A human thymus primary cell isolation kit and isolation method

Abstract

The present invention relates to a human thymic primary cell separation kit and a separation method, wherein the kit includes an extraction reagent for preparing a thymic tissue single cell suspension and a sorting reagent for purification, and through the reagent combination in the kit and the corresponding operation steps, one or more combinations of thymic epithelial cells, thymic dendritic cells, thymic T cells and thymic B cells can be accurately extracted from human thymic tissue. The beneficial effects of the present invention are: the target primary cells in the thymic tissue sample can be separated, the specificity is strong, and through the combination of different reagents, different primary cell components can be separated in one operation to obtain a variety of target primary cells with higher purity; and the success rate of separating the primary cell components in the thymic tissue sample using this method is as high as 90%, reducing sample loss.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a human thymus primary cell separation kit and a separation method. Background Art

[0002] The thymus is an important central immune organ. It is not only the site for T cell development and maturation, but also mediates the body's immune tolerance through positive selection, negative selection and the production of Treg cells. This process is sophisticated and complex, requiring the coordinated participation of thymic epithelial cells, dendritic cells, B cells and thymocytes. The thymus is relatively large in infancy and reaches 35 to 40 grams during human adolescence. Thereafter, the thymus begins to degenerate and gradually atrophy and is replaced by adipose tissue. The resulting reduction in T cell output is associated with age-related cancer, infection and autoimmunity.

[0003] T cell precursors from the fetal liver or bone marrow migrate to the thymus and then differentiate into different types of mature T cells. The sophisticated microenvironment of the thymus synergistically supports T cell differentiation. Although thymic epithelial cells provide key cues to promote T cell fate, other cell types are also involved in this process, such as dendritic cells for antigen presentation and medullary B cells that support TEC differentiation and maintenance. Since the 1960s, groundbreaking experiments in animal models have provided important insights into the function and cellular composition of the thymus. However, the pattern and rhythm of human organ maturation are unique to our species, which requires more in-depth research on the human thymus. Research on normal thymic function is difficult to advance due to the preciousness of thymic tissue samples, the low success rate of primary thymic culture, and the lack of cell lines available for experimental use.

[0004] Since the mechanism of thymus-mediated immune tolerance is subtle and complex, any disease that affects the normal structure and function of the thymus may lead to the failure of immune tolerance, that is, the generation of autoimmunity. A large number of studies have shown that thymic diseases are associated with the occurrence of a variety of autoimmune diseases, such as myasthenia gravis, Sjögren's syndrome, pure red cell aplasia, etc. Studies have shown that there are as many as 123 autoimmune diseases associated with thymic diseases. Therefore, studying the mechanism by which thymic diseases lead to the generation of autoimmune diseases has important clinical and social significance. However, although people have discovered that thymectomy can improve the symptoms of patients with myasthenia gravis since 1960, due to the lack of mature thymic primary cell culture methods and alternative cell lines, there is still no clear answer to the relationship between thymic diseases and autoimmune diseases.

[0005] Whether it is the exploration of the normal function of the thymus or the study of the relationship between thymic diseases and autoimmune diseases, there is an important technical problem - the separation and culture of normal thymic cell components. At present, the most commonly used research methods are trypsin digestion and direct tissue block culture. The former is to directly use the tissue for culture after trypsin digestion. This method uses trypsin to digest the tissue, which is easy to damage the primary cells, and the separated cells are not easy to survive, and the success rate is low; the latter has a slightly higher success rate than the former, but it takes a long time. Successful separation often takes more than 2 weeks, and the successfully cultured primary cells are difficult to separate from the tissue blocks, which is not conducive to subsequent experiments. In addition, because the thymus contains a large number of T lymphocytes and their precursors, resulting in a low proportion of other cell components, these two methods cannot accurately purify and separate various cell components, so they are not suitable for primary culture of thymic tissue. These two methods have many problems in terms of success rate, sensitivity, specificity and repeatability. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a human thymus primary cell isolation kit and a separation method.

[0007] The technical solution adopted by the present invention is: a thymus tissue cell separation kit, including an extraction reagent and a sorting reagent, the extraction reagent includes:

[0008] Extraction reagent A, including 2-3 g / L Hepes, 0.5-1 g / L EDTA, 50-100 U / ml penicillin-streptomycin, pH 7.0-7.5;

[0009] Extraction reagent B: including 10-20ml / L HEPES, 2.5-5g / L bovine serum albumin, 50-75mg / L trypsin inhibitor, 0.1-0.2g / L butyric acid, 1-2mmol / L L-glutamine, 1-2mg / ml linoleic acid; pH value is 7.0-7.5;

[0010] Extraction reagent C: including 3-4 mg / ml collagenase I, 5-10 ug / ml hyaluronidase, 0.1-0.2 mg / ml deoxyribonuclease I, pH 7.0-7.5;

[0011] Extraction reagent D: containing 50%-60% percoll stock solution and 1-2 mol / L sodium chloride by volume, with a pH value of 7.0-7.5;

[0012] The sorting reagent includes a combination of two or more of sorting reagent A, sorting reagent B, sorting reagent C, sorting reagent D and sorting reagent E, wherein:

[0013] Sorting reagent A is a biotin-labeled anti-CD45 monoclonal antibody;

[0014] Sorting reagent B is a biotin-labeled anti-CD19 monoclonal antibody;

[0015] Sorting reagent C is superparamagnetic nanoparticles coupled with streptavidin;

[0016] Sorting reagent D is a phycoerythrin-labeled anti-CD11C monoclonal antibody;

[0017] Sorting reagent E: anti-phycoerythrin superparamagnetic nanoparticles.

[0018] Preferably, the sorting reagents include sorting reagent A, sorting reagent B, sorting reagent C, sorting reagent D and sorting reagent E;

[0019] Or the sorting reagent includes sorting reagent A and sorting reagent C;

[0020] Or the sorting reagent includes sorting reagent A, sorting reagent C, sorting reagent D and sorting reagent E;

[0021] Alternatively, the sorting reagent includes sorting reagent B and sorting reagent C.

[0022] The method of separating cells in thymic tissue using a kit comprises first treating the thymic tissue with an extraction reagent to prepare a thymic tissue single cell suspension, and then enriching the thymic epithelial cells, thymic dendritic cells, thymic T cells and thymic B cells with an immunomagnetic bead method using a sorting reagent.

[0023] Preferably, the specific steps for preparing the thymus tissue single cell suspension are as follows:

[0024] Step 1: Place the thymus tissue sample in a container, add extraction reagent A to wash, and aseptically chop into pieces of approximately 1 mm. 3 After repeated pipetting, transfer to a 50 ml centrifuge tube, tilt and let stand for 10 min, and discard the supernatant.

[0025] Step 2: Add 10 mL of extraction reagent B, mix well, let stand for 10 min, and discard the supernatant;

[0026] Step 3: Add 10 ml of extraction reagent C, add a sterile magnetic rotor, place on a magnetic stirrer at 37°C, stir at a speed of 100-120 rpm / min for 10 min;

[0027] Step 4: Tilt and let stand for 15 minutes, discard the supernatant through a Pasteur tube, add 10 ml of extraction reagent A, pipette repeatedly and let stand for 15 minutes;

[0028] Step 5: Discard the supernatant, add 10 ml of extraction reagent C, add a sterile magnetic rotor, place on a magnetic stirrer at 37°C, stir at a speed of 100-120 rpm / min for 30 min;

[0029] Step 6: Filter the resulting solution through a 70 μm sterile cell sieve and centrifuge at 800-1000 rpm for 5-10 min at room temperature;

[0030] Step 7: Discard the supernatant, add 15 ml of extraction reagent A again, mix thoroughly with a pipette, centrifuge at 800-1000 rpm for 5-10 min at room temperature, discard the supernatant, add 5-10 ml of extraction reagent B and mix thoroughly;

[0031] Step 8: Slowly add the obtained solution dropwise to the extraction reagent D, the volume ratio of the solution to the extraction reagent D is 0.75-1:1, and centrifuge at 2000 rpm for 30-50 min at 4°C, with the lifting speed being 0;

[0032] Step 9: Aseptically aspirate the cells of the low-density cell layer and transfer them to a centrifuge tube containing extraction reagent B for later use. The obtained cells are thymic stromal cells containing thymic epithelial cells and thymic dendritic cells;

[0033] The cells in the high-density cell layer are aseptically aspirated and transferred to a centrifuge tube containing extraction reagent B for later use. The obtained cells contain thymic T cells and thymic B cells at various developmental stages.

[0034] Preferably, the step of isolating primary thymic epithelial cells comprises:

[0035] Step 10: Add an appropriate amount of sorting reagent A to the low-density cell layer cells obtained in step 9 and mix thoroughly. Incubate at 4°C in the dark for 20 minutes, then add an appropriate amount of sorting reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 minutes, then add to the magnetic sorting column rinsed with PBS. The cells that are not bound to the sorting column are primary thymic epithelial cells.

[0036] Preferably, the step of isolating primary thymic dendritic cells comprises:

[0037] Step 10: Add an appropriate amount of sorting reagent A to the low-density cell layer cells obtained in step 9 and mix thoroughly. Incubate at 4°C in the dark for 20 minutes, then add an appropriate amount of sorting reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 minutes, then add to the magnetic separation column rinsed with PBS to collect the cells bound to the separation column.

[0038] Step 11: Collect the cells bound to the sorting column in step 10, add an appropriate amount of sorting reagent D thereto and mix thoroughly, incubate at 4°C in the dark for 20 minutes, then add an appropriate amount of sorting reagent E, incubate at 4°C in the dark for 30 minutes, then add to the magnetic sorting column rinsed with PBS, and collect the cells bound to the sorting column, which are the primary thymic dendritic cells;

[0039] Preferably, the step of separating thymic T cells and thymic B cells comprises:

[0040] Step 12: Add an appropriate amount of sorting reagent B to the high-density cell layer cells obtained in step 9 and mix thoroughly. After incubating at 4°C in the dark for 20 minutes, add an appropriate amount of sorting reagent C and mix thoroughly. After incubating at 4°C in the dark for 30 minutes, add to the magnetic sorting column rinsed with PBS. The cells bound to the sorting column are the primary thymic B lymphocytes, and the cells not bound to the sorting column are the T lymphocytes at various developmental stages in the thymus.

[0041] Preferably, the amount of sorting reagent A, sorting reagent B, sorting reagent C, sorting reagent D and sorting reagent E is 10 7 10-20ul per cell.

[0042] A combination of one or more of thymic epithelial cells, thymic dendritic cells, thymic T cells and thymic B cells separated by a method for separating human thymic primary cells.

[0043] The kit or the method for isolating primary human thymic cells is used for isolating and purifying B lymphocytes, T lymphocytes, epithelial cells and dendritic cells in human thymic tissue.

[0044] The advantages and positive effects of the present invention are: the method provided by the present invention can separate the target primary cells in the thymus tissue sample with high specificity, and through the combination of different reagents, different primary cell components can be separated in one operation to obtain a variety of target primary cells with high purity;

[0045] Thymus tissue samples are relatively precious. This method can be used to efficiently separate primary cells from thymus tissue samples and reduce sample loss.

[0046] The operation process is simple, which is conducive to promotion. It does not require special instruments or complicated operation steps, and has low requirements for laboratory conditions, which is conducive to experiments in grassroots laboratories. The purification mechanism of this method is scientific, and the obtained target cells have strong activity, are less damaged, and have a high proliferation rate, which is conducive to various subsequent scientific experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1The cell status image of the primary human thymic epithelial cells isolated in Example 2 on the 3rd day (taken from the field of view under a 100x optical microscope);

[0048] Figure 2 Cell status diagram of human thymic primary dendritic cells isolated in Example 3 on day 7 (all taken from the field of view under a 400x optical microscope);

[0049] Figure 3 Cell status diagram of human thymic primary B cells isolated in Example 4 on day 3 (all taken from the field of view under a 200x optical microscope);

[0050] Figure 4 Cell status diagram of human thymic primary T cells isolated in Example 4 on day 3 (all taken from the field of view under a 200x optical microscope);

[0051] Figure 5 Cell immunofluorescence images of primary human thymic epithelial cells isolated in Example 2 (all taken from the field of view under a 400x optical microscope). DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0053] In view of the problems of low success rate of traditional thymic primary cell culture and lack of primary cell lines, the present invention relates to a human thymic primary cell isolation kit and a separation method. Through the combination of reagents in the kit and corresponding operating steps, one or more combinations of thymic epithelial cells, thymic dendritic cells, thymic T cells and thymic B cells can be accurately extracted from human thymic tissue.

[0054] The kit includes an extraction reagent for preparing a single cell suspension of thymus tissue and a sorting reagent for purification. The specific components and configuration methods are as follows:

[0055] Extraction reagent A: includes 2-3g / L Hepes, 0.5-1g / L EDTA, 50-100U / ml penicillin-streptomycin, and pH value is 7.0-7.5. When preparing extraction reagent A, the solvent is sterile ultrapure water. After Hepes, EDTA and penicillin-streptomycin are fully dissolved in sterile ultrapure water, the pH value is adjusted, filtered with a 0.22μm filter, and stored at 4°C for later use.

[0056] Extraction reagent B: including 10-20ml / L HEPES, 2.5-5g / L bovine serum albumin, 50-75mg / L trypsin inhibitor, 0.1-0.2g / L butyric acid, 1-2mmol / L L-glutamine, 1-2mg / ml linoleic acid; pH value is 7.0-7.5. When preparing extraction reagent B, the solvent is Ham's F-12 medium. After HEPES, bovine serum albumin, trypsin inhibitor, butyric acid, L-glutamine and linoleic acid are fully dissolved in Ham's F-12 medium, the pH is adjusted, filtered with a 0.22μm filter, and stored at 4℃ for later use.

[0057] Extraction reagent C: includes 3-4mg / ml collagenase I, 5-10ug / ml hyaluronidase, 0.1-0.2mg / ml deoxyribonuclease I; pH value is 7.0-7.5. When preparing extraction reagent C, fully dissolve collagenase I, hyaluronidase and deoxyribonuclease I in extraction reagent A, filter with a 0.22μm filter, and retain; preheat at 37℃ before use.

[0058] Extraction reagent D: contains 50%-60% (volume ratio) percoll stock solution and 1-2 mol / L sodium chloride; the pH value of extraction reagent D is 7.0-7.5. When preparing extraction reagent D, the percoll stock solution and sodium chloride solution are fully dissolved in sterile ultrapure water, and then the mass percentage of percoll is adjusted to 80-90%, filtered with a 0.22μm filter, and stored at 4℃ for later use.

[0059] Sorting reagent A: biotin-labeled anti-CD45 monoclonal antibody.

[0060] Sorting reagent B: biotin-labeled anti-CD19 monoclonal antibody.

[0061] Sorting reagent C: Superparamagnetic nanoparticles coupled with streptavidin.

[0062] Sorting reagent D: anti-CD11C monoclonal antibody labeled with phycoerythrin.

[0063] Sorting reagent E: anti-phycoerythrin superparamagnetic nanoparticles.

[0064] When isolating various types of cells in thymic tissue using the above-mentioned human thymic primary cell isolation kit, first prepare a single cell suspension of the thymic sample; then enrich and purify primary thymic epithelial cells and primary thymic dendritic cells by immunomagnetic beads; or enrich and purify primary thymic B cells and T cells at various developmental stages by immunomagnetic beads. Specifically, the following methods can be used:

[0065] Step 1: Place the human thymus tissue sample in a culture dish and add extraction reagent A for washing. The purpose is to remove the blood vessels in the thymus tissue, separate the surface membrane-like connective tissue and adipose tissue, and aseptically cut the gland into pieces of about 1 mm 3 After repeated pipetting, transfer to a 50 ml centrifuge tube, tilt and let stand for 10 min, and discard the supernatant.

[0066] Step 2: Add 10 mL of extraction reagent B, mix well, let stand for 10 min, and discard the supernatant;

[0067] Step 3: Add 10 ml of extraction reagent C, add a sterile magnetic rotor, place on a magnetic stirrer at 37°C, stir at a speed of 100-120 rpm / min for 10 min;

[0068] Step 4: Tilt and let stand for 15 minutes, discard the supernatant through a Pasteur tube, add 10 ml of extraction reagent A, pipette repeatedly and let stand for 15 minutes.

[0069] Step 5: Discard the supernatant, add 10 ml of extraction reagent C, add a sterile magnetic rotor, place on a magnetic stirrer at 37°C, stir at a speed of 100-120 rpm / min for 30 min;

[0070] Step 6: Filter the resulting solution through a 70 μm sterile cell sieve and centrifuge at 800-1000 rpm for 5-10 min at room temperature;

[0071] Step 7: Discard the supernatant, add 15 ml of extraction reagent A again, mix thoroughly with a pipette, centrifuge at 800-1000 rpm for 5-10 min at room temperature, discard the supernatant, add 5-10 ml of extraction reagent B and mix thoroughly;

[0072] Step 8: Slowly add the obtained solution dropwise to the extraction reagent D, the volume ratio of the solution to the extraction reagent D is 0.75-1:1, and centrifuge at 2000 rpm for 30-50 min at 4°C, with the lifting speed being 0;

[0073] Step 9: Aseptically aspirate the cells of the low-density cell layer and transfer them to a centrifuge tube containing extraction reagent B for later use. The obtained cells are thymic stromal cells containing thymic epithelial cells and thymic dendritic cells;

[0074] Aseptically aspirate the cells in the high-density cell layer and transfer them to a centrifuge tube containing extraction reagent B for later use. The obtained cells contain thymic T cells and thymic B cells at various developmental stages.

[0075] Step 10: Add an appropriate amount of sorting reagent A to the low-density cell layer cells obtained in step 9 and mix thoroughly. Incubate at 4°C in the dark for 20 minutes, then add an appropriate amount of sorting reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 minutes, then add to the magnetic sorting column rinsed with PBS, collect the cells bound to the sorting column for later use, and the cells not bound to the sorting column are the primary thymic epithelial cells;

[0076] Step 11: Collect the cells bound to the sorting column in step 10, add an appropriate amount of sorting reagent D thereto and mix thoroughly, incubate at 4°C in the dark for 20 minutes, then add an appropriate amount of sorting reagent E, incubate at 4°C in the dark for 30 minutes, then add to the magnetic sorting column rinsed with PBS, and collect the cells bound to the sorting column, which are the primary thymic dendritic cells;

[0077] Step 12: Add an appropriate amount of sorting reagent B to the high-density cell layer cells obtained in step 9 and mix thoroughly. After incubating at 4°C in the dark for 20 minutes, add an appropriate amount of sorting reagent C and mix thoroughly. After incubating at 4°C in the dark for 30 minutes, add to the magnetic sorting column rinsed with PBS. The cells bound to the sorting column are the primary thymic B lymphocytes, and the cells not bound to the sorting column are the T lymphocytes at various developmental stages in the thymus.

[0078] Among them, in steps 10-12, the dosage of sorting reagent A, sorting reagent B, sorting reagent C, sorting reagent D and sorting reagent E is 10 7 10-20ul per cell.

[0079] By using some or all of the steps in the above method, one or more of primary thymic epithelial cells, primary thymic dendritic cells, thymic stromal cells containing thymic epithelial cells and thymic dendritic cells, primary thymic B lymphocytes, T lymphocytes at various developmental stages in the thymus, and mixed samples containing thymic T cells and thymic B cells at various developmental stages can be isolated from thymic tissue. The human thymic primary cell isolation kit can be divided into different models according to the purpose of separating cells. The kits of different models include extraction reagent A, extraction reagent B, extraction reagent C and extraction reagent D, and also include a combination of two or more of sorting reagent A, sorting reagent B, sorting reagent C, sorting reagent D and sorting reagent E. In certain embodiments of the present invention, the kit is used to separate and obtain one or more of primary thymic epithelial cells, primary thymic dendritic cells, primary thymic B lymphocytes, and T lymphocytes at various developmental stages in the thymus, and the sorting reagents in the kit include all of sorting reagents A, sorting reagents B, sorting reagents C, sorting reagents D, and sorting reagents E. In certain embodiments of the present invention, the kit is used to separate and obtain primary epithelial cells and / or primary thymic dendritic cells, and the sorting reagents in the kit include sorting reagents A, sorting reagents C, sorting reagents D, and sorting reagents E. In certain embodiments of the present invention, the kit is used to separate and obtain primary B lymphocytes and / or T lymphocytes at various developmental stages in the thymus, and the sorting reagents in the kit include sorting reagents B and sorting reagents C. In certain embodiments of the present invention, the kit is only used to separate and obtain primary epithelial cells, and the sorting reagents in the kit include sorting reagents A and sorting reagents C.

[0080] The use of the above-mentioned kit and separation method can efficiently separate thymic tissue and can separate the target primary cells in the thymic tissue sample. Thymic tissue samples are relatively precious. The success rate of separating primary cell components in thymic tissue samples using this method is as high as 90%, reducing sample loss.

[0081] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0082] Materials and reagents used in the embodiments of the present invention: the human thymus tissue is derived from fresh thymus tissue samples obtained from thoracotomy of children with congenital heart disease in the Department of Cardiac Surgery of Tianjin Children's Hospital; CD45 monoclonal antibody is a commercial product purchased from abcam in the UK, the product name is recombinant Anti-CD45 antibody [EP322Y], and the product number is ab40763; CD19 monoclonal antibody is a commercial product purchased from abcam in the UK, the product name is recombinant Anti-CD19 antibody [EPR5906], and the product number is ab134114; phycoerythrin-labeled anti-CD11C monoclonal antibody is a commercial product purchased from Biolegend in the United States, the product name is PE anti-human CD11c antibody, and the product number is AB_2564135; super-paramagnetic nanoparticles coupled with streptavidin are commercial products purchased from Miltenyi Biotec in Germany, the product name is Anti-Biotin MicroBeads UltraPure, product number 130-105-637; anti-phycoerythrin ultra-smooth nanomagnetic beads are commercial products purchased from Miltenyi Biotec, Germany, product name Anti-PE MicroBeads, product number 130-048-801.

[0083] Example 1: Preparation of a human thymus primary cell isolation kit

[0084] The kit includes extraction reagent A, extraction reagent B, extraction reagent C and extraction reagent D, and sorting reagent A, sorting reagent B, sorting reagent C, sorting reagent D and sorting reagent E.

[0085] The preparation method of extraction reagent A is as follows: Hepes, EDTA and penicillin-streptomycin are fully dissolved in sterile ultrapure water, wherein Hepes 2g / L, EDTA 1g / L, penicillin-streptomycin 60U / ml, the pH value is adjusted to 7.4, and the extraction reagent A is obtained by filtering with a 0.22μm filter and stored at 4°C for future use.

[0086] The preparation method of extraction reagent B is as follows: HEPES, bovine serum albumin, trypsin inhibitor, butyric acid, L-glutamine and linoleic acid are fully dissolved in Ham's F-12 medium, wherein HEPES 15ml / L, bovine serum albumin 3g / L, trypsin inhibitor 75mg / L, butyric acid 0.2g / L, L-glutamine 1mmol / L, linoleic acid 1mg / ml; the pH value is adjusted to 7.4, filtered with a 0.22μm filter to obtain extraction reagent B, and stored at 4°C for future use.

[0087] The preparation method of extraction reagent C is as follows: collagenase I, hyaluronidase and deoxyribonuclease I are fully dissolved in extraction reagent A, wherein collagenase I 4 mg / ml, hyaluronidase 10 ug / ml, deoxyribonuclease I 0.1 mg / ml; the pH value is adjusted to 7.4, and extraction reagent C is obtained by filtering with a 0.22 μm filter; and preheating at 37° C. before use.

[0088] The preparation method of extraction reagent D is as follows: fully dissolve the Percoll stock solution and sodium chloride solution in sterile ultrapure water, wherein the sodium chloride is 2 mol / L; adjust the pH value to 7.4, and then adjust the mass percentage of Percoll to 80%, filter with a 0.22 μm filter to obtain extraction reagent D, and store at 4°C for future use.

[0089] Biotin-labeled anti-CD45 monoclonal antibody was prepared as sorting reagent A; biotin-labeled anti-CD19 monoclonal antibody was prepared as sorting reagent B; super-paramagnetic nanoparticles coupled with streptavidin were used as sorting reagent C; phycoerythrin-labeled anti-CD11C monoclonal antibody was used as sorting reagent D. Super-paramagnetic nanoparticles with anti-phycoerythrin were used as sorting reagent E.

[0090] The prepared kit can be used for separation and purification of primary human thymus cells.

[0091] Example 2: Isolation, purification and culture of primary human thymic epithelial cells

[0092] 2.1 Fresh thymus tissue samples were obtained from thoracotomy of children with congenital heart disease in the Department of Cardiac Surgery, Tianjin Children's Hospital;

[0093] 2.2 Move the tissue to a culture dish, add extraction reagent A to wash, remove blood vessels, separate the surface membrane-like connective tissue and adipose tissue, and aseptically chop the gland into 1 mm 3 , repeatedly pipette and transfer to a 50ml centrifuge tube, tilt and let stand for 10 minutes, and discard the supernatant;

[0094] 2.3 Add 10 mL of extraction reagent B, mix well, let stand for 10 min, and discard the supernatant;

[0095] 2.4 Add 10 ml of extraction reagent C, add a sterile magnetic rotor, place on a magnetic stirrer at 37°C, stir at a speed of 100-120 rpm / min for 10 min;

[0096] 2.5 Tilt and let stand for 15 minutes, discard the supernatant in the Pasteur tube, add 10 ml of extraction reagent A, pipette repeatedly and let stand for 15 minutes;

[0097] 2.6 Discard the supernatant, add 10 ml of extraction reagent C, add a sterile magnetic rotor, place on a magnetic stirrer at 37°C, stir at a speed of 100-120 rpm / min for 30 min;

[0098] 2.7 Filter the resulting solution through a 70 μm sterile cell sieve and centrifuge at 800-1000 rpm for 10 min at room temperature;

[0099] 2.8 Discard the supernatant, add 15 ml of extraction reagent A again, mix thoroughly with a pipette, centrifuge at 1000 rpm for 10 min at room temperature, discard the supernatant, add 10 ml of extraction reagent B and mix thoroughly;

[0100] 2.9 Slowly add the obtained solution dropwise onto an equal amount of extraction reagent D liquid, centrifuge at 2000 rpm for 50 min at 4 degrees Celsius, with the lifting speed being 0;

[0101] 2.10 Aseptically aspirate the cells of the low-density cell layer and transfer them to a centrifuge tube containing extraction reagent B for later use. The obtained cells are thymic stromal cells containing thymic epithelial cells and thymic dendritic cells;

[0102] 2.11 Add 20 μl of separation reagent A to the above low-density cell layer cells and mix thoroughly. Incubate at 4°C in the dark for 20 min, then add an appropriate amount of separation reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 min, then add to the magnetic separation column rinsed with PBS. The cells that are not bound to the separation column are the primary thymic epithelial cells.

[0103] Count the cell suspension obtained above, wash it 2-3 times with PBS, resuspend it in DMEM-F12 medium, transfer it to an appropriate culture bottle and culture it at 37℃, 5% CO2. Figure 1 The figure shows the cell status of the isolated human thymic primary epithelial cells on the third day. It can be seen from the figure that the thymic epithelial cells are spindle-shaped, well adherent, and in good cell condition. Figure 5 This is the Dapi immunofluorescence staining image of human thymic primary epithelial cells. It can be seen that the survival rate of the isolated human thymic primary epithelial cells is extremely high and can be effectively used in subsequent research processes.

[0104] Example 3: Isolation, purification and culture of primary human thymic dendritic cells

[0105] 3.1 Take the thymic stromal cells containing thymic epithelial cells and thymic dendritic cells prepared in step 2.10 of Example 2;

[0106] 3.2 Add 20 μl of separation reagent A to the above low-density cell layer cells and mix thoroughly. Incubate at 4°C in the dark for 20 min, then add an appropriate amount of separation reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 min, then add to the magnetic separation column rinsed with PBS, and collect the cells bound to the separation column for later use;

[0107] 3.3 Collect the cells bound to the above separation column, add 20 μl of reagent D to it and mix thoroughly, incubate at 4°C in the dark for 20 minutes, then add 20 μl of separation reagent E, incubate at 4°C in the dark for 30 minutes, add to the magnetic separation column rinsed with PBS, and collect the cells bound to the separation column, which are the primary thymic dendritic cells;

[0108] Count the cell suspension obtained above, wash it 2-3 times with PBS, resuspend it in RPMI 1640 medium, transfer it to an appropriate culture flask and culture it at 37℃, 5% CO2. Figure 2 The figure shows the cell status of the isolated human thymic primary dendritic cells on day 7. The thymic dendritic cells are round suspension cells with extended burr-like protrusions, and they are growing well.

[0109] Example 4: Isolation, purification and culture of primary human thymic B cells and T lymphocytes

[0110] 4.1 Take the sample obtained by centrifugation in step 9 of Example 2;

[0111] 4.2 Aseptically aspirate the cells from the high-density cell layer and transfer them to a centrifuge tube containing extraction reagent B for later use. The resulting cells contain thymic T cells and thymic B cells at various developmental stages;

[0112] 4.3 Add 20 μl of separation reagent B to the above high-density cell layer cells and mix thoroughly. Incubate at 4°C in the dark for 20 min, then add 20 μl of separation reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 min, then add to the magnetic separation column rinsed with PBS. The cells bound to the separation column are the primary thymic B lymphocytes, and the cells not bound to the separation column are the T lymphocytes at various developmental stages in the thymus.

[0113] Count the cell suspensions obtained above, wash them 2-3 times with PBS, resuspend them in RPMI 1640 medium, transfer them to appropriate culture bottles and culture them at 37°C, 5% CO2. Figure 3 and Figure 4 As shown, they are the cell state diagrams of human thymic primary B cells on the 3rd day, and the cell state diagrams of human thymic primary T cells on the 3rd day, respectively. Both thymic B cells and thymic T cells are round suspension cells, and some cells can be seen in clusters, indicating good growth.

[0114] It can be seen from the above embodiments that the kit reagent combination disclosed in the present invention, combined with the separation and purification method, can efficiently achieve the separation of thymic primary epithelial cells, thymic primary dendritic cells, thymic primary B lymphocytes and thymic T lymphocytes in thymic tissue, and ensure that the separated cells have a high survival rate, high cell activity, and a high proliferation rate, and can be used for various subsequent scientific experiments.

[0115] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A human thymus primary cell isolation kit, characterized in that: Including extraction reagents and sorting reagents, the extraction reagents include: Extraction reagent A, including 2-3 g / L Hepes, 0.5-1 g / L EDTA, 50-100 U / ml penicillin-streptomycin, pH 7.0-7.5; Extraction reagent B: including 10-20ml / L HEPES, 2.5-5g / L bovine serum albumin, 50-75mg / L trypsin inhibitor, 0.1-0.2g / L butyric acid, 1-2mmol / L L-glutamine, 1-2mg / ml linoleic acid; pH value is 7.0-7.5; Extraction reagent C: including 3-4 mg / ml collagenase I, 5-10 ug / ml hyaluronidase, 0.1-0.2 mg / ml deoxyribonuclease I, pH 7.0-7.5; Extraction reagent D: containing 50%-60% percoll stock solution, 1-2 mol / L sodium chloride, pH 7.0-7.5; The sorting reagent specifically consists of sorting reagent A, sorting reagent B, sorting reagent C, sorting reagent D and sorting reagent E; Or the sorting reagent consists of sorting reagent A and sorting reagent C; Or the sorting reagent consists of sorting reagent A, sorting reagent C, sorting reagent D and sorting reagent E; Or the sorting reagent consists of sorting reagent B and sorting reagent C; in, Sorting reagent A is a biotin-labeled anti-CD45 monoclonal antibody; Sorting reagent B is a biotin-labeled anti-CD19 monoclonal antibody; Sorting reagent C is superparamagnetic nanoparticles coupled with streptavidin; Sorting reagent D is a phycoerythrin-labeled anti-CD11C monoclonal antibody; Sorting reagent E: anti-phycoerythrin superparamagnetic nanoparticles.

2. A method for isolating primary human thymic cells using the kit according to claim 1, characterized in that: First, the thymic tissue is treated with an extraction reagent to prepare a thymic tissue single cell suspension, and then one or more of thymic epithelial cells, thymic dendritic cells, thymic T cells and thymic B cells are enriched with a sorting reagent by an immunomagnetic bead method; The specific steps for preparing single cell suspension of thymus tissue are as follows: Step 1: Place the thymus tissue sample in a container, add extraction reagent A to wash, chop aseptically, pipette repeatedly and transfer to a centrifuge tube, tilt and let stand, and discard the supernatant; Step 2: Add extraction reagent B and mix well, let stand and discard the supernatant; Step 3: Add extraction reagent C, add a sterile magnetic rotor, place on a magnetic stirrer at 37°C, stir at a speed of 100-120 rpm / min for 10 min; Step 4: Tilt and let stand, discard the supernatant through a Pasteur tube, add extraction reagent A, pipette repeatedly and let stand; Step 5: Discard the supernatant, add extraction reagent C, add a sterile magnetic rotor, place on a magnetic stirrer at 37°C, stir at a speed of 100-120 rpm / min for 30 min; Step 6: Filter the resulting solution through a 70 μm sterile cell sieve and centrifuge at 800-1000 rpm for 5-10 min at room temperature; Step 7: Discard the supernatant, add extraction reagent A again, mix thoroughly with a pipette, centrifuge at 800-1000 rpm for 5-10 min at room temperature, discard the supernatant, add extraction reagent B and mix thoroughly; Step 8: Slowly add the obtained solution dropwise to the extraction reagent D, the volume ratio of the solution to the extraction reagent D is 0.75-1:1, and centrifuge at 2000 rpm for 30-50 min at 4°C, with the lifting speed being 0; Step 9: Aseptically aspirate the cells of the low-density cell layer and transfer them to a centrifuge tube containing extraction reagent B for later use. The obtained cells are thymic stromal cells containing thymic epithelial cells and thymic dendritic cells; The cells in the high-density cell layer are aseptically aspirated and transferred to a centrifuge tube containing extraction reagent B for later use. The obtained cells contain thymic T cells and thymic B cells at various developmental stages.

3. The method for isolating primary human thymic cells according to claim 2, characterized in that: The steps for isolating primary thymic epithelial cells include: Step 10: Add an appropriate amount of sorting reagent A to the low-density cell layer cells obtained in step 9 and mix thoroughly. Incubate at 4°C in the dark for 20 minutes, then add an appropriate amount of sorting reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 minutes, then add to the magnetic sorting column rinsed with PBS. The cells that do not bind to the sorting column are primary thymic epithelial cells.

4. The method for isolating primary human thymic cells according to claim 2, characterized in that: The steps for isolating primary thymic dendritic cells include: Step 10: Add sorting reagent A to the low-density cell layer cells obtained in step 9 and mix thoroughly. Incubate at 4°C in the dark for 20 minutes, then add an appropriate amount of sorting reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 minutes, then add to the magnetic separation column rinsed with PBS to collect the cells bound to the separation column. Step 11: Collect the cells bound to the sorting column, add sorting reagent D to them and mix thoroughly, incubate at 4°C in the dark for 20 minutes, then add sorting reagent E, incubate at 4°C in the dark for 30 minutes, add to the magnetic sorting column rinsed with PBS, and collect the cells bound to the sorting column, which are the thymic primary dendritic cells.

5. The method for isolating primary human thymic cells according to claim 2, characterized in that: The steps for isolating thymic T cells and thymic B cells include: Step 12: Add sorting reagent B to the high-density cell layer cells obtained in step 9 and mix thoroughly. Incubate at 4°C in the dark for 20 minutes, then add sorting reagent C and mix thoroughly. Incubate at 4°C in the dark for 30 minutes, then add to the magnetic sorting column rinsed with PBS. The cells bound to the sorting column are the primary thymic B lymphocytes, and the cells not bound to the sorting column are the T lymphocytes at various developmental stages in the thymus.

6. The method for isolating primary human thymic cells according to any one of claims 3 to 5, characterized in that: The dosage of sorting reagent A, sorting reagent B, sorting reagent C, sorting reagent D and sorting reagent E is 10 7 10-20ul per cell.

7. Use of the kit according to claim 1 in separating and purifying B lymphocytes, T lymphocytes, epithelial cells and dendritic cells in human thymus tissue.

8. Use of the method for separating primary human thymic cells according to claim 3 in separating and purifying epithelial cells in human thymic tissue.

9. Use of the method for isolating primary human thymic cells according to claim 4 in isolating and purifying dendritic cells in human thymic tissue.

10. Use of the method for isolating primary human thymic cells according to claim 5 in isolating and purifying B lymphocytes and / or T lymphocytes in human thymic tissue.

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