0319] If any anti-CD90 antibody is present in the target eluate and / or non-target eluate, a signal is expected to appear in the FITC channel during flow cytometry analysis. The percentages of FITC-negative and FITC-positive cells detected are shown in Table 3.

[0320] As shown in Table 3, no anti-CD90 antibody was present in the target eluate and the non-target eluate. As above, very low FITC signals were detected in the negative control group, which represented background noise. Samples stained with the target eluate and the non-target eluate produced the same signal as the negative control group, i.e., the eluate was essentially free of anti-CD90 antibody.

[0321] Table 3: Shows flow cytometric analysis of Kasumi (CD90-) and Jurkat (CD90+) cells stained with non-target and target eluates followed by anti-human Fc FITC affinity secondary antibody (Invitrogen), percentage of FITC positive and negative cells.

[0322] Cell samples Negative% Positive% Kasumi (CD90-) cells - unstained negative control 99％ 1％ Kasumi (CD90-) cells - stained with secondary antibody only - negative control 99％ 1％ Kasumi (CD90-) cells - stained with non-target eluate 99％ 1％ Kasumi (CD90-) cells - stained with target eluate 99％ 1％ Jurkat (CD90+) cells - unstained negative control 99％ 1％ Jurkat (CD90+) cells - stained with Ab only - negative control 99％ 1％ Jurkat (CD90+) cells - stained with anti-CD90 antibody - positive control 0％ 100％ Jurkat (CD90+) cells - stained with non-target eluate 99％ 1％ Jurkat (CD90+) cells - stained with target eluate 99％ 1％

[0323] Example 10: Manual cell separation method according to the present invention for isolating CD19+ B lymphocytes from peripheral blood mononuclear cell (PBMC) samples

[0324] Isolation of PBMCs from whole blood samples

[0325] Prepare 50ml PBMC culture medium: 10% FBS in RPMI. Add 10ml of Histopaque-1077 to a 50ml centrifuge tube and carefully layer 10mL of whole blood sample on the histopaque. Centrifuge at room temperature for exactly 30 minutes at 400g. After centrifugation, carefully aspirate the upper layer to within the 0.5cm opaque interface containing mononuclear cells with a Pasteur pipette. Discard the upper layer and carefully transfer the opaque interface to a clean 50ml tube with a Pasteur pipette. Wash the cells by adding HBSS (up to 50ml at most), mix by gently aspirating up and down using a Pasteur pipette, and centrifuge the cell suspension at 250g for 10 minutes.

[0326] Draw and discard the supernatant. The cell pellet is resuspended in 1 ml of red blood cell lysis buffer (Roche, catalog number 11814389001) and incubated at room temperature for 5 minutes. Add 30 mL of HBSS and mix by gently aspirating up and down using a Pasteur pipette, and the cell suspension is centrifuged at 250 g for 10 minutes. Repeat these steps twice. The cell pellet is resuspended in PBMC culture medium for freezing or using the PBMC cell sample of separation immediately.

[0327] Isolation of CD19+ B lymphocytes from PBMC samples

[0328] Prepare cells and wash buffer. Prepare cells and wash buffer by warming 10× 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 times. Resuspend the PBMC pellet in 3 mL of diluted buffer. Count the cells and prepare 3 mL of a cell suspension at a concentration of 5×105 cells / mL. Add 1 mL of cells to a sterile commercial centrifuge tube. Add 20 mg of non-porous silica microparticles activated with anti-CD19 antibodies. Gently mix five times to allow contact between the cells and the non-porous microparticles and leave the mixture at room temperature for 30 minutes to incubate. Place a filter with 30 μm pores on a sterile commercial centrifuge tube and pour the mixture of cells and microparticles on the filter. Wash the cell / microparticle complex with 16 mL of wash buffer by pouring the buffer on the complex to remove all unbound substances and non-target cells (which are small enough to pass through the filter). Close the opening on the filter so that liquid cannot pass through and place all the materials in a clean, sterile commercial centrifuge tube. Add 2 mL of wash buffer and pipette up and down 20 times to mechanically disrupt the target cell / particle complex. Elute the target cells through the filter into a collection tube and wash the filter with another 10 mL of wash buffer. Prepare a collection tube containing non-target cells and target cells for analysis on a MACS Quant flow cytometer.

[0329] In a human PBMC sample having a starting content of 2% CD19+ B lymphocytes in the sample, the cell separation method according to the present invention using non-porous microparticles containing anti-CD19+ antibodies resulted in an enrichment of CD19+ B lymphocytes by about 20 times (purity 40%) with a viability of 78%. In addition, the results of another cell separation using a PBMC sample from another (healthy) donor were Fig.14 Shown in.

[0330] A schematic diagram of the method according to the invention is shown in Figure 8 and 9 Drawn in.

[0331] Example 11: Manual method for cell separation of human leukapheresis samples according to the present invention

[0332] Human Leukapheresis Sample Preparation and Freezing:

[0333] After the leukapheresis collection (the process in which the donor's blood is passed through a machine to remove the white blood cells and return all other blood cells and plasma to the bloodstream) is completed, the collection bag containing the cell suspension is stored in a refrigerator at 4°C. For 100mL of apheresis, prepare 50mL of apheresis medium (RPMI+10% FBS), 400mL of DPBS containing 2% FBS for washing, and RPMI freezing medium (RPMI+20% FBS+20% DMSO). Sterilize the scissors with ethanol and cut a tube coming out of the collection bag. Pour the cell suspension into a sterile container. Prepare a 10mL aliquot of apheresis cells in a 50mL tube and add cooled DPBS+2% FBS to the top. Centrifuge at 300×g for 10 minutes at 10°C. Discard the supernatant and resuspend the pellet in 5mL of cooled apheresis medium. If necessary, filter the cells through a 70μm filter into a new 50mL tube. The cells can be frozen at -80°C at this time.

[0334] Thawing leukapheresis samples:

[0335] Thaw the sample, for example in a 37°C water bath or by manual warming. Add 10 mL of DPBS while ice crystals are still present in the tube. Transfer the cell suspension to a 50 mL tube and fill with DPBS. Centrifuge at 300 x g for 10 minutes and resuspend the pellet in 10 mL of wash buffer. Pass the cell suspension through a 70 μm filter to remove any clumping cells.

[0336] Cell separation process:

[0337] Prepare the following samples: a suspension containing viable target cells and an isotonic animal protein-free physiological pH buffer (e.g., DPBS supplemented with 0.1% human albumin). Thaw the human leukapheresis sample and centrifuge at 300 rpm for 10 minutes. Count the cells and prepare 3 mL of a 5×10 5A cell suspension of 10 cells / mL was prepared. 1 mL of cells was added to a sterile commercial centrifuge tube. 20 mg of nonporous silica microparticles activated by anti-CD4 antibodies prepared using the procedure provided in Example 2a was added. Gently mix five times to allow contact between the cells and the nonporous microparticles and the mixture was left to incubate at room temperature for 30 minutes. A filter with 30 μm pores was placed in a sterile commercial centrifuge tube and the solution containing the target cell / microparticle complex was poured onto the filter. The target cell / microparticle complex was washed with 16 mL of wash buffer by pouring the buffer over the target cell / microparticle complex to remove all unbound and non-target substances. The opening on the filter was closed so that liquid could not pass through and all substances were placed in a clean, sterile commercial centrifuge tube. 2 mL of wash buffer was added and pipetted up and down 20 times to mechanically disrupt the target cell / microparticle complex and elute the target cells. The filter was washed with an additional 10 mL of wash buffer. The purity and viability in the target fraction and non-target fraction were measured. The results are shown in Table 1. Fig.10 a and Fig.10 b shows the purity of target cells in the sample and the improvement of the viability of the sample after separation. Cell purity was determined using a VYB flow cytometer. Cells were stained using Miltenyi VioGreen CD4 dye (Catalog No. 130-113-223) at a concentration of 1:100 and PI at a concentration of 1:1000. They were incubated in the dark for 10 minutes at room temperature and washed with a buffer containing 1× PBS, 0.5% BSA, and 2mM EDTA following the manufacturer's protocol. 20'000 events per sample were read in 450 μL in gated P4 (live single cells). Using Miltenyi Cell viability was determined using a VYB flow cytometer. Cells were stained with propidium iodide solution (Miltenyi, catalog number: 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. They were incubated in the dark for 10 minutes at room temperature and washed with MACS buffer. 20'000 events per sample were read in 450 μL. MACSQuantify TM Software for data analysis.

[0338] Example 12: Manual method for cell isolation of human stromal vascular fraction samples according to the present invention

[0339] Human stromal vascular fraction vascular Fraction, SVF) sample preparation:

[0340] 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 to 3 minutes for the fat to separate from the remaining liquid. Remove and discard the lower liquid phase and aliquot 45 mL of the lipoaspirate into 50 mL tubes. Centrifuge the lipoaspirate at 430×g for 10 minutes. Push the tip of a 10 mL pipette (stripette) through the fat layer and remove the top PBS and oil layer. Add PBS to a full volume of 50 mL and centrifuge again; repeat this three times. Remove PBS after the last centrifugation. Add a volume of collagenase solution in digestion buffer warmed at 37°C, which is equivalent to the volume of adipose tissue. Mix well and incubate on a tilted roller mixer in an incubator at 37°C for 30 minutes. Centrifuge at 600×g for 10 minutes and discard the supernatant. Remove as much fat layer as possible. Resuspend the SVF pellet in 5 mL of DPBS from each tube. Transfer the pellet through a 100 μm filter into a 50 mL tube. Centrifuge at 600 × g for 10 minutes, remove the supernatant and resuspend the pellet in 10 mL of 1× red blood cell lysis buffer from each tube. Incubate at room temperature in the dark for 10 minutes. Centrifuge at 600 × 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 use in the isolation process.

[0341] Cell separation process:

[0342] Prepare the following sample: a suspension containing viable target cells from a human stromal vascular fraction sample and an isotonic animal protein-free physiological pH buffer (e.g., DPBS supplemented with 0.5% human albumin). Count the cells and prepare 3 mL of a 5×10 5A cell suspension of 10 cells / mL was prepared. 1 mL of cells was added to a sterile commercial centrifuge tube. 20 mg of nonporous silica microparticles activated with anti-CD90 (or anti-CD34) antibodies prepared using the procedures provided in Examples 1 and 2b, respectively, was added. Gently mix five times to allow contact between the cells and the nonporous microparticles and the mixture was left at room temperature for 30 minutes for incubation. A filter with 30 μm pores was placed on a sterile commercial centrifuge tube and the solution containing the target cell / microparticle complex was poured onto the filter. The target cell / microparticle complex was washed with 16 mL of wash buffer by pouring the buffer over the target cell / microparticle complex to remove all unbound and non-target substances. The opening on the filter was closed so that liquid could not pass through and all substances were placed in a clean, sterile commercial centrifuge tube. 2 mL of wash buffer was added and pipetted up and down 20 times to mechanically disrupt the target cell / microparticle complex and elute the target cells. The filter was washed with an additional 10 mL of wash buffer. The purity and viability in the target fraction and the non-target fraction were measured. The results were as follows when anti-CD90 antibody was used. Fig.11 a and Fig.11 b is shown, and when anti-CD34 antibody was used the results were Fig.12 a and Fig.12 As shown in b, the purity of target cells in the sample and the viability of the sample increased after separation. Cell purity was determined using a VYB flow cytometer. Cells were stained using Miltenyi VioGreen CD90 dye (Catalog No.: 130-112-683) at a concentration of 1:50 (or in the case of anti-CD34 antibody, Miltenyi anti-human CD34 antibody (Catalog No.: 130-113-179) at a concentration of 1:50). In both cases, they were incubated in the dark for 10 minutes at room temperature and washed with a buffer containing 1×PBS, 0.5% BSA and 2mM EDTA following the manufacturer's protocol. 20'000 events per sample were read in 400 μL in gated P4 (live single cells). Using Miltenyi Cell viability was determined using a VYB flow cytometer. Cells were stained with propidium iodide solution (Miltenyi, catalog number: 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. They were incubated in the dark for 10 minutes at room temperature and washed with MACS buffer. 20'000 events per sample were read in 450 μL. MACSQuantify TM Software for data analysis.

[0343] Example 13: Automated method for cell separation of human leukapheresis samples according to the present invention

[0344] Prepare the following samples: a suspension containing viable target cells and an isotonic physiological pH buffer without animal protein (e.g., DPBS supplemented with 0.1% human albumin). According to the method provided in Example 11, thaw the human leukapheresis sample and centrifuge at 300 rpm for 10 minutes. Count the cells and prepare 4 mL of a 1×10 6 cells / mL of cell suspension.

[0345] Two 10 mL syringes were prepared: 3 mL of cells suspended in buffer were transferred in the first syringe and 160 mg of nonporous silica microparticles activated with anti-CD4 antibody (prepared by the procedure provided in Example 2a) suspended in 3 mL of buffer were transferred in the second syringe.

[0346] Attach the syringe to the cartridge ( Figure 7 ):

[0347] 1 – empty 30 mL syringe;

[0348] 3 – empty 50 mL syringe;

[0349] 5 – empty 50 mL syringe;

[0350] 7 – 50 mL syringe containing 50 mL of wash buffer;

[0351] 9 and 10 – 10 mL syringes containing microparticles and cells, respectively.

[0352] The program for the specific cell type is selected on the device and the cartridge with the syringe is firmly attached to the device and the program is run. The device according to the invention is programmed so that in a first step the cells are mixed with the microparticles and transferred to the tube (element 12, Figure 7 ) for incubation at room temperature. After 15 minutes of incubation, washing of unbound material and non-target cells was performed by washing the tube with 30 mL of wash buffer, the same buffer used to prepare the cell suspension. After this step, elution of the cells was performed by mechanical disruption of the cell / particle complexes by adding 7 mL of wash buffer and pushing the liquid through the tube in 5 "back and forth" steps. The final cell suspension (of viable target cells) was collected in syringe 1.

[0353] After the device has finished the procedure, syringes 1, 3, and 5 are collected and the purity and viability in the target and non-target fractions are measured. Fig.13 a and Fig.13 The purity and viability of target cells in samples before and after separation are shown in b. Cell purity was determined using a VYB flow cytometer. Cells were stained using Miltenyi anti-human CD4 antibody (Catalog No. 130-113-230) at a concentration of 1:300. They were incubated in the dark for 10 minutes at room temperature and washed with a buffer containing 1× PBS, 0.5% BSA and 2mM EDTA following the manufacturer's protocol. 20'000 events per sample were read in 450 μL gated P4 (live single cells). Miltenyi Cell viability was determined using a VYB flow cytometer. Cells were stained with propidium iodide solution (Miltenyi, catalog number: 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. They were incubated in the dark for 5 minutes at room temperature and washed with MACS buffer. 20'000 events per sample were read in 400 μL. MACSQuantify TM Software for data analysis.

[0354] Example 14: Manual method for cell separation of DPS cells mixed with human leukapheresis samples according to the present invention

[0355] Sample preparation:

[0356] Human leukapheresis samples were thawed using the procedure described in Example 11. DPS (dental pulp stem) cells (CD90+) were added to human leukapheresis samples to give a ratio of 50% DPS cells to 50% of all leukapheresis cells.

[0357] Cell separation process:

[0358] Prepare the following samples: a suspension of viable target cells containing human leukapheresis cells and DPS cells, and an isotonic physiological pH buffer without animal protein (e.g., DPBS supplemented with 0.1% human albumin). Add 1 mL of cells to a sterile commercial centrifuge tube. Add 20 mg of nonporous silica microparticles activated by anti-CD90 antibodies prepared using the procedure provided in Example 1. Gently mix five times to allow contact between the cells and the nonporous microparticles and leave the mixture at room temperature for 30 minutes for incubation. Place a filter with 30 μm pores on a sterile commercial centrifuge tube and pour the solution containing the target cell / microparticle complex on the filter. Wash the target cell / microparticle complex with 16 mL of wash buffer by pouring the buffer on the target cell / microparticle complex to remove all unbound substances and non-target substances. Close the opening on the filter so that the liquid cannot pass and place all substances in a clean, sterile commercial 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 in the target and non-target fractions. Fig.15 a and Fig.15 b shows the purity of target cells in the sample and the improvement of the viability of the sample after separation. Cell purity was determined using a VYB flow cytometer. Cells were stained using Miltenyi VioGreen CD90 dye (Catalog No.: 130-112-683) at a concentration of 1:50. They were incubated in the dark for 10 minutes at room temperature and washed with a buffer containing 1×PBS, 0.5% BSA and 2mM EDTA following the manufacturer's protocol. 20'000 events per sample were read in gated P4 (live single cells) in 400 μL. MACSQuantify TM Data analysis was performed using MACSQuantify software. Cells were stained with propidium iodide solution (Miltenyi, catalog number: 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. They were incubated in the dark for 5 minutes at room temperature and read directly. 20'000 events per sample were read in 450 μL using MACSQuantify TM Software for data analysis.

[0359] Example 15: Automated method for cell separation of MS (mesenchymal stem) cells mixed with human leukapheresis samples according to the present invention

[0360] Sample preparation:

[0361] Human leukapheresis samples were thawed using the procedure described in Example 11. MS (mesenchymal stem) cells (CD90+) were added to human leukapheresis samples to give a ratio of 33% MS cells to 66% of all leukapheresis cells.

[0362] Cell separation process:

[0363] Prepare the following sample: a suspension of viable target cells containing human leukapheresis cells and MS cells, and an isotonic physiological pH buffer without animal protein (e.g., DPBS supplemented with 0.1% human albumin). According to the method provided in Example 11, thaw the human leukapheresis sample and centrifuge at 300 rpm for 10 minutes. Count the cells and prepare 4 mL of a 1×10 6 cells / mL of cell suspension.

[0364] Two 10 mL syringes were prepared: in the first syringe 3 mL of cells suspended in buffer were transferred and in the second syringe 160 mg of nonporous silica microparticles activated with anti-CD4 antibody (prepared by the procedure provided in Example 2a) suspended in 3 mL of buffer were transferred.

[0365] Attach the syringe to the cartridge ( Figure 7 ):

[0366] 1 – empty 30 mL syringe;

[0367] 3 – empty 50 mL syringe;

[0368] 5 – empty 50 mL syringe;

[0369] 7 – 50 mL syringe containing 50 mL of wash buffer;

[0370] 9 and 10 – 10 mL syringes containing microparticles and cells, respectively.

[0371] The program for the specific cell type is selected on the device and the cartridge with the syringe is firmly attached to the device and the program is run. The device according to the invention is programmed so that in a first step the cells are mixed with the microparticles and transferred to the tube (element 12, Figure 7) for incubation at room temperature. After 15 minutes of incubation, washing of unbound material and non-target cells was performed by washing the tube with 30 mL of wash buffer, the same buffer used to prepare the cell suspension. After this step, elution of the cells was performed by mechanical disruption of the cell / particle complexes by adding 7 mL of wash buffer and pushing the liquid through the tube in 5 "back and forth" steps. The final cell suspension (of viable target cells) was collected in syringe 1.

[0372] After the device has finished the procedure, syringes 1, 3, and 5 are collected and the purity and viability in the target and non-target fractions are measured. Fig.16 a and Fig.16 The purity and viability of target cells in samples before and after separation are shown in b. Cell purity was determined by VYB flow cytometry. Miltenyi anti-human CD90 Cells were stained with the 770 antibody (Catalog No. 130-114-904) at a concentration of 1:400. They were incubated in the dark for 10 minutes at room temperature and washed with a buffer containing 1× PBS, 0.5% BSA and 2mM EDTA following the manufacturer's protocol. 20'000 events per sample were read in gated P4 (live single cells) in 450 μL. Miltenyi Cell viability was determined using a VYB flow cytometer. Cells were stained with propidium iodide solution (Miltenyi, catalog number: 130-093-233) at a concentration of 1:1000 according to the manufacturer's protocol. They were incubated in the dark for 5 minutes at room temperature and washed with MACS buffer. 20'000 events per sample were read in 400 μL. MACSQuantify TM Software for data analysis.

Claims

1. A method for isolating viable target cells from a sample, comprising the following steps: a. contacting a sample comprising a suspension of viable target cells displaying molecules on the cell surface with nonporous microparticles and a capture ligand covalently immobilized to the surface of the microparticles, wherein the density of the nonporous microparticles is about 1.45 g / cm 3 or larger, with a diameter of about 10 μm to 200 μm, and the capture ligand is capable of specifically binding to the molecule; b. incubating the sample substantially without agitation to form a target cell / particle complex; c. separating the target cell / microparticle complex from the unbound material in the sample by washing the unbound material through a filter while retaining the target cell / microparticle complex; d. Mechanically dissociating the target cell / microparticle complexes and eluting the viable target cells through the filter while retaining the microparticles with the capture ligands covalently immobilized to the microparticle surface.

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

3. The method according to claim 1 or claim 2, wherein the capture ligand is 10 -5 Up to 10 -12 , more preferably 10 -9 Up to 10 -11 or more preferably 10 -7 Up to 10 -10 , and more preferably about 10 -9 The molecule specifically binds to the surface of the target cell with a dissociation constant of the order of magnitude.

4. The method according to any one of claims 1 to 3, wherein the capture ligand is selected from the following group: antibody, FabFc2, Fab, Fv, Fd, F(ab')2, Fv fragment containing only the light chain and heavy chain variable regions, Fab or F(ab')2 fragment containing the variable region and part of the constant region, single-chain antibody, scFv, CDR-grafted antibody, dAb and / or nanobody.

5. The method according to any one of claims 1 to 4, 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 any one of claims 1 to 5, wherein the method further comprises obtaining a medicament comprising a viable target cell suspension of eluted viable target cells, wherein the eluted viable target cell suspension is substantially free of capture ligand.

7. The method according to any one of claims 1 to 6, wherein the target cells are selected from the group consisting of: human granulocytes; T lymphocytes; monocytes; T regulatory cells; T helper cells; cytotoxic T cells; B lymphocytes; tumor infiltrating lymphocytes; coagulocytes; 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; liver stem cells; dental pulp cells; Periodontal ligament cells; retinal pigment epithelial cells; adipose-derived stem cells 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; fibroblasts from any tissue or organ, including heart, liver, kidney, colon, intestine, esophagus, stomach, neural tissue from brain and spinal cord, lung, vascular tissue from arteries, veins and capillaries, lymphatic tissue from lymph glands, adenoids, tonsils, bone marrow and blood, spleen; muscle cells; pancreatic cells; cardiac cells and cells established by such primary cells.

8. The method according to any one of claims 1 to 7, 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 any one of claims 1 to 8, wherein the sample is selected from the group consisting of human whole blood, apheresis, bone marrow aspirate, biopsy, liquefied tissue such as enzymatically degraded lipoaspirate, cell culture, bioreactor culture, tumor cells, single cell suspension.

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

11. The closed housing cartridge according to claim 10, comprising: - a filter enclosed in a unit having an inlet end and an outlet end, a fluid conduit connected to the filter inlet and having one or more valves capable of regulating the flow of a source of a sample containing target cells, a source of a suspension of non-porous microparticles, a source of a washing solution and a source of an elution solution, and - a fluid conduit connected to the filter outlet, comprising one or more valves, wherein one valve is capable of transferring the solution of target cells through the fluid conduit to a container for collecting target cells or to a different container for washing solution and unbound substances.

12. A kit comprising a container and a closed housing cartridge according to claim 10 or 11, wherein the container comprises a suspension of non-porous microparticles and a capture ligand covalently fixed to the surface of the microparticles, wherein the density of the non-porous microparticles is about 1.45 g / cm 3 or larger, with a diameter of about 10 μm to 200 μm, and the capture ligand is capable of specifically binding to molecules on the cell surface.

13. The closed housing cartridge according to claim 10 or 11 or the kit according to claim 12, wherein the components of the closed housing cartridge or the kit are contained in a sealed sterile package.

14. The method of any one of claims 1 to 9 or the kit of claims 12 or 13, wherein the density of the non-porous microparticles is about 1.95 g / cm 3 or larger.

15. The method of any one of claims 1 to 9 and 14 or the kit of any one of claims 12 to 14, wherein the non-porous microparticles have a diameter of about 35 to 50 μm.

16. The method according to any one of claims 1 to 9, 14 and 15 or the kit according to any one of claims 12 to 15, wherein the non-porous microparticles are made of silicon dioxide (SiO2).

17. The method of any one of claims 1 to 9, wherein the eluted viable target cell suspension is substantially free of capture ligand antibodies that specifically bind to molecules displayed on the surface of target cells.

18. A device configured to separate viable target cells from a sample according to the method of any one of claims 1 to 9, the device comprising: - means for firmly fixing a closed housing cartridge according to claim 10 or 11 so that the valve of the cartridge can be mechanically operated by an electronic control system, - means for connecting the cartridge to one or more containers with one or more solutions and / or suspensions, - components for moving solutions and / or suspensions, and - a control system configured to set, measure and / or regulate the flow of the solution and / or suspension through the valves of the cartridge when performing the method according to any one of claims 1 to 9.

19. The device of claim 18, wherein the device comprises one or more pre-treatment filters, a keyboard, a touch screen, a barcode reading device, a communication element for WIFI, near field communication (NFC) or Bluetooth, a motor, a vibration isolation unit for any or all pumps or motors, sensors or electrodes for measuring pressure, flow, impedance, conductivity, resistivity, 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 port for a data storage device such as a CD, a USB flash drive stick, or NFC.

20. A medicament comprising a viable target cell suspension obtainable according to the method of any one of claims 6 to 9, wherein the eluted viable target cell suspension is substantially free of capture ligands.

21. A viable target cell suspension obtainable according to the method of any one of claims 6 to 9 for use as a medicament, wherein the eluted viable target cell suspension is substantially free of capture ligands.

22. A method of treating a subject in need of a viable target cell suspension obtainable according to the method of any one of claims 6 to 9, comprising administering to the subject the viable target cell suspension substantially free of capture ligands for the prevention and / or treatment of a disease.

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