Method for aseptically separating myocardial cells from adult mouse heart

Abstract

The invention discloses a method for aseptically separating myocardial cells from the heart of an adult mouse. The method comprises the following steps: (1) transferring the heart into a super clean bench; (2) connecting a flow guide tube at the ascending aorta, and ligating and fixing; (3) injecting an EDTA (Ethylene Diamine Tetraacetic Acid) buffer solution from the flow guide tube, wherein the injection rate is 1.5-2.5 ml / min; (4) injecting a perfusion buffer solution from the flow guide tube at the same injection rate; (5) injecting a digestive enzyme buffer solution from the diversion tube, wherein the injection rate is 1.0-1.5 ml / min; (6) repeating the step (5) until the heart is completely digested; (7) removing aorta and atrium tissues, gently shredding the remaining part, continuing to add the digestive enzyme buffer solution, and gently blowing and beating until the ventricular tissues are dissociated into single cells; (8) adding a digestion stop solution, and gently blowing and beating; and filtering and centrifuging the cell suspension to obtain the myocardial cells. The separation process does not depend on a langendorff perfusion system.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for aseptically separating myocardial cells from the heart of an adult mouse. Background Art

[0002] Cardiovascular disease is one of the most important causes of death worldwide. In order to simulate cardiac pathology or physiological state, conduct related genomics, epigenomics, transgenic and gene therapy research, and provide treatment options for cardiovascular disease, it is necessary to culture cardiomyocytes in vitro.

[0003] However, adult mammalian cardiomyocytes are not only closely connected to adjacent cells and extracellular matrix, but also have intercalated disc structures between adjacent cardiomyocytes, in which the cell membrane is concave and convex, and is specially differentiated to form desmosomes, which are closely connected to each other; this makes it very difficult to separate intact adult mammalian cardiomyocytes. At the same time, adult mammalian cardiomyocytes are susceptible to mechanical damage, enzyme damage, hypoxia, pH and ion fluctuations during the separation process, resulting in separation failure or reduced activity of the isolated cardiomyocytes.

[0004] In order to improve the isolation efficiency of cardiomyocytes, Langendorff O invented the heart perfusion method in 1898 for the isolation of adult mouse cardiomyocytes. The implementation process of the heart perfusion method is roughly as follows: the excised heart is mounted on the Langendorff perfusion system, a cannula is inserted from the ascending aorta, and the digestive enzyme buffer is injected from the cannula, and enters the coronary circulation through the coronary sinus at the root of the aorta to achieve complete digestion of the heart. During the perfusion process, the flow rate, temperature and oxygen content of the digestive enzyme buffer need to be closely monitored. To date, the Langendorff heart perfusion method is still the best method for isolating and studying adult rodent cardiomyocytes.

[0005] However, the Langendorff heart perfusion method has the following shortcomings: (1) The Langendorff perfusion system it relies on is not only expensive, but also requires the operator to have professional operating knowledge, and the cost of use is high; (2) The Langendorff perfusion system it relies on is large in size and cannot be used to separate cardiomyocytes in an ultra-clean bench. The operation process is difficult to meet the sterility requirements, which brings great difficulties to the subsequent cardiomyocyte culture; (3) It is usually required to inject anticoagulants such as heparin into the animals in advance, which may interfere with the subsequent polymerase chain reaction (PCR) reaction.

[0006] To solve the above problems, Roger S.Foo et al. proposed a method for isolating adult mouse cardiomyocytes without the need for Langendorff equipment. The implementation process of this method is roughly as follows: clamp the ascending aorta of the heart, and inject digestive enzyme buffer into the left ventricle from the apex of the heart, so that the digestive enzyme will pass through the aortic valve retrogradely through the coronary circulation, thereby achieving digestion of the heart. Although this method does not require Langendorff perfusion equipment and professional operation technology training, it requires repeated puncture of the left ventricle with a syringe, which results in a larger and larger puncture hole in the left ventricle during the digestion process. The digestive enzyme buffer injected into the left ventricle cannot maintain sufficient pressure to enter the coronary circulation to achieve effective cardiomyocyte dissociation, which ultimately leads to a decrease in the efficiency of cardiomyocyte dissociation. At the same time, the entire digestion (15-30 minutes) and collection of cardiomyocytes (about 30 minutes) process of this method takes about 45-60 minutes, which greatly prolongs the hypoxia time of cardiomyocytes, which may lead to changes in cardiomyocyte function and gene expression, and is not conducive to the use of dissociated cardiomyocytes for subsequent research.

[0007] Therefore, it is urgent to develop a stable and efficient method that does not rely on the Langendorff perfusion system to isolate cardiomyocytes. Summary of the invention

[0008] The purpose of the invention is to provide a method for aseptically isolating cardiomyocytes from the heart of an adult mouse, which method can be performed without relying on a Langendorff perfusion system and is low-cost. The method utilizes the characteristics that the weight and volume of cardiomyocytes are much larger than those of non-cardiomyocytes, and the cardiomyocytes are quickly collected by low-speed centrifugation, which is simple and efficient to operate. The entire separation operation process can be completed in an ultra-clean bench, and the separated cardiomyocytes can be aseptically cultured for a long time.

[0009] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0010] A method for aseptically isolating cardiomyocytes from the heart of an adult mouse, the method comprising the following steps in sequence:

[0011] (1) Transfer the heart of an adult mouse to a clean bench;

[0012] (2) Connect the catheter to the ascending aorta of the heart and tie it in place;

[0013] (3) Inject EDTA buffer into the heart through the catheter at a rate of 1.5-2.5 ml / min;

[0014] (4) Inject perfusion buffer into the heart through the catheter at a rate of 1.5-2.5 ml / min;

[0015] (5) Inject digestive enzyme buffer into the heart through the drainage tube at a rate of 1.0-1.5 ml / min;

[0016] (6) Repeat step (5) until the heart is completely digested;

[0017] (7) Remove the aorta and atrial tissue of the heart, gently tear up the remaining ventricular tissue, continue to add digestion enzyme buffer and gently blow until the ventricular tissue is dissociated into single cells;

[0018] (8) Add digestion termination solution and gently blow to terminate digestion; then filter and centrifuge the cell suspension to obtain cardiomyocytes.

[0019] The present invention injects each reagent from the ascending aorta, and each reagent can flow through the entire heart through the coronary artery circulation through the coronary artery orifice at the root of the ascending aorta. On the one hand, the same coronary artery circulation perfusion efficiency as the Langendorff scheme can be achieved. On the other hand, the consumables required for the entire separation process are cheap and easy to obtain, and do not rely on the expensive and complicated Langendorff perfusion system. The entire separation process is simpler and easier to implement, and the implementation cost is greatly reduced without the need for professional technical support.

[0020] In addition, the present invention utilizes the characteristics that the weight and volume of cardiomyocytes are much larger than those of non-cardiomyocytes, and quickly collects cardiomyocytes by low-speed centrifugation, which greatly shortens the time required for collecting cardiomyocytes, and the operation is simple and efficient; not only that, after eliminating the dependence on the Langendorff perfusion system, the entire cardiomyocyte isolation process can be carried out in an ultra-clean bench with a sterile environment, which enables the isolated cardiomyocytes to be sterilely cultured for a long time, and the isolated cardiomyocytes have normal contraction and relaxation functions, can be stimulated by a variety of drugs and adenovirus transfection and other operations, etc., providing a good solution for the aseptic isolation of adult mouse cardiomyocytes for the study of cardiovascular diseases.

[0021] Preferably, in step (1) of the method for aseptically isolating myocardial cells from the heart of an adult mouse, the experimental animal is killed and then immersed in alcohol, and then transferred to a clean bench. The alcohol immersion is for skin disinfection.

[0022] Preferably, in step (1) of the above method for aseptically isolating cardiomyocytes from the heart of an adult mouse, after the heart is removed, it is quickly placed in an EDTA buffer to prevent blood coagulation.

[0023] Preferably, in step (3) of the above method for aseptically isolating cardiomyocytes from the heart of an adult mouse, the composition of EDTA buffer is: NaCl 130mM, KCl 5mM, NaH2PO4 0.5mM, HEPES 10mM, Glucose 10mM, BDM10mM, Taurine 10mM, EDTA 5mM, and the pH is adjusted to 7.80 after dissolving in dddH2O.

[0024] EDTA buffer can inhibit the aggregation of residual blood cells in the coronary arteries, avoiding the formation of thrombi in the coronary arteries, which may lead to insufficient perfusion of the digestive enzyme buffer and affect the digestion efficiency. In addition, EDTA buffer can inhibit the contraction of cardiomyocytes and loosen the intercellular connections of cardiomyocytes, which helps to quickly digest cardiomyocytes.

[0025] Preferably, in step (3) of the above method for aseptically isolating cardiomyocytes from the heart of an adult mouse, the injection volume of EDTA buffer is 4-6 mL.

[0026] Preferably, in step (4) of the above method for aseptically isolating cardiomyocytes from the heart of an adult mouse, the composition of the Perfusion buffer is: NaCl 130mM, KCl 5mM, NaH2PO4 0.5mM, HEPES 10mM, Glucose 10mM, BDM 10mM, Taurine 10mM, MgCl2 1mM; and the pH is adjusted to 7.80 after dissolving with dddH2O.

[0027] EDTA can chelate calcium ions necessary for the activity of type II collagenase (the main component of digestive juice), thereby indirectly inhibiting collagenase. Therefore, before perfusing the digestive enzyme buffer, perfusing the Perfusion buffer through the coronary circulation can remove residual EDTA Buffer to avoid affecting the digestion efficiency of the subsequent digestive enzyme buffer.

[0028] Preferably, in step (4) of the above method for aseptically isolating cardiomyocytes from the heart of an adult mouse, the injection volume of the Perfusion buffer is 4-6 mL.

[0029] Preferably, in step (5) of the above method for aseptically isolating cardiomyocytes from the heart of an adult mouse, the digestive enzyme buffer is a perfusion buffer containing 0.5 mg / ml type II collagenase.

[0030] Preferably, in step (8) of the above method for aseptically isolating cardiomyocytes from the heart of an adult mouse, the composition of the digestion termination solution is calculated by volume as follows: 5% fetal bovine serum, 1% penicillin or streptomycin, and 94% perfusion buffer.

[0031] Preferably, in step (8) of the above method for aseptically isolating cardiomyocytes from the heart of an adult mouse, the cell suspension is filtered using a 100-mesh filter to remove undigested clumps or connective tissue; the cardiomyocytes collected by filtration and centrifugation are resuspended in a digestion stop solution and centrifuged three times for purification.

[0032] Note that the centrifugation mentioned in the present invention is short-term low-speed centrifugation (100×g for 1 min), which will not damage the cell structure and can speed up the collection process of cardiomyocytes. Cardiomyocytes with a purity close to 100% can be obtained by resuspending with digestion stop solution and centrifuging at low speed three times.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) In the present invention, each reagent is injected into the ascending aorta, and each reagent can flow through the entire heart through the coronary artery orifice at the root of the ascending aorta and the coronary artery circulation. On the one hand, the same coronary artery circulation perfusion efficiency as the Langendorff scheme can be achieved. On the other hand, the consumables required for the entire separation process are cheap and easy to obtain, and the aseptic separation of adult mouse cardiomyocytes can be achieved without relying on the Langendorff perfusion system. The entire separation process is simpler and easier to implement, and the implementation cost is greatly reduced without the need for professional technical support.

[0035] (2) The present invention utilizes the characteristics that the weight and volume of cardiomyocytes are much larger than those of non-cardiomyocytes, and quickly collects cardiomyocytes by low-speed centrifugation, which greatly shortens the time required for collecting cardiomyocytes, and is simple and efficient to operate. Moreover, after eliminating the dependence on the Langendorff perfusion system, the entire cardiomyocyte isolation process can be carried out in a clean bench with a sterile environment, which enables the isolated cardiomyocytes to be sterilely cultured for a long time, and the isolated cardiomyocytes have normal contraction and relaxation functions, and can be subjected to various drug stimulations and adenovirus transfection and other operations, etc., providing a good solution for the aseptic isolation of adult mouse cardiomyocytes for the study of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results of analysis of the number of viable cells in cardiomyocytes isolated by different methods;

[0037] Wherein, Method 1 represents the separation method invented by Roger S. Foo (Comparative Example 1), Method 2 represents the separation method of Langendorff (Comparative Example 2), Method 3 represents the separation method of the present invention, Total rod-shaped cells represents the total number of viable cells, ns represents no difference, *** represents P < 0.001, the same below;

[0038] Figure 2 The results of optical microscopy observation of cardiomyocytes isolated by different methods are shown;

[0039] Figure 3 The results of the analysis of the mRNA expression level of the myocardial hypertrophy-related gene ANP after myocardial cells isolated by different methods were given Ang II to induce myocardial cell hypertrophy.

[0040] Among them, Method 1-Ctrl represents the control group of cardiomyocytes obtained by Roger S. Foo's isolation method, Method 1-Ang II represents the angiotensin (Ang II)-induced group of cardiomyocytes obtained by Roger S. Foo's isolation method, and the same applies to Method 2-Ctrl, Method 2-Ang II, Method 3-Ctrl, and Method 3-Ang II; Relative ANPmRNA expression represents the relative ANP mRNA expression level; the same below;

[0041] Figure 4 The results of the analysis of the relative BNP mRNA expression levels of cardiomyocytes isolated by different methods;

[0042] Figure 5 The results of relative Myh7 mRNA expression level analysis of cardiomyocytes isolated by different methods;

[0043] Herein, Ang II represents angiotensin II.

[0044] Figure 6 The results of the analysis of total protein and phosphorylation levels of AKT and PLN in cardiomyocytes isolated by different methods;

[0045] Here, ISO stands for isoproterenol. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0047] Example 1

[0048] This example provides a method for aseptically isolating cardiomyocytes from adult mouse hearts.

[0049] Specifically, the method for aseptically isolating cardiomyocytes from the heart of an adult mouse in this embodiment comprises the following steps:

[0050] (1) Transfer the heart of an adult mouse to a clean bench;

[0051] Specifically, mice were killed by cervical dislocation after deep anesthesia with 3% isoflurane, and then immersed in 75% alcohol for 10 seconds and transferred to a clean bench; the skin and chest cavity were quickly cut open with sterile scissors and forceps, and the heart was removed after cutting along the ascending aorta, and placed in a 6-cm dish filled with EDTA buffer to keep the heart in a completely infiltrated state to prevent blood coagulation and thrombus formation;

[0052] The composition of EDTA buffer is: NaCl 130mM, KCl 5mM, NaH2PO4 0.5mM, HEPES10mM, Glucose 10mM, BDM 10mM, Taurine 10mM, EDTA 5mM, and the pH is adjusted to 7.80 after dissolving in dddH2O;

[0053] (2) Connect the catheter to the ascending aorta of the heart and tie it in place;

[0054] Specifically, a clinical No. 7 lumbar puncture needle shortened to 3 cm was carefully inserted into the ascending aorta as a guide tube and fixed with 6-0 silk suture;

[0055] (3) Inject EDTA buffer into the heart through the catheter at a rate of 1.5-2.5 ml / min;

[0056] Specifically, use a 5 ml syringe to inject 5 ml of 37°C preheated EDTA buffer into the ascending aorta from the outer end of the drainage tube. Try to inject slowly and evenly, and control the injection time between 2-3 minutes.

[0057] (4) Inject perfusion buffer into the heart through the catheter at a rate of 1.5-2.5 ml / min;

[0058] Specifically, use another syringe filled with 5 ml of Perfusion buffer preheated at 37°C, connect the drainage tube and inject as slowly and evenly as possible, and control the injection time between 2-3 minutes;

[0059] The composition of the perfusion buffer is as follows: NaCl 130mM, KCl 5mM, NaH2PO4 0.5mM, HEPES10mM, Glucose 10mM, BDM 10mM, Taurine 10mM, MgCl2 1mM; the pH is adjusted to 7.80 after dissolving with dddH2O;

[0060] (5) Inject digestive enzyme buffer into the heart through the drainage tube at a rate of 1.0-1.5 ml / min;

[0061] Specifically, use another syringe filled with 5 ml of digestive enzyme buffer preheated at 37°C, connect the drainage tube and inject as slowly and evenly as possible until the heart digestion is complete;

[0062] The digestion enzyme buffer is Perfusion buffer containing 0.5 mg / ml type II collagenase (Worthington, Cat. No: LS004176);

[0063] (6) Repeat step (5) until the heart is completely digested;

[0064] When the heart becomes pale, soft, and inelastic, and the gritty myocardial cells begin to fall off, it indicates that the heart is completely digested;

[0065] (7) Remove the aorta and atrial tissue of the heart, gently tear up the remaining ventricular tissue, continue to add digestion enzyme buffer and gently blow until the ventricular tissue is dissociated into single cells;

[0066] Specifically, the lumbar puncture needle was removed, the heart was placed in a new 6 cm dish, excess blood vessels and atrial tissue were removed, the remaining ventricular tissue was gently torn into pieces with forceps, 3 ml of digestive enzyme buffer was added, and the heart was gently blown with a Pasteur tube for 2-3 min until the ventricular tissue was dissociated into single cells;

[0067] (8) Add digestion termination solution and gently pipette to terminate digestion; then filter and centrifuge the cell suspension to obtain cardiomyocytes;

[0068] The composition of the digestion termination solution, by volume fraction, is: 5% fetal bovine serum, 1% penicillin, 94% Perfusion buffer;

[0069] Specifically, add 7 ml of digestion stop solution to the cell suspension in step (7), and gently blow to mix to stop digestion; then filter the cell suspension with a 100-mesh cell strainer (to remove undigested cell clumps and connective tissue) and collect it in a 15 ml centrifuge tube, centrifuge at 100×g for 1 min, discard the supernatant and add 10 ml of digestion stop solution again, gently resuspend the cell clumps, and centrifuge at 100×g for 1 min again; repeat 3 times to obtain cardiomyocytes with a purity close to 100%.

[0070] The obtained cardiomyocytes can be directly lysed and then used for subsequent molecular biology operations such as Western Blot and qPCR. If sterile culture and recalcification operations are required, they can be carried out step by step according to the protocols reported in previous literature.

[0071] Comparative Example 1

[0072] In this comparative example, the Langendorff separation method was used to isolate cardiomyocytes from the heart of adult mice. For detailed steps, please refer to the literature: Huiliang Zhang, Peter S Rabinovitch. Protocol for Isolation of Cardiomyocyte from Adult Mouse and Rat. Bio-protocol Journal. 2022 May 20; 12(10), e4412. DOI: 10.21769 / BioProtoc.4412. PMID: 35813022.

[0073] Comparative Example 2

[0074] In this comparative example, the method of Roger S. Foo was used to isolate cardiomyocytes from the adult mouse heart. For detailed steps, please refer to the literature: Matthew Ackers-Johnson, Peter Yiqing Li, Andrew P Holmes, Sian-Marie O'Brien, Davor Pavlovic, Roger S Foo. A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart. Circulation Research. 2016 Sep 30; 119 (8), 909-20. DOI: 10.1161 / CIRCRESAHA.116.309202. PMID: 27502479.

[0075] The number of surviving cells, cell morphology and cell activity after adherent culture of the cardiomyocytes isolated and obtained in Example 1 and Comparative Examples 1-2 were tested. The test method and test results are as follows:

[0076] 1. Analysis of the number of isolated cardiomyocytes

[0077] The number of isolated cardiomyocytes obtained in Example 1 and Comparative Examples 1-2 was detected by the following method: the isolated cardiomyocytes were counted using a hemacytometer. When counting, only the cells within the grid line squares or on the right and lower borders were counted. The rod-shaped cardiomyocytes in each group (16) of corner squares were recorded, and the total number of surviving cells was calculated.

[0078] Test results see Figure 1 .

[0079] Depend on Figure 1 It can be seen that the number of cardiomyocytes isolated from each mouse by Roger S. Foo’s method is approximately 2.57×10 6 The number of cardiomyocytes per mouse isolated by the Langendorff method and the method of the present invention was approximately 3.17×10 6 The number of cardiomyocytes isolated by the Langendorff method and the method of the present invention was much greater than that by Roger S. Foo's method (P < 0.001), and the method of the present invention does not require the use of a Langendorff device.

[0080] 2. Cell Morphology Analysis

[0081] The cell morphology of the isolated cardiomyocytes obtained in Example 1 and Comparative Example 1-2 was observed under an optical microscope. Figure 2 shown.

[0082] Depend on Figure 2 It can be seen that in the representative images of adult mouse cardiomyocytes isolated by the three methods, rod-shaped cardiomyocytes with normal morphology and clear sarcomere stripes can be seen.

[0083] 3. Real-time qPCR analysis of the expression levels of myocardial hypertrophy-related genes

[0084] Real-time qPCR was used to analyze the mRNA expression levels of ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide), and MYH7 (myosin heavy chain 7) in the cardiomyocytes isolated and obtained in Example 1 and Comparative Examples 1-2 under angiotensin (Ang II) induced stimulation conditions. The analysis method is as follows:

[0085] The animals in the model group were implanted with an osmotic pump (AngII 1000 ng / kg / min) subcutaneously to induce myocardial hypertrophy; the animals in the control group were implanted with an osmotic pump subcutaneously with physiological saline inside the pump. After 28 days, cardiomyocytes were isolated and obtained using the three separation methods of Example 1 and Comparative Examples 1-2, respectively, and cardiomyocyte RNA was extracted and subjected to Real-time qPCR analysis.

[0086] The analysis results are as follows Figure 3 , Figure 4 and Figure 5 shown.

[0087] Depend on Figure 3 , Figure 4 and Figure 5 It can be seen that the mRNA expression levels of ANP, BNP, and MYH7 in cardiomyocytes obtained by different separation methods were higher than those in the control group under Ang II-induced stimulation, indicating that the cardiomyocytes obtained by the three different separation methods all retained the characteristic transcriptional characteristics of myocardial hypertrophy.

[0088] 4. Western blot analysis of total protein and phosphorylation levels of AKT and PLN in cardiomyocytes

[0089] Different doses of isoproterenol were used to stimulate the cardiomyocytes isolated by the three methods. After obtaining the cardiomyocyte lysates, Western blotting analysis was performed using specific antibodies to detect the total protein and phosphorylation levels of AKT and PLN in cardiomyocytes under isoproterenol (ISO)-induced stimulation.

[0090] The analysis results are as follows Figure 6 shown.

[0091] Depend on Figure 6 It can be seen that under the conditions of ISO induction stimulation of different concentrations, the phosphorylation levels of AKT and PLN in the cardiomyocytes obtained by the three separation methods were increased compared with the control treatment group (without isoproterenol stimulation), while the total protein levels of AKT and PLN in each cardiomyocyte remained unchanged. This suggests that the cardiomyocytes obtained by the three different separation methods can respond normally to drug stimulation.

Claims

1. A method for aseptically isolating cardiomyocytes from the heart of an adult mouse, characterized in that: The following steps are included in sequence: (1) Remove the heart of an adult mouse in a clean bench; (2) Connect the catheter to the ascending aorta of the heart and tie it in place; (3) Inject EDTA buffer into the heart through the catheter at a rate of 1.5-2.5 ml / min; (4) Inject perfusion buffer into the heart through the catheter at a rate of 1.5-2.5 ml / min; (5) Inject digestive enzyme buffer into the heart through the drainage tube at a rate of 1.0-1.5 ml / min; (6) Repeat step (5) until the heart is completely digested; (7) Remove the aorta and atrial tissue, gently tear up the remaining ventricular tissue, continue to add digestion enzyme buffer and gently blow until the ventricular tissue is dissociated into single cells; (8) Add digestion termination solution and gently blow to terminate digestion; then filter and centrifuge the cell suspension to obtain cardiomyocytes.

2. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (1), the experimental animals are killed and immersed in alcohol, and then transferred to a clean bench.

3. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (1), after the heart is removed, it is quickly placed in EDTA buffer to prevent blood clotting.

4. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (3), the composition of EDTA buffer is: NaCl 130mM, KCl 5mM, NaH2PO40.5mM, HEPES10mM, Glucose10mM, BDM 10mM, Taurine 10mM, EDTA 5mM, and the pH is adjusted to 7.80 after dissolving in dddH2O.

5. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (3), the injection volume of EDTA buffer is 4-6 mL.

6. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (4), the composition of the perfusion buffer is: NaCl 130mM, KCl 5mM, NaH2PO4 0.5mM, HEPES 10mM, Glucose 10mM, BDM 10mM, Taurine 10mM, MgCl2 1mM; after dissolving with dddH2O, the pH is adjusted to 7.

80.

7. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (4), the injection volume of Perfusion buffer is 4-6 mL.

8. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (5), the digestion enzyme buffer is a perfusion buffer containing 0.5 mg / ml type II collagenase.

9. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (8), the composition of the digestion termination solution is calculated by volume fraction as follows: 5% fetal bovine serum, 1% penicillin and streptomycin, and 94% perfusion buffer.

10. The method for aseptically isolating cardiomyocytes from the heart of an adult mouse according to claim 1, characterized in that: In step (8), the cell suspension is filtered using a 100-mesh filter to remove undigested clumps or connective tissue; the cardiomyocytes collected by filtration and centrifugation are resuspended in a digestion stop solution and centrifuged three times for purification.

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