A magnetic microsphere and its application in cell sorting

Abstract

The present invention relates to a magnetic microsphere and its application in cell sorting. The magnetic microsphere includes magnetite magnetic nanoparticles with dextran modified on the outer surface and specific antibodies coupled to the dextran. Organic acids are mixed in the magnetite magnetic nanoparticles; wherein, the organic acid is selected from one or more of tartaric acid, malic acid, and citric acid. The magnetic microsphere of the present invention has good sorting effects in the field of cell sorting.

Description

Technical Field

[0001] The present invention relates to the technical field of magnetic microspheres, and particularly to a magnetic microsphere and its application in cell sorting. Background Art

[0002] Cell sorting technology is a key technology in modern biomedical research, which allows researchers to isolate specific cell subsets from complex cell populations based on cell surface markers or cell intrinsic properties. This technology is of great significance for the research of disease mechanisms, drug screening, cell therapy, and the discovery of biomarkers.

[0003] Traditional cell sorting methods include flow cytometry (FACS) and magnetic bead sorting technology. Flow cytometry relies on the physical properties of cells, such as size, granularity, and electromagnetic properties, for rapid sorting, but its operation is complex, the cost is high, and it may cause damage to cells. Magnetic bead sorting technology utilizes the property of specific binding between magnetic microspheres and cell surfaces to achieve cell separation by applying an external magnetic field. This method has the advantages of simple operation, good separation effect, and little damage to cells. Therefore, magnetic bead sorting technology has become a commonly used technology for cell sorting.

[0004] The preparation and surface modification technology of magnetic microspheres are the core of magnetic bead sorting technology. Early magnetic microspheres were mostly micron-sized, and the surface modification technology was relatively simple, but there were problems such as low separation efficiency and the risk of cell damage. With the development of nanotechnology, superparamagnetic nanomicrospheres have gradually become a research hotspot in the field of cell sorting due to their unique physical and chemical properties, such as high magnetism, small size, and good biocompatibility. These nanomicrospheres can carry specific antibodies through surface modification to achieve specific recognition and separation of target cells.

[0005] Currently, there are various types of magnetic microspheres applied to cell sorting. Which specific type of magnetic microspheres is more suitable for cell sorting and can achieve an ideal sorting effect of purity and recovery rate is still a challenge for magnetic microspheres used in cell sorting and an urgent problem to be solved in current cell sorting technology. Summary of the Invention

[0006] Aiming at the defects of the above-mentioned prior art, the present invention aims to provide a magnetic microsphere (magnetic bead) with a simple preparation process, environmental friendliness, and safety for application in cell sorting.

[0007] Solutions for Solving the Problem:

[0008] The present invention provides an application of magnetic microspheres in the preparation of cell sorting reagent products. The magnetic microspheres include iron oxide magnetic nanoparticles with dextran modified on the outer surface and specific antibodies conjugated to the dextran. Organic acids are mixed in the iron oxide magnetic nanoparticles; wherein, the organic acids are selected from one or more of tartaric acid, malic acid, and citric acid, preferably citric acid and / or tartaric acid.

[0009] Preferably, the molar ratio of the dextran to the organic acid in the feed is 1:(5 - 15), preferably 1:(7 - 15), more preferably 1:(10 - 15).

[0010] Preferably, the particle size of the magnetic microspheres is 40 - 70 nm, preferably 45 - 65 nm.

[0011] Preferably, the specific antibody is selected from one or more of CD8, CD4, CD45, CD3, CD56, and CD34.

[0012] Preferably, the preparation method of the magnetic microspheres includes:

[0013] (1) Reacting ferric salt and ferrous salt in the presence of dextran, organic acid, and base to obtain magnetic nanoparticles;

[0014] (2) Reacting the magnetic nanoparticles obtained in step (1) in the presence of a carboxylating reagent to obtain carboxylated magnetic nanoparticles;

[0015] (3) Conjugating the carboxylated magnetic nanoparticles with an antibody to obtain the magnetic microspheres;

[0016] Wherein, the organic acids are selected from one or more of tartaric acid, malic acid, and citric acid, preferably citric acid and / or tartaric acid.

[0017] Preferably, in step (1), the total molar concentration ratio of the ferrous salt, ferric salt, dextran, and organic acid is (1 - 10):(5 - 15):1, preferably (2 - 6):(5 - 12):1, more preferably (3 - 5):(6 - 10):1, and most preferably 4:8:1.

[0018] Preferably, the molar ratio of the dextran to the organic acid is 1:(5 - 15), preferably 1:(7 - 15), more preferably 1:(10 - 15).

[0019] Preferably, the weight - average molecular weight of the dextran is 4000 - 8000.

[0020] Preferably, the ferric salt is ferric chloride or ferric sulfate, preferably ferric chloride hexahydrate, and the ferrous salt is ferrous chloride or ferrous sulfate, preferably ferrous chloride tetrahydrate.

[0021] Preferably, the base in step (1) is sodium hydroxide, and the concentration of the sodium hydroxide is 0.05 - 0.3 M, preferably 0.05 - 0.2 M.

[0022] Preferably, the reaction in step (1) is carried out in the presence of solvent 1, and the solvent 1 is selected from one or more of ethylene glycol, propylene glycol, butylene glycol, glycerol, and hexanediol.

[0023] Preferably, the reaction in step (1) is controlled to be carried out at 140 - 180 °C for 10 - 20 hours.

[0024] Preferably, in step (2), the mass ratio of the magnetic nanoparticles to the carboxylating agent is (1 - 20):1, preferably (1 - 10):1, and more preferably (2 - 8):1.

[0025] Preferably, the carboxylating agent is selected from one or more of dihydro - 2,5 - furandione, maleic anhydride, acrylic anhydride, and hexanoic anhydride.

[0026] Preferably, the reaction in step (2) is carried out in the presence of solvent 2, and the solvent 2 is selected from one or more of DMF, tetrahydrofuran, and acetonitrile.

[0027] Preferably, the reaction in step (2) is controlled to be carried out at 50 - 100 °C for 10 - 40 hours.

[0028] Preferably, the reaction in step (2) is carried out in the presence of a catalyst, and the catalyst is selected from one or more of triethylamine, ethylenediamine, and 1,6 - hexanediamine.

[0029] Preferably, in steps (1) and (2), a post - treatment step is included after the reaction.

[0030] Preferably, the post - treatment step in step (1) includes centrifuging and washing the reaction product to obtain the magnetic nanoparticles.

[0031] Preferably, the post - treatment step in step (2) includes centrifuging, dialyzing, and sorting the reaction product to obtain the carboxylated magnetic nanoparticles.

[0032] Preferably, the coupling ratio of the carboxylated magnetic nanoparticles to the specific antibody is (1 - 5) mg:200 μg, preferably (1 - 3) mg:200 μg.

[0033] Effects of the invention

[0034] The magnetic microspheres provided by the present invention have the following advantages: (1) They can be applied to the field of cell sorting and can solve the problem of low purity and recovery rate in current cell sorting; (2) They can be prepared by simple and low-cost methods, are easy to scale up production, and make the preparation and application of magnetic beads more convenient and economical; (3) There are no toxic reagents in the preparation process, and the magnetic microspheres have biodegradability and no pollution to the environment, meeting the current requirements of green and environmental protection for scientific research and industrial production.

[0035] The characteristics of high purity and high recovery rate of the magnetic microspheres of the present invention in cell sorting applications enable them to be used as a preferred domestic alternative to cell sorting magnetic beads, meeting the cell sorting needs of biomedical research and clinical applications, and thus contributing to the development of fields such as cell biology, disease mechanism research, drug screening, and cell therapy, and having important value and significance in practical applications. Brief Description of the Drawings

[0036] Figure 1 It is the electron microscope image of the magnetic nanoparticles prepared in Example 3;

[0037] Figure 2 It is the scatter plot of cell sorting of the magnetic microspheres prepared in Examples 4, 9, 12, and 15, where Figure 2 a) is the scatter plot of cell sorting of the magnetic microspheres prepared in Example 4, b) is the scatter plot of cell sorting of the magnetic microspheres prepared in Example 9, c) is the scatter plot of cell sorting of the magnetic microspheres prepared in Example 12, and d) is the scatter plot of cell sorting of the magnetic microspheres prepared in Example 15. Detailed Description of the Embodiments

[0038] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following is a detailed description by listing specific examples. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical and scientific fields to which this application belongs.

[0039] The present invention provides an application of magnetic microspheres in the preparation of cell sorting reagent products. The magnetic microspheres include magnetite magnetic nanoparticles with dextran modified on the outer surface and specific antibodies coupled to the dextran. Organic acids are mixed in the magnetite magnetic nanoparticles; wherein, the organic acids are selected from one or more of tartaric acid, malic acid, and citric acid.

[0040] In some embodiments, the organic acid is citric acid and / or tartaric acid.

[0041] In certain embodiments, the molar ratio of the dextran to the organic acid in the feed is 1:(5 - 15), such as 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc.

[0042] In certain embodiments, the molar ratio of the dextran to the organic acid in the feed is 1:(7 - 15).

[0043] In certain embodiments, the molar ratio of the dextran to the organic acid in the feed is 1:(10 - 15).

[0044] In certain embodiments, the particle size of the magnetic microspheres is 40 - 70 nm, such as 42 nm, 44 nm, 46 nm, 48 nm, 50 nm, 52 nm, 54 nm, 56 nm, 58 nm, 60 nm, 62 nm, 64 nm, 66 nm, 68 nm, 70 nm, etc.

[0045] In certain embodiments, the particle size of the magnetic microspheres is 45 - 65 nm.

[0046] In the present application, the antibody can specifically bind to the antigen on the surface of target cells. Based on the sorting requirements of different types of cells, different antibodies can be adaptively conjugated to the surface of the magnetic microspheres provided in the present application. For example, in certain embodiments, the specific antibody is selected from one or more of the antibodies CD8, CD4, CD45, CD3, CD56, and CD34. In certain embodiments, if the magnetic microspheres provided by the present invention are used for sorting T cells expressing different marker molecules, then the specific antibody is selected from one or more of the antibodies CD8, CD4, and CD3.

[0047] In certain embodiments, the method for preparing the magnetic microspheres includes:

[0048] (1) Reacting an iron salt and a ferrous salt in the presence of dextran, an organic acid, and a base to obtain magnetic nanoparticles;

[0049] (2) Reacting the magnetic nanoparticles obtained in step (1) in the presence of a carboxylating agent to obtain carboxylated magnetic nanoparticles;

[0050] (3) Conjugating the carboxylated magnetic nanoparticles with an antibody to obtain the magnetic microspheres;

[0051] Wherein, the organic acid is selected from one or more of tartaric acid, malic acid, and citric acid.

[0052] In certain embodiments, the organic acid is selected from citric acid and / or tartaric acid.

[0053] In some embodiments, in step (1), the total molar concentration ratio of the ferrous salt, ferric salt, dextran, and organic acid is (1 to 10):(5 to 15):1.

[0054] In some embodiments, the total molar concentration ratio of the ferrous salt, ferric salt, dextran, and organic acid is (2 to 6):(5 to 12):1.

[0055] In some embodiments, the total molar concentration ratio of the ferrous salt, ferric salt, dextran, and organic acid is (3 to 5):(6 to 10):1.

[0056] In some embodiments, the total molar concentration ratio of the ferrous salt, ferric salt, dextran, and organic acid is 4:8:1.

[0057] In some embodiments, the molar ratio of the dextran to the organic acid is 1:(5 to 15), such as 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc.

[0058] In some embodiments, the molar ratio of the dextran to the organic acid is 1:(7 to 15).

[0059] In some embodiments, the molar ratio of the dextran to the organic acid is 1:(10 to 15).

[0060] In some embodiments, the weight-average molecular weight of the dextran is 4000 to 8000, such as 4500, 5000, 5500, 6000, 6500, 7000, 7500, etc.

[0061] In some embodiments, the ferric salt is ferric chloride or ferric sulfate.

[0062] In some embodiments, the ferric salt is ferric chloride hexahydrate.

[0063] In some embodiments, the ferrous salt is ferrous chloride or ferrous sulfate.

[0064] In some embodiments, the ferrous salt is ferrous chloride tetrahydrate.

[0065] In some embodiments, in step (1), the base is sodium hydroxide, and the concentration of the sodium hydroxide is 0.05 to 0.3 M, such as 0.06 M, 0.08 M, 0.1 M, 0.12 M, 0.14 M, 0.16 M, 0.18 M, 0.2 M, 0.25 M, 0.3 M, etc.

[0066] In some embodiments, the concentration of the sodium hydroxide is 0.05 to 0.2 M.

[0067] In some embodiments, the concentration of the sodium hydroxide is 0.1 M.

[0068] In some embodiments, the reaction in step (1) is carried out in the presence of solvent 1, and the solvent 1 is selected from one or more of ethylene glycol, propylene glycol, butylene glycol, glycerol, and hexanediol.

[0069] In some embodiments, the solvent 1 is selected from ethylene glycol.

[0070] In some embodiments, the reaction in step (1) is controlled to be carried out at 140 - 180 °C (such as 145 °C, 150 °C, 155 °C, 160 °C, 165 °C, 170 °C, 175 °C, etc.) for 10 - 20 hours (such as 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, etc.).

[0071] In some embodiments, in step (2), the feeding mass ratio of the magnetic nanoparticles to the carboxylating agent is (1 - 20):1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, etc.

[0072] In some embodiments, the feeding mass ratio of the magnetic nanoparticles to the carboxylating agent is (1 - 10):1.

[0073] In some embodiments, the feeding mass ratio of the magnetic nanoparticles to the carboxylating agent is (2 - 8):1.

[0074] In some embodiments, the feeding mass ratio of the magnetic nanoparticles to the carboxylating agent is (3 - 6):1.

[0075] In some embodiments, the feeding mass ratio of the magnetic nanoparticles to the carboxylating agent is 5:1.

[0076] In some embodiments, the carboxylating agent is selected from one or more of dihydro - 2,5 - furandione, maleic anhydride, acrylic anhydride, and hexanoic anhydride.

[0077] In some embodiments, the carboxylating agent is selected from dihydro - 2,5 - furandione.

[0078] In some embodiments, the reaction in step (2) is carried out in the presence of solvent 2, and the solvent 2 is selected from one or more of DMF, tetrahydrofuran, and acetonitrile.

[0079] In certain embodiments, the solvent 2 is selected from DMF.

[0080] In certain embodiments, the reaction in step (2) is carried out at 50-100 °C (such as 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 95 °C, etc.) for 10-40 hours (such as 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, etc.).

[0081] In certain embodiments, the reaction in step (2) is carried out in the presence of a catalyst, and the catalyst is selected from one or more of triethylamine, ethylenediamine, and 1,6-hexanediamine.

[0082] In certain embodiments, the catalyst in step (2) is selected from triethylamine.

[0083] In certain embodiments, the reaction in step (2) is carried out in the presence of a co-solvent, and the co-solvent is pyridine.

[0084] In certain embodiments, in steps (1) and (2), after the reaction, a post-treatment step is included.

[0085] In certain embodiments, the post-treatment step in step (1) includes centrifuging and washing the reaction product to obtain the magnetic nanoparticles.

[0086] In certain embodiments, the post-treatment step in step (2) includes centrifuging, dialyzing, and sorting the reaction product to obtain the carboxylated magnetic nanoparticles.

[0087] In certain embodiments, step (3) specifically includes the following steps:

[0088] Activating the carboxylated magnetic nanoparticles;

[0089] Coupling the carboxylated magnetic nanoparticles with a specific antibody and blocking to obtain the magnetic microspheres.

[0090] In certain embodiments, the coupling ratio of the carboxylated magnetic nanoparticles to the specific antibody is (1-5) mg: 200 μg, such as 1 mg: 200 μg, 2 mg: 200 μg, 3 mg: 200 μg, 4 mg: 200 μg, 5 mg: 200 μg, etc.

[0091] In certain embodiments, the coupling ratio of the carboxylated magnetic nanoparticles to the specific antibody is (1-3) mg: 200 μg.

[0092] In some embodiments, the coupling ratio of the carboxylated magnetic nanoparticles to the specific antibody is 2 mg: 200 μg.

[0093] In some embodiments, the activation is carried out using an activation reagent, and the components contained in the activation reagent and the concentration of each component are: 50 - 80 mg / ml EDC, 50 - 80 mg / ml NHS, 3 - 5 g / L MES, and 20 - 35 g / L NaCl.

[0094] In some embodiments, the components contained in the activation reagent and the concentration of each component are: 60 mg / ml EDC, 60 mg / ml NHS, 4.265 g / L MES, and 28 g / L NaCl.

[0095] In some embodiments, the coupling is carried out using a coupling reagent, and the components contained in the coupling reagent and the concentration of each component are 5 - 10 g / L NaCl, 0.1 - 1 g / L KCl, 2 - 5 g / L Na2HPO4·12H2O, and 0.1 - 1 g / L KH2PO4.

[0096] In some embodiments, the components contained in the coupling reagent and the concentration of each component are: 8 g / L NaCl, 0.2 g / L KCl, 3.63 g / L Na2HPO4·12H2O, and 0.24 g / L KH2PO4.

[0097] In some embodiments, the pH of the coupling reagent is 7.4.

[0098] In some embodiments, the blocking is carried out using a blocking solution, and the components contained in the blocking solution and the concentration of each component are: 5 - 10 g / L Tris, 5 - 15 g / L BSA, 0.1 - 2 mL / L PC300, and 1 - 5 mL / L ethanolamine.

[0099] In some embodiments, the components contained in the blocking solution and the concentration of each component are: 6.6 g / L Tris, 10 g / L BSA, 1 mL / L PC300, and 3 mL / L ethanolamine.

[0100] In some embodiments, the pH of the blocking solution is 7.4.

[0101] Reagents used in the following examples:

[0102] Activation buffer: MES 4.265 g / L, NaCl 28 g / L, pH 6.0;

[0103] Activation solution (activation reagent): EDC (60 mg / ml), NHS (60 mg / ml) (prepared with activation buffer, freshly prepared before use);

[0104] Coupling buffer (coupling reagent): NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4·12H2O 3.63 g / L, KH2PO4 0.24 g / L, pH 7.4;

[0105] Blocking solution: Tris 6.6 g / L, BSA 10 g / L, PC300 1 mL / L, ethanolamine 3 mL / L, pH 7.4;

[0106] Magnetic bead storage solution: Tris 6.6 g / L, BSA 0.5 g / L, Tween 20 0.5 mL / L, PC300 1 mL / L, pH 7.4.

[0107] The present invention will be further described below through specific examples. In this article, if not otherwise specified, "%" represents mass percentage. The materials and reagents in the following examples, unless otherwise specified, are commonly used materials or reagents in the art, and can all be obtained commercially or synthesized by known methods. The experimental methods without specified conditions in the following implementation cases are usually carried out according to conventional experimental conditions or the conditions recommended by the manufacturers of relevant reagents (kits). Example 1

[0108] (1) Preparation of magnetic nanoparticles

[0109] Take ferrous chloride tetrahydrate and ferric chloride hexahydrate and dissolve them in 200 ml of ethylene glycol (the concentration of ferrous chloride tetrahydrate is 8 mmol / L, and the concentration of ferric chloride hexahydrate is 16 mmol / L), add dextran with a weight average molecular weight of 1000 (concentration of 2 mmol / L); after stirring evenly, pour it into a 500 ml four-necked flask, add 0.1 M NaOH and stir evenly, carry out condensation reflux, heat up to 160 °C, keep the temperature constant for reaction for 16 hours, cool to room temperature, then centrifuge and wash the reaction solution with ethanol and deionized water, concentrate to 25 mg / ml, add 0.05% ProClin300, and store at 4 °C for later use.

[0110] It was determined by transmission electron microscopy that the diameter of the prepared magnetic nanoparticles was 16 ± 3 nm.

[0111] (2) Carboxyl modification of magnetic nanoparticles

[0112] Weigh 20 ml of the above-mentioned magnetic nanoparticles after dehydration, add 200 ml of DMF and 3 ml of pyridine, stir evenly, pour into a reaction kettle, then add 0.1 g of dihydro-2,5-furandione (dissolved in 50 ml of DMF) and 1 ml of triethylamine, heat up to 80 °C, react for 24 hours, cool to room temperature, centrifuge at 2000 rpm to remove large precipitate particles, pour the supernatant into a dialysis bag (cut-off molecular weight is 14000), dialyze for 3 days, take out and collect by passing through a Miltenyi sorting column (MACS LS Columns) to obtain carboxyl-modified magnetic nanoparticles, adjust the concentration to 2 mg / ml, add 0.05% of ProClin300, and store at 4 °C for later use.

[0113] (3)Antibody labeling (taking CD8 antibody as an example, antibody clone number is HIT8a (purchased from Biolegend, catalog number 300902))

[0114] Take 1 ml of the magnetic nanoparticles prepared in step (2), add them into a sorting column, place it on a magnetic sorting rack, rinse three times with activation buffer, remove the magnetic sorting rack, collect the magnetic nanoparticles in the sorting column into a 5 ml EP tube, add 2 ml of activation buffer and 50 μl of activation solution (EDC and NHS), place it on a constant temperature oscillator at 37 °C for activation for 30 min; pour the magnetic nanoparticle suspension into the sorting column, place it on a magnetic sorting rack, rinse three times with coupling buffer, remove the magnetic sorting rack, collect the magnetic nanoparticles in the sorting column into a 5 ml EP tube, add 2 ml of coupling buffer and 200 μg of CD8 antibody, place it on a constant temperature oscillator at 37 °C for activation for 2 h; pour the magnetic bead suspension into the sorting column, place it on a magnetic sorting rack, rinse three times with blocking solution, remove the magnetic sorting rack, collect the magnetic beads in the sorting column into a 5 ml EP tube, add 2 ml of blocking solution, place it on a constant temperature oscillator at 37 °C for blocking for 30 min; pour the magnetic bead suspension into the sorting column, place it on a magnetic sorting rack, rinse three times with magnetic bead storage solution, remove the magnetic sorting rack, collect the magnetic beads in the sorting column into a 5 ml EP tube, adjust the concentration to 1 mg / mL, store at 4 °C for later use to obtain magnetic microspheres conjugated with specific antibodies. Example 2

[0115] The conditions and methods for preparing magnetic beads in Example 2 are basically the same as those in Example 1, and the difference from Example 1 is that the weight-average molecular weight of dextran is 2000. Example 3

[0116] The conditions and methods for preparing magnetic beads in Example 3 are basically the same as those in Example 1, and the difference from Example 1 is that the weight-average molecular weight of dextran is 4000. The electron micrograph of the magnetic beads prepared in Example 3 is as Figure 1 shown. Example 4

[0117] Example 4 The method for preparing magnetic beads is basically the same as that in Example 1, except that the weight-average molecular weight of dextran is 6000. Example 5

[0118] Example 5 The method for preparing magnetic beads is basically the same as that in Example 1, except that the weight-average molecular weight of dextran is 8000. Example 6

[0119] Example 6 The method for preparing magnetic beads is basically the same as that in Example 1, except that the weight-average molecular weight of dextran is 20000. Serious aggregation of the prepared magnetic nanoparticles occurred in this example. Example 7

[0120] Example 7 The method for preparing magnetic beads is basically the same as that in Example 1, except that the weight-average molecular weight of dextran is 40000. Serious aggregation of the prepared magnetic nanoparticles occurred in this example.

[0121] The following method was used to verify the cell sorting performance of the magnetic microspheres prepared in Examples 1-5 for human peripheral blood mononuclear cells (PBMC) (the cell sorting results of different individuals will vary, and the same sample was used in the following performance verification of the present invention):

[0122] Resuspend human peripheral blood mononuclear cells in MACS buffer, take a sample for counting, and transfer 1x10 7 cells into a 1.5 mL EP tube and centrifuge at 1500 rpm for 5 min. Discard the supernatant, resuspend the cell pellet in 100 μL of MACS buffer solution, then add 20 μL of the CD8 sorting magnetic beads from Examples 1-5 above, mix well, and place it in a 2-8 °C refrigerator for incubation for 15 min. Place the sorting column on the magnetic separation rack and rinse it twice with 1 mL of MACS buffer. Take out the incubated sample from the 2-8 °C refrigerator, add 1 mL of MACS buffer, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Add 1 mL of MACS buffer to resuspend the sample, and then add the sample to the sorting column. Collect the effluent with a 15 mL centrifuge tube, and wash the sorting column 3 times with 2 mL of MACS buffer, collect the effluent to obtain unlabeled cells. After all the MACS buffer has flowed out, remove the sorting column from the magnetic separation rack and place it in another new 15 mL centrifuge tube. Add 2 mL of MACS buffer to the sorting column and push the piston of the LS sorting column to directly wash down the magnetically labeled cells. Place the 15 mL centrifuge tube containing the collected liquid in a horizontal centrifuge and centrifuge at 1500 rpm for 5 min. After centrifugation, pour out the supernatant to obtain the sorted cells.

[0123] The results of the effects of different dextran weight-average molecular weights on the sorting purity and yield of CD8+ T cells are shown in Table 1 below.

[0124] Table 1

[0125]

[0126] As can be seen from Table 1, as the dextran weight-average molecular weight increases, the particle size of the magnetic microspheres shows an increasing trend (the particle size in Table 1 is the particle size of the microsphere solution measured by a laser particle size analyzer), but when the weight-average molecular weight is too large, severe particle aggregation occurs. At the same time, it was found in the cell sorting experiment that different dextran weight-average molecular weights have an impact on both the purity and recovery rate of cell sorting.

[0127] However, the sorting data in Table 1 have not yet reached the ideal sorting results, and the composition of the magnetic microspheres needs to be further optimized. From the results in Table 1, it was found that as the particle size decreases, the recovery rate shows a downward trend, mainly because the magnetic beads with a smaller particle size are prone to loss during the process of passing through the sorting column, resulting in a low final recovery rate. Therefore, magnetic beads with a smaller particle size are not suitable for cell sorting.

[0128] In order to further optimize the sorting performance of the cell sorting magnetic beads, based on the preparation processes of Examples 3, 4, and 5, further optimization was carried out on this basis. Example 8

[0129] The conditions and methods for preparing magnetic beads in Example 8 are basically the same as those described in Example 3, and the difference between it and Example 3 is that in Example 8, dextran and malic acid are added, and their total molar concentration is 2 mmol / L, where the molar ratio of dextran to malic acid is 1:5. Example 9

[0130] The conditions and methods for preparing magnetic beads in Example 9 are basically the same as those described in Example 4, and the difference between it and Example 4 is that in Example 9, dextran and malic acid are added, and their total molar concentration is 2 mmol / L, where the molar ratio of dextran to malic acid is 1:10. Example 10

[0131] The conditions and methods for preparing magnetic beads in Example 10 are basically the same as those described in Example 5, and the difference between it and Example 5 is that in Example 10, dextran and malic acid are added, and their total molar concentration is 2 mmol / L, where the molar ratio of dextran to malic acid is 1:15. Example 11

[0132] Example 11 The method for preparing magnetic beads is basically the same as that in Example 3, except that in Example 11, dextran and tartaric acid are added, and the total molar concentration of the two is 2 mmol / L. Among them, the molar ratio of dextran to tartaric acid is 1:5. Example 12

[0133] Example 12 The method for preparing magnetic beads is basically the same as that in Example 4, except that in Example 12, dextran and tartaric acid are added, and the total molar concentration of the two is 2 mmol / L. Among them, the molar ratio of dextran to tartaric acid is 1:10. Example 13

[0134] Example 13 The method for preparing magnetic beads is basically the same as that in Example 5, except that in Example 13, dextran and tartaric acid are added, and the total molar concentration of the two is 2 mmol / L. Among them, the molar ratio of dextran to tartaric acid is 1:15. Example 14

[0135] Example 14 The method for preparing magnetic beads is basically the same as that in Example 3, except that in Example 14, dextran and citric acid are added, and the total molar concentration of the two is 2 mmol / L. Among them, the molar ratio of dextran to citric acid is 1:5. Example 15

[0136] Example 15 The method for preparing magnetic beads is basically the same as that in Example 4, except that in Example 15, dextran and citric acid are added, and the total molar concentration of the two is 2 mmol / L. Among them, the molar ratio of dextran to citric acid is 1:10. Example 16

[0137] Example 16 The method for preparing magnetic beads is basically the same as that in Example 5, except that in Example 16, dextran and citric acid are added, and the total molar concentration of the two is 2 mmol / L. Among them, the molar ratio of dextran to citric acid is 1:15.

[0138] Referring to the above cell sorting method, the CD8+ T cell sorting performance of the magnetic microspheres prepared in Examples 8-16 was measured, and the measurement results are shown in Table 2 and Figure 2 as shown below, Figure 2 Scatter plots of cell sorting using the magnetic microspheres prepared in Examples 4, 9, 12, and 15 are respectively shown.

[0139] Table 2

[0140]

[0141] As can be seen from Table 2, adding organic acids on the basis of the corresponding preferred embodiments in Table 1 can significantly improve the purity and recovery rate of CD8+ T cell sorting. Currently, there are no industry standard requirements for the purity and recovery rate of cell sorting. The sorting data achieved by Miltenyi Biotec's similar products (generally, purity greater than 90% and recovery rate greater than 70%) are mainly used as a reference. The sorting effects of the above embodiments of this application using CD8 antibody to sort T cells can all reach the level of Miltenyi Biotec's similar products, and are far higher than the above general requirements.

[0142] Due to different types of sorted cells and different sorting application requirements, the requirements for the purity and recovery rate of sorted cells are different. Therefore, the dosage of organic acids can be adjusted based on different needs. As can be seen from Table 2, within the range of dextran:organic acid of 1:(5 - 15), the addition of tartaric acid has the best sorting effect on CD8+ T cells. In order to further analyze whether the ratio of dextran to tartaric acid affects the sorting effect, the following embodiments were further carried out. Example 17

[0143] The conditions and methods for preparing magnetic nanoparticles in Example 17 are basically the same as those described in Example 12, and the difference from Example 12 is that the molar ratio of dextran to tartaric acid is 1:2. Example 18

[0144] The conditions and methods for preparing magnetic nanoparticles in Example 18 are basically the same as those described in Example 12, and the difference from Example 12 is that the molar ratio of dextran to tartaric acid is 1:20.

[0145] Referring to the above cell sorting method, the CD8+ cell sorting performance of the magnetic microspheres prepared in Examples 17 - 18 was measured, and the measurement results are shown in Table 3.

[0146] Table 3

[0147] Example 19

[0148] The conditions and methods for preparing magnetic beads in Example 19 are basically the same as those described in Example 12, and the difference from Example 12 is that the CD8 antibody clone number in Example 19 is SK1 (purchased from Biolegend, catalog number 344702). Example 20

[0149] The conditions and methods for preparing magnetic beads in Example 20 are basically the same as those described in Example 10, and the difference from Example 10 is that the CD8 antibody clone number in Example 20 is SK1 (purchased from Biolegend, catalog number 344702). Example 21

[0150] Example 21 The method for preparing magnetic beads was basically the same as that in Example 12, except that the antibody used in Example 21 was a CD4 antibody with the clone number HIT4a (purchased from Beijing Kuangbo Biotech Co., Ltd., product number A6081). Example 22

[0151] Example 22 The method for preparing magnetic beads was basically the same as that in Example 13, except that the antibody used in Example 22 was a CD4 antibody with the clone number HIT4a (purchased from Beijing Kuangbo Biotech Co., Ltd., product number A6081). Example 23

[0152] Example 23 The method for preparing magnetic beads was basically the same as that in Example 15, except that the antibody used in Example 23 was a CD4 antibody with the clone number RPA-T4 (purchased from Beijing Kuangbo Biotech Co., Ltd., product number A7601). Example 24

[0153] Example 24 The method for preparing magnetic beads was basically the same as that in Example 13, except that the antibody used in Example 24 was a CD4 antibody with the clone number RPA-T4 (purchased from Beijing Kuangbo Biotech Co., Ltd., product number A7601).

[0154] The magnetic microspheres prepared in Examples 19 - 24 were subjected to the measurement of the sorting performance of CD8+ or CD4+ T cells according to the above cell sorting method, and the measurement results are shown in Table 4.

[0155] Table 4

[0156]

[0157] In addition, the difference in the sorting of the same magnetic microspheres for different samples was tested. The test scheme was as follows: 10 PBMC samples from different individuals were taken. First, the CD8+ T cell sorting magnetic microspheres prepared in Example 12 were used for sorting, and the sorting results of the magnetic microspheres prepared in Example 12 for the 10 samples were tested. Similarly, the magnetic microspheres prepared in Examples 19, 21, and 23 were used for sorting the 10 samples respectively, and the sorting results were tested. The sorting results of the corresponding T cells showed that both the sorting purity and the recovery rate reached over 90%, indicating that the difference in the sorting of the same magnetic microspheres for different samples was small, and indirectly reflecting that the coupling of magnetic nanoparticles with the same type of antibodies with different sequences had little effect on the cell sorting results.

[0158] In summary, the present invention confirms that the iron oxide magnetic microspheres added with dextran and organic acid are suitable for cell sorting. Even when different antibodies are conjugated, ideal cell sorting purity and recovery rate can be obtained, and there are no special requirements for the selection of antibodies and samples. Moreover, based on the different cells to be sorted and the different purity and recovery rate requirements for different application scenarios of the sorted cells, the corresponding sorting requirements can be achieved by adjusting the dosage of the organic acid relative to the dextran.

[0159] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can be made on the basis of the above embodiments. Similarly, any combination of the technical features of the above embodiments can be made to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. Use of magnetic microspheres in the preparation of cell sorting reagent products, characterized in that, The magnetic microspheres include magnetite magnetic nanoparticles with dextran modified on the outer surface and specific antibodies conjugated to the dextran, and organic acids are mixed in the magnetite magnetic nanoparticles; The magnetic microspheres are obtained by the following preparation method, including: (1) Ferric salts and ferrous salts react in the presence of dextran, organic acids, and bases in solvent 1 at 140 - 180 °C to obtain magnetic nanoparticles; (2) The magnetic nanoparticles obtained in step (1) react in the presence of a carboxylating reagent to obtain carboxylated magnetic nanoparticles; (3) The carboxylated magnetic nanoparticles are conjugated with specific antibodies to obtain the magnetic microspheres; Among them, the weight-average molecular weight of the dextran is 4000 - 8000; In step (1), the molar concentration ratio of the ferrous salt: ferric salt: dextran and organic acid is (1 - 10):(5 - 15):1; the organic acid is selected from one or more of tartaric acid, malic acid, and citric acid; the molar ratio of the dextran and the organic acid in the feed is 1:(5 - 15); solvent 1 is selected from one or more of ethylene glycol, propylene glycol, butylene glycol, glycerol, and hexanediol; In step (2), the mass ratio of the magnetic nanoparticles to the carboxylating reagent in the feed is (2 - 8):

1.

2. The application according to claim 1, characterized in that, The particle size of the magnetic microspheres is 40 - 70 nm; And / or, the specific antibody is selected from one or more of CD8, CD4, CD45, CD3, CD56, and CD34 antibodies.

3. The application according to claim 1, characterized in that, In step (1), the molar concentration ratio of the ferrous salt: ferric salt: dextran and organic acid is 4:8:1; And / or, the molar ratio of the dextran and the organic acid is 1:(7 - 15).

4. The application according to claim 1, characterized in that, The ferric salt is ferric chloride or ferric sulfate, and the ferrous salt is ferrous chloride or ferrous sulfate; And / or, in step (1), the base is sodium hydroxide, and the concentration of the sodium hydroxide is 0.05 - 0.3 M; And / or, control the reaction in step (1) to proceed for 10 - 20 hours.

5. The application according to claim 1, wherein The carboxylating reagent is selected from one or more of dihydro-2,5-furandione, maleic anhydride, acrylic anhydride, and hexanoic anhydride; And / or, the reaction in step (2) is carried out in the presence of solvent 2, and the solvent 2 is selected from one or more of DMF, tetrahydrofuran, and acetonitrile; And / or, control the reaction in step (2) to proceed at 50 - 100 °C for 10 - 40 hours; And / or, the reaction in step (2) is carried out in the presence of a catalyst, and the catalyst is selected from one or more of triethylamine, ethylenediamine, and 1,6-hexanediamine.

6. The application according to claim 1, wherein In steps (1) and (2), after-treatment steps are included after the reaction; among them, the after-treatment step in step (1) includes centrifuging and washing the reaction product to obtain the magnetic nanoparticles; the after-treatment step in step (2) includes centrifuging, dialyzing, and sorting the reaction product to obtain the carboxylated magnetic nanoparticles.

7. The application according to claim 1, wherein The conjugation ratio of the carboxylated magnetic nanoparticles to the specific antibody is (1 - 5) mg: 200 μg.

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