Myocardial cell freezing solution and application thereof

Abstract

The present invention belongs to the field of cell cryopreservation technology, specifically relating to a myocardial cell cryopreservation solution and its application. The myocardial cell cryopreservation solution comprises: human serum albumin, a cryoprotectant, a rock inhibitor, and electrolytes. The cryopreservation solution is serum-free and free of xenobiotics, and can maintain a high level of myocardial cell viability (above 80%) after recovery. After recovery, the cells can still adhere normally to the wall and exhibit normal pulsation within about 7 days. It has no effect on the expression of myocardial cell markers, APA, and FPD, and has a good effect on maintaining the electrophysiological activity of myocardial cells. It also has no effect on the therapeutic effect of myocardial cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell cryopreservation, and in particular relates to a myocardial cell cryopreservation solution and application thereof. Background Art

[0002] In recent decades, cell therapy has been widely studied and applied in cardiovascular diseases. Among them, cardiomyocytes (hPSC-CMs) derived from human pluripotent stem cells (hPSCs) have great application potential in cardiac tissue repair, cardiovascular disease modeling, new drug screening, and cardiac toxicity evaluation. In the process of large-scale promotion and application of mature cardiomyocytes, the storage and transportation of cardiomyocytes are very important. The storage and transportation of cardiomyocytes are inseparable from the cryopreservation and recovery process. Because mature cardiomyocytes have a complex sarcomere network structure and unique electrophysiological properties, the preparation of cardiomyocyte cryopreservation fluid is very critical. It is necessary to ensure the recovery viability of cardiomyocytes and ensure that their cellular properties are not significantly affected.

[0003] Currently available cardiomyocyte cryopreservation solutions are primarily targeted at early-stage cardiomyocytes differentiated from iPSCs, such as those differentiated for around 20 days. Since cardiomyocyte maturity increases with increasing differentiation time, the viability of cardiomyocytes decreases significantly after cryopreservation using conventional cardiomyocyte cryopreservation solutions. Currently, there is no solution that can cryopreserve cardiomyocytes at later stages of iPSC differentiation, such as those differentiated for around 30 to 60 days. Therefore, it is necessary to develop a cryopreservation solution that can maintain a high viability of mature, differentiated cardiomyocytes. Summary of the Invention

[0004] The first aspect of the present invention aims to provide a myocardial cell freezing solution.

[0005] The second aspect of the present invention aims to provide a use of the cardiomyocyte cryopreservation solution of the first aspect of the present invention.

[0006] The third aspect of the present invention aims to provide a method for cryopreservation of cardiomyocytes.

[0007] The fourth aspect of the present invention aims to provide a cardiomyocyte preparation.

[0008] The fifth aspect of the present invention aims to provide a use of the cardiomyocyte preparation according to the fourth aspect of the present invention.

[0009] The sixth aspect of the present invention aims to provide a product.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] A first aspect of the present invention provides a myocardial cell freezing solution comprising: human serum albumin, a cryoprotectant, a Rock inhibitor, and electrolytes.

[0012] Preferably, the cryoprotectant comprises at least one of dimethyl sulfoxide, ethylene glycol, polyvinyl pyrrolidone and trehalose; further preferably, the cryoprotectant comprises dimethyl sulfoxide.

[0013] Preferably, the electrolyte comprises at least one of compound electrolyte injection, normal saline and glucose saline; further preferably, the electrolyte comprises compound electrolyte injection.

[0014] Preferably, the Rock inhibitor comprises at least one of Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, and Narciclasine; further preferably, the ROCK inhibitor comprises Y-27632.

[0015] Preferably, the concentration of the human serum albumin in the freezing solution is 0.25-5 w / v%; further 1-3 w / v%; further 1.25-2.5 w / v%; further 2.5 w / v%.

[0016] Preferably, the concentration of the cryoprotectant in the freezing solution is 4-16% by volume; further 5-15%; further 8-12%; further 10% by volume.

[0017] Preferably, the concentration of the electrolyte in the freezing solution is 64-95% by volume; further 73-91%; further 78-87%; further 80% by volume.

[0018] Preferably, the concentration of the Rock inhibitor in the freezing solution is 1 to 20 μM; further 8 to 12 μM; further 9 to 11 μM; further 10 μM.

[0019] Preferably, the cardiomyocytes are stem cell-derived cardiomyocytes (i.e., cardiomyocytes differentiated from stem cells); further, they are stem cell-derived cardiomyocytes in the late stage of differentiation (i.e., cardiomyocytes differentiated from stem cells in the late stage of differentiation); further, they are stem cell-derived cardiomyocytes at the 30th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells at the 30th to 60th day of differentiation); further, they are stem cell-derived cardiomyocytes at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells at the 50th to 60th day of differentiation); further, they are stem cell-derived cardiomyocytes at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells and induced to mature by maturation reagents at the 50th to 60th day of differentiation). Further, they are stem cell-derived cardiomyocytes induced to mature by maturation reagents at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells and induced to mature by maturation reagents at the 50th to 60th day of differentiation).

[0020] Preferably, the stem cells are stem cells with multidirectional differentiation potential.

[0021] Preferably, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; further preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0022] Preferably, the stem cells are derived from mammals; further from primates, and even further from humans.

[0023] Preferably, the cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0024] Preferably, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0025] Preferably, the human embryonic stem cells are stem cells isolated or obtained from a human embryo within 14 days of fertilization that has not undergone in vivo development.

[0026] The second aspect of the present invention provides the use of the cardiomyocyte freezing solution of the first aspect of the present invention in any one of (a1) to (a2):

[0027] (a1) Cryopreserved cardiomyocytes;

[0028] (a2) Preparation of cardiomyocyte preparation.

[0029] Preferably, the cardiomyocytes are stem cell-derived cardiomyocytes (i.e., cardiomyocytes differentiated from stem cells); further, they are stem cell-derived cardiomyocytes in the late stage of differentiation (i.e., cardiomyocytes differentiated from stem cells in the late stage of differentiation); further, they are stem cell-derived cardiomyocytes at the 30th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells at the 30th to 60th day of differentiation); further, they are stem cell-derived cardiomyocytes at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells at the 50th to 60th day of differentiation); further, they are stem cell-derived cardiomyocytes at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells and induced to mature by maturation reagents at the 50th to 60th day of differentiation). Further, they are stem cell-derived cardiomyocytes induced to mature by maturation reagents at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells and induced to mature by maturation reagents at the 50th to 60th day of differentiation).

[0030] Preferably, the stem cells are stem cells with multidirectional differentiation potential.

[0031] Preferably, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; further preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0032] Preferably, the stem cells are derived from mammals; further from primates, and even further from humans.

[0033] Preferably, the cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0034] Preferably, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0035] Preferably, the human embryonic stem cells are stem cells isolated or obtained from a human embryo within 14 days of fertilization that has not undergone in vivo development.

[0036] A third aspect of the present invention provides a method for freezing cardiomyocytes, wherein the cardiomyocytes are frozen using the cardiomyocyte freezing solution of the first aspect of the present invention.

[0037] Preferably, the density of the cardiomyocytes in the cardiomyocyte freezing solution is 5×10 5 ~2×10 6 pieces / mL.

[0038] Preferably, the cardiomyocytes are stem cell-derived cardiomyocytes (i.e., cardiomyocytes differentiated from stem cells); further, they are stem cell-derived cardiomyocytes in the late stage of differentiation (i.e., cardiomyocytes differentiated from stem cells in the late stage of differentiation); further, they are stem cell-derived cardiomyocytes at the 30th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells at the 30th to 60th day of differentiation); further, they are stem cell-derived cardiomyocytes at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells at the 50th to 60th day of differentiation); further, they are stem cell-derived cardiomyocytes at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells and induced to mature by maturation reagents at the 50th to 60th day of differentiation). Further, they are stem cell-derived cardiomyocytes induced to mature by maturation reagents at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells and induced to mature by maturation reagents at the 50th to 60th day of differentiation).

[0039] Preferably, the stem cells are stem cells with multidirectional differentiation potential.

[0040] Preferably, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; further preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0041] Preferably, the stem cells are derived from mammals; further from primates, and even further from humans.

[0042] Preferably, the cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0043] Preferably, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0044] Preferably, the human embryonic stem cells are stem cells isolated or obtained from a human embryo within 14 days of fertilization that has not undergone in vivo development.

[0045] A fourth aspect of the present invention provides a cardiomyocyte preparation comprising cardiomyocytes and the cardiomyocyte freezing solution of the first aspect of the present invention.

[0046] Preferably, the density of the cardiomyocytes in the cardiomyocyte preparation is 5×10 5 ~2×10 6 pieces / mL.

[0047] Preferably, the cardiomyocytes are stem cell-derived cardiomyocytes (i.e., cardiomyocytes differentiated from stem cells); further, they are stem cell-derived cardiomyocytes in the late stage of differentiation (i.e., cardiomyocytes differentiated from stem cells in the late stage of differentiation); further, they are stem cell-derived cardiomyocytes at the 30th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells at the 30th to 60th day of differentiation); further, they are stem cell-derived cardiomyocytes at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells at the 50th to 60th day of differentiation); further, they are stem cell-derived cardiomyocytes at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells and induced to mature by maturation reagents at the 50th to 60th day of differentiation). Further, they are stem cell-derived cardiomyocytes induced to mature by maturation reagents at the 50th to 60th day of differentiation (i.e., cardiomyocytes differentiated from stem cells and induced to mature by maturation reagents at the 50th to 60th day of differentiation).

[0048] Preferably, the stem cells are stem cells with multidirectional differentiation potential.

[0049] Preferably, the stem cells with multidirectional differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells; further preferably, the stem cells with multidirectional differentiation potential include induced pluripotent stem cells.

[0050] Preferably, the stem cells are derived from mammals; further from primates, and even further from humans.

[0051] Preferably, the cells are human embryonic stem cells (hESCs) (eg, H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (eg, WC50, IMR90).

[0052] Preferably, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0053] Preferably, the human embryonic stem cells are stem cells isolated or obtained from a human embryo within 14 days of fertilization that has not undergone in vivo development.

[0054] The fifth aspect of the present invention provides the use of the cardiomyocyte preparation according to the fourth aspect of the present invention in any one of (b1) to (b4):

[0055] (b1) preparing drugs for preventing and treating cardiovascular diseases;

[0056] (b2) cardiovascular disease modeling;

[0057] (b3) Screening for drugs to prevent and treat cardiovascular diseases;

[0058] (b4) Cardiotoxicity evaluation.

[0059] Preferably, the cardiovascular disease comprises at least one of heart failure, acute coronary heart disease, and myocardial infarction; further comprising myocardial infarction.

[0060] Preferably, the heart failure is heart failure with reduced ejection fraction (HFrEF).

[0061] The sixth aspect of the present invention provides a product comprising the cardiomyocyte preparation according to the fourth aspect of the present invention.

[0062] Preferably, the product is used in any one of (c1) to (c4):

[0063] (c1) prevention and treatment of cardiovascular diseases;

[0064] (c2) Cardiovascular disease modeling;

[0065] (c3) prevention and treatment of cardiovascular diseases;

[0066] (c4) Cardiac toxicity evaluation.

[0067] Preferably, the product is a drug or a cell model.

[0068] Preferably, the cardiovascular disease comprises at least one of heart failure, acute coronary heart disease, and myocardial infarction; further comprising myocardial infarction.

[0069] Preferably, the heart failure is heart failure with reduced ejection fraction (HFrEF).

[0070] The beneficial effects of the present invention are:

[0071] The present invention provides a myocardial cell freezing solution, comprising: human serum albumin, a cryoprotectant, a rock inhibitor and electrolytes; the freezing solution is serum-free and free of xenoproteins, and can maintain a high level of myocardial cell viability (above 80%) after recovery; the myocardial cells can still adhere normally to the wall after recovery, and exhibit normal pulsation in about 7 days; it has no effect on the expression of myocardial cell markers; has no effect on APA and FPD, has a good maintenance effect on the electrophysiological activity of myocardial cells, and has no effect on the therapeutic effect of myocardial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 3 is a comparison of the adhesion of cardiomyocytes before and after cryopreservation in Example 2, with a scale bar of 200 μm.

[0073] Figure 2 3 is a comparison chart of the cTnt positive rates of cardiomyocytes before and after cryopreservation in Effect Example 2.

[0074] Figure 3 3 is a comparison diagram of the field potential of the cardiomyocytes before freezing in Example 2.

[0075] Figure 4 This is a comparison diagram of the field potential of the myocardial cells after recovery in Effect Example 2.

[0076] Figure 5 This figure shows the results of the effect of the cardiomyocyte cryopreservation solution on the therapeutic effect of cardiomyocytes in Example 3. The data in this figure are expressed as mean ± standard deviation (SD). DETAILED DESCRIPTION

[0077] The present invention is further described in detail below through specific examples.

[0078] Explanation of terms

[0079] Cardiomyocytes: human muscle cells that contain abundant myofibrils, striations and mitochondria, and have excitability, conductivity, autonomy and contractility.

[0080] cTnt: Cardiac myocyte-specific troponin, a classic marker used to detect myocardial cell sarcomere structure.

[0081] Field potential duration (FPD): The time from depolarization to repolarization is called field potential duration (FPD), which is a key indicator for predicting cardiotoxicity screening tests.

[0082] Action potential amplitude (APA): The action potential amplitude (APA) is defined as the voltage difference between the resting potential value and the action potential overshoot value.

[0083] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0084] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0085] The human albumin solution in the following examples and effect examples is a 25 w / v (g / mL)% human albumin concentrate purchased from Takeda Pharmaceutical (Drug Registration Certificate No. S20181006); the compound electrolyte injection was purchased from Shanghai Baxter Medical Products Co., Ltd., DMSO was purchased from Sigma, and Y-27632 was purchased from Selleck.

[0086] The detection method of myocardial cell viability in the following examples and effect examples refers to Appendix A of the group standard human myocardial cells T / CSCB0007-2021.

[0087] Example 1 A cardiomyocyte cryopreservation solution

[0088] A cardiomyocyte cryopreservation solution comprises the following components: a human albumin solution, a compound electrolyte injection, dimethyl sulfoxide (DMSO), and Y-27632; wherein the concentration of the human albumin solution in the cryopreservation solution is 10% by volume (equivalent to a human albumin concentration in the cryopreservation solution of 2.5% by mass volume (g / mL)), the concentration of the compound electrolyte injection in the cryopreservation solution is 80% by volume, the concentration of DMSO in the cryopreservation solution is 10% by volume, and the concentration of Y-27632 in the cryopreservation solution is 10 μM.

[0089] Example 2 A cardiomyocyte cryopreservation solution

[0090] A cardiomyocyte cryopreservation solution comprises the following components: a human albumin solution, a compound electrolyte injection, dimethyl sulfoxide (DMSO), and Y-27632; wherein the concentration of the human albumin solution in the cryopreservation solution is 5% by volume (equivalent to a human albumin concentration in the cryopreservation solution of 1.25% by mass volume (g / mL)), the concentration of the compound electrolyte injection in the cryopreservation solution is 85% by volume, the concentration of DMSO in the cryopreservation solution is 10% by volume, and the concentration of Y-27632 in the cryopreservation solution is 10 μM.

[0091] Comparative Example 1 A cardiomyocyte freezing solution

[0092] A cardiomyocyte freezing solution consists of the following components: dimethyl sulfoxide (DMSO) and fetal bovine serum; wherein the concentration of DMSO in the freezing solution is 10% by volume, and the concentration of fetal bovine serum in the freezing solution is 90% by volume.

[0093] Comparative Example 2: A cardiomyocyte cryopreservation solution

[0094] A cardiomyocyte freezing solution consists of the following components: dimethyl sulfoxide (DMSO) and compound electrolyte injection; wherein the concentration of DMSO in the freezing solution is 5% by volume, and the concentration of the compound electrolyte injection in the freezing solution is 95% by volume.

[0095] Comparative Example 3: A cardiomyocyte cryopreservation solution

[0096] A cardiomyocyte cryopreservation solution comprises the following components: a human albumin solution, dimethyl sulfoxide (DMSO), and a compound electrolyte injection; wherein the concentration of the human albumin solution in the cryopreservation solution is 5% by volume (equivalent to a human albumin concentration in the cryopreservation solution of 1.25% by mass-to-volume (g / mL) ratio), the concentration of DMSO in the cryopreservation solution is 10% by volume, and the concentration of the compound electrolyte injection in the cryopreservation solution is 85% by volume.

[0097] Comparative Example 4: A cardiomyocyte cryopreservation solution

[0098] A cardiomyocyte cryopreservation solution comprises the following components: a human albumin solution, dimethyl sulfoxide (DMSO), and a compound electrolyte injection; wherein the concentration of the human albumin solution in the cryopreservation solution is 10% by volume (equivalent to a human albumin concentration in the cryopreservation solution of 2.5% by mass-to-volume (g / mL) ratio), the concentration of DMSO in the cryopreservation solution is 10% by volume, and the concentration of the compound electrolyte injection in the cryopreservation solution is 80% by volume.

[0099] Effect Example 1 Effect of Myocardial Cell Cryopreservation Solution on Cell Viability

[0100] Cardiomyocytes differentiated from iPSC cells for 55 days were cryopreserved using the cardiomyocyte cryopreservation solutions of Examples 1 to 2 and Comparative Examples 1 to 4, respectively (for specific differentiation methods, see the literature: Sijia Ji, Xin Xie, The Aurora Kinase Inhibitor CYC116 Promotes the Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells, Mol. Cells 2022; 45(12): 923-934, specifically the induction and maturation method in Figure 1A of the literature, wherein the Aurora inhibitor is 5 μM CYC116), and the cryopreservation density was 2×10 6 / mL, the cell viability was 92% before freezing, and the cells were cooled to -90℃ using a thermo 7452 series CryoMed programmed cooling instrument. The specific freezing procedure was as follows: Step 1 Wait at 4.0℃, Step 2 1.0℃ / m S (sample) to -4.0℃, Step 3 25.0℃ / m C (chamber) to -40℃, Step 4 10.0℃ / m C to -12.0℃, Step 5 1.0℃ / m C to -40℃, Step 6 10.0℃ / m C to -90℃, Step 7 End; then the cells were transferred to a liquid nitrogen tank for long-term storage. After 2 weeks, the cryopreserved cardiomyocytes were placed in a 37°C water bath for about 2 minutes and 20 seconds. When the cryopreserved cardiomyocytes melted into a small piece of ice, they were taken out and 4°C resuscitation solution (containing 5v / v% human albumin solution (equivalent to a concentration of human albumin in the cryopreservation solution of 1.25w / v% by mass volume (g / mL)) and 95% compound electrolyte injection) was added. The resuscitation solution was added dropwise to the cryopreservation tube at a speed of 2-3 seconds / 100μL. The cryopreservation solution was gently shaken clockwise while adding the solution to mix the two evenly. Each milliliter of cryopreservation solution corresponded to 10mL of resuscitation solution. After the resuspension solution was added, the cells were immediately centrifuged at 300g for 2 minutes at 4°C, the supernatant was removed, and the cells were resuspended with resuspension solution. The cell density after resuspension was 5×10 6 The viability of cardiomyocytes was tested, and the results are shown in Table 1: The cardiomyocyte cryopreservation solution provided by the present application can maintain a high viability (above 80%) of differentiated and mature cardiomyocytes after recovery.

[0101] Table 1 Recovery rate of different cryopreservation solutions

[0102] Cryopreservation solution Survival rate% Comparative Example 1 26.10 Comparative Example 2 36.87 Comparative Example 3 68.62 Comparative Example 4 75.81 Example 2 80.34 Example 1 88.34 Before freezing 92.00

[0103] Effect Example 2 Effect of Myocardial Cell Cryopreservation Solution on Myocardial Cell Characteristics

[0104] Effect Example 1 Myocardial cells frozen with the myocardial cell freezing solution of Example 1 can still adhere normally after recovery ( Figure 1 ), and normal pulsation appears in about 7 days.

[0105] The cell purity of cTnt protein expressed on the surface of myocardial cells before freezing and after recovery of myocardial cells frozen in the myocardial cell freezing solution of Example 1 was detected by flow cytometry. The detection method is shown in Appendix B of the group standard human myocardial cells T / CSCB0007-2021. The flow cytometry results are as follows: Figure 2 As shown: the purity of cTnt+ cells before freezing was 96.18%, and the purity of cTnt+ cells after thawing was 96.73%, indicating that the cardiomyocyte freezing solution provided in the present application has no effect on the expression level of cardiomyocyte markers.

[0106] The myocardial cell field potential was tested on the myocardial cells before freezing and after recovery of the myocardial cells frozen in the myocardial cell freezing solution of Example 1 in Example 1. The field potential detection method is shown in Appendix D of the group standard human myocardial cells T / CSCB0007-2021. The results are as follows Figure 3 and Figure 4 As shown: the APA of the myocardial cells before freezing was about 1.632 mV, and the APA of the myocardial cells after resuscitation was about 1.764 mV; the FPD of the myocardial cells before freezing was about 0.478 ms, and the FPD of the myocardial cells after resuscitation was about 0.416 ms; this indicates that the myocardial cell freezing solution provided in the present application has no effect on APA and FPD, and has a good maintaining effect on the electrophysiological activity of myocardial cells.

[0107] Effect Example 3 Effect of Myocardial Cell Cryopreservation Solution on the Therapeutic Effect of Myocardial Cells

[0108] Myocardial infarction (MI) and acute coronary heart disease are among the most prominent causes of death in cardiovascular disease. The MI mouse model with permanent ligation of the left anterior descending (LAD) coronary artery is very similar to human MI. The mouse surgical model of myocardial infarction through permanent LAD coronary artery ligation is highly reproducible and is currently the most widely used method for creating a mouse myocardial infarction model, providing a stable model basis for product efficacy verification. After the MI model is successfully created, blood flow to most of the left ventricular myocardium stops. Insufficient myocardial oxygen supply leads to ischemic death of cardiomyocytes. This pathological condition triggers a response in the ventricular tissue, ultimately leading to ventricular dysfunction, remodeling, and heart failure.

[0109] Mice were anesthetized by intraperitoneal injection of 1.25% tribromoethanol at a dose of 10 μL / g, and a mouse MI model was established (for preparation methods, see Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction; April 2017; Journal of Visualized Experiments, Karla Reichert et al.). Specifically, the thoracic cavity was opened between the 3rd and 4th ribs using blunt forceps; the left anterior descending (LAD) coronary artery was located; and in both the model group (vehicle) and the treatment group (fresh, thawed), a 7-0 silk suture was passed under the LAD 2-3 mm below the left atrium and slowly tightened to ligate the LAD vessel while avoiding tearing of the heart tissue. (In the control group (sham) mice, the suture was pulled under the LAD and slowly removed to avoid tissue tearing, i.e., the suture was not tightened, the LAD vessel was not ligated, and the LAD was patent). The third rib was then grasped with forceps and sutured twice under the third and fourth ribs with 5-0 silk sutures.

[0110] After modeling, the model was confirmed according to the method described in LAD-Ligation: A Murine Model of Myocardial Infarction; Journal of Visualized Experiments, 2009, Mandy VV Kolk et al. Four days after modeling, mice with successful modeling underwent a second thoracotomy. Treatment groups (fresh and thawed, where fresh injections were performed using the myocardial cell cryopreservation solution of Example 1 before cryopreservation (i.e., untreated) and thawed injections were performed using the myocardial cell cryopreservation solution of Example 1 after resuscitation) were injected at three locations around the infarct, yielding approximately 2 million viable cells. The vehicle group received an equal volume of vehicle (95 v / v% compound electrolytes + 5 v / v% human albumin). The sham control group received no treatment (no cell and / or vehicle injections, no second thoracotomy). Cardiac ultrasound function testing was performed two months after injection. Echocardiography can assess cardiac structure and function in vivo. Relevant measurements include left ventricular wall thickness, inner diameter, mass, fractional shortening, and ejection fraction. After depilating the chest and abdomen of the mouse, the specific procedures are as follows:

[0111] The left ventricle was imaged in B-mode in the parasternal long-axis view. The mouse ultrasound stage was carefully adjusted to position the long axis of the left ventricle in the same plane as the ultrasound beam. The aortic valve and apex of the left ventricle were placed in the same plane as the ultrasound beam during imaging. The aortic valve and apex served as fixed points and remained unchanged between serial studies in the same animal. M-mode imaging of the left ventricle was performed to image the ventricular wall and interior. After imaging, the following parameters were calculated based on the maximum ventricular contraction and relaxation: ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic volume (LV Vol,S), and left ventricular end-diastolic volume (LV Vol,D).

[0112] The efficacy of the drug was evaluated in mice with myocardial infarction using cardiomyocytes before and after cryopreservation. The efficacy of the cardiomyocytes before and after cryopreservation in treating myocardial infarction was compared. There were 4 mice in the control group (sham), 14 mice in the model group (vehicle), 4 mice in the treatment group (fresh), and 4 mice in the treatment group (thawed). Figure 5 As shown in the results, both fresh and thawed cardiomyocytes had a significant effect on the recovery of cardiac function in mice (P < 0.05), and there was no significant difference between the two groups (the differences between the fresh, thawed, and vehicle groups were calculated using Student's t-test, and differences were considered statistically significant when P < 0.05). The EF value of the fresh cell treatment group was 39.7%, while the EF value of the thawed cell treatment group was 34.9%; the FS value of the fresh cell treatment group was 18.9%, while the FS value of the thawed cell treatment group was 16.8%; the LV Vol,S value of the fresh cell treatment group was 34.0 μL, while the LV Vol,S value of the thawed cell treatment group was 46.4 μL; the LV Vol,D value of the fresh cell treatment group was 56.7 μL, while the LV Vol,D value of the thawed cell treatment group was 70.5 μL. It can be seen that freezing with the cardiomyocyte cryopreservation solution of the present invention does not affect the therapeutic effect of cardiomyocytes.

[0113] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A cardiomyocyte cryopreservation solution, comprising human serum albumin, dimethyl sulfoxide, Rock inhibitor, and compound electrolyte injection; the cardiomyocytes are stem cell-derived cardiomyocytes at day 30 to 60 of differentiation.

2. The cardiomyocyte freezing solution according to claim 1, wherein: The Rock inhibitor comprises at least one of Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, and Narciclasine.

3. The cardiomyocyte freezing solution according to claim 2, wherein: The concentration of the human serum albumin in the freezing solution is 0.25-5 w / v%.

4. The cardiomyocyte freezing solution according to claim 2, wherein: The concentration of the cryoprotectant in the freezing solution is 4 to 16% by volume.

5. The cardiomyocyte freezing solution according to claim 2, wherein: The concentration of the electrolyte in the freezing solution is 64-95% by volume.

6. The cardiomyocyte freezing solution according to claim 2, characterized in that: The concentration of the Rock inhibitor in the freezing solution is 1-20 μM.

7. Use of the cardiomyocyte freezing solution according to any one of claims 1 to 6 in freezing cardiomyocytes; The cardiomyocytes are stem cell-derived cardiomyocytes that are at the 30th to 60th day of differentiation.

8. A method for freezing cardiomyocytes, comprising freezing cardiomyocytes using the cardiomyocyte freezing solution according to any one of claims 1 to 6; the cardiomyocytes are stem cell-derived cardiomyocytes at day 30 to 60 of differentiation.

9. The cryopreservation method according to claim 8, wherein: The density of the cardiomyocytes in the cardiomyocyte freezing solution is 5×10 5 ~2×10 6 pieces / mL.

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