Composition for cryopreservation of cardiomyocytes

Abstract

It relates to a composition for cryopreserving cardiomyocytes, which contains BLEB and / or PAB (or its physiologically acceptable salt) and a cryoprotectant. It also relates to a cardiomyocyte cryopreservation kit containing the composition, a method for cryopreserving mammalian cardiomyocytes with the aforementioned composition, and the use of the aforementioned composition in the preparation of a reagent for cryopreserving mammalian cardiomyocytes.

Description

[0001] The scientific research project involved in this application is the Medical and Health Science and Technology Innovation Project of the Chinese Academy of Medical Sciences, with the project number 2017-I2M-1-003. Technical Field

[0002] The present invention relates to a composition for cryopreserving cardiomyocytes, which contains BLEB and / or PAB (or its physiologically acceptable salt) and a cryoprotectant. The present invention also relates to a cardiomyocyte cryopreservation kit containing the composition, a method for cryopreserving mammalian cardiomyocytes using the foregoing composition, and the use of the foregoing composition in the preparation of a reagent for cryopreserving mammalian cardiomyocytes. Background Art

[0003] Deaths caused by cardiovascular diseases account for more than 40% of the total number of deaths from diseases, higher than tumors and other diseases, making it the biggest killer of human life and health. "China Cardiovascular Disease Report 2018" shows that the prevalence of cardiovascular diseases in China continues to rise, and the mortality rate still ranks first, higher than tumors and other diseases (Non-patent Document 1). Therefore, the prevention and treatment of cardiovascular diseases are of crucial importance to human health, and the importance of scientific research on the prevention and treatment of cardiovascular diseases is self-evident.

[0004] However, in the research on the pathological mechanisms and the development of new drugs in the cardiovascular field, there has been no breakthrough for many years. The progress of traditional cardiovascular drug treatment has been very slow, and clinical trials of myocardial protection drugs have also frequently failed. One of the main reasons for the above phenomena is the limitation of the cell models used in clinical research.

[0005] To study the prevention and treatment of any disease, a good cell model is required. The research model for cardiovascular diseases is mammalian, especially human, cardiomyocytes. However, different from other somatic cells, cardiomyocytes themselves have the characteristics of strong motility and high oxygen consumption. This characteristic determines that they need to consume a large amount of energy and produce a large amount of metabolites during their survival process, resulting in the difficulty of obtaining cardiomyocytes through cultivation; in addition, although theoretically cardiomyocytes can be obtained by the directed differentiation of human embryonic stem cells or induced pluripotent stem cells, this method has problems such as incomplete differentiation and lack of epigenetic modification (Non-patent Documents 2 and 3), thus unable to truthfully reflect the true state of the disease. This makes cardiomyocytes can only be obtained from animals or humans, and must be used immediately after extraction. For animals, the animals from which cardiomyocytes are extracted cannot survive naturally; for humans, cardiomyocytes can only be obtained from subjects, and their sources are even more precious. Moreover, the survival period of cardiomyocytes is much shorter than that of other somatic cells, and these freshly extracted cells often cannot be used up at one time and sometimes cannot be used immediately, resulting in inevitable waste, which further exacerbates the shortage of cardiomyocytes.

[0006] In particular, compared with cardiomyocytes of other mammals (such as rodents), cryopreservation and resuscitation of human cardiomyocytes are extremely challenging tasks. Compared with other species, human cardiomyocytes have an even lower tolerance to ischemia and hypoxia, which means that cryopreservation and resuscitation of human cardiomyocytes are more difficult than those of other species. If the cryopreservation and resuscitation process is slightly mishandled, cell apoptosis or necrosis may occur even within just a few minutes. Due to the great technical difficulty, there has never been any precedent for cryopreserving and successfully resuscitating human cardiomyocytes so far.

[0007] To solve the above practical problems and ensure an adequate supply of cardiomyocytes for clinical research and new drug development related to cardiovascular diseases, it is necessary to develop an efficient cryopreservation method for cardiomyocytes of mammals, especially humans, and develop corresponding compositions (such as cryopreservation reagents) for cryopreservation, so as to make it possible to recycle cardiomyocytes of mammals, especially humans, and save precious resources.

[0008] For ordinary cells, the process of cell cryopreservation and resuscitation is "slow freezing and rapid thawing". On the one hand, "slow freezing" is required for cell cryopreservation. If the cell temperature suddenly drops below zero, cell organelles will undergo dehydration, and the concentration of soluble substances in the cell will increase, forming ice crystals inside the cell and causing cell damage; while cooling and cryopreserving at a slow gradient can enable the cells to slowly dehydrate, and no large ice crystals will be formed inside the cells. In addition, cryoprotectants (such as Ficoll, dextrans, etc.) can be added to the cryopreservation reagent. By binding of the cryoprotectant to water molecules in the solution, the freezing point can be lowered, the formation of ice crystals inside the cell can be reduced, and the concentration of electrolytes in the unfrozen solution can be decreased, thereby inhibiting cell damage and making it possible to preserve cells at ultra-low temperature. Currently commonly used cryoprotectants include polyvinyl pyrrolidone (PVP), Ficoll, and some dextran substances, etc. On the other hand, "rapid thawing" should be carried out for cell resuscitation, which can ensure that the extracellular crystals melt within a very short time, avoiding damage to the cells caused by intracellular recrystallization due to slow thawing that allows water to penetrate into the cells (Non-Patent Documents 4 and 5).

[0009] However, the above content is only the general theory for cryopreserving and resuscitating ordinary somatic cells. For cardiomyocytes, especially human cardiomyocytes, there is no existing technology that can cryopreserve and resuscitate with high survival rate and maintain cell morphology, which seriously hinders the development of related scientific research work and cannot meet the need to provide a research basis for clinical disease diagnosis and treatment.

[0010] Related literature

[0011] Non-patent Document 1: Hu Shengshou, Gao Runlin, Liu Lisheng, etc., "Summary of China Cardiovascular Disease Report 2018", Chinese Circulation Journal, 2019, 34(3): 209-220.

[0012] Non-patent Document 2: D.M. DeLaughter, A.G. Bick, H. Wakimoto, D. McKean, J.M. Gorham, I.S. Kathiriya, J.T. Hinson, J. Homsy, J. Gray, W. Pu, B.G. Bruneau, J.G. Seidman, and C.E. Seidman, "Single-Cell Resolution of Temporal Gene Expression During Heart Development", Dev Cell, 39(2016), 480-90.

[0013] Non-patent Document 3: X. Yang, L. Pabon, and C.E. Murry, "Engineering Adolescence: Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes", Circ Res, 114(2014), 511-23.

[0014] Non-patent Document 4: LOVELOCK JE, BISHOP MW "Prevention of freezing damage to living cells by dimethyl sulphoxide", Nature. 1959 May 16; 183(4672): 1394-5.

[0015] Non-patent Document 5: Meryman HT, "Cryopreservation of living cells: principles and practice", Transfusion, 2007 May; 47(5): 935-45. Summary of the Invention

[0016] In consideration of the actual clinical needs and the characteristics of human cardiomyocytes, the inventors of the present application have conducted in-depth research and continuous optimization on the cryopreservation technology and related reagents for human cardiomyocytes in order to solve the problem of cryopreservation and resuscitation of human cardiomyocytes, and thus developed a composition for cryopreserving cardiomyocytes.

[0017] Therefore, the present invention includes but is not limited to the following technical contents:

[0018] 1. A composition comprising BLEB and / or PAB or a physiologically acceptable salt thereof, and an antifreeze agent

[0019]

[0020] 2. The composition according to technical item 1, further comprising one or more components selected from the following: an energy component, a metabolic regulator, an acid-base regulator, an isotonic agent.

[0021] 3. The composition according to any one of the foregoing technical items, comprising the following components: BLEB and / or PAB or a physiologically acceptable salt thereof, hydroxyethyl starch, DMSO, glucose, creatine, adenosine, allopurinol, reduced glutathione, taurine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride.

[0022] 4. The composition according to any one of the foregoing technical items, comprising the following components: 1 to 50 μM of BLEB or 1 to 100 μM of PAB, and 20 to 100 mg / ml of hydroxyethyl starch, 1 to 30% by volume of DMSO, 5 to 50 mM of glucose, 0.5 to 20 mM of creatine, 0.1 to 20 mM of adenosine, 0.1 to 5 mM of allopurinol, 0.5 to 10 mM of reduced glutathione, 5 to 50 mM of taurine, 0.5 to 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1 to 20 mM of magnesium sulfate, 5 to 100 mM of potassium dihydrogen phosphate, 0.5 to 5 mM of calcium chloride.

[0023] 5. The composition according to any one of the foregoing technical items, having a pH value of 7.0 to 7.8.

[0024] 6. A cryopreservation kit for cardiomyocytes, comprising the composition according to any one of technical items 1 to 5.

[0025] 7. A method for cryopreserving mammalian cardiomyocytes, wherein the composition according to any one of technical items 1 to 5 is used.

[0026] 8. The method according to technical item 7, wherein the mammal is a human.

[0027] 9. Use of the composition according to any one of technical items 1 to 5 in the preparation of a reagent for cryopreserving mammalian cardiomyocytes.

[0028] 10. The use according to technical item 9, wherein the mammal is a human.

[0029] By using the composition of the present invention, mammalian cardiomyocytes can be efficiently cryopreserved and revived, and the cells after cryopreservation and revival can still maintain an ideal survival rate and morphology. In particular, by using the composition of the present invention, the revival of human cardiomyocytes after cryopreservation is achieved for the first time, and good survival rate and cell morphology are obtained after revival. Thus, the present invention can provide a solid foundation for cardiovascular disease research, clinical translation, drug development and personalized treatment. Description of the Drawings

[0030] Figure 1 : The revival rate of cardiomyocytes after the cryopreservation and revival operations of Examples 1 to 2.

[0031] Figure 2 : Comparison diagrams of the states of cardiomyocytes before and after the cryopreservation and revival operations of Examples 1 to 2.

[0032] Figure 3 : The revival rate of cardiomyocytes obtained when cryopreservation and revival are carried out using cryopreservation reagents containing different concentrations of HES in Example 3.

[0033] Figure 4 : A diagram showing the influence of BLEB and PAB on the rod-shaped rate of cardiomyocytes. Detailed Description of the Invention

[0034] The inventors of the present application have conducted a large number of extensive and in-depth studies, carried out a large number of experiments and explorations, and unexpectedly found that BLEB or its derivative PAB can well solve the technical problems to be solved by the present invention.

[0035] BLEB is also known as "(-)-Blebbistatin", which is a non-muscle myosin II inhibitor and a cell-permeable inhibitor, and is known to be able to inhibit the contraction of mouse cardiomyocytes; in addition, in cardiovascular physiology research, BLEB has been widely used as a specific uncoupler. PAB is also known as "para-aminoblebbistatin", which is a derivative of BLEB and has a structure and properties similar to those of BLEB. However, BLEB has some adverse chemical characteristics, for example, it is unstable to light, has phototoxicity and cytotoxicity, high fluorescence and low water solubility (the solubility is only 10.9 ± 0.9 μM), etc. Because of these adverse characteristics, researchers have biases against the application of BLEB and its derivative PAB, and there has been no report on the use of BLEB and / or PAB for the cryopreservation and revival of cardiomyocytes, especially human cardiomyocytes, so far.

[0036] However, the inventors of the present application surprisingly found that BLEB and PAB can unexpectedly maintain the original morphology and survival rate of cardiomyocytes after cryopreservation and resuscitation. On this basis, the inventors of the present application used BLEB and / or PAB or their physiologically acceptable salts as key components in the cryopreservation composition and combined them with other specific components to perform cryopreservation and resuscitation, thereby well solving the above-mentioned problems to be solved by the present invention and thus completing the present invention.

[0037] The first aspect of the present invention relates to a composition containing BLEB and / or PAB and an antifreeze agent.

[0038] In one embodiment, when used alone or in combination, the concentration of BLEB in the composition of the present invention is 1 to 50 μM, preferably 3 to 20 μM, more preferably 5 to 15 μM, particularly preferably 8 to 12 μM, and more particularly preferably about 10 μM; the concentration of PAB is 1 to 100 μM, preferably 3 to 60 μM, more preferably 5 to 50 μM, particularly preferably about 10 to 20 μM.

[0039] As used in the context of this specification, the term "about" means a range of plus or minus 10% of the corresponding value. For example, if the concentration of a certain component is about 5 mM, it means its concentration is 4.5 to 5.5 mM; if the concentration range of a certain component is about 5 to 10 mM, it means its concentration range is 4.5 to 11 mM.

[0040] As described above, the antifreeze agent used in the present invention can reduce the formation of intracellular ice crystals under low temperature conditions and reduce the concentration of electrolytes in the unfrozen solution, thereby inhibiting cell damage. In one embodiment, the antifreeze agent in the composition of the present invention is selected from polyvinyl pyrrolidone (PVP), hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), Ficoll, and dextran-based antifreeze agents, or any combination thereof. In a particularly preferred embodiment, the antifreeze agent is hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), or a combination thereof. When used alone or in combination, the concentration of HES is 20 to 100 mg / ml, preferably 25 to 60 mg / ml, more preferably 30 to 40 mg / ml, and more preferably about 36 mg / ml; the concentration of DMSO is 1 to 30% by volume, more preferably 3 to 20% by volume, more preferably 5 to 15% by volume, more preferably 8 to 12% by volume, and more preferably about 10% by volume.

[0041] In one embodiment, the present invention also optionally contains one or more components selected from the following: energy components, metabolic regulators, acid-base regulators, and isotonic agents.

[0042] The composition of the present invention optionally contains an energy substance, which provides necessary energy reserves for cells during cryopreservation and resuscitation. In a preferred embodiment, the energy substance is glucose, and its concentration is, for example, 5 - 50 mM, preferably 10 - 30 mM, more preferably 20 - 25 mM, and particularly preferably about 22 mM.

[0043] The composition of the present invention optionally contains metabolic regulators, such as adenosine, allopurinol, reduced glutathione, taurine, sodium pyruvate, insulin, creatine, taurine, L-carnitine, etc., which help cells regulate energy metabolism (such as sugar metabolism) during cryopreservation and resuscitation. In a preferred embodiment, the metabolic regulator is adenosine, allopurinol, reduced glutathione, taurine, creatine or any combination thereof. When used alone or in combination, the concentration of adenosine is 0.1 - 20 mM, preferably 1 - 10 mM, more preferably 3 - 8 mM, and particularly preferably about 5 mM; the concentration of allopurinol is 0.1 - 5 mM, preferably 0.5 - 2 mM, more preferably about 0.8 - 1.5 mM, and particularly preferably about 1 mM; the concentration of reduced glutathione is 0.5 - 10 mM, preferably 1 - 8 mM, more preferably 2 - 5 mM, and particularly preferably about 3 mM; the concentration of taurine is 5 - 50 mM, preferably 10 - 30 mM, more preferably 15 - 25 mM, and particularly preferably about 20 mM; the concentration of creatine is 0.5 - 20 mM, preferably 1 - 10 mM, more preferably 3 - 8 mM, and particularly preferably about 5 mM.

[0044] The composition of the present invention optionally contains acid-base regulators, which include buffer substances for maintaining the pH value stability of the composition, such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and acid-base substances for directly adjusting the pH value of the composition, such as potassium hydroxide, sodium hydroxide, etc. In a preferred embodiment, the acid-base regulator is HEPES. In another preferred embodiment, the acid-base regulator is a combination of HEPES, potassium hydroxide, and sodium hydroxide. When used alone or in combination, the concentration of HEPES is 0.5 - 10 mM, preferably 1 - 8 mM, more preferably 3 - 6 mM, and particularly preferably about 5 mM; the concentration of potassium hydroxide or sodium hydroxide (or their total concentration) is, for example, 10 - 200 mM, preferably 50 - 150 mM, more preferably 80 - 120 mM, and particularly preferably about 100 mM. Additionally, by using the acid-base regulator, the final pH value of the composition is adjusted to 7.0 - 7.8, preferably 7.2 - 7.6, more preferably 7.3 - 7.5, and particularly preferably about 7.4. Referring to the above components and their concentration ranges, those skilled in the art can select the acid-base regulator and its dosage according to specific circumstances.

[0045] The composition of the present invention optionally contains an osmotic agent, which is a substance for maintaining the osmotic pressure of the composition to ensure the water and electrolyte balance of cells. In a preferred embodiment in this regard, the osmotic agent is magnesium sulfate (such as the hydrate of magnesium sulfate, such as its heptahydrate), potassium dihydrogen phosphate, calcium chloride, or a combination thereof. When used alone or in combination, the concentration of magnesium sulfate is 1 to 20 mM, preferably 2 to 10 mM, more preferably 3 to 8 mM, and particularly preferably about 5 mM; the concentration of potassium dihydrogen phosphate is 5 to 100 mM, preferably 10 to 50 mM, more preferably 20 to 30 mM, and particularly preferably about 25 to 26 mM; the concentration of calcium chloride is 0.5 to 5 mM, preferably 1 to 3 mM, more preferably 1.5 to 2 mM, and particularly preferably about 1.8 mM.

[0046] It should be noted that some components may have more than one function in the cryopreservation preparation of the present invention. For example, although potassium dihydrogen phosphate is listed as an osmotic agent in this specification, it also has the function of pH adjustment and buffering and can also be regarded as an acid-base regulator. Similarly, although calcium chloride is listed as a metabolic regulator and glucose is listed as an energy substance in this specification, these substances also play a role in maintaining osmotic pressure (i.e., osmotic effect) and can also be regarded as osmotic agents, and so on.

[0047] The composition of the present invention may also optionally contain other components known in the art or commonly used for cell preservation. For example, the composition of the present invention may contain an apoptosis inhibitor, thereby increasing the survival rate of cells during cryopreservation and resuscitation. For example, the apoptosis inhibitor may be Z-VAD-FMK, Emericase, Belnacasan, or a combination thereof.

[0048] In one embodiment, the composition of the present invention contains the following components: BLEB and / or PAB (or its physiologically acceptable salt), and hydroxyethyl starch, DMSO, glucose, creatine, adenosine, allopurinol, reduced glutathione, taurine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride. Optionally, the composition of the present invention may also contain other acid-base regulators, such as potassium hydroxide, sodium hydroxide, etc.

[0049] In a preferred embodiment, the composition of the present invention contains the following components: 1 to 50 μM of BLEB and / or 1 to 100 μM of PAB, and 20 to 100 mg / ml of hydroxyethyl starch, 1 to 30% by volume of DMSO, 5 to 50 mM of glucose, 0.5 to 20 mM of creatine, 0.1 to 20 mM of adenosine, 0.1 to 5 mM of allopurinol, 0.5 to 10 mM of reduced glutathione, 5 to 50 mM of taurine, 0.5 to 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1 to 20 mM of magnesium sulfate, 5 to 100 mM of potassium dihydrogen phosphate, 0.5 to 5 mM of calcium chloride. Optionally, the composition of the present invention may further contain other acid-base regulators, such as 10 to 200 mM of potassium hydroxide and / or an appropriate amount of sodium hydroxide, to adjust the final pH value of the composition to 7.0 to 7.8.

[0050] In a more preferred embodiment, the composition of the present invention contains the following components: 3 to 20 μM of BLEB and / or 3 to 60 μM of PAB, and 25 to 60 mg / ml of hydroxyethyl starch, 3 to 20% by volume of DMSO, 10 to 30 mM of glucose, 1 to 10 mM of creatine, 1 to 10 mM of adenosine, 0.5 to 2 mM of allopurinol, 1 to 8 mM of reduced glutathione, 10 to 30 mM of taurine, 1 to 8 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 to 10 mM of magnesium sulfate, 10 to 50 mM of potassium dihydrogen phosphate, 1 to 3 mM of calcium chloride. Optionally, the composition of the present invention may further contain other acid-base regulators, such as 50 to 150 mM of potassium hydroxide and / or an appropriate amount of sodium hydroxide, to adjust the final pH value of the composition to 7.2 to 7.6.

[0051] In a particularly preferred embodiment, the composition of the present invention contains the following components: 5 to 15 μM (preferably 8 to 12 μM, more preferably about 10 μM) of BLEB and / or 5 to 50 μM (preferably about 10 to 20 μM) of PAB, and 30 to 40 mg / ml of hydroxyethyl starch, 5 to 15% by volume of DMSO, 20 to 25 mM of glucose, 3 to 8 mM of creatine, 3 to 8 mM of adenosine, 0.8 to 1.5 mM of allopurinol, 2 to 5 mM of reduced glutathione, 15 to 25 mM of taurine, 3 to 6 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 3 to 8 mM of magnesium sulfate, 20 to 30 mM of potassium dihydrogen phosphate, 1.5 to 2 mM of calcium chloride. Optionally, the composition of the present invention may further contain other acid-base regulators, such as 80 to 120 mM of potassium hydroxide and / or an appropriate amount of sodium hydroxide, to adjust the final pH value of the composition to 7.3 to 7.5.

[0052] In one embodiment, the composition of the present invention is used as a cryopreservation reagent for mammalian cardiomyocytes, and the mammals include but are not limited to mice, rats, dogs, monkeys, and humans. In a preferred embodiment, the composition of the present invention is used as a cryopreservation reagent for human cardiomyocytes.

[0053] Another aspect of the present invention relates to a cardiomyocyte cryopreservation kit, which contains any one of the foregoing compositions. In one embodiment, the box body of the cryopreservation kit is made of a low-temperature resistant material to ensure that it will not be damaged during the process of freezing and rewarming as a container for holding the composition. In one embodiment, the cryopreservation kit is a gradient cooling box. In one embodiment, a cryopreservation tube for containing the composition is placed in the cryopreservation kit (such as the gradient cooling box produced by Corning, Inc., USA).

[0054] Another aspect of the present invention relates to a method for cryopreserving mammalian cardiomyocytes, in which any one of the foregoing compositions is used. In one embodiment, the cryopreservation method includes the following steps:

[0055] · Pre-cooling step: Pre-cool the composition of the present invention at 0 - 5°C (such as 4°C), and optionally, the cardiomyocytes can be recalcified to maintain their viability;

[0056] · Transfer step: Centrifuge the cardiomyocytes placed in the culture medium and remove the supernatant, and suspend the precipitated cell mass obtained by centrifugation in the pre-cooled composition so that the cell density in the composition is, for example, 0.5×10 6 / ml to 5×10 6 / ml, preferably 1×10 6 / ml to 2×10 6 / ml, and then transfer it into a cryopreservation container (such as transfer it into a cryopreservation tube placed in the cryopreservation kit);

[0057] · Cooling step: Slowly lower the temperature to the target temperature (such as -80°C), and the temperature can be lowered by gradient cooling, and the gradient is, for example, about -0.8 to -2°C / minute, preferably about -1 to -1.2°C / minute. It should be noted that the cooling rate should not be too fast, otherwise the cells are likely to die.

[0058] After cryopreserving the cells for a period of time (e.g., 1 to 72 hours, preferably 12 to 48 hours) by the aforementioned method, the cryopreserved cells can be placed in a constant temperature water bath at an appropriate temperature (e.g., human cardiomyocytes should be placed in a constant temperature water bath at human body temperature, i.e., 37°C) to revive the cells. Subsequently, they can be stored in a known culture medium (e.g., one or more culture media selected from the M199 series, MEM series, DMEM series) by known methods for subsequent use. In one embodiment, the culture medium also contains an appropriate amount of serum (e.g., fetal bovine serum) or serum protein (e.g., bovine serum albumin) and antibiotics (e.g., penicillin and / or streptomycin). In a preferred embodiment, the culture medium also contains BLEB and / or PAB. Among them, when used alone or in combination, the aforementioned serum (e.g., fetal bovine serum) can account for a volume fraction of 1% - 20% of the culture medium, preferably 2% - 15%, more preferably 3% - 12%, and particularly preferably about 5% - 10%; the concentration of the aforementioned serum protein (e.g., bovine serum albumin) in the culture medium can be 0.1 - 10 g / ml, preferably 0.2 - 5 g / ml, more preferably 0.3 - 1 g / ml, and particularly preferably about 0.5 g / ml; the concentration of penicillin in the culture medium can be 10 - 500 U / ml, preferably 20 - 400 U / ml, more preferably 50 - 300 U / ml, and particularly preferably about 100 - 200 U / ml, about 100 U / ml, or about 200 U / ml; the concentration of streptomycin in the culture medium can be 10 - 500 μg / ml, preferably 20 - 400 μg / ml, more preferably 50 - 300 μg / ml, and particularly preferably about 100 - 200 μg / ml, about 100 μg / ml, or about 200 μg / ml; the concentration of BLEB in the culture medium can be 1 - 50 μM, preferably 3 - 20 μM, more preferably 5 - 15 μM, particularly preferably 8 - 12 μM, and even more particularly preferably about 10 μM; the concentration of PAB in the culture medium can be 1 - 100 μM, preferably 3 - 60 μM, more preferably 5 - 50 μM, particularly preferably about 10 - 20 μM.

[0059] More specific embodiments of the present invention will be illustratively explained by the following examples, but it should be recognized that these examples are not intended to limit the scope of the present invention.

[0060] Examples

[0061] Example 1: Cryopreservation of cardiomyocytes

[0062] First, prepare the cell cryopreservation reagent according to the following formula:

[0063]

[0064]

[0065] Note: The osmotic pressure is about 300 mOsm / kg;

[0066] Pre-cool the prepared cryopreservation reagent, gradient cooling box (produced by Corning, USA) and low-temperature centrifuge (Eppendorf centrifuge 5804R, Germany) to 4°C in advance. Centrifuge the freshly isolated and collected cardiomyocytes (100×g, 4°C, 1 minute), discard the supernatant, and slowly add the cell cryopreservation reagent at a cell density of about 1×10 6 / ml, gently pipette to mix evenly, transfer the cell suspension to a cell cryopreservation tube, place it in a 4°C refrigerator, let it stand for 15 minutes, then put it in the gradient cooling box, place the cooling box in an -80°C refrigerator for gradient cooling, and transfer it to -196°C liquid nitrogen after 12 hours.

[0067] The human cardiomyocytes used in this example were collected from the left atrial appendage of three male patients (aged 51±4 years) undergoing mitral valvuloplasty, mitral valve replacement, or coronary artery bypass grafting.

[0068] Example 2: Recovery of cardiomyocytes

[0069] Adjust the water temperature of the constant temperature water bath (Shanghai Boxun, SSW-420-2S, China) to 37°C for preheating. Prepare a certain volume of culture medium according to the volume ratio of cell suspension to culture medium of 1 / 10. The culture medium contains the following components: MEM-HEPES-GlutaMAX (Thermo, 42360032), 10% (v / v) fetal bovine serum (10099141C), 10 μM BLEB (Selleck, S7099), 100 U / ml penicillin, and 100 μg / ml streptomycin (Gibco, 15240062).

[0070] Next, take out the cryopreservation tube containing the cryopreserved cells stored for 60 hours in Example 1 from liquid nitrogen, quickly place it in a 37°C constant temperature water bath and shake it moderately to melt the cell suspension within about 1.5 minutes. Pipette (Eppendorf, Germany) a volume of culture medium equal to the cell suspension, slowly add it to the cryopreservation tube, gently mix evenly, then transfer all the cell suspension to the pre-prepared culture medium, insert the pipette tip below the liquid surface and gently drip it in at about 2 - 4 drops / second. After dripping, gently mix evenly, place it in a centrifuge and centrifuge (100×g, 4°C, 2 - 3 minutes), discard the supernatant, and take 1 - 2 ml of culture medium to gently resuspend the precipitated cell mass.

[0071] As Figure 1As shown, human cardiomyocytes were counted before and after the cryopreservation and resuscitation operations in Examples 1-2, and the cell resuscitation rate (i.e., the ratio of the number of rod-shaped cells after cryopreservation and resuscitation to the number of cells before cryopreservation) was calculated to be 63.5±12.05%. In addition, it could be clearly seen under a microscope (Leica, DMI4000B, 10X) that the cells after resuscitation basically maintained the cell morphology before cryopreservation ( Figure 2 ). The above results indicate that the cell cryopreservation reagent of the present invention can successfully cryopreserve and resuscitate cardiomyocytes and well maintain the cell morphology.

[0072] Example 3: Effect of HES concentration on cryopreservation and recovery

[0073] Experiments were conducted to study the effect of adding different concentrations of HES to the cardiomyocyte cryopreservation reagent on the cryopreservation and resuscitation effects. This experiment was carried out in three parallel groups. The cardiomyocytes used were collected from the left atrial appendage of 3 male patients (aged 61±6 years) undergoing coronary artery bypass grafting. The concentrations of the cryopreservation reagents used were 36 mg / ml, 60 mg / ml, and 100 mg / ml, respectively. Other components of the cryopreservation reagent and the operation steps were the same as those described in Examples 1 and 2. The rod-shaped cell rates obtained from the three groups of experiments were 52.69±1.44%, 51.09±6.49%, and 53.68±2.82%, respectively, indicating that the concentration of HES had no significant effect on the resuscitation effect of cardiomyocytes.

[0074] Example 4: Comparison of the effects of BLEB and PAB

[0075] The purpose of this experiment was to investigate the effects of BLEB and PAB on cell viability and morphology during the ex vivo survival of cardiomyocytes, so as to predict the similarity of their roles in the cryopreservation and resuscitation process of cardiomyocytes.

[0076] 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin were added to M199 medium (purchased from Sigma), and the resulting medium was used as the basal medium. The experiment was divided into 6 groups, and DMSO (as the control group) and 10 μM BLEB and 5 μM, 10 μM, 20 μM, and 50 μM PAB were added to the basal medium respectively. The isolated human cardiomyocytes were divided into 6 groups, and the cells were seeded on a 48-well cell culture plate pre-coated with 200 μg / ml laminin using the 6 kinds of media, and placed in a 37°C cell culture incubator (5% CO 2 , with a relative saturated humidity of 95%) for 7 days, and the rod-shaped rate of cardiomyocytes was calculated. The results are as Figure 4 shown.

[0077] From Figure 4It can be seen that the PAB group at 5 - 50 μM achieved an effect close to or even better than that of the BLEB group at 10 μM, that is, the effects of the two on the viability of human cardiomyocytes were roughly equivalent. Thus, it can be known that PAB, as a BLEB derivative, can also achieve an effect similar to that of BLEB during the cryopreservation and recovery of cardiomyocytes using the method of the present invention.

[0078] The foregoing examples show that the cryopreservation reagent and cryopreservation method of the present invention can achieve a good survival rate even for the most difficult - to - cryopreserve and recover human cardiomyocytes, and can well maintain the cell morphology.

Claims

1. A method for cryopreserving mammalian cardiomyocytes, which uses a composition, wherein the composition contains the following components: 5-15 μM of BLEB, and 20-100 mg / ml of hydroxyethyl starch, 3-20% by volume of DMSO, 10-30 mM of glucose, 1-10 mM of creatine, 1-10 mM of adenosine, 0.5-2 mM of allopurinol, 1-8 mM of reduced glutathione, 10-30 mM of taurine, 1-8 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-10 mM of magnesium sulfate, 10-50 mM of potassium dihydrogen phosphate, 1-3 mM of calcium chloride; 2. The method according to claim 1, wherein the composition consists of the following components : 5-15 μM of BLEB, and 20-100 mg / ml of hydroxyethyl starch, 3-20% by volume of DMSO, 10-30 mM of glucose, 1-10 mM of creatine, 1-10 mM of adenosine, 0.5-2 mM of allopurinol, 1-8 mM of reduced glutathione, 10-30 mM of taurine, 1-8 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-10 mM of magnesium sulfate, 10-50 mM of potassium dihydrogen phosphate, 1-3 mM of calcium chloride, 50-150 mM of NaOH and / or KOH, with the balance being water.

3. The method according to claim 1 or 2, wherein the pH value is 7.0-7.

8.

4. The method according to claim 1 or 2, wherein the mammal is a human.

5. A cryopreservation composition for mammalian cardiomyocytes, the composition contains the following components: 5-15 μM of BLEB, and 20-100 mg / ml of hydroxyethyl starch, 3-20% by volume of DMSO, 10-30 mM of glucose, 1-10 mM of creatine, 1-10 mM of adenosine, 0.5-2 mM of allopurinol, 1-8 mM of reduced glutathione, 10-30 mM of taurine, 1-8 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-10 mM of magnesium sulfate, 10-50 mM of potassium dihydrogen phosphate, 1-3 mM of calcium chloride 6. The composition according to claim 5, which consists of the following components: 5-15 μM of BLEB, and 20-100 mg / ml of hydroxyethyl starch, 3-20% by volume of DMSO, 10-30 mM of glucose, 1-10 mM of creatine, 1-10 mM of adenosine, 0.5-2 mM of allopurinol, 1-8 mM of reduced glutathione, 10-30 mM of taurine, 1-8 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-10 mM of magnesium sulfate, 10-50 mM of potassium dihydrogen phosphate, 1-3 mM of calcium chloride, 50-150 mM of NaOH and / or KOH, with the balance being water.

7. The composition according to claim 5 or 6, wherein the pH value is 7.0-7.

8.

8. The composition according to claim 5 or 6, wherein the mammal is a human.

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