Method for constructing myocarditis organ disease model

Abstract

The invention relates to the technical field of disease model construction, and discloses a method for constructing a myocarditis organ disease model, which comprises the following steps: carrying out first-stage culture on human induced pluripotent stem cells in a culture medium M1 containing a GSK-3beta inhibitor, and then carrying out second-stage culture by using a culture medium M2 containing VEGFA (Vascular Endothelial Growth Fatty Acid) to obtain a stably pulsatile cell aggregate; and performing third-stage culture on the cell aggregate in a lipopolysaccharide-containing culture medium M3 to obtain the myocarditis organ disease model. According to the method, three specific culture media are sequentially adopted, construction from hiPSC to the myocarditis organ disease model can be achieved, and the construction process is efficient, rapid, simple and convenient.

Description

Technical Field

[0001] The present invention relates to the technical field of disease model construction, and in particular to a method for constructing a myocarditis organoid disease model. Background Art

[0002] The triggering factors of myocarditis usually include infectious factors and non-infectious factors. Non-infectious factors include autoimmunity, hypersensitivity reactions, drugs, toxic substances, etc. Infectious factors include viruses, bacteria, fungi, etc. Among them, viral infection induces myocarditis through direct damage to myocardial cells and immune pathogenesis, and has been proven to be the most common cause of myocarditis. If the cause of myocarditis is not completely eliminated or the inflammatory response persists after elimination, it will eventually develop into inflammatory cardiomyopathy, and if it is not effectively treated, it will progress to heart failure.

[0003] In the past, in vitro models of myocarditis were often based on ordinary 2D culture of cardiomyocytes. Although it has shown certain reference value for the study of myocarditis mechanisms and drug screening, ordinary monolayer culture cannot well simulate the microenvironment of the heart under physiological and pathological conditions, such as the lack of interaction between cells and between cells and extracellular matrix. Organoid technology is used to establish an in vitro model with multiple cell types, which is close to the corresponding in vivo structure in function and structure. This technology provides an ideal platform for studying the physiological and pathological states of tissues and organs, as well as drug screening. However, due to the differences between organoids and 2D culture in vitro models, the methods for inducing myocarditis in the two models cannot be universal. When the method of inducing myocarditis in 2D culture is used in organoids, the heart organoids may disintegrate severely or a large number of cells may fall off, resulting in the failure of model construction, which brings great difficulties to the construction of organoid disease models of myocarditis.

[0004] Patent CN118834823A discloses a myocarditis organoid model and its construction method and application, which uses TeSR-E8 medium containing ROCK inhibitor, TeSR-E8 medium without ROCK inhibitor, STEMdiff cardiomyocyte differentiation kit, and cardiomyocyte maintenance medium for culture, and human induced pluripotent stem cells (hiPSC) are induced to differentiate into mature cardiomyocytes, and then co-cultured with activated CD4+ and / or CD8+T cells isolated from human peripheral blood. Although this method can realize the construction of myocarditis organoid model, the process of inducing hiPSC to differentiate into mature cardiomyocytes requires multiple steps, and activated CD4+ and / or CD8+T cells need to be isolated from human peripheral blood. The whole process is relatively complicated and difficult to operate. Summary of the invention

[0005] In order to solve the technical problems of the existing method for constructing myocarditis organoids, which has complex steps and high difficulty in operation, the present invention provides a method for constructing a myocarditis organoid disease model. The present invention sequentially uses three specific culture media to achieve the construction of a myocarditis organoid disease model from hiPSCs, and the construction process is efficient, fast and simple.

[0006] The specific technical scheme of the present invention is: A method for constructing a myocarditis organoid disease model, comprising the following steps: (1) After the human induced pluripotent stem cells are cultured in a medium M1 containing a GSK-3β inhibitor for the first stage, the medium M2 containing VEGFA (vascular endothelial growth factor A) is used for the second stage of culture to obtain stably beating cell aggregates; (2) The cell aggregates were cultured in the third stage in the culture medium M3 containing lipopolysaccharide to obtain a myocarditis organoid disease model.

[0007] The present invention found that by sequentially using a culture medium M1 containing a GSK-3β inhibitor, a culture medium M2 containing VEGFA, and a culture medium M3 containing lipopolysaccharide for culturing, a cardiac organoid can be formed from human induced pluripotent stem cells (hiPSCs), and symptoms of myocarditis can be caused. Compared with the 2D model, this myositis organoid disease model is closer to the corresponding in vivo structure in function and structure, can better simulate the in vivo 3D microenvironment, and has a good application prospect in the study of the physiological and pathological state of myocarditis and the evaluation of its therapeutic drugs.

[0008] Preferably, in step (2), the content of lipopolysaccharide in the culture medium M3 is 5-10 mg / mL; the temperature of the third stage culture is 35-37° C., and the time is 20-24 h.

[0009] The concentration of lipopolysaccharide will affect the construction of the myocarditis organoid disease model. When the lipopolysaccharide concentration is too high, it will cause the cell aggregates to disintegrate and a large number of cells to fall off, resulting in the failure of the construction of the myocarditis organoid disease model.

[0010] Preferably, in step (2), the culture medium M3 also contains CD lipid, NEAA (non-essential amino acids), L-AA2P (L-ascorbic acid-2-phosphate), α-MTG (1-thioglycerol), insulin and FGF2 (fibroblast growth factor 2).

[0011] Preferably, in step (2), the preparation process of the culture medium M3 comprises: mixing an additive solution containing CD lipids, NEAA, L-AA2P, α-MTG, insulin and FGF2 with RPMI 1640 culture medium, and adding lipopolysaccharide to obtain culture medium M3.

[0012] Furthermore, in the additive solution, the contents of CD lipid, NEAA, L-AA2P, α-MTG, insulin and FGF2 are 0.5×~2×, 0.5×~2×, 100~200 ng / mL, 350~450 μM, 4~6 μg / mL, and 150~250 ng / mL, respectively; and the volume ratio of the additive solution to the RPMI 1640 culture medium is 1:10~15.

[0013] Preferably, after step (2), immunofluorescence technology is used to detect the distribution of p38 MAPK and NF-κB p65 in the cell aggregates, thereby determining whether the myocarditis organoid disease model is successfully constructed.

[0014] The P38 MAPK signaling pathway plays an important role in cell malignancy, tumor invasion and metastasis through various pathways; NF-κB p65 is a subunit of NF-κB, which is of great significance to the transcriptional activation of proteins. It participates in the initiation regulation of gene transcription and is related to cell proliferation and apoptosis. By detecting the distribution of p38 MAPK and NF-κB p65, the proliferation and apoptosis of cells in cell aggregates can be better reflected. When the proliferating cells are significantly reduced and the apoptotic cells are significantly increased, it indicates that the myocarditis organoid disease model has been successfully constructed.

[0015] Furthermore, the steps of the immunofluorescence technique include: fixing, slicing and blocking the cell aggregates after completing the third stage of culture, dividing them into two groups, incubating them with p38 MAPK antibody and NF-κB p65 antibody respectively, incubating them with secondary antibodies with fluorescent groups after washing, washing, and detecting fluorescent signals.

[0016] Furthermore, the steps of fixation, slicing and blocking include: washing the cell aggregates after completing the third stage of culture, fixing with a paraformaldehyde solution, washing, and then dehydrating with a sucrose solution, removing the sucrose solution, soaking in a mixture of a sucrose solution and a tissue cryogen, and then with a tissue cryogen, freezing and slicing, and adding Triton-X and goat serum for blocking.

[0017] Preferably, in step (1), the formula of the culture medium M1 is as shown in Table 1 below.

[0018] Table 1 Formulation of medium M1 Preferably, in step (1), the formula of the culture medium M2 is as shown in Table 2 below.

[0019] Table 2 Formulation of medium M2 Element content DMEM / F12 medium As solvent CD lipids 0.5×～2× ITS-X 0.5×～2× L-AA2P 100–200 ng / mL α-MTG 350~450μM insulin 1–5 μg / mL FGF2 10–30 ng / mL VEGFA 100–200 ng / mL Preferably, in step (1), the temperature of the first stage culture is 35-37°C, and the time is 36-40 hours; the temperature of the second stage culture is 35-37°C, and the time is 240-280 hours, and the culture medium M2 is replaced every 45-50 hours.

[0020] By using the above-mentioned specific formulas of medium M1 and medium M2 for culture in sequence, and replacing medium M1 with medium M2 at a specific time point (the first stage of culture is 36 to 40 hours), hiPSCs can grow and differentiate according to ideal conditions to form stable beating cell aggregates (cardiac organoids). The cardiac organoids obtained by this method can cooperate well with the subsequent addition of lipopolysaccharide, and lipopolysaccharide is used to make the cardiac organoids form a myocarditis organoid disease model.

[0021] In addition, using the above method, only two culture media are needed, and cardiac organoids can be constructed from hiPSCs in a short culture time, which simplifies the construction steps of cardiac organoids and shortens the construction time. Moreover, under the above method, cardiac organoids can be constructed from discrete hiPSCs, and there is no need to pre-form hiPSCs into cell spheres and maintain them in the form of cell spheres before culturing using the culture medium M1. This can simplify the construction process of cardiac organoids to a greater extent and reduce the difficulty of operation.

[0022] Preferably, before step (1), human induced pluripotent stem cells are cultured until the cell density reaches 73-78%, and then digested, and then step (1) is performed.

[0023] Preferably, in step (1), the first stage culture and the second stage culture are carried out in an ultra-low adsorption cell culture plate with a U-shaped bottom hole.

[0024] Preferably, in step (1), before the first stage of culture, human induced pluripotent stem cells are suspended in medium M1 at a rate of 0.5×10 4 ~1.0×10 4 Pieces / cm 2 The cells were inoculated into the ultra-low attachment cell culture plate at a density of 100 cells / mL to start the first stage of culture.

[0025] Compared with the prior art, the present invention has the following advantages: (1) The present invention can construct a myocarditis organoid disease model from hiPSCs by sequentially using three culture media with specific formulas. The construction process is efficient, rapid, and simple. The obtained myocarditis organoid disease model has good application prospects in the study of the physiological and pathological state of myocarditis and the evaluation of its therapeutic drugs.

[0026] (2) The present invention adopts a specific method to prepare stably beating cell aggregates from hiPSCs, which can not only cooperate well with the lipopolysaccharide used subsequently, but also successfully induce cell aggregates to form a myocarditis organoid disease model using lipopolysaccharide. The process of preparing stably beating cell aggregates also has the advantages of simple preparation steps, short time required, and low operation difficulty. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : is the immunofluorescence staining image of the cardiac organoids in the experimental group and the control group in Example 1. Note: Blue fluorescence indicates the cell nucleus, red fluorescence in the "p38 MAPK" group indicates p38 MAPK, and red fluorescence in the "NF-κB p65" group indicates NF-κB p65; since it is impossible to distinguish red fluorescence from blue fluorescence in the non-colored image, Figure 1 Color illustrations are used.

[0028] Figure 2 This is the process of constructing the cardiac organoid in Example 1. Wherein: Figure 2 A is an image of iPS cells just seeded into a cell culture plate; Figure 2 B is a microscope image of cell aggregates at day 2 of culture in the first stage; Figure 2 C is a microscope image of cell aggregates at day 10 of the second stage of culture.

[0029] Figure 3 : is a diagram showing the identification results of the cardiac organoid constructed in Example 1. Figure 3 A is the HE staining identification result; Figure 3 B is the result of immunofluorescence staining. Note: Red fluorescence indicates endothelial cells, and blue fluorescence indicates cell nuclei; since it is impossible to distinguish red fluorescence from blue fluorescence in the non-colored image, Figure 3 Color illustrations are used.

[0030] Figure 4 This is a microscope image of cell aggregates at the 10th day of the second stage culture in Example 2.

[0031] Figure 5 : is a diagram of the identification results of the myocarditis organoid constructed in Example 2. Among them, Figure 5 A is a bright field image of myocarditis organoid; Figure 5 B is the immunofluorescence staining of myocarditis organoids. Figure 5 In B, blue fluorescence indicates the cell nucleus, red fluorescence in the “p38MAPK” group indicates p38 MAPK, and red fluorescence in the “NF-κB p65” group indicates NF-κB p65; since it is impossible to distinguish red fluorescence from blue fluorescence in the non-colored graph, Figure 5 B uses color images.

[0032] Figure 6 This is a microscope image of cell aggregates at day 10 of the second stage culture in Example 3.

[0033] Figure 7 : is a diagram of the identification results of the myocarditis organoid constructed in Example 3. Among them, Figure 7 A is a bright field image of myocarditis organoid; Figure 7 B is the immunofluorescence staining of myocarditis organoids. Figure 7 In B, blue fluorescence indicates the cell nucleus, red fluorescence in the “p38MAPK” group indicates p38 MAPK, and red fluorescence in the “NF-κB p65” group indicates NF-κB p65; since it is impossible to distinguish red fluorescence from blue fluorescence in the non-colored graph, Figure 7 B uses color images.

[0034] Figure 8 This is a microscope image of cell aggregates after culturing in medium M3 for 24 hours in Comparative Example 1.

[0035] Fig. 9 This is a microscope image of cell aggregates after culturing in culture medium M3 for 24 hours in Comparative Example 2.

[0036] Fig.10 This is a microscope image of cell aggregates in Comparative Example 3 at the 10th day of the second stage of culture.

[0037] Fig.11 This is a microscope image of cell aggregates in Comparative Example 4 at the 10th day of the second stage of culture.

[0038] Fig.12 This is a microscope image of cell aggregates in Comparative Example 5 at the 10th day of the second stage of culture. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be imagined by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0040] In the following examples and comparative examples, the sources of the materials used are shown in Table 3, but the protection scope of the present invention is not limited thereto.

[0041] Table 3 Sources of materials used in Examples and Comparative Examples Example 1 The following steps were performed to construct a myocarditis organoid disease model: Step 1: Construction of cardiac organoids (1.1) Inoculate human induced pluripotent stem cells into mTeSR Plus medium and place in an incubator with the temperature controlled at 37°C and CO 2 The concentration was 5%, and the culture medium was replaced every day. When the cell density of human induced pluripotent stem cells reached 73%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0042] (1.2) The iPS cells digested in step (1.1) were resuspended in medium M1 (formula see Table 4), and then cultured at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped bottom. Figure 2 A. Aggregates were observed on the second day of culture in this cell culture plate (see Figure 2 B), at 36h of culture, the first stage of culture is completed.

[0043] Table 4 Formula of medium M1 in Example 1 Element content DMEM / F12 medium As solvent CD lipid 2× ITS-X 2× L-AA2P 100ng / mL α-MTG 400μM insulin 5 μg / mL FGF2 30 ng / mL CHIR99021 10μM (1.3) After the first stage of culture, the medium M1 in the cell culture plate was removed and the medium M2 (formula see Table 5) was added and culture was continued. The medium M2 was replaced every 2 days. From the time when the medium M1 was replaced with the medium M2, on the 10th day of culture, a stable beating cell aggregate (i.e., cardiac organoid) was obtained, and its morphology was as follows: Figure 2 C, the results of pathological staining and immunofluorescence staining are shown in Figure 3 , indicating that the cardiac organoids obtained in this example conform to the physiological structure of normal cardiac organs.

[0044] Table 5 Formulation of medium M2 in Example 1 Element content DMEM / F12 medium As solvent CD lipid 0.5× ITS-X 0.5× L-AA2P 100ng / mL α-MTG 400μM insulin 5 μg / mL FGF2 30 ng / mL VEGFA 200 μg / mL Step 2: Constructing myocarditis organoids (2.1) Mix solution A (RPMI 1640 culture medium) and solution B (formula see Table 6) at a mass ratio of 12:1 to obtain culture medium M3'. Add lipopolysaccharide to culture medium M3' and mix well to obtain culture medium M3 with a lipopolysaccharide content of 5 mg / mL.

[0045] Table 6 Formula of Liquid B in Example 1 Element content Deionized water (sterile) As solvent CD lipid 1× NEAA 1× L-AA2P 150 ng / mL α-MTG 400μM insulin 5 μg / mL FGF2 200ng / mL (2.2) Take the cardiac organoids obtained in step (1.3), remove the culture medium M2 in the cell culture plate, add 200 μL of culture medium M3 to each well, place in an incubator, control the temperature in the incubator to 37°C, and culture for 24 hours as the experimental group. Take the cardiac organoids obtained in step (1.3), remove the culture medium M2 in the cell culture plate, add 200 μL of culture medium M3' to each well, place in an incubator, control the temperature in the incubator to 37°C, and culture for 24 hours as the control group.

[0046] Step 3. Test the myocarditis organoid disease model (3.1). Use a gun tip with the tip cut off to transfer the cardiac organoids after step (2.2) from the cell culture plate to a 1.5 mL centrifuge tube, aspirate the residual culture medium in the centrifuge tube, wash with PBS buffer, add 2 mL of 4% paraformaldehyde solution to each centrifuge tube, fix at room temperature for 2 h, wash with PBS buffer, add 1 mL of 30% sucrose solution to each centrifuge tube, and dehydrate at 4°C overnight.

[0047] (3.2) After completing step (3.1), transfer the cardiac organoids to a 15 mL centrifuge tube, remove the residual sucrose solution in the tube, add the same volume of 2% sucrose solution and tissue cryogen, place on a shaker and soak for 3 hours, and repeat this operation. Use a pipette to remove the solution in the centrifuge tube, add tissue cryogen, place on a shaker and soak for 3 hours, and repeat this operation. Then embed the cardiac organoids in tissue cryogen and store at -80°C.

[0048] (3.3) Use a freezing microtome to cut the cardiac organoids embedded in the tissue cryogen into slices with a thickness of 20 μm. Use a histochemical pen to draw circles around the cardiac organoid slices. After drying, wash them with PBS buffer, and then prepare PBS buffer containing 0.1% Triton-X and 10% goat serum, and block the slices for 1 hour.

[0049] (3.4) The cardiac organoid slices obtained after completing step (3.3) were washed with PBS buffer, and then PBS buffer containing primary antibody and 10% goat serum was prepared, and the slices were incubated (incubated overnight in a 4°C refrigerator). The primary antibodies used, their species, and the dilution ratio when mixed with PBS buffer are shown in Table 7 (the slices were divided into two groups, and each group was incubated with one primary antibody).

[0050] Table 7 Primary antibody species and their dilution ratios Antibody Name Species Dilution ratio p38 MAPK Antibody rabbit 1:200 NF-κB p65 Antibody rabbit 1:200 (3.5) The cardiac organoid slices after step (3.4) were washed with PBS buffer, and then a PBS buffer containing a secondary antibody and 3% goat serum was prepared, and the slices were incubated at room temperature for 2 hours. The secondary antibodies used, their species, and the dilution ratio when mixed with PBS buffer are shown in Table 8.

[0051] Table 8 Secondary antibody species and dilution ratio (3.6) After completing step (3.5), the cardiac organoid slices were washed with PBS buffer, sealed with sealing agent, and the fluorescence signal was detected. The immunofluorescence staining results obtained in the experimental group ("LPS") and the control group ("control") are shown in Figure 1 Compared with the control group, cavities appeared in the cardiac organoids of the experimental group, the number of proliferating cells was significantly reduced, and the number of apoptotic cells was significantly increased, indicating that the myocarditis organoid disease model of the experimental group was successfully constructed.

[0052] Example 2 The following steps were performed to construct a myocarditis organoid disease model: Step 1: Construction of cardiac organoids (1.1) Inoculate human induced pluripotent stem cells into mTeSR Plus medium and place in an incubator with the temperature controlled at 37°C and CO 2 The concentration is 5%, and the culture medium is replaced every day. When the cell density of human induced pluripotent stem cells reaches 75%, they are digested with stem cell digestion solution, and the digestion is terminated to obtain digested iPS cells.

[0053] (1.2) The iPS cells digested in step (1.1) were resuspended in medium M1 (formula see Table 9), and then cultured at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low adsorption cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of culture was completed on the 36th hour.

[0054] Table 9 Formulation of medium M1 in Example 2 Element content DMEM / F12 medium As solvent CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL CHIR99021 4μM (1.3) After the first stage of culture, the medium M1 in the cell culture plate was removed and the medium M2 (formula see Table 10) was added. The culture was continued and the medium M2 was replaced every 2 days. From the time when the medium M1 was replaced with the medium M2, on the 10th day of culture, a stable beating cell aggregate (i.e., cardiac organoid) was obtained, and its morphology was as follows: Figure 4 shown.

[0055] Table 10 Formulation of medium M2 in Example 2 Element content DMEM / F12 medium As solvent CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL VEGFA 100 μg / mL Step 2. Constructing myocarditis organoids (2.1) Mix solution A (RPMI 1640 culture medium) and solution B (formula see Table 11) at a mass ratio of 15:1, add lipopolysaccharide, mix well, and obtain culture medium M3 with a lipopolysaccharide content of 10 mg / mL.

[0056] Table 11 Formula of Liquid B in Example 2 Element content Deionized water (sterile) As solvent CD lipid 0.5× NEAA 2× L-AA2P 200ng / mL α-MTG 450μM insulin 4 μg / mL FGF2 150 ng / mL (2.2) Take the heart organoids obtained in step (1.3), remove the culture medium M2 in the cell culture plate, add 200 μL of culture medium M3 to each well, place in an incubator, control the temperature in the incubator to 37°C, and culture for 24 hours. The morphology of the heart organoids is as follows: Figure 5 As shown in A.

[0057] Step 3. Test the myocarditis organoid disease model (3.1). Use a gun tip with the tip cut off to transfer the cardiac organoids after step (2.2) from the cell culture plate to a 1.5 mL centrifuge tube, aspirate the residual culture medium in the centrifuge tube, wash with PBS buffer, add 2 mL of 4% paraformaldehyde solution to each centrifuge tube, fix at room temperature for 2 h, wash with PBS buffer, add 1 mL of 30% sucrose solution to each centrifuge tube, and dehydrate at 4°C overnight.

[0058] (3.2) After completing step (3.1), transfer the cardiac organoids to a 15 mL centrifuge tube, remove the residual sucrose solution in the tube, add the same volume of 2% sucrose solution and tissue cryogen, place on a shaker and soak for 3 hours, and repeat this operation. Use a pipette to remove the solution in the centrifuge tube, add tissue cryogen, place on a shaker and soak for 3 hours, and repeat this operation. Then embed the cardiac organoids in tissue cryogen and store at -80°C.

[0059] (3.3) Use a freezing microtome to cut the cardiac organoids embedded in the tissue cryogen into slices with a thickness of 20 μm. Use a histochemical pen to draw circles around the cardiac organoid slices. After drying, wash them with PBS buffer, and then prepare PBS buffer containing 0.1% Triton-X and 10% goat serum, and block the slices for 1 hour.

[0060] (3.4) The cardiac organoid slices obtained after completing step (3.3) were washed with PBS buffer, and then PBS buffer containing primary antibody and 10% goat serum was prepared, and the slices were incubated (incubated overnight in a 4°C refrigerator). The primary antibodies used, their species, and the dilution ratio when mixed with PBS buffer are shown in Table 7 above (the slices were divided into two groups, and each group was incubated with one primary antibody).

[0061] (3.5) The cardiac organoid slices after step (3.4) were washed with PBS buffer, and then a PBS buffer containing a secondary antibody and 3% goat serum was prepared, and the slices were incubated at room temperature for 2 hours. The secondary antibodies used, their species, and the dilution ratio when mixed with PBS buffer are shown in Table 8 above.

[0062] (3.6) After completing step (3.5), the heart organoid slices were washed with PBS buffer, sealed with sealing agent, and the fluorescence signal was detected. The obtained immunofluorescence staining results are shown in Figure 5 B. From Figure 5 A and Figure 5 B shows that after treatment with culture medium M3, the integrity of the cardiac organoids was damaged to a certain extent, with cavities, blurred edges, and shedding of apoptotic cells. In addition, the number of proliferating cells was significantly reduced, while the number of apoptotic cells was significantly increased, indicating that the myocarditis organoid disease model of the experimental group was successfully constructed.

[0063] Example 3 The following steps were performed to construct a myocarditis organoid disease model: Step 1: Construction of cardiac organoids (1.1) Inoculate human induced pluripotent stem cells into mTeSR Plus medium and place in an incubator with the temperature controlled at 37°C and CO 2 The concentration is 5%, and the culture medium is replaced every day. When the cell density of human induced pluripotent stem cells reaches 78%, they are digested with stem cell digestion solution, and the digestion is terminated to obtain digested iPS cells.

[0064] (1.2) The iPS cells digested in step (1.1) were resuspended in medium M1 (formula see Table 12), and then cultured at a cell density of 1.0×10 4 Pieces / cm 2The cells were inoculated into a 96-well ultra-low adsorption cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of culture was completed on the 40th hour.

[0065] Table 12 Formulation of medium M1 in Example 3 Element content DMEM / F12 medium As solvent CD lipid 0.5× ITS-X 0.5× L-AA2P 150ng / mL α-MTG 350μM insulin 3 μg / mL FGF2 10 ng / mL CHIR99021 1μM (1.3) After the first stage of culture, the medium M1 in the cell culture plate was removed and the medium M2 (formula see Table 13) was added. The culture was continued and the medium M2 was replaced every 2 days. From the time when the medium M1 was replaced with the medium M2, the culture was continued until the 10th day, and the stable beating cell aggregates (i.e., cardiac organoids) were obtained. Their morphology was as follows: Figure 6 shown.

[0066] Table 13 Formulation of medium M2 in Example 3 Element content DMEM / F12 medium As solvent CD lipid 2× ITS-X 2× L-AA2P 150ng / mL α-MTG 350μM insulin 3 μg / mL FGF2 10 ng / mL VEGFA 150 μg / mL Step 2. Construction of myocarditis organoids (2.1) Mix solution A (RPMI 1640 culture medium) and solution B (formula see Table 13) at a mass ratio of 10:1, add lipopolysaccharide, mix well, and obtain culture medium M3 with a lipopolysaccharide content of 5 mg / mL.

[0067] Table 13 Formula of Liquid B in Example 3 Element content Deionized water (sterile) As solvent CD lipid 2× NEAA 0.5× L-AA2P 100ng / mL α-MTG 350μM insulin 6 μg / mL FGF2 250ng / mL (2.2) Take the heart organoids obtained in step (1.3), remove the culture medium M2 in the cell culture plate, add 200 μL of culture medium M3 to each well, place in an incubator, control the temperature in the incubator to 37°C, and culture for 24 hours. The morphology of the heart organoids is as follows: Figure 7 As shown in A.

[0068] Step 3. Test the myocarditis organoid disease model (3.1). Use a gun tip with the tip cut off to transfer the cardiac organoids after step (2.2) from the cell culture plate to a 1.5 mL centrifuge tube, aspirate the residual culture medium in the centrifuge tube, wash with PBS buffer, add 2 mL of 4% paraformaldehyde solution to each centrifuge tube, fix at room temperature for 2 h, wash with PBS buffer, add 1 mL of 30% sucrose solution to each centrifuge tube, and dehydrate at 4°C overnight.

[0069] (3.2) After completing step (3.1), transfer the cardiac organoids to a 15 mL centrifuge tube, remove the residual sucrose solution in the tube, add the same volume of 2% sucrose solution and tissue cryogen, place on a shaker and soak for 3 hours, and repeat this operation. Use a pipette to remove the solution in the centrifuge tube, add tissue cryogen, place on a shaker and soak for 3 hours, and repeat this operation. Then embed the cardiac organoids in tissue cryogen and store at -80°C.

[0070] (3.3) Use a freezing microtome to cut the cardiac organoids embedded in the tissue cryogen into slices with a thickness of 20 μm. Use a histochemical pen to draw circles around the cardiac organoid slices. After drying, wash them with PBS buffer, and then prepare PBS buffer containing 0.1% Triton-X and 10% goat serum, and block the slices for 1 hour.

[0071] (3.4) The cardiac organoid slices obtained after completing step (3.3) were washed with PBS buffer, and then PBS buffer containing primary antibody and 10% goat serum was prepared, and the slices were incubated (incubated overnight in a 4°C refrigerator). The primary antibodies used, their species, and the dilution ratio when mixed with PBS buffer are shown in Table 7 above (the slices were divided into two groups, and each group was incubated with one primary antibody).

[0072] (3.5) The cardiac organoid slices after step (3.4) were washed with PBS buffer, and then a PBS buffer containing a secondary antibody and 3% goat serum was prepared, and the slices were incubated at room temperature for 2 hours. The secondary antibodies used, their species, and the dilution ratio when mixed with PBS buffer are shown in Table 8 above.

[0073] (3.6) After completing step (3.5), the heart organoid slices were washed with PBS buffer, sealed with sealing agent, and the fluorescence signal was detected. The obtained immunofluorescence staining results are shown in Figure 7 B. From Figure 7 A and Figure 7 B shows that after treatment with culture medium M3, the integrity of the cardiac organoids was damaged to a certain extent, with cavities, blurred edges, and shedding of apoptotic cells. In addition, the number of proliferating cells was significantly reduced, while the number of apoptotic cells was significantly increased, indicating that the myocarditis organoid disease model of the experimental group was successfully constructed.

[0074] Comparative Example 1 The difference between this comparative example and Example 1 is that the content of lipopolysaccharide in the culture medium M3 is increased in this comparative example. Specifically, the steps of constructing the myocarditis organoid disease model in this comparative example are as follows: Step 1: Constructing cardiac organoids The steps for constructing cardiac organoids are the same as those in Example 1.

[0075] Step 2. Constructing myocarditis organoids (2.1) Mix solution A (RPMI 1640 culture medium) and solution B (the formula is shown in Table 6 above) in a mass ratio of 12:1, add lipopolysaccharide, and mix well to obtain culture medium M3 with a lipopolysaccharide content of 50 mg / mL.

[0076] (2.2) Take the heart organoids obtained in step (1.3), remove the culture medium M2 in the cell culture plate, add 200 μL of culture medium M3 to each well, place in an incubator, control the temperature in the incubator to 37°C, and culture for 24 hours. The morphology of the heart organoids is as follows: Figure 8 As shown. Figure 8 It can be seen that the organoids were severely disintegrated and a large number of cells fell off, making it impossible to conduct subsequent experiments, resulting in the failure of constructing the myocarditis organoid disease model.

[0077] Comparative Example 2 The difference between this comparative example and Example 1 is that lipopolysaccharide is replaced with azithromycin in this comparative example. Specifically, the process of constructing the myocarditis organoid disease model in this comparative example is as follows: Step 1: Constructing cardiac organoids The steps for constructing cardiac organoids are the same as those in Example 1.

[0078] Step 2. Construction of myocarditis organoids (2.1) Mix solution A (RPMI 1640 culture medium) and solution B (formula see Table 6 above) in a mass ratio of 12:1, add azithromycin, mix well, and obtain culture medium M3 with an azithromycin content of 5 mg / mL.

[0079] (2.2) Take the heart organoids obtained in step (1.3), remove the culture medium M2 in the cell culture plate, add 200 μL of culture medium M3 to each well, place in an incubator, control the temperature in the incubator to 37°C, and culture for 24 hours. The morphology of the heart organoids is as follows: Fig. 9 As shown. Fig. 9 It can be seen that most of the cells began to fall off the aggregates and die, the size of the organoids became smaller, and subsequent experiments could not be carried out, resulting in the failure of constructing the myocarditis organoid disease model.

[0080] Comparative Example 3 The difference between this comparative example and Example 2 is that in the process of constructing cardiac organoids in step 1, this comparative example changes the timing of replacing the culture medium M1 with the culture medium M2. Specifically, the process of constructing cardiac organoids in this comparative example is as follows: (1.1) Inoculate human induced pluripotent stem cells into mTeSR Plus medium and place them in an incubator with the temperature controlled at 37°C and CO 2 The concentration is 5%, and the culture medium is replaced every day. When the cell density of human induced pluripotent stem cells reaches 75%, they are digested with stem cell digestion solution, and the digestion is terminated to obtain digested iPS cells.

[0081] (1.2) The iPS cells digested in step (1.1) were resuspended in medium M1 (formula see Table 9 above) and then plated at a cell density of 1.0×10 4 Pieces / cm2 The cells were inoculated into a 96-well ultra-low adsorption cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of culture was completed on the 96th hour.

[0082] (1.3) After the first stage of culture, remove the medium M1 from the cell culture plate and add medium M2 (see Table 10 above for the formula) and continue culturing. During this period, medium M2 is replaced every 2 days. From the time medium M1 is replaced with medium M2, on the 10th day of culture, the morphology of the cell aggregates is as follows: Fig.10 As shown. Fig.10 It can be seen that most of the cells detached from the aggregates and died, which resulted in the inability to undergo subsequent differentiation and the failure of cardiac organoid construction.

[0083] Comparative Example 4 The difference between this comparative example and Example 2 is that in the process of constructing cardiac organoids in step 1, ITS-X in culture medium M1 and culture medium M2 is replaced with ITS-G. Specifically, the process of constructing cardiac organoids in this comparative example is as follows: (1.1) Inoculate human induced pluripotent stem cells into mTeSR Plus culture medium and place them in an incubator with the temperature in the incubator controlled at 37°C and CO 2 The concentration is 5%, and the culture medium is replaced every day. When the cell density of human induced pluripotent stem cells reaches 75%, they are digested with stem cell digestion solution, and the digestion is terminated to obtain digested iPS cells.

[0084] (1.2) The iPS cells digested in step (1.1) were resuspended in medium M1 (formula see Table 16), and then cultured at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low adsorption cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of culture was completed on the 36th hour.

[0085] Table 16 Formula of medium M1 in comparative example 4 Element content DMEM / F12 medium As solvent CD lipid 1× ITS-G 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL CHIR99021 4μM (1.3) After the first stage of culture, remove the medium M1 from the cell culture plate and add the medium M2 (see Table 17 for the formula) and continue culturing. During this period, the medium M2 is replaced every 2 days. From the time when the medium M1 is replaced with the medium M2, the morphology of the cell aggregates is as follows on the 10th day of culture: Fig.11 As shown. Fig.11 It can be seen that black plaques appear in the aggregates and a large number of dead cells fall off, resulting in the failure of cardiac organoid construction.

[0086] Table 17 Formula of medium M2 in comparative example 4 Element content DMEM / F12 medium As solvent CD lipid 1× ITS-G 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL VEGFA 100 μg / mL Comparative Example 5 The difference between this comparative example and Example 2 is that in the process of constructing cardiac organoids in step 1, VEGFA in the culture medium M2 is replaced with IL-5 in this comparative example. Specifically, the process of constructing cardiac organoids in this comparative example is as follows: (1.1) Inoculate human induced pluripotent stem cells into mTeSR Plus medium and place them in an incubator with the temperature controlled at 37°C and CO 2 The concentration is 5%, and the culture medium is replaced every day. When the cell density of human induced pluripotent stem cells reaches 75%, they are digested with stem cell digestion solution, and the digestion is terminated to obtain digested iPS cells.

[0087] (1.2) The iPS cells digested in step (1.1) were resuspended in medium M1 (formula see Table 9 above) and then plated at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low adsorption cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of culture was completed on the 36th hour.

[0088] (1.3) After the first stage of culture, remove the medium M1 from the cell culture plate and add the medium M2 (see Table 18 for the formula) and continue culturing. During this period, the medium M2 is replaced every 2 days. From the time when the medium M1 is replaced with the medium M2, the morphology of the cell aggregates is as follows on the 10th day of culture: Fig.12 As shown. Fig.12 It can be seen that the aggregates are adhered and dead cells fall off, resulting in the failure of cardiac organoid construction.

[0089] Table 18 Formula of medium M2 in comparative example 5 Element concentration DMEM / F12 medium As solvent CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL IL-5 100 μg / mL Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0090] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing a myocarditis organoid disease model, characterized in that: The following steps are involved: (1) After the human induced pluripotent stem cells are cultured in a medium M1 containing a GSK-3β inhibitor for the first stage, the medium M2 containing VEGFA is used for the second stage to obtain stably beating cell aggregates; (2) The cell aggregates were cultured in the third stage in the culture medium M3 containing lipopolysaccharide to obtain a myocarditis organoid disease model.

2. The method according to claim 1, characterized in that In step (2), the content of lipopolysaccharide in the culture medium M3 is 5-10 mg / mL; the temperature of the third stage culture is 35-37° C., and the time is 20-24 h.

3. The method according to claim 1, characterized in that In step (2), the culture medium M3 also contains CD lipids, NEAA, L-AA2P, α-MTG, insulin and FGF2.

4. The method according to claim 3, characterized in that In step (2), the preparation process of the culture medium M3 includes: mixing the additive solution containing CD lipid, NEAA, L-AA2P, α-MTG, insulin and FGF2 with RPMI 1640 culture medium, adding lipopolysaccharide, and obtaining the culture medium M3.

5. The method according to claim 4, characterized in that In the additive solution, the contents of CD lipid, NEAA, L-AA2P, α-MTG, insulin and FGF2 are 0.5×~2×, 0.5×~2×, 100~200 ng / mL, 350~450 μM, 4~6 μg / mL and 150~250 ng / mL respectively; the volume ratio of the additive solution to the RPMI 1640 culture medium is 1:10~15.

6. The method according to claim 1, characterized in that After step (2), immunofluorescence technology is used to detect the distribution of p38 MAPK and NF-κB p65 in the cell aggregates to determine whether the myocarditis organoid disease model is successfully constructed.

7. The method according to claim 6, characterized in that The immunofluorescence technique comprises the following steps: fixing, slicing and blocking the cell aggregates after completing the third stage of culture, dividing them into two groups, incubating them with p38 MAPK antibody and NF-κBp65 antibody respectively, washing them, incubating them with secondary antibodies with fluorescent groups, washing them, and detecting fluorescent signals.

8. The method according to claim 1, characterized in that In step (1), the formula of the culture medium M1 is as follows: 。 9. The method according to claim 1, characterized in that: In step (1), the formula of the culture medium M2 is as follows: 。 10. The method according to claim 8 or 9, characterized in that: In step (1), the temperature of the first stage culture is 35-37°C, and the time is 36-40 hours; the temperature of the second stage culture is 35-37°C, and the time is 240-280 hours, and the culture medium M2 is replaced every 45-50 hours.

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