Co-culture microsphere model with core-shell structure and preparation method of co-culture microsphere model

Abstract

The invention relates to a co-culture microsphere model with a core-shell structure and a preparation method of the co-culture microsphere model. The co-culture microsphere model comprises a core-shell microsphere, the core-shell microsphere is composed of an inner core and an outer shell, the inner core is an organoid loaded by matrigel, the outer shell is formed by crosslinking a photo-crosslinking hydrogel material, co-culture cells are doped in the photo-crosslinking hydrogel material, and the inner core is wrapped by the outer shell. According to the co-culture microsphere model, double-layer structure microspheres of co-culture cell wrapped organoids are formed, immune infiltration and in-vivo characteristics can be simulated, and a similar in-vivo three-dimensional tumor immune microenvironment is provided. And the types of cells loaded on the inner layer and the outer layer can be flexibly replaced to form a microsphere model for co-culture of single or multiple cells and organoids. The co-culture microsphere model is more stable in structure, the size of the diameter of the core-shell microsphere can be optimized by changing 3D printing parameters, the operability of subsequent research is high, and the co-culture microsphere model is suitable for different research application scenes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a core-shell structured co-cultured microsphere model and its preparation method. Background Technology

[0002] Immune co-culture is a technique for culturing different types of immune cells or other related cells together in an in vitro environment. This method is primarily used to study cell-cell interactions and their roles in the function and regulation of the immune system. Immune co-culture has broad application prospects in immunological research, disease treatment, and drug development; however, the main challenge currently lies in the lack of high-throughput in vitro models applicable to immunotherapy research. Existing immune co-culture models still face challenges and limitations, such as cell type compatibility, culture medium selection, structural complexity, and throughput issues. Summary of the Invention

[0003] The purpose of this invention is to disclose a core-shell structured co-cultured microsphere model and its preparation method, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0004] The first aspect of the present invention is to provide a co-culture microsphere model with a core-shell structure.

[0005] A second aspect of the present invention is to provide a method for preparing the co-cultured microsphere model.

[0006] The co-culture microsphere model described in the first aspect of this invention includes core-shell microspheres, each consisting of a core and an outer shell. The core is an organoid supported by a matrix gel, and the outer shell is formed by cross-linking a photocrosslinked hydrogel material doped with co-cultured cells. The outer shell encapsulates the core. This co-culture microsphere model is a relatively independent and standardized micro-unit. After preparation, it can be segmented and seeded into well plates according to subsequent experimental needs, and stained with molecular markers, etc., exhibiting enhanced mobility, operability, and stability. Furthermore, because the outer shell is formed by cross-linking a photocrosslinked hydrogel material, it possesses a certain mechanical strength, preventing damage to the organoids or the entire co-culture system even in culture environments with liquid shear, such as suspension culture.

[0007] In some embodiments of the first aspect of the invention, the organoids are cultured from normal visceral cells or cancer cells.

[0008] In some embodiments of the first aspect of the present invention, the co-cultured cells are selected from immune cells, fibroblasts, or vascular endothelial cells. Further, the immune cells are selected from at least one of T cells, B cells, NK cells, TILs cells, or CAR-T cells.

[0009] Common combinations of organoids and co-cultured cells include co-culturing various cancer organoids such as those from breast cancer and colorectal cancer with various immune cells such as T cells, which can provide reliable in vitro models for tumor research, immunotherapy and other fields; or co-culturing organoids from normal internal organs with vascular endothelial cells, which can form vascularized co-cultured microsphere models for use in organ repair and other research projects.

[0010] In some application embodiments of the first aspect of the present invention, the photocrosslinked hydrogel material is selected from at least one of methacrylamide hyaluronic acid (HAMA), methacrylamide polylysine (PLMA), carboxymethyl chitosan (CMCS), methacrylamide sodium alginate (AlgMA), or methacrylamide dextran (DexMA). Depending on the different photocrosslinked hydrogel materials, the corresponding illumination conditions will vary, requiring replacement with blue light, ultraviolet light, etc.

[0011] The preparation method of the co-cultured microsphere model according to the second aspect of the present invention includes the following steps: preparing core material: culturing organoids and mixing the organoids with the matrix gel to form core material; preparing shell material: obtaining co-cultured cells and mixing the co-cultured cells with the photocrosslinked hydrogel material to form shell material; 3D printing the co-cultured microsphere model: filling the core material and the shell material into the corresponding hopper of the droplet microfluidic printing device, performing 3D printing of core-shell microspheres, and achieving crosslinking and molding of the printed product by light irradiation, thereby obtaining the co-cultured microsphere model.

[0012] In some application embodiments of the second aspect of the present invention, the steps of culturing the organoids during the preparation of the core raw materials include: (1-1) cleaning the tissue sample, wetting the tissue sample with PBS buffer, physically removing excess tissue to obtain a tissue block; (1-2) placing the tissue block on an ice box, cutting it into small pieces with a side length of 1 mm, adding PBS buffer and mixing well, and transferring it to a centrifuge tube; (1-3) centrifuging, discarding the supernatant, adding 3-5 mL of organoid digestion solution I, and digesting at 37 ℃ on a shaker for 1-2 minutes; (1-4) pipetting 2-3 times, centrifuging at 1500 rpm for 3 minutes, and discarding the supernatant; (1-5) adding 2-3 mL of organoid digestion solution II and mixing well, and digesting at 37 ℃ on a shaker for 8-10 minutes; (1-6) adding 4-6 mL of digestion termination solution, mixing well to terminate digestion, centrifuging, and discarding the supernatant; (1-7) resuspending the cells in PBS buffer, filtering the cell suspension through a 100 μm pore size filter, collecting the filtrate, and centrifuging at 1500 rpm. Centrifuge at rpm for 3 minutes, discard the supernatant, and add organoid culture medium to obtain cell resuspension; (1-8) Mix the matrix gel with the cell resuspension to maintain the cell density at 1000~2000 cells / μL to obtain matrix gel cell suspension; (1-9) Take 20~30 μL of the matrix gel cell suspension and seed it into a well plate, and incubate at 37 ℃ until fully solidified; (1-10) Take out the well plate, add 1~2 mL of organoid culture medium to the solidified gel droplet, and incubate at 37 ℃ for 5~7 days to obtain the organoid.

[0013] After obtaining organoids in good condition, they can be passaged using the following steps: (1-11) Discard the old organoid culture medium in the well plate; (1-12) Add 1-2 mL of organoid passage digestion solution to each well, pipette a few times to disperse the droplets, and digest at 37°C for 5-10 minutes to digest the organoids into single cells; (1-13) Add 1-2 mL of digestion stop solution, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant; (1-14) Resuspend 20-40 μL of organoid culture medium, add an appropriate amount of matrix gel and mix thoroughly; (1-15) Seed 20-30 μL of the matrix gel cell suspension into the well plate, and incubate upside down at 37°C until fully solidified; (1-16) Remove the well plate, add 1-2 mL of organoid culture medium to the solidified droplets, and incubate at 37°C for 5-7 days to complete the passage of the organoids.

[0014] In some embodiments of the second aspect of the invention, the organoid digestion solution I is prepared in DMEMF-12 medium containing 10 μMY27632, 1 mg / mL Collagenase type II and 10 ng / mL EGF.

[0015] In some embodiments of the second aspect of the invention, the organoid digestion solution II is prepared in DMEMF-12 medium containing 1 U / mL Dispase and 100 μg / mL DNase enzyme I.

[0016] In some application embodiments of the second aspect of the present invention, in the step of preparing the shell material, the co-cultured cells are immune cells, and the step of obtaining the immune cells includes: (2-1) taking blood that has been anticoagulated and diluting it with PBS buffer; (2-2) rinsing the centrifuge tube wall with lymphocyte separation solution, then slowly adding the diluted blood, centrifuging at 2000 rpm for 20 minutes with a horizontal centrifuge, and carefully aspirating the middle layer of white flocculent material with a dropper; (2-3) washing the white flocculent material twice with PBS buffer, centrifuging at 1500 rpm for 10 minutes after each wash, and discarding the supernatant; (2-4) adding red blood cell lysis buffer, lysing and removing red blood cells, washing with PBS buffer, centrifuging at 1800 rpm for 10 minutes, and collecting the lymphocytes at the bottom of the tube; (2-5) resuspending the lymphocytes in immune cell-specific culture medium, seeding them into well plates, and changing half or all of the medium every 2-3 days.

[0017] In some implementations of the second aspect of the present invention, the immune cells are T cells, and the corresponding immune cell-specific culture medium consists of 10% FBS, 1% penicillin-dextrin antibodies, and 100 IU / mL IL-2.

[0018] In some application embodiments of the second aspect of the present invention, the co-cultured microsphere model obtained after cross-linking is placed in a special culture medium for co-cultured microspheres. When the organoid in the co-cultured microsphere model is a breast cancer organoid and T cell co-culture system, the special culture medium for co-cultured microspheres is ImmunoCult-XF T Cell Exp Medium containing 1% penicillin antibody, 1% GlutaMAX, 1×B27, 1×N-2, 500 ng / mL R-spondin 1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 100 ng / mL A83-01, 50 ng / mL EGF, 100 nM β-estradiol, 5 nM Heregulinβ-1, 50 ng / mL IL-2, 25 ng / mL IL-15 and 20 μg / mL PD-L1.

[0019] Compared with existing detection technologies, the present invention has the following advantages: The co-cultured microsphere model forms a bilayered structure of microspheres containing co-cultured cells and organoids, which can simulate immune infiltration and recreate in vivo characteristics, providing a three-dimensional tumor immune microenvironment similar to that in vivo. The cell types loaded in the inner and outer layers can also be flexibly changed to form microsphere models co-cultured with single or multiple cell types and organoids. Furthermore, the co-cultured microsphere model has a more stable structure, and the diameter of the core-shell microspheres can be optimized by changing the 3D printing parameters. It also offers strong operability for subsequent research and is suitable for various research applications. Attached Figure Description

[0020] Figure 1 These are photographs of co-cultured microsphere models prepared using different brands of matrix adhesives in Example 1; Figure 2 This is a flowchart of the preparation of the breast cancer organoid and T cell co-culture microsphere model in Example 2; Figure 3 These are photos of the cultured breast cancer organoids and expanded cultured T cells in Example 2; Figure 4 This is a photograph of the co-cultured microsphere model D1 in Example 2; Figure 5 This is a photograph of the co-cultured microsphere model D5 from Example 2; Figure 6 This is a photograph of the co-culture microsphere model of colorectal cancer organoids and immune cells prepared in Example 3; Figure 7 This is a photograph of the co-cultured microsphere model prepared from mouse lung organoids and mouse lung microvascular endothelial cells in Example 4. Detailed Implementation

[0021] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0022] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0023] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the kit and product instructions. Unless otherwise specified, the biological materials used in the kits are commercially available.

[0024] Example 1: Preparation of core-shell microspheres To verify the effects of different brands and models of matrix adhesives on the preparation of co-cultured microsphere models, a foreign brand matrix adhesive (product number: 356231) and a domestic brand matrix adhesive (product number: VM004-PRF-10) were selected as core materials to prepare core-shell microspheres.

[0025] Specifically, a core material containing 60% matrix adhesive and a shell material containing 2% HAMA solution (containing 0.3% LAP) were each drawn into a 1 mL syringe, and corn oil (containing 1% Span-80) was drawn into a 50 mL syringe. These three syringes were loaded onto the corresponding hoppers of the droplet microfluidic printing device for the core, shell, and oil phase, respectively, to perform 3D printing of core-shell microspheres. The printed products were then cross-linked and formed using UV curing. The resulting core-shell microspheres are shown below. Figure 1 As shown, the core-shell microspheres prepared by the two matrix adhesives are round and uniform in size (the outer shell is about 400 μm in size and the core is about 300 μm in size). The core structure can be clearly observed inside. Therefore, it can be concluded that using different brands of matrix adhesives as core raw materials does not affect the microsphere structure and both can form core-shell microspheres.

[0026] Example 2: Co-culture microsphere model of breast cancer organoids and T cells according to Figure 2 The process shown is for co-culturing microsphere models. The specific implementation steps are as follows: S1. Passage and expansion culture of breast cancer organoids: Obtain breast cancer surgical samples from patients, mechanically cut the tissue samples, carry out primary culture, and then passage and expand. During the culture process, observe the growth status of breast cancer organoids and count the organoids until a sufficient number of cells are reached for the preparation of co-cultured microsphere models. S2. Extraction of blood immune cells: Blood from the same patient is collected into an anticoagulant tube. After simple dilution of the anticoagulated blood, it is mixed with the upper layer of the lymphocyte separation solution. After low-speed centrifugation to form cell stratification, the middle layer of white flocculent PBMC cell precipitate is aspirated, thus completing the extraction of immune cells. S3, T cell sorting and amplification activation: Immunomagnetic beads were used for sorting, and antibody-coated magnetic beads were used to separate T cell subsets. They were then added to a special amplification medium for T cell culture and cultured. During the culture process, immune cells were counted. After a sufficient number of cells were reached, antibodies were added to activate the immune cells. S4. 3D Printing of Co-culture Microspheres of Breast Cancer Organoids (Nucleus) and T Cells (Shell): Breast cancer organoids are mixed in matrix gel to form the core material; immune-activated T cells are mixed in HAMA to form the shell material; and microfluidic chips are used to print the core-shell microspheres.

[0027] The specific implementation method for S1 breast cancer organoid culture mainly includes the following steps: (1) Wash breast cancer tissue samples 20 times with 5-10 mL of cold PBS buffer (containing 2% penicillin + 1× amphotericin B). During the washing process, the tissues will settle naturally by gravity. Remove the supernatant of the washing solution with a Papanicolaou pipette. (2) After cleaning, use tweezers to transfer the tissue block to a new 6 cm culture dish, add a small amount of PBS buffer to moisten the tissue block, and use scissors and tweezers to remove obvious adipose tissue, normal tissue, blood vessels and damaged tissue from the sample. (3) Place the tissue block on an ice box, cut it into small pieces of 1 mm in size, then use a Pasteur pipette to draw 3-5 mL of PBS buffer to mix the tissue pieces, and transfer them to a 15 mL centrifuge tube; (4) Centrifuge, discard the supernatant, and add 3-5 mL of organoid digestion solution I (components: DMEM F-12, 10 μMY27632, 1 mg / mL Collagenase type II, 10 ng / mL EGF). (5) Place it on a shaker in a 37 ℃ incubator and digest for 2 hours; (6) After digestion, blow the mixture 2-3 times with a Pasteur pipette, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant; (7) Add 2-3 mL of organoid digestion solution II (components: DMEM F-12, 1 U / mL Dispase, 100 μg / mL DNase enzyme I), mix well, and place on a shaker in a 37 ℃ incubator for 10 minutes to digest; (8) Add 4-6 mL of digestion termination solution, mix well to terminate digestion, centrifuge, and discard the supernatant; (9) Add 10 mL of PBS buffer to resuspend the cells, filter the cell suspension through a 100 μm filter, collect the filtrate, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant; (10) If red blood cell precipitation is obvious, add 2-3 mL of red blood cell lysis buffer to the cell precipitation according to the amount of red blood cells, mix well by pipetting, let stand at room temperature for 3-5 minutes, add 6-10 mL of PBS buffer to stop lysis, centrifuge, and discard the supernatant. (11) Resuspend the cells in an appropriate amount of breast cancer organoid culture medium. Mix the matrix gel and cell resuspension in a 3:2 ratio (the final matrix gel concentration is about 5.5 mg / mL). At this time, the cell density should be maintained at 1000~2000 cells / μL. Take care to avoid generating bubbles. (12) Take 20-30 μL of matrix gel cell suspension and seed it into a 12-well plate. Carefully invert the 12-well plate and place it in a 37 ℃ incubator. After the gel droplets have fully solidified, add 1.5 mL of breast cancer organoid culture medium. (13) Place the organoids in a 37 ℃ incubator for 5-7 days, and observe under a microscope that the primary single cells have grown into well-formed breast cancer organoids, which can then be passaged. (14) The passage steps for breast cancer organoids include: (14-1) Discard the old organoid culture medium in the 12-well plate, being careful not to remove the matrix gel droplets; (14-2) Add 1 mL of organoid passage digestion solution to each well, pipette a few times to break up the droplets, and place in a 37 ℃ incubator for 5-10 minutes to digest the organoids into single cells; (14-3) After digestion is complete, add 2 mL of organoid digestion termination solution to stop digestion, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant; (14-4) Take 20~40 μL of breast cancer organoid culture medium to resuspend organoid cells, add an appropriate amount of matrix gel and mix them thoroughly; (14-5) Take 20-30 μL of the matrix gel cell suspension and seed it into a 12-well plate. Carefully invert the 12-well plate and place it in a 37 ℃ incubator. After the gel droplets have fully solidified, add 1.5 mL of breast cancer organoid culture medium.

[0028] The digestive fluid used for organoid passage in this invention consists of: 50% TrypLE Express (containing phenol red) + 50% trypsin-EDTA (0.25%), containing phenol red + 10 μM Y27.

[0029] The specific implementation method for extracting S2 blood immune cells mainly includes the following steps: (1) Take about 5 mL of anticoagulated blood and use a dropper to add the same volume of PBS buffer to a centrifuge tube to dilute the blood; (2) Take a new 15 mL centrifuge tube and add 1 mL of lymphocyte separation solution Ficoll. You can slowly rinse the centrifuge tube wall with Ficoll. Then slowly add diluted whole blood. At this time, the blood is spread evenly on the top layer of Ficoll liquid (be careful not to mix or shake, the volume ratio of whole blood to separation solution is 1:1). (3) Centrifuge at 2000 rpm for 20 minutes using a horizontal centrifuge (note that the centrifuge speed should be set to 1 for increasing speed and 0 for decreasing speed). Carefully remove the test tube. At this time, the diluted blood flowing down the test tube wall will be superimposed on the separation liquid and form a clear interface with the separation liquid. (4) Carefully aspirate the white flocculent material in the middle layer with a flat-mouthed dropper, wash twice with PBS buffer, and centrifuge at 1500 rpm for 10 minutes each time; (5) Discard the supernatant, add red blood cell lysis buffer, lyse and remove red blood cells, add PBS buffer to wash cells, centrifuge at 1800 rpm for 10 minutes to collect lymphocytes at the bottom of the tube; (6) Add 2 mL of immune cell-specific culture medium, resuspend the centrifuged cell pellet, seed it into a 12-well plate, and observe and record the cell status under a microscope (the immune cell-specific culture medium consists of 10% FBS, 1% penicillin antibody and 100 IU / mL IL-2). (7) During cell culture, the culture status should be monitored in a timely manner. During the culture process, half or all of the medium should be replaced every 2 to 3 days depending on the cell status, and the sample status should be recorded by taking photos.

[0030] The specific implementation method for sorting and expanding T cells in S3 mainly includes the following steps: (1) After culturing lymphocytes for 2-3 days, the lymphocytes were washed and counted. T cells were sorted using Miltenyi's T cell sorting kit according to the instructions. (2) After the T cells are sorted, the collected T cells are seeded into 24-well plates and cultured in T cell culture medium. The T cell culture medium used at this time is Stem cell ImmunoCult-XF T Cell Exp Medium (containing 10% FBS, 1% penicillin antibody, 100 IU / mL IL-2 and 50 IU / mL IL-15).

[0031] Images of breast cancer organoids and unsorted peripheral blood mononuclear cells obtained on days 1 and 4 are recorded as follows: Figure 3 As shown in the figure, the culture method and culture medium of the present invention can be used to expand the culture of breast cancer organoids and T cells required for constructing a co-culture system.

[0032] The specific implementation method for 3D printing breast cancer organoid (nucleus)-T cell (shell) co-culture microspheres in S4 mainly includes the following steps: (1) T cell activation (1-1) Antibody coating for co-culture plates (24-well plates, 1 day before co-culture): Add 150 μL of PBS buffer (containing 50 μg / mL CD8 antibody) to each well of the 24-well plate and place the plate in a 4 ℃ refrigerator for overnight incubation. (1-2) After the T cells have been counted, take an appropriate amount of T cells and transfer them to a 24-well plate. Add T cell culture medium (containing 10% FBS, 1% penicillin antibody, 100 IU / mL IL-2, and 50 IU / mL IL-15) and incubate overnight at 37 ℃. (2) Enhancement of breast cancer organoid antigen (2-1) In a 12-well plate containing organoids, carefully aspirate the old culture medium without damaging the matrix gel droplets. Add 1 mL of Tryple Express to each well, break up and dissipate the matrix gel with a 1 mL pipette tip, and incubate at 37°C for 3-5 minutes to allow the organoids to digest into cell spheres containing 2-3 cells. Then transfer the organoids from the plate to a 15 mL centrifuge tube, add twice the volume of organoid digestion stop solution and mix well. At the same time, rinse the corresponding wells of the culture dish with 1 mL of DMEM F-12 and transfer it to the 15 mL centrifuge tube. (2-2) Centrifuge at 1200 rpm for 5 minutes and discard the supernatant; (2-3) Use 300 μL of organoid culture medium (add IFN-γ factor to a final concentration of 50 μg / mL to enhance antigen; considering the problem of organoid adhesion, 5% matrix gel can be added to the culture medium in this step), resuspend the organoid pellet, inoculate it into well plate, and incubate it in a 37 ℃ incubator for 24 hours; (3) Preparation of core-shell co-cultured microspheres (3-1) Take the antibody-coated plate out of the 4 ℃ refrigerator 1 hour in advance, discard the original liquid, add 100 μL of PBS buffer, and wash the wells twice. (3-2) The preparation of core-shell co-culture microspheres was carried out using a 3D organoid printer, wherein the concentration of the outer shell immune cells was 5000 cells / μL and the concentration of the core organoids was 40000 cells / μL; (3-3) After the preparation of breast cancer organoids and T cell co-culture microspheres, they were transferred to antibody-coated plates and 500 μL of special culture medium for breast cancer organoids and T cell co-culture microspheres were added.

[0033] The culture medium for breast cancer and T cell co-culture microspheres in this invention consists of: ImmunoCult-XF TCell Exp Medium, 1% penicillin antibody, 1% GlutaMAX, 1×B27, 1×N-2, 500 ng / mL R-spondin 1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 100 ng / mL A83-01, 50 ng / mL EGF, 100 nM β-estradiol, 5 nM Heregulinβ-1, 50 ng / mL IL-2, 25 ng / mL IL-15, and 20 μg / mL PD-L1.

[0034] To better observe the cell distribution within the core-shell microspheres, cell tracers were used to stain breast cancer organoids (green fluorescence) and T cells (red fluorescence) before preparing the co-cultured microspheres. The culture status of the prepared co-cultured microsphere model on day 1 (D1) is as follows. Figure 4 As shown, the culture status on day 5 (D5) is as follows. Figure 5 As shown, at D1, breast cancer organoid cells (green fluorescence) are concentrated in the core of the microspheres, while T cells (red fluorescence) are distributed in the outer shell, forming a microenvironment structure in which immune cells encapsulate the tumor organoids. At D5, it can be clearly observed that tumor cells self-assemble into clusters in the core, while immune cells are partially distributed in the outer shell and partially enter the core, forming a tendency to infiltrate the tumor organoids.

[0035] Example 3: Co-culture microsphere model of colorectal cancer organoids and T cells The preparation method was basically the same as in Example 2, except that colorectal cancer tissue was used instead of breast cancer tissue. The culture status of the co-cultured microsphere model obtained on the day of preparation (D0) and 9 days later (D9) is as follows: Figure 6 As shown, the co-cultured microsphere model is clear and complete, and the green fluorescent colorectal cancer organoid cells and red fluorescent T cells can be clearly observed using cell tracers.

[0036] Example 4: Co-culture microsphere model of mouse lung organoids and mouse lung microvascular endothelial cells The preparation method is basically the same as in Example 2, except that mouse lung organoids are mixed with matrix gel as the core material, and mouse lung microvascular endothelial cells (MPMVECs) are mixed with HAMA to prepare the outer shell material. The culture status of the co-cultured microsphere model on day 0 (D0) and day 8 (D8) is as follows: Figure 7As shown, mouse lung organoids rapidly proliferate and self-assemble, forming organoid clusters of 100–300 μm within the core, while MPMVECs grow normally within the outer shell. An AMPI (Amphipathic Fluorescence Injection) assay revealed extremely strong green fluorescence in this co-cultured microsphere model, indicating high cell viability.

[0037] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A co-culture microsphere model, characterized in that, The core-shell microsphere is composed of an inner core and an outer shell, the inner core is an organoid loaded by Matrigel, and the outer shell is formed by cross-linking a photo-cross-linking hydrogel material, and the photo-cross-linking hydrogel material is doped with co-cultured cells, and the outer shell wraps the inner core.

2. The co-culture microsphere model of claim 1, wherein, The organoid is cultured by normal internal organ cells or cancer cells.

3. The co-culture microsphere model of claim 1, wherein, The co-cultured cells are selected from immune cells, fibroblasts or vascular endothelial cells; preferably, the immune cells are selected from at least one of T cells, B cells, NK cells, TILs cells or CAR-T cells.

4. The co-culture microsphere model according to any one of claims 1 to 3, wherein, The photo-cross-linking hydrogel material is selected from at least one of methacrylated hyaluronic acid, methacrylated polylysine, carboxymethyl chitosan, methacrylated sodium alginate or methacrylated dextran.

5. A method for preparing the co-culture microsphere model according to any one of claims 1 to 4, characterized in that, The steps include: Preparation of inner core raw material: culture organoids, mix the organoids with Matrigel into inner core raw material; Preparation of outer shell raw material: obtain co-cultured cells, mix the co-cultured cells with the photo-cross-linking hydrogel material into outer shell raw material; 3D printing of co-cultured microsphere model: load the inner core raw material and the outer shell raw material into the corresponding hoppers of the droplet microfluidic printing device respectively, 3D print the core-shell microsphere, and cross-link the printing product by light to form a model, thereby obtaining the co-cultured microsphere model.

6. The preparation method according to claim 5, characterized in that, In the step of preparing the inner core raw material, the step of culturing the organoids includes: (1-1) washing the tissue sample, wetting the tissue sample with PBS buffer, physically removing excess tissue, and obtaining a tissue block; (1-2) placing the tissue block on an ice box, cutting it into small pieces with a side length of 1 mm, adding PBS buffer and mixing, and transferring it to a centrifuge tube; (1-3) centrifugation, discarding the supernatant, adding 3-5 mL of organoid digestion solution I, and shaking at 37°C for 1-2 minutes; (1-4) blow 2-3 times, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant; (1-5) add 2-3 mL of organoid digestion solution II and mix, shake at 37°C for 8-10 minutes; (1-6) add 4-6 mL of digestion termination solution, mix to terminate digestion, centrifuge, and discard the supernatant; (1-7) resuspend the cells with PBS buffer, filter the cell suspension with a filter screen with a pore size of 100 μm, collect the filtrate, centrifuge at 1500 rpm for 3 minutes, discard the supernatant, and add organoid culture medium to obtain a cell resuspension; (1-8) mix Matrigel with the cell resuspension to maintain a cell density of 1000-2000 cells / μL, and obtain a Matrigel cell suspension; (1-9) take 20-30 μL of the Matrigel cell suspension and inoculate it into a well plate, and incubate at 37°C until the gel drops are fully solidified; (1-10) take out the well plate, add 1-2 mL of organoid culture medium to the solidified gel drops, and incubate at 37°C for 5-7 days, thereby obtaining the organoids.

7. The preparation method according to claim 6, characterized in that, The organoid digestion solution I is prepared from DMEM F-12 medium containing 10 μM Y27632, 1 mg / mL Collagenase enzyme type II and 10 ng / mL EGF; preferably, the organoid digestion solution II is prepared from DMEM F-12 medium containing 1 U / mL Dispase and 100 μg / mL DNase enzyme I.

8. The method of manufacture according to any one of claims 5 to 7, wherein, In the preparation of the shell raw material step, the co-cultured cells are immune cells, and the step of obtaining the immune cells comprises: (2-1) Take the anticoagulated blood, add PBS buffer for dilution, (2-2) Use lymphocyte separation medium to rinse the wall of the centrifuge tube, then slowly add the diluted blood, use a horizontal centrifuge to centrifuge at 2000 rpm for 20 minutes, and carefully suck the middle layer white flocculent material with a flat dropper; (2-3) Wash the white flocculent material twice with PBS buffer, centrifuge at 1500 rpm for 10 minutes after each washing, and discard the supernatant; (2-4) Add red blood cell lysis solution, lyse and remove red blood cells, add PBS buffer for washing, centrifuge at 1800 rpm for 10 minutes, and collect the lymphocytes at the bottom of the tube; (2-5) Add immune cell special culture medium to resuspend the lymphocytes, inoculate into a well plate, and perform half-volume or full-volume medium replacement every 2-3 days.

9. The preparation method according to claim 8, characterized in that, The composition of the immune cell special culture medium is ImmunoCult-XF T Cell Exp Medium containing 10 % FBS, 1 % double antibody, 100 IU / mL IL-2 and 50 IU / mL IL-5.

10. The preparation method according to claim 8, characterized in that, The co-cultured microsphere model obtained after crosslinking is placed in a co-cultured microsphere special culture medium, and the co-cultured microsphere special culture medium is ImmunoCult-XF T Cell Exp Medium containing 1 % double antibody, 1 % GlutaMAX, 1 × B27, 1 × N-2, 500 ng / mL R-spondin 1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 100 ng / mL A83-01, 50 ng / mL EGF, 100 nM β-estradiol, 5 nM Heregulin β-1, 50 ng / mL IL-2, 25 ng / mL IL-15 and 20 μg / mL PD-L1.

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