Construction method of heart-liver symbiotic organ and function maintenance culture medium thereof

Abstract

The invention relates to the technical field of organoid culture, in particular to a construction method of a heart-liver symbiotic organoid, which comprises the following steps: performing directional differentiation on human induced pluripotent stem cells by using Activin A, BMP4, a B27 culture additive (insulin removal type) and double antibodies to obtain endoderm / mesoderm; in the process of inducing endoderm / mesoderm to the progenitor cells, VEGF-A, bFGF, FGF4, BMP2 and double antibodies are added into a culture medium at the same time, and HUVECs are added when differentiation is carried out to the eighth day, so that a culture in which the hepatic progenitor cells and endothelial progenitor cells are symbiotic is obtained. The construction method provided by the invention not only can realize simultaneous differentiation of symbiotic myocardial organs and liver organs in the same differentiation system, but also comprises other non-parenchymal cell types. The invention further provides a supplement composition which can be used for maintaining the functions of the heart-liver symbiotic organs and prolonging the survival time of the heart-liver symbiotic organs. By adding the supplement composition, the contraction time and the liver functions of the heart-liver symbiotic organs can be remarkably increased.

Description

Technical Field

[0001] This application relates to the technical field of organoid culture, and particularly to a method for constructing a heart-liver symbiotic organoid and a functional maintenance culture medium therefor. Background Art

[0002] The heart and the liver are extremely important organs in the human body. The interaction between the two is a complex physiological process, involving functional cooperation in multiple aspects. The liver is an important metabolic center in the body, responsible for the synthesis and decomposition of glycogen and regulating blood sugar levels, which is crucial for the energy supply of the heart. When liver function is impaired, it may lead to blood sugar fluctuations and affect the energy supply of the heart. At the same time, heart diseases, such as heart failure, can also affect liver health. For example, heart failure may cause acute or chronic liver diseases. Conversely, patients with cirrhosis may also face a higher risk of heart diseases due to increased heart load. The liver is also the main site for the synthesis of coagulation factors, which are crucial for the blood coagulation process. Heart injury or diseases may lead to abnormalities in the coagulation system and affect the coagulation function of the liver.

[0003] Despite such a close interaction between the heart and the liver, there are still no reports on heart-liver symbiotic organoids at present. This severely restricts the research in the fields of the occurrence and development mechanisms of heart-liver multi-system diseases and drug screening.

[0004] Existing methods for constructing organoids mainly include: ① Traditional organoid induction: By combining stem cells (such as induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs) or adult stem cells (ASCs)) with Matrigel, factors required to maintain the organoid ecosystem, and factors required for differentiation, an organoid model with specific functions is constructed. ② Self-assembly: By utilizing the self-organizing characteristics of cells, cells are prompted to spontaneously form organoids in a suitable environment. This method is relatively simple to operate, but the generated organoids are usually small. ③ 3D printing: Using 3D printing technology, organoids with specific shapes and complex structures are printed layer by layer. This method allows for precise control of the morphology and cell distribution of organoids and is suitable for tissue models requiring complex geometries. ④ Microfluidic chips: Organoids constructed through microfluidic technology can be cultured under precisely controlled biological, physical, and chemical conditions.

[0005] As early as in 2013, the team of Takanori Takebe first reported in Nature the successful cultivation of vascularized liver organoids from hiPSCs, achieving a major breakthrough in the field of organoid research. In 2021, the team of Sasha Mendjan activated all six known signaling pathways involved in embryonic heart development in a specific order to induce stem cells to self-organize. After one week of development, the organoids were structurally equivalent to the heart of a 25-day embryo. The heart organoids beat at a regular rhythm, with a diameter of about 2 millimeters, and included the main cell types common to this stage of development: cardiomyocytes, epithelial cells, fibroblasts, and epicardium. They also had a distinct ventricle that beat 60 to 100 times per minute, which was the same rate as the embryonic heart of the same age.

[0006] 3D bioprinting mainly uses substances such as cells, biological hormones, growth factors, and extracellular matrix to print living tissues with biological functions. 3D bioprinting can obtain precise liver structures, including the specific spatial structure and vascular network of the liver. Some scholars have used bioprinting technology to create a human liver model containing primary hepatocytes, hepatic stellate cells, and endothelial cells to simulate the fibrosis caused by liver injury induced by methotrexate and thioacetamide. After exposure to these compounds, liver injury, including hepatocyte injury, as well as the deposition and accumulation of fibrous collagen, was detected in this model, indicating that the 3D bioprinted liver reproduced the compound-induced liver injury response.

[0007] As a solution, the organ-on-a-chip (OoC) reconstructs the 3D microenvironment and functions of organs and has become an alternative candidate for cell experiments and drug screening. The Liver Chip platform generalizes the physiological and pathological characteristics of the human liver by reproducing the liver structure, maintaining high cell viability and cell phenotype, and simulating natural liver functions. Therefore, it is usually more capable of predicting the results of applying candidate drugs to humans. Parker KK designed a specific microfluidic device: first, cover the edge of the solid carrier surface with a protective film to generate the expected contour substrate; then spin-coat with acrylamide; and peel off the protective film to obtain the coated independent body. And the gene expression profiles generated by cardiomyocyte alignment and contractile function were demonstrated. Domestic scholars used a microfluidic device to construct a cell culture chamber and seeded mouse cardiomyocytes into the chamber. By precisely adjusting the flow rate of the culture medium in the chamber through the lateral channel, the physiological state of cardiomyocytes could be highly simulated. It should be noted that due to the difficulty in obtaining cardiomyocytes and the difficulty in long-term culture, currently, embryonic stem cells or induced pluripotent stem cells are often differentiated into cardiomyocytes by chemical methods, so as to simulate the structure and function of cardiomyocytes on the microdevice.

[0008] In summary, although the prior art has obtained liver and heart organoids with structures and functions gradually approaching the in vivo real microenvironment through traditional culture methods, 3D printing, and organ-on-a-chip technology, and has made remarkable progress, and combined organoid technology with 3D printing technology and microfluidic chip technology to construct organoids that are close to the real microenvironment in the human body in terms of cell composition and structure, however, this traditional organoid culture method usually cannot fully replicate the complex microenvironment of in vivo organs, including the extracellular matrix, vascular network, and immune cells, etc. This results in differences between the generated organoids and real organs in terms of structure and function. For example, currently, 3D-printed organoids are usually very unstable in structure and easily become too fragile, which limits their application in subsequent transplantation surgeries.

[0009] In addition, even though liver organoids and cardiac organoids derived from hiPSCs have been reported successively, independent organoids are not sufficient to simulate the real in vivo microenvironment and the complex interaction relationships between different organs. To address this defect, scientists have made some attempts. The only reported case of a heart-liver symbiotic organoid is the co-culture of mature cardiac organoids and liver organoids derived from induced pluripotent stem cells through a microfluidic chip with an artificial upper and lower chamber. This method is a multi-organ chip system derived from hiPSCs, which can be used to evaluate the cardiac safety of antidepressant drugs and perform liver metabolism. This liver-heart organoid chip device includes chambers separated by a porous membrane, and 3D liver organoids can be co-cultured in the upper porous chamber, and cardiac organoids can be co-cultured in the bottom microcolumn array at the same time. The co-cultured liver and heart organoids on the chip respectively maintain good viability and human organ-specific functions, including the synthesis of albumin and urea in liver organoids, and the beating function of cardiac organoids. However, this culture method combines the cardiac organoids and liver organoids after they are induced to mature separately, so it cannot be used to explore the embryonic development mechanism of mesendoderm symbiotic organoids. Therefore, a method for inducing symbiotic liver organoids and cardiac organoids in the same differentiation system is still urgently needed to be studied. Summary of the Invention

[0010] The present invention provides a method for constructing a heart-liver symbiotic organoid, and the heart-liver symbiotic organoid includes hepatocytes, non-parenchymal cells, and cardiomyocytes.

[0011] To achieve the above object, the technical solution of the present invention is realized as follows:

[0012] A method for constructing a heart-liver symbiotic organoid includes:

[0013] S1: Differentiate human induced pluripotent stem cells in a medium containing Activin A, BMP4, B27 culture additive (insulin-free type), and penicillin-streptomycin to obtain an endoderm / mesoderm co-differentiated endoderm / mesoderm culture;

[0014] S2: Induce the endoderm / mesoderm to differentiate into progenitor cells: Add VEGF-A and bFGF to the medium simultaneously to obtain a culture in which hepatic progenitor cells and endothelial progenitor cells coexist;

[0015] S3: Differentiate the progenitor cells into cardio-hepatoblasts, and then transform the culture into mature cardio-hepatic co-existing organoids, where step S3 includes:

[0016] S31: Use a cell culture medium supplemented with HGF, KGF, and B27 culture additive to induce the progenitor cells to continue differentiating to produce cardio-hepatoblasts;

[0017] S32: Use a medium containing OSM and DEX to induce the transformation of cardio-hepatoblasts into mature cells, and finally construct a cardio-hepatic organoid in which cardiomyocytes, hepatocytes, and non-parenchymal cells coexist.

[0018] Furthermore, the endoderm / mesoderm obtained in step S1 contains endoderm cells and mesoderm cells.

[0019] Furthermore, in the medium of step S1, the concentration of Activin A is 50 - 200 ng / ml, the concentration of BMP4 is 10 - 30 ng / ml, the volume concentration of the B27 culture additive (insulin-free type) is 1% - 3%, and the volume concentration of penicillin-streptomycin is 0.5% - 2%.

[0020] Furthermore, the step of adding VEGF-A and bFGF to the medium simultaneously is carried out on the 5th day of the initial culture of human pluripotent stem cells.

[0021] Furthermore, the medium used in step S2 also includes BMP2, FGF4, B27 culture additive, and penicillin-streptomycin.

[0022] Furthermore, in step S2, the concentration of VEGF-A is 5 - 20 ng / ml, the concentration of bFGF is 5 - 20 ng / ml, the concentration of BMP2 is 5 - 20 ng / ml, the concentration of FGF4 is 10 - 40 ng / ml, the volume concentration of the B27 culture additive is 1% - 3%, and the volume concentration of penicillin-streptomycin is 0.5% - 2%.

[0023] Furthermore, step S2 also includes adding HUVECs to the medium on the 8th day of differentiation, and the addition amount of human umbilical vein endothelial cells is 1 / 2 - 1 / 5 of the cell amount at this time.

[0024] Further, the step S32 includes or does not include the MIX composition.

[0025] On the other hand, the embodiment of the present invention also provides a heart-liver symbiotic organoid prepared by using the above construction method, and the heart-liver symbiotic organoid includes hepatocytes, cardiomyocytes and non-parenchymal cells.

[0026] In the third aspect, the embodiment of the present invention also provides a supplement composition for maintaining heart-liver function, and the supplement composition includes Non-Essential Amino Acids Solution (NEAA), GlutaMAX Supplement, insulin-like growth factor-1 receptor (IGF-1R) and triiodothyronine (T3).

[0027] Further, the volume concentration of the NEAA is 25-75%, the volume concentration of the GlutaMAX Supplement is 25-75%, the concentration of the IGF-1R is 0.5-1 μg / ml, and the concentration of the T3 is 0.1-0.2 μm / ml.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The construction method of the heart-liver symbiotic organoid provided by the present invention can realize the symbiotic differentiation of cardiomyocytes and liver organoids. This differentiation scheme can not only realize the simultaneous differentiation of symbiotic heart-liver symbiotic organoids in the same differentiation system, but also include other non-parenchymal cell types. The heart-liver symbiotic organoid cultured and differentiated by this differentiation scheme not only provides a good research model for drug screening and drug metabolism of multi-system diseases of the heart and liver, but also provides a physiological model for exploring the embryonic development of the mesoderm-derived heart and the endoderm-derived liver of non-parenchymal cells.

[0030] The supplement provided by the present invention can be used to maintain the function of the heart-liver symbiotic organoid and extend its survival time. The addition of this supplement can significantly increase the contraction time of the cardiomyoid and liver function. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. The drawings in the following description are only used to explain some embodiments of the present invention.

[0032] Figure 1It is a flowchart of the method for constructing heart-liver symbiotic organoids provided in Example 1 of the present invention.

[0033] Figure 2 It is a cell morphology diagram of the heart-liver symbiotic organoids induced to the 30th day in Example 1 of the present invention, taken by an optical microscope.

[0034] Figure 3 It is an overlay immunofluorescence staining diagram (cardiomyocytes positive for cTnT, hepatocytes positive for CYP3A4, and nuclei positive for DAPI) of cardiomyocytes and hepatocytes in the heart-liver symbiotic organoids differentiated to the 30th day in Example 1 of the present invention.

[0035] Figure 4 It is an immunofluorescence staining diagram of cardiomyocytes (cTnT-positive cells) differentiated to the 30th day in Example 1 of the present invention.

[0036] Figure 5 It is an immunofluorescence staining diagram of hepatocytes (CYP3A4-positive cells) differentiated to the 30th day in Example 1 of the present invention.

[0037] Figure 6 It is an immunofluorescence staining diagram of nuclei (DAPI-positive cells) differentiated to the 30th day in Example 1 of the present invention.

[0038] Figure 7 Overlay immunofluorescence staining diagram (cardiomyocytes positive for MYH1, non-parenchymal cells positive for αSMA, and nuclei positive for DAPI) of cardiomyocytes and non-parenchymal cells in the heart-liver symbiotic organoids differentiated to the 30th day in Example 1 of the present invention.

[0039] Figure 8 It is an immunofluorescence staining diagram of non-parenchymal cells (αSMA-positive cells) differentiated to the 30th day in Example 1 of the present invention.

[0040] Figure 9 It is an immunofluorescence staining diagram of cardiomyocytes (MYH1-positive cells) differentiated to the 30th day in Example 1 of the present invention.

[0041] Figure 10 It is another field immunofluorescence staining diagram of nuclei (DAPI-positive cells) differentiated to the 30th day in Example 1 of the present invention.

[0042] Figure 11 It is the real-time fluorescence quantitative PCR detection result of the cardiomyocyte marker gene (MYH6) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 1 of the present invention.

[0043] Figure 12It is the result of real-time fluorescence quantitative PCR detection of the cardiomyocyte marker gene (PLB) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 1 of the present invention.

[0044] Figure 13 It is the result of real-time fluorescence quantitative PCR detection of the hepatocyte marker gene (ALB) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 1 of the present invention.

[0045] Figure 14 It is the result of real-time fluorescence quantitative PCR detection of the hepatocyte marker gene (CYP3A4) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 1 of the present invention.

[0046] Figure 15 It is the result of real-time fluorescence quantitative PCR detection of the non-parenchymal cell marker gene (HGF) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 1 of the present invention.

[0047] Figure 16 It is the result of real-time fluorescence quantitative PCR detection of the non-parenchymal cell marker gene (Desmin) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 1 of the present invention.

[0048] Figure 17 It is the flow chart of the method for constructing heart-liver symbiotic organoids provided in Example 2 of the present invention;

[0049] Figure 18 It is the cell morphology diagram of the heart-liver symbiotic organoids induced to the 30th day in Example 2 of the present invention taken by an optical microscope. Bright-field picture of heart-liver symbiotic organoids;

[0050] Figure 19 It is the superimposed immunofluorescence staining map of cardiomyocytes and hepatocytes in the heart-liver symbiotic organoids differentiated to the 30th day in Example 2 of the present invention. (Cardiomyocytes are positive for cTnT, hepatocytes are positive for CYP3A4, and cell nuclei are positive for DAPI);

[0051] Figure 20 It is the immunofluorescence staining map of cell nuclei (DAPI-positive cells) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 2 of the present invention;

[0052] Figure 21 It is the immunofluorescence staining map of hepatocytes (CYP3A4-positive cells) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 2 of the present invention;

[0053] Figure 22 It is the immunofluorescence staining map of cardiomyocytes (cTnT-positive cells) in the heart-liver symbiotic organoids differentiated to the 30th day in Example 2 of the present invention;

[0054] Figure 23 It is a comparison chart of the number of days of contraction of myocardial organoids in the supplement-added group or the non-supplement-added group in Example 1 of the present invention. Detailed implementation manners

[0055] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0056] The embodiment of the present invention provides a method for constructing a heart-liver symbiotic organoid, and the heart-liver symbiotic organoid includes hepatocytes, non-parenchymal cells and cardiomyocytes. This method effectively regulates the signaling pathway during the differentiation of human induced pluripotent stem cells into definitive endoderm, enabling the human induced pluripotent stem cells to achieve co-differentiation of the endoderm and mesoderm, so that some mesoderm cells can be retained during the definitive endoderm stage. According to the construction method of the present invention, as the hepatocytes derived from the endoderm differentiate, the non-parenchymal cells and cardiomyocytes derived from the mesoderm also differentiate accordingly, and finally break through the barrier to achieve the symbiosis of hepatocytes, cardiomyocytes and non-parenchymal cells. This heart-liver symbiotic organoid differentiation protocol realizes the simultaneous differentiation of symbiotic myocardial organoids and liver organoids in the same differentiation system for the first time, which not only provides a good research model for drug screening and drug metabolism of multi-system diseases of the heart and liver, but also provides a physiological model for exploring the embryonic development of the mesoderm-derived heart and the endoderm-derived non-parenchymal cell liver.

[0057] The embodiment of the present invention provides a method for constructing a heart-liver symbiotic organoid, and this method includes: in the initial stage of culture, adding Activin A, BMP4, B27 culture additive (insulin-free type) and double antibody to the culture medium to direct the differentiation of human pluripotent stem cells to form an endoderm / mesoderm with co-differentiation of the endoderm and mesoderm.

[0058] Specifically, the culture of the endoderm / mesoderm lasts until the 4th day; wherein the concentration of Activin A is 50-200 ng / ml, the concentration of BMP4 is 10-30 ng / ml, the volume concentration of the B27 culture additive (insulin-free type) is 1%-3%, and the culture medium also contains double antibody with a volume concentration of 0.5%-2%.

[0059] The differentiation of heart-liver symbiotic organoids goes through multiple differentiation stages, including the process of "human induced pluripotent stem cells → endoderm / mesoderm → progenitor cell stage → heart-liver blast cell stage → mature heart-liver organoids". Hepatocytes are derived from the endoderm, while cardiomyocytes and non-parenchymal liver cells are derived from the mesoderm. To achieve the symbiosis of hepatocytes, cardiomyocytes and non-parenchymal cells, it is necessary to ensure that during the formation of the definitive endoderm, some mesodermal cells are retained. In subsequent differentiation stages, ensure that the remaining mesodermal cells can differentiate into cardiomyocytes and non-parenchymal cells along with hepatocytes.

[0060] In addition, the differentiation of cardiomyocytes requires the participation of endothelial cells. To fully ensure that myocardial progenitor cells can complete differentiation relying on endothelial cells, on the 8th day of differentiation, HUVECs and progenitor cells are mixed at a ratio of 1:2 - 1:5.

[0061] The construction method of the present invention can be divided into the following stages:

[0062] (1) Establish a differentiation protocol for the stage of "human induced pluripotent stem cells → endoderm / mesoderm" of co-differentiation of mesoderm and endoderm.

[0063] To enable human induced pluripotent stem cells to achieve co-differentiation of endoderm / mesoderm, the present application uses Activin A, BMP4, B27 culture additive (insulin-free type) and double antibody in the culture system of this stage to direct the differentiation of human pluripotent stem cells, so that some mesodermal cells can be retained during the differentiation of human induced pluripotent stem cells into the definitive endoderm stage.

[0064] (2) Establish a differentiation protocol for the stage of "endoderm / mesoderm → progenitor cells" of symbiosis of hepatic progenitor cells and endothelial progenitor cells

[0065] To retain more vascular endothelial progenitor cells during the hepatic progenitor cell stage, the present application adds the necessary VEGF-A and bFGF for vascular endothelial cells on the basis of the culture in the stage of "endoderm / mesoderm → progenitor cells", in order to obtain more endothelial progenitor cells and non-parenchymal cells derived from the mesoderm. VEGF-A is a protein family required for angiogenesis, and it plays an important role in the formation and growth of blood vessels. bFGF is a key cell growth factor belonging to the fibroblast growth factor family. The addition of the two can effectively promote the differentiation of some mesodermal cells retained during the definitive endoderm stage into non-parenchymal cells and cardiomyocytes. Specifically, the addition amount of VEGF-A is 5 - 20 ng / ml, and the addition amount of bFGF is 5 - 20 ng / ml. In this stage, HUVECs also need to be added to the culture medium on the 8th day of differentiation, and the addition amount of the HUVECs is 1 / 2 - 1 / 5 of the cell amount at this time.

[0066] The process of the construction method of the heart-liver symbiotic organoids provided by the present invention is as Figure 1As shown. The construction method specifically includes three stages: The first stage (Stage I): Use RPMI-1640 cell culture medium supplemented with Activin A, BMP4, B27 culture additive (insulin-free type), and double antibody to induce human induced pluripotent stem cells to differentiate into endoderm / mesoderm (Day1-4); S2 (Stage II): On the basis of the endoderm / mesoderm cells generated in S1, use RPMI-1640 cell culture supplemented with VEGF-A, BMP2, FGF4, bFGF, B27 culture additive, and double antibody to induce the differentiation of endoderm / mesoderm into progenitor cells (Day5-9); S3: First, use RPMI-1640 cell culture supplemented with HGF, KGF, B27 culture additive, and double antibody to induce the progenitor cells to continue to differentiate to produce cardio-hepatoblasts (Day10-15); Then use the medium of HCM with OSM, DEX, MIX composition, and double antibody to induce the transformation of cardio-hepatoblasts into mature cells, and finally construct a cardio-hepatic organoid in which cardiomyocytes, hepatocytes, and non-parenchymal cells coexist (Day16-30). The MIX is a solution obtained by dissolving 9.81 mg of DEX in 1 ml of DMSO and 4 ml of HCM medium without added components.

[0067] The following specifically describes the three stages experienced by this method in combination with specific examples.

[0068] Example 1 Construction of cardio-hepatic symbiotic organoid

[0069] Prepare the culture media corresponding to different stages according to the content shown in Table 1.

[0070] Table 1: Details of culture media at different stages

[0071]

[0072] The specific operation process of the construction method in this example is as follows:

[0073] S1: Use RPMI 1640 medium containing Activin A, BMP4, B27 culture additive, and double antibody to induce human pluripotent stem cells to differentiate into endoderm / mesoderm ( Figure 1 Day0-4 in

[0074] Among them: Culture human pluripotent stem cells in RPMI 1640 medium supplemented with 100 ng / ml Activin A, 10 ng / ml BMP4, 2% B27 culture additive (insulin-free type), and 1% double antibody, and culture for 4 days to obtain endoderm / mesoderm.

[0075] S2: Induce the differentiation of endoderm / mesoderm into progenitor cells using RPMI 1640 medium containing VEGF-A, bFGF, BMP2, FGF4, B27 culture additive and double antibody,

[0076] wherein: on the 5th day, transfer the cells successfully differentiated in S1 into RPMI 1640 medium supplemented with 10 ng / ml vascular endothelial growth factor, 10 ng / ml bFGF, 10 ng / ml BMP2, 30 ng / ml FGF4, 2% B27 culture additive and 1% double antibody, and continue the culture. On the 8th day of differentiation, add HUVECs to the medium and mix them with the progenitor cells at a ratio of 1:4, and continue the culture until the 9th day.

[0077] S3: First, use RPMI 1640 medium containing HGF, KGF, B27 culture additive and 1% double antibody to induce the differentiation of progenitor cells to generate cardio-hepatoblasts; then use HCM medium containing OSM, DEX, MIX composition and double antibody to induce the transformation of hepatoblasts into mature hepatocytes and cardiomyocytes, and construct cardio-hepatic symbiotic organoids.

[0078] Specifically, step S3 further includes 2 sub-steps:

[0079] S31: On the 10th day: Transfer the cells successfully differentiated in S2 above into RPMI 1640 medium supplemented with 20 ng / ml HGF, 20 ng / ml KGF, 2% B27 culture additive and 1% double antibody, and continue the culture until the 15th day;

[0080] S32: On the 16th day: The cells successfully differentiated in S31 continue to be induced to differentiate into cardio-hepatic symbiotic organoids coexisting with hepatocytes, non-parenchymal cells and cardiomyocytes. Specifically, transfer the cells successfully differentiated in S31 above into HCM medium supplemented with 20 ng / ml OSM, 0.5 μM DEX, 0.5 μM MIX and 1% double antibody, and continue the culture until the 30th day.

[0081] The suppliers and catalog numbers of the related reagents used in this example are shown in Table 2.

[0082] Table 2: Reagent Information List

[0083]

[0084]

[0085] The inventors also tested the cardio-hepatic symbiotic organoids obtained in the above steps, and the test content and process are as follows:

[0086] (1) Bright - field observation: The heart - liver symbiotic organoids prepared in this example were continuously cultured. After differentiation and maturation, their bright - field morphology was observed under an ordinary optical microscope. The experimental results are shown in Figure 2 . It can be observed that the hepatocytes in the heart - liver symbiotic organoids obtained in this example are polygonal and have a binuclear or multinuclear structure, while the cardiomyocytes aggregate into round cell clusters.

[0087] (2) Immunofluorescence detection: Immunofluorescence labeling was performed on hepatocytes (CYP3A4+), cardiomyocytes (MYH1+, cTnT+), and non - parenchymal cells (αSMA+) at the mature stage, and the cells of different cell types were observed under a fluorescence microscope. The detection results are shown in Figures 3 - 10 , where Figure 3 is an overlay image of cardiomyocytes and hepatocytes in the heart - liver symbiotic organoids differentiated to day 30; Figure 4 is the morphology image of cardiomyocytes (cTnT - positive cells) differentiated to day 30 in the example of the present invention; Figure 5 is the morphology image of hepatocytes (CYP3A4 - positive cells) differentiated to day 30 in the example of the present invention; Figure 6 is the staining image of cell nuclei (DAPI - positive cells) differentiated to day 30 in the example of the present invention; Figure 7 An overlay image of cardiomyocytes and non - cells in the heart - liver symbiotic organoids differentiated to day 30 in the example of the present invention; Figure 8 is the morphology image of non - parenchymal cells (αSMA - positive cells) differentiated to day 30 in the example of the present invention; Figure 9 is the morphology image of cardiomyocytes (MYH1 - positive cells) differentiated to day 30 in the example of the present invention; Figure 10 is another field staining image of cell nuclei (DAPI - positive cells) differentiated to day 30 in the example of the present invention. It can be seen from Figures 3 - 10 that the heart - liver symbiotic organoids cultured by the culture method of the present invention simultaneously have hepatic parenchymal cells, non - parenchymal cells, and cardiomyocytes.

[0088] (3) Fluorescent quantitative PCR: In this example, by detecting the expression of marker genes related to cardiomyocytes, hepatic parenchymal cells, and non - parenchymal cells, the above immunofluorescence results were verified. The detection results are shown in Figures 11 - 16 . Among them, Figure 11 and Figure 12 are respectively the expression detection results of cardiomyocyte marker genes (MYH6 and PLB) during the induction process of the heart - liver symbiotic organoids prepared in this example; Figure 13 and Figure 14 are respectively the expression detection results of hepatocyte marker genes (ALB and CYP3A4) during the induction process of the heart - liver symbiotic organoids prepared in this example; Figure 15 and Figure 16They are respectively the detection results of the expression of non-parenchymal cell marker genes (HGF and Desmin) during the induction process of the heart-liver symbiotic organoids prepared in this example. From Figures 11 - 16 It can be seen that on the 30th day of the heart-liver symbiotic organoids constructed by the present invention, the expression of related genes of cardiomyocytes, hepatocytes and non-parenchymal cells is stronger than that of human induced pluripotent stem cells (undifferentiated period), indicating that cardiomyocytes, hepatocytes and non-parenchymal cells coexist in the heart-liver symbiotic organoids, further verifying the results of immunofluorescence detection.

[0089] Example 2 Construction of heart-liver symbiotic organoids

[0090] The flow chart of constructing the heart-liver symbiotic organoids in this example is as Figure 17 shown, and its difference from Example 1 is only that the MIX composition is not added on the 16th - 30th days.

[0091] Test the obtained heart-liver symbiotic organoids according to the method of Example 1, and the test results are as Figures 18 - 22 shown. It can be seen from the above results that heart-liver symbiotic organoids containing cardiomyocytes, hepatocytes and non-parenchymal cells can also be cultured without adding the MIX composition.

[0092] Example 3 Preparation of supplement composition for maintaining heart-liver symbiotic organoids

[0093] Configure the supplement composition according to the content shown in Table 3.

[0094] Table 3 Raw materials for supplement preparation (1 ml)

[0095]

[0096] In line with the fact that the addition of this supplement composition can effectively promote the function and lifespan of heart / liver organoids, while not restricting the function and lifespan of the other type of organoids, non-essential amino acids (NEAA), GlutaMAX Supplement, IGF-IR and T3 are selected as the main components of the supplement. Among them, insulin-like growth factor-1 receptor (IGF-1R) is an important factor regulating cardiomyocyte proliferation, development and promoting heart function maturation. Triiodothyronine (T3) is a hormone secreted by thyroid follicles and is indispensable for the normal development of the heart.

[0097] To verify the effect of the supplement composition prepared above on the survival time of the heart-liver symbiotic organoids, the inventors conducted a comparative experiment. On the 30th day of culturing the heart-liver symbiotic organoids, 1 ml of the supplement composition was added to the culture medium of the experimental group, and the control group was not treated with anything. The culture was continued, and the contraction of the organoids in the culture medium was observed. The experimental results are as follows Figure 23 shown. The supplement composition provided by the present invention can significantly increase the survival time and contraction duration of the myocardial organoids.

[0098] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a heart-liver symbiotic organoid, comprising: S1: Differentiating human pluripotent stem cells in a culture medium containing Activin A, BMP4, and B27 culture additive to obtain co-differentiated endoderm / mesoderm; S2: Directing the induction of endoderm / mesoderm into progenitor cells: Adding vascular endothelial growth factor and bFGF to the culture medium simultaneously to obtain a culture of symbiotic hepatic progenitor cells and endothelial progenitor cells; S3: Differentiating progenitor cells into heart-liver blast cells, and then transforming the culture into a mature heart-liver symbiotic organoid, wherein step S3 includes: S31: Inducing the continuous differentiation of progenitor cells using a cell culture medium supplemented with HGF, KGF, and B27 culture additive to generate heart-liver blast cells; S32: Inducing the transformation of heart-liver blast cells into mature cells using a culture medium of OSM and DEX, and finally constructing a heart-liver organoid in which cardiomyocytes, hepatocytes, and non-parenchymal cells coexist.

2. The method for constructing the heart-liver symbiotic organoid according to claim 1, wherein In the culture medium of step S1, the concentration of Activin A is 50 - 200 ng / ml, the concentration of BMP4 is 10 - 30 ng / ml, and the volume concentration of B27 culture additive is 1% - 3%.

3. The construction method of the heart-liver symbiotic organoid according to claim 1, wherein, The step of adding vascular endothelial growth factor and bFGF to the culture medium simultaneously is carried out on the 5th day of the initial culture of human pluripotent stem cells.

4. The method for constructing the heart-liver symbiotic organoid according to claim 1, wherein, The culture medium used in step S2 further includes BMP2, FGF4, and B27 culture additive.

5. The method for constructing the heart-liver symbiotic organoid according to claim 1, characterized in that, Step S2 further includes adding human umbilical vein endothelial cells to the culture medium, and the addition amount of human umbilical vein endothelial cells is 1 / 2 - 1 / 5 of the amount of progenitor cells.

6. The method for constructing a heart-liver symbiotic organoid according to claim 1, wherein Step S32 includes or does not include a MIX composition.

7. A heart-liver symbiotic organoid prepared by using the construction method according to any one of claims 1 - 6, wherein the heart-liver symbiotic organoid includes hepatocytes, cardiomyocytes, and non-parenchymal cells.

8. A supplement composition for maintaining heart-liver function, comprising Non-Essential Amino Acids Solution, GlutaMAX Supplement, insulin-like growth factor receptor-1, and triiodothyronine.

9. The supplement composition for maintaining heart-liver function according to claim 8, characterized in that, The volume concentration of the Non-Essential Amino Acids Solution is 25 - 75%, the volume concentration of the GlutaMAX Supplement is 25 - 75%, the concentration of insulin-like growth factor receptor-1 is 0.5 - 1 μg / ml, and the concentration of triiodothyronine is 0.1 - 0.2 μm / ml.

10. Use of the supplement composition for maintaining heart-liver function according to claim 8 or 9 in maintaining the function of the heart-liver symbiotic organoid and prolonging its survival time.

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