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 heart-liver symbiotic organoid construction method which comprises the following steps: performing directional differentiation on human pluripotent stem cells by using Activin A, BMP4 and B27 culture additives to obtain a directional endoderm; in the process of inducing the directional endoderm into the hepatic progenitor cells, the vascular endothelial growth factor and the bFGF are simultaneously added into a culture medium, and a culture in which the hepatic progenitor cells and the 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] The present application relates to the technical field of organoid culture, and in particular to a method for constructing heart-liver symbiotic organoids and a culture medium for maintaining their functions. Background Art

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

[0003] Despite the close interaction between the heart and liver, there are still no reports on heart-liver symbiotic organoids, which seriously restricts the research on the occurrence, development mechanism and drug screening of multi-system heart-liver diseases.

[0004] The existing organoid construction methods mainly include ① traditional organoid induction: by combining stem cells (such as iPSC, embryonic stem cells (ESC) or adult stem cells (ASC)) and matrix gel, factors required to maintain the organoid ecology and factors required for differentiation, an organoid model with specific functions is constructed. ② Self-assembly: through the characteristics of cell self-organization, cells are prompted to spontaneously form organoids in an appropriate 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 precise control of the morphology and cell distribution of organoids, and is suitable for tissue models that require complex geometric shapes. ④ Microfluidic chips: Organoids constructed by microfluidic technology can be cultured under precisely controlled biological, physical and chemical conditions.

[0005] As early as 2013, Takanori Takebe's team reported in Nature for the first time that vascularized liver organoids were successfully cultivated using hiPSCs, making a major breakthrough in the field of organoid research. In 2021, Sasha Mendjan's team activated all six known signaling pathways involved in embryonic heart development in a specific order to induce stem cell self-organization. After one week of development, the organoid was structurally equivalent to the heart of a 25-day embryo. The heart organoid beats with a regular rhythm, has a diameter of about 2 mm, and includes the main cell types common to this stage of development: cardiomyocytes, epithelial cells, fibroblasts, and epicardium. They also have a clear ventricle that beats 60 to 100 times per minute, which is the same rate as the heart of an embryo of the same age.

[0006] 3D bioprinting mainly uses cells, biological hormones, growth factors, intercellular matrix and other substances to print out living tissues with biological functions. 3D bioprinting can obtain precise liver structure, 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 fibrosis caused by methotrexate and thioacetamide-induced liver damage. After exposure to these compounds, the model detected liver damage, including hepatocyte damage, and the deposition and accumulation of fibrillar collagen, indicating that the 3D bioprinted liver reproduced the compound-induced liver damage response.

[0007] As a solution, organ-on-a-chip (OoC) reconstructs the 3D microenvironment and function of organs. It has become an alternative candidate for cell experiments and drug screening. The Liver Chip platform recapitulates the physiological and pathological characteristics of human liver by reproducing liver structure, maintaining high cell viability and cell phenotype, and simulating natural liver function. Therefore, it is usually more predictive of the results of candidate drugs in humans. Parker KK designed a specific microfluidic device: first, the edge of the solid carrier surface is covered with a protective film to generate the expected contour base; then the acrylamide is spin-coated; the protective film is peeled off to obtain the coated independent body. And the gene expression profile generated by the arrangement and contractile function of cardiomyocytes was demonstrated. Domestic scholars used microfluidic devices to construct a cell culture chamber, and implanted mouse cardiomyocytes into the culture chamber. The flow rate of the culture medium in the chamber was precisely adjusted through the lateral channel, which can highly simulate the physiological state of cardiomyocytes. It is worth noting that due to the difficulty in obtaining cardiomyocytes and difficulty in long-term culture, chemical methods are often used to differentiate embryonic stem cells or induced pluripotent stem cells into cardiomyocytes, thereby simulating the structure and function of cardiomyocytes on microdevices.

[0008] In summary, although the existing technology has made significant progress in obtaining liver and heart organoids whose structure and function are gradually close to the real microenvironment in the body through traditional culture methods, 3D printing and organ chip technology, and combined organoid technology with 3D printing technology and microfluidic chip technology to construct organoids whose cell composition and structure are close to the real microenvironment in the human body, this traditional organoid culture method is usually unable to completely replicate the complex microenvironment of organs in the body, including extracellular matrix, vascular network and immune cells. This leads to differences in structure and function between the generated organoids and real organs. For example, the current 3D printed organoids are usually very unstable in structure and easily become too fragile, which limits their application in subsequent transplantation operations.

[0009] In addition, even though hiPSCs-derived liver organoids and myocardial organoids have been reported one after another, independent organoids are not enough to simulate the complex interactions between real microcircuits and different organs in vivo. To address this defect, scientists have made some attempts. At present, the only report on heart-liver symbiotic organoids is to artificially co-culture mature myocardial organoids and liver organoids derived from induced pluripotent stem cells through upper and lower chambers through microfluidic chips. This method is a multi-organ chip system derived from hiPSCs, which can be used to evaluate the cardiac safety of antidepressant drugs and conduct liver metabolism. The liver-heart organoid chip device contains chambers separated by porous membranes, which can simultaneously culture 3D liver organoids in the upper porous chamber and heart organoids in the bottom microcolumn array. The co-cultured liver and heart organoids on the chip maintain good vitality and human organ-specific functions, including the synthesis of albumin and urea in liver organoids, and the beating function of heart organoids. However, this culture method combines myocardial organoids and liver organoids together after inducing them to mature, so it cannot be used as a tool to explore the embryonic development mechanism of mesendodermal symbiotic organoids. Therefore, methods to induce symbiotic liver organoids and heart organoids in the same differentiation system need to be studied urgently. Summary of the invention

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

[0011] To achieve the above object, the technical solution of the present invention is achieved as follows: A method for constructing a heart-liver symbiotic organoid, comprising: S1: Human pluripotent stem cells are directed to differentiate using a medium containing Activin A, BMP4 and B27 culture supplement to obtain directed endoderm with co-differentiation of endoderm and mesoderm; S2: Inducing the endoderm to be oriented toward hepatic progenitor cells: Vascular endothelial growth factor (VEGF-A) and bFGF are added to the culture medium at the same time to obtain a symbiotic culture of hepatic progenitor cells and endothelial progenitor cells.

[0012] Furthermore, the directed endoderm obtained in step S1 comprises endoderm cells and mesoderm cells.

[0013] Furthermore, the concentration of Activin A in the culture medium of step S1 is 50-200 ng / ml, the concentration of BMP4 is 10-30 ng / ml, the volume concentration of B27 culture additive is 1%-3%, and the culture medium also contains bispecific antibodies with a volume concentration of 0.5%-2%.

[0014] Furthermore, the step of simultaneously adding VEGF-A and bFGF to the culture medium is performed on the 5th day of initial culture of human pluripotent stem cells.

[0015] Furthermore, the culture medium used in step S2 also includes BMP2, FGF4, B27 culture additives and bispecific antibodies.

[0016] 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 B27 culture additive is 1%-3%, and the volume concentration of bispecific antibody is 0.5%-2%.

[0017] Furthermore, the step S2 further comprises adding human umbilical vein endothelial cells (HUVECs) into the culture medium, wherein the amount of the human umbilical vein endothelial cells added is 1 / 2-1 / 5 of the amount of the hepatic progenitor cells.

[0018] Furthermore, after step S2, the method further includes: S3: Differentiate hepatic progenitor cells into hepatoblasts and then convert the culture into mature heart-liver symbiotic organoids.

[0019] On the other hand, an embodiment of the present invention also provides a heart-liver symbiotic organoid prepared by the above-mentioned construction method, wherein the heart-liver symbiotic organoid comprises hepatic parenchymal cells, non-parenchymal cells and cardiomyocytes.

[0020] In a third aspect, an embodiment of the present invention further provides a supplement composition for maintaining heart-liver function, the supplement composition comprising Non-Essential Amino Acids Solution (NEAA), GlutaMAX Supplement, insulin-like growth factor-1 receptor (IGF-1R) and triiodothyronine (T3).

[0021] Furthermore, 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.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The method for constructing the heart-liver symbiotic organoid provided by the present invention can realize the symbiotic differentiation of myocardial and liver organoids. The differentiation scheme can not only realize the simultaneous differentiation of symbiotic myocardial organoids and liver organoids in the same differentiation system, but also include other non-parenchymal cell types. The heart-liver symbiotic organoids cultured and differentiated by the differentiation scheme not only provide a good research model for drug screening and drug metabolism for multi-system diseases of the heart and liver, but also provide a physiological model for exploring the embryonic development of the heart derived from the mesoderm and the liver derived from the non-parenchymal cell endoderm.

[0023] The present invention provides a supplement that can be used to maintain the function of heart-liver symbiotic organoids and prolong their survival time. The addition of the supplement can significantly increase the contraction time and liver function of myocardial organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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 are briefly introduced below. The drawings in the following description are only used to explain some embodiments of the present invention.

[0025] Figure 1 It is a flow chart of the method for constructing heart-liver symbiotic organoids provided by the present invention.

[0026] Figure 2 This is a cell morphology image of the heart-liver symbiotic organoid induced to day 30 in an example of the present invention, taken using an optical microscope.

[0027] Figure 3 This is an overlay of cardiomyocytes and hepatocytes of the heart-liver symbiotic organoid differentiated to day 30 in an embodiment of the present invention.

[0028] Figure 4 This is a morphological diagram of cardiomyocytes (cTnT positive cells) differentiated to day 30 in an embodiment of the present invention.

[0029] Figure 5 This is a morphological diagram of hepatocytes (CYP3A4-positive cells) differentiated to day 30 in an example of the present invention.

[0030] Figure 6 This is a staining image of cell nuclei (DAPI-positive cells) differentiated to day 30 in an embodiment of the present invention.

[0031] Figure 7 Overlay of cardiomyocytes and non-cells of heart-liver symbiotic organoids differentiated to day 30 in an embodiment of the present invention.

[0032] Figure 8 This is a morphological diagram of non-parenchymal cells (αSMA-positive cells) differentiated to day 30 in an example of the present invention.

[0033] Fig. 9 This is a morphological diagram of cardiomyocytes (MYH1 positive cells) differentiated to day 30 in an embodiment of the present invention.

[0034] Fig.10 This is another visual field staining image of the cell nuclei (DAPI positive cells) differentiated to the 30th day in the embodiment of the present invention.

[0035] Fig.11 This is the result of detecting the expression of the cardiomyocyte marker gene (MYH6) in the heart-liver symbiotic organoid differentiated to day 30 in the example of the present invention.

[0036] Fig.12 This is the result of detecting the expression of the cardiomyocyte marker gene (PLB) in the heart-liver symbiotic organoid differentiated to day 30 in the example of the present invention.

[0037] Fig.13 This is the result of detecting the expression of the hepatocyte marker gene (ALB) in the heart-liver symbiotic organoid differentiated to day 30 in the example of the present invention.

[0038] Fig.14 This is the result of detecting the expression of the hepatocyte marker gene (CYP3A4) in the heart-liver symbiotic organoid differentiated to the 30th day in the example of the present invention.

[0039] Fig.15 This is the result of detecting the expression of the non-parenchymal cell marker gene (HGF) in the heart-liver symbiotic organoids differentiated to day 30 in the example of the present invention.

[0040] Fig.16This is the result of detecting the expression of the non-parenchymal cell marker gene (Desmin) in the heart-liver symbiotic organoid differentiated to day 30 in the example of the present invention.

[0041] Fig.17 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 the embodiment of the present invention. DETAILED DESCRIPTION

[0042] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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 intended to limit the present invention.

[0043] An embodiment of the present invention provides a method for constructing a heart-liver symbiotic organoid, wherein the heart-liver symbiotic organoid includes hepatocytes, non-parenchymal cells, and cardiomyocytes. The method effectively regulates the signaling pathways in the process of hiPSC differentiation into directed endoderm, so that hiPSC can achieve co-differentiation of endoderm and mesoderm, thereby retaining some mesoderm cells during the directed endoderm period. According to the construction method of the present invention, as the endoderm-derived hepatocytes differentiate, the mesoderm-derived non-parenchymal cells and cardiomyocytes also differentiate, and finally break through the barriers to achieve the symbiosis of hepatocytes, cardiomyocytes, and non-parenchymal cells. This heart-liver symbiotic organoid differentiation scheme 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 for exploring 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 non-parenchymal cell endoderm-derived liver.

[0044] An embodiment of the present invention provides a method for constructing a heart-liver symbiotic organoid, the method comprising: in the early stage of culture, adding Activin A, BMP4 and B27 culture additives to the culture medium to direct the differentiation of human pluripotent stem cells to form a directed endoderm that co-differentiates endoderm and mesoderm.

[0045] Specifically, the culture of directed endoderm continues 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 B27 culture additive is 1%-3%, and the culture medium also contains bispecific antibodies with a volume concentration of 0.5%-2%.

[0046] The differentiation of heart-liver symbiotic organoids requires multiple differentiation stages, including the process of "hiPSC→directed endoderm→hepatic progenitor cell stage→hepatic blast stage→mature heart-liver organoids". Liver parenchymal cells are derived from the endoderm, while cardiomyocytes and liver non-parenchymal cells are derived from the mesoderm. In order to achieve the symbiosis of liver cells, cardiomyocytes and non-parenchymal cells, it is first necessary to ensure the formation of the directed endoderm and retain some mesoderm cells. In the subsequent differentiation stage, ensure that the preserved mesoderm cells can differentiate into cardiomyocytes and non-parenchymal cells along with liver cells.

[0047] In addition, the differentiation of cardiomyocytes requires the participation of endothelial cells. In order to fully ensure that cardiomyocyte progenitor cells can complete differentiation dependent on endothelial cells, human umbilical vein endothelial cells and liver progenitor cells are mixed in a ratio of 1:2-1:5.

[0048] The construction method of the present invention can be divided into the following stages: (1) Establish a differentiation protocol for the "hiPSC→directed endoderm" stage of co-differentiation between mesoderm and endoderm.

[0049] In order to enable hiPSC to achieve co-differentiation of endoderm and mesoderm, the present application uses Activin A, BMP4 and B27 culture additives in the culture system at this stage to direct the differentiation of human pluripotent stem cells, so that hiPSC can retain some mesoderm cells during the differentiation process to the directed endoderm period.

[0050] (2) Establish a differentiation strategy for the "directed endoderm → hepatic progenitor cell" stage of symbiosis between hepatic progenitor cells and endothelial progenitor cells.

[0051] In order to retain more vascular endothelial progenitor cells in the liver progenitor cell stage, the present application adds vascular endothelial growth factor (VEGF-A) and bFGF necessary for vascular endothelial cells on the basis of the culture of the "directed endoderm → liver progenitor cell" stage, in order to obtain more endothelial progenitor cells and non-parenchymal cells from the mesoderm. VEGF-A is a family of proteins required for angiogenesis, which 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 mesoderm cells retained in the directed endoderm period into non-parenchymal cells and cardiomyocytes. Specifically, the amount of VEGF-A added is 5-20ng / ml, and the amount of bFGF added is 10 ng / ml. In this stage, human umbilical vein endothelial cells (HUVECs) need to be added to the culture medium, and the amount of human umbilical vein endothelial cells added is 1 / 2-1 / 5 of the amount of liver progenitor cells.

[0052] The method for constructing the heart-liver symbiotic organoid provided by the present invention is as follows Figure 1As shown. The construction method specifically includes three stages: S1 (Stage I): using Activin A, BMP4 and B27 culture additives in the cell culture medium to induce human pluripotent stem cells to differentiate into directed endoderm (Day 1-4); S2 (Stage II): based on the directed endoderm cells produced in S1, using VEGF-A, BMP2, FGF4, bFGF and B27 culture additives to induce the directed endoderm to differentiate into hepatic progenitor cells (Day 5-9); S3: first using HGF, KGF and B27 culture additives to induce hepatic progenitor cells to continue to differentiate and produce hepatoblasts (Day 10-15); then using OSM, DEX and MIX composition to induce the transformation of hepatoblasts into mature cells, and finally constructing heart and liver organoids in which cardiomyocytes, hepatocytes and non-parenchymal cells coexist (Day 16-30).

[0053] The following is a detailed description of the three stages of the method in conjunction with specific embodiments.

[0054] Example 1 Construction of heart-liver symbiotic organoids Prepare the culture medium corresponding to different stages according to the contents shown in Table 1.

[0055] Table 1: Details of culture medium at different stages The specific operation process of the construction method of this embodiment is as follows: S1: Using medium containing Activin A, BMP4 and B27 culture supplement to induce human pluripotent stem cells to differentiate into committed endoderm ( Figure 1 Day 0-4 in the Wherein: human pluripotent stem cells were cultured in RPMI 1640 medium supplemented with 100 ng / ml Activin A, 10 ng / ml BMP4, 2% B27 culture supplement (insulin-depleted type) and 1% double antibody, and directed endoderm was obtained on the 4th day of culture.

[0056] S2: Use medium containing vascular endothelial growth factor, bFGF, BMP2, FGF4 and B27 culture supplement to induce directed endoderm differentiation into hepatic progenitor cells. Among them: On the 5th day, the S1 successfully differentiated cells were transferred 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 additives and 1% double antibody for continued culture, and human umbilical vein endothelial cells were added to the culture medium and mixed with hepatic progenitor cells at a ratio of 1:4 and cultured until the 9th day.

[0057] S3: First, a culture medium containing HGF, KGF and B27 culture additives is used to induce the differentiation of liver progenitor cells into hepatoblasts; then a culture medium containing a combination of OSM, DEX and MIX is used to induce the transformation of hepatoblasts into mature hepatocytes and cardiomyocytes to construct heart-liver symbiotic organoids. Among them, step S3 specifically also includes 2 sub-steps: S31: Day 10: The cells successfully differentiated in S2 were transferred into RPMI 1640 medium supplemented with 20 ng / ml HGF, 20 ng / ml KGF, 2% B27 culture additives and 1% double antibody and continued to be cultured until day 15; S32: Day 16: The cells successfully differentiated in S31 were further induced to differentiate into heart-liver symbiotic organoids in which hepatocytes, non-parenchymal cells and cardiomyocytes coexisted. Specifically, the cells successfully differentiated in S31 were transferred into HCM culture medium supplemented with 20 ng / ml OSM, 0.5 μM DEX, 0.5 μM MIX and 1% dual antibody and continued to be cultured until day 30.

[0058] The suppliers and product numbers of the relevant reagents used in this example are shown in Table 2.

[0059] Table 2: Reagent information list The inventors also tested the heart-liver symbiotic organoids obtained in the above steps. The test content and process are as follows: (1) Bright field observation: The heart-liver symbiotic organoids prepared in this example were cultured continuously and their bright field morphology was observed under a common optical microscope after differentiation and maturation. 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.

[0060] (2) Immunofluorescence detection: Immunofluorescence labeling of mature hepatocytes (ALB+, CYP3A4+), cardiomyocytes (MYH1+, cTnT+), non-parenchymal cells (Desmin+, Vimentin+, αSMA+) and vascular endothelial cells (CD31+) was performed, and cells of different cell types were observed under a fluorescence microscope. The test results are shown in Figure 3-Figure 10 ,in Figure 3 This is an overlay of cardiomyocytes and hepatocytes in the heart-liver symbiotic organoid differentiated to day 30; Figure 4 This is a morphological diagram of cardiomyocytes (cTnT positive cells) differentiated to day 30 in an embodiment of the present invention; Figure 5is a morphological diagram of hepatocytes (CYP3A4 positive cells) differentiated to day 30 in an embodiment of the present invention; Figure 6 This is a staining image of cell nuclei (DAPI-positive cells) differentiated to day 30 in an embodiment of the present invention; Figure 7 Overlay of myocardial cells and non-cells of heart-liver symbiotic organoids differentiated to day 30 in the embodiment of the present invention; Figure 8 This is a morphological diagram of non-parenchymal cells (αSMA-positive cells) differentiated to day 30 in an embodiment of the present invention; Fig. 9 This is a morphological diagram of cardiomyocytes (MYH1 positive cells) differentiated to day 30 in an embodiment of the present invention; Fig.10 This is another field of view staining of the cell nuclei (DAPI positive cells) differentiated to day 30 in the embodiment of the present invention. Figure 3-Figure 10 It can be seen that the heart-liver symbiotic organoids cultured using the culture method of the present invention simultaneously have hepatic parenchymal cells, non-parenchymal cells and cardiomyocytes.

[0061] (3) Fluorescence quantitative PCR: This example verifies the above immunofluorescence results by detecting the expression of marker genes related to cardiomyocytes, hepatocytes and non-parenchymal cells. The test results are shown in Figure 11-Figure 16 .in Fig.11 and Fig.12 They are respectively the expression detection results of cardiomyocyte marker genes (MYH6 and PLB) during the induction process of the heart-liver symbiotic organoid prepared in this example; Fig.13 and Fig.14 They are respectively the expression detection results of hepatocyte marker genes (ALB and CYP3A4) during the induction process of the heart-liver symbiotic organoid prepared in this example; Fig.15 and Fig.16 They are respectively the expression detection results of non-parenchymal cell marker genes (HGF and Desmin) during the induction of the heart-liver symbiotic organoids prepared in this example. Figure 11-Figure 16 It can be seen that at D30, the expression of related genes of cardiomyocytes, hepatocytes and non-parenchymal cells in the heart-liver symbiotic organoids constructed by the present invention is stronger than that at D0, indicating that cardiomyocytes, hepatocytes and non-parenchymal cells exist simultaneously in the heart-liver symbiotic organoids, further verifying the results of immunofluorescence detection.

[0062] Example 2 Preparation of Supplement Composition for Maintaining Heart-Liver Symbiotic Organoids The supplement composition was prepared according to the contents shown in Table 3.

[0063] Table 3 Raw materials for supplement preparation (1 ml) In order to effectively promote the function and lifespan of heart / liver organoids without limiting the function and lifespan of another organoid, NEAA, GlutaMAX Supplement, IGF-IR and T3 were selected as the main ingredients of the supplement. Among them, insulin-like growth factor-1 receptor (IGF-1R) is an important factor in regulating cardiomyocyte proliferation, development and promoting cardiac function maturation. Triiodothyronine (T3) is a hormone secreted by thyroid follicles, which is essential for the normal development of the heart.

[0064] In order to verify the effect of the supplement composition prepared above on the survival time of heart-liver symbiotic organoids, the inventors conducted a comparative experiment. On D30 of the heart-liver symbiotic organoid culture, 1 ml of the supplement composition was added to the culture medium of the experimental group, and the control group was not treated and continued to culture to observe the contraction of the organoids in the culture medium. The experimental results are as follows Fig.17 As shown, the supplement composition provided by the present invention can significantly increase the survival time and contraction duration of myocardial organoids.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a heart-liver symbiotic organoid, comprising: S1: Human pluripotent stem cells are directed to differentiate using a medium containing Activin A, BMP4 and B27 culture supplement to obtain directed endoderm with co-differentiation of endoderm and mesoderm; S2: Direction induction of endoderm into hepatic progenitor cells: Vascular endothelial growth factor and bFGF are added to the culture medium simultaneously to obtain a symbiotic culture of hepatic progenitor cells and endothelial progenitor cells.

2. The method for constructing a heart-liver symbiotic organoid according to claim 1, characterized in that: 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, the volume concentration of B27 culture additive is 1%-3%, and the culture medium also contains bispecific antibodies with a volume concentration of 0.5%-2%.

3. The method for constructing a heart-liver symbiotic organoid according to claim 1, characterized in that: The step of simultaneously adding the vascular endothelial growth factor and the bFGF to the culture medium is performed on the 5th day of initial culture of human pluripotent stem cells.

4. The method for constructing a heart-liver symbiotic organoid according to claim 1, characterized in that: The culture medium used in step S2 also includes BMP2, FGF4, B27 culture additives and double antibodies.

5. The method for constructing a heart-liver symbiotic organoid according to claim 1, characterized in that: The step S2 further comprises adding human umbilical vein endothelial cells into the culture medium, wherein the amount of human umbilical vein endothelial cells added is 1 / 2-1 / 5 of the amount of hepatic progenitor cells.

6. The method for constructing a heart-liver symbiotic organoid according to claim 1, characterized in that: After step S2, the method further includes: S3: Differentiate hepatic progenitor cells into hepatoblasts and then convert them into mature heart-liver symbiotic organoids.

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

8. A supplement composition for maintaining the heart-liver function of the heart-liver symbiotic organoid of claim 7, comprising Non-Essential Amino Acids Solution, GlutaMAX Supplement, insulin-like growth factor receptor-1 and triiodothyronine.

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

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

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