Three-level lymphoid structure model and preparation method thereof

Abstract

The invention relates to a three-level lymphoid structure model and a preparation method thereof, and belongs to the technical field of cell biology. The method for preparing the three-stage lymphoid structure model comprises the following steps: S1, inducing and activating peripheral blood mononuclear cells by using an extracellular matrix to obtain activated peripheral blood mononuclear cells; s2, the peripheral blood mononuclear cells and the mesenchymal stem cells activated in the step S1 are coated with hydrogel, and hydrogel microspheres are obtained; and S3, culturing T cells and B cells by using the hydrogel microspheres obtained in the step S2 to obtain the three-stage lymphatic structure. The three-level lymphoid structure model obtained by the invention maintains long-term stability in vitro, the form is normal, and the activity of T cells and B cells is strong. The method can be used for dynamically observing the three-level lymphoid structure in the subsequent research immunotherapy and drug screening process, is used for high-throughput experiments, effectively improves the efficiency of drug screening experiments, and is high in accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a three-level lymphoid structure model and a preparation method thereof. Background Art

[0002] With the increasing incidence of malignant tumors, researchers have realized the importance of tumor microecology in improving the effectiveness of tumor treatment and improving patient prognosis. As an important cellular component of tumor microecology, immune cells in tumors are closely related to the effect of tumor treatment, especially immunotherapy. Tertiary lymphoid structures (TLS) refer to organized immune cell aggregates formed in non-lymphoid tissues under non-physiological conditions, which often appear in autoimmune diseases, chronic inflammatory tissues or tumor tissues. TLS is mainly composed of T cell regions and B cell regions, and also contains a small number of follicular dendritic cells, macrophages and endothelial cells. Previous basic research on TLS has confirmed that adaptive immunity can be generated or enhanced in TLS, and B cells in TLS can produce disease-related antibodies, thereby playing a regulatory role or antibody-dependent cytotoxic effect in anti-tumor immunity. Previous clinical studies on tertiary lymphoid structures have confirmed that TLS in tumors are often closely related to the prognosis of patients with immunotherapy. The above research findings make TLS an attractive target in the field of tumor immunotherapy.

[0003] The model of in vitro lymphocyte separation and culture refers to the separation of immune cells from peripheral blood or tumor in situ by differential centrifugation and other means, and the culture is carried out in vitro. By means of additional cytokines in the culture medium, the cells can be kept alive for a certain period of time. By adding immunotherapy drugs to the culture medium, indicators such as the activation state and viability of immune cells during immunotherapy are dynamically monitored. Most existing technologies can only separate and culture a single type of immune cell in vitro, or conduct co-culture experiments at the two-dimensional culture level. They cannot induce the formation of functional immune cell aggregates, cannot induce lymphocyte aggregates with three-dimensional structures in vitro, and cannot completely simulate the complex immune microenvironment in the tumor microecology. Therefore, it is impossible to establish a powerful model in in vitro experiments to dynamically monitor the complex phenomena such as the interaction of immune cells and the subsequent effects of antibody production in the immune microecology during immunotherapy.

[0004] The mouse tumor immunotherapy model is based on the mouse in vitro tumor-bearing model. After the mouse is subcutaneously loaded with a tumor, the drug is injected into the tail vein or intraperitoneally, and then the mouse is immunotherapy-treated. The tumor size and the proportion of peripheral blood immune cells are then dynamically monitored. The mouse model cannot dynamically monitor the changes in immune cells in the tumor after drug treatment: in tumor treatment, it is necessary to dynamically monitor the changes in immune cells, but the immune cells in the tumor can only be detected by isolating primary tumor cells when the experimental endpoint is reached, and dynamic observation during the treatment process cannot be performed. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a three-level lymphoid structure model that can be dynamically monitored and a preparation method thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for preparing a three-level lymphoid structure model, comprising the following steps:

[0008] S1: Induce and activate peripheral blood mononuclear cells with extracellular matrix to obtain activated peripheral blood mononuclear cells;

[0009] S2: using hydrogel to encapsulate the peripheral blood mononuclear cells and mesenchymal stem cells activated in step S1 to obtain hydrogel microspheres;

[0010] S3: Cultivate T cells and B cells using the hydrogel microspheres described in step S2 to obtain a three-level lymphoid structure model.

[0011] The present invention further induces the differentiation of mesenchymal stem cells through activated peripheral blood mononuclear cells, releases cytokines and adhesion molecules, and promotes T cells and B cells to adhere to the hydrogel through cell-cell interaction, forming structured lymphocyte infiltration, and constructing a three-level lymphoid structure model.

[0012] Furthermore, in step S1, the method of inducing activation of peripheral blood mononuclear cells with extracellular matrix comprises the following steps:

[0013] S11: cross-linking the gelatin solution and the glutaraldehyde solution, quenching the glutaraldehyde with a glycine solution, adding a fibroblast culture medium for incubation, and obtaining a pretreated fibroblast culture medium; the fibroblast culture medium is a cell culture medium containing antibiotics and serum;

[0014] S12: culturing fibroblasts with the pretreated fibroblast culture medium of step S11 until the cell density reaches 100%, adding ascorbic acid for culturing, and obtaining cultured fibroblasts;

[0015] S13: Lyse the cultured fibroblasts of step S12 with an extracellular matrix extract, extract the extracellular matrix, remove the extracellular matrix extract, and use DNA enzyme to remove the remaining DNA to obtain the extracellular matrix attached to the bottom, wherein the extracellular matrix extract contains phosphate buffer, NH4OH and polyethylene glycol octylphenyl ether.

[0016] S14: Peripheral blood mononuclear cells were inoculated into the extracellular matrix described in step S13, 1×10 5 ~2×10 5 cells / well and cultured at 35-37°C for 24-48h to obtain activated peripheral blood mononuclear cells.

[0017] The extracellular matrix acquisition method used in the present invention is the membrane permeation method, which obtains extracellular matrix proteins by punching holes in the cell membrane and using a small amount of denaturant. Other methods, such as the use of SDS strong denaturant, do not retain extracellular matrix components as well as the method described in the present invention. When cells are cultured in an incubator, they generally settle due to gravity, so the extracellular matrix attached to the bottom can better contact with the cells.

[0018] Furthermore, in step S11, the concentration of the gelatin solution is 0.2% to 0.25% (w / v), the concentration of the glutaraldehyde solution is 1% to 2% (w / v), and the concentration of the glycine solution is 1 to 2M.

[0019] In a specific embodiment of the present invention, the concentration of the gelatin solution is 0.2% (w / v), the concentration of the glutaraldehyde solution is 1% (w / v), and the concentration of the glycine solution is 1M.

[0020] Furthermore, in step S11, the volume ratio of the gelatin solution, the glutaraldehyde solution and the glycine solution is gelatin solution: glutaraldehyde solution: glycine solution = 1-2:1:1.

[0021] In a specific embodiment of the present invention, the ratio of gelatin solution: glutaraldehyde solution: glycine solution is 2:1:1.

[0022] Furthermore, in step S11, the cross-linking time is 30 to 60 minutes. In a specific embodiment of the present invention, the cross-linking time is 30 minutes.

[0023] Furthermore, in step S11, the incubation time is 1 to 3 hours. In a specific embodiment of the present invention, the incubation time is 1 hour.

[0024] Furthermore, in step S11, the fibroblast culture medium is a high-glucose DMEM culture medium (glucose content is 4.5 g / L) containing penicillin, streptomycin, serum and DMEM. The DMEM culture medium is Dulbecco's Modified Eagle Medium, which is a conventional culture medium containing various amino acids and glucose.

[0025] Furthermore, in step S11, the fibroblast culture medium is a high-glucose DMEM culture medium containing 100-200 U / mL penicillin, 100-200 μg / mL streptomycin, and 10%-15% (v / v) serum.

[0026] Furthermore, in step S11, the fibroblast culture medium is a high-glucose DMEM culture medium containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% (v / v) serum.

[0027] Furthermore, in step S11, after the gelatin solution and the glutaraldehyde solution are cross-linked and / or the glutaraldehyde is quenched by the glycine solution, the bottom of the well plate is washed 3 to 5 times with a phosphate buffer solution. In a specific embodiment of the present invention, the number of washing times is 3 times.

[0028] Furthermore, in step S12, the final concentration of ascorbic acid is 50-100 μg / mL. In a specific embodiment of the present invention, the concentration of ascorbic acid is 50 μg / mL.

[0029] Furthermore, in step S12, the volume of ascorbic acid is 0.8-1 mL per well, and in a specific embodiment of the present invention, 1 mL per well.

[0030] Furthermore, in step S12, ascorbic acid is added and cultured for 5 to 9 days, and in a specific embodiment of the present invention, the culture time is 7 days, so that the extracellular matrix skeleton structure is more solid.

[0031] Furthermore, in step S13, the DNase is a phosphate buffer solution containing DNase I.

[0032] Furthermore, in step S13, the concentration of the DNase I is 0.1-0.2 mg / mL. In a specific embodiment of the present invention, the concentration is 0.1 mg / mL.

[0033] Furthermore, in step S13, the mixture is incubated with DNase I at 35-37° C. for 30-60 min. In a specific embodiment of the present invention, the mixture is incubated with DNase I at 37° C. for 30 min.

[0034] Furthermore, in step S13, after removing the extracellular matrix extract and / or removing the remaining DNA with DNase, the bottom of the well plate is washed 2 to 5 times with a phosphate buffer solution. In a specific embodiment of the present invention, the number of washing times is 3 times.

[0035] Furthermore, in step S13, the volume ratio of phosphate buffer, NH4OH and polyethylene glycol octyl phenyl ether in the extracellular matrix extract is phosphate buffer: NH4OH: polyethylene glycol octyl phenyl ether = 47.75-48.75: 1-2: 0.25-1.25. In a specific embodiment of the present invention, phosphate buffer: NH4OH: polyethylene glycol octyl phenyl ether = 48.75: 1: 0.25.

[0036] In a specific embodiment of the present invention, in step S14, 1×10 5 cells / well and cultured at 37°C for 48 h.

[0037] Furthermore, the hydrogel contains serum, and the volume ratio of the hydrogel to the serum is hydrogel: serum = 3 to 9:1.

[0038] Preferably, the volume ratio of the hydrogel to serum is hydrogel: serum = 9: 1. When the hydrogel concentration is too low, the cell spheroids cannot be tightly bound for a long time, the spheroids break, and the cells are released from the spheroids and adhere to the wall, and cannot form a three-dimensional structure.

[0039] Furthermore, the serum is fetal bovine serum, and the type of the fetal bovine serum can be Australian fetal bovine serum, Brazilian fetal bovine serum or New Zealand fetal bovine serum. In a specific embodiment of the present invention, the experiment is conducted by taking Brazilian fetal bovine serum as an example.

[0040] Furthermore, the concentration of the fetal bovine serum is 10% to 15% (v / v). In a specific embodiment of the present invention, the concentration is 10% (v / v).

[0041] Furthermore, in step S2, the peripheral blood mononuclear cells and mesenchymal stem cells are digested with 1×Tryple digestion solution and then coated with hydrogel.

[0042] Furthermore, in step S2, the content of activated peripheral blood mononuclear cells is 5×10 5 ~1×10 6 cells / mL. In a specific embodiment of the present invention, the content of activated peripheral blood mononuclear cells is 1×10 6 cells / mL.

[0043] Furthermore, in step S2, the ratio of the number of activated peripheral blood mononuclear cells to the number of mesenchymal stem cells is activated peripheral blood mononuclear cells: mesenchymal stem cells=(50-100):1.

[0044] In a specific embodiment of the present invention, the ratio of activated peripheral blood mononuclear cells to mesenchymal stem cells is 100:1, and the content of mesenchymal stem cells is 1×10 4 cells / mL.

[0045] Further, in step S2, the hydrogel, activated peripheral blood mononuclear cells and mesenchymal stem cells are mixed and co-cultured at a temperature of 35 to 37° C. for 30 to 60 min. In a specific embodiment of the present invention, the culture temperature is 37° C. for 30 min.

[0046] Further, in step S2, the types of hydrogels include Hydrogel Matrix and / or MSC, in a specific embodiment of the present invention, the hydrogel is Hydrogel Matrix.

[0047] In a specific embodiment of the present invention, in step S2, the mesenchymal stem cells are derived from bone marrow.

[0048] Further, in step S3, the hydrogel microspheres are mixed with T cells and B cells and co-cultured at a temperature of 35 to 37° C. for 72 to 144 h. In a specific embodiment of the present invention, the culture temperature is 37° C. for 72 h.

[0049] Further, in step S3, the hydrogel microspheres are mixed with a cell suspension containing T cells and B cells for co-culture, and the content of T cells and / or B cells in the cell suspension is 1 to 2×10 6 cells / mL.

[0050] In a specific embodiment of the present invention, the content of T cells or B cells is 1×10 6 cells / mL.

[0051] Further, T cells and B cells were resuspended in PRIM-1640 medium containing interleukin-2, T cell-B cell activation molecule, interleukin-4, streptomycin, penicillin and fetal bovine serum to obtain a mixed cell suspension.

[0052] As a preferred embodiment, T cells and B cells are resuspended in PRIM-1640 medium containing 50-100 U / mL interleukin-2, 5-10 μg / mL T cell-B cell activation molecule, 10-20 ng / mL interleukin-4, 100-200 U / mL streptomycin, 100-200 U / mL penicillin and 10%-15% (v / v) fetal bovine serum.

[0053] In a specific embodiment of the present invention, T cells and B cells are resuspended in PRIM-1640 medium containing 50 U / mL interleukin-2, 5 μg / mL T cell-B cell activation molecule, 10 ng / mL interleukin-4, 100 U / mL penicillin, 100 μg / mL streptomycin and 10% (v / v) fetal bovine serum.

[0054] Furthermore, in step S3, the volume ratio of the hydrogel microspheres to the cell mixed suspension is 1:95-105.

[0055] In a specific embodiment of the present invention, the volume ratio of the hydrogel microspheres to the cell mixed suspension is 1:100.

[0056] In a second aspect, the present invention provides a three-level lymphoid structure model prepared by the preparation method.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The tertiary lymphoid structure model obtained by the present invention maintains long-term stability in vitro, has normal morphology, and has strong activity of T cells and B cells. It can be used for dynamic observation of tertiary lymphoid structures in subsequent research on immunotherapy and drug screening, and for high-throughput experiments, effectively improving the efficiency of drug screening experiments with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Laser confocal microscopy was used to observe the formation of T cell and B cell immune aggregates in hydrogel microspheres.

[0060] Figure 2 Flow chart for constructing an in vitro model of induced tertiary lymphoid structures.

[0061] Figure 3 It is the T cell viability in the in vitro induced tertiary lymphoid structure model.

[0062] Figure 4 This is the activation of T cells in the in vitro induced tertiary lymphoid structure model.

[0063] Figure 5 This is the activation of B cells in the in vitro induced tertiary lymphoid structure model.

[0064] Figure 6 The effect of the ratio of fetal bovine serum to hydrogel on the culture effect.

[0065] Figure 7 It is a hydrogel microsphere coated with cytokines only. DETAILED DESCRIPTION

[0066] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0067] Example 1 Activation of peripheral blood mononuclear cells (PBMC)

[0068] 1. Isolation of PBMCs

[0069] (1) Wild-type C57BL / 6J mice aged 6 to 8 weeks were anesthetized with pentobarbital to obtain anesthetized mice.

[0070] (2) Use a 1 mL syringe to puncture the heart from the diaphragm of the anesthetized mouse in step (1) to collect blood, and place the blood in an anticoagulant EP tube (small centrifuge tube).

[0071] (3) Blood dilution: dilute the blood in step (2) with physiological saline, wherein the volume ratio of physiological saline to blood is 1:1, to obtain diluted blood.

[0072] (4) Adding separation solution: Carefully spread the blood diluted in step (3) on 3 mL of mouse lymphocyte separation solution (brand: Dakoway, product number: DKW33-R0100), keeping the interface between the two liquids clear and avoiding mixing, to obtain blood with added separation solution.

[0073] (5) Centrifuge the blood in step (4) at room temperature at 400g for 20 minutes. Adjust the centrifuge speed to 0. After centrifugation, the liquid in the centrifuge tube is divided into several layers, among which the ring-shaped milky white lymphocyte layer is the target to be separated. Use a pipette to carefully draw the mononuclear cells in this layer into another centrifuge tube.

[0074] (6) After adding physiological saline to the mononuclear cells in step (5), the cells were dispersed and centrifuged at 300 g for 10 min. The supernatant was removed and the mixture was repeated twice to collect the cells.

[0075] (7) Resuspend the cells in step (6) in high-glucose DMEM medium (Dulbecco's Modified Eagle Medium, a culture medium containing various amino acids and glucose, with a glucose content of 4.5 g / L) containing 1% (w / v) penicillin-streptomycin dual antibody (100 U / mL penicillin, 100 μg / mL streptomycin) and 10% (v / v) fetal bovine serum, and culture in an incubator at 37°C and 5% CO2 to obtain PBMCs.

[0076] 2. Extraction of Extracellular Matrix

[0077] (1) After anesthetizing, mmTv-pymt mice (a human breast cancer animal model) with tumors were killed by cervical dislocation. The mouse tumors were bluntly dissected and completely removed. The tumors were repeatedly rinsed with saline containing 2% (w / v) streptomycin and penicillin (100 U / mL penicillin, 100 μg / mL streptomycin) and 10% (v / v) fetal bovine serum to remove hair and blood vessels attached to the tumors. The tumor tissues were cut into pieces of 1 mm using ophthalmic scissors. 3 size and obtain tumor tissue blocks.

[0078] (2) The tumor tissue block from step (1) was transferred into a C tube for tissue digestion (brand: Miltenyi Biotec, item number: 130-096-334) using a Pasteur tube, 10 mL of digestion solution (containing 1 mg / mL type I collagenase, 1 mg / mL type IV collagenase, 0.1 mg / mL DNase I and 10% (v / v) fetal bovine high-glucose DMEM medium) was added, and the C tube for tissue digestion was placed on a test tube mixer (brand: Miltenyi Biotec), digested at 37°C for 40 min, and 10 mL of pre-cooled PBS (phosphate buffer solution) was added to terminate the digestion to obtain a digested tissue homogenate.

[0079] (3) Filter the tissue homogenate after digestion in step (2) through a 100 μm filter, centrifuge at 400 g for 5 min, discard the supernatant, and collect the cells; add 10 mL of PBS to the cells and resuspend them again, centrifuge at 300 g for 10 min, discard the supernatant, and collect the cells.

[0080] (4) Count the cells in step (3) and calculate the number of cells per 1×10 7 The cells were resuspended in 90 μL MACS (magnetic activated cell sorting) buffer (brand: Miltenyi, catalog number: 130-091-222), and 10 μL CD326 (EPCAM) magnetic beads (brand: Miltenyi, catalog number: 130-105-958) were added. After mixing, the mixture was placed in a 2-8°C refrigerator for incubation for 15 min. 1-2 mL MACS buffer was added, mixed, and centrifuged at 300 g for 10 min. The supernatant was discarded to obtain magnetic bead-labeled cells.

[0081] (5) Place the LS Separation columns in the magnetic field of a suitable MACS magnetic bead separator and rinse the LS columns with 3 mL of MACS buffer; suspend the cells labeled with magnetic beads in step (4) in 500 μL of MACS buffer to obtain a magnetic bead-labeled cell suspension, add the magnetic bead-labeled cell suspension to the LS columns, and rinse the LS columns again with 3 mL of MACS buffer. Repeat this process three times and collect the cell (fibroblast) suspension that has not been labeled with magnetic beads.

[0082] (6) The cell suspension that was not labeled with magnetic beads discharged from step (5) was centrifuged at 300 g for 10 min to obtain fibroblasts, which were resuspended in high-glucose DMEM fibroblast culture medium containing 1% (w / v) penicillin-streptomycin dual antibody (100 U / mL penicillin, 100 μg / mL streptomycin) and 10% (v / v) fetal bovine serum, and placed in a culture flask and cultured in a constant temperature incubator at 37° C. and 5% CO2.

[0083] (7) Add 0.2% (w / v) gelatin solution (1 mL / well) to a 12-well plate and treat at 37°C for 1 h. Add 1% (w / v) glutaraldehyde solution (500 μL / well) and cross-link at room temperature for 30 min. Rinse the bottom of the plate three times with PBS. Add 1 M glycine solution (500 μL / well) to quench the glutaraldehyde. Rinse the bottom of the plate three times with PBS and add fibroblast culture medium for pretreatment for 1 h.

[0084] (8) After digesting and counting the fibroblasts in step (6), the fibroblasts were inoculated into the 12-well plate treated in step (7) at a density of 10,000 cells / well and cultured in a constant temperature incubator at 37° C. and 5% CO 2 . The density of fibroblasts was dynamically observed. When the cell density was close to 100%, ascorbic acid at a final concentration of 50 μg / mL was added to the fibroblast culture medium. The culture was continued for 7 days with the medium changed every day to make the extracellular matrix skeleton structure produced by the fibroblasts more solid.

[0085] (9) After the fibroblasts were cultured in step (8), 1 mL of preheated extracellular matrix extract was added to each well to extract the extracellular matrix. The extracellular matrix extract was prepared by mixing 48.75 mL of PBS, 1 mL of NH4OH, and 250 μL of Triton X-100 (polyethylene glycol octylphenyl ether) and sterilizing it by filtering it with a 0.45 μm filter.

[0086] (10) Observe the fibroblasts in step (9) under a microscope. When all the fibroblasts are lysed, gently aspirate the extracellular matrix extract containing the cell lysate. The part attached to the bottom of the plate is the extracellular matrix. Rinse gently three times with PBS. Add PBS solution containing 0.1 mg / mL DNase I, 1 mL / well, and incubate at 37°C for 30 min to remove the DNA remaining on the bottom of the plate. Rinse three times with PBS to obtain the extracellular matrix attached to the bottom of the plate.

[0087] 3. Induce activation of PBMC cells

[0088] The PBMCs from step 1 were digested with 1×Tryple digestion solution and counted. The PBMCs were resuspended in high-glucose DMEM medium containing 10% (v / v) fetal bovine serum and 1% (w / v) penicillin-streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin) and inoculated into 12-well plates with extracellular matrix attached to the bottom of the plate. 1×10 5 cells / well, and cultured in a constant temperature incubator at 37°C, 5% CO2 for 48 hours to induce PBMC activation and obtain activated PBMC.

[0089] Example 2 Extraction of bone marrow-derived mesenchymal stem cells (MSC)

[0090] 1. After anesthetization, 6-8 week old wild-type C57BL / 6J mice were killed by cervical dislocation. The femur and pelvis of the mice were cut open, and the attached skin and muscle tissue were peeled off with scissors and tweezers. DMEM medium was extracted with a 20 mL syringe, and the needle of the 20 mL syringe was replaced with a 1 mL syringe needle. The two ends of the femur and tibia of the mice were cut open with a clean pair of ophthalmic scissors. The syringe needle was inserted into the bone marrow cavity and repeatedly flushed until most of the bone marrow was flushed out and the bone turned white. The flushed bone marrow cells were repeatedly aspirated with a Pasteur pipette to disperse the clustered cells, and filtered with a 70 μm filter to remove hair and muscle tissue, collect cells, centrifuge at 300 g for 10 min, remove the supernatant, and obtain cell pellets.

[0091] 2. Resuspend the cell pellet from step 1 in low-glucose DMEM medium (glucose content is 1 g / L) containing 1% (w / v) penicillin-streptomycin double antibody (100 U / mL penicillin, 100 μg / mL streptomycin) and 10% (v / v) fetal bovine serum, count the cells, plant the cells in a 6-well plate, and culture them in an incubator at 37°C, 5% CO2. Change the medium every 8 hours to remove most of the immune cells. After 72 hours of culture, change the medium every 2 days. MSC colony formation can be seen in about 7 days.

[0092] Example 3 Isolation of T cells and B cells

[0093] 1. Isolation of lymphocytes

[0094] (1) The spleen of a 10-week-old tumor-bearing C57 mouse was taken and placed on a rinsed 40 μm sieve. The spleen was ground with the soft tip of a 5 mL syringe piston and sieved.

[0095] (2) After washing the sieve in step (1) with 10 mL of PRMI 1640 medium (Roswell Park Memorial Institute, a medium for cell culture and tissue culture), collect the filtrate and transfer it into a 15 mL centrifuge tube. Centrifuge at 4°C, 300 g for 10 min, discard the supernatant, and collect the spleen cell pellet.

[0096] (3) Resuspend the spleen cell pellet from step (2) with 4 mL of mouse lymphocyte separation solution to obtain a spleen cell separation solution resuspension. The viscosity of the resuspension is not too high. Tilt the tube 45° and slowly add 8 mL of PRMI 1640 medium to the upper layer of the spleen cell separation solution resuspension using a Pasteur pipette. Be careful not to damage the interface between the two liquids. Centrifuge at 800 g for 3 min at room temperature. It can be seen that the lymphocyte layer is located between the lymph node cell separation solution and the PRMI 1640 medium.

[0097] (4) Use a Pasteur pipette to draw the lymphocyte layer from step (3) into a new 15 mL centrifuge tube, add PRMI 1640 medium to dilute to 10 mL, centrifuge at 300 g at 4°C for 10 min, remove the supernatant, and collect the precipitate, which is the lymphocytes.

[0098] 2. Sorting T cells and B cells

[0099] (1) The lymphocytes obtained in step 1 were resuspended in 200 μL sterile PBS and divided into two equal parts, blocked with Fc receptor (receptor for the C-terminus of the Fc part of immunoglobulin), and resuspended in 100 μL PBS to obtain two lymphocyte resuspensions with a cell content of 1×10 6 cells / mL; 5 μL APC-anti mouse CD90.2 (Brand: biolegend; Catalog No.: 140312) antibody and 4 μL FITC-anti mouse CD19 / CD20 (Brand: biolegend; Catalog No.: 152404 / 980202) antibody were added to the lymphocyte resuspension, and incubated at 4°C in the dark for 20 min.

[0100] (2) Wash the lymphocytes incubated with APC-anti mouse CD90.2 and FITC-anti mouse CD19 / CD20 in step (1) with 5 mL of pre-cooled PBS, centrifuge at 300 g for 10 min at 4°C, discard the supernatant, repeat 3 times, and collect the lymphocytes.

[0101] (3) Resuspend the lymphocytes in step (2) with 40 μL MACS buffer to obtain a lymphocyte resuspension incubated with APC-anti mouse CD90.2 and a lymphocyte resuspension incubated with FITC-anti mouse CD19 / CD20; add 10 μL APC-labeled magnetic beads (brand: Miltenyi Biotec, catalog number: 130-090-855) to the lymphocyte resuspension incubated with APC-anti mouse CD90.2, and add 20 μL FITC-labeled magnetic beads (brand: Miltenyi Biotec, catalog number: 130-048-701) to the lymphocyte resuspension incubated with FITC-anti mouse CD19 / CD20, incubate at 4°C for 20 min, wash with 2 mL MACS buffer respectively, centrifuge at 4°C and 300 g for 10 min, discard the supernatant, and resuspend the precipitate with 50 μL MACS buffer respectively to obtain magnetic bead-labeled APC-anti mouse Lymphocyte resuspension incubated with CD90.2 and lymphocyte resuspension incubated with magnetic beads and FITC-anti mouse CD19 / CD20.

[0102] (4) Place the MS column (Separation columns) in the magnetic field of a suitable MACS magnetic bead separator, rinse the MS column with 1 mL of MACS buffer, add the two magnetic bead-labeled lymphocyte resuspensions in step (3) to the MS column respectively, and rinse the column again with 0.5 mL of MACS buffer. Repeat 3 times and discard the lymphocytes that have flowed out and are not labeled with magnetic beads.

[0103] (5) Add 2 mL of MACS buffer to the column in step (4) and use the piston to quickly push out the positive lymphocytes labeled with magnetic beads. Among them, CD90.2 positive cells are T cells, and CD19 / CD20 positive cells are B cells.

[0104] 3. Staining of T cells and B cells

[0105] (1) The T cells and B cells separated in step 2 were centrifuged at 300 g for 10 min at 4°C, and then resuspended in PRMI 1640 medium at room temperature to obtain T cell resuspension and B cell resuspension.

[0106] (2) Add cell-tracker deep red (live cell tracing probe (deep red); brand: Thermo, catalog number: C34565) to the T cell suspension, the volume ratio of cell-tracker deep red to T cell suspension is 1:1000, and place in a 37°C incubator away from light for staining for 20 min; add CMFDA (5-chloromethylfluorescein diacetate, a green fluorescent dye that can freely penetrate the living cell membrane to trace cells; brand: Thermo, catalog number: C7025) to the B cell suspension, the volume ratio of CMFDA to B cell suspension is 1:200, and place in a 37°C constant temperature incubator away from light for staining for 20 min.

[0107] (3) The cells stained in step (2) were washed three times with PRIM-1640 medium containing 10% (v / v) fetal bovine serum, and then counted. The cells were resuspended in T&B medium (PRIM-1640 medium containing 50 U / mL IL-2, 5 μg / mL CD40L, 10 ng / mL IL-4, 1% (w / v) streptomycin-penicillin dual antibody (100 U / mL penicillin, 100 μg / mL streptomycin) and 10% (v / v) fetal bovine serum) to a total cell content of 2×10 6 cells / mL, of which the T cell content was 1×10 6 cells / mL, and the B cell content was 1×10 6 cells / mL to obtain a mixed suspension of T&B cells.

[0108] Example 4 In vitro induction of a functional tertiary lymphoid structure model

[0109] 1. Hydrogel microspheres coated with activated PBMC and MSC

[0110] (1) The activated PBMCs of Example 1 and the MSCs of Example 2 were digested with 1×Tryple digestion solution and then counted.

[0111] (2) Use 10% (v / v) fetal bovine serum Resuspend the cells in step (1) with Hydrogel Matrix (manufacturer: TheWellBioscience, catalog number: TWG010) to obtain a hydrogel containing activated PBMC and MSC (PBMC cell content: 1×10 6 cells / mL, MSC cell content is 1×10 4 cells / mL).

[0112] (3) Add the hydrogel containing activated PBMC and MSC to the bottom of a 12-well confocal plate using a small pipette tip, 5 μL / well, to form a microsphere structure. Place the confocal plate in a constant temperature incubator at 37°C and 5% CO2 for 30 min to obtain hydrogel microspheres coated with activated PBMC and MSC.

[0113] 2. Add T&B cell mixed suspension for co-culture

[0114] (1) Add the mixed suspension of T and B cells in Example 3 to the confocal plate in step 1, 500 μL / well, and place in a constant temperature incubator at 37° C. and 5% CO 2 for 72 hours.

[0115] (2) Laser confocal microscopy was used to observe the formation of T cell and B cell immune aggregates in hydrogel microspheres. Figure 1 ), T cells and B cells form immune aggregates to obtain tertiary lymphoid structures. Figure 2 Shown is a flow chart for constructing an in vitro induced tertiary lymphoid structure model.

[0116] Example 5 Viability and activation of T cells and B cells in the three-level lymphoid structure model

[0117] 1. Experimental Methods

[0118] The tertiary lymphoid structure of Example 4 was cultured in an incubator at 37°C with 5% CO2 for 5 days and then treated with a dissolving agent ( Organoid Recovery, brand: The Well, item number: MS04-100) was used to separate T cells and B cells from the in vitro induced tertiary lymphoid structure model, and the cell viability and activation degree were detected using Beckman cytofles s flow cytometer. The control group was T cells and B cells cultured alone and subjected to the same experimental treatment.

[0119] 2. Experimental Results

[0120] After 5 days of culture, the viability of T cells in the in vitro induced tertiary lymphoid structure model ( Figure 3 ) and activation status ( Figure 4 ) were better than the control group cultured alone.

[0121] After 5 days of culture, some B cells in the in vitro induced tertiary lymphoid structure model can be induced to germinal center B cell phenotype, while the control group B cells cannot tolerate long-term in vitro culture under the same in vitro culture conditions, showing death and disintegration, and cannot be detected by flow cytometry ( Figure 5 ). BCL6 is a marker of germinal center B cells. The induction of BCL6+ B cells indicates that the in vitro induced tertiary lymphoid structure model has a germinal center structure unique to the tertiary lymphoid structure.

[0122] Example 6 Effect of the ratio of fetal bovine serum to hydrogel on the culture effect

[0123] 1. Experimental Methods

[0124] The volume ratios of fetal bovine serum and hydrogel in the hydrogel were set to 3:1, 5:1 and 9:1 respectively, and a three-level lymphoid structure model was prepared according to the method of Example 4, and photographed using a laser scanning confocal (zeiss LSM880).

[0125] 2. Experimental Results

[0126] When the hydrogel concentration is too low, the cell spheroids cannot be tightly bound for a long time, the spheroids break, and the cells are released from the spheroids and adhere to the wall, and cannot form a three-dimensional structure ( Figure 6 ).

[0127] Comparative Example 1

[0128] 1. Experimental Methods

[0129] Cytokines were mixed with 10% (v / v) fetal bovine serum Hydrogel Matrix hydrogel (manufacturer: TheWell Bioscience, item number: TWG010) was mixed to obtain a cytokine-coated hydrogel. Cytokine-coated hydrogel microspheres were obtained according to the method of Example 4, and then co-cultured with a mixed suspension of T&B cells to construct a tertiary lymphoid structure.

[0130] The cytokines consist of interleukin-2 (IL-2; brand: Nearshore protein; item number: CK24), interleukin-4 (IL-4; brand: Nearshore protein; item number: CK74), interleukin-21 (IL-21; brand: Nearshore protein; item number: CK10), B cell activating factor (BAFF; brand: Nearshore protein; item number: C06U) and T cell-B cell activation molecule (CD40L; brand: Nearshore protein; item number: CU78). Among them, the content of IL-2 is 30U / mL, the content of IL-4 is 20ng / mL, the content of IL-21 is 10ng / mL, the content of BAFF is 50ng / mL, and the content of CD40L is 2μg / mL.

[0131] 2. Experimental Results

[0132] like Figure 7 As shown, the hydrogel microspheres only contained cytokines and lacked PBMC and MSC cell components, and could not withstand long-term culture. The hydrogel of the cells in the 72h culture system had disintegrated and the formed structure could not be observed.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a tertiary lymphoid structure model, characterized in that: The following steps are involved: S1: Induce and activate peripheral blood mononuclear cells with extracellular matrix to obtain activated peripheral blood mononuclear cells; S2: using hydrogel to encapsulate the peripheral blood mononuclear cells and mesenchymal stem cells activated in step S1 to obtain hydrogel microspheres; S3: Cultivate T cells and B cells using the hydrogel microspheres described in step S2 to obtain tertiary lymphoid structures.

2. The preparation method according to claim 1, characterized in that In step S1, the method of inducing activation of peripheral blood mononuclear cells with extracellular matrix comprises the following steps: S11: cross-linking the gelatin solution and the glutaraldehyde solution, quenching the glutaraldehyde with a glycine solution, adding a fibroblast culture medium for incubation, and obtaining a pretreated fibroblast culture medium; the fibroblast culture medium is a cell culture medium containing antibiotics and serum; S12: culturing fibroblasts with the pretreated fibroblast culture medium of step S11 until the cell density reaches 100%, adding ascorbic acid for culturing, and obtaining cultured fibroblasts; S13: Lysing the cultured fibroblasts in step S12 with an extracellular matrix extract, extracting the extracellular matrix, removing the extracellular matrix extract, and removing the remaining DNA with a DNA enzyme to obtain the extracellular matrix attached to the bottom, wherein the extracellular matrix extract contains a phosphate buffer, NH4OH, and polyethylene glycol octylphenyl ether; S14: Inoculate peripheral blood mononuclear cells into the extracellular matrix described in step S13, 2 to 8×10 5 cells / well and cultured at 35-37°C for 24-72h to obtain activated peripheral blood mononuclear cells.

3. The preparation method according to claim 1, characterized in that: In step S2, the hydrogel contains serum, and the volume ratio of the hydrogel to the serum is hydrogel: serum = 3 to 9:

1.

4. The preparation method according to claim 1, characterized in that: In step S2, the content of activated peripheral blood mononuclear cells is 5×10 5 ~1×10 6 cells / mL.

5. The preparation method according to claim 1, characterized in that: In step S2, the ratio of the number of activated peripheral blood mononuclear cells to that of mesenchymal stem cells is activated peripheral blood mononuclear cells:mesenchymal stem cells=(50-100):

1.

6. The preparation method according to claim 1, characterized in that: In step S2, the hydrogel, activated peripheral blood mononuclear cells and mesenchymal stem cells are mixed and co-cultured at a temperature of 35 to 37° C. for 30 to 60 minutes.

7. The preparation method according to claim 1, characterized in that: In step S2, the hydrogel type includes at least one of the following.

8. The preparation method according to claim 1, characterized in that: In step S2, the mesenchymal stem cells are derived from bone marrow.

9. The preparation method according to claim 1, characterized in that: In step S3, the hydrogel microspheres are mixed with T cells and B cells and co-cultured at a temperature of 35 to 37° C. for 72 to 144 hours.

10. A three-level lymphoid structure model, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 9.

Images