Nanometer magnetic bead for cell sorting and preparation method thereof

Abstract

The invention discloses a preparation method of nano magnetic beads for cell sorting. According to the method, hemicellulose, arabinogalactan, FeCl3, FeCl2 and Na2CO3 are taken as raw materials, DMSO is taken as a solvent, monodispersed hemicellulose / arabinogalactan coated Fe3O4 magnetic nano magnetic beads are generated through a solvothermal method reaction, then the nano magnetic beads are further activated by 4-fluorobenzene sulfonyl chloride, a fluorobenzene sulfonyl group is introduced, then the fluorobenzene sulfonyl group is coupled with a cell sorting antibody CD3, the cell sorting antibody CD3 is separated, and the cell sorting antibody CD3 is obtained. And purifying and sorting the cells by using coupled antibody magnetic beads.

Description

Technical Field

[0001] The invention belongs to the field of antibody and magnetic bead coupling, and specifically relates to a nanomagnetic bead for cell sorting and a preparation method thereof, in particular to a nanomagnetic bead of a biodegradable hemicellulose matrix for cell sorting and a preparation method thereof. Background Art

[0002] Immunomagnetic beads, also known as magnetic beads for short, have good superparamagnetism, are easy to operate, and have a large specific surface area, making them a good separation material. As a solid material, magnetic beads are modified to have chemical groups on their surface, which can be covalently coupled to protein molecules. Because magnetic beads have a large specific surface area, they can bind more proteins and are used for cell sorting.

[0003] The most effective and popular magnetic beads on the market are the ones developed by Metronidex for sorting cells using sorting columns. There is little information about the coupling technology of these sorting magnetic beads, and there are very few such magnetic beads, which are all trade secrets. Most of the products on the market currently use carboxyl group-coupled antibodies, but this method cannot be used for Metronidex cell sorting. The magnetic beads cannot stay in the buffer for a long time without sinking to the bottom, and cannot pass Metronidex's sorting columns. Other conventional magnetic beads are too large in size and have poor hydrophilicity, and are prone to clogging when passing through sorting columns. Summary of the invention

[0004] The purpose of the present invention is to provide a nanomagnetic bead for cell sorting and a preparation method thereof in view of the defects and shortcomings of the prior art.

[0005] The present invention coats hemicellulose / arabinogalactan on the surface of Fe3O4 nanoparticles because hemicellulose / arabinogalactan is rich in hydroxyl groups. After coating the magnetic beads, the magnetic beads are more hydrophilic and better dispersed in water. The magnetic beads do not sink to the bottom in the buffer for a long time and can pass through the sorting column of Mei Tianni. At the same time, the hydroxyl group is also used for the modification of the fluorobenzenesulfonyl group later. Compared with other coating materials, such as PVA, which is a high molecular polymer material, it is obtained by chemical synthesis, and has partial toxicity and cannot be biodegraded. Since the cells obtained after the present invention is used for cell sorting are generally used for clinical Car-T cell therapy of cancer, all the cell sorting magnets used are preferably non-toxic biodegradable materials, and hemicellulose and arabinogalactan meet the requirements. Among them, the main function of hemicellulose is to provide hydroxyl groups and subsequent modifications, and arabinogalactan as a surfactant sugar is mainly used as a magnetic bead surfactant, making the magnetic beads more easily dispersed in water.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a nanomagnetic bead for cell sorting, wherein the nanomagnetic bead is a core-shell structure; the shell is antibody-functionalized hemicellulose (Hemicellulose) / arabinogalactan, and the core is Fe3O4 nanoparticles;

[0008] Antibody-functionalized hemicellulose / arabinogalactan is prepared by activating the hydroxyl groups on hemicellulose and arabinogalactan using 4-fluorobenzenesulfonyl chloride and triethylamine to obtain magnetic nanoparticles coated with fluorobenzenesulfonyl groups, which are then coupled with antibodies to obtain antibody-coated hemicellulose and arabinogalactan, i.e., antibody-functionalized hemicellulose / arabinogalactan.

[0009] Preferably, the nanomagnetic beads are monodisperse particles with a transmission electron microscope particle size of 8-12 nanometers. The nanomagnetic beads obtained by the present invention are the best monodisperse, smallest particle size, and non-sinking magnetic beads in the current prior art, which solves the problem of precipitation after long-term placement of magnetic beads and antibodies after coupling, and at the same time allows the magnetic beads coupled with antibodies to more fully bind to the target biomacromolecules, thereby improving the binding efficiency.

[0010] The Fe3O4 nanoparticles of the present invention are subjected to an activation reaction of 4-fluorobenzenesulfonyl chloride and triethylamine on the hydroxyl groups on hemicellulose / arabinogalactan to obtain magnetic nanoparticle beads modified with fluorobenzenesulfonyl groups; the fluorobenzenesulfonyl groups on the surfaces of the obtained magnetic nanoparticle beads are coupled with T cell recognition antibodies CD3 to obtain CD3 antibody-coated nanoparticle beads; and then the CD3 antibody nanoparticle beads are used to enrich immune cells containing CD3.

[0011] In a second aspect, the present invention relates to a method for preparing biodegradable hemicellulose-based nanomagnetic beads for cell sorting, the preparation method comprising the following steps:

[0012] S1, using hemicellulose, arabinogalactan, ferric chloride, ferrous chloride, and sodium carbonate as raw materials, adding them into a solvent, and generating monodisperse, core-shell structured hemicellulose / arabinogalactan@Fe3O4 nanoparticles by solvothermal reaction;

[0013] S2, using 4-fluorobenzenesulfonyl chloride and triethylamine to activate the hemicellulose / arabinogalactan@Fe3O4 nanoparticles to obtain hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups;

[0014] S3. The obtained hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups are coupled with antibodies to obtain antibody-coated hemicellulose / arabinogalactan@Fe3O4 nanoparticles, namely the nanomagnetic beads for cell sorting.

[0015] Preferably, in step S1, the usage ratio of hemicellulose, arabinogalactan, ferric chloride, ferrous chloride, and sodium carbonate is 0.2-0.8 g: 0.1-0.4: 0.02-0.62 g: 0.012-0.25 g: 0.4-0.65 g. The molar ratio of ferric chloride or its hydrate and ferrous chloride or its hydrate is preferably fixed at 2:1.

[0016] Preferably, in step S1, the usage ratio of solvent, hemicellulose, arabinogalactan, ferric chloride, ferrous chloride and sodium carbonate is 0.1-2 ml: 0.2-0.8 g: 0.1-0.4: 0.02-0.62 g: 0.012-0.25 g: 0.4-0.65 g.

[0017] Preferably, in step S1, ferric chloride is added in the form of ferric chloride or its hydrate, ferrous chloride is added in the form of ferrous chloride or its hydrate, and sodium carbonate is added in the form of sodium carbonate or its hydrate. Ferric chloride hydrate is preferably FeCl3·6H2O, ferrous chloride hydrate is preferably FeCl2·4H2O, and sodium carbonate hydrate is preferably Na2CO3·10H2O.

[0018] Preferably, in step S1, the solvent is DMSO.

[0019] Preferably, in step S1, the reaction temperature of the solvent thermal reaction is 120 to 220° C., and the reaction time is 2 to 48 hours.

[0020] Preferably, in step S1, the hemicellulose / arabinogalactan@Fe3O4 nanoparticles are hemicellulose / arabinogalactan-coated Fe3O4 nanoparticles.

[0021] Preferably, in step S2, the activation reaction is to add hemicellulose / arabinogalactan@Fe3O4 nanoparticles, 4-fluorobenzenesulfonyl chloride, and triethylamine to a solvent, and react at 25 to 80°C for 2 to 8 hours to obtain hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups. The solvent is DMSO. The activation is to perform an activation reaction of 4-fluorobenzenesulfonyl chloride and triethylamine on the hydroxyl groups on the hemicellulose / arabinogalactan to obtain magnetic nanoparticles coated with fluorobenzenesulfonyl groups.

[0022] The present invention couples antibodies through fluorobenzenesulfonyl groups, which reduces the antibody coupling time and increases the coupling efficiency compared with other coupling methods.

[0023] Preferably, in step S2, the ratio of hemicellulose / arabinogalactan@Fe3O4 nanoparticles, 4-fluorobenzenesulfonyl chloride, and triethylamine is 1-5 g: 0.5-5 ml: 0.1-0.4 ml. Triethylamine acts as an organic base catalyst.

[0024] Preferably, in step S3, the antibody coupling is achieved by reacting the hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups with the antibody in a sodium carbonate-sodium bicarbonate solution (pH 9.5) at 4 to 50° C. for 1 to 3 hours.

[0025] Preferably, in step S3, the mass ratio of hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups and antibodies is 0.1-5:0.1-5.

[0026] Preferably, in step S3, the antibody is an antibody against CD3 protein, which recognizes CD3 protein on the surface of T cells.

[0027] In a third aspect, the present invention also relates to a use of the nanomagnetic beads in purifying and sorting cells.

[0028] Preferably, the application is the use of nanomagnetic beads in a sorting column of Mei Tianni to purify and sort cells.

[0029] Preferably, the application comprises the following steps:

[0030] A1. Prepare cell samples, resuspend cell pellets with buffer, add nanomagnetic beads and mix well;

[0031] A2. Incubate the mixed cell sample, wash the cells with buffer, centrifuge, completely aspirate the supernatant, and resuspend in buffer;

[0032] A3. Perform magnetic cell separation on the resuspended cell sample.

[0033] In step A1, the buffer is 1X PBS pH 7.2-7.4 including 2 mM EDTA, 0.5% HSA buffer (without Ca 2 + ).

[0034] In step A1, before using the cell sample, the cell suspension was centrifuged at 300 g for 10 minutes, and the supernatant was completely aspirated.

[0035] In step A1, resuspend the cell pellet in buffer at a rate of 1x10 7Resuspend the cell pellet with 80 μL buffer (you can use a 30 μm cell strainer to remove adherent and large clumps of cells in advance), and add 1x10 7 Add 20 μL of nanomagnetic bead dispersion (the amount of magnetic beads is 5x10 10 indivual).

[0036] Each 1x10 7 For each cell, resuspend the cell pellet in 60 μL buffer and add 20 μL of the nanomagnetic bead suspension corresponding to the CD3 protein.

[0037] In step A2, the incubation time is 15 minutes at a temperature of 2-8° C. If fluorescent labeling is performed, 10 μL of fluorescent antibody can be added after incubation and incubated for 5 minutes in the dark.

[0038] In step A2, wash cells with buffer at 1x10 7 The cells were washed with 1-2 mL of buffer and centrifuged at 300 g for 10 minutes.

[0039] In step A2, resuspend no more than 1x10 cells in 500 μL of buffer. 8 The number of cells does not exceed 1x10 7 For higher cell numbers, increase the buffer volume accordingly.

[0040] In step A3, magnetic cell separation is performed using Miltenyi MS or LS columns.

[0041] In step A3, the magnetic cell separation step is:

[0042] (1) Magnetic separation using Miltenyi MS or LS columns;

[0043] (2) Place the separation column in the separation magnet of the Miltenyi MACS Separator;

[0044] (3) Rinse the column with buffer (MS: 500 μL, LS: 3 mL);

[0045] (4) dropping the cell suspension onto a separation column and collecting the effluent containing unlabeled cells;

[0046] (5) Rinse the separation column with buffer (MS: 3X500 μL, LS: 3x3 mL), collect the unlabeled cells that pass through, and mix them with the effluent from step (3);

[0047] (6) Remove the separation column from the separation magnet, place it in a collection tube, transfer the buffer to the separation column, push the plunger into the separation column and flush out the magnetically labeled cells (MS: 1 mL, LS: 5 mL);

[0048] (7) Finally, flow cytometry analysis was performed.

[0049] In step (5), once the separation column reservoir is empty, an equal amount of buffer is immediately added for a washing step.

[0050] Hemicellulose used in the present invention is a type of natural polysaccharide, which is widely present in the cell walls of plants and constitutes the main component of plant cell walls together with cellulose and lignin. Different from the linear structure of cellulose, hemicellulose has a branched structure, a small molecular weight, and a diverse structure, often consisting of a variety of different sugar units, such as xylose, mannose, glucose, galactose and arabinose. The present invention uses hemicellulose to coat magnetic beads, which are easily degraded by microorganisms during use, will not accumulate in the environment for a long time, and it is a natural polysaccharide, which is also harmless to the human body.

[0051] The nanomagnetic beads of the present invention have higher sorting efficiency, are simpler, produce purer cells, cause less damage to cells, and have a higher recovery rate.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The nanomagnetic beads obtained by the present invention have the best monodispersity, the smallest particle size, and are non-sinking in water. They solve the problem of precipitation after long-term storage of magnetic beads and antibodies after coupling. At the same time, they allow the magnetic beads coupled with antibodies to more fully bind to the target biomacromolecules, thereby improving the binding efficiency.

[0054] (2) Higher sorting efficiency, higher recovery rate, simple process and easy operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0056] Figure 1 This is a projection electron micrograph (10 nm) of the nanomagnetic beads used for cell sorting obtained in Example 1 of the present invention.

[0057] Figure 2 This is a projection electron micrograph (10 nm) of the nanomagnetic beads used for cell sorting obtained in Example 1 of the present invention.

[0058] Figure 3 The PBMC cells were sorted using the CD3 antibody cell sorting magnetic beads obtained in Example 1 of the present invention; wherein a is the purity of CD3 cells in PBMC before cell sorting (69.77%), and b is the purity after enrichment with the sorting magnetic beads (97.34%).

[0059] Figure 4 1 is a scanning electron microscope image of the failed example magnetic beads obtained in comparative example 1 of the present invention.

[0060] Figure 5 1 is a scanning electron microscope image of the failed example magnetic beads obtained in comparative example 1 of the present invention.

[0061] Figure 6 3 is a scanning electron microscope image of the failed example magnetic beads obtained in comparative example 2 of the present invention.

[0062] Figure 7 3 is a scanning electron microscope image of the failed example magnetic beads obtained in comparative example 2 of the present invention.

[0063] Figure 8 3 is a scanning electron microscope image of the failed example magnetic beads obtained in comparative example 3 of the present invention.

[0064] Fig. 9 3 is a scanning electron microscope image of the failed example magnetic beads obtained in comparative example 3 of the present invention. DETAILED DESCRIPTION

[0065] The present invention is described in detail below in conjunction with specific embodiments and accompanying drawings. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0066] The molecular weight of the hemicellulose used in the experiment of the present invention is 7500 g / mol, and its components, calculated by mass fraction, mainly include 17% glucose, 65% mannose, and 15% galactose.

[0067] Example 1

[0068] This embodiment relates to a novel preparation and application method of nanomagnetic beads for cell sorting, which is as follows:

[0069] 1. The preparation method of magnetic beads comprises the following steps:

[0070] In the first step, the reactants 0.5 g hemicellulose, 0.25 g (+)-arabinogalactan, 0.5 g FeCl3·6H2O, 0.368 g FeCl2·4H2O, and 1.5 g Na2CO3·10H2O were mixed in 20 ml DMSO solvent, and mechanically stirred at 300 rpm until completely dissolved. The above solution was transferred into a 50 ml reactor and reacted at 195°C for 25 hours. After cooling to room temperature, the reaction product was transferred to a 50 ml centrifuge tube, washed with deionized water, collected with a magnet, and stored in a vacuum dryer to obtain black Fe3O4 nanoparticles.

[0071] In the second step, the magnetic beads are surface-modified to obtain nanomagnetic beads modified with fluorobenzenesulfonyl groups: 3 grams of hemicellulose-coated nanomagnetic beads, 0.4 milliliters of triethylamine and 2 milliliters of 4-fluorobenzenesulfonyl chloride are reacted in 30 milliliters of DMSO solvent at 50°C for 3 hours, and then washed several times with anhydrous ethanol and deionized water to obtain nanomagnetic beads modified with fluorobenzenesulfonyl groups.

[0072] The third step is to introduce CD3 antibodies on the surface of magnetic beads: 2 mg of nanomagnetic beads modified with fluorobenzenesulfonyl groups are reacted with 1 mg of CD3 antibodies in 8 ml of 0.5 M sodium bicarbonate, pH 9.5 solution at 37°C for 16 hours, and then washed with deionized water several times to obtain CD3 antibody-modified nanomagnetic beads.

[0073] 2. The CD3 antibody nanomagnetic beads prepared in this example are used to purify and sort CD3 immune cells. The purification method includes the following steps:

[0074] Sample preparation:

[0075] Peripheral blood cells PBMC cells, buffer 1X PBS pH7.2-7.4 containing 2mM EDTA, 0.5% HSA buffer without Ca 2+ ;

[0076] 1. Determine the cell number.

[0077] 2. Centrifuge the cell suspension at 300 g for 10 minutes and completely aspirate the supernatant.

[0078] 3. According to every 1x10 7 Resuspend the cell pellet with 80 μL buffer (you can use a 30 μm cell strainer to remove adherent and large clumps of cells in advance), and add 1x10 7 Add 20 μL CD3 nanoparticles dispersion (the amount of magnetic beads is 5x10 10 ), used for CD3+ cell separation.

[0079] 4. Mix well and incubate in refrigerator (2-8°C) for 15 minutes.

[0080] 5. Every 1x10 7 The cells were washed by adding 1-2 mL of buffer, then centrifuged at 300 g for 10 min and the supernatant was completely aspirated.

[0081] 6. Resuspend up to 1x10 in 500 μL of buffer. 8 cells, for 1x10 7Or less cells resuspend in 50 μL of buffer, for higher cell numbers increase the buffer volume accordingly.

[0082] 7. Perform magnetic separation.

[0083] Magnetic cell separation:

[0084] 1. Magnetic separation using Miltenyi MS or LS columns.

[0085] 2. Place the separation column in the separation magnet of an appropriate Miltenyi MACS Separator.

[0086] 3. Rinse the column with an appropriate amount of buffer: MS: 500 μL, LS: 3 mL.

[0087] 4. Drop the cell suspension onto the separation column and collect the flow-through containing unlabeled cells.

[0088] 5. Rinse the separation column with an appropriate amount of buffer, collect the unlabeled cells that pass through, and mix with the effluent from step 3. MS: 3X500μL, LS: 3x3 mL.

[0089] Note: Once the separation column reservoir is empty, immediately add an equal amount of buffer for the wash step.

[0090] 6. Remove the separation column from the separation magnet and place it in a suitable collection tube. Pipette an appropriate amount of buffer onto the separation column. Push the plunger into the separation column and flush out the magnetically labeled cells. MS: 1mL, LS: 5mL.

[0091] Then flow cytometry analysis was performed.

[0092] The projection electron micrograph (10 nm) of the nanomagnetic beads used for cell sorting obtained in Example 1 is shown in Figure 1 , 2 As shown. CD3 antibody cell sorting magnetic bead sorting PBMC cells as shown Figure 3 As shown; wherein, a is the purity of CD3 cells in PBMC before cell sorting (69.77%), and b is the purity after enrichment with sorting magnetic beads (97.34%).

[0093] Comparative Example 1

[0094] This embodiment relates to a novel preparation and application method of nanomagnetic beads for cell sorting, which is as follows:

[0095] The method for preparing magnetic beads comprises the following steps:

[0096] In the first step, the reactants 0.5 g hemicellulose, 0.25 g (+)-arabinogalactan, 0.5 g FeCl3·6H2O, 0.368 g FeCl2·4H2O, and 2 g Na2CO3·10H2O were mixed in 20 ml DMSO solvent, and mechanically stirred at 300 rpm until completely dissolved. The above solution was transferred into a 50 ml reactor and reacted at 195°C for 25 hours. After cooling to room temperature, the reaction product was transferred to a 50 ml centrifuge tube, washed with deionized water, collected with a magnet, and stored in a vacuum dryer to obtain black Fe3O4 nanoparticles.

[0097] In the second step, the magnetic beads are surface-modified to obtain nanomagnetic beads modified with fluorobenzenesulfonyl groups: 3 grams of hemicellulose-coated nanomagnetic beads, 0.4 milliliters of triethylamine and 2 milliliters of 4-fluorobenzenesulfonyl chloride are reacted in 30 milliliters of DMSO solvent at 50°C for 3 hours, and then washed several times with anhydrous ethanol and deionized water to obtain nanomagnetic beads modified with fluorobenzenesulfonyl groups.

[0098] The third step is to introduce CD3 antibodies on the surface of magnetic beads: 2 mg of nanomagnetic beads modified with fluorobenzenesulfonyl groups are reacted with 1 mg of CD3 antibodies in 8 ml of 0.5 M sodium bicarbonate, pH 9.5 solution at 37°C for 16 hours, and then washed with deionized water several times to obtain CD3 antibody-modified nanomagnetic beads.

[0099] The CD3 antibody nanomagnetic beads prepared in this example are used to purify and sort CD3 immune cells. The purification method includes the following steps:

[0100] A1. Buffer:

[0101] Sample preparation:

[0102] Peripheral blood cells PBMC cells, 1X PBS pH7.2-7.4 containing 2mM EDTA, 0.5% HSA buffer without Ca 2+ ;

[0103] 1. Determine the cell number.

[0104] 2. Centrifuge the cell suspension at 300 g for 10 minutes and completely aspirate the supernatant.

[0105] 3. According to every 1x10 7 Resuspend the cell pellet with 80 μL buffer (you can use a 30 μm cell strainer to remove adherent and large clumps of cells in advance), and add 1x10 7 Add 20 μL CD3 nanoparticles dispersion (the amount of magnetic beads is 5x10 10), used for CD3+ cell separation.

[0106] 4. Mix well and incubate in refrigerator for 15 minutes (2-8°C).

[0107] 5. Every 1x10 7 The cells were washed by adding 1-2 mL of buffer, then centrifuged at 300 g for 10 min and the supernatant was completely aspirated.

[0108] 6. Resuspend up to 1x10 in 500 μL of buffer. 8 cells, for 1x10 7 Or less cells resuspend in 50 μL of buffer, for higher cell numbers increase the buffer volume accordingly.

[0109] 7. Perform magnetic separation.

[0110] Magnetic cell separation

[0111] 1. Magnetic separation using Miltenyi MS or LS columns.

[0112] 2. Place the separation column in the separation magnet of an appropriate Miltenyi MACS Separator.

[0113] 3. Rinse the column with an appropriate amount of buffer: MS: 500 μL, LS: 3 mL.

[0114] 4. Drop the cell suspension onto the separation column and collect the flow-through containing unlabeled cells.

[0115] 5. Rinse the separation column with an appropriate amount of buffer, collect the unlabeled cells that pass through, and mix with the effluent from step 3. MS: 3X500μL, LS: 3x3 mL.

[0116] Note: Once the separation column reservoir is empty, immediately add an equal amount of buffer for the wash step.

[0117] 6. Remove the separation column from the separation magnet and place it in a suitable collection tube. Pipette an appropriate amount of buffer onto the separation column, push the plunger into the separation column and flush out the magnetically labeled cells, MS: 1mL, LS: 5mL.

[0118] The scanning electron microscope image of the failed magnetic beads obtained in Example 2 is as follows: Figure 4 , 5 As shown in the figure, the magnetic beads have aggregated. Due to the aggregation of magnetic beads, the separation column is clogged and the plunger cannot flush out the magnetically labeled cells.

[0119] Comparative Example 2

[0120] This embodiment relates to a novel preparation and application method of nanomagnetic beads for cell sorting, which is as follows:

[0121] The method for preparing magnetic beads comprises the following steps:

[0122] In the first step, the reactants 1 gram of hemicellulose, 0.5 gram of (+)-arabinogalactan, 0.5 gram of FeCl3·6H2O, 0.368 gram of FeCl2·4H2O, and 2 gram of Na2CO3·10H2O were mixed in 20 ml of DMSO solvent, and mechanically stirred at 300 rpm until completely dissolved. The above solution was transferred into a 50 ml reactor and reacted at 195°C for 25 hours. After cooling to room temperature, the reaction product was transferred to a 50 ml centrifuge tube, washed with deionized water, collected with a magnet, and stored in a vacuum dryer to obtain black Fe3O4 nanoparticles.

[0123] In the second step, the magnetic beads are surface-modified to obtain nanomagnetic beads modified with fluorobenzenesulfonyl groups: 3 grams of hemicellulose-coated nanomagnetic beads, 0.4 milliliters of triethylamine and 2 milliliters of 4-fluorobenzenesulfonyl chloride are reacted in 30 milliliters of DMSO solvent at 50°C for 3 hours, and then washed several times with anhydrous ethanol and deionized water to obtain nanomagnetic beads modified with fluorobenzenesulfonyl groups.

[0124] The third step is to introduce CD3 antibodies on the surface of magnetic beads: 2 mg of nanomagnetic beads modified with fluorobenzenesulfonyl groups are reacted with 1 mg of CD3 antibodies in 8 ml of 0.5 M sodium bicarbonate, pH 9.5 solution at 37°C for 16 hours, and then washed with deionized water several times to obtain CD3 antibody-modified nanomagnetic beads.

[0125] The CD3 antibody nanomagnetic beads prepared in this example are used to purify and sort CD3 immune cells. The purification method includes the following steps:

[0126] A1. Buffer:

[0127] Sample preparation:

[0128] Peripheral blood cells PBMC cells, 1X PBS pH7.2-7.4 containing 2mM EDTA, 0.5% HSA buffer without Ca 2+ ;

[0129] 1. Determine the cell number.

[0130] 2. Centrifuge the cell suspension at 300 g for 10 minutes and completely aspirate the supernatant.

[0131] 3. According to every 1x10 7Resuspend the cell pellet with 80 μL buffer (you can use a 30 μm cell strainer to remove adherent and large clumps of cells in advance), and add 1x10 7 Add 20 μL CD3 nanoparticles dispersion (the amount of magnetic beads is 5x10 10 ), used for CD3+ cell separation.

[0132] 4. Mix well and incubate in a refrigerator for 15 minutes (+2 to +8°C). If fluorescent labeling is performed, add 10 μL of fluorescent antibody after 15 minutes and incubate for 5 minutes in the dark.

[0133] 5. Every 1x10 7 The cells were washed by adding 1-2 mL of buffer, then centrifuged at 300 g for 10 min and the supernatant was completely aspirated.

[0134] 6. Resuspend up to 1x10 in 500 μL of buffer. 8 cells, for 1x10 7 Or less cells resuspend in 50 μL of buffer, for higher cell numbers increase the buffer volume accordingly.

[0135] 7. Perform magnetic separation.

[0136] Magnetic cell separation

[0137] 1. Magnetic separation using Miltenyi MS or LS columns.

[0138] 2. Place the separation column in the separation magnet of an appropriate Miltenyi MACS Separator.

[0139] 3. Rinse the column with an appropriate amount of buffer: MS: 500 μL, LS: 3 mL.

[0140] 4. Drop the cell suspension onto the separation column and collect the flow-through containing unlabeled cells.

[0141] 5. Rinse the separation column with an appropriate amount of buffer, collect the unlabeled cells that pass through, and mix with the effluent from step 3. MS: 3X500μL, LS: 3x3 mL.

[0142] Note: Once the separation column reservoir is empty, immediately add an equal amount of buffer for the wash step.

[0143] 6. Remove the separation column from the separation magnet and place it in a suitable collection tube. Pipette an appropriate amount of buffer onto the separation column, push the plunger into the separation column and flush out the magnetically labeled cells, MS: 1mL, LS: 5mL.

[0144] The scanning electron microscope image of the failed magnetic beads obtained in Example 3 is as follows Figure 6, 7 The magnetic beads shown have aggregated. Due to the aggregation of magnetic beads, the separation column is clogged and the plunger cannot flush out the magnetically labeled cells.

[0145] Comparative Example 3

[0146] This embodiment relates to a novel preparation and application method of nanomagnetic beads for cell sorting, which is as follows:

[0147] The method for preparing magnetic beads comprises the following steps:

[0148] In the first step, the reactants 1 gram of hemicellulose, 0.25 grams of (+)-arabinogalactan, 0.5 grams of FeCl3·6H2O, 0.368 grams of FeCl2·4H2O, and 2 grams of Na2CO3·10H2O were mixed in 20 milliliters of DMSO solvent, and mechanically stirred at 300 rpm until completely dissolved. The above solution was transferred into a 50-ml reactor and reacted at 195°C for 25 hours. After cooling to room temperature, the reaction product was transferred to a 50-ml centrifuge tube, washed with deionized water, collected with a magnet, and stored in a vacuum dryer to obtain black Fe3O4 nanoparticles.

[0149] In the second step, the magnetic beads are surface-modified to obtain nanomagnetic beads modified with fluorobenzenesulfonyl groups: 3 grams of hemicellulose-coated nanomagnetic beads, 0.4 milliliters of triethylamine and 2 milliliters of 4-fluorobenzenesulfonyl chloride are reacted in 30 milliliters of DMSO solvent at 50°C for 3 hours, and then washed several times with anhydrous ethanol and deionized water to obtain nanomagnetic beads modified with fluorobenzenesulfonyl groups.

[0150] The third step is to introduce CD3 antibodies on the surface of magnetic beads: 2 mg of nanomagnetic beads modified with fluorobenzenesulfonyl groups are reacted with 1 mg of CD3 antibodies in 8 ml of 0.5 M sodium bicarbonate, pH 9.5 solution at 37°C for 16 hours, and then washed with deionized water several times to obtain CD3 antibody-modified nanomagnetic beads.

[0151] Step 5.

[0152] The CD3 antibody nanomagnetic beads prepared in this example are used to purify and sort CD3 immune cells. The purification method includes the following steps:

[0153] A1. Buffer:

[0154] Sample preparation:

[0155] Peripheral blood cells PBMC cells, 1X PBS pH7.2-7.4 containing 2mM EDTA, 0.5% HSA buffer without Ca 2+ ;

[0156] 1. Determine the cell number.

[0157] 2. Centrifuge the cell suspension at 300 g for 10 minutes and completely aspirate the supernatant.

[0158] 3. According to every 1x10 7 Resuspend the cell pellet with 80 μL buffer (you can use a 30 μm cell strainer to remove adherent and large clumps of cells in advance), and add 1x10 7 Add 20 μL CD3 nanoparticles dispersion (the amount of magnetic beads is 5x10 10 ) were used for CD3+ cell isolation.

[0159] 4. Mix well and incubate in refrigerator (2-8°C) for 15 minutes.

[0160] 5. Every 1x10 7 The cells were washed by adding 1-2 mL of buffer, then centrifuged at 300 g for 10 min and the supernatant was completely aspirated.

[0161] 6. Resuspend up to 1x10 in 500 μL of buffer. 8 cells, for 1x10 7 Or less cells resuspend in 50 μL of buffer, for higher cell numbers increase the buffer volume accordingly.

[0162] 7. Perform magnetic separation.

[0163] Magnetic cell separation

[0164] 1. Magnetic separation using Miltenyi MS or LS columns.

[0165] 2. Place the separation column in the separation magnet of an appropriate Miltenyi MACS Separator.

[0166] 3. Rinse the column with an appropriate amount of buffer: MS: 500 μL, LS: 3 mL.

[0167] 4. Drop the cell suspension onto the separation column and collect the flow-through containing unlabeled cells.

[0168] 5. Rinse the separation column with an appropriate amount of buffer, collect the unlabeled cells that pass through, and mix with the effluent from step 3. MS: 3X500μL, LS: 3x3 mL.

[0169] Note: Once the separation column reservoir is empty, immediately add an equal amount of buffer for the wash step.

[0170] 6. Remove the separation column from the separation magnet and place it in a suitable collection tube. Pipette an appropriate amount of buffer onto the separation column, push the plunger into the separation column and flush out the magnetically labeled cells, MS: 1mL, LS: 5mL.

[0171] The scanning electron microscope image of the failed magnetic beads obtained in Example 4 is as follows: Figure 8 , 9 As shown in the figure, the magnetic beads have aggregated. Due to the aggregation of magnetic beads, the separation column is clogged and the plunger cannot flush out the magnetically labeled cells.

[0172] Comparative Example 4

[0173] This comparative example relates to a novel preparation and application method of nanomagnetic beads for cell sorting, and the steps are basically the same as those in Example 1, except that the reactants 0.5 g of hemicellulose and 0.25 g of (+)-arabinogalactan are replaced with 0.75 g of hemicellulose. The magnetic beads agglomerated, causing clogging of the sorting column.

[0174] Comparative Example 5

[0175] This comparative example relates to a novel preparation and application method of nanomagnetic beads for cell sorting, and the steps are basically the same as those in Example 1, except that the reactants 0.5 g of hemicellulose and 0.25 g of (+)-arabinogalactan are replaced with 0.75 g of (+)-arabinogalactan. The magnetic beads agglomerated, causing clogging of the sorting column.

[0176] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A nanomagnetic bead for cell sorting, characterized in that: The nanomagnetic beads are of core-shell structure; the outer shell is antibody-functionalized hemicellulose / arabinogalactan, and the inner core is Fe3O4 nanoparticles; The antibody-functionalized hemicellulose / arabinogalactan is prepared by activating the hydroxyl groups on the hemicellulose and arabinogalactan using 4-fluorobenzenesulfonyl chloride and triethylamine to obtain magnetic nanoparticles coated with fluorobenzenesulfonyl groups, which are then coupled with antibodies to obtain antibody-coated hemicellulose and arabinogalactan, i.e., antibody-functionalized hemicellulose / arabinogalactan.

2. The nanomagnetic beads for cell sorting according to claim 1, characterized in that: The nanomagnetic beads are monodisperse particles with a particle size of 8-12 nanometers.

3. A method for preparing nanomagnetic beads for cell sorting, characterized in that: The preparation method comprises the following steps: S1, using hemicellulose, arabinogalactan, ferric chloride, ferrous chloride, and sodium carbonate as raw materials, adding them into a solvent, and generating monodisperse, core-shell structured hemicellulose / arabinogalactan@Fe3O4 nanoparticles by solvothermal reaction; S2, using 4-fluorobenzenesulfonyl chloride and triethylamine to activate the hemicellulose / arabinogalactan@Fe3O4 nanoparticles to obtain hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups; S3. The obtained hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups are coupled with antibodies to obtain antibody-coated hemicellulose / arabinogalactan@Fe3O4 nanoparticles, namely the nanomagnetic beads for cell sorting.

4. The method for preparing nano magnetic beads according to claim 3, characterized in that: In step S1, the usage ratio of hemicellulose, arabinogalactan, ferric chloride, ferrous chloride and sodium carbonate is 0.2-0.8g: 0.1-0.4: 0.02-0.62g: 0.012-0.25g: 0.4-0.65g.

5. The method for preparing nano magnetic beads according to claim 3, characterized in that: In step S1, the solvent includes DMSO.

6. The method for preparing nano magnetic beads according to claim 3, characterized in that: In step S1, the reaction temperature of the solvent thermal reaction is 120 to 220° C., and the reaction time is 2 to 48 hours.

7. The method for preparing nano magnetic beads according to claim 3, characterized in that: In step S2, the activation reaction is to add hemicellulose / arabinogalactan@Fe3O4 nanoparticles, 4-fluorobenzenesulfonyl chloride, and triethylamine into a solvent, and react at 25 to 80° C. for 2 to 8 hours to obtain hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups; And / or, the usage ratio of hemicellulose / arabinogalactan@Fe3O4 nanoparticles, 4-fluorobenzenesulfonyl chloride and triethylamine is 1-5g:0.5-5ml:0.1-0.4ml.

8. The method for preparing nano magnetic beads according to claim 3, characterized in that: In step S3, the antibody coupling is achieved by reacting the hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups with the antibody in a sodium carbonate-sodium bicarbonate solution at 4-50°C for 1-3 hours; and / or, the mass ratio of hemicellulose / arabinogalactan@Fe3O4 nanoparticles modified with fluorobenzenesulfonyl groups to the antibody is 0.1-5:0.1-5; And / or, the antibody is an antibody against CD3 protein, recognizing CD3 protein on the surface of T cells.

9. Use of the nanomagnetic beads as claimed in claim 1 or 2 in purifying and sorting cells.

10. The use according to claim 9, characterized in that: The application includes the following steps: A1. Prepare cell samples, resuspend cell pellets with buffer, add nanomagnetic beads and mix well; A2. Incubate the mixed cell sample, wash the cells with buffer, centrifuge, completely aspirate the supernatant, and resuspend in buffer; A3. Perform magnetic cell separation on the resuspended cell sample.

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