A method for culturing a cardiac organoid, and a culture reagent

Abstract

The application provides a culture method and culture reagent of a heart organoid, and the scheme of the application uses a culture medium with clear components to perform four-stage induction differentiation culture, so that the heart organoid can be constructed from dispersed human induced pluripotent stem cells, the construction process of the heart organoid is simplified, the construction period of the heart organoid is shortened, and the use of the culture medium system with clear components greatly improves the success rate of heart organoid induction, and can increase the success rate of the traditional heart organoid construction method to 80% to 100%.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for culturing cardiac organoids and a culture reagent. Background Technology

[0002] Cardiac organoids, as three-dimensional cell aggregates that mimic the structure and function of heart tissue, have broad application prospects in fields such as heart disease research, drug screening, and regenerative medicine. However, the induction and differentiation of cardiac organoids currently faces challenges such as complex culture components, long time cycles, and low induction success rates. Firstly, the complexity of the culture components is mainly reflected in the cardiac organoid culture system, which currently involves two aspects: First: mTeSR TM 1. As a serum-free and allogeneic-free culture medium, it is commonly used for the maintenance culture of human pluripotent stem cells (hESC / iPSC), maintaining the cells in an undifferentiated state and laying the foundation for subsequent directed differentiation of cardiac organoids. 2. The RPMI 1640 + B27 additive combination is often used as the core basal culture medium for the induction differentiation stage of cardiac organoids. RPMI 1640 provides basic nutrients such as amino acids and glucose, while B27 (with or without vitamin A), as a serum-free supplement, contains 20 components including vitamins, BSA, and insulin, which can support the survival and differentiation of cardiomyocytes. Vitamin A-free B27 is more suitable for early cardiac lineage induction. Furthermore, cardiac organoids typically exhibit myocardial rhythm phenomena between 10 and 14 days during the induction differentiation stage, while the maturation cycle of cardiac organoids requires 14 to 28 days. Therefore, generally, when studying the functional characteristics of cardiac organoids, cardiac organoids that have undergone 14 days of induction differentiation and exhibit myocardial rhythm phenomena should be selected. However, for experiments involving the study of cardiac organoid disease models and drug screening, cardiac organoids induced to differentiate for 21-28 days are required. Finally, although there are numerous references and patents on the induction and differentiation of cardiac organoids, the success rate for obtaining structurally complete cardiac organoids suitable for disease modeling or drug screening is generally 30%-60%. If the goal is to further pursue highly mature organoids that closely resemble the real heart, the success rate is generally 10%-30%. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a culture reagent and corresponding construction method for constructing cardiac organoids with clearly defined components and high efficiency, so as to solve the problems existing in the current research status, namely, the culture components for constructing cardiac organoids from human induced pluripotent stem cells are complex, the time cycle is long and the induction success rate is low.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] The present invention provides a composition comprising Advanced DMEM / F12 medium, BSA, ITS, lipid concentrate, thioglycerol (MTG), and L-ascorbic acid 2-phosphate.

[0006] The present invention also provides the use of the above composition in the preparation of a basic culture medium for constructing cardiac organoids.

[0007] The present invention also provides a culture reagent for constructing cardiac organoids, comprising a basal culture medium;

[0008] The basal culture medium includes: Advanced DMEM / F12 medium, BSA, ITS, lipid concentrate, thioglycerol, and L-ascorbic acid 2-phosphate.

[0009] The present invention also provides the application of the basal culture medium of the above-mentioned culture reagent in the preparation of mesoderm differentiation culture medium, cardiac mesoderm differentiation culture medium, cardiomyocyte differentiation culture medium and / or cardiac organoid maturation culture medium.

[0010] In some specific embodiments of the present invention, the above-mentioned culture reagent further includes at least one of culture medium A, culture medium B, culture medium C, and culture medium D;

[0011] The culture medium A includes: the basal culture medium, FGF2, LY294002, activator A, BMP4, CHIR-99021, and Y-27632;

[0012] The culture medium B includes: the basal culture medium, IWP-2, FGF2, vascular endothelial growth factor A, BMP4, insulin, and retinoic acid;

[0013] The culture medium C includes: the basal culture medium, FGF2, vascular endothelial growth factor A, BMP4, and insulin;

[0014] The culture medium D includes: the basal culture medium, vascular endothelial growth factor A, and insulin.

[0015] In some specific embodiments of the present invention, the above-mentioned culture reagent:

[0016] The concentration of the Advanced DMEM / F12 medium is 1×; and / or

[0017] The BSA content in the basal culture medium is 4-6 mg / mL; and / or

[0018] The ITS content in the basal culture medium is 0.5×~2×; and / or

[0019] The lipid concentrate has a concentration of 0.5×~2× in the basal culture medium; and / or

[0020] The thioglycerol content in the basal culture medium is 400-500 μM; and / or

[0021] The L-ascorbic acid 2-phosphate in the basal culture medium is 25~100 μg / mL; and / or

[0022] The FGF2 content in culture medium A is 25~35 ng / mL; and / or

[0023] The concentration of LY294002 in culture medium A is 4-6 μM; and / or

[0024] The activator A is present in culture medium A at a concentration of 45-55 ng / mL; and / or

[0025] The BMP4 content in culture medium A, culture medium B, or culture medium C is 5-15 ng / mL; and / or

[0026] The concentration of CHIR-99021 in culture medium A is 3~5 μM; and / or

[0027] The concentration of Y-27632 in culture medium A is 2.5~7.5 μM; and / or

[0028] The concentration of IWP-2 in culture medium B is 4.5–5.5 μM; and / or

[0029] The FGF2 content in culture medium B or culture medium C is 7-9 ng / mL; and / or

[0030] The concentration of vascular endothelial growth factor A in culture medium B is 150-250 ng / mL; and / or

[0031] The insulin concentration in culture medium B, culture medium C, or culture medium D is 8–15 μg / mL; and / or

[0032] The retinoic acid content in the culture medium B is 0.45~0.55 μM; and / or

[0033] The content of vascular endothelial growth factor A in culture medium C or culture medium D is 50~150 ng / mL.

[0034] In some specific embodiments of the present invention, the above-mentioned culture reagent:

[0035] The concentration of the Advanced DMEM / F12 medium is 1×; and / or

[0036] The BSA content in the basal culture medium is 5 mg / mL; and / or

[0037] The ITS content in the basal culture medium is 1×; and / or

[0038] The lipid concentrate is a CD lipid concentrate, and its concentration in the basal culture medium is 1×; and / or

[0039] The thioglycerol content in the basal culture medium is 450 μM; and / or

[0040] The L-ascorbic acid 2-phosphate in the basal medium is present at a concentration of 50 μg / mL; and / or

[0041] The FGF2 content in culture medium A is 30 ng / mL; and / or

[0042] The concentration of LY294002 in culture medium A is 5 μM; and / or

[0043] The activator A is present at a concentration of 50 ng / mL in culture medium A; and / or

[0044] The BMP4 content in culture medium A, culture medium B, or culture medium C is 10 ng / mL; and / or

[0045] The concentration of CHIR-99021 in culture medium A is 4 μM; and / or

[0046] The concentration of Y-27632 in culture medium A is 5 μM; and / or

[0047] The concentration of IWP-2 in culture medium B is 5 μM; and / or

[0048] The FGF2 content in culture medium B or culture medium C is 8 ng / mL; and / or

[0049] The concentration of vascular endothelial growth factor A in culture medium B is 200 ng / mL; and / or

[0050] The insulin concentration in culture medium B, culture medium C, or culture medium D is 10 μg / mL; and / or

[0051] The retinoic acid content in the culture medium B is 0.5 μM; and / or

[0052] The concentration of vascular endothelial growth factor A in culture medium C or culture medium D is 100 ng / mL.

[0053] The present invention also provides the application of the above-mentioned culture reagent in the construction or culture of cardiac organoids.

[0054] The present invention also provides a method for culturing cardiac organoids, which is based on the above-mentioned culture reagent.

[0055] In some specific embodiments of the present invention, the above-mentioned cultivation method includes:

[0056] Based on the above-mentioned basic culture media, mesodermal differentiation medium, cardiac mesodermal differentiation medium, cardiomyocyte differentiation medium and / or cardiac organoid maturation medium were prepared.

[0057] Human induced pluripotent stem cells were cultured using the aforementioned mesodermal differentiation medium, cardiac mesodermal differentiation medium, cardiomyocyte differentiation medium, and / or cardiac organoid maturation medium to obtain cardiac organoids.

[0058] In some specific embodiments of the present invention, the above-mentioned cultivation method includes:

[0059] S1. Prepare mesodermal differentiation medium, cardiac mesodermal differentiation medium, cardiomyocyte differentiation medium, and cardiac organoid maturation medium using the above-mentioned basic culture medium;

[0060] S2. Culture hiPS cells in a U-shaped ultra-low adsorption cell culture plate using the mesodermal differentiation medium until aggregates appear;

[0061] S3. Replace the culture medium with the cardiac mesodermal differentiation medium, and culture until day 6 to complete the cardiac mesodermal differentiation culture;

[0062] S4. Replace the culture medium with the cardiomyocyte differentiation culture medium, and culture until day 8 to complete the cardiomyocyte differentiation culture;

[0063] S5. Replace the culture medium with the heart organoid maturation culture medium, and obtain the heart organoid on day 12.

[0064] In some specific embodiments of the present invention, the above-described cultivation method is as follows:

[0065] The mesodermal differentiation medium is the aforementioned medium A; and / or

[0066] The cardiac mesodermal differentiation culture medium is culture medium B mentioned above; and / or

[0067] The cardiomyocyte differentiation culture medium is the aforementioned culture medium C; and / or

[0068] The heart organoid maturation culture medium is the aforementioned culture medium D.

[0069] In some specific embodiments of the present invention, the above-mentioned cultivation method includes:

[0070] S1. Culture hiPS cells in ultra-low adsorption cell culture plates with a U-shaped bottom using the above-mentioned culture medium A until aggregates appear;

[0071] S2. Replace the culture medium with the above-mentioned culture medium B, and culture until day 6 to complete the differentiation culture of the cardiac mesoderm;

[0072] S3. Replace the culture medium with the above-mentioned culture medium C, and culture until day 8 to complete the differentiation culture of cardiomyocytes;

[0073] S4. Replace the culture medium with the above-mentioned culture medium D, and culture until day 12 to obtain heart organoids.

[0074] The present invention has the following beneficial effects:

[0075] 1. A culture medium formulation for constructing heart organoids with clearly defined components is provided (instead of using the traditional RPMI 1640 + B27 system, a more clearly defined component is used to replace B27).

[0076] 2. Digested and discrete human induced pluripotent stem cells can be resuspended in mesotherapy medium to directly form spheres for subsequent heart organoid construction. This method can skip the embryoid formation stage, shorten the heart organoid induction differentiation cycle to 7-10 days, and the heart organoid maturation stage to 10-14 days, greatly shortening the heart organoid construction cycle.

[0077] 3. By using a clearly defined heart organoid culture medium formula, the success rate of traditional heart organoid construction methods (30%~60%) can be increased to 80%~100%.

[0078] In summary, this invention provides a method for constructing cardiac organoids with well-defined components and high efficiency. By using a culture medium with well-defined components for four-stage induction and differentiation culture, cardiac organoids can be constructed from digested and discrete human induced pluripotent stem cells. This simplifies the construction process of cardiac organoids, shortens the construction cycle, and greatly improves the success rate of cardiac organoid induction by using a culture medium system with well-defined components. It can increase the success rate of traditional cardiac organoid construction methods to 80%~100%. Attached Figure Description

[0079] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0080] Figure 1 Schematic diagram of heart organoid culture;

[0081] Figure 2 Example 1 shows human induced pluripotent stem cells grown to a density of 70%~80%;

[0082] Figure 3 This shows an immunofluorescence staining identification image from Example 1;

[0083] Figure 4 Example 2 shows the growth of human induced pluripotent stem cells to a density of 70%~80%;

[0084] Figure 5 Example 2 shows an immunofluorescence staining identification image;

[0085] Figure 6 Example 3 shows the growth of human induced pluripotent stem cells to a density of 70%~80%;

[0086] Figure 7 This shows an immunofluorescence staining identification image from Example 3;

[0087] Figure 8 The control group showed that induced pluripotent stem cells in one person grew to a density of 70%~80%;

[0088] Figure 9 Comparative diagram showing the embryoid formation induced by culture medium A in Comparative Example 1 (mT1, E8, and A);

[0089] Figure 10 The control group showed that induced pluripotent stem cells in two individuals grew to a density of 70%–80%.

[0090] Figure 11 This image shows a comparison between cardiac organoids induced by the formulation of this invention and cardiac organoids induced by formulations in the literature. Detailed Implementation

[0091] This invention discloses a culture reagent and corresponding construction method for constructing cardiac organoids with clearly defined components and high efficiency. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0092] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0093] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0094] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0095] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0096] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0097] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0098] In one implementation, a schematic diagram of heart organoid culture is shown below. Figure 1 As shown. In practice, follow these steps:

[0099] S1. Culture hiPSCs in mT1 medium, discard the supernatant, wash twice with 1 mL DPBS, discard the supernatant again, then digest with 1× Tryple for 5 min, centrifuge to collect hiPSCs, resuspend in appropriate medium A, and repeat at 8×10⁻⁶. 3To adjust the density of the wells, add 100 μL of cell suspension to the bottom of a U-shaped ultra-low adsorption 96-well plate, centrifuge at 200g for 5 min, and then incubate the U-shaped ultra-low adsorption 96-well plate in an incubator for 2 days without disturbing the plate, allowing the cells to spontaneously form aggregates.

[0100] S2. After aggregates form, aspirate culture medium A along the sidewall of the well plate and add 100 μL of culture medium B. Replace with fresh culture medium B daily for 4 consecutive days.

[0101] S3. On day 6, aspirate culture medium B along the side wall of the well plate, then add 100 μL of culture medium C, and repeat this process for 2 days.

[0102] S4. Gently aspirate culture medium C along the wall, then add 100 μL of culture medium D. Change the medium every two days thereafter, and the heart organoid model will be obtained on day 12.

[0103] In one example, the method for preparing human induced pluripotent stem cells in step S1 includes: culturing human induced pluripotent stem cells (IPSC-DYR0100, Ubigene Biosciences, Guangzhou) until the cell density reaches 70%~80% before digestion and plating can begin.

[0104] In one example, in step S1, human induced pluripotent stem cells suspended in culture medium A are added at a ratio of 7.5 × 10⁻⁶. 3 ~9×10 3 Cells were seeded at a density of 1 cell per well into ultra-low adsorption cell culture plates with a U-shaped bottom.

[0105] In practice, culture media A, B, C and D all contain basal culture media.

[0106] The basal culture medium specifically includes: using Advanced DMEM / F12 medium as the solvent, a solution containing 4~6 mg / mL BSA, 0.5×~2× ITS, 0.5×~2× CD lipid concentrate, 400~500 μM MTG, and 25~75 μg / mL L-ascorbic acid 2-phosphate.

[0107] The culture medium A specifically includes: using Advanced DMEM / F12 medium as the solvent, a solution containing 4~6 mg / mL BSA, 0.5×~2× ITS, 0.5×~2× CD lipid concentrate, 400~500 μM MTG, 25~100 μg / mL L-ascorbic acid 2-phosphate, 25~35 ng / mL FGF2, 4~6 μM LY294002, 45~55 ng / mL activin A, 5~15 ng / mL BMP4, 3~5 μM CHIR-99021, and 2.5~7.5 μM Y-27632.

[0108] The culture medium B specifically includes: using Advanced DMEM / F12 medium as solvent, a solution containing 4~6 mg / mL BSA, 0.5×~2× ITS, 0.5×~2× CD lipid concentrate, 400~500 μM MTG, 25~100 μg / mL L-ascorbic acid 2-phosphate, 4.5~5.5 μM IWP-2, 7~9 ng / mL FGF2, 150~250 ng / mL vascular endothelial growth factor A (VEGF-A), 5~15 ng / mL BMP4, 8~15 μg / mL insulin, and 0.45~0.55 μM retinoic acid.

[0109] The culture medium C specifically includes: using Advanced DMEM / F12 medium as solvent, a solution containing 4~6 mg / mL BSA, 0.5×~2× ITS, 0.5×~2× CD lipid concentrate, 400~500 μM MTG, 25~100 μg / mL L-ascorbic acid 2-phosphate, 7~9 ng / mL FGF2, 50~150 ng / mL vascular endothelial growth factor A (VEGF-A), 5~15 ng / mL BMP4, and 8~15 μg / mL insulin.

[0110] The culture medium D specifically includes: using Advanced DMEM / F12 medium as the solvent, a solution containing 4~6 mg / mL BSA, 0.5×~2× ITS, 0.5×~2× CD lipid concentrate, 400~500 μM MTG, 25~100 μg / mL L-ascorbic acid 2-phosphate, 50~150 ng / mL vascular endothelial growth factor A (VEGF-A), and 8~15 μg / mL insulin.

[0111] In one example, the basal culture medium comprises: Advanced DMEM / F12 medium, BSA (5 mg / mL), ITS (1×), CD lipid concentrate (1×), MTG (450 μM), and L-Ascorbic Acid 2-Phosphate (50 μg / mL).

[0112] In one example, culture medium A includes: Advanced DMEM / F12 medium, BSA (5 mg / mL), ITS (1×), CD lipid concentrate (1×), MTG (450 μM), L-Ascorbic Acid 2-Phosphate (50 μg / mL), FGF2 (30 ng / mL), LY294002 (5 μM), Activin A (50 ng / mL), BMP4 (10 ng / mL), CHIR-99021 (4 μM), and Y-27632 (5 μM).

[0113] In one example, culture medium B includes: Advanced DMEM / F12 medium, BSA (5 mg / mL), ITS (1×), CD lipid concentrate (1×), MTG (450 μM), L-Ascorbic Acid 2-Phosphate (50 μg / mL), IWP-2 (5 μM), FGF2 (8 ng / mL), vascular endothelial growth factor A (VEGF-A, 200 ng / mL), BMP4 (10 ng / mL), insulin (10 μg / mL), and retinoic acid (0.5 μM).

[0114] In one example, culture medium C includes: Advanced DMEM / F12 medium, BSA (5 mg / mL), ITS (1×), CD lipid concentrate (1×), MTG (450 μM), L-Ascorbic Acid 2-Phosphate (50 μg / mL), FGF2 (8 ng / mL), vascular endothelial growth factor A (VEGF-A, 100 ng / mL), BMP4 (10 ng / mL), and insulin (10 μg / mL).

[0115] In one example, culture medium D includes: Advanced DMEM / F12 medium, BSA (5 mg / mL), ITS (1×), CD lipid concentrate (1×), MTG (450 μM), L-Ascorbic Acid 2-Phosphate (50 μg / mL), vascular endothelial growth factor A (VEGF-A, 100 ng / mL), and insulin (10 μg / mL).

[0116] Table I provides the sources (brands and part numbers) of the materials used in each embodiment, but the scope of protection of this invention is not limited thereto.

[0117] Table I

[0118]

[0119] In the example, the product information for the 96-well ultra-low adsorption cell culture plate with a U-shaped bottom is as follows: Consumable name: 96-well Clear Round Bottom Ultra-Low Attachment Microplate, Brand: Corning, Item number: 7007.

[0120] The present invention will be further illustrated below with reference to the embodiments.

[0121] Example 1

[0122] Step 1: Prepare the basic culture medium

[0123] BSA, ITS, CD lipid concentrate, MTG, and L-Ascorbic Acid 2-Phosphate were added to Advanced DMEM / F12 medium and mixed thoroughly to obtain the basal medium for later use. The concentrations of each component in the medium are shown in Table 1.

[0124] Table 1: Basic Culture Medium Formulation

[0125]

[0126] Step 2: Prepare culture medium A

[0127] FGF2, LY294002, Activin A, BMP4, CHIR99021, and Y-27632 were added to the basal medium and mixed thoroughly to obtain medium A, which was then set aside. The concentrations of each component in medium A are shown in Table 2.

[0128] Table 2: Culture Medium A Formulation

[0129]

[0130] Step 3: Prepare culture medium B

[0131] IWP2, FGF2, VEGF-A, BMP4, Insulin, and RA were added to the basal medium and mixed thoroughly to obtain medium B, which was then set aside. The concentrations of each component in medium B are shown in Table 3.

[0132] Table 3: Culture Medium B Formulation

[0133]

[0134] Step 4: Prepare culture medium C

[0135] FGF2, VEGF-A, BMP4, and Insulin were added to the basal medium and mixed thoroughly to obtain medium C, which was then set aside. The concentrations of each component in medium C are shown in Table 4.

[0136] Table 4: Culture Medium C Formulation

[0137]

[0138] Step 5: Prepare culture medium D

[0139] VEGF-A and insulin were added to the basal medium and mixed thoroughly to obtain medium D, which was then set aside. The concentrations of each component in medium D are shown in Table 5.

[0140] Table 5: Culture Medium D Formulation

[0141]

[0142] Step Six: Culture hiPS cells

[0143] Human induced pluripotent stem cells were seeded into mTeSR1 medium and placed in an incubator with the temperature controlled at 37°C and the CO2 concentration at 5%. The medium was changed daily. The growth of the human induced pluripotent stem cells was observed as follows: Figure 2 As shown. When the cell density of human induced pluripotent stem cells reaches 70%~80%, they are digested with Tryple E for 5 minutes, and then digestion is terminated to obtain digested hiPS cells.

[0144] Step 7: Mesodermal Differentiation Culture

[0145] Resuspend the digested hiPS cells from step six in culture medium A, at a cell density of 8 × 10⁶ cells / mL. 3 Cells / well were seeded into 96-well ultra-low adsorption cell culture plates with a U-shaped bottom. Aggregates were observed on day 2 of culture in these plates (see...). Figure 1 Day 2), complete the mesodermal differentiation culture.

[0146] Step 8: Cardiac Mesodermal Differentiation Culture

[0147] After the mesodermal differentiation culture was completed, culture medium A was aspirated from the cell culture plate, and culture medium B was added. The culture continued, with culture medium B being changed daily. From the time culture medium A was replaced with culture medium B, the cardiac mesodermal differentiation culture was completed on day 6.

[0148] Step Nine: Cardiac Cell Differentiation Culture

[0149] After completing the cardiac mesodermal differentiation culture, culture medium B was aspirated from the cell culture plate, and culture medium C was added. Culture continued, with culture medium C being changed daily. From the time the medium was changed to medium C, the cardiomyocyte differentiation culture was completed on day 8.

[0150] Step 10: Maturation Culture of Heart Organoids

[0151] After completing the differentiation and culture of cardiomyocytes, culture medium C was aspirated from the cell culture plate, and culture medium D was added. Culture continued, with culture medium D changed daily. From the time the culture medium was changed to medium D, cardiac organoids were obtained on day 12, at which point pulsation of the aggregates was observed. Immunofluorescence staining identification of the obtained cardiac organoids was performed; the results are shown below. Figure 3 The blue fluorescence represents the cell nucleus, the red fluorescence represents the vascular endothelial cells, and the yellow fluorescence represents the cardiomyocytes, indicating that the heart organoids obtained in this embodiment conform to the physiological structure of normal heart organs.

[0152] Example 2

[0153] The steps for constructing a heart organoid in this embodiment are as follows.

[0154] Step 1: Prepare the basic culture medium

[0155] BSA, ITS, CD lipid concentrate, MTG, and L-Ascorbic Acid 2-Phosphate were added to Advanced DMEM / F12 medium and mixed thoroughly to obtain the basal medium for later use. The concentrations of each component in the medium are shown in Table 6.

[0156] Table 6: Basic Culture Medium Formulation

[0157]

[0158] Step 2: Prepare culture medium A

[0159] FGF2, LY294002, Activin A, BMP4, CHIR99021, and Y-27632 were added to the basal medium and mixed thoroughly to obtain medium A, which was then set aside. The concentrations of each component in medium A are shown in Table 7.

[0160] Table 7: Culture Medium A Formulation

[0161]

[0162] Step 3: Prepare culture medium B

[0163] IWP2, FGF2, VEGF-A, BMP4, Insulin, and RA were added to the basal medium and mixed thoroughly to obtain medium B, which was then set aside. The concentrations of each component in medium B are shown in Table 8.

[0164] Table 8: Culture Medium B Formulation

[0165]

[0166] Step 4: Prepare culture medium C

[0167] FGF2, VEGF-A, BMP4, and Insulin were added to the basal medium and mixed thoroughly to obtain medium C, which was then set aside. The concentrations of each component in medium C are shown in Table 9.

[0168] Table 9: Culture Medium C Formulation

[0169]

[0170] Step 5: Prepare culture medium D

[0171] VEGF-A and Insulin were added to the basal medium and mixed thoroughly to obtain medium D, which was then set aside. The concentrations of each component in medium D are shown in Table 10.

[0172] Table 10: Culture Medium D Formulation

[0173]

[0174] Step Six: Culture hiPS cells

[0175] Human induced pluripotent stem cells were seeded into mTeSR1 medium and placed in an incubator with the temperature controlled at 37°C and the CO2 concentration at 5%. The medium was changed daily. The growth of the human induced pluripotent stem cells was observed as follows: Figure 4 As shown. When the cell density of human induced pluripotent stem cells reaches 70%~80%, they are digested with Tryple E for 5 minutes, and then digestion is terminated to obtain digested hiPS cells.

[0176] Step 7: Mesodermal Differentiation Culture

[0177] Resuspend the digested hiPS cells from step six in culture medium A, at a cell density of 8 × 10⁶ cells / mL. 3 Cells were seeded per well into 96-well ultra-low adsorption cell culture plates with a U-shaped bottom. Aggregates were observed on day 2 of culture in these plates, indicating completion of mesodermal differentiation.

[0178] Step 8: Cardiac Mesodermal Differentiation Culture

[0179] After the mesodermal differentiation culture was completed, culture medium A was aspirated from the cell culture plate, and culture medium B was added. The culture continued, with culture medium B being changed daily. From the time culture medium A was replaced with culture medium B, the cardiac mesodermal differentiation culture was completed on day 6.

[0180] Step Nine: Cardiac Cell Differentiation Culture

[0181] After completing the cardiac mesodermal differentiation culture, culture medium B was aspirated from the cell culture plate, and culture medium C was added. Culture continued, with culture medium C being changed daily. From the time the medium was changed to medium C, the cardiomyocyte differentiation culture was completed on day 8.

[0182] Step 10: Maturation Culture of Heart Organoids

[0183] After completing the differentiation and culture of cardiomyocytes, culture medium C was aspirated from the cell culture plate, and culture medium D was added. Culture continued, with culture medium D changed daily. From the time the culture medium was changed to medium D, cardiac organoids were obtained on day 12, at which point pulsation of the aggregates was observed. Immunofluorescence staining identification of the obtained cardiac organoids was performed; the results are shown below. Figure 5 The blue fluorescence represents the cell nucleus, the red fluorescence represents the vascular endothelial cells, and the yellow fluorescence represents the cardiomyocytes, indicating that the heart organoids obtained in this embodiment conform to the physiological structure of normal heart organs.

[0184] Example 3

[0185] The steps for constructing a heart organoid in this embodiment are as follows.

[0186] Step 1: Prepare the basic culture medium

[0187] BSA, ITS, CD lipid concentrate, MTG, and L-Ascorbic Acid 2-Phosphate were added to Advanced DMEM / F12 medium and mixed thoroughly to obtain the basal medium for later use. The concentrations of each component in the medium are shown in Table 11.

[0188] Table 11: Basic Culture Medium Formulation

[0189]

[0190] Step 2: Prepare culture medium A

[0191] FGF2, LY294002, Activin A, BMP4, CHIR99021, and Y-27632 were added to the basal medium and mixed thoroughly to obtain medium A, which was then set aside. The concentrations of each component in medium A are shown in Table 12.

[0192] Table 12: Culture Medium A Formulation

[0193]

[0194] Step 3: Prepare culture medium B

[0195] IWP2, FGF2, VEGF-A, BMP4, Insulin, and RA were added to the basal medium and mixed thoroughly to obtain medium B, which was then set aside. The concentrations of each component in medium B are shown in Table 13.

[0196] Table 13: Culture Medium B Formulation

[0197]

[0198] Step 4: Prepare culture medium C

[0199] FGF2, VEGF-A, BMP4, and insulin were added to the basal medium and mixed thoroughly to obtain medium C, which was then set aside. The concentrations of each component in medium C are shown in Table 14.

[0200] Table 14: Culture Medium C Formulation

[0201]

[0202] Step 5: Prepare culture medium D

[0203] VEGF-A and insulin were added to the basal medium and mixed thoroughly to obtain medium D, which was then set aside. The concentrations of each component in medium D are shown in Table 15.

[0204] Table 15: Culture Medium D Formulation

[0205]

[0206] Step Six: Culture hiPS cells

[0207] Human induced pluripotent stem cells were seeded into mTeSR1 medium and placed in an incubator with the temperature controlled at 37°C and the CO2 concentration at 5%. The medium was changed daily. The growth of the human induced pluripotent stem cells was observed as follows: Figure 6 As shown. When the cell density of human induced pluripotent stem cells reaches 70%~80%, they are digested with Tryple E for 5 minutes, and then digestion is terminated to obtain digested hiPS cells.

[0208] Step 7: Mesodermal Differentiation Culture

[0209] Resuspend the digested hiPS cells from step six in culture medium A, at a cell density of 8 × 10⁶ cells / mL. 3 Cells were seeded per well into 96-well ultra-low adsorption cell culture plates with a U-shaped bottom. Aggregates were observed on day 2 of culture in these plates, indicating completion of mesodermal differentiation.

[0210] Step 8: Cardiac Mesodermal Differentiation Culture

[0211] After the mesodermal differentiation culture was completed, culture medium A was aspirated from the cell culture plate, and culture medium B was added. The culture continued, with culture medium B being changed daily. From the time culture medium A was replaced with culture medium B, the cardiac mesodermal differentiation culture was completed on day 6.

[0212] Step Nine: Cardiac Cell Differentiation Culture

[0213] After completing the cardiac mesodermal differentiation culture, culture medium B was aspirated from the cell culture plate, and culture medium C was added. Culture continued, with culture medium C being changed daily. From the time the medium was changed to medium C, the cardiomyocyte differentiation culture was completed on day 8.

[0214] Step 10: Maturation Culture of Heart Organoids

[0215] After completing the differentiation and culture of cardiomyocytes, culture medium C was aspirated from the cell culture plate, and culture medium D was added. Culture continued, with culture medium D changed daily. From the time the culture medium was changed to medium D, cardiac organoids were obtained on day 12, at which point pulsation of the aggregates was observed. Immunofluorescence staining identification of the obtained cardiac organoids was performed; the results are shown below. Figure 7 The blue fluorescence represents the cell nucleus, the red fluorescence represents the vascular endothelial cells, and the yellow fluorescence represents the cardiomyocytes, indicating that the heart organoids obtained in this embodiment conform to the physiological structure of normal heart organs.

[0216] Comparative Example 1

[0217] By comparing the effects of directly forming embryoids using mesodermal differentiation medium with forming embryoids using traditional mT1 and E8 media, this invention yielded heart organoids with higher beating rates.

[0218] The sources (brands and item numbers) of the materials used in this example are shown in Table II.

[0219] Table II

[0220]

[0221] The steps for constructing a heart organoid in this example are as follows.

[0222] Step 1: Prepare the basic culture medium, culture medium A, culture medium B, culture medium C and culture medium D (prepare according to Example 2).

[0223] Step 2: Culture hiPS cells

[0224] Human induced pluripotent stem cells were seeded into mTeSR1 medium and placed in an incubator with the temperature controlled at 37°C and the CO2 concentration at 5%. The medium was changed daily. The growth of the human induced pluripotent stem cells was observed as follows: Figure 8 As shown. When the cell density of human induced pluripotent stem cells reaches 70%~80%, they are digested with Tryple E for 5 minutes, and then digestion is terminated to obtain digested hiPS cells.

[0225] Step 3: Embryomorph formation and mesodermal differentiation culture

[0226] The digested hiPS cells from step two were resuspended in mT1, E8, and culture medium A, respectively, at a cell density of 8 × 10⁶ cells / year. 3 Cells / well were seeded into 96-well ultra-low adsorption cell culture plates with a U-shaped bottom. Aggregates were observed on day 1 or 2 of culture in these plates, indicating completion of embryoid / mesoderm differentiation. The culture medium was then changed according to the stage in Table 16.

[0227] Table 16: Heart Organoid Construction Process

[0228]

[0229] Step 4: Mesodermal / Cardiodermal Differentiation Culture

[0230] After completing the embryoid / mesoderm differentiation culture, remove mT1, E8 or culture medium A from the cell culture plate, add culture medium A or culture medium B, and continue culturing for 36h or 96h. During this period, change culture medium B once a day to complete the mesodermal / cardiac mesodermal differentiation culture.

[0231] Step 5: Differentiation and Culture of Cardiac Mesodermal / Cardiomyocytes

[0232] After completing the differentiation culture of mesoderm / cardiac mesoderm, remove culture medium A or culture medium B from the cell culture plate, add culture medium B or culture medium C, and continue culturing for 96h or 48h. During this period, change culture medium B or culture medium C once a day to complete the differentiation culture of cardiac mesoderm / cardiac cells.

[0233] Step Six: Cardiomyocyte Differentiation / Heart Organoid Maturation Culture

[0234] After completing the differentiation culture of cardiac mesodermal / cardiomyocytes, aspirate culture medium B or C from the cell culture plate and add culture medium C or D, continuing culture for 48h or 96h, changing culture medium C or D daily during this period. Starting from the time culture medium C is changed to culture medium D, culture for 96h yields cardiac organoids, at which point pulsation of the aggregates is observed. A comparison of cardiac organoid culture stages is shown below. Figure 9 As shown, it can be observed that the heart organoids induced to differentiate after cell resuspension in culture medium A to form embryoid bodies have a better morphology than those formed by cell resuspension in mT1 and E8 cultures.

[0235] The number of cardiac organoids exhibiting myocardial rhythm phenomena in this batch (totaling 30) was counted. The success rate was calculated as: number of cardiac organoids beating / total number of cardiac organoids, as shown in Table 17. By analyzing the beating success rate, it was found that cardiac organoids induced to differentiate using culture medium A (cells resuspended in medium A to form embryoid bodies) were functionally superior to those induced by mT1 and E8 culture (if cardiac organoids did not exhibit beating by Day 12.5, they were considered incapable of beating).

[0236] Table 17: Comparison of the number of heart organoid beats

[0237]

[0238] Comparative Example 2

[0239] Comparing the effects of using the cardiac organoid formulation of this invention or the existing cardiac organoid formulation (Cardioids reveal self-organizing principles of human cardiogenesis), this invention yields cardiac organoids with a higher beating rate.

[0240] See Table III for the sources of materials used (brand and item number).

[0241] Table III

[0242]

[0243] The present invention provides a reference embodiment 2 for the construction of cardiac organoids. The steps for constructing cardiac organoids in the prior art are as follows.

[0244] Step 1: Prepare CDM culture medium

[0245] Insulin, Transferrin, MTG, and BSA were added to IMDM and F12 media and mixed thoroughly to obtain CDM media, which were then set aside. The concentrations of each component in the media are shown in Table 18.

[0246] Table 18: CDM Culture Medium Formulation

[0247]

[0248] Step 2: Prepare culture medium A

[0249] FGF2, LY294002, Activin A, BMP4, CHIR99021, and Y-27632 were added to CDM medium and mixed thoroughly to obtain medium A, which was then set aside. The concentrations of each component in medium A are shown in Table 19.

[0250] Table 19: Culture Medium A Formulation

[0251]

[0252] Step 3: Prepare culture medium B

[0253] IWP2, FGF2, VEGF-A, BMP4, Insulin, and RA were added to CDM medium and mixed thoroughly to obtain medium B, which was then set aside. The concentrations of each component in medium B are shown in Table 20.

[0254] Table 20: Culture Medium B Formulation

[0255]

[0256] Step 4: Prepare culture medium C

[0257] FGF2, VEGF-A, BMP4, and Insulin were added to CDM medium and mixed thoroughly to obtain medium C, which was then set aside. The concentrations of each component in medium C are shown in Table 21.

[0258] Table 21: Culture Medium C Formulation

[0259]

[0260] Step 5: Prepare culture medium D

[0261] VEGF-A and Insulin were added to CDM medium and mixed thoroughly to obtain medium D, which was then set aside. The concentrations of each component in medium D are shown in Table 22.

[0262] Table 22: Culture Medium D Formulation

[0263]

[0264] Step Six: Culture hiPS cells

[0265] Human induced pluripotent stem cells were seeded into mTeSR1 medium and placed in an incubator with the temperature controlled at 37°C and the CO2 concentration at 5%. The medium was changed daily. The growth of the human induced pluripotent stem cells was observed as follows: Figure 10 As shown. When the cell density of human induced pluripotent stem cells reaches 70%~80%, they are digested with Tryple E for 5 minutes, and then digestion is terminated to obtain digested hiPS cells.

[0266] Step 7: Formation of the embryoid body

[0267] The digested hiPS cells from step six were resuspended in E8 medium and cultured at a cell density of 8 × 10⁶ cells / year. 3 Cells / well were seeded into 96-well ultra-low adsorption cell culture plates with a U-shaped bottom. On day 1 of culture in these plates, aggregates were observed, indicating the completion of embryoid formation. The culture medium was then changed according to the stage in Table 23.

[0268] Table 23: Heart Organoid Construction Process

[0269]

[0270] Step 8: Mesodermal Differentiation Culture

[0271] After the embryoid body formation was completed, the E8 medium in the cell culture plate was aspirated, medium A was added, and the culture was continued for 36 hours to complete the mesodermal differentiation culture.

[0272] Step Nine: Cardiac Mesodermal Differentiation Culture

[0273] After the mesodermal differentiation culture was completed, culture medium A was aspirated from the cell culture plate, culture medium B was added, and the culture was continued for 96 hours. During this period, culture medium B was changed once a day to complete the cardiac mesodermal differentiation culture.

[0274] Step 10: Cardiac cell differentiation culture

[0275] After completing the cardiac mesodermal differentiation culture, the culture medium B in the cell culture plate was aspirated, culture medium C was added, and the cells were cultured for another 48 hours. During this period, culture medium C was changed once a day to complete the cardiomyocyte differentiation culture.

[0276] Step 11: Maturation Culture of Heart Organoids

[0277] After completing the differentiation culture of cardiomyocytes, culture medium C was aspirated from the cell culture plate, culture medium D was added, and the cells were cultured for another 96 hours to obtain cardiac organoids. At this point, the aggregates were observed to pulsate. A comparison of the different stages of cardiac organoid culture is shown below. Figure 11 As shown, it can be observed that the heart organoids induced by the heart organoid formulation of the present invention have a better morphology than the heart organoid formulations in the literature.

[0278] The number of cardiac organoids exhibiting myocardial rhythm phenomena in this batch (totaling 30) was counted. The success rate was calculated as: number of cardiac organoids beating / total number of cardiac organoids, as shown in Table 24. By analyzing the beating success rate, it was found that cardiac organoids induced by the cardiac organoid formulation of this invention are functionally superior to those induced by the cardiac organoid formulation in the literature (if cardiac organoids do not exhibit beating by Day 12.5, they are considered to be unable to beat).

[0279] Table 24: Comparison of the number of heart organoid beats

[0280]

[0281] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A culture reagent, characterized in that, Including basal culture medium; The basal culture medium includes: Advanced DMEM / F12 medium, BSA, ITS, lipid concentrate, thioglycerol, and L-ascorbic acid 2-phosphate.

2. The culture reagent as described in claim 1, characterized in that, It also includes at least one of culture medium A, culture medium B, culture medium C, and culture medium D; The culture medium A includes: the basal culture medium, FGF2, LY294002, activator A, BMP4, CHIR-99021, and Y-27632; The culture medium B includes: the basal culture medium, IWP-2, FGF2, vascular endothelial growth factor A, BMP4, insulin, and retinoic acid; The culture medium C includes: the basal culture medium, FGF2, vascular endothelial growth factor A, BMP4, and insulin; The culture medium D includes: the basal culture medium, vascular endothelial growth factor A, and insulin.

3. The culture reagent as described in claim 2, characterized in that, include: The concentration of the Advanced DMEM / F12 medium is 1×; and / or The BSA content in the basal culture medium is 4-6 mg / mL; and / or The ITS content in the basal culture medium is 0.5×~2×; and / or The lipid concentrate has a concentration of 0.5×~2× in the basal culture medium; and / or The thioglycerol content in the basal culture medium is 400-500 μM; and / or The L-ascorbic acid 2-phosphate in the basal medium is present at a concentration of 25–100 μg / mL; and / or The FGF2 content in culture medium A is 25~35 ng / mL; and / or The concentration of LY294002 in culture medium A is 4-6 μM; and / or The activator A is present in culture medium A at a concentration of 45-55 ng / mL; and / or The BMP4 content in culture medium A, culture medium B, or culture medium C is 5-15 ng / mL; and / or The concentration of CHIR-99021 in culture medium A is 3~5 μM; and / or The concentration of Y-27632 in culture medium A is 2.5~7.5 μM; and / or The concentration of IWP-2 in culture medium B is 4.5–5.5 μM; and / or The FGF2 content in culture medium B or culture medium C is 7-9 ng / mL; and / or The concentration of vascular endothelial growth factor A in culture medium B is 150-250 ng / mL; and / or The insulin concentration in culture medium B, culture medium C, or culture medium D is 8–15 μg / mL; and / or The retinoic acid content in culture medium B is 0.45~0.55 μM; and / or The content of vascular endothelial growth factor A in culture medium C or culture medium D is 50~150 ng / mL.

4. The culture reagent as described in claim 2, characterized in that, include: The concentration of the Advanced DMEM / F12 medium is 1×; and / or The BSA content in the basal culture medium is 5 mg / mL; and / or The ITS content in the basal culture medium is 1×; and / or The lipid concentrate is a CD lipid concentrate, and its concentration in the basal culture medium is 1×; and / or The thioglycerol content in the basal culture medium is 450 μM; and / or The L-ascorbic acid 2-phosphate in the basal medium is present at a concentration of 50 μg / mL; and / or The FGF2 content in culture medium A is 30 ng / mL; and / or The concentration of LY294002 in culture medium A is 5 μM; and / or The activator A is present at a concentration of 50 ng / mL in culture medium A; and / or The BMP4 content in culture medium A, culture medium B, or culture medium C is 10 ng / mL; and / or The concentration of CHIR-99021 in culture medium A is 4 μM; and / or The concentration of Y-27632 in culture medium A is 5 μM; and / or The concentration of IWP-2 in culture medium B is 5 μM; and / or The FGF2 content in culture medium B or culture medium C is 8 ng / mL; and / or The concentration of vascular endothelial growth factor A in culture medium B is 200 ng / mL; and / or The insulin concentration in culture medium B, culture medium C, or culture medium D is 10 μg / mL; and / or The retinoic acid content in the culture medium B is 0.5 μM; and / or The concentration of vascular endothelial growth factor A in culture medium C or culture medium D is 100 ng / mL.

5. The use of the culture reagent as described in any one of claims 1 to 4 in the construction or culture of cardiac organoids.

6. A method for culturing cardiac organoids, characterized in that, Cultured using the culture reagent according to any one of claims 1 to 4.

7. The cultivation method as described in claim 6, characterized in that, include: Mesodermal differentiation medium, cardiac mesodermal differentiation medium, cardiomyocyte differentiation medium and / or cardiac organoid maturation medium are prepared based on the culture reagent described in claim 1. Human induced pluripotent stem cells were cultured using the aforementioned mesodermal differentiation medium, cardiac mesodermal differentiation medium, cardiomyocyte differentiation medium, and / or cardiac organoid maturation medium to obtain cardiac organoids.

8. The cultivation method as described in claim 7, characterized in that, include: S1. Prepare mesodermal differentiation medium, cardiac mesodermal differentiation medium, cardiomyocyte differentiation medium, and cardiac organoid maturation medium using the culture reagent described in claim 1; S2. Culture hiPS cells in a U-shaped ultra-low adsorption cell culture plate using the mesodermal differentiation medium until aggregates appear; S3. Replace the culture medium with the cardiac mesodermal differentiation medium, and culture until day 6 to complete the cardiac mesodermal differentiation culture; S4. Replace the culture medium with the cardiomyocyte differentiation culture medium, and culture until day 8 to complete the cardiomyocyte differentiation culture; S5. Replace the culture medium with the heart organoid maturation culture medium, and obtain the heart organoid on day 12.

9. The cultivation method as described in claim 8, characterized in that, include: The mesodermal differentiation medium is the culture medium A in the culture reagent of claim 2; and / or The cardiac mesodermal differentiation culture medium is culture medium B in the culture reagent of claim 2; and / or The cardiomyocyte differentiation culture medium is culture medium C in the culture reagent of claim 2; and / or The heart organoid maturation culture medium is the culture medium D in the culture reagent of claim 2.

10. The cultivation method as described in claim 6, characterized in that, include: S1. Culture hiPS cells in a U-shaped cell culture plate with culture medium A until aggregates appear; S2. Replace the culture medium with culture medium B, and culture until day 6 to complete the differentiation culture of the cardiac mesoderm; S3. Replace the culture medium with culture medium C, and culture until day 8 to complete the differentiation culture of cardiomyocytes; S4. Replace the culture medium with culture medium D, and on day 12, obtain heart organoids; The culture medium A, culture medium B, culture medium C, and culture medium D are, in order, the culture medium A, culture medium B, culture medium C, and culture medium D in the culture reagent of claim 2.

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