An induced medium and method for quickly obtaining spontaneously beating myocardial spheres

Abstract

The application relates to the technical field of cell differentiation and the technical field of myocardial tissue regeneration medicine, in particular to an induced culture medium and method for quickly obtaining a spontaneously beating myocardial ball, wherein the myocardial cells of a human induced pluripotent stem cell (hiPSC) source obtained through co-culture with hMSCs are combined with a differentiation culture medium, so that the myocardial cell clones harvested on day 7 are more independent and naturally separated from other non-myocardial cells, the myocardial clones can be quickly screened and purified through physical and non-enzyme reagents, the step of medium screening and purification in the traditional method is omitted, and time and differentiation cost are saved.

Description

Technical Field

[0001] This invention relates to the fields of cell differentiation technology and myocardial tissue regeneration medicine technology, and particularly to an induction culture medium and method for rapidly obtaining spontaneously beating myocardial bulbs. This invention also relates to a method for inducing differentiation into myocardial tissue by co-culturing with mesenchymal stem cells in a three-dimensional matrix gel environment in vitro. Background Technology

[0002] Human pluripotent stem cells (hPSCs) include human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), which have the ability to differentiate into any somatic cell type. To date, hPSCs have demonstrated strong functionality in in vitro disease modeling, drug screening, and developmental biology. Furthermore, in regenerative medicine, in vitro-derived induced pluripotent stem cells (iPSCs) such as pancreatic islets and neurons have been used in human clinical trials to treat diseases such as diabetes and epilepsy.

[0003] In mammals, cardiomyocytes essentially exit the cell cycle shortly after birth, switching from proliferation mode to terminal differentiation mode. This means that the human heart muscle is one of the tissues with the worst regenerative capacity in the body. When a myocardial infarction occurs, a large number of damaged cardiomyocytes die, and the heart cannot regenerate new cardiomyocytes to replace them. To address this deficiency of non-regenerative cardiomyocytes, scientists have come up with the idea of ​​transplanting external, functional cardiomyocytes to the damaged area of ​​the heart to directly replace the dead cells when the heart is injured.

[0004] Several problems exist with currently disclosed in vitro cardiomyocyte induction methods. For example, the in vitro induction time is long, and the culture medium composition is complex (e.g., patents with application numbers CN 202411392887 and CN 202311111730). Alternatively, shortening the induction time may result in insufficient cardiomyocyte yield (e.g., patent with application number CN 202510140533). Furthermore, the currently disclosed methods only obtain cardiomyocytes at a relatively early stage of development; compared to real cardiomyocytes in vivo, their maturity and performance are insufficient.

[0005] Therefore, in order to solve the above problems, it is particularly important to provide a simple, easy-to-operate method that can significantly improve the in vitro yield and maturity of hPSC-derived cardiomyocytes. Summary of the Invention

[0006] To address the technical problems mentioned in the background section, this invention provides an induction culture medium and method for rapidly obtaining spontaneously beating myocardial bulbs. The specific technical solution is as follows:

[0007] An induction culture medium for rapidly obtaining spontaneously beating myocardial bulbs includes basal medium, differentiation medium A, differentiation medium B, differentiation medium C, and differentiation medium D;

[0008] in,

[0009] Basic culture medium: DMEM / F12 medium + 50 μg / mL vitamin C + 1X B27-insulin + 100X trace element B;

[0010] Differentiation medium A: basal medium + 6 μM Chir99021;

[0011] Differentiation medium B: basal medium + 2.5 μM IWR1 + 2.5 μM IWP2 + 4 mM hydrochloric acid;

[0012] Differentiation medium C: basal medium + 4 mM hydrochloric acid;

[0013] Differentiation medium D: DMEM / F12 medium + 50 μg / mL vitamin C + 1X B27 + 100X trace element B.

[0014] Preferably, the induction culture medium for rapidly obtaining spontaneously beating myocardial bulbs further includes:

[0015] Cardiac cell maintenance medium: RPMI 1640 medium + 1X B27 + 50μg / mL vitamin C + 5ng / mL FGF2.

[0016] A rapid induction culture method for obtaining spontaneously beating myocardial spheres, using an induction culture medium for rapidly obtaining spontaneously beating myocardial spheres.

[0017] Preferably, the method for rapidly obtaining spontaneously beating myocardial bulbs through induction culture includes the following stages:

[0018] The first stage involves co-culturing hiPSCs and hMSCs in 2D culture to obtain spontaneously beating cardiomyocytes.

[0019] Preferably, the starting cell count of hiPSC is 50,000 per square centimeter, and the starting cell count of hMSC is 30,000 per square centimeter.

[0020] Preferably, the first stage includes the following steps:

[0021] 1) MSC plate-coating stage: Prepare stem cell matrix gel-coated cell culture plates in advance, select human umbilical cord isolated hMSCs from passages P3 to P5, seed them in the coated well plates, add MSC amplification medium MH01 and culture overnight.

[0022] 2) hPSC monolayer seeding: Select healthy hiPSCs and detect their pluripotency marker OCT4 / TRA-1-60 positivity rate greater than 90% by flow cytometry; digest them into single cells and prepare single-cell suspension for later use; take out the MSC well plate that has been cultured overnight, discard the original MSC expansion medium MH01 and add pluripotent stem cell expansion medium mTeSR1; seed an appropriate amount of hiPSC single-cell suspension into the wells with hMSCs as the bottom layer cells and add 10 μM Y27632;

[0023] 3) Induction Phase: The day after hPSC inoculation is marked as the start of the induction phase, or day 0 (D0) of differentiation. Discard the old culture medium from the wells containing hMSCs and hiPSCs and add differentiation medium A. On day 1 (D1) of induction differentiation, discard differentiation medium A and replace the basal medium. At the end of induction on D1, hiPSCs can be induced into mesodermal precursor cells. On day 2 (D2) of induction differentiation, add differentiation medium B. This phase continues for two days, with fresh differentiation medium B added daily. On day 4 (D4) of induction differentiation, replace the differentiation medium with C. This phase continues for two days, with fresh differentiation medium C added daily. On day 6 of induction differentiation, replace the differentiation medium with D. This phase continues for two days. Induction ends on day 7, at which point a large area of ​​spontaneously beating cardiomyocytes can be observed.

[0024] Preferably, the second stage is a 3D culture stage, in which the spontaneously beating cardiomyocytes induced in the first stage are digested and separated by a short digestion with an enzyme-free reagent and then selected by a pipette tip to obtain spontaneously beating myocardial bulbs.

[0025] Preferably, the second stage includes the following steps:

[0026] 1) Remove the cardiomyocytes induced to D7, discard the old culture medium, wash with DPBS, add 0.5mM EDTA digestion solution, incubate at room temperature, discard the EDTA digestion solution, add cardiomyocyte maintenance culture medium, at this time, small independent clumps can be seen in the well plate. Use the pipette tip to gently peel these clones along the edge of the clones in the well plate, transfer the peeled cardiomyocyte clones to EP tubes with a pipette, mix by pipetting several times, and then place on ice to stand.

[0027] 2) After standing, the cell suspension shows cell stratification. Larger myocardial clones (200-500 μm in diameter) settle at the bottom, while the upper layer contains other cells (single cells with a diameter of 10 μm and smaller fragments with a diameter of 10-200 μm). The upper layer of cells is aspirated, leaving only the small myocardial clumps at the bottom. Completely melted organoid matrix gel is quickly added to ice, and the mixture is mixed by pipetting. The mixture is then dispensed into well plates, with a mixture of myocardial clumps and organoid matrix gel added to each well. The mixture is added dropwise from top to bottom to form a dome structure. After dispensing, the matrix gel is solidified in the well plates. After solidification, myocardial cell maintenance medium is added to each well. After culturing for 5 days, independent, spontaneously beating 3D myocardial spheres can be obtained.

[0028] Compared with other solutions, the beneficial effects, innovations, and improvements of this invention are as follows:

[0029] 1) This invention creatively induces different ratios of hiPSCs and hMSCs through co-culture, and spontaneously beating small myocardial clones can appear on just day 6. The efficiency is greatly improved on day 7, accounting for more than 80% of the bottom area of ​​the entire well plate. The cardiomyocytes derived from hiPSCs obtained by co-culturing with hMSCs benefit from the paracrine effect of hMSCs, resulting in better maturity, more viable cells remaining at harvest on day 7, and healthier cardiomyocytes.

[0030] 2) The co-culture induction method provided by this invention, combined with the differentiation medium, makes the cardiomyocyte clones harvested from D7 more independent and naturally separated from other non-cardiomyocytes. Cardiomyocyte clones can be quickly screened and purified using physical and enzyme-free reagents. This eliminates the traditional method of medium screening and purification, saving time and differentiation costs.

[0031] 3) The method of obtaining myocardial spheres by reculturing myocardial masses using the matrix gel dome method provided by this invention transforms traditional two-dimensional cardiomyocytes into three-dimensional myocardial spheres with better maturity, higher purity, and higher cell yield. These myocardial spheres have shown great potential in the study of early cardiac development, regeneration and repair of heart diseases, and drug screening.

[0032] This invention, through extensive experiments and comparative studies, yielded an optimized differentiation medium and induction protocol. Firstly, it was discovered that the ratio of hiPSCs to hMSCs at the initial stage of the induction protocol is a crucial factor, determining whether cardiomyocytes can be successfully induced and the time to initial spontaneous beating, as demonstrated in Example 1 and Comparative Examples 1-7. Secondly, regarding the method for isolating and purifying beating cardiomyocyte clones, our enzyme-free reagent + physical dissection protocol (Example 2) is superior to the purely enzyme-free digestion solution protocol (Comparative Example 8) and the purely physical dissection protocol (Comparative Example 9). Attached Figure Description

[0033] The accompanying drawings are provided to further illustrate the present application and form part of the specification. They are used together with the embodiments of the present application to explain the application and do not constitute a limitation thereof. In the drawings:

[0034] Figure 1 This is a three-dimensional myocardial bulb induction flowchart;

[0035] Figure 2 These are morphological images of the induction stage (D0 is a 40x electron microscope image, D2 is a 40x electron microscope image, D4 ​​is a 40x electron microscope image, D6 is a 40x and 100x electron microscope image, and D13 is a 100x electron microscope image).

[0036] Figure 3 This is a statistical chart showing the timeline of the first spontaneously beating clones appearing during the induction phase.

[0037] Figure 4 This is a statistical chart of the number of spontaneously jumping clones in the induction phase;

[0038] Figure 5 These are comparison images of myocardial spheres obtained after different purification methods;

[0039] Figure 6 This indicates the cTnT expression status at the end of the first induction phase. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0041] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for inducing human pluripotent stem cells (including hESC or hiPSC) to differentiate into spontaneously beating cardiomyocytes in vitro.

[0042] To achieve the above objectives, the present invention is divided into two stages: as follows Figures 1-2 As shown.

[0043] Example 1

[0044] The first stage involves rapidly inducing spontaneously beating cardiomyocytes by co-culturing hiPSCs and hMSCs under 2D culture conditions.

[0045] The specific steps are as follows:

[0046] 1. MSC plating stage: Prepare stem cell matrix gel (manufacturer: Suzhou Yikesai, catalog number: RF010-N012) in advance to coat the cell culture plates. The coating amount is 0.01~0.02 mg / cm². 2The coated MSCs were incubated at 37°C for 1 hour. Human umbilical cord isolates from generations P3 to P5 were seeded into coated plates, and MSC amplification medium MH01 (manufacturer: Suzhou Yikesai Biotechnology, catalog number: MH000-N012) was added before incubation overnight at 37°C in a 5% CO2 incubator.

[0047] 2. hPSC monolayer seeding: Select healthy hiPSCs, with a flow cytometry result showing a positive rate of over 90% for the pluripotency marker OCT4 / TRA-1-60. Digest them into single cells and prepare a single-cell suspension for later use. Remove the overnight cultured MSC plate, discard the original MSC expansion medium MH01, and add pluripotent stem cell expansion medium mTeSR1 (manufacturer: Canadian Stem Cell Technology Co., Ltd., catalog number: 85850). Add an appropriate amount (e.g., 1 mL) of hiPSC single-cell suspension (10 5 Cells (number / mL) were seeded in wells with hMSCs as the base cells and 10 μM Y27632 was added.

[0048] 3. Induction Phase: The day after hPSC inoculation is designated as the start of the induction phase, or day 0 (D0) of differentiation. Discard the old culture medium from the wells containing hMSCs and hiPSCs and add differentiation medium A. On day 1 (D1) of induction differentiation, discard differentiation medium A and replace the basal medium. At the end of induction on D1, hiPSCs can be induced into mesodermal precursor cells. On day 2 (D2) of induction differentiation, add differentiation medium B. This phase continues for two days, with fresh differentiation medium B added daily. On day 4 (D4) of induction differentiation, replace the differentiation medium with C. This phase continues for two days, with fresh differentiation medium C added daily. On day 6 of induction differentiation, replace the differentiation medium with D. This phase continues for two days. Induction ends on day 7, at which point a large area of ​​spontaneously beating cardiomyocytes can be observed.

[0049] The training described in the first stage is specifically as follows:

[0050] Basic culture medium: DMEM / F12 medium + 50 μg / mL vitamin C + 1X B27-insulin + 100X trace element B;

[0051] Differentiation medium A: basal medium + 6 μM Chir99021;

[0052] Differentiation medium B: basal medium + 2.5 μM IWR1 + 2.5 μM IWP2 + 4 mM hydrochloric acid;

[0053] Differentiation medium C: basal medium + 4 mM hydrochloric acid;

[0054] Differentiation medium D: DMEM / F12 medium + 50 μg / mL vitamin C + 1X B27 + 100X trace element B.

[0055] Example 2

[0056] The second stage is the 3D culture stage. The spontaneously beating cardiomyocytes induced in the first stage are digested and separated, then coated and cultured with organoid matrix gel (manufacturer: Suzhou Yikesai, catalog number: RF010-N022) in cardiomyocyte maintenance medium.

[0057] Cardiac cell maintenance medium: RPMI 1640 medium + 1X B27 + 50μg / mL vitamin C + 5ng / mL FGF2.

[0058] The specific steps are as follows:

[0059] 1. Prepare organoid matrix gel: Take one tube of organoid matrix gel out of the -80℃ freezer the night before and place it in the 4℃ freezer overnight to melt;

[0060] Remove cardiomyocytes induced to day 7, discard the old culture medium, wash once with DPBS, add 0.5 mM EDTA digestion solution, incubate at room temperature for 1 minute, gently tap the bottom of the cell culture plate, at this time the remaining hMSCs in the well plate can be detached, discard the EDTA digestion solution, add cardiomyocyte maintenance culture medium, at this time small clusters of independent clones can be seen with the naked eye in the well plate. Use a 100 μL pipette tip to gently peel these clones along the edge of the clones in the well plate, transfer the peeled cardiomyocyte clones to a 1.5 mL EP tube with a 2 mL pipette, mix by pipetting 5 times, and place on ice for 5 minutes.

[0061] 2. After standing for five minutes, the cell suspension showed cell stratification. Larger myocardial clones settled at the bottom, while other cells were on top. The upper layer of cells was aspirated, leaving only the small myocardial clumps at the bottom. 500 μL of completely melted organoid matrix gel was quickly added on ice, and the mixture was mixed by pipetting. The mixture was then dispensed into 24-well plates, with 50 μL of the mixture of myocardial clumps and organoid matrix gel added to each well. The mixture was added dropwise from top to bottom to form a dome structure. After dispensing, the plates were placed in a 37°C incubator to cure the matrix gel for 10 minutes. After curing, the plates were removed and placed in a biosafety cabinet. 500 μL of myocardial cell maintenance medium was added to each well. After culturing for 5 days, independent, spontaneously beating 3D myocardial spheres could be obtained.

[0062] Comparative Examples 1-9

[0063] Below are the detailed steps and data display for the comparison scale:

[0064] 1. Cell preparation

[0065] hPSCs are derived from the pluripotent stem cell bank established by Suzhou Ekosei Biotechnology Co., Ltd., and can contain hiPSCs from different somatic cell sources and hESCs from different cell lines. The culture system is pluripotent stem cell expansion medium mTeSR1 + Matrigel medium (manufacturer: Corning Life Sciences, catalog number: 356234), and the passage reagent is EDTA digestion solution. hMSCs are derived from the mesenchymal stem cell bank isolated from human umbilical cord established by Suzhou Ekosei Biotechnology Co., Ltd., and the culture system is MSC expansion medium MH01, and the passage reagent is recombinant trypsin digestion solution.

[0066] 2. Reagent preparation and culture medium preparation

[0067] Table 1 Reagent List

[0068] Reagent Name brand Item number Use concentration DMEM / F12 medium Ikosai M102462 1X B27-insulin Thermo Fisher A1895601 1X B27 Thermo Fisher A3582801 1X Trace element B Corning 25-022-CI 500X Chir99021 Merck SML1046 6μM hydrochloric acid Love Chemistry 7647-01-0 8mM IWR1 Merck I0161 2.5μM IWP2 Merck I0536 2.5μM Vitamin C Merck 1043003 50ug / ml RPMI 1640 medium Thermo Fisher 61870036 1X FGF2 Nearshore proteins C046 5ng / ml EDTA enzyme-free digestion reagent Azure Sky ST066 0.5mM Recombinant pancreatic enzyme digestion solution Ikosai RF000-N031 1X

[0069] 3. Induction phase:

[0070] The first stage of the induction process is as described in the specific steps of the invention above. The induction plate is a 12-well plate, and 1 mL of culture medium is added to each well. Here, only the differences between the examples and comparative examples are listed in detail.

[0071] Table 2. First-stage examples and comparative examples

[0072] Experimental protocol hiPSC starting cell quantity hMSC initial cell quantity Example 1 50,000 per square centimeter 30,000 per square centimeter Comparative Example 1 15,000 per square centimeter 0 Comparative Example 2 15,000 per square centimeter 10,000 per square centimeter Comparative Example 3 15,000 per square centimeter 20,000 per square centimeter Comparative Example 4 15,000 per square centimeter 30,000 / square centimeter Comparative Example 5 50,000 per square centimeter 10,000 per square centimeter Comparative Example 6 50,000 per square centimeter 20,000 per square centimeter Comparative Example 7 50,000 per square centimeter 0

[0073] Table 3. Second-stage examples and comparative examples

[0074] Experimental protocol Purification and isolation protocol for myocardial clones Example 2 Enzyme-free reagent brief digestion + pipette tip selection Comparative Example 8 Recombinant trypsin reagent digests into single cells Comparative Example 9 Pipette tip separation of myocardial clones

[0075] The specific steps for different purification and separation methods in the second stage are as follows:

[0076] Comparative Example 8: Cardiomyocytes induced to day 7 were removed, old culture medium was discarded, and the cells were washed once with DPBS. 500 μL of recombinant trypsin reagent was added, and the cells were incubated at 37°C for 5 minutes. After incubation, 500 μL of cardiomyocyte maintenance medium was added. The cells in the entire well plate were completely pipetted and transferred to a 1.5 mL EP tube. After centrifugation at 250 g for 5 minutes at room temperature, the supernatant was discarded, leaving only the cell pellet at the bottom. 500 μL of completely melted organoid matrix gel was quickly added on ice, and the mixture was pipetted and mixed thoroughly. The mixture was then aliquoted into 24-well plates, with 50 μL of the cell and organoid matrix gel mixture added to each well, dripping from top to bottom to form a dome structure. After aliquoting, the plates were placed in a 37°C incubator to cure the matrix gel for 10 minutes. After curing, 500 μL of cardiomyocyte maintenance medium was added to each well, and the plates were returned to the incubator for static culture.

[0077] Comparative Example 9: Cardiomyocytes induced to day 7 were removed, the old culture medium was discarded, and the cells were washed once with DPBS. 500 μL of cardiomyocyte maintenance medium was added. Using a 10 μL pipette tip, cross lines were drawn at the bottom of the wells to divide the cells into small squares. Then, using a 1 mL pipette tip, the cells were gently pipetted down from the wells and transferred to 1.5 mL EP tubes. After gently mixing five times, the tubes were placed on ice. After standing for five minutes, the upper cell suspension was discarded, leaving only the cell clumps at the bottom. 500 μL of completely melted organoid matrix gel was quickly added on ice. After mixing, the mixture was aliquoted into 24-well plates. 50 μL of the cell clump and organoid matrix gel mixture was added to each well, dripping from top to bottom to form a dome structure. After aliquoting, the plates were placed in a 37°C incubator to cure the matrix gel for 10 minutes. After curing, 500 μL of cardiomyocyte maintenance medium was added to each well, and the plates were returned to the incubator for static culture.

[0078] Experimental conclusions

[0079] 1. In the first stage of induction, the spontaneous jumping time in Example 1 was significantly longer than in other comparative examples;

[0080] pass Figure 3 It can be seen that the induction efficiency and the time to first spontaneous beat of hiPSC and hMSC co-culture in the myocardium are better than those of iPSC alone. The best performance was in Example 1, which was on the 6th day after induction, while the worst performance was in Comparative Example 7, which had the first spontaneous beat on the 13th day after induction, when the first stage of induction was over.

[0081] 2. In the first stage of induction, the number of clones exhibiting spontaneous jumping in Example 1 was significantly higher than in other comparative examples;

[0082] After spontaneously beating cardiac clones appeared in each experimental group, we counted the number of spontaneously beating clones in each well plate under a microscope (statistical method: a cross was drawn on the bottom of each individual well in the plate with a black pen, dividing an individual culture well into four independent regions; clones stuck on the black lines were counted only on one side). The final statistical data is as follows: Figure 4 The results showed that the experimental group with the most spontaneously beating myocardial clones was Example 1, with 735 clones, while the experimental group with the worst performance was Comparative Example 7, with 280 clones. Note: The well plates used in this invention for counting myocardial clones were 12-well plates.

[0083] 3. The three-dimensional myocardial spheres obtained by the purification method in Example 2 have higher purification efficiency and better growth status;

[0084] Traditional myocardial cell screening methods involve subtracting glucose and adding sodium lactate to obtain cardiomyocytes. While this method is highly efficient, it is time-consuming, and the resulting cardiomyocytes tend to be aged after culture medium screening. To address this issue, we creatively improved the purification method. We select beating myocardial clones as soon as they begin to spontaneously beat and transfer them to three-dimensional culture to obtain pure three-dimensional myocardial spheres. The screening method is specifically implemented in Examples 2, 8, and 9. Figure 5 The images show that the well plate obtained by the purification method in Example 2 contains only pure three-dimensional myocardial spheres without any other cells; the myocardial spheres obtained by the purification methods in Comparative Examples 8 and 9 contain a large number of other impurity cells, and the purification efficiency is significantly lower than that in Example 2.

[0085] 4. The myocardial clone obtained in this invention highly expresses troponin (cTnT).

[0086] The success of myocardial clone induction can be verified not only by its spontaneous beating characteristic but also by the expression of cTnT. We used flow cytometry to detect the myocardial clones obtained in the first stage (D6) of Example 1, and the results are as follows: Figure 6 As shown, its cTnT positivity rate was 81.3%.

[0087] Any other improvements and variations that can be directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the scope of protection of the present invention.

Claims

1. An induction culture medium for rapidly obtaining spontaneously beating myocardial bulbs, characterized in that, The medium includes MSC expansion medium MH01, pluripotent stem cell expansion medium mTeSR1, 10 μM Y27632, cardiomyocyte maintenance medium, basal medium, differentiation medium A, differentiation medium B, differentiation medium C, and differentiation medium D. in, Basic culture medium: DMEM / F12 medium + 50 μg / mL vitamin C + 1X B27-insulin + 100X trace element B; Differentiation medium A: basal medium + 6 μM Chir99021; Differentiation medium B: basal medium + 2.5 μM IWR1 + 2.5 μM IWP2 + 4 mM hydrochloric acid; Differentiation medium C: basal medium + 4 mM hydrochloric acid; Differentiation medium D: DMEM / F12 medium + 50 μg / mL vitamin C + 1X B27 + 100X trace element B; Cardiac cell maintenance medium: RPMI 1640 medium + 1X B27 + 50μg / mL vitamin C + 5ng / mL FGF2.

2. A method for rapidly obtaining spontaneously beating myocardial bulbs through induction culture, characterized in that, Induction culture was performed using an induction medium as described in claim 1 for rapidly obtaining spontaneously beating myocardial bulbs.

3. The method for rapidly obtaining spontaneously beating myocardial bulbs through induction culture as described in claim 2, characterized in that, Includes the following stages: The first stage involves co-culturing hiPSCs and hMSCs in 2D culture to obtain spontaneously beating cardiomyocytes.

4. The method for rapidly obtaining spontaneously beating myocardial bulbs through induction culture as described in claim 3, characterized in that, The ratio of the initial cell quantity of hiPSC to the initial cell quantity of hMSC is 5:

3.

5. The method for rapidly obtaining spontaneously beating myocardial bulbs through induction culture as described in claim 4, characterized in that, The initial cell count for hiPSCs is 50,000 per square centimeter, and the initial cell count for hMSCs is 30,000 per square centimeter.

6. The method for rapidly obtaining spontaneously beating myocardial bulbs through induction culture as described in claim 3, characterized in that, The first stage includes the following steps: 1) MSC plate-coating stage: Prepare stem cell matrix gel-coated cell culture plates in advance, select human umbilical cord isolated hMSCs from passages P3 to P5, seed them in the coated well plates, add MSC amplification medium MH01 and culture overnight. 2) hPSC monolayer seeding: Select healthy hiPSCs and detect their pluripotency marker OCT4 / TRA-1-60 positivity rate greater than 90% by flow cytometry; digest them into single cells and prepare single-cell suspension for later use; take out the MSC well plate that has been cultured overnight, discard the original MSC expansion medium MH01 and add pluripotent stem cell expansion medium mTeSR1; seed an appropriate amount of hiPSC single-cell suspension into the wells with hMSCs as the bottom layer cells and add 10 μM Y27632; 3) Induction Phase: The day after hPSC inoculation is marked as the start of the induction phase, or day 0 (D0) of differentiation. Discard the old culture medium from the wells containing hMSCs and hiPSCs and add differentiation medium A. On day 1 (D1) of induction differentiation, discard differentiation medium A and replace the basal medium. At the end of induction on D1, hiPSCs can be induced into mesodermal precursor cells. On day 2 (D2) of induction differentiation, add differentiation medium B. This phase continues for two days, with fresh differentiation medium B added daily. On day 4 (D4) of induction differentiation, replace the differentiation medium with C. This phase continues for two days, with fresh differentiation medium C added daily. On day 6 of induction differentiation, replace the differentiation medium with D. This phase continues for two days. Induction ends on day 7, at which point a large area of ​​spontaneously beating cardiomyocytes can be observed.

7. The method for rapidly obtaining spontaneously beating myocardial bulbs through induction culture as described in claim 2, characterized in that, Includes the following stages: The second stage is the 3D culture stage, in which the spontaneously beating cardiomyocytes induced in the first stage are digested and separated by a short digestion with an enzyme-free reagent and then selected by a pipette tip to obtain spontaneously beating cardiomyocytes.

8. The method for rapidly obtaining spontaneously beating myocardial bulbs through induction culture as described in claim 7, characterized in that, The second stage includes the following steps: 1) Remove the cardiomyocytes induced to D7, discard the old culture medium, wash with DPBS, add 0.5mM EDTA digestion solution, incubate at room temperature, discard the EDTA digestion solution, add cardiomyocyte maintenance culture medium, at this time, small independent clumps can be seen in the well plate. Use the pipette tip to gently peel these clones along the edge of the clones in the well plate, transfer the peeled cardiomyocyte clones to EP tubes with a pipette, mix by pipetting several times, and then place on ice to stand. 2) After standing, the cell suspension shows cell stratification. Larger myocardial clones settle at the bottom, while other cells are on the top. The upper layer of cells is aspirated, leaving only the small myocardial clumps at the bottom. Completely melted organoid matrix gel is quickly added to ice, and the mixture is mixed by pipetting. The mixture is then dispensed into well plates, with a mixture of myocardial clumps and organoid matrix gel added to each well. The mixture is added dropwise from top to bottom to form a dome structure. After dispensing, the matrix gel is solidified in the well plates. After solidification, myocardial cell maintenance medium is added to each well. After culturing for 5 days, independent, spontaneously beating 3D myocardial spheres can be obtained.

Images