Method for in-vitro amplification and passage of myocardial cells

Abstract

The invention relates to the technical field of biology, in particular to a myocardial cell in-vitro amplification and passage method which comprises the following steps: S1, culturing human induced pluripotent stem cells until the cell confluence degree reaches 70%-80%; s2, differentiating the human induced pluripotent stem cells into myocardial cells; s3, observing the number of pulsated myocardial cells, selecting holes with the pulsation percentage of 50%-90%, digesting and re-suspending the myocardial cells in the holes, counting the cells, laying the cells into a six-hole plate according to 1 / 20-1 / 10 of the total cell number, and culturing for 24 hours by using an RPMI / B-27 insulin-free culture medium containing Y-27632; wherein the concentration of the Y-27632 is 2 to 10 [mu] M; s4, replacing the culture medium with an RPMI / B-27 insulin-free culture medium containing Chir99021, replacing the culture medium every day, culturing for 3-5 days until the cell density is 70%-80%, and arranging next passage and amplification; s5, the steps S3 and S4 are repeated according to needs for amplification and passage, the cardiac muscle cells with the increased number are obtained, and the problem that large-scale preparation is limited during in-vitro amplification of the human pluripotent stem cell-derived cardiac muscle cells is solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for in vitro expansion and passage of cardiomyocytes. Background Technology

[0002] Cardiac cardiomyocytes in adult mammals are considered terminally differentiated cells, with extremely limited self-renewal and regeneration capabilities. Once damaged, myocardial tissue often repairs itself through fibrotic scarring rather than regenerating functional myocardium. This irreversible damage severely impairs the heart's pumping function and may eventually lead to heart failure, placing a heavy burden on patients, families, and society.

[0003] Therefore, repairing or regenerating damaged myocardial tissue is one of the most pressing issues for cardiovascular researchers and regenerative medicine. The core challenge in achieving this goal lies in obtaining a large quantity of functional and safe cardiomyocytes. While traditional organ transplantation offers hope to patients with end-stage heart failure, it consistently faces insurmountable bottlenecks such as severe donor shortages, immune rejection, and ethical constraints. This has driven the scientific community to focus on in vitro expansion technology for cardiomyocytes—that is, using stem cell biology, tissue engineering, and regenerative medicine to prepare mature, functional cardiomyocytes on a large scale in a laboratory environment.

[0004] Therefore, the importance and necessity of achieving efficient and large-scale in vitro expansion of cardiomyocytes are self-evident. First, whether it's cardiomyocyte patch transplantation based on cell patches or direct intracardiac injection of cell suspensions, hundreds of millions of functional cardiomyocytes are needed as "seeds." Without a stable and sufficient source of cells, any cell replacement therapy will be a pipe dream. Second, cardiomyocytes derived from patient somatic cells (such as induced pluripotent stem cells, iPSCs) can construct highly personalized disease models for precisely analyzing pathological mechanisms, screening and evaluating the efficacy and cardiotoxicity of novel drugs, greatly accelerating the drug development process and promoting the development of personalized medicine. However, the in vitro expansion of cardiomyocytes still faces many severe challenges, such as how to maintain cell purity, how to induce them to reach a highly mature phenotype comparable to adult cardiomyocytes, and how to ensure their survival rate and electrophysiological integration capacity after transplantation. Solving these problems highly depends on our in-depth understanding and technological innovation in cardiac developmental biology, electrophysiology, and biomaterials.

[0005] To this end, many studies have been dedicated to screening molecules that promote the proliferation of differentiated cardiomyocytes derived from PSCs. However, when these molecules are applied to induce the in vitro expansion of cardiomyocytes derived from human pluripotent stem cells (hiPSCs), they can only produce slight proliferation capacity, and the degree of proliferation is limited, which in turn limits the application of cardiomyocytes in cell therapy. Summary of the Invention

[0006] To address the aforementioned shortcomings, the present invention aims to propose a method for in vitro expansion and passage of cardiomyocytes, thereby solving the problem of limited large-scale production when expanding cardiomyocytes derived from human pluripotent stem cells in vitro.

[0007] To achieve this objective, the present invention adopts the following technical solution: A method for in vitro expansion and passage of cardiomyocytes includes the following steps: S1. Human induced pluripotent stem cells are seeded into 12-well plates at a density of 0.5-1.5 million cells / well and cultured until the cell confluence reaches 70%-80%. S2. Human induced pluripotent stem cells with a cell confluence of 70%-80% are induced and cultured to differentiate into cardiomyocytes; S3. Observe the number of beating cardiomyocytes. Select wells with a beating percentage of 50%-90%, digest and resuspend the cardiomyocytes in the wells, and count the cells. Seed the cells into six-well plates at 1 / 20-1 / 10 of the total number of cells and culture them in RPMI / B-27 insulin-free medium containing Y-27632 for 24 hours. The concentration of Y-27632 was 2-10 μM. S4. Replace with RPMI / B-27 insulin-free medium containing Chir99021. Change the medium daily and culture for 3-5 days until the cell density reaches 70%-80%. Then, arrange for the next passage and expansion. S5. Repeat steps S3 and S4 as needed for amplification and passage to obtain an increased number of cardiomyocytes.

[0008] Preferably, in step S4, the content of Chir99021 is 0.2-6 μM.

[0009] Preferably, in step S3, the operation of digesting and resuspending cardiomyocytes is as follows: Discard the original culture medium, wash the cells with phosphate buffer, incubate the cardiomyocytes with TrypLE Select digestion solution for 13-17 minutes, add RPMI-1640 medium, and mix to obtain a cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge the cell suspension at 200×g for 3 minutes, discard the supernatant, and resuspend the cell suspension in RPMI / B-27 insulin-free medium containing 2-10 μM of Y-27632.

[0010] Preferably, the induction culture operation in step S2 is as follows: (1) On day 0 of differentiation, the culture medium was discarded, myocardial induction culture medium was added for culture, and the time was recorded. The myocardial induction medium is RPMI / B-27 insulin-free medium supplemented with activin A, basic fibroblast growth factor and KnockOut serum substitute; (2) On the second day of differentiation, the myocardial induction medium was discarded and RPMI / B-27 insulin-free medium containing BMP4 and basic fibroblast growth factor was added for culture. (3) On the 5th day of differentiation, the old culture medium was discarded and RPMI / B-27 insulin-free culture medium was added for continued culture; (4) On the 7th day of differentiation, discard the old culture medium and add RPMI / B-27 complete culture medium for culture. Change the RPMI / B-27 complete culture medium every three days until the 10th-11th day of differentiation. If spontaneous cell beating is observed, the differentiation is successful and the required cardiomyocytes are obtained.

[0011] Furthermore, in the myocardial induction culture medium, the concentration of activin A is 50-200 ng / ml, the concentration of basic fibroblast growth factor is 10-20 ng / ml, and the volume percentage of KnockOut serum substitute is 0.5%-2%.

[0012] Preferably, in step (2), the concentration of BMP4 is 5 ng / ml and the concentration of basic fibroblast growth factor is 5 ng / ml.

[0013] Preferably, in step S1, the inoculation operation is as follows: Take human induced pluripotent stem cells with a confluence of 80%-90%, discard the old culture medium, add Accutase digestion solution and incubate for 8 minutes, add mTeSR1 medium, resuspend and collect all cells, count the cells, centrifuge and discard the supernatant, resuspend the cells in mTeSR1 medium containing 5µM Y27632, and seed them in 12-well plates at a density of 0.5-1.5 million cells per well.

[0014] Preferably, in step S1, after inoculation, the culture is carried out using mTeSR1 medium containing 1 µM CHIR99021.

[0015] The technical solution provided by this invention may include the following beneficial effects: This invention provides a method for in vitro expansion and passage of cardiomyocytes. Human induced pluripotent stem cells are directed to differentiate into cardiomyocytes. Wells with a pulsation percentage of 50%-90% are selected, and cells are plated and cultured at 1 / 20-1 / 10 of the total number of cells in the corresponding wells. The cell contact inhibition is relieved, and the mitogen Chir99021 is added to promote cardiomyocyte division and expansion, significantly improving the in vitro expansion capacity of cardiomyocytes and facilitating the preparation of large quantities of cardiomyocytes. Attached Figure Description

[0016] Figure 1 This is a cell morphology diagram of Example 1 of the present invention.

[0017] Figure 2 This is a cell morphology diagram of Example 1 of the present invention.

[0018] Figure 3 This is a cell morphology diagram of Example 1 of the present invention.

[0019] Figure 4 This is an immunofluorescence detection pattern according to an embodiment of the present invention.

[0020] Figure 5 This is a cell morphology diagram of Comparative Example 1 of the present invention.

[0021] Figure 6 This is a quantity detection statistics chart according to an embodiment of the present invention.

[0022] Figure 7 This is a cell morphology diagram completed in Embodiment 1 of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be further illustrated below through specific embodiments.

[0024] To facilitate understanding of the present invention, a more complete description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0025] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] A method for in vitro expansion and passage of cardiomyocytes includes the following steps: S1. Human induced pluripotent stem cells are seeded into 12-well plates at a density of 0.5-1.5 million cells / well and cultured until the cell confluence reaches 70%-80%. S2. Human induced pluripotent stem cells with a cell confluence of 70%-80% are induced and cultured to differentiate into cardiomyocytes; S3. Observe the number of beating cardiomyocytes. Select wells with a beating percentage of 50%-90%, digest and resuspend the cardiomyocytes in the wells, and count the cells. Seed the cells into six-well plates at 1 / 20-1 / 10 of the total number of cells and culture them in RPMI / B-27 insulin-free medium containing Y-27632 for 24 hours. The concentration of Y-27632 was 2-10 μM. S4. Replace with RPMI / B-27 insulin-free medium containing Chir99021. Change the medium daily and culture for 3-5 days until the cell density reaches 70%-80%. Then, arrange for the next passage and expansion. S5. Repeat steps S3 and S4 as needed for amplification and passage to obtain an increased number of cardiomyocytes.

[0027] Human pluripotent stem cells (hiPSCs) possess the capacity for self-renewal and multi-directional differentiation. Theoretically, they can provide an unlimited source of cardiomyocytes. Although highly efficient directed differentiation protocols exist in practice, obtaining the required number of cardiomyocytes for treatment remains an extremely time-consuming and costly process. This is because the natural proliferation rate of cardiomyocytes obtained through in vitro directed differentiation is relatively low. To promote the proliferation of cardiomyocytes in vitro, many studies are currently dedicated to screening molecules that promote the proliferation of PSC-derived differentiated cardiomyocytes. However, when these molecules are applied to induce the in vitro expansion of human pluripotent stem cell (hiPSC)-derived cardiomyocytes, proliferation... The extent of this effect is limited, generally 3-5 times. To address the problems existing in the prior art, this invention proposes a method for in vitro expansion and passage of cardiomyocytes. Through steps S1 and S2, human induced pluripotent stem cells differentiate into cardiomyocytes. In step S3, the number of beating cardiomyocytes is observed, and wells with a beating percentage of 50%-90% are selected. The cardiomyocytes in these wells are digested and resuspended, and then the cells are counted. Cells are seeded into six-well plates at 1 / 20-1 / 10 of the total number of cells per well. This effectively facilitates cell contact and relieves inhibition. Contact inhibition is a factor affecting normal tissue morphogenesis, homeostasis, and... The natural regulatory mechanisms in the regeneration process reveal that the limited in vitro proliferation of many somatic cells is affected by cell-cell contact inhibition. Generally, the higher the cell density, the greater the likelihood of contact inhibition. When human induced pluripotent stem cells undergo targeted cardiac specialization and differentiation in vitro, the initial cell quantity is large, and successfully differentiated cells contract tightly during differentiation and completely merge in the culture dish, triggering contact inhibition and blocking cell cycle progression. Therefore, this invention, by seeding cells into six-well plates at 1 / 20 to 1 / 10 of the total number of cells per well, effectively reduces the probability of triggering contact inhibition and provides sufficient proliferation. The method provides a proliferative space to avoid cell cycle arrest caused by contact inhibition and to prevent apoptosis caused by low cell density, ensuring necessary signal exchange between cells to maintain the physiological phenotype of cardiomyocytes. Furthermore, in step S4, RPMI / B-27 insulin-free medium containing Chir99021 is selected for culturing human induced pluripotent stem cell-derived cardiomyocytes. Chir99021 is a mitogen that can promote cardiomyocyte division and expansion, effectively promoting the initiation of division in cardiomyocytes with significantly reduced mitotic capacity, thereby improving the proliferative capacity of cardiomyocytes. In summary, this invention provides a method for in vitro expansion and passage of cardiomyocytes. Human induced pluripotent stem cells are directed to differentiate into cardiomyocytes. Wells with a pulsation percentage of 50%-90% are selected, and cells are plated at 1 / 20-1 / 10 of the total cell count to relieve cell contact inhibition. The addition of the mitogen Chir99021 promotes cardiomyocyte division and expansion, significantly improving the in vitro expansion capacity of cardiomyocytes and facilitating the preparation of large quantities of cardiomyocytes. Figure 6It is evident that after one passage expansion, the number of cells significantly increases compared to the initial generation, with a proliferation rate of more than five times. This allows for continued passage expansion as needed, addressing the limitation on large-scale production when expanding human pluripotent stem cell-derived cardiomyocytes in vitro.

[0028] In step S3, a microscope is used under bright field to determine whether 50%-90% of the cells exhibit regional pulsation to select the desired culture wells. Cell pulsation is a unique marker of cardiomyocytes derived from human induced pluripotent stem cells, indicating successful differentiation into cardiomyocytes with contractile function. Therefore, the desired culture wells are selected based on regional pulsation, and the proportion of pulsating cells is limited to 50%-90%. Wells with a proportion less than 50% contain a large number of undifferentiated human induced pluripotent stem cells or other miscellaneous cells, resulting in a low proportion of cardiomyocytes. This leads to material waste in subsequent expansion and fails to meet the required number of cardiomyocytes. At the same time, wells with a pulsation percentage greater than 90% are excluded to avoid excessive cell aggregation, which can cause cell proliferation to stop due to contact inhibition, making it difficult to achieve the required efficient expansion.

[0029] In step S3, freshly inoculated cardiomyocytes are cultured for 24 hours in RPMI / B-27 insulin-free medium containing 2-10 μM Y-27632 to ensure cell inoculation quality, reduce cell apoptosis, and ensure the large-scale expansion and preparation of cardiomyocytes.

[0030] In step S1, the density of the human induced pluripotent stem cells is 0.5-1.5 million per well. This avoids the human induced pluripotent stem cells being too far apart, which would prevent them from forming an effective signaling network and thus affecting differentiation efficiency. It also prevents the cells from being too crowded during the culture of human induced pluripotent stem cells, which would trigger contact inhibition, affect the activity of human induced pluripotent stem cells, and interfere with the cell cycle, thus affecting the uniformity of subsequent differentiation.

[0031] In step S2, human induced pluripotent stem cells with a cell confluence of 70%-80% are induced and cultured. A cell confluence of 70%-80% ensures a sufficient number of cells and that the cells are in the logarithmic growth phase, which is conducive to the successful advancement of myocardial differentiation.

[0032] It is worth noting that the RPMI / B-27 insulin-free medium was prepared by adding B-27 insulin-free additive to RPMI-1640 basal medium at a ratio of 1:50.

[0033] Preferably, in step S4, the content of Chir99021 is 0.2-6 μM.

[0034] Specifically, to meet the needs of cardiomyocyte proliferation, the mitogen Chir99021 is added. Within this content range, it can ensure that Chir99021 works with cardiomyocytes after the contact inhibition is relieved to act as a mitogen, meet the needs of stable signal regulation, maintain cardiomyocyte growth, and not affect cardiomyocyte morphology.

[0035] Preferably, in step S3, the operation of digesting and resuspending cardiomyocytes is as follows: Discard the original culture medium, wash the cells with phosphate buffer, incubate the cardiomyocytes with TrypLE Select digestion solution for 13-17 minutes, add RPMI-1640 medium, and mix to obtain a cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge the cell suspension at 200×g for 3 minutes, discard the supernatant, and resuspend the cell suspension in RPMI / B-27 insulin-free medium containing 2-10 μM of Y-27632.

[0036] Specifically, human induced pluripotent stem cell-derived cardiomyocytes were digested and resuspended to obtain a homogeneous cell suspension, facilitating subsequent cell plating. Impurities remaining after the original culture medium was washed away with phosphate-buffered saline (PFS) to maintain stable osmotic pressure in the cardiomyocytes. Then, TrypLE Select digestion solution was added to specifically break down cell adhesion proteins, allowing adherent cells to detach from the matrix and disperse. Preheated RPMI-1640 medium was added for mixing, stopping the digestion with TrypLE Select and obtaining a cell suspension. After centrifugation to remove TrypLE Select digestion solution, RPMI-1640 medium, and cell debris, the cell suspension was resuspended in preheated RPMI / B-27 insulin-free medium for subsequent cell counting. Preheated RPMI / B-27 insulin-free medium is suitable for the nutritional requirements of subsequent cardiomyocyte expansion, and its 2-10 μM Y-27632 content can inhibit cardiomyocyte apoptosis and maintain cell viability, facilitating subsequent plating culture of human induced pluripotent stem cells.

[0037] The incubation temperature using TrypLE Select digestion solution was 37°C, which is the optimal reaction temperature for the enzyme. This temperature is conducive to the occurrence of enzymatic digestion to disperse cardiomyocytes. The RPMI-1640 medium and RPMI / B-27 insulin-free medium used subsequently need to be preheated to avoid low temperature stimulation of cell stress response.

[0038] Preferably, the induction culture operation in step S2 is as follows: (1) On day 0 of differentiation, the culture medium was discarded, myocardial induction culture medium was added for culture, and the time was recorded. The myocardial induction medium is RPMI / B-27 insulin-free medium supplemented with activin A, basic fibroblast growth factor and KnockOut serum substitute; (2) On the second day of differentiation, the myocardial induction medium was discarded and RPMI / B-27 insulin-free medium containing BMP4 and basic fibroblast growth factor was added for culture. (3) On the 5th day of differentiation, the old culture medium was discarded and RPMI / B-27 insulin-free culture medium was added for continued culture; (4) On the 7th day of differentiation, discard the old culture medium and add RPMI / B-27 complete culture medium for culture. Change the RPMI / B-27 complete culture medium every three days until the 10th-11th day of differentiation. If spontaneous cell beating is observed, the differentiation is successful and the required cardiomyocytes are obtained.

[0039] Specifically, it is necessary to differentiate human induced pluripotent stem cells into cardiomyocytes. This requires differentiation from human induced pluripotent stem cells, progenitor cells, and cardiomyocyte precursor cells into cardiomyocytes. On the 10th-11th day of differentiation, it is necessary to observe whether the cells can contract and beat spontaneously to determine whether the differentiation is successful. This corresponds to 50%-90% of the cells exhibiting regional beating, which meets the requirements for subsequent passage expansion.

[0040] To obtain a large number of cardiomyocytes, it is necessary to ensure the high efficiency and purity of cardiomyocyte induction differentiation in step S2. Therefore, the induction differentiation process based on step S2 can efficiently obtain cardiomyocytes with high purity, which facilitates subsequent passage expansion and thus meets the needs of obtaining a large number of cardiomyocytes to meet the application requirements.

[0041] On day 0 of differentiation, the cells were cultured in myocardial induction medium to induce the directed differentiation of human induced pluripotent stem cells into cardiomyocytes. This myocardial induction medium was RPMI / B-27 insulin-free medium supplemented with activin A, basic fibroblast growth factor (FGF), and KnockOut serum substitute. Activin A and basic fibroblast growth factor activate the TGF-β and FGF signaling pathways, promoting the differentiation of human pluripotent stem cells into protozoan cells. On day 2 of differentiation, the medium was replaced with RPMI / B-27 insulin-free medium containing BMP4 and basic fibroblast growth factor. Basic fibroblast growth factor activates the BMP and FGF signaling pathways, promoting the transformation of the proto-cells induced in the previous step into cardiomyocyte precursor cells. On day 5 of differentiation, the medium is replaced with RPMI / B-27 insulin-free medium without the addition of exogenous growth factors to promote the autonomous differentiation of cardiomyocyte precursor cells. On day 7 of differentiation, the medium is replaced with RPMI / B-27 complete medium to activate the PI3K / Akt signaling pathway, maintain a stable culture environment, promote the functional maturation of cardiomyocytes, and develop spontaneous beating ability, ultimately obtaining functional cardiomyocytes and realizing the directed differentiation of human induced pluripotent stem cells into cardiomyocytes.

[0042] It is worth noting that the RPMI / B-27 complete medium was prepared by adding B-27 additive to RPMI-1640 basal medium at a ratio of 1:50.

[0043] Furthermore, in the myocardial induction culture medium, the concentration of activin A is 50-200 ng / ml, the concentration of basic fibroblast growth factor is 10-20 ng / ml, and the volume percentage of KnockOut serum substitute is 0.5%-2%.

[0044] Specifically, activin A is the core factor driving the differentiation of human induced pluripotent stem cells into mesoderm. The concentration of activin A is limited to 50-200 ng / ml to ensure that human induced pluripotent stem cells can differentiate into protosthenes while avoiding deviation from the differentiation lineage, which is beneficial for subsequent differentiation into cardiomyocytes. Basic fibroblast growth factor can synergistically promote differentiation with activin A and maintain high cell viability. The volume percentage of KnockOut serum substitute is 0.5%-2%, which meets the nutritional needs of cell differentiation while ensuring that it does not interfere with the targeting effects of other factors, thus ensuring the stability of human induced pluripotent stem cells differentiating into protosthenes.

[0045] Preferably, in step (2), the concentration of BMP4 is 5 ng / ml and the concentration of basic fibroblast growth factor is 5 ng / ml.

[0046] Specifically, BMP4 can specifically induce protosthenes to differentiate into cardiomyocyte precursor cells, limit the concentration of BMP4 and basic fibroblast growth factor, and the two work synergistically to promote the transformation of protosthenes into cardiomyocyte precursor cells, thereby improving differentiation efficiency and population uniformity.

[0047] Preferably, in step S1, the inoculation operation is as follows: Take human induced pluripotent stem cells with a confluence of 80%-90%, discard the old culture medium, add Accutase digestion solution and incubate for 8 minutes, add mTeSR1 medium, resuspend and collect all cells, count the cells, centrifuge and discard the supernatant, resuspend the cells in mTeSR1 medium containing 5µM Y27632, and seed them in 12-well plates at a density of 0.5-1.5 million cells per well.

[0048] Specifically, human induced pluripotent stem cells with a fusion rate of 80%-90% are in the optimal state for maintaining stemness and proliferative activity. Adding Accutase digestion solution and allowing static culture for 8 minutes ensures that the human induced pluripotent stem cells can completely detach from the cell wall. Adding mTeSR1 medium terminates the enzymatic activity of Accutase digestion solution. Resuspending the cells in mTeSR1 medium containing 5µM Y27632 ensures the quality of stem cell seeding and reduces cell apoptosis.

[0049] The centrifugation conditions were as follows: centrifugation at 200g for 4 minutes at room temperature.

[0050] Preferably, in step S1, after inoculation, the culture is carried out using mTeSR1 medium containing 1 µM CHIR99021.

[0051] Specifically, mTeSR1 medium is a serum-free stem cell maintenance medium specifically for human induced pluripotent stem cells, which can provide standardized nutritional support and stem cell maintenance signals. The addition of 1µM CHIR99021 can target and activate the Wnt pathway, avoiding differentiation deviation and loss of cell stemness.

[0052] The technical solution of the present invention will be further illustrated below through specific embodiments.

[0053] Operating procedures: S1. Take human induced pluripotent stem cells cultured in mTeSR1 medium with a confluence of 80%-90%, discard the old medium, add 1 ml of room temperature Accutase digestion solution to each well, place the culture plate in a 37℃, 5% CO2 incubator and incubate for 8 minutes, add 0.5 ml of mTeSR1 medium to each well, resuspend and collect all cells, perform cell technology, centrifuge at 200g for 4 minutes at room temperature, discard the supernatant, resuspend the cells in mTeSR1 medium containing 5 µM MY 27632, and seed them in a 12-well plate at a density of 0.5-1.5 million cells / well. Replace each well with 2 ml of mTeSR1 medium containing 1 µM CHIR99021 and culture until the cell confluence reaches 80%. S2. Human induced pluripotent stem cells with a cell confluence of 80% were induced and cultured to differentiate into cardiomyocytes. (1) On day 0 of differentiation, the culture medium was discarded, and 1 ml of myocardial induction culture medium was added to each well. The wells were incubated at 37°C in a 5% CO2 incubator, and the time was recorded. The myocardial induction medium was RPMI / B-27 insulin-free medium supplemented with 100 ng / ml activin A, 10 ng / ml basic fibroblast growth factor and 1% KnockOut serum substitute; (2) On the second day of differentiation, the myocardial induction medium was aspirated, and 2 ml of RPMI / B-27 insulin-free medium containing 5 ng / ml BMP4 and 5 ng / ml basic fibroblast growth factor was added to each well for culture. (3) On the 5th day of differentiation, the old culture medium was discarded, and 2 ml of RPMI / B-27 insulin-free culture medium was added to each well for continued culture; (4) On the 7th day of differentiation, discard the old culture medium and add 2 ml of RPMI / B-27 complete culture medium to each well for culture. Change the RPMI / B-27 complete culture medium every three days. By the 10th-11th day of differentiation, obvious spontaneous pulsation can be seen, and the required cardiomyocytes can be obtained. S3. Observe the number of beating cardiomyocytes under a microscope, select wells with a beating percentage of 80%, discard the culture medium, wash the cells with phosphate buffer, and then incubate the human induced pluripotent stem cell-derived cardiomyocytes in the wells with TrypLE Select digestion solution at 37°C for 15 minutes. Gently shake to detach the cardiomyocytes, add preheated RPMI-1640 culture medium, and pipette the plate surface until all cells are detached. Gently pipette the resuspension five times. Transfer the cell suspension to centrifuge tubes and centrifuge at 200×g for 3 minutes. Discard the supernatant and resuspend the cell suspension in preheated RPMI / B-27 insulin-free medium containing 5 μM Y-27632, 1 mL of medium per culture flask. Perform cell counting, seeding 1 / 10 of the total cell count into two six-well plates. Add 2 mL of RPMI / B-27 insulin-free medium containing 5 μM Y-27632 to each well. Incubate for 24 hours, then perform cell count analysis. Cell counts are shown below. Figure 6 As shown, this corresponds to D0.

[0054] Example 1 Based on the above operating procedures: S4. For the six-well plates, replace the medium with RPMI / B-27 insulin-free medium containing 3 μM Chir99021. Change the medium daily and culture for 3 days. The cell morphology images on the first, second, and third days of culture correspond to the following values: Figure 1 , Figure 2 and Figure 3 ; On the second day of culture, immunofluorescence assay was performed, such as... Figure 4 As shown; On the third day of culture, cell count was measured, such as... Figure 6 As shown, this corresponds to D3+chir; Once the cell density reaches 80%, the next passaging and expansion can be scheduled. Repeat steps S3 and S4, passage to 5 generations, to obtain cardiomyocytes, such as Figure 7 As shown.

[0055] Comparative Example 1 Based on the above operating procedures: S4. For another six-well plate, use RPMI / B-27 insulin-free medium, changing the medium daily, and culture for 3 days. The cell morphology on the third day is shown in the image below. Figure 5 As shown; On the third day of culture, cell count was measured, such as... Figure 6 As shown, this corresponds to D3 no chir; Once the cell density reaches 80%, the next passaging and expansion can be scheduled. Repeat steps S3 and S4, passage to the 5th generation, to obtain cardiomyocytes.

[0056] from Figure 1 , Figure 2 and Figure 3 As can be seen, the growth of cardiomyocytes is clearly visible, proving that the method in Example 1 can achieve cardiomyocyte proliferation. Figure 4As can be seen from the immunofluorescence assay of the passaged and amplified cardiomyocytes, DAPI binds to DNA, showing the specific location of the cells. Cardiac troponin T (cTnT) is a cardiomyocyte-specific marker and a protein unique to cardiomyocytes. It can be seen that most of the cells are cardiomyocytes and are in a healthy growth state.

[0057] contrast Figure 5 and Figure 3 It is evident that, under the same culture time, the cell morphology of Comparative Example 1 could not be maintained normally, and from... Figure 6 It is evident that although Comparative Example 1 relieved contact inhibition, it did not add mitogens, and the number of cardiomyocytes obtained was not as high as in Example 1.

[0058] The technical principles of the present invention have been described above with reference to specific embodiments. These descriptions are merely for explaining the principles of the invention and should not be construed as limiting the scope of protection of the invention in any way. Based on this explanation, those skilled in the art can readily conceive of other specific embodiments of the invention without inventive effort, and these embodiments will all fall within the scope of protection of the present invention.

Claims

1. A method for in vitro expansion and passage of cardiomyocytes, characterized in that, Includes the following steps: S1. Human induced pluripotent stem cells are seeded into 12-well plates at a density of 0.5-1.5 million cells / well and cultured until the cell confluence reaches 70%-80%. S2. Human induced pluripotent stem cells with a cell confluence of 70%-80% are induced and cultured to differentiate into cardiomyocytes; S3. Observe the number of beating cardiomyocytes. Select wells with a beating percentage of 50%-90%, digest and resuspend the cardiomyocytes in the wells, and count the cells. Seed the cells into six-well plates at 1 / 20-1 / 10 of the total number of cells and culture them in RPMI / B-27 insulin-free medium containing Y-27632 for 24 hours. The concentration of Y-27632 was 2-10 μM. S4. Replace with RPMI / B-27 insulin-free medium containing Chir99021. Change the medium daily and culture for 3-5 days until the cell density reaches 70%-80%. Then, arrange for the next passage and expansion. S5. Repeat steps S3 and S4 as needed for amplification and passage to obtain an increased number of cardiomyocytes.

2. The method for in vitro expansion and passage of cardiomyocytes according to claim 1, characterized in that: In step S4, the content of Chir99021 is 0.2-6 μM.

3. The method for in vitro expansion and passage of cardiomyocytes according to claim 1, characterized in that, In step S3, the procedure for digesting and resuspending cardiomyocytes is as follows: Discard the original culture medium, wash the cells with phosphate buffer, incubate the cardiomyocytes with TrypLE Select digestion solution for 13-17 minutes, add RPMI-1640 medium, and mix to obtain a cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge the cell suspension at 200×g for 3 minutes, discard the supernatant, and resuspend the cell suspension in RPMI / B-27 insulin-free medium containing 2-10 μM of Y-27632.

4. The method for in vitro expansion and passage of cardiomyocytes according to claim 1, characterized in that, The induction culture procedure in step S2 is as follows: (1) On day 0 of differentiation, the culture medium was discarded, myocardial induction culture medium was added for culture, and the time was recorded. The myocardial induction medium is RPMI / B-27 insulin-free medium supplemented with activin A, basic fibroblast growth factor and KnockOut serum substitute; (2) On the second day of differentiation, the myocardial induction medium was discarded and RPMI / B-27 insulin-free medium containing BMP4 and basic fibroblast growth factor was added for culture. (3) On the 5th day of differentiation, the old culture medium was discarded and RPMI / B-27 insulin-free culture medium was added for continued culture; (4) On the 7th day of differentiation, discard the old culture medium and add RPMI / B-27 complete culture medium for culture. Change the RPMI / B-27 complete culture medium every three days until the 10th-11th day of differentiation. If spontaneous cell beating is observed, the differentiation is successful and the required cardiomyocytes are obtained.

5. The method for in vitro expansion and passage of cardiomyocytes according to claim 4, characterized in that: The myocardial induction medium contains 50-200 ng / ml of activin A, 10-20 ng / ml of basic fibroblast growth factor, and 0.5%-2% KnockOut serum substitute by volume.

6. The method for in vitro expansion and passage of cardiomyocytes according to claim 4, characterized in that: In step (2), the concentration of BMP4 is 5 ng / ml and the concentration of basic fibroblast growth factor is 5 ng / ml.

7. The method for in vitro expansion and passage of cardiomyocytes according to claim 1, characterized in that: In step S1, the inoculation procedure is as follows: Take human induced pluripotent stem cells with a confluence of 80%-90%, discard the old culture medium, add Accutase digestion solution and incubate for 8 minutes, add mTeSR1 medium, resuspend and collect all cells, count the cells, centrifuge and discard the supernatant, resuspend the cells in mTeSR1 medium containing 5µM Y27632, and seed them in 12-well plates at a density of 0.5-1.5 million cells per well.

8. The method for in vitro expansion and passage of cardiomyocytes according to claim 1, characterized in that: In step S1, after inoculation, the culture is carried out using mTeSR1 medium containing 1 µM CHIR99021.

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