Cell separation method

Abstract

This application discloses a cell separation method, which includes providing a cell sample solution comprising at least a first type of cell; mixing the cell sample solution and first type of magnetic beads in a separation cup, wherein the first type of magnetic beads are coated with a first type of antibody for binding to the first type of cell, and the diameter of the first type of magnetic beads is 500-4500 nanometers; placing the separation cup containing the first type of magnetic beads and the cell sample solution above a separation seat with a magnet, so that the magnet adsorbs the first type of magnetic beads to the bottom of the separation cup; and separating the first type of magnetic beads from the cell supernatant. The cell separation method of this application can improve cell separation efficiency and purity.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method for cell separation. Background Technology

[0002] Lymphocytes are a type of white blood cell, the smallest of which, produced by lymphoid organs, and are an important cellular component of the body's immune response. Lymphocytes are a cell lineage with immune recognition functions, and based on their origin, migration, surface molecules, and functions, they can be divided into T lymphocytes (also known as T cells), B lymphocytes (also known as B cells), and natural killer (NK) cells. Both T cells and B cells are antigen-specific lymphocytes, and they share the same initial origin: hematopoietic tissue. T lymphocytes circulate in the bloodstream to the thymus, where they mature under the influence of thymic hormones, while B cells differentiate and mature in the bone marrow.

[0003] When stimulated by antigens, T lymphocytes transform into lymphoblasts, which then differentiate into sensitized T lymphocytes to participate in cell-mediated immunity. Their immune function is mainly to fight intracellular infections, tumor cells, and allogeneic cells. B lymphocytes first transform into plasmablasts, then differentiate into plasma cells, which produce and secrete immunoglobulins (antibodies) to participate in humoral immunity. Their functions include producing antibodies, presenting antigens, and secreting intracellular factors to participate in immune regulation. NK cells spontaneously exert cytotoxic effects without relying on antigen stimulation and have the function of killing target cells.

[0004] Two decades ago, the emergence of biologics based on recombinant hormones, soluble receptors, and antibodies brought about a significant revolution in the pharmaceutical industry, which had long been dominated by small-molecule drugs. Now, with the exploration of the multi-faceted potential of microbial and human cell therapies, biomedicine is once again at the forefront, triggering a new revolution. Continuous advancements in cell engineering provide a systematic framework for developing safe and predictable cell therapies. Microbes and human cells are also being used as therapeutic entities, with the potential to address some important, currently unmet needs and treat some of the deadliest diseases, including cancer and autoimmune diseases.

[0005] Researchers at the University of California, San Francisco, published an article in the "Perspective" section of the journal *Science Translational Medicine* outlining the prospects of cell therapy. The article states that cell therapy will become the "third pillar of medicine," widely used to treat patients, much like drugs currently made from engineered proteins, antibodies, or smaller chemicals. The researchers likened it to small molecules and biopharmaceuticals as tools, and cells as carpenters, architects, and engineers. Cells can perform functions that small molecule drugs and targeted therapies cannot. For example, cells are highly adaptable; they can migrate to specific sites, perceive their environment better than current drugs, and then make correct decisions, altering their responses to better adapt to physiological conditions.

[0006] First, cells are inherently responsible for many therapeutic tasks, such as macrophages engulfing pathogens, recruiting adaptive immune cells, hematopoietic stem cells generating myeloid and lymphoid lineage cells, and chondrocytes producing chondrocyte extracellular matrix.

[0007] Secondly, cellular behavior is selective. Small molecules and biologics don't have an on / off switch; they exert their biological effects as long as they bind to a target. However, cells can sense their surroundings and only function when specific signaling molecules are triggered. Therefore, cell therapy is better able to avoid off-target effects, and for this reason, it can deliver medication more effectively, making it a good carrier for other therapies.

[0008] Third, cell therapy can better adapt to the genetic diversity of humans. For example, due to individual differences, drugs are metabolized differently in the human body, resulting in varying therapeutic effects. However, by modifying cells to automatically adjust concentration changes like a resistive circuit, they can adapt to the metabolism of different hosts, thereby achieving better therapeutic effects.

[0009] Finally, cellular function can be regulated by altering cell genes. For example, T lymphocytes can be modified using biosynthetic techniques to enable them to sense blood sugar and secrete insulin, thus relieving type 1 diabetes patients from their dependence on blood sugar. Immune responses to cancer are often weak, but by manipulating and cultivating populations of immune cells that can target specific molecules on cancer cells, anti-tumor responses can be enhanced. These new experimental studies represent a significant breakthrough for clinical treatment.

[0010] With the continuous maturation of cell therapy technology, the separation of large quantities of lymphocytes is a key step.

[0011] Currently, the most mature and widely used cell separation technologies include density gradient centrifugation, immunomagnetic bead separation, and flow cytometry. Density gradient centrifugation separates specific cell types based on cell density; it is simple to operate and low-cost, but can only perform coarse separation of cell populations, failing to meet the complex cell separation needs of clinical and research applications. Immunomagnetic bead separation and flow cytometry are both based on the principle of antibody-captured cells, utilizing specific markers on the cell surface in combination with other techniques to achieve the separation of specific cell types. These two techniques can separate specific subpopulations of cells, offering greater accuracy and higher purity.

[0012] Immunomagnetic bead separation of cells uses two types of magnetic beads: large (0.1mm–0.45mm) and small (approximately 50nm–5000nm). Currently, the commercially available method using large magnetic beads is simple, fast, and low-cost, but results in low purity, mechanical stress on cells affecting their biological activity, and is not conducive to post-separation culture. The large magnetic beads must also be sheared after separation. The method using small magnetic beads primarily employs a separation column, which offers high purity and good cell viability. The beads do not need to be sheared and can be directly fed into flow cytometry. However, the separation speed is slow, the separation column is disposable, resulting in high cost, and post-column washing of negative and positive cells is required, making the process more cumbersome.

[0013] Existing cell separation methods suffer from low cell separation efficiency. Summary of the Invention

[0014] This application proposes a cell separation method that can improve cell separation efficiency and purity.

[0015] To address the above problems, this application provides a cell separation method, which includes providing a cell sample solution comprising at least a first type of cells; mixing the cell sample solution and first type of magnetic beads in a separation cup, wherein the first type of magnetic beads are coated with a first type of antibody for binding to the first type of cells, and the diameter of the first type of magnetic beads is 500–4500 nanometers; placing the separation cup containing the first type of magnetic beads and the cell sample solution above a separation seat with a magnet, so that the magnet adsorbs the first type of magnetic beads to the bottom of the separation cup; and separating the first type of magnetic beads from the cell supernatant.

[0016] In this process, after separating the first type of magnetic beads from the cell supernatant, the separation cup is removed from the separation seat to obtain the first target cell, which is the first type of cell coupled with the first type of magnetic beads that has precipitated in the separation cup.

[0017] After removing the separation cup from the separation seat, cell culture medium is added to the separation cup to obtain the first target cell sample solution, which can be directly used for the next step.

[0018] The separation of the first type of magnetic beads from the cell supernatant includes: inverting the separation cup and separation seat, and pouring out the cell supernatant.

[0019] The process of mixing cell sample solution and type I magnetic beads in a separation cup includes: adding cell sample solution to a separation cup, adding 0.3 to 2.0 times the amount of type I magnetic beads to the cell sample solution to obtain a cell mixture; or mixing cell sample solution with 0.3 to 2.0 times the amount of type I magnetic beads to obtain a cell mixture, and adding the cell mixture to a separation cup.

[0020] After separating the first type of magnetic beads from the cell supernatant, the separation cup is removed from the separation seat, cell culture medium is added to the separation cup, and the first type of magnetic beads and the first type of cells are resuspended; the separation cup is then placed above the separation seat with the magnet again; the first type of magnetic beads and the cell supernatant are then separated.

[0021] The cell sample solution also includes a second type of cell. After separating the first type of magnetic beads from the cell supernatant, the process includes: collecting the cell supernatant; mixing the cell supernatant and the second type of magnetic beads in another separation cup; the second type of magnetic beads are bound with a second type of antibody for binding to the second type of cell; placing the separation cup containing the second type of magnetic beads and the cell supernatant above a separation seat with a magnet; separating the second type of magnetic beads from the cell supernatant; and removing the separation cup from the separation seat to obtain the second target cell, which is a second type of cell precipitated in the separation cup and coupled with the second type of magnetic beads.

[0022] The process of mixing the cell sample solution and the first type of magnetic beads in the separation cup also includes incubating the cell sample solution mixed with the first type of magnetic beads at 2-8°C for 10-40 minutes.

[0023] Among them, the first type of cells are positive cells, and the first type of magnetic beads are magnetic beads with antibodies corresponding to the positive cells.

[0024] The separation cup is a one-piece molded separation cup assembly containing at least two separation cups, which can hold at least two cell mixtures.

[0025] The cell separation method of this application eliminates the need for a separation column when separating cells using small magnetic beads. Instead, it uses a separation cup and a separation seat to directly separate the magnetic beads from the cell supernatant. This method can improve the separation speed, reduce the separation cost, and improve the separation purity. At the same time, the separation seat can be reused, and the manufacturing cost of the separation cup is relatively low. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of an embodiment of the cell separation device of this application;

[0027] Figure 2 yes Figure 1 A schematic diagram of the cell separation device from another perspective;

[0028] Figure 3 yes Figure 1 A schematic diagram of the separation cup assembly of the cell separation device shown in one embodiment;

[0029] Figure 4 This is a flowchart illustrating the first embodiment of the cell separation method of this application. Detailed Implementation

[0030] The cell separation device of this application includes a separation seat, at least one separation cup, and at least one magnet. The separation cup is used to contain a cell mixture with magnetic beads. The separation cup is detachably mounted on the separation seat, and the magnet is located below the separation cup to adsorb the magnetic beads to the bottom of the separation cup. The cell mixture with magnetic beads can be magnetic beads coupled with antibodies corresponding to the target cells and cell sample solution. In other embodiments, it can also be magnetic beads coupled with substances capable of specifically binding to the target cells and cell sample solution. The separation cup allows the magnetic beads with adsorbed target cells to be firmly adsorbed to the bottom of the separation cup without sticking to the walls, thereby improving the yield.

[0031] The cell separation device of this application includes a separation seat, a separation cup, and a magnet. The separation seat is reusable, while the separation cup is preferably disposable. The target cells inside the separation cup can be attracted by the magnet, thereby achieving cell separation. The cell separation device of this application has a simple structure, low cost, and high separation efficiency.

[0032] Please refer to the following: Figures 1-3 , Figure 1 This is a schematic diagram of the structure of an embodiment of the cell separation device of this application; Figure 2 yes Figure 1 A schematic diagram of the cell separation device from another perspective; Figure 3 yes Figure 1 The diagram shows a structural schematic of an embodiment of the cell separation device's separation cup assembly. In this embodiment, the cell separation device includes a separation seat 10, a separation cup assembly 20, and a magnet 30.

[0033] The separating seat 10 includes a partition 101, a side plate 102, and a sealing plate 103. The partition 101 is a horizontal plate, and a plurality of receiving slots 104 are provided below the partition 101 for accommodating magnets 30. The plurality of receiving slots 104 are spaced apart along the length of the partition 101, and each magnet 30 corresponds to a separating cup of the separating cup assembly 20. Preferably, the receiving slots 104 are square, and the magnets 30 are square magnets, which are galvanized neodymium iron boron magnets of grade N35 with dimensions of 7mm × 10mm × 10mm. Preferably, the separating seat 10 is formed of white ABS material.

[0034] Above the partition 101, there is a guide plate assembly perpendicular to the partition 101. The guide plate assembly includes a first guide plate 105 and a second guide plate 106 arranged in parallel and spaced apart.

[0035] The side plate 102 is located on one side of the partition 101 and is perpendicular to the partition 101 and the guide plate assembly. The side plate 102 is provided with a limiting hole 107.

[0036] The encapsulation plate 103 is parallel to the side plate 102 and is used to encapsulate the magnet 30. After the magnet 30 is installed in the receiving groove 104, it can be sealed with the encapsulation plate 103 to prevent the magnet 30 from falling out. The encapsulation plate 103 can be glued or snapped onto the open side of the receiving groove 104.

[0037] The separation cup assembly 20 is used to contain cell mixtures with magnetic beads. The separation cup assembly 20 is detachably mounted on the separation seat 10, and magnets 30 correspond one-to-one with the separation cups at the bottom of the separation cup assembly 20. The cell mixture with magnetic beads can be magnetic beads coupled with antibodies corresponding to the target cells and cell sample solution. The diameter of the magnetic beads is preferably 500 nm to 5000 nm. In other embodiments, it can also be magnetic beads coupled with substances capable of specifically binding to the target cells and cell sample solution. The separation cup assembly 20 enables the magnetic beads adsorbed with the target cells to be firmly adsorbed at the bottom of the separation cup assembly 20 without sticking to the walls, thereby improving the yield. Specifically, the separation cup assembly 20 is formed by combining two adjacent separation cups, and the separation cup assembly 20 is preferably integrally molded. The separating cup assembly 20 includes a first separating cup 201, a second separating cup 202, and a connecting block 203 connecting the first separating cup 201 and the second separating cup 202. The connecting block 203 is inverted V-shaped. A first engaging groove 204 and a second engaging groove 205 are provided at the connection points between the connecting block 203 and the first separating cup 201 and the second separating cup 202, respectively. The first engaging groove 204 engages with a first guide plate 105, and the second engaging groove 205 engages with a second guide plate 106. In this embodiment, the first separating cup 201, the second separating cup 202, and the connecting block 203 are integrally formed. This integrally formed separating cup assembly 20 reduces processing costs and increases structural stability and sealing. The connecting block 203 has a protruding limiting rod 206, and a limiting hole 107 is adapted to the limiting rod 206. The engagement of the first snap-fit ​​groove 204 and the first guide plate 105, as well as the engagement of the second snap-fit ​​groove 205 and the second guide plate 106, allows the separating cup assembly 20 to be limited along the length of the partition 101. The engagement of the limiting hole 107 and the limiting rod ensures that the separating seat 10 and the separating cup assembly 20 do not separate when the whole assembly is inverted. Of course, in other embodiments, the separating cup assembly 20 and the separating seat 10 can also be detachably connected by snap-fit ​​or adhesive. It should be noted that the limiting rod 206 can also be provided on the first separating cup 201 and / or the second separating cup 202, with the limiting hole 107 on the side plate 102 corresponding one-to-one with the limiting rod 206. This three-point positioning structure in this embodiment can stably fix the separating cup assembly 20 to the separating seat 10.

[0038] Those skilled in the art will readily conceive of equivalent embodiments of the above positioning structure, which should fall within the scope of protection of this application. For example, a limiting rod can be provided on the side plate 102, and a limiting hole can be provided on the connecting block 203.

[0039] In this embodiment, the magnet 30 is designed to be removable or insertable. In other embodiments, the magnet 30 can be integrally formed with the separation seat 10 to increase the stability of the structure.

[0040] Preferably, the sides of the separating cup assembly 20 are all planar, and the bottoms of the first separating cup 201 and the second separating cup 202 are planar to abut against the partition 101. The contact between the planar surfaces allows the separating cup assembly 20 to be more stably secured on the partition 101.

[0041] In this embodiment, the separation cup assembly 20 is made of transparent PC material. Transparent PC material is beneficial for observing the cell mixture inside the separation cup. Preferably, the capacity of each separation cup is 1 mL.

[0042] Preferably, the upper surfaces of both the first guide plate 105 and the second guide plate 106 are rounded, and both the first snap-fit ​​groove 204 and the second snap-fit ​​groove 205 are rounded. Rounding allows for better fit between the snap-fit ​​groove and the guide plate, and reduces processing difficulty.

[0043] It is worth mentioning that in this embodiment, a total of three separation cup assemblies 20 are provided, comprising six containers, enabling the separation of six types of cells at once. In other embodiments, those skilled in the art can set multiple sets of separation cup assemblies 20 according to actual conditions, and this application does not limit this.

[0044] The cell separation device of this embodiment has the following advantages:

[0045] 1. The separation cup assembly is W-shaped, and the magnet can firmly adhere the magnetic beads with the target cells to the bottom of the separation cup without sticking to the wall, thereby improving the yield;

[0046] 2. The separation seat is reusable, while the separation cup is disposable, which significantly reduces costs compared to existing disposable separation columns.

[0047] 3. Compared with existing small magnetic bead separation technology, the separation procedure reduces the steps of rinsing the magnetic bead separation column, three repeated rinsing, target cell rinsing, and centrifugation. Compared with large magnetic beads, it reduces the steps of magnetic bead cell separation, aspiration of target cell supernatant, and centrifugation, thus shortening the reaction time and reducing labor, material, and time costs.

[0048] 4. The entire cell separation process is completed in one cup, reducing cross-contamination caused by changing test tubes or containers during the separation process in existing technologies;

[0049] 5. The entire device has a simple structure, is easy to assemble and disassemble, and is easy to clean and disinfect.

[0050] It should be noted that the above embodiment combines two adjacent separating cups to form a separating cup assembly. In other embodiments, three or four separating cups can also be combined according to the actual situation, with multiple guide plate assemblies correspondingly set for snap-fit ​​engagement. As long as the separating cups are arranged in a V-shape, they should fall within the protection scope of this application. The following is a brief description of the case where three separating cups are combined to form a separating cup assembly.

[0051] Three adjacent separation cups are combined to form a separation cup assembly, which is integrally molded.

[0052] The separating seat includes a partition, with multiple receiving slots below the partition for accommodating magnets. Above the partition are a first guide plate assembly and a second guide plate assembly perpendicular to the partition. The first guide plate assembly includes a first guide plate and a second guide plate arranged in parallel and spaced apart. The second guide plate assembly includes a third guide plate and a fourth guide plate arranged in parallel and spaced apart. The separating cup assembly includes a first separating cup, a second separating cup, a third separating cup, a first connecting block, and a second connecting block. The first connecting block connects between the first separating cup and the second separating cup, and the second connecting block connects between the second separating cup and the third separating cup. Both the first connecting block and the second connecting block are inverted V-shaped. The first connecting block has a first locking groove and a second locking groove at the connection points with the first separating cup and the second separating cup, respectively. The first locking groove engages with the first guide plate, and the second locking groove engages with the second guide plate. The second connecting block has a third locking groove and a fourth locking groove at the connection points with the second separating cup and the third separating cup, respectively. The third locking groove engages with the third guide plate, and the fourth locking groove engages with the fourth guide plate.

[0053] In one embodiment, the steps for separating cells using the above-described separation device are as follows:

[0054] The mixture of magnetic beads conjugated with the target cell-corresponding antibody and cell sample solution is incubated at 2–8°C for 10–40 minutes, or 30 minutes on ice. Preferably, the incubation temperature is 3°C–8°C, and more preferably, the incubation temperature is 4°C.

[0055] Add the incubated magnetic beads and cell sample solution to the separation cup, insert the separation cup into the separation seat, and let it stand at room temperature for 1-3 minutes. At this time, the magnetic beads coupled with the target cells will precipitate to the bottom of the separation cup under the action of the magnetic field.

[0056] Line the biosafety cabinet with sterile gauze, invert the separation cup and separation seat, and use the absorbency of the gauze to absorb the solution in the separation cup. Compared to using a pipette to aspirate the supernatant, this step removes as much supernatant as possible from the separation cup, improving the purity of the target cells.

[0057] Remove the separation cup; the positive cells coupled with the magnetic beads that have precipitated in the separation cup can be used directly for the next step.

[0058] By implementing this method, the separation process, compared to methods using separation columns, reduces the steps of rinsing the small magnetic bead separation column, three repeated rinsings, target cell rinsing, and centrifugation. Compared to large magnetic bead separation methods, it reduces the steps of magnetic bead shearing, aspiration of supernatant containing target cells, and centrifugation. This shortens the separation time and reduces labor, material, and time costs. Furthermore, the device is simple, easy to clean and sterilize. The separation unit is also reusable, offering greater flexibility.

[0059] To improve cell purity, 50–500 μL of cell culture medium can be added to the precipitated magnetic beads and coupled cells to resuspend them. This process is repeated to complete the washing step. Washing 1–3 times is acceptable; this washing step can increase cell purity by 5%.

[0060] In another embodiment, the steps of separating cells using the above-described separation device are as follows:

[0061] A mixture of one or more magnetic beads conjugated with antibodies corresponding to non-target cells and cell sample solution is incubated at 2–8°C for 10–40 minutes, or 30 minutes on ice. Preferably, the incubation temperature is 3°C–8°C, and more preferably, the incubation temperature is 4°C.

[0062] Add the incubated magnetic beads and cell sample solution to the separation cup, insert the separation cup into the separation seat, and let it stand at room temperature for 1-3 minutes. At this time, the magnetic beads coupled with non-target cells will precipitate to the bottom of the separation cup under the action of the magnetic field.

[0063] Collect the cells from the supernatant in the separation cup, and discard any non-target cells that have adsorbed onto the magnetic beads and precipitated at the bottom of the separation cup due to magnetic attraction. Carefully aspirate the supernatant with a pipette without touching the magnetic beads and transfer it to another separation cup (or sterile container). The cells contained in the supernatant are the target cells.

[0064] Similarly, to improve cell purity, magnetic beads conjugated with antibodies corresponding to non-target cells can be added to the supernatant again, and the above steps can be repeated to complete the washing step. Washing 1 to 3 times is acceptable.

[0065] Please see Figure 4 , Figure 4 This is a schematic flowchart of the first embodiment of the cell separation method of this application. In this embodiment, the cell separation method uses the cell separation device of any of the above embodiments or implementations for separation, and includes the following steps:

[0066] S410: Provide cell sample solution, which includes at least type I cells.

[0067] Cell sample solutions can be obtained by processing collected biological samples. Different biological samples can be processed according to existing corresponding processing methods, and no limitation is made here.

[0068] S420: Mix the cell sample solution and type I magnetic beads in a separation cup. Type I antibodies are coupled to the type I magnetic beads for binding to type I cells.

[0069] The first type of magnetic beads has a diameter of 500–5000 nanometers, such as 500 nm, 1000 nm, 1500 nm, 2000 nm, and 4000 nm. This method uses magnetic beads of medium size, does not damage cells, and achieves high separation purity.

[0070] S430: Place the separation cup containing the first type of magnetic beads and cell sample solution above the separation seat with a magnet, so that the magnet attracts the first type of magnetic beads to the bottom of the separation cup.

[0071] The first type of magnetic beads utilizes the first type of antibody to bind to the first cell.

[0072] S440: Separate the first type of magnetic beads from the cell supernatant.

[0073] Among them, magnetic fields are used to adsorb magnetic beads, so that cells bound to magnetic beads can be separated from other cells, and the cell supernatant can be removed by direct pouring.

[0074] By implementing this method, when separating cells using small magnetic beads, a separation column is no longer needed. Instead, the magnetic beads are directly separated from the cell supernatant, which can improve the separation speed, reduce the separation cost, and improve the separation purity.

[0075] After separating the first type of magnetic beads from the cell supernatant, the first target cells can be obtained. These first target cells are the first type of cells coupled with the first type of magnetic beads and precipitated in the separation vessel. These first type of cells coupled with the first type of magnetic beads can be directly used in the next step. Specifically, the separation vessel is removed from the separation holder, and cell culture medium is added to the separation vessel to obtain the first target cell sample solution, which can be directly used in the next step. Alternatively, the first type of magnetic beads can be separated from the first type of cells before use in the next step.

[0076] In one embodiment, the cell sample solution can be added to a separation cup, and 0.3 to 2.0 times the amount of first-type magnetic beads can be added to the cell sample solution. The mixture is then prepared in the separation cup to obtain a cell mixture. Alternatively, the cell sample solution can be mixed with 0.3 to 2.0 times the amount of first-type magnetic beads first to obtain a cell mixture, and then the cell mixture can be added to the separation cup. This is not a limitation.

[0077] In one embodiment, after adding the first type of magnetic beads to the cell sample solution, a shaker or mixer can be used to thoroughly mix the first type of magnetic beads with the first type of cells, thereby improving separation efficiency. During the mixing process, the shaking or mixing speed should be controlled to ensure that the cells are not damaged. After thoroughly mixing the first type of magnetic beads with the first type of cells, the separation cup containing the cell mixture is then placed onto the separation seat.

[0078] In one embodiment, the amount of the first type of magnetic beads added is 0.3 to 2.0 times that of the cell sample solution, such as 0.3, 0.5, 1.0, 1.3, 1.8, 2.0, etc. The magnetic beads used in this method have small particle sizes and strong adsorption capacity, thus requiring a smaller amount of magnetic beads, reducing the amount used and lowering costs.

[0079] In one embodiment, after removing the cell supernatant, cell culture medium can be added to the first type of magnetic beads to wash the first type of cells. Adding cell culture medium resuspends the magnetic beads and cells, and then placing them in a magnetic field to adsorb and remove the supernatant, completing the washing step. Washing can be performed 1 to 3 times. Washing can improve the purity of the separated cells, increasing the purity by at least 5%.

[0080] After washing, cell culture medium is added to the first type of magnetic beads; the magnetic beads and cells are then resuspended to obtain the first type of cells. The magnetic beads are composed of non-toxic iron oxide and polysaccharides, and are biodegradable, so subsequent operations can be performed directly without separation.

[0081] In one embodiment, this method can be used to separate positive cells, where the magnetic beads used are magnetic beads carrying antibodies corresponding to the positive cells. It can also be used to separate negative cells. Direct magnetic cell labeling can be used to bind the magnetic beads to the target cells. Indirect magnetic cell labeling can also be used, where the magnetic beads bind to unwanted cells. In this case, the target cells are in the cell supernatant, which can be further sorted to obtain the target cells. The cells in the cell supernatant can also be further sorted. Specifically, the cell sample solution also includes a second type of cells. The cell supernatant and the second type of magnetic beads are mixed in another separation cup. The second type of magnetic beads are coupled with a second type of antibody for binding to the second type of cells. The separation cup containing the second type of magnetic beads and the cell supernatant is placed above a separation seat with a magnet. The second type of magnetic beads are separated from the cell supernatant. The separation cup is removed from the separation seat to obtain the second target cells. The second target cells are second type of cells precipitated in the separation cup and coupled with the second type of magnetic beads. The second target cells obtained at this time can be directly used for cell applications and other operations.

[0082] The above description is merely an embodiment of this application and does not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A cell separation method, characterized in that, The cell separation method utilizes a cell separation device for the separation step, the cell separation device comprising: A separation seat, comprising a partition and a side plate, wherein a plurality of receiving grooves are provided below the partition and a guide plate assembly perpendicular to the partition is provided above the partition, the guide plate assembly comprising a first guide plate and a second guide plate arranged in parallel and spaced apart, the side plate being disposed on one side of the partition and perpendicular to the partition and the guide plate assembly, and the side plate being provided with a limiting hole; Multiple separation cups are provided, with two adjacent separation cups connected by a connecting block to form a separation cup assembly. The separation cup assembly is integrally formed, and the connecting block is inverted V-shaped. The connecting block is provided with a first locking groove and a second locking groove at the connection points with two adjacent separation cups. The first locking groove is engaged with the first guide plate, and the second locking groove is engaged with the second guide plate. The connecting block is provided with a limiting rod protruding outward, and the limiting hole is adapted to the limiting rod. Multiple magnets are correspondingly disposed in the receiving groove, and each magnet corresponds to a separation cup; The cell separation method includes: Provide a cell sample solution, wherein the cell sample solution comprises at least type I cells; The cell sample solution and the first type of magnetic beads are mixed in a separation cup. The first type of magnetic beads are bound with a first type of antibody for binding to the first type of cells. The diameter of the first type of magnetic beads is 500 to 4500 nanometers. The separation cup containing the first type of magnetic beads and the cell sample solution is placed above the separation seat with a magnet, so that the magnet attracts the first type of magnetic beads to the bottom of the separation cup; Invert the separation cup and the separation seat, and pour out the cell supernatant to separate the first type of magnetic beads from the cell supernatant.

2. The cell separation method according to claim 1, characterized in that, The process of separating the first type of magnetic beads from the cell supernatant also includes: The separation cup is removed from the separation seat to obtain the first target cell, which is a first type of cell that is precipitated in the separation cup and coupled with a first type of magnetic beads.

3. The cell separation method according to claim 2, characterized in that, After removing the separation cup from the separation seat, the process further includes: Cell culture medium is added to the separation cup to obtain the first target cell sample solution, which can be directly used for the next step.

4. The cell separation method according to claim 1, characterized in that, The step of mixing the cell sample solution and the first type of magnetic beads in a separation cup includes: Add the cell sample solution to the separation cup, and add 0.3 to 2.0 times the amount of the first type of magnetic beads to the cell sample solution to obtain a cell mixture; or The cell sample solution is mixed with 0.3 to 2.0 times the amount of the first type of magnetic beads to obtain a cell mixture, which is then added to the separation cup.

5. The cell separation method according to claim 1, characterized in that, The process of separating the first type of magnetic beads from the cell supernatant also includes: Remove the separation cup from the separation seat, add cell culture medium to the separation cup, and resuspend the first type of magnetic beads and the first type of cells; Place the separation cup above the separation seat with the magnet again; Separate the first type of magnetic beads from the cell supernatant.

6. The cell separation method according to claim 1, characterized in that, The cell sample solution also includes a second type of cells, and after separating the first type of magnetic beads from the cell supernatant, the following steps are also included: Collect the cell supernatant, and mix the cell supernatant and the second type of magnetic beads in another separation cup. The second type of magnetic beads are bound with a second type of antibody for binding to the second type of cells. The separation cup containing the second type of magnetic beads and the cell supernatant is placed above the separation seat with the magnet; Separate the second type of magnetic beads from the cell supernatant; The separation cup is removed from the separation seat to obtain the second target cell, which is a second type of cell that has been deposited in the separation cup and coupled with a second type of magnetic beads.

7. The cell separation method according to claim 1, characterized in that, The process of mixing the cell sample solution and the first type of magnetic beads in the separation cup also includes: The cell sample solution containing the first type of magnetic beads was incubated at 2–8°C for 10–40 minutes.

8. The cell separation method according to claim 1, characterized in that, The first type of cells are positive cells, and the first type of magnetic beads are magnetic beads with antibodies corresponding to the positive cells.

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