WO2021203911A1 - 用于心肌细胞冻存的组合物 - Google Patents

用于心肌细胞冻存的组合物 Download PDF

Info

Publication number
WO2021203911A1
WO2021203911A1 PCT/CN2021/080535 CN2021080535W WO2021203911A1 WO 2021203911 A1 WO2021203911 A1 WO 2021203911A1 CN 2021080535 W CN2021080535 W CN 2021080535W WO 2021203911 A1 WO2021203911 A1 WO 2021203911A1
Authority
WO
WIPO (PCT)
Prior art keywords
cryopreservation
cardiomyocytes
composition
bleb
pab
Prior art date
Application number
PCT/CN2021/080535
Other languages
English (en)
French (fr)
Inventor
胡盛寿
周冰莹
侯永凤
师珣
唐晓丽
Original Assignee
中国医学科学院阜外医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国医学科学院阜外医院 filed Critical 中国医学科学院阜外医院
Priority to US17/995,409 priority Critical patent/US20230189794A1/en
Priority to EP21783704.6A priority patent/EP4134428A4/en
Priority to CN202180027339.9A priority patent/CN115698263A/zh
Publication of WO2021203911A1 publication Critical patent/WO2021203911A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0278—Physical preservation processes
    • A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the scientific research project involved in this application is the medical and health technology innovation project of the Chinese Academy of Medical Sciences, and the project number is 2017-I2M-1-003.
  • the present invention relates to a composition for freezing cardiomyocytes, which contains BLEB and/or PAB (or a physiologically acceptable salt thereof) and an antifreeze.
  • the present invention also relates to a cardiomyocyte cryopreservation kit containing the composition, a method for cryopreserving mammalian cardiomyocytes using the aforementioned composition, and the use of the aforementioned composition in the preparation of a reagent for cryopreserving mammalian cardiomyocytes use.
  • Cardiovascular diseases account for more than 40% of deaths due to diseases, higher than tumors and other diseases, and are the biggest killer of human life and health.
  • “China Cardiovascular Disease Report 2018” shows that the prevalence of cardiovascular disease in China continues to rise, and the mortality rate is still the first, higher than that of tumors and other diseases (Non-Patent Document 1). Therefore, the prevention and treatment of cardiovascular diseases is very important to human health, and the importance of scientific research on the prevention and treatment of cardiovascular diseases is self-evident.
  • cardiomyocytes of mammals, especially humans.
  • cardiomyocytes have the characteristics of strong mobility and high oxygen consumption. This feature determines that they need to consume a lot of energy and produce a large amount of metabolites in the process of survival, which makes cardiomyocytes difficult to obtain through culture.
  • this method has problems such as incomplete differentiation and lack of epigenetic modification (Non-Patent Documents 2 and 3), making it impossible Reflect the true state of the disease truthfully.
  • cardiomyocytes can only be obtained from animals or humans, and they must be taken off-the-shelf. For animals, the animals from which the cardiomyocytes are taken out cannot survive; for humans, the cardiomyocytes can only be obtained from the subject, and its source is even more precious. Moreover, compared with other somatic cells, the survival time of cardiomyocytes is much shorter, and these ready-to-use cells often cannot be used up all at once, sometimes even cannot be used immediately, resulting in unavoidable waste, which makes the supply of cardiomyocytes in short supply. situation.
  • human cardiomyocytes have even lower tolerance to ischemia and hypoxia, which means that the cryopreservation and recovery of human cardiomyocytes is more difficult than that of other species. Improper handling may even cause cell apoptosis or necrosis in just a few minutes. Due to the great technical difficulty, so far there has never been any precedent for cryopreserving and successfully resuscitating human cardiomyocytes.
  • cryopreserved composition e.g., cryopreservation reagent
  • cryopreservation and recovery is a "slow freezing and fast thawing" process.
  • the cryopreservation of cells requires “slow freezing”. If the cell temperature suddenly drops below zero, the organelles will be dehydrated, and the concentration of soluble substances in the cells will increase, forming ice crystals in the cells, causing cell damage; and cooling and storing with a slow gradient can slowly dehydrate the cells. There will be no large ice crystals inside.
  • antifreeze agents such as polysucrose, dextran, etc.
  • the freezing point can be reduced by the combination of the antifreeze agent and the water molecules in the solution, reducing the formation of intracellular ice crystals, and reducing the The concentration of electrolyte in the freezing solution inhibits cell damage and enables ultra-low temperature storage of cells.
  • antifreeze agents include polyvinylpyrrolidone (PVP), polysucrose (Ficoll), and some dextran substances.
  • Non-Patent Literature 4 the recovery of cells should be "fast-melted", which can ensure that the extracellular crystals melt in a short period of time, and prevent the slow melting of water from penetrating into the cells to form intracellular recrystallization and causing damage to the cells.
  • Non-Patent Document 1 Hu Shengshou, Gao Runlin, Liu Lisheng, etc., Summary of “Chinese Cardiovascular Disease Report 2018", Chinese Journal of Circulation, 2019, 34(3): 209-220.
  • Non-Patent Document 2 DMDelaughter, AGBick, H. Wakimoto, D. McKean, JMGorham, ISKathiriya, JTHinson, J. Homsy, J. Gray, W. Pu, BGBruneau, JGSeidman, and CESeidman, "Single-Cell Resolution of Temporal Gene Expression During Heart Development", Dev Cell, 39(2016), 480-90.
  • 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.
  • 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.
  • Non-Patent Document 5 Meryman HT, "Cryopreservation of living cells: principles and practice", Transfusion, 2007 May; 47(5):935-45.
  • the inventor of this application has conducted in-depth research on the cryopreservation technology of human cardiomyocytes and related reagents and has continued Optimized to develop a composition for freezing cardiomyocytes.
  • the present invention includes but is not limited to the following technical content:
  • composition containing BLEB and/or PAB or a physiologically acceptable salt thereof, and an antifreeze
  • composition according to item 1 which further contains one or more components selected from the group consisting of energy components, metabolic regulators, acid-base regulators, and isotonic agents.
  • composition according to any one of the preceding technical items which contains the following ingredients: BLEB and/or PAB or its physiologically acceptable salt, hydroxyethyl starch, DMSO, glucose, creatine, adenine nucleus Glycoside, allopurinol, reduced glutathione, taurine, 4-hydroxyethylpiperazine ethanesulfonic acid, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride.
  • composition according to any one of the foregoing technical items, which contains the following ingredients: 1-50 ⁇ M BLEB or 1-100 ⁇ M PAB, and 20-100mg/ml hydroxyethyl starch, 1-30% by volume DMSO, 5-50mM glucose, 0.5-20mM creatine, 0.1-20mM adenosine, 0.1-5mM allopurinol, 0.5-10mM reduced glutathione, 5-50mM taurine Acid, 0.5-10mM 4-hydroxyethylpiperazine ethanesulfonic acid, 1-20mM magnesium sulfate, 5-100mM potassium dihydrogen phosphate, 0.5-5mM calcium chloride.
  • composition according to any one of the preceding technical items which has a pH value of 7.0 to 7.8.
  • a kit for cryopreservation of cardiomyocytes comprising the composition according to any one of technical items 1 to 5.
  • composition according to any one of technical items 1 to 5 in preparing a reagent for cryopreserving mammalian cardiomyocytes.
  • mammalian cardiomyocytes can be efficiently frozen and resuscitated, and the cells after freezing and resuscitation can still maintain an ideal survival rate and morphology.
  • the freezing of human cardiomyocytes was realized for the first time, and a good survival rate and cell morphology were obtained after the resuscitation. Therefore, the present invention can provide a solid foundation for cardiovascular disease research, clinical translation, drug development and individualized treatment.
  • Figure 1 Resuscitation rate of cardiomyocytes after cryopreservation and resuscitation in Examples 1-2.
  • Figure 2 Comparison of the state of cardiomyocytes before and after cryopreservation and resuscitation in Examples 1-2.
  • Figure 3 The recovery rate of cardiomyocytes obtained in Example 3 using cryopreservation reagents containing different concentrations of HES for cryopreservation and resuscitation.
  • Figure 4 is a graph showing the influence of BLEB and PAB on the rod rate of cardiomyocytes.
  • the inventor of the present application has conducted a large number of extensive and in-depth research, conducted a large number of experiments and explorations, and unexpectedly found that BLEB or its derivative PAB can well solve the technical problem to be solved by the present invention.
  • BLEB also known as "(-)-Blebbistatin”
  • PAB also known as "para-aminoblebbistatin”
  • PAB is a derivative of BLEB and has similar structure and properties to BLEB.
  • BLEB has some unfavorable chemical properties, such as light instability, phototoxicity and cytotoxicity, high fluorescence and low water solubility (solubility is only 10.9 ⁇ 0.9 ⁇ M), etc.
  • the inventor of the present application was surprised to find that BLEB and PAB can unexpectedly maintain the original shape and survival rate of cardiomyocytes after cryopreservation and resuscitation.
  • the inventor of the present application uses BLEB and/or PAB or its physiologically acceptable salt as a key ingredient in the cryopreservation composition, and combines it with other specific ingredients to implement cryopreservation and recovery.
  • the first aspect of the present invention relates to a composition containing BLEB and/or PAB and an antifreeze agent.
  • the concentration of BLEB in the composition of the present invention is 1-50 ⁇ M, preferably 3-20 ⁇ M, more preferably 5-15 ⁇ M, particularly preferably 8-12 ⁇ M, and more particularly about 10 ⁇ M; the concentration of PAB is 1-100 ⁇ M, preferably 3-60 ⁇ M, more preferably 5-50 ⁇ M, particularly preferably about 10-20 ⁇ M.
  • the term "about” used in the context of this specification means a range that fluctuates 10% above and below the corresponding value. For example, if the concentration of a certain component is about 5mM, it means that its concentration is 4.5-5.5mM; if the concentration of a certain component is about 5-10mM, it means that its concentration is in the range of 4.5-11mM.
  • the antifreeze used in the present invention can reduce the formation of ice crystals in cells under low temperature conditions and reduce the electrolyte concentration in the uniced solution, thereby inhibiting cell damage.
  • the antifreeze in the composition of the present invention is selected from polyvinylpyrrolidone (PVP), hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), polysucrose (Ficoll) And dextran antifreeze, or any combination thereof.
  • the antifreeze agent is hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) or a combination thereof.
  • the concentration of HES is 20-100 mg/ml, preferably 25-60 mg/ml, more preferably 30-40 mg/ml, more preferably about 36 mg/ml;
  • the concentration of DMSO is 1-30 % By volume, more preferably 3-20% by volume, more preferably 5-15% by volume, more preferably 8-12% by volume, more preferably about 10% by volume.
  • the present invention optionally further contains one or more components selected from the following: energy components, metabolic regulators, acid-base regulators, and isotonic agents.
  • the composition of the present invention optionally contains energy substances, which provide necessary energy reserves for cells in the process of freezing and resuscitation.
  • 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.
  • composition of the present invention optionally contains a metabolic regulator, 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.
  • a metabolic regulator such as adenosine, allopurinol, reduced glutathione, taurine, creatine or any combination thereof.
  • the concentration of adenosine is 0.1-20 mM, preferably 1-10 mM, more preferably 3-8 mM, particularly preferably about 5 mM;
  • the concentration of allopurinol is 0.1-5 mM, preferably 0.5 ⁇ 2 mM, more preferably about 0.8 to 1.5 mM, particularly preferably about 1 mM;
  • the concentration of reduced glutathione is 0.5 to 10 mM, preferably 1 to 8 mM, more preferably 2 to 5 mM, particularly preferably about 3 mM; taurine
  • the concentration of creatine is 5-50mM, preferably 10-30mM, more preferably 15-25mM, especially about 20mM;
  • the concentration of creatine is 0.5-20mM, preferably 1-10mM, more preferably 3-8mM, particularly preferably about 5mM.
  • the composition of the present invention optionally contains acid-base regulators, which include buffer substances for maintaining a stable pH value of the composition, such as 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), and for directly adjusting the composition Acid-base substances with pH value, such as potassium hydroxide, sodium hydroxide, etc.
  • the acid-base regulator is HEPES.
  • the acid-base regulator is a combination of HEPES, potassium hydroxide, and sodium hydroxide.
  • the concentration of HEPES is 0.5-10 mM, preferably 1-8 mM, more preferably 3-6 mM, particularly preferably about 5 mM; the concentration of potassium hydroxide or sodium hydroxide (or the total concentration thereof) For example, it is 10 to 200 mM, preferably 50 to 150 mM, more preferably 80 to 120 mM, and particularly preferably about 100 mM.
  • the final pH 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.
  • the composition of the present invention optionally contains an isotonic agent, which is a substance that maintains the osmotic pressure of the composition to ensure the balance of moisture and electrolytes of the cells.
  • the isotonicity agent is magnesium sulfate (e.g., a hydrate of magnesium sulfate, such as its heptahydrate), potassium dihydrogen phosphate, calcium chloride, or a combination thereof.
  • the concentration of magnesium sulfate is 1-20 mM, preferably 2-10 mM, more preferably 3-8 mM, particularly preferably about 5 mM; the concentration of potassium dihydrogen phosphate is 5-100 mM, preferably 10 ⁇ 50mM, more preferably 20-30mM, particularly preferably about 25 ⁇ 26mM; the concentration of calcium chloride is 0.5-5mM, preferably 1-3mM, more preferably 1.5-2mM, particularly preferably about 1.8mM.
  • potassium dihydrogen phosphate is listed as an isotonic agent in this specification, it also has the function of pH adjustment and buffering, and can also be regarded as an acid-base regulator.
  • 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., isotonic), and can also be regarded as isotonic. Agent, etc.
  • composition of the present invention may optionally contain other ingredients known in the art or commonly used for cell preservation.
  • the composition of the present invention may include an apoptosis inhibitor, thereby improving the survival rate of cells during cryopreservation and resuscitation.
  • the apoptosis inhibitor can be Z-VAD-FMK, Emericase, Belnacasan, or a combination thereof.
  • the composition of the present invention contains the following ingredients: BLEB and/or PAB (or a physiologically acceptable salt thereof), and hydroxyethyl starch, DMSO, glucose, creatine, adenosine, other Purinol, reduced glutathione, taurine, 4-hydroxyethylpiperazine ethanesulfonic acid, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride.
  • the composition of the present invention may also contain other acid-base regulators, such as potassium hydroxide, sodium hydroxide and the like.
  • the composition of the present invention contains the following ingredients: 1-50 ⁇ M BLEB and/or 1-100 ⁇ M PAB, and 20-100 mg/ml hydroxyethyl starch, 1-30 vol% DMSO, 5-50mM glucose, 0.5-20mM creatine, 0.1-20mM adenosine, 0.1-5mM allopurinol, 0.5-10mM reduced glutathione, 5-50mM taurine, 0.5 ⁇ 10mM 4-hydroxyethylpiperazine ethanesulfonic acid, 1-20mM magnesium sulfate, 5-100mM potassium dihydrogen phosphate, 0.5-5mM calcium chloride.
  • the composition of the present invention may also contain other acid-base regulators, such as 10-200 mM potassium hydroxide and/or an appropriate amount of sodium hydroxide, to adjust the final pH of the composition to 7.0-7.8.
  • the composition of the present invention contains the following ingredients: 3-20 ⁇ M BLEB and/or 3-60 ⁇ M PAB, and 25-60mg/ml hydroxyethyl starch, 3-20% by volume DMSO, 10 ⁇ 30mM glucose, 1 ⁇ 10mM creatine, 1 ⁇ 10mM adenosine, 0.5 ⁇ 2mM allopurinol, 1 ⁇ 8mM reduced glutathione, 10 ⁇ 30mM taurine , 1-8mM 4-hydroxyethylpiperazine ethanesulfonic acid, 2-10mM magnesium sulfate, 10-50mM potassium dihydrogen phosphate, 1-3mM calcium chloride.
  • the composition of the present invention may also contain other acid-base regulators, such as 50-150 mM potassium hydroxide and/or an appropriate amount of sodium hydroxide, to adjust the final pH of the composition to 7.2-7.6.
  • the composition of the present invention contains the following ingredients: 5-15 ⁇ M (preferably 8-12 ⁇ M, more preferably about 10 ⁇ M) BLEB and/or 5-50 ⁇ M (preferably about 10-20 ⁇ M) PAB, And 30 ⁇ 40mg/ml hydroxyethyl starch, 5 ⁇ 15% by volume DMSO, 20 ⁇ 25mM glucose, 3 ⁇ 8mM creatine, 3 ⁇ 8mM adenosine, 0.8 ⁇ 1.5mM allopurinol , 2 ⁇ 5mM reduced glutathione, 15 ⁇ 25mM taurine, 3 ⁇ 6mM 4-hydroxyethylpiperazine ethanesulfonic acid, 3 ⁇ 8mM magnesium sulfate, 20 ⁇ 30mM potassium dihydrogen phosphate , 1.5-2mM calcium chloride.
  • the composition of the present invention may also contain other acid-base regulators, such as 80-120 mM potassium hydroxide and/or appropriate amount of sodium hydroxide, to adjust the final
  • the composition of the present invention is used as a cryopreservation reagent for cardiomyocytes of mammals, including but 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.
  • a cardiomyocyte cryopreservation kit which comprises any of the aforementioned compositions.
  • the box body of the cryopreservation kit is made of low-temperature resistant materials to ensure that no damage occurs during freezing and rewarming as a container for holding the composition.
  • the cryopreservation kit is a gradient cooling kit.
  • the cryopreservation kit (for example, a gradient cooling kit produced by Corning, USA) is provided with a cryopreservation tube for containing the composition.
  • Another aspect of the present invention relates to a method for cryopreserving mammalian cardiomyocytes, wherein any one of the aforementioned compositions is used.
  • the freezing method includes the following steps:
  • Pre-cooling step pre-cooling the composition of the present invention at 0 to 5°C (for example, 4°C), wherein the cardiomyocytes can be optionally recalcified to maintain their activity;
  • ⁇ Transfer step Centrifuge the cardiomyocytes placed in the culture medium and remove the supernatant, and suspend the pelleted cell mass obtained by centrifugation in the pre-cooled composition, so that the density of the cells 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 transferred to a cryopreservation container (for example, transferred to a cryopreservation tube placed in a cryopreservation kit);
  • a cryopreservation container for example, transferred to a cryopreservation tube placed in a cryopreservation kit
  • ⁇ Cooling step slowly lowering the temperature to the target temperature (for example -80°C), wherein the temperature can be lowered by a gradient cooling method, the gradient is, for example, about -0.8 to -2°C/min, preferably about -1 to- 1.2°C/min. It should be noted that the cooling rate should not be too fast, otherwise the cells will die easily.
  • the cells in the frozen state can be placed in a constant temperature water bath at an appropriate temperature (for example, human cardiomyocytes should be placed in The human body temperature is 37°C in a constant temperature water bath) to resuscitate the cells, and then store them in a known medium (for example, one or more selected from M199 series, MEM series, and DMEM series) by known methods. Seed culture medium), save it for subsequent use.
  • a known medium for example, one or more selected from M199 series, MEM series, and DMEM series
  • the medium also contains an appropriate amount of serum (for example, fetal bovine serum) or serum protein (for example, bovine serum albumin) and antibiotics (for example, penicillin and/or streptomycin).
  • serum for example, fetal bovine serum
  • serum protein for example, bovine serum albumin
  • antibiotics for example, penicillin and/or streptomycin
  • the culture medium also contains BLEB and/or PAB.
  • the volume fraction of the aforementioned serum (such as fetal bovine serum) in the medium may be 1%-20%, preferably 2%-15%, more preferably 3%-12%, Especially preferably about 5%-10%;
  • the concentration of the aforementioned serum protein (for example, bovine serum albumin) in the medium may be 0.1-10g/ml, preferably 0.2-5g/ml, more preferably 0.3-1g/ml, Particularly preferably about 0.5g/ml;
  • the concentration of penicillin in the medium may be 10-500U/ml, preferably 20-400U/ml, more preferably 50-300U/ml, particularly preferably about 100-200U/ml, about 100 U/ml or about 200 U/ml;
  • the concentration of streptomycin in the medium may be 10-500 ⁇ g/ml, preferably 20-400 ⁇ g/ml, more preferably 50-300 ⁇ g/ml, particularly preferably about 100-200 ⁇ g/ml, about
  • Element concentration Element concentration HES 36mg/ml Reduced glutathione 3mM DMSO 10v/v% Taurine 20mM Potassium Dihydrogen Phosphate 25.8mM Creatine 5mM Magnesium sulfate 7 hydrate 5mM glucose 22mM Calcium chloride 1.8mM HEPES 5mM BLEB 10 ⁇ M Potassium hydroxide 100mM
  • the osmotic pressure is about 300mOsm/kg
  • gradient cooling box produced by Corning, USA
  • the human cardiomyocytes used in this example were collected from the left atrial appendages of three male patients (aged 51 ⁇ 4 years) undergoing mitral valvuloplasty, mitral valve replacement, or coronary artery bypass grafting.
  • the medium contains the following components: MEM-HEPES-GlutaMAX (Thermo, 42360032), 10% by volume fetal bovine serum (10099141C), 10 ⁇ M BLEB (Selleck, S7099), 100U/ml penicillin and 100 ⁇ g/ml streptomycin ( Gibco, 15240062).
  • Example 3 The effect of HES concentration on the effect of cryopreservation and resuscitation
  • the purpose of this experiment is to investigate the effects of BLEB and PAB on the survival rate and morphology of cardiomyocytes in vitro, so as to predict the similarity of their roles in the process of cryopreservation and resuscitation of cardiomyocytes.
  • fetal bovine serum 100 U/ml penicillin and 100 ⁇ g/ml streptomycin were added to the M199 medium (purchased from Sigma), and the medium thus obtained was used as the basic medium.
  • the experiment was divided into 6 groups, and DMSO (as a control group) and 10 ⁇ M BLEB and 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, 50 ⁇ M PAB were added to the basal medium.
  • the isolated human cardiomyocytes were divided into 6 groups, and the cells were plated on a 48-well cell culture plate coated with 200 ⁇ g/ml laminin in advance using 6 culture media, and placed in a 37°C cell incubator (5% CO 2 , The relative saturated humidity is 95%) cultured for 7 days, the rod-shaped rate of cardiomyocytes was calculated, and the results are shown in Figure 4.
  • PAB which is a BLEB derivative
  • PAB can also achieve similar effects to BLEB during the freezing and resuscitation of cardiomyocytes using the method of the present invention.
  • cryopreservation reagent and cryopreservation method of the present invention can achieve a good survival rate even for human cardiomyocytes that are most difficult to be cryopreserved and resuscitated, and maintain the cell morphology well.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Rheumatology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Cardiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Physiology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

涉及用于冻存心肌细胞的组合物，其含有BLEB和/或PAB(或其生理学上可接受的盐)和抗冻剂。还涉及包含所述组合物的心肌细胞冻存试剂盒，用前述组合物进行哺乳动物心肌细胞的冻存的方法，以及前述组合物在制备用于冻存哺乳动物心肌细胞的试剂中的用途。

Description

用于心肌细胞冻存的组合物
本申请所涉及的科研项目系中国医学科学院医学与健康科技创新工程项目，项目编号为2017-I2M-1-003。
技术领域
本发明涉及用于冻存心肌细胞的组合物，其含有BLEB和/或PAB(或其生理学上可接受的盐)和抗冻剂。本发明还涉及包含所述组合物的心肌细胞冻存试剂盒，用前述组合物进行哺乳动物心肌细胞的冻存的方法，以及前述组合物在制备用于冻存哺乳动物心肌细胞的试剂中的用途。
背景技术
心血管疾病导致的死亡占因病死亡总数的40％以上，高于肿瘤及其他疾病，是人类生命健康的最大杀手。《中国心血管病报告2018》显示我国心血管病患病率持续上升，死亡率仍居首位，高于肿瘤及其他疾病(非专利文献1)。因此，心血管疾病的防治对人类健康而言至关重要，针对心血管疾病防治的科学研究的重要性自然不言而喻。
然而，在心血管领域的病理机制研究与新型药物研发中，已有多年未能取得突破，传统心血管病药物治疗进展甚微，心肌保护药物临床试验也频告失败。造成以上现象的主要原因之一为临床研究所用细胞模型的局限性。
研究任何一种疾病的预防和治疗，都要有好的细胞模型，心血管疾病的研究模型即为哺乳动物、特别是人的心肌细胞。然而，不同于其它体细胞，心肌细胞本身具有运动性强、耗氧高的特点，这一特点决定了其存活过程中需要消耗大量能量并且产生大量代谢产物，导致心肌细胞不易通过培养的方式获得；另外，虽然理论上可以通过人胚胎干细胞或诱导多能干细胞的定向分化来获得心肌细胞，但该方法存在分化不完全、缺少表观遗传修饰等问题(非专利文献2、3)，从而无法如实反映疾病的真实状态。这使得心肌细胞只能从动物体或人体获取，而且必须是现用现取。对于动物而言，取出心肌细胞的动物自然无法存活；而对于人类而言，心肌细胞只能从受试者获得，其来源更是非常珍贵的。况且，心肌细胞相比于其他体细胞存活期短得多，而这些现取的细胞又常 常不能一次性用完、有时甚至无法立即使用，导致浪费在所难免，这更加剧了心肌细胞供不应求的局面。
特别是，与其它哺乳动物(例如啮齿类动物)的心肌细胞相比，人心肌细胞的冻存和复苏更是挑战性极高的工作。与其它物种相比，人类的心肌细胞对缺血缺氧耐受力甚至更低，这意味着人心肌细胞的冻存复苏比其它物种的心肌细胞难度更大，一旦冻存复苏过程中稍有处理不当，甚至可能在短短数分钟内即发生细胞凋亡或坏死。由于存在极大的技术难度，使得到目前为止还从未有任何将人心肌细胞进行冻存并成功复苏的先例。
为解决上述现实问题，为心血管疾病相关药物的临床研究和新药开发保障充足的心肌细胞供应，需要针对哺乳动物、特别是人类的心肌细胞开发一种高效的冻存方法，并研发相应的用于冻存的组合物(例如冻存试剂)，以使得哺乳动物、特别是人的心肌细胞的回收利用成为可能，节约宝贵的资源。
对于普通的细胞来说，细胞冻存复苏是“慢冻快融”的过程。一方面，细胞的冻存需要“慢冻”。若细胞温度骤然降至零度以下，细胞器会发生脱水，并且细胞中可溶性物质浓度升高，在细胞内形成冰晶，造成细胞损伤；而以缓慢的梯度进行降温冻存，可使细胞缓慢脱水，细胞内不会产生大的冰晶。另外，可在冻存试剂中加入抗冻剂(如聚蔗糖、右旋糖苷类等)，通过抗冻剂与溶液中的水分子的结合来降低冰点，减少细胞内冰晶的形成，并降低未结冰溶液中电解质的浓度，由此抑制细胞损伤，使细胞的超低温保存成为可能。目前常用的抗冻剂包括聚乙烯吡咯烷酮(polyvinyl pyrrolidone，PVP)、聚蔗糖(Ficoll)以及一些右旋糖苷类物质等。另一方面，细胞的复苏应“快融”，这样可以保证细胞外结晶在很短的时间内即融化，避免由于缓慢融化使水分渗入细胞内形成胞内再结晶对细胞造成损伤(非专利文献4、5)。
然而，上述内容仅是针对普通的体细胞进行冻存和复苏的一般理论。对于心肌细胞、特别是人心肌细胞而言，现有技术不存在任何能够以高存活率进行冻存和复苏并保持细胞形态的技术，这严重地阻碍了相关科研工作的开展，无法满足为临床上的疾病诊断和治疗提供研究基础的需要。
相关文献
非专利文献1：胡盛寿、高润霖、刘力生等，《中国心血管病报告2018》概要，中国循环杂志，2019，34(3)：209-220.
非专利文献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.
非专利文献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.
非专利文献4：LOVELOCK JE,BISHOP MW“Prevention of freezing damage to living cells by dimethyl sulphoxide”,Nature.1959 May 16；183(4672):1394-5.
非专利文献5：Meryman HT,“Cryopreservation of living cells:principles and practice”,Transfusion,2007 May；47(5):935-45.
发明内容
本申请的发明人考虑到临床上的实际需要以及人心肌细胞的特点，为解决人心肌细胞的冻存复苏这一课题，对人心肌细胞的冻存技术以及相关的试剂进行了深入研究并不断优化，从而开发了一种用于冻存心肌细胞的组合物。
因此，本发明包括但不限于以下技术内容：
1、一种组合物，其含有BLEB和/或PAB或其生理学上可接受的盐，以及抗冻剂
Figure PCTCN2021080535-appb-000001
2、如技术项1所述的组合物，其还含有选自以下的一种或多种成分：能量成分、代谢调节剂、酸碱调节剂、等渗剂。
3、如前述技术项中任一项所述的组合物，其含有以下成分：BLEB和/或PAB或其生理学上可接受的盐、羟乙基淀粉、DMSO、葡萄糖、肌酸、腺嘌呤核苷、别嘌呤醇、还原型谷胱甘肽、牛磺酸、4-羟乙基哌嗪乙磺酸、硫酸镁、磷酸二氢钾、氯化钙。
4、如前述技术项中任一项所述的组合物，其含有以下成分：1～50μM的BLEB或1～100μM的PAB，以及20～100mg/ml的羟乙基淀粉、1～30体积％的DMSO、5～50mM的葡萄糖、0.5～20mM的肌酸、0.1～20mM的腺嘌呤核苷、0.1～5mM的别嘌呤醇、0.5～10mM的还原型谷胱甘肽、5～50mM的牛磺酸、0.5～10mM的4-羟乙基哌嗪乙磺酸、1～20mM的硫酸镁、5～100mM的磷酸二氢钾、0.5～5mM的氯化钙。
5、如前述技术项中任一项所述的组合物，其pH值为7.0～7.8。
6、一种心肌细胞冻存试剂盒，其包含如技术项1～5中任一项所述的组合物。
7、一种对哺乳动物心肌细胞进行冻存的方法，其中使用如技术项1～5中任一项所述的组合物。
8、如技术项7所述的方法，其中所述哺乳动物为人。
9、如技术项1～5中任一项所述的组合物在制备用于冻存哺乳动物心肌细胞的试剂中的用途。
10、如技术项9所述的用途，其中所述哺乳动物为人。
通过使用本发明的组合物，能够高效地冻存并复苏哺乳动物心肌细胞，并且使经冻存并复苏后的细胞仍能够维持理想的存活率和形态。特别是，通过使用本发明的组合物，首次实现了人心肌细胞的冻存后复苏，并且在复苏后获得了很好的存活率及细胞形态。由此，本发明可以为心血管疾病研究、临床转化、药物研发和个体化治疗提供坚实的基础。
附图说明
图1：实施例1～2的冻存复苏操作后心肌细胞的复苏率。
图2：实施例1～2的冻存复苏操作前后心肌细胞状态的对比图。
图3：实施例3中使用含不同浓度HES的冻存试剂进行冻存复苏的情况下获得的心肌细胞复苏率。
图4：是示出BLEB和PAB对心肌细胞杆状率影响的图。
具体实施方式
本申请发明人对进行了大量广泛而深入的研究，进行了大量的实验与摸索，出乎意料地发现BLEB或其衍生物PAB能够很好地解决本发明所要解决的技术课题。
BLEB又称为“(-)-Blebbistatin”，是一种非肌肉肌球蛋白II抑制剂和细胞渗透性抑制剂，已知其可以用于抑制小鼠心肌细胞收缩；另外，在心血管生理学研究中，BLEB作为特异性的解偶联剂已经被广泛应用。PAB又称为“para-aminoblebbistatin”，是BLEB的衍生物，具有与BLEB类似的结构和性质。但BLEB存在一些不利的化学特性，例如，对光不稳定、具有光毒性和细胞毒性、高荧光和低水溶性(溶解度仅为10.9±0.9μM)等，正因为存在这些不利特性，研究人员对BLEB及其衍生物PAB的应用存在偏见，迄今为止尚未有将BLEB和/或PAB用于心肌细胞、特别是人心肌细胞的冻存复苏的报道。
然而，本申请发明人却惊讶地发现，BLEB及PAB能够使心肌细胞在冻存并复苏后出人意料地维持原有的形态和存活率。在此基础上，本申请发明人将BLEB和/或PAB或其生理学上可接受的盐作为冻存组合物中的关键成分，与其他特定成分进行组合来实施冻存复苏，由此很好地解决本发明所要解决的上述课题，从而完成了本发明。
本发明的第一个方面涉及一种组合物，其含有BLEB和/或PAB和抗冻剂。
在一个实施方式中，在单独或组合使用的情况下，本发明的组合物中BLEB的浓度为1～50μM，优选3～20μM，更优选5～15μM，特别优选8～12μM，更特别优选约10μM；PAB的浓度为1～100μM，优选3～60μM，更优选5～50μM，特别优选约10～20μM。
本说明书上下文中所用的术语“约”表示在相应数值上下浮动10％的范围。例如，若某种成分的浓度为约5mM，表明其浓度为4.5～5.5mM；若某种成分的浓度范围为约5～10mM，表明其浓度范围为4.5～11mM。
如前文所述，本发明所用的抗冻剂可在低温条件下减少细胞内冰晶的形成，并降低未结冰溶液中电解质的浓度，由此抑制细胞损伤。在一个实施方式中，本发明的组合物中的抗冻剂选自聚乙烯吡咯烷酮(polyvinyl pyrrolidone，PVP)、羟乙基淀粉(HES)、二甲基亚砜(DMSO)、聚蔗糖(Ficoll)以及右旋糖苷类抗冻剂，或其任意组合。在一个特别优选的实施方式中，抗冻剂为羟乙基淀粉(HES)、二甲基亚砜(DMSO)或其组合。在单独或组合使用的情况下，HES的浓度为20～100mg/ml，优选为25～60mg/ml，更优选为30～40mg/ml，更优选约36mg/ml；DMSO的浓度为1～30体积％，更优选3～20体积％，更优选5～15体积％，更优选8～12体积％，更优选约10体积％。
在一个实施方式中，本发明的还任选含有选自以下的一种或多种成分：能量成分、代谢调节剂、酸碱调节剂、等渗剂。
本发明的组合物中任选含有能量物质，其为细胞在冻存和复苏过程中提供必要的能量储备。在一个优选实施方式中，所述能量物质为葡萄糖，其浓度例如为5～50mM，优选为10～30mM，更优选为20～25mM，特别优选约22mM。
本发明的组合物中任选含有代谢调节剂，例如腺嘌呤核苷、别嘌呤醇、还原型谷胱甘肽、牛磺酸、丙酮酸钠、胰岛素(insulin)、肌酸(Creatine)、β-氨基乙磺酸(Taurine)、L-左旋肉碱(L-carnitine)等，其有助于细胞在冻存和复苏过程中调节能量代谢(如糖代谢)。在一个优选实施方式中，所述代谢调节剂为腺嘌呤核苷、别嘌呤醇、还原型谷胱甘肽、牛磺酸、肌酸或其任意组合。在单独或组合使用的情况下，腺嘌呤核苷的浓度为0.1～20mM，优选1～10mM，更优选为3～8mM，特别优选约5mM；别嘌呤醇的浓度为0.1～5mM，优选为0.5～2mM，更优选约0.8～1.5mM，特别优选约1mM；还原型谷胱甘肽的浓度为0.5～10mM，优选为1～8mM，更优选为2～5mM，特别优选约3mM；牛磺酸的浓度为5～50mM，优选为10～30mM，更优选为15～25mM，特别优选约20mM；肌酸的浓度为0.5～20mM，优选为1～10mM，更优选为3～8mM，特别优选约5mM。
本发明的组合物中任选含有酸碱调节剂，其包括用于维持组合物pH值稳定的缓冲物质，如4-羟乙基哌嗪乙磺酸(HEPES)，以及用于直接调节组合物pH值的酸碱物质，如氢氧化钾、氢氧化钠等。在一个优选实施方式中，所述酸碱调节剂为HEPES。在另一个优选实施方式中，所述酸碱调节剂为HEPES、氢氧化钾、氢氧化钠的组合。在单独或组合使用的情况下，HEPES的浓度为0.5～10mM，优选为1～8mM，更优选为3～6mM，特别优选约5mM；氢氧化钾或氢氧化钠的浓度(或其总浓度)例如为10～200mM，优选为50～150mM，更优选为80～120mM，特别优选为约100mM。另外，通过酸碱调节剂的使用，将组合物的最终pH值调节至7.0～7.8，优选为7.2～7.6，更优选为7.3～7.5，特别优选为约7.4。参考上述各成分及其浓度范围，本领域技术人员能够根据具体情况选择所用的酸碱调节剂及其用量。
本发明的组合物中任选含有等渗剂，其为维持组合物渗透压、以确保细胞的水分和电解质平衡的物质。在该方面的一个优选实施方式中，所述等渗剂为 硫酸镁(例如硫酸镁的水合物，例如其七水合物)、磷酸二氢钾、氯化钙或其组合。在单独或组合使用的情况下，硫酸镁的浓度为1～20mM，优选为2～10mM，更优选为3～8mM，特别优选约5mM；磷酸二氢钾的浓度为5～100mM，优选为10～50mM，更优选为20～30mM，特别优选约25～26mM；氯化钙的浓度为0.5～5mM，优选为1～3mM，更优选为1.5～2mM，特别优选约1.8mM。
需要说明，某些成分在本发明的冻存制剂中可能具有不止一种作用。例如本说明书中虽然将磷酸二氢钾列为等渗剂，但其也具有pH调节缓冲的作用，也可视为酸碱调节剂。类似地，本说明书中虽然将氯化钙列为代谢调节剂、将葡萄糖列为能量物质，但这些物质同时也会起到维持渗透压的作用(即等渗作用)，也可视为等渗剂，等等。
本发明的组合物中还可任选含有其它本领域已知的或通常用于细胞保存的成分。例如，本发明的组合物中可包含凋亡抑制剂，由此提高细胞在冻存和复苏过程中的存活率。例如，凋亡抑制剂可为Z-VAD-FMK、Emericase、Belnacasan或其组合。
在一个实施方式中，本发明的组合物含有以下成分：BLEB和/或PAB(或其生理学上可接受的盐)，以及羟乙基淀粉、DMSO、葡萄糖、肌酸、腺嘌呤核苷、别嘌呤醇、还原型谷胱甘肽、牛磺酸、4-羟乙基哌嗪乙磺酸、硫酸镁、磷酸二氢钾、氯化钙。任选地，本发明的组合物还可含有其它酸碱调节剂，例如氢氧化钾、氢氧化钠等。
在一个优选实施方式中，本发明的组合物含有以下成分：1～50μM的BLEB和/或1～100μM的PAB，以及20～100mg/ml的羟乙基淀粉、1～30体积％的DMSO、5～50mM的葡萄糖、0.5～20mM的肌酸、0.1～20mM的腺嘌呤核苷、0.1～5mM的别嘌呤醇、0.5～10mM的还原型谷胱甘肽、5～50mM的牛磺酸、0.5～10mM的4-羟乙基哌嗪乙磺酸、1～20mM的硫酸镁、5～100mM的磷酸二氢钾、0.5～5mM的氯化钙。任选地，本发明的组合物还可含有其它酸碱调节剂，例如10～200mM的氢氧化钾和/或适量的氢氧化钠，以将组合物的最终pH值调节至7.0～7.8。
在一个更优选的实施方式中，本发明的组合物含有以下成分：3～20μM的BLEB和/或3～60μM的PAB，以及25～60mg/ml的羟乙基淀粉、3～20体积％的DMSO、10～30mM的葡萄糖、1～10mM的肌酸、1～10mM的腺嘌呤核苷、 0.5～2mM的别嘌呤醇、1～8mM的还原型谷胱甘肽、10～30mM的牛磺酸、1～8mM的4-羟乙基哌嗪乙磺酸、2～10mM的硫酸镁、10～50mM的磷酸二氢钾、1～3mM的氯化钙。任选地，本发明的组合物还可含有其它酸碱调节剂，例如50～150mM的氢氧化钾和/或适量的氢氧化钠，以将组合物的最终pH值调节至7.2～7.6。
在一个特别优选的实施方式中，本发明的组合物含有以下成分：5～15μM(优选8～12μM、更优选约10μM)的BLEB和/或5～50μM(优选约10～20μM)的PAB，以及30～40mg/ml的羟乙基淀粉、5～15体积％的DMSO、20～25mM的葡萄糖、3～8mM的肌酸、3～8mM的腺嘌呤核苷、0.8～1.5mM的别嘌呤醇、2～5mM的还原型谷胱甘肽、15～25mM的牛磺酸、3～6mM的4-羟乙基哌嗪乙磺酸、3～8mM的硫酸镁、20～30mM的磷酸二氢钾、1.5～2mM的氯化钙。任选地，本发明的组合物还可含有其它酸碱调节剂，例如80～120mM的氢氧化钾和/或适量的氢氧化钠，以将组合物的最终pH值调节至7.3～7.5。
在一个实施方式中，本发明的组合物用作哺乳动物心肌细胞的冻存试剂，所述哺乳动物包括但不限于小鼠、大鼠、狗、猴和人。在一个优选实施方式中，本发明的组合物用作人心肌细胞的冻存试剂。
本发明的另一个方面涉及一种心肌细胞冻存试剂盒，其包含前述任一种组合物。在一个实施方式中，该冻存试剂盒的盒体由耐低温材料制成，以确保在作为盛放组合物的容器进行冷冻与回温的过程中不会发生损坏。在一个实施方式中，该冻存试剂盒为梯度降温盒。在一个实施方式中，该冻存试剂盒(例如美国Corning公司生产的梯度降温盒)中放置有用于容纳组合物的冻存管。
本发明的另一个方面涉及一种对哺乳动物心肌细胞进行冻存的方法，其中使用前述任一种组合物。在一个实施方式中，所述冻存方法包括以下步骤：
·预冷步骤：在0～5℃(例如4℃)将本发明的组合物进行预冷，其中可任选对心肌细胞进行复钙以保持其活度；
·转移步骤：将置于培养基中的心肌细胞离心并除去上清液，并将离心得到的沉淀细胞团悬浮于预冷的组合物中，使组合物中细胞的密度例如为0.5×10 6/ml至5×10 6/ml、优选1×10 6/ml至2×10 6/ml，然后转移至冻存容器内(例如转移至置于冻存试剂盒中的冻存管内)；
·降温步骤：缓慢将温度降至目标温度(例如-80℃)，其中可以通过梯 度降温的方式进行降温，所述梯度例如为约-0.8～-2℃/分钟，优选为约-1～-1.2℃/分钟。要注意降温的速度不宜过快，否则细胞容易死亡。
在通过前述方法将细胞冻存一段时间(例如1～72小时、优选12～48小时)之后，可将冷冻状态下的细胞置于适当温度的恒温水浴锅中(例如，人心肌细胞应置于人体体温即37℃的恒温水浴锅中)即可使细胞复苏，其后通过已知的方法将其保存于已知的培养基(例如选自M199系列、MEM系列、DMEM系列的一种或多种培养基)中，留待后续使用即可。在一个实施方式中，所述培养基中还含有适量的血清(例如胎牛血清)或血清蛋白(例如牛血清白蛋白)及抗生素(例如青霉素和/或链霉素)。在一个优选实施方式中，所述培养基中还含有BLEB和/或PAB。其中，在单独或组合使用的情况下，前述血清(例如胎牛血清)占所述培养基的体积分数可为1％-20％，优选2％-15％，更优选3％-12％，特别优选约5％-10％；前述血清蛋白(例如牛血清白蛋白)在所述培养基中的浓度可为0.1-10g/ml，优选0.2-5g/ml，更优选0.3-1g/ml，特别优选约0.5g/ml；青霉素在所述培养基中的浓度可为10-500U/ml，优选20-400U/ml，更优选50-300U/ml，特别优选约100-200U/ml、约100U/ml或约200U/ml；链霉素在所述培养基中的浓度可为10-500μg/ml，优选20-400μg/ml，更优选50-300μg/ml，特别优选约100-200μg/ml、约100μg/ml或约200μg/ml；BLEB在所述培养基中的浓度可为1～50μM，优选3～20μM，更优选5～15μM，特别优选8～12μM，更特别优选约10μM；PAB在所述培养基中的浓度可为1～100μM，优选3～60μM，更优选5～50μM，特别优选约10～20μM。
本发明的更具体的实施方式将通过以下实施例进行例示性解释说明，但应认识到这些实施例并非意在限制本发明的范围。
实施例
实施例1：心肌细胞的冻存
首先，按照如下配方来配制细胞冻存试剂：
成分 浓度 成分 浓度
HES 36mg/ml 还原型谷胱甘肽 3mM
DMSO 10v/v％ 牛磺酸 20mM
磷酸二氢钾 25.8mM 肌酸 5mM
7水硫酸镁 5mM 葡萄糖 22mM
氯化钙 1.8mM HEPES 5mM
BLEB 10μM 氢氧化钾 100mM
腺嘌呤核苷 5mM 氢氧化钠 适量至pH为7.4
别嘌呤醇 1mM 去离子水  
注：渗透压为约300mOsm/kg；
提前将配制好的冻存试剂、梯度降温盒(美国Corning公司生产)和低温离心机(Eppendorf centrifuge 5804R，德国)预冷至4℃。将新鲜分离、收集到的心肌细胞离心(100×g，4℃，1分钟)，去掉上清，按约1×10 6/ml细胞密度缓慢加入细胞冻存试剂，轻轻吹打混匀，将细胞悬液转移至细胞冻存管中，置于4℃冰箱，静置15分钟后，放在梯度降温盒内，将降温盒放在-80℃冰箱梯度降温，12小时后转移至-196℃液氮中。
本实施例中所使用的人心肌细胞通过三位进行二尖瓣成形术、二尖瓣置换术或冠状动脉旁路移植术的男性患者(年龄为51±4岁)的左心耳采集。
实施例2：心肌细胞的复苏
将恒温水浴锅(上海博讯，SSW-420-2S，中国)的水温调节为37℃进行预热。按照细胞悬液体积与培养基体积为1/10准备一定体积培养基。所述培养基包含以下成分：MEM-HEPES-GlutaMAX(Thermo，42360032)，10体积％的胎牛血清(10099141C)，10μM BLEB(Selleck，S7099)，100U/ml青霉素和100μg/ml链霉素(Gibco，15240062)。
接下来，从液氮中取出在实施例1中保存60小时的盛放冻存细胞的冻存管，迅速将其放入37℃恒温水浴锅中振荡，力度适中，在约1.5分钟之内融化细胞悬液。用移液器(Eppendorf，德国)吸取与细胞悬液等体积的培养基，缓慢加入冻存管中，轻轻混匀，然后将所有细胞悬液转移至提前准备好的培养基中，移液器枪头伸入液面下轻轻滴入，约2-4滴/秒，滴完后轻轻混匀，放入离心机中离心(100×g，4℃，2-3分钟)，弃上清，取1-2ml培养基轻轻吹匀沉淀细胞团。
如图1所示，在实施例1～2的冻存及复苏操作之前和之后对人心肌细胞进行计数，并通过计算得出细胞复苏率(即冻存复苏后杆状细胞数与冻存前细胞数之比)为63.5±12.05％。另外，显微镜(莱卡，DMI4000B，10X)下可以明显看出复苏后的细胞基本保持了冻存前的细胞形态(图2)。以上结果说明，本发明的细胞冻存试剂能够成功冻存和复苏心肌细胞，并很好地保持细胞形态。
实施例3：HES浓度对冻存复苏效果的影响
通过实验来研究向心肌细胞冻存试剂中加入不同浓度HES对冻存复苏效 果的影响。该实验平行三组进行，所用心肌细胞通过3位冠状动脉旁路移植术男性患者(年龄为61±6岁)的左心耳采集，所用的冻存试剂浓度分别为36mg/ml、60mg/ml、100mg/ml，其它冻存试剂成分及操作步骤与实施例1和2中所述相同。三组实验所获得的杆状细胞率分别为52.69±1.44％、51.09±6.49％、53.68±2.82％，表明在HES浓度对心肌细胞复苏效果无显著影响。
实施例4：BLEB与PAB的效果对比
本实验的目的是考察BLEB与PAB在心肌细胞离体存活过程中对细胞存活率和形态的影响，从而预测两者在心肌细胞冻存复苏过程中的作用的相似性。
在M199培养基(购自Sigma公司)中加入10％胎牛血清、100U/ml青霉素和100μg/ml链霉素，将由此得到的培养基作为基础培养基。实验分为6组，在基础培养基中分别加入DMSO(作为对照组)以及10μM的BLEB和5μM、10μM、20μM、50μM的PAB。将分离得到的人心肌细胞分为6组，使用6种培养基将细胞铺于提前使用200μg/ml层粘连蛋白包被的48孔细胞培养板，置于37℃细胞培养箱(5％CO 2，相对饱和湿度为95％)中培养7天，计算心肌细胞杆状率，结果如图4所示。
由图4可知，5～50μM的PAB组达到了与10μM的BLEB组接近甚至更优的效果，也即，两者对人心肌细胞存活能力的影响是大致相当的。由此可知，作为BLEB衍生物的PAB在使用本发明的方法对心肌细胞进行冻存复苏过程中也能够取得与BLEB类似的效果。
前述实施例表明，本发明的冻存试剂及冻存方法即使对于最难冻存复苏的人心肌细胞而言都能够实现良好的存活率，并且很好地维持细胞的形态。

Claims (10)

  1. 一种组合物，其含有BLEB和/或PAB或其生理学上可接受的盐，以及抗冻剂
    Figure PCTCN2021080535-appb-100001
  2. 如权利要求1所述的组合物，其还含有选自以下的一种或多种成分：能量成分、代谢调节剂、酸碱调节剂、等渗剂。
  3. 如前述权利要求中任一项所述的组合物，其含有以下成分：BLEB和/或PAB或其生理学上可接受的盐、羟乙基淀粉、DMSO、葡萄糖、肌酸、腺嘌呤核苷、别嘌呤醇、还原型谷胱甘肽、牛磺酸、4-羟乙基哌嗪乙磺酸、硫酸镁、磷酸二氢钾、氯化钙。
  4. 如前述权利要求中任一项所述的组合物，其含有以下成分：1～50μM的BLEB或1～100μM的PAB，以及20～100mg/ml的羟乙基淀粉、1～30体积％的DMSO、5～50mM的葡萄糖、0.5～20mM的肌酸、0.1～20mM的腺嘌呤核苷、0.1～5mM的别嘌呤醇、0.5～10mM的还原型谷胱甘肽、5～50mM的牛磺酸、0.5～10mM的4-羟乙基哌嗪乙磺酸、1～20mM的硫酸镁、5～100mM的磷酸二氢钾、0.5～5mM的氯化钙。
  5. 如前述权利要求中任一项所述的组合物，其pH值为7.0～7.8。
  6. 一种心肌细胞冻存试剂盒，其包含如权利要求1～5中任一项所述的组合物。
  7. 一种对哺乳动物心肌细胞进行冻存的方法，其中使用如权利要求1～5中任一项所述的组合物。
  8. 如权利要求7所述的方法，其中所述哺乳动物为人。
  9. 如权利要求1～5中任一项所述的组合物在制备用于冻存哺乳动物心肌细胞的试剂中的用途。
  10. 如权利要求9所述的用途，其中所述哺乳动物为人。
PCT/CN2021/080535 2020-04-09 2021-03-12 用于心肌细胞冻存的组合物 WO2021203911A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US17/995,409 US20230189794A1 (en) 2020-04-09 2021-03-12 Composition for cryopreservation of myocardial cells
EP21783704.6A EP4134428A4 (en) 2020-04-09 2021-03-12 COMPOSITION FOR CRYOPRESERVATION OF MYOCARDIAL CELLS
CN202180027339.9A CN115698263A (zh) 2020-04-09 2021-03-12 用于心肌细胞冻存的组合物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNPCT/CN2020/083872 2020-04-09
CN2020083872 2020-04-09

Publications (1)

Publication Number Publication Date
WO2021203911A1 true WO2021203911A1 (zh) 2021-10-14

Family

ID=78024012

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/080535 WO2021203911A1 (zh) 2020-04-09 2021-03-12 用于心肌细胞冻存的组合物

Country Status (4)

Country Link
US (1) US20230189794A1 (zh)
EP (1) EP4134428A4 (zh)
CN (1) CN115698263A (zh)
WO (1) WO2021203911A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103201377A (zh) * 2010-11-09 2013-07-10 隆萨科隆有限公司 控制细胞与基质结合的方法
US20150191697A1 (en) * 2009-10-19 2015-07-09 Cellular Dynamics International, Inc. Cardiomyocyte production
CN107636149A (zh) * 2015-04-03 2018-01-26 普罗帕格尼克斯公司 上皮细胞的离体增殖
WO2019144968A1 (zh) * 2018-01-29 2019-08-01 中国科学院动物研究所 一种细胞诱导的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111778205B (zh) * 2019-04-04 2022-09-30 青岛百洋智心科技有限公司 人心肌细胞分离试剂、培养基、分离方法和培养方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150191697A1 (en) * 2009-10-19 2015-07-09 Cellular Dynamics International, Inc. Cardiomyocyte production
CN103201377A (zh) * 2010-11-09 2013-07-10 隆萨科隆有限公司 控制细胞与基质结合的方法
CN107636149A (zh) * 2015-04-03 2018-01-26 普罗帕格尼克斯公司 上皮细胞的离体增殖
WO2019144968A1 (zh) * 2018-01-29 2019-08-01 中国科学院动物研究所 一种细胞诱导的方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
D. M. DELAUGHTERA. G. BICKH. WAKIMOTOD. MCKEANJ. M. GORHAMI. S. KATHIRIYAJ. T. HINSONJ. HOMSYJ. GRAYW. PU: "Single-Cell Resolution of Temporal Gene Expression During Heart Development", DEV CELL, vol. 39, 2016, pages 480 - 90, XP029819206, DOI: 10.1016/j.devcel.2016.10.001
HU SHENGSHOUGAO RUNLINLIU LISHENG ET AL.: "Summary of Report on Cardiovascular Diseases in China 2018", CHINESE CIRCULATION JOURNAL, vol. 34, no. 3, 2019, pages 209 - 220
LOVELOCK JEBISHOP MW: "Prevention of freezing damage to living cells by dimethyl sulphoxide", NATURE, vol. 183, no. 4672, 16 May 1959 (1959-05-16), pages 1394 - 5
MERYMAN HT: "Cryopreservation of living cells: principles and practice", TRANSFUSION, vol. 47, no. 5, May 2007 (2007-05-01), pages 935 - 45, XP007916229, DOI: 10.111/j.1537-2995-2007.01212.x
RAUSCHER ANNA Á.; GYIMESI MáTé; KOVáCS MIHáLY; MáLNáSI-CSIZMADIA ANDRáS: "Targeting Myosin by Blebbistatin Derivatives: Optimization and Pharmacological Potential", TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER, AMSTERDAM, NL, vol. 43, no. 9, 26 July 2018 (2018-07-26), AMSTERDAM, NL , pages 700 - 713, XP085447981, ISSN: 0968-0004, DOI: 10.1016/j.tibs.2018.06.006 *
See also references of EP4134428A4
VÁRKUTI BOGLÁRKA H., KÉPIRÓ MIKLÓS, HORVÁTH ISTVÁN ÁDÁM, VÉGNER LÁSZLÓ, RÁTI SZILVIA, ZSIGMOND ÁRON, HEGYI GYÖRGY, LENKEI ZSOLT, V: "A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative", SCIENTIFIC REPORTS, vol. 6, no. 1, 1 September 2016 (2016-09-01), XP055856920, DOI: 10.1038/srep26141 *
X. YANGL. PABONC. E. MURRY: "Engineering Adolescence: Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes", CIRC RES, vol. 114, 2014, pages 511 - 23, XP055831814, DOI: 10.1161/CIRCRESAHA.114.300558

Also Published As

Publication number Publication date
EP4134428A1 (en) 2023-02-15
CN115698263A (zh) 2023-02-03
US20230189794A1 (en) 2023-06-22
EP4134428A4 (en) 2024-05-15

Similar Documents

Publication Publication Date Title
Ma et al. Advanced biomaterials in cell preservation: Hypothermic preservation and cryopreservation
Buchanan et al. Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage
KR20210052587A (ko) 세포의 동결보존 및 캡슐화 방법
Rajan et al. Development and application of cryoprotectants
TW201821610A (zh) 哺乳動物細胞凍結保存液
US20030077655A1 (en) Physiological medium for perfusing, preserving and storing isolated cell, tissue and organ samples
US20190274300A1 (en) Preservative solution for live cells or composition containing live cells
WO2020200272A1 (zh) 人心肌细胞分离试剂、培养基、分离方法和培养方法
US20220354108A1 (en) Preservation methods using trehalose with other cryoprotectants being absent from the cryopreservation protocol
WO2021203911A1 (zh) 用于心肌细胞冻存的组合物
Wu et al. Intracellular calcium chelating agent (BAPTA‐AM) aids stallion semen cooling and freezing–thawing
Baust et al. Concepts in biopreservation
JP2009219376A (ja) 医療用細胞の保護用液
Görbe et al. Cytoprotection by the NO-donor SNAP against ischemia/reoxygenation injury in mouse embryonic stem cell-derived cardiomyocytes
Al-Attar et al. Lessons from nature: Leveraging the freeze-tolerant wood frog as a model to improve organ cryopreservation and biobanking
Ozawa et al. Successful pig embryonic development in vitro outside a CO2 gas-regulated incubator: effects of pH and osmolality
KR101635042B1 (ko) 배아배양 조성물
Hosseinzadeh et al. Coenzyme Q10 improves developmental competence of mice Pre_antral follicle derived from vitrified ovary
US11523607B2 (en) Sperm storage media comprising MHY 1485 for enhancing sperm quality and function
JP6042099B2 (ja) 細胞増殖抑制剤
Magalhaes et al. The use of vitrification to preserve primary rat hepatocyte monolayer on collagen-coated poly (ethylene-terephthalate) surfaces for a hybrid liver support system
Mourouzis et al. Effects of T3 Administration on Ex Vivo Rat Hearts Subjected to Normothermic Perfusion: Therapeutic Implications in Donor Heart Preservation and Repair
JP2008104407A (ja) 細胞保存方法
RU2731065C1 (ru) Состав криоконсерванта для длительного хранения первичных кератиноцитов
Chen et al. Polyethylene Glycol and Caspase Inhibitor Emricasan Alleviates Cold Injury in Primary Rat Hepatocytes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21783704

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021783704

Country of ref document: EP

Effective date: 20221109