WO2020200272A1 - 人心肌细胞分离试剂、培养基、分离方法和培养方法 - Google Patents

人心肌细胞分离试剂、培养基、分离方法和培养方法 Download PDF

Info

Publication number
WO2020200272A1
WO2020200272A1 PCT/CN2020/082949 CN2020082949W WO2020200272A1 WO 2020200272 A1 WO2020200272 A1 WO 2020200272A1 CN 2020082949 W CN2020082949 W CN 2020082949W WO 2020200272 A1 WO2020200272 A1 WO 2020200272A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
culture
blebbistatin
cardiomyocytes
concentration
Prior art date
Application number
PCT/CN2020/082949
Other languages
English (en)
French (fr)
Inventor
胡盛寿
周冰莹
侯永凤
唐晓丽
师珣
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 US17/594,072 priority Critical patent/US20220169989A1/en
Priority to EP20781916.0A priority patent/EP3967751A4/en
Publication of WO2020200272A1 publication Critical patent/WO2020200272A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the invention relates to a separation reagent and a separation method for separating mammalian cardiomyocytes, especially human cardiomyocytes.
  • the invention also relates to a culture medium and a culture method for culturing mammalian cardiomyocytes, especially human cardiomyocytes.
  • cardiomyocyte model can only be obtained from biological subjects, its source is very rare and precious (especially for human cardiomyocytes), and the existing isolation and culture methods cannot guarantee the survival rate. Therefore, the supply of cardiomyocytes, especially human cardiomyocytes, is far from meeting the needs of clinical research. Especially, compared with the cardiomyocytes of other mammals (such as rodents), the culture and isolation of human cardiomyocytes is a very challenging task. Compared with other species, humans have obvious species differences, which are reflected in the transcriptome, proteome, epigenetic modification, and electrophysiology (Non-Patent Documents 1-4).
  • Non-Patent Documents 7, 8 2,3-butanedione monoxime (2,3-butanedione monoxime, hereinafter referred to as "BDM"
  • BDM 2,3-butanedione monoxime
  • hPCM human primary cardiomyocytes
  • BDM is a skeletal muscle myosin II ATPase inhibitor, which can inhibit the contraction of skeletal muscle and cardiomyocytes.
  • BDM as a chemical phosphatase, is presumed to affect cell contraction by affecting calcium ion channels, transient outward potassium channels, sodium-calcium exchange, and blocking intercellular gap junction communication.
  • Studies have shown that the addition of BDM during the isolation and culture of adult mouse and rat cardiomyocytes can significantly increase the number of living cells and extend the in vitro culture time.
  • BDM can protect cardiomyocytes by stabilizing cell membrane structure and maintaining cytoskeleton stability to increase glycolytic ATP products and negatively regulate myocardial contractility.
  • Non-Patent Document 1 Stephane N. Hatem and Martin Morad*James SKSham, "Species Differences in the Activity of the Na(+)-Ca 2+ Exchanger in Mammalian Cardiac Myocytes", Journal of Physiology (1995).
  • Non-Patent Document 2 KWLINZ*R.MEYER*t,R.SURGES*,S.MEINARDUS*,J.VEES*,A.HOFFMANN*,0.WINDHOLZ*AND C.GROHEt,"Rapid Modulation of L-Type Calcium Current by Acutely Applied Oestrogens in Isolated Cardiac Myocytes from Human, Guinea-Pig and Rat", Experimental Physiology (1998).
  • Non-Patent Document 3 S. Werfel, S. Nothjunge, T. Schwarzmayr, TM Strom, T. Meitinger, and S. Engelhardt, "Characterization of Circular Rnas in Human, Mouse and Rat Hearts", J Mol Cell Cardiol, 98( 2016), 103-7.
  • Non-Patent Document 4 Jr. William, A. Clark, Richard, A. Chizzonite, Alan, W. Everett, Murray, Rabinowitz, and Radovan Zak, “Species Correlations between Cardiac Isomyosins. A Comparison of Electronic MIS and Immunological JOURSAL” (1981).
  • Non-Patent Document 5 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 6 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 7 M. Ackers-Johnson, PYLi, APHolmes, SMO'Brien, D. Pavlovic, and RSFoo, “A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and NonmyAdocytes from theult Mouse Heart",Circ Res,119(2016),909-20.
  • Non-Patent Document 8 E.J.Sharpe, J.R.St Clair, and C.Proenza, “Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice", J Vis Exp (2016).
  • the inventor of the present application has conducted in-depth research and continuous optimization on the separation and culture technology of cardiomyocytes, taking into account the actual needs in clinical practice. It is discovered that the technical solution of the present invention can obtain the technical effect of more truly reflecting the state of the cardiomyocytes and making a higher clinical conversion rate possible.
  • the purpose of the present invention is to provide a technical solution that can be used to effectively separate and culture cardiomyocytes, including separation reagents, culture media, and separation and culture methods.
  • the cells can be maintained in an optimal state during culture, and the morphology, survival rate and purity of the cells can be maintained.
  • the inventor of the present application has conducted a large number of extensive and in-depth studies, and unexpectedly found that (-)-Blebbistatin or its derivative para-aminoblebbistatin can be used in the isolation and culture of mammalian cardiomyocytes (especially human cardiomyocytes). Maintain the morphology and survival rate of cardiomyocytes, which plays a key role in the separation and culture of cardiomyocytes.
  • the inventor of the present application also found that combining (-)-Blebbistatin or para-aminoblebbistatin with other components, and implementing separation and cultivation under certain conditions, can well solve the above technical problems to be solved by the present invention, thus completing the present invention. invention.
  • the first embodiment of the present invention relates to a composition that can be used as a reagent for separating cardiomyocytes, which contains (-)-Blebbistatin and/or para-aminoblebbistatin, and optionally one or more components selected from the following : Energy components, metabolic regulators, acid-base regulators, antibiotics, isotonic agents, among them,
  • (-)-Blebbistatin is a compound represented by the following formula (1),
  • Para-aminoblebbistatin is a compound represented by the following formula (2).
  • the composition contains (-)-Blebbistatin and/or para-aminoblebbistatin, NaCl, KCl, MgCl 2 (such as its hexahydrate), NaH 2 PO 4 , 4-hydroxyethyl piperidine Ethanesulfonic acid, glucose, ⁇ -aminoethanesulfonic acid, creatine, sodium pyruvate, and penicillin and streptomycin.
  • the second embodiment of the present invention relates to a method for separating cardiomyocytes, which includes decalcification, digestion, and cell collection steps, wherein the composition as described in the first embodiment is used as a calcium-free solution in the decalcification step.
  • the third embodiment of the present invention relates to a composition, which can be used for the culture of cardiomyocytes, which is obtained by adding (-)-Blebbistatin and/or para-aminoblebbistatin, antibiotics and serum to known cell culture Medium (preferably one or more medium selected from M199 series, MEM series, and DMEM series).
  • known cell culture Medium preferably one or more medium selected from M199 series, MEM series, and DMEM series.
  • the fourth embodiment of the present invention relates to a kit, which can be used for the culture of cardiomyocytes, which comprises:
  • a coating composition comprising Matrigel or Laminin.
  • the fifth embodiment of the present invention relates to a cardiomyocyte cell culture method in which the composition described in the aforementioned third embodiment is used as a culture medium.
  • the culture plate is coated with a coating composition before culturing the cells, and the coating composition contains Matrigel or laminin.
  • the sixth embodiment of the present invention relates to the use of (-)-Blebbistatin and/or para-aminoblebbistatin for preparing cardiomyocyte separation reagents or cardiomyocyte culture reagents.
  • human cardiomyocytes can be maintained in the best state, cell morphology, survival rate and purity during the isolation and culture process, which can be used for cardiovascular disease research, clinical transformation, drug development and individual Chemical treatment provides a solid foundation.
  • FIG. 1 A is a graph showing the rod-shaped rate of cardiomyocytes separated from the 20mM BDM group, 5 ⁇ M and 10 ⁇ M(-)-Blebbistatin group, and B and C show the 20mM BDM group, 5 ⁇ M and 10 ⁇ M(-)-Blebbistatin group
  • the graph of the length of cardiomyocytes D is a photomicrograph of human cardiomyocytes after separation
  • E is a culture of 20mM BDM and 10 ⁇ M(-)-Blebbistatin group after separation of cardiomyocytes with 20mM BDM and 10 ⁇ M(-)-Blebbistatin Graph of rod-shaped rate of cardiomyocytes after 5 days of basal culture.
  • FIG. 2 A is a graph showing the effect of different concentrations of (-)-Blebbistatin on the rod rate of cardiomyocytes, B is a graph showing the effect of different concentrations of (-)-Blebbistatin on the state of cardiomyocytes, and C is a graph showing The graph of the effect of different concentrations of para-aminoblebbistatin on the survival rate of cardiomyocytes.
  • FIG. 3 is a graph showing the effects of different coating compositions on the number of cardiomyocytes attached.
  • FIG. 4 is a graph showing the effect of different serum concentrations on the average rod-shaped cell rate of cardiomyocytes.
  • FIG. 5 is a graph showing the effects of different types of finished media on the survival rate of cardiomyocytes.
  • Fig. 6 is a graph showing the effects of myosin inhibitors N-benzyl-p-toluenesulfonamide (BTS) and MYK-461 on the survival rate of cardiomyocytes.
  • BTS N-benzyl-p-toluenesulfonamide
  • FIG. 7 A graph showing the survival rate of cardiomyocytes in the (-)-Blebbistatin group and the PICCT-added group.
  • FIG. 8 is a graph showing the comparison result of the medium of the present invention and the ACCIT medium.
  • FIG. 9 A: is a graph showing the change in the rod-shaped rate of cardiomyocytes with the culture time; B: is a graph showing the change in the length of the cardiomyocytes with the culture time; C: is the change of the cardiomyocytes after different incubation times Microscopic image.
  • FIG. 10 A: is a graph showing the change in the rod-shaped ratio of cardiomyocytes with the culture time; B: is a graph showing the change in the length of the cardiomyocytes with the culture time; C: is a graph showing the width of the cardiomyocytes with the culture time The graph of change; D: is the microscopic image of cardiomyocytes after different incubation time.
  • Fig. 11 is a graph showing the trend of up- or down-regulation of cardiomyocyte genes.
  • the inventor of the present application has conducted a lot of extensive and in-depth research, conducted a lot of experiments and explorations, and unexpectedly discovered that (-)-Blebbistatin or its para-aminoblebbistatin can well solve the technical problem to be solved by the present invention.
  • (-)-Blebbistatin is a known non-muscle myosin II inhibitor and cell permeability inhibitor, while para-aminoblebbistatin is a derivative of (-)-Blebbistatin and has a similar structure to (-)-Blebbistatin And nature.
  • (-)-Blebbistatin is usually used as a specific uncoupling agent, etc., but (-)-Blebbistatin is known to have some unfavorable chemical properties, for example, it is unstable to light, has phototoxicity and cytotoxicity , High fluorescence and low water solubility (its solubility is only 10.9 ⁇ 0.9 ⁇ M), etc.
  • 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, indicating that its concentration is 4.5-5.5mM; if the concentration of a certain component is in the range of about 5-10 mM, indicating that the concentration range is 4.5-11mM.
  • any component may have more than one effect, and their actual effect in the overall composition may be unpredictable.
  • sodium 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.
  • glucose is listed as an energy substance in this specification, the substance also plays a role in maintaining osmotic pressure (i.e., isotonic), and can also be regarded as an isotonic agent, and so on.
  • cardiomyocytes described herein are mammalian cardiomyocytes, preferably human cardiomyocytes, such as human primary cardiomyocytes.
  • the composition for separation of cardiomyocytes of the present invention contains (-)-Blebbistatin and/or para-aminoblebbistatin.
  • the concentration of (-)-Blebbistatin in the composition is 1-50 ⁇ M, more preferably 2.5-20 ⁇ M, more preferably 3-15 ⁇ M, more preferably about 5-10 ⁇ M.
  • the concentration of para-aminoblebbistatin in the composition is 1-100 ⁇ M, more preferably 5-50 ⁇ M, more preferably 8-25 ⁇ M, more preferably about 10-20 ⁇ M.
  • composition for separation optionally further contains one or more components selected from the group consisting of energy components, metabolic regulators, acid-base regulators, antibiotics, and isotonic agents.
  • the composition for separation optionally contains energy substances, which provide necessary energy reserves for the cells during the separation process.
  • the energy substance is glucose, and its concentration is, for example, 5-50 mM, preferably 10-30 mM, more preferably 20-25 mM, particularly preferably about 22 mM.
  • the composition for separation optionally contains metabolic regulators, such as sodium pyruvate, insulin, creatine, ⁇ -aminoethanesulfonic acid (Taurine), and L-carnitine (L-carnitine). carnitine), etc., which help cells regulate energy metabolism during the separation process.
  • the metabolic regulator is sodium pyruvate, creatine and/or ⁇ -aminoethanesulfonic acid.
  • the concentration of sodium pyruvate may be 0.1-20 mM, preferably 1-10 mM, more preferably 3-8 mM, particularly preferably about 5 mM;
  • the concentration of creatine may be 0.1-20 mM, preferably 1-10 mM, more preferably 3-8 mM, particularly preferably about 5 mM;
  • the concentration of ⁇ -aminoethanesulfonic acid may be 5-50 mM, preferably 10-30 mM, more preferably 15-25 mM, particularly preferably about 20 mM.
  • the composition for separation optionally contains acid-base regulators, which include buffer substances for maintaining a stable pH of the composition, such as 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), and/or Acid-base substances used to directly adjust the pH of the composition, such as potassium hydroxide, sodium hydroxide, etc.
  • the acid-base regulator is HEPES and/or 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 sodium hydroxide is not specifically limited, as long as the final composition
  • the pH value 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 for separation optionally contains antibiotics, which function to inhibit the growth of bacteria.
  • Antibiotics include but are not limited to penicillin, streptomycin, and Primocin.
  • the concentration of penicillin may be 10-500 U/ml, preferably 20-400 U/ml, more preferably 50-300 U/ml, particularly preferably about 100-200 U/ml, about 100 U/ml or About 200U/ml
  • the concentration of streptomycin 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 100 ⁇ g/ml or about 200 ⁇ g/ ml
  • the concentration of Primocin 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 100 ⁇ g/ml or about 200 ⁇ g/ml.
  • the composition for separation optionally contains isotonic agents, which can maintain the osmotic pressure to ensure the balance of water and electrolytes of the cells.
  • Isotonic agents include, but are not limited to, magnesium chloride (e.g., its hydrate, such as hexahydrate), potassium chloride, sodium chloride, sodium dihydrogen phosphate, or a combination thereof.
  • the concentration of magnesium chloride is 0.1-20 mM, preferably 1-10 mM, more preferably 3-8 mM, and particularly preferably about 5 mM;
  • the concentration of potassium chloride is 0.1-20 mM, preferably 1-10 mM , More preferably 3-8mM, particularly preferably about 4-5mM;
  • the concentration of sodium chloride is 10-500mM, preferably about 50-200mM, more preferably about 100-150mM, particularly preferably about 120-130mM;
  • the concentration is 0.5-30mM, preferably 1-20mM, more preferably 3-15mM, particularly preferably about 5-10mM.
  • the composition for separation contains the following ingredients: 1-50 ⁇ M (-)-Blebbistatin, 5-50mM glucose, 0.1-20mM sodium pyruvate, 0.1-20mM muscle Acid, 5-50mM ⁇ -aminoethanesulfonic acid, 0.5-10mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 10-500U/ml penicillin, 10-500 ⁇ g/ml streptomycin, 0.1 -20mM magnesium chloride, 0.1-20mM potassium chloride, 10-500mM sodium chloride, 0.5-30mM sodium dihydrogen phosphate, and an appropriate amount of NaOH, so that the pH of the composition is 7.0-7.8 (hereinafter referred to as "separation Composition 1").
  • the composition for separation contains the following ingredients: 2.5-20 ⁇ M (-)-Blebbistatin, 10-30mM glucose, 1-10mM sodium pyruvate, 1-10mM Creatine, 10-30mM ⁇ -aminoethanesulfonic acid, 1-8mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 20-400U/ml penicillin, 20-400 ⁇ g/ml streptomycin, 1-10mM magnesium chloride, 1-10mM potassium chloride, 50-200mM sodium chloride, 1-20mM sodium dihydrogen phosphate, and an appropriate amount of NaOH, so that the pH of the composition is 7.2-7.6 (hereinafter referred to as " Separate composition 2").
  • the composition for separation contains the following ingredients: 3-15 ⁇ M (-)-Blebbistatin, 20-25mM glucose, 3-8mM sodium pyruvate, 3-8mM Creatine, 15-25mM ⁇ -aminoethanesulfonic acid, 3-6mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 50-300U/ml penicillin, 50-300 ⁇ g/ml streptomycin, 3-8mM magnesium chloride, 3-8mM potassium chloride, 100-150mM sodium chloride, 3-15mM sodium dihydrogen phosphate, and an appropriate amount of NaOH, so that the pH of the composition is 7.3-7.5 (hereinafter referred to as " Separate composition 3").
  • HEPES 4-hydroxyethylpiperazine ethanesulfonic acid
  • 50-300U/ml penicillin 50-300 ⁇ g/ml streptomycin
  • 3-8mM magnesium chloride 3-8mM potassium chloride
  • the composition for separation contains the following ingredients: 1-100 ⁇ M para-aminoblebbistatin, 5-50 mM glucose, 0.1-20 mM sodium pyruvate, 0.1-20 mM creatine, 5-50mM ⁇ -aminoethanesulfonic acid, 0.5-10mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 10-500U/ml penicillin, 10-500 ⁇ g/ml streptomycin, 0.1-20mM Magnesium chloride, 0.1-20mM potassium chloride, 10-500mM sodium chloride, 0.5-30mM sodium dihydrogen phosphate, and an appropriate amount of NaOH, so that the pH of the composition is 7.0-7.8 (hereinafter referred to as "separation composition 4”).
  • the composition for separation contains the following ingredients: 5-50 ⁇ M para-aminoblebbistatin, 10-30mM glucose, 1-10mM sodium pyruvate, 1-10mM creatine , 10-30 mM ⁇ -aminoethanesulfonic acid, 1-8mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 20-400U/ml penicillin, 20-400 ⁇ g/ml streptomycin, 1 -10mM magnesium chloride, 1-10mM potassium chloride, 50-200mM sodium chloride, 1-20mM sodium dihydrogen phosphate, and an appropriate amount of NaOH, so that the pH of the composition is 7.2-7.6 (hereinafter referred to as "separation Composition 5").
  • the composition for separation contains the following ingredients: 8-25 ⁇ M para-aminoblebbistatin, 20-25mM glucose, 3-8mM sodium pyruvate, 3-8mM creatine , 15-25mM ⁇ -aminoethanesulfonic acid, 3-6mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 50-300U/ml penicillin, 50-300 ⁇ g/ml streptomycin, 3- 8mM magnesium chloride, 3-8mM potassium chloride, 100-150mM sodium chloride, 3-15mM sodium dihydrogen phosphate, and an appropriate amount of NaOH, so that the pH of the composition is 7.3-7.5 (hereinafter referred to as "separation combination ⁇ 6”).
  • the method for separating cardiomyocytes includes the steps of decalcification, digestion and cell collection.
  • any of the composition for cardiomyocyte separation described in the present invention can be used as a calcium-free solution; in the digestion step, the calcium-free solution can be added to the calcium-free solution for cell digestion.
  • the remaining operations, reagents and conditions are the same as those used in general cell separation methods in the prior art.
  • the composition used as a digestive juice in the digestion step is to add an enzyme solution and a chlorinated solution to the calcium-free solution (that is, any composition for cardiomyocyte separation according to the present invention).
  • the enzyme solution is obtained by calcium, for example, protease (for example, protease XXIV) and/or collagenase (for example, collagenase I, collagenase II).
  • the concentration of the protease (such as protease XXIV) in the digestive juice is 0.1-5 U/ml, preferably 0.5-3 U/ml, more preferably 1-1.5 U/ml , Particularly preferably about 1.2U/ml;
  • the concentration of collagenase (such as collagenase I, collagenase II) is, for example, 50-800U/ml, preferably 100-600U/ml, more preferably 200-400U/ml, particularly preferably about 300U /ml.
  • the concentration of calcium chloride in the digestive juice is 0.002-0.1mM, preferably 0.005-0.05mM, more preferably 0.01-0.02mM, particularly preferably about 0.015mM.
  • the composition for digestion (its pH is 7.0-7.8), in addition to the components described for the aforementioned separation composition 1 or 4, also contains the following components: 50-800 U/ ml collagenase II and 0.002-0.1 mM calcium chloride; in another specific embodiment, it additionally contains 0.1-5 U/ml protease XXIV.
  • the composition for digestion (with a pH of 7.2-7.6) contains the following components in addition to the components described for the aforementioned separation composition 2 or 5: 100-600U /ml collagenase II and 0.005-0.05mM calcium chloride; in another specific embodiment, it additionally contains 0.5-3U/ml protease XXIV.
  • the composition for digestion (its pH is 7.3-7.5), in addition to the components described in the aforementioned separation composition 3 or 6, also contains the following components: 200-400 U/ ml collagenase II and 0.01-0.02mM calcium chloride; in another specific embodiment, it additionally contains 1-1.5U/ml protease XXIV.
  • the digestion steps of the present invention can be carried out in stages, and the same or different compositions described in the above embodiments can be used as digestive juice in each stage.
  • the composition used as the cell transport fluid in the cell transport step is to add serum to the calcium-free fluid (that is, any composition for cardiomyocyte separation according to the present invention) And obtained.
  • serum can refer to any serum commonly used in the field of cell culture, or its main components (for example, serum albumin, etc.).
  • the serum is fetal bovine serum (FBS).
  • the serum is bovine serum albumin (BSA).
  • the serum is fetal bovine serum
  • the volume fraction of the fetal bovine serum in the composition used as a cell transport fluid is 1%-20%, preferably 2%-15%, more preferably 3% -12%, particularly preferably about 5%-10%
  • the serum is bovine serum albumin
  • the concentration of bovine serum albumin in the composition used as a cell transport fluid is 0.1-10 g/ml, It is preferably 0.2-5 g/ml, more preferably 0.3-1 g/ml, particularly preferably about 0.5 g/ml.
  • the composition (with a pH of 7.0-7.8) used as a cell transport fluid is obtained by adding fetal bovine serum and/or bovine serum albumin to the aforementioned separation composition 1 or 4
  • the volume fraction of fetal bovine serum in the cardiomyocyte culture composition is 1%-20%
  • the concentration of bovine serum albumin in the cardiomyocyte culture composition is 0.1-10 g/ml.
  • the composition (with a pH of 7.2-7.6) used as a cell transport fluid is obtained by adding fetal bovine serum and/or bovine serum albumin to the aforementioned separation composition 2 or 5.
  • the volume fraction of fetal bovine serum in the cardiomyocyte culture composition is 2%-15%
  • the concentration of bovine serum albumin in the cardiomyocyte culture composition is 0.2-5 g/ml.
  • the composition (with a pH of 7.3-7.5) used as a cell transport fluid is obtained by adding fetal bovine serum and/or bovine serum albumin to the aforementioned separation composition 3 or 6.
  • the volume fraction of fetal bovine serum in the cardiomyocyte culture composition is 3%-12%
  • the concentration of bovine serum albumin in the cardiomyocyte culture composition is 0.3-1 g/ml.
  • the composition for cardiomyocyte culture of the present invention is obtained by adding (-)-Blebbistatin and/or para-aminoblebbistatin, serum and antibiotics to the medium conventionally used for cell culture in the art.
  • the medium is, for example, one or more medium selected from M199 series, MEM series, and DMEM series; preferably, the medium can be selected from M199 medium, M199-glutamine-free medium, M199-4 -Hydroxyethylpiperazine ethanesulfonic acid-glutamine-free medium, MEM medium, MEM-GlutaMAX medium, MEM-4-hydroxyethylpiperazine ethanesulfonic acid-GlutaMAX medium, DMEM-high glucose medium , DMEM-low sugar medium; more preferably, the medium may be M199 medium, MEM-GlutaMAX medium or MEM-4-hydroxyethylpiperazine ethanesulfonic acid-GlutaMAX medium.
  • the composition for culturing cardiomyocytes is added to any one of the above-mentioned media (such as M199, MEM-GlutaMAX or MEM-4-hydroxyethylpiperazine ethanesulfonic acid).
  • -GlutaMAX medium is obtained by adding the following ingredients (hereinafter collectively referred to as "culture composition 1"):
  • Penicillin, and/or streptomycin, and/or Primocin wherein the concentration of penicillin in the composition is 10-500 U/ml, and the concentration of streptomycin in the composition is 10-500 ⁇ g/ml, The concentration of Primocin in the composition is 10-500 ⁇ g/ml;
  • Fetal bovine serum and/or bovine serum albumin wherein the volume fraction of fetal bovine serum in the composition is 1%-20%, and the concentration of bovine serum albumin in the composition is 0.1-10 g/ml.
  • composition for culturing cardiomyocytes is added to any one of the above-mentioned media (such as M199, MEM-GlutaMAX or MEM-4-hydroxyethylpiperazine ethyl sulfonate).
  • Acid-GlutaMAX medium is obtained by adding the following ingredients (hereinafter collectively referred to as "culture composition 2"):
  • Penicillin, and/or streptomycin, and/or Primocin wherein the concentration of penicillin in the composition is 20-400 U/ml, and the concentration of streptomycin in the composition is 20-400 ⁇ g/ml, The concentration of Primocin in the composition is 20-400 ⁇ g/ml;
  • Fetal bovine serum and/or bovine serum albumin wherein the volume fraction of fetal bovine serum in the composition is 2%-15%, and the concentration of bovine serum albumin in the composition is 0.2-5 g/ml.
  • composition for culturing cardiomyocytes is added to any one of the above-mentioned media (such as M199, MEM-GlutaMAX or MEM-4-hydroxyethylpiperazine sulfonate).
  • Acid-GlutaMAX medium is obtained by adding the following ingredients (hereinafter collectively referred to as "culture composition 3"):
  • Penicillin, and/or streptomycin, and/or Primocin wherein the concentration of penicillin in the composition is 50-300 U/ml (for example, about 100 U g/ml or 200 U/ml), and streptomycin is in the composition
  • concentration in the composition is 50-300 ⁇ g/ml (for example, about 100 ⁇ g/ml or 200 ⁇ g/ml)
  • concentration of Primocin in the composition is 50-300 ⁇ g/ml (for example, about 100 ⁇ g/ml or 200 ⁇ g/ml);
  • ⁇ Fetal bovine serum and/or bovine serum albumin wherein the volume fraction of fetal bovine serum in the composition is 5%-10% (for example, about 5%), and the concentration of bovine serum albumin in the composition is 0.3-1 g/ml (for example, about 0.5 g/ml).
  • the kit for myocardial culture of the present invention includes:
  • a coating composition comprising Matrigel and/or Laminin, and the function of Matrigel and/or Laminin is to promote the growth of cells adhering to the wall.
  • laminin when laminin is used, its concentration is 10-500 ⁇ g/ml, preferably 20-300 ⁇ g/ml, more preferably 50-200 ⁇ g/ml, particularly preferably about 50 ⁇ g/ml or about 200 ⁇ g/ml.
  • the kit includes:
  • Coating composition 1 said coating composition 1 comprising Matrigel, and/or 10-500 ⁇ g/ml laminin.
  • the kit includes:
  • Coating composition 2 said coating composition 2 comprising Matrigel, and/or 20-300 ⁇ g/ml laminin.
  • the kit comprises:
  • Coating composition 3 said coating composition 3 comprising Matrigel, and/or 50-200 ⁇ g/ml laminin.
  • the amount of Matrigel can be adapted to the actual volume of the hole, for example, it can be 150-200 ⁇ l/well.
  • the cells are cultured using the composition described in part (3) or the kit described in part (4) above.
  • the culture plate is coated with the coating composition described in section (4) above, and then the composition described in section (3) above is used as The medium cultivates the cells.
  • the present invention also relates to the use of (-)-Blebbistatin and/or para-aminoblebbistatin in the preparation of cardiomyocyte separation reagents or cardiomyocyte culture reagents, and the usage mode is as described in the previous part (1) to (5).
  • the separation process of cardiomyocytes generally includes tissue transport and sectioning, decalcification, enzyme digestion and cell collection.
  • the calcium-free bench-top solution (calcium-free solution) is the main reagent used in the separation process for decalcification, enzyme digestion and cell collection.
  • the literature Liang Chen, Guang-ran Guo, Man Rao, Kai Chen, Jiang-ping Song and Sheng-shou Hu, "A Modified Method for Isolation of Human Cardiomyocytes to Model Cardiac Diseases.”, Journal of Translational (2018).
  • the cardiomyocyte isolation experimental protocol described in the operation Specific steps are as follows.
  • the isolated left atrial appendage immediately in the organ preservation solution (manufactured by the University of Wisconsin, USA) (about 4°C), quickly transfer it to a 60mm cell culture dish in the ultra-clean table, and add the organ preservation solution (about 4°C).
  • organ preservation solution about 4°C
  • the shredded auricles were divided into three groups, and the tissues were decalcified using the calcium-free solution of the present invention, three times in total.
  • calcium-free solution containing 5 ⁇ M(-)-Blebbistatin, 10 ⁇ M(-)-Blebbistatin and 20mM BDM were used.
  • the composition of calcium-free solution and enzyme solution 1 and 2 are shown in Table 1-3 below.
  • Protease XXIV 1.2U/ml Collagenase II 300U/ml CaCl 2 0.015mM
  • FIG. 1 The result is shown in Figure 1. It can be seen from Figure 1A that the rod-shaped rate of the cells isolated in the 20mM BDM group was standardized to 1.0, and the standardized rod-shaped cell rate obtained in the 5 ⁇ M and 10 ⁇ M (-)-Blebbistatin groups reached 1.2-1.5 (p ⁇ 0.05), which significantly improved Cardiomyocyte viability. It can be seen from Figure 1B and Figure 1C that the length of human cardiomyocytes isolated from the 5 ⁇ M and 10 ⁇ M (-)-Blebbistatin groups is also significantly longer than that of the 20mM BDM group (p ⁇ 0.05).
  • Figure 1D is a photomicrograph of isolated human cardiomyocytes (Lycra, DMI4000B, 10X).
  • the rod-shaped cell morphology of the 20mM BDM group is not well maintained, and a large number of spherical cells can be seen in the field of view; while 5 ⁇ M and 10 ⁇ M
  • the (-)-Blebbistatin group can maintain the long rod-like shape of human cardiomyocytes, and the 10 ⁇ M(-)-Blebbistatin group has better effect.
  • the above results show that the group containing 5-10 ⁇ M Blebbistatin separation reagent can well maintain the morphology of human cardiomyocytes and increase the rod-shaped rate during cell separation.
  • the present invention uses a 5-step recalcification method to restore the calcium ion concentration of human cardiomyocytes.
  • the concentration gradient of CaCl 2 (stock concentration 30 mM) added to the cell collection solution was 0.08 mM (5 min), 0.25 mM (5 min), 0.5 mM (5 min), 1 mM (5 min), 1.8 mM (15 min).
  • the human cardiomyocytes isolated from the 20mM BDM group use M199 medium, add 10% fetal bovine serum (FBS) (Note: the concentration percentage of fetal bovine serum in the examples and corresponding figures is volume percentage), 100U/ml penicillin and 100 ⁇ g /ml streptomycin and 20mM BDM for culture.
  • FBS fetal bovine serum
  • the human cardiomyocytes isolated from the 10 ⁇ M(-)-Blebbistatin group were cultured using M199 medium, adding 10% fetal bovine serum, 100U/ml penicillin, 100 ⁇ g/ml streptomycin and 10 ⁇ M(-)-Blebbistatin. Specifically, the cells were placed in a 37°C incubator (5% CO 2 , relative saturated humidity 95%, Thermo Scientific Heracell VIOS CO 2 160i, USA) and cultured for 6 days.
  • composition of the cell collection solution (Table 1-3) is shown in Table 4 below.
  • Example 2.1 The effect of different concentrations of (-)-Blebbistatin and para-aminoblebbistatin on the rod-shaped rate of human cardiomyocytes
  • the experiment was divided into 9 groups. 0, 2.5, 5, 10, 20 ⁇ M (-)-Blebbistatin and 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, 50 ⁇ M para-aminoblebbistatin were added to the basal medium.
  • the isolated human cardiomyocytes were divided into 9 groups.
  • the cells were plated on a 48-well cell culture plate coated with 200 ⁇ g laminin in advance using 9 culture media, and placed in a 37 °C cell incubator (5% CO 2 , Saturated humidity 95%) cultured for 7 days, calculated the rod-shaped rate of cardiomyocytes, the results are shown in Figure 2.
  • Example 2.2 Effect of the coating composition on the number of adherent human cardiomyocytes
  • the medium used in this experiment was obtained by adding 20 mM BDM, 10% fetal bovine serum, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin to M199 medium.
  • the concentration of laminin used as a coating composition in the prior art is usually about 10 ⁇ g/ml, and the inventor of the present application found that when the concentration of laminin in the present invention is 50-200 ⁇ g/ml, cardiomyocytes can be effectively attached. wall.
  • the average rod-shaped cell rate (the proportion of rod-shaped cells in the total cells) of the experimental group was significantly higher than that of the control group, and the results were statistically different (p ⁇ 0.05). However, there was no statistical difference between the three experimental groups with different serum concentrations (p>0.05).
  • the average rod-shaped cell rate of the 10% fetal bovine serum group was slightly higher than that of the 5% fetal bovine serum group and the 20% fetal bovine serum group . From this, it can be determined that the suitable fetal bovine serum concentration during cardiomyocyte culture is 5-20%, preferably 10%.
  • Example 2.4 The effect of different types of finished culture media on the survival rate of human cardiomyocytes
  • the following 8 commonly used finished culture media were selected, namely MEM (purchased from Thermo Company), MEM-GlutaMAX (purchased from Thermo Company), MEM-GlutaMAX-4-hydroxyethylpiperazine ethanesulfonic acid (purchased from Thermo Company) ), M199 (purchased from Sigma company), M199-glutamine-free (purchased from Thermo Company), M199-4-hydroxyethylpiperazine ethanesulfonic acid-glutamine-free (purchased from Thermo Company), DMEM-high Sugar (purchased from Thermo Company) and DMEM-low sugar-sodium pyruvate (purchased from Thermo Company).
  • the isolated human cardiomyocytes were spread on a 48-well cell culture plate coated with 200 ⁇ g of laminin in advance, placed in a 37°C cell incubator (5% CO 2 , relative saturated humidity 95%), and cultured for 5 days.
  • the eight kinds of culture media have reached a rod-shaped cell rate of about 50%-70% or more, so they can all be used for human cardiomyocyte culture.
  • several other media M199-glutamine-free medium, M199-4-hydroxyethylpiperazineethanesulfonic acid-glutamine-free medium , MEM medium, MEM-GlutaMAX medium, MEM-4-hydroxyethylpiperazine ethanesulfonic acid-GlutaMAX medium, DMEM-high-sugar medium, DMEM-low-sugar medium
  • a flat or even higher rod-shaped cell rate indicates that the choice of medium has little effect on the survival rate of human cardiomyocytes.
  • Example 2.5 The effect of myosin inhibitors N-benzyl-p-toluenesulfonamide (BTS) and MYK-461 on the survival rate of human cardiomyocytes
  • the metabolic regulators sodium pyruvate (2mM), insulin (10mg/L), creatine (5mM), ⁇ -aminoethanesulfonic acid (taurine, 5mM) and L-carnitine ( L-carnitine, 2mM) was mixed and added to the medium, and the mixture of the aforementioned metabolic regulators was referred to as "PICCT" for short.
  • the experimental group was divided into different concentrations of (-)-Blebbistatin (0 ⁇ M, 5 ⁇ M, 10 ⁇ M) according to the basal medium (M199 medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100 ⁇ g/ml streptomycin)
  • Basal medium M199 medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100 ⁇ g/ml streptomycin
  • the calibrated cardiomyocyte survival rate cell survival rate on day 7/cell survival rate on day 0 ⁇ 100%).
  • some cells lose their original long rod-shaped cell morphology and grow adherently. These cells are regarded as dead cells and are not counted as living cells.
  • Example 2.7 Comparison of the medium of the present invention and ACCIT medium
  • the preferred culture conditions for human cardiomyocytes of the present invention are: Matrigel (37°C, 30min) or 50-200 ⁇ g/ml laminin (incubate overnight in a 37°C incubator, dry) in advance for coating culture Plate, used in selected from MEM-4-hydroxyethylpiperazine ethanesulfonic acid-GlutaMAX, M199-glutamine-free, M199-4-hydroxyethylpiperazine ethanesulfonic acid-non-glutamine, MEM, MEM- GlutaMAX, DMEM-high glucose is added to any of the finished medium of 10%-20% fetal bovine serum, 2.5-20 ⁇ M(-)-Blebbistatin, 100U/ml penicillin and 100 ⁇ g/ml streptomycin. Medium.
  • Example 2.8 Influence of culture time on cell morphology
  • the isolated human primary cardiomyocytes were divided into two groups, and they were resuspended in a basal medium containing 1% BSA and 10% FBS, respectively, using a layer of 200 ⁇ g/ml
  • the zonulin-coated 96-well cell culture plate was cultured with 6000 cells per well and placed in a 37°C cell incubator (5% CO 2 , relative saturated humidity 95%) for 7 days.
  • the medium was changed every two days, and the Incucyte (S3) instrument was used to automatically take pictures every 4 hours.
  • S3 instrument was used to automatically take pictures every 4 hours.
  • the length and width of each cell are measured, and cells with a ratio of length to width greater than 2 are counted as rod-shaped cells.
  • the rod-shaped rate of cells under each culture condition was calculated, and the experiment was repeated 6 times. The results are shown in Figure 9.
  • Figure 9A shows that the rod-shaped rate of cardiomyocytes cultured in the two mediums decreased with time
  • Figure 9B shows that the cell length of the 10% FBS group maintained relatively well during the time period tested
  • Figure 9C It shows that the survival rate of rod-shaped cells in the 1% BSA group after the third day is relatively good.
  • the present inventors further designed a plan for using BSA and FBS in cell culture at the same time to detect changes in cell rod rate, cell length, and cell width at 3 and 6 days of culture, hoping that the simultaneous application of BSA and FBS would reduce the damage The influence of cell morphology.
  • the specific grouping is as follows: 0%BSA+0%FBS, 1%BSA, 10%FBS, 1%BSA+10%FBS, 0.5%BSA+10%FBS, 1%BSA+5 %FBS, 0.5%BSA+5%FBS, a total of 7 groups, repeated 3 times. Other experimental conditions remain unchanged. The results are shown in Figure 10, indicating that each group maintained the cell morphology at about 3 days and 6 days.
  • Example 2.9 The effect of continuous culture on cell transcription level
  • the human primary cardiomyocytes were cultured with the basal medium containing 1% BSA and 10% FBS as described in Example 2.8, and compared with the day of isolation one day and three days after culture. Specifically, the cell samples thus obtained were centrifuged first, and then the cell pellet with the supernatant slurry discarded was stored in a -80°C refrigerator.
  • the kit GeneJET RNA Purification Kit (Thermo Scientific, K0731) was used to extract RNA, and NanoDrop 2000 (Thermo Scientific) was used to determine the RNA concentration and quality. Then KAPA mRNA HyperPrep Kit (Illumina, KK8581) was used to construct a cDNA library with 500 ng of RNA.
  • the size and concentration of the cDNA library were measured using 2100 Bioanalyzer (Agilent) and Qubit (Thermo Fisher Scientific). Dilute the library to 2-3pM and use the Illumina NextSeq 500 sequencing platform for sequencing.
  • the corresponding kit is 75cycles of NextSeq 500/550 High Output Kit v2.5kit (Illumina, 20024906). According to the sequencing results, a heat map is produced, and the number of up-regulated and down-regulated genes and the amount of up-regulated gene change (
  • the t-test was used to compare the changes of 1% BSA and 10% FBS in D1 up-regulation, D1 down-regulation, D3 up-regulation, D3 down-regulation. It can be seen from Figure 11A that 1% BSA and 10% FBS have basically the same trend of gene up-regulation changes for 1 day and 3 days in culture, indicating that both can maintain the original cell state of cardiomyocytes.
  • Figure 11B shows that the number of up-regulated genes in the 1 and 3 days of culture shows that the number of genes in the 1% BSA group is even less than that in the 10% FBS group;
  • Figure 11C shows that in the up-regulated genes, the number of up-regulated genes is 1 day and 3 days 1
  • the degree of change in the %BSA group is also less than that of the 10%FBS group, which indicates that the effect of the 1%BSA group is even better than that of the 10%FBS group.

Landscapes

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

Abstract

提供了一种含有（-）-Blebbistain和/或para-Blebbistain的组合物，包含该组合物的试剂盒，以及使用该组合物作为培养基的心肌细胞培养方法。还提供了（-）-Blebbistain和/或para-Blebbistain在用于制备心肌细胞分离试剂或心肌细胞培养试剂中的用途。

Description

人心肌细胞分离试剂、培养基、分离方法和培养方法
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
本发明涉及用于分离哺乳动物心肌细胞、特别是人心肌细胞的分离试剂和分离方法。本发明还涉及用于培养哺乳动物心肌细胞、特别是人心肌细胞的培养基和培养方法。
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。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。
研究任何一种疾病的预防和治疗，都要有好的细胞模型。然而，不同于其他体细胞，心血管疾病的研究模型即心肌细胞本身具有运动性强、耗氧高的特点，这一特点决定了其分离与培养过程均需要消耗大量能量并且产生大量代谢产物，导致分离培养条件极难控制，也极难保证分离培养过程中的细胞存活率和纯度。这在很大程度上对心血管疾病的研究带来了局限性。
此外，由于心肌细胞模型只能从生物体受试者获取，因此其来源非常稀少且珍贵(对于人心肌细胞而言尤其如此)，现有的分离培养方法又不能保证存活率。因此心肌细胞、特别是人心肌细胞的供应远远不能满足临床研究的需要。特别是，与其它哺乳动物(例如啮齿类动物)的心肌细胞相比，人心肌细胞的培养分离更是挑战性极高的工作。与其它物种相比，人类存在明显的物种差异，该差异体现在转录组、蛋白组、表观遗传修饰、电生理等各个层面(非专利文献1-4)，这些差异使得人类的心肌细胞对体外培养分离条件的耐受力比其它物种更低，导致人心肌细胞的培养分离比其它物种的心肌细胞难度更大。尽管在其他细胞的培养中可以采用人类组织样本来消除物种差异，但由于现有细胞培养方法无法保证细胞纯度，导致实验结果中出现背景噪音大、或因无生物活性而无法用于功能学实验等不足，无法通过使用组织样本的方式将动物心肌细胞的实验结果类推到人类。现有技术中另外一种获得人心肌细胞的方法是人胚胎干细胞或诱导多能干细胞的定向分化，这一方法虽然部分地克服了上述障碍，但仍存在分化不完全、缺少表观遗传修饰等问题(非专利文献5、6)，从而无法如实反映疾病真实状态，无法推动心血管疾病的研究工作。
综上，现有技术虽然为哺乳动物、特别是人的心肌细胞的获得提供了多种途径，但目前还没有任何一条途径能够以高存活率、高纯度获得哺乳动物、特别是人的人心肌细胞或其有效模拟物，这严重地阻碍了相关科研工作的开展，无法为临床上的疾病诊断和治疗提 供研究基础。
如前文所述，在现有技术中，尽管很早就有研究人员开始探索心肌细胞的分离并培养，但迄今为止只有少数的有关非人哺乳动物心肌细胞培养的报道(非专利文献7、8)，其中主要使用2,3-丁二酮单肟(2,3-butanedione monoxime，以下简称“BDM”)。此外，还没有任何培养或分离人心肌细胞、特别是人原代心肌细胞(Human Primary Cardiomyocyte，hPCM)的报道。
BDM为骨骼肌肌球蛋白II ATP酶抑制剂，可以抑制骨骼肌和心肌细胞收缩。BDM作为化学磷酸酶，据推测是通过影响钙离子通道、瞬时外向钾通道、钠钙交换以及阻塞细胞间间隙连接通信等途径来影响细胞的收缩。有研究表明，成年小鼠、大鼠心肌细胞分离和培养时加入BDM能够明显提高活细胞数量，并延长体外培养时间。也有报道称BDM通过稳定细胞膜结构、维持细胞骨架稳定来增加糖酵解ATP产物以及负向调节心肌收缩力，由此可以保护心肌细胞。然而，由于BDM的培养分离效果并不足以满足科研和产业上的需求，因此使用BDM培养分离哺乳动物心肌细胞的构想目前还无法付诸实施，特别是对于人心肌细胞的培养分离而言更是如此。
现有技术文献
非专利文献1：Stephane N.Hatem and Martin Morad*James S.K.Sham,“Species Differences in the Activity of the Na(+)-Ca 2+Exchanger in Mammalian Cardiac Myocytes”,Journal of Physiology(1995).
非专利文献2：K.W.LINZ*R.MEYER*t,R.SURGES*,S.MEINARDUS*,J.VEES*,A.HOFFMANN*,0.WINDHOLZ*AND C.GROHEt,“Rapid Modulation of L-Type Calcium Current by Acutely Applied Oestrogens in Isolated Cardiac Myocytes from Human,Guinea-Pig and Rat”,Experimental Physiology(1998).
非专利文献3：S.Werfel,S.Nothjunge,T.Schwarzmayr,T.M.Strom,T.Meitinger,and S.Engelhardt,“Characterization of Circular Rnas in Human,Mouse and Rat Hearts”,J Mol Cell Cardiol,98(2016),103-7.
非专利文献4：Jr.William A.Clark,Richard A.Chizzonite,Alan W.Everett,Murray Rabinowitz,and Radovan Zak,“Species Correlations between Cardiac Isomyosins.A Comparison of Electrophoretic and Immunological Properties”,THE JOURNAL OF BIOLOGICACL CHEMISTRY(1981).
非专利文献5：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6：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7：M.Ackers-Johnson,P.Y.Li,A.P.Holmes,S.M.O’Brien,D.Pavlovic,and  R.S.Foo,“A Simplified,Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes from the Adult Mouse Heart”,Circ Res,119(2016),909-20.
非专利文献8：E.J.Sharpe,J.R.St Clair,and C.Proenza,“Methods for the Isolation,Culture,and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice”,J Vis Exp(2016).
发明内容
本申请发明人考虑到临床上的实际需要，为解决哺乳动物心肌细胞(特别是人心肌细胞)的分离与培养这一课题，对心肌细胞的分离和培养技术进行了深入研究并不断优化，从而发现了可以获得更真实地反映心肌细胞状态、使更高临床转化率成为可能的技术效果的本发明技术方案。
本发明目的是提供能够用于有效地分离及培养心肌细胞的技术方案，包括分离试剂、培养基及分离和培养方法。通过采用本发明的技术方案，可以在培养中将细胞维持在最佳状态、保持细胞形态、存活率和纯度。
本申请发明人进行了大量广泛而深入的研究，结果意外地发现(-)-Blebbistatin或其衍生物para-aminoblebbistatin可以在哺乳动物心肌细胞(特别是人心肌细胞)的分离和培养中很好地维持心肌细胞的形态和存活率，其对心肌细胞的分离和培养起到了关键作用。本申请发明人还发现，将(-)-Blebbistatin或para-aminoblebbistatin与其他成分进行组合，在一定条件下实施分离和培养，能够很好地解决本发明所要解决的上述技术问题，从而完成了本发明。
本发明的第一实施方式涉及一种组合物，其可用作心肌细胞分离试剂，其含有(-)-Blebbistatin和/或para-aminoblebbistatin，以及任选的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、抗生素、等渗剂，其中，
(-)-Blebbistatin是由下述式(1)表示的化合物，
Figure PCTCN2020082949-appb-000001
para-aminoblebbistatin是由下述式(2)表示的化合物。
Figure PCTCN2020082949-appb-000002
在一个优选实施方式中，所述组合物中含有(-)-Blebbistatin和/或para-aminoblebbistatin、NaCl、KCl、MgCl 2(例如其六水合物)、NaH 2PO 4、4-羟乙基哌嗪乙磺酸、葡萄糖、β-氨基乙磺酸、肌酸、丙酮酸钠、以及青霉素和链霉素。
本发明第二实施方式涉及心肌细胞分离方法，其包括脱钙、消化和细胞收集步骤，其中在脱钙步骤中使用如前述第一实施方式所述的组合物作为无钙液。
本发明第三实施方式涉及一种组合物，其可用于心肌细胞的培养，其通过如下获得：将(-)-Blebbistatin和/或para-aminoblebbistatin、抗生素和血清添加于已知用于细胞培养的培养基(优选选自M199系列、MEM系列、DMEM系列的一种或多种培养基)中。
本发明第四实施方式涉及一种试剂盒，其可用于心肌细胞的培养，其包含：
(1)前述第三实施方式中所述的组合物；和
(2)包被组合物，所述包被组合物包含基质胶或层粘连蛋白。
本发明第五实施方式涉及心肌细胞培养方法，其中使用前述第三实施方式中所述的组合物作为培养基。
在该实施方式中，在培养细胞之前使用包被组合物对培养板进行包被，所述包被组合物中包含基质胶或层粘连蛋白。
本发明第六实施方式涉及涉及(-)-Blebbistatin和/或para-aminoblebbistatin在用于制备心肌细胞分离试剂或心肌细胞培养试剂中的用途。
通过采用本发明的技术方案，能够在分离和培养过程中将人心肌细胞维持在最佳状态、保持细胞形态、存活率和纯度，由此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]A是示出20mM BDM组、5μM和10μM(-)-Blebbistatin组分离得到心肌细胞杆状率的图，B和C是示出20mM BDM组、5μM和10μM(-)-Blebbistatin组的心肌细胞长度的图，D是分离后人心肌细胞的显微照片，E是20mM BDM和10μM(-)-Blebbistatin组的心肌细胞分离后分别使用含有20mM BDM和10μM(-)-Blebbistatin的培养基培养5天后心肌细胞杆状率的图。
[图2]A是示出不同浓度的(-)-Blebbistatin对心肌细胞杆状率影响的图，B是示出不同 浓度的(-)-Blebbistatin对心肌细胞状态影响的图，C是示出不同浓度的para-aminoblebbistatin对心肌细胞存活率影响的图。
[图3]是示出不同包被组合物对心肌细胞贴壁数量影响的图。
[图4]是示出不同血清浓度对心肌细胞平均杆状细胞率影响的图。
[图5]是示出不同类型成品培养基对心肌细胞存活率影响的图。
[图6]是示出肌球蛋白抑制剂N-苄基-对甲苯磺酰胺(BTS)及MYK-461对心肌细胞存活率影响的图。
[图7]是示出(-)-Blebbistatin组和加入PICCT组的心肌细胞存活率的图。
[图8]是示出本发明培养基与ACCIT培养基对比结果的图。
[图9]A：是示出心肌细胞杆状率随培养时间而变化的图；B：是示出心肌细胞长度随培养时间而变化的图；C：是经不同的培养时间后心肌细胞的显微图像。
[图10]A：是示出心肌细胞杆状率随培养时间而变化的图；B：是示出心肌细胞长度随培养时间而变化的图；C：是示出心肌细胞宽度随培养时间而变化的图；D：是经不同的培养时间后心肌细胞的显微图像。
[图11]是示出心肌细胞基因上调或下调趋势的图。
注：附图中的“BLEB”指“(-)-Blebbistatin”，“PAB”指“para-aminoblebbistatin”。
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(-)-Blebbistatin或其para-aminoblebbistatin能够很好地解决本发明所要解决的技术课题。
(-)-Blebbistatin是一种已知的非肌肉肌球蛋白II抑制剂和细胞渗透性抑制剂，而para-aminoblebbistatin是(-)-Blebbistatin的衍生物，具有与(-)-Blebbistatin类似的结构和性质。在心血管生理学研究中，(-)-Blebbistatin通常用作特异性解偶联剂等，但已知(-)-Blebbistatin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(其溶解度仅为10.9±0.9μM)等。正因为存在这些不利特性，研究人员对(-)-Blebbistatin及其衍生物para-aminoblebbistatin的应用存在偏见，迄今为止尚未有将(-)-Blebbistatin或para-aminoblebbistatin用于心肌细胞、特别是人心肌细胞分离或培养的报道。然而，本申请发明人却惊讶地发现，(-)-Blebbistatin和para-aminoblebbistatin能够使心肌细胞在培养和分离的过程中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(-)-Blebbistatin和/或para-aminoblebbistatin作为培养和分离组合物中的关键成分，与其他特定成分进行组合来实施培养分离，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。
以下分别对本发明各实施方式中的具体实施方式进行描述。
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5-10 mM，表明其浓度范围为4.5-11mM。
在下文中归纳了各组合物中一些成分的作用。需要说明的是，任一成分都可能具有不止一种作用，且它们在整体组合物中实际的作用可能是无法预测的。例如本说明书中虽然将磷酸二氢钠列为等渗剂，但其同时具有pH调节缓冲的作用，也可视为酸碱调节剂。类似地，本说明书中虽然将葡萄糖列为能量物质，但该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。
除非另外指明，否则本文所述的心肌细胞为哺乳动物心肌细胞，优选为人心肌细胞，例如人原代心肌细胞。
(一)用于心肌细胞分离的组合物
在本发明的用于心肌细胞分离的组合物中含有(-)-Blebbistatin和/或para-aminoblebbistatin。在一个实施方式中，所述组合物中(-)-Blebbistatin的浓度为1-50μM，更优选2.5-20μM，更优选3-15μM，更优选约5-10μM。在一个实施方式中，所述组合物中para-aminoblebbistatin的浓度为1-100μM，更优选5-50μM，更优选8-25μM，更优选约10-20μM。
在一个实施方式中，所述用于分离的组合物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、抗生素、等渗剂。
所述用于分离的组合物中任选含有能量物质，其为细胞在分离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。
所述用于分离的组合物中任选含有代谢调节剂，例如丙酮酸钠、胰岛素(insulin)、肌酸(Creatine)、β-氨基乙磺酸(Taurine)、L-左旋肉碱(L-carnitine)等，其有助于细胞在分离过程中调节能量代谢。在一个优选实施方式中，所述代谢调节剂为丙酮酸钠、肌酸和/或β-氨基乙磺酸。在单独或组合使用的情况下，丙酮酸钠的浓度可为0.1-20mM，优选为1-10mM，更优选为3-8mM，特别优选约5mM；肌酸的浓度可为0.1-20mM，优选为1-10mM，更优选为3-8mM，特别优选约5mM；β-氨基乙磺酸的浓度可为5-50mM，优选为10-30mM，更优选为15-25mM，特别优选约20mM。
所述用于分离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)，和/或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。在另一个优选实施方式中，所述酸碱调节剂为HEPES和/或氢氧化钠。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；氢氧化钠的浓度没有明确限制，只要将组合物的最终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即可。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。
所述用于分离的组合物任选含有抗生素，其作用是抑制细菌生长。抗生素包括但不限于青霉素、链霉素和Primocin。在单独或组合使用的情况下，青霉素的浓度可为10-500 U/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；Primocin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。
所述用于分离的组合物中任选含有等渗剂，其能够维持渗透压，以确保细胞的水分和电解质平衡。等渗剂包括但不限于氯化镁(例如其水合物，如六水合物)、氯化钾、氯化钠、磷酸二氢钠或其组合。在单独或组合使用的情况下，氯化镁的浓度为0.1-20mM，优选为1-10mM，更优选为3-8mM，特别优选约5mM；氯化钾的浓度为0.1-20mM，优选为1-10mM，更优选为3-8mM，特别优选约4-5mM；氯化钠的浓度为10-500mM，优选约50-200mM，更优选约100-150mM，特别优选约120-130mM；磷酸二氢钠的浓度为0.5-30mM，优选为1-20mM，更优选为3-15mM，特别优选约5-10mM。
在一个优选的实施方式中，所述用于分离的组合物中包含如下成分：1-50μM的(-)-Blebbistatin，5-50mM的葡萄糖，0.1-20mM的丙酮酸钠，0.1-20mM的肌酸，5-50mM的β-氨基乙磺酸，0.5-10mM的4-羟乙基哌嗪乙磺酸(HEPES)，10-500U/ml的青霉素，10-500μg/ml的链霉素，0.1-20mM的氯化镁，0.1-20mM的氯化钾，10-500mM的氯化钠，0.5-30mM的磷酸二氢钠，以及适量的NaOH，使得组合物的pH值为7.0-7.8(下称“分离组合物1”)。
在一个更优选的实施方式中，所述用于分离的组合物中包含如下成分：2.5-20μM的(-)-Blebbistatin，10-30mM的葡萄糖，1-10mM的丙酮酸钠，1-10mM的肌酸，10-30mM的β-氨基乙磺酸，1-8mM的4-羟乙基哌嗪乙磺酸(HEPES)，20-400U/ml的青霉素，20-400μg/ml的链霉素，1-10mM的氯化镁，1-10mM的氯化钾，50-200mM的氯化钠，1-20mM的磷酸二氢钠，以及适量的NaOH，使得组合物的pH值为7.2-7.6(下称“分离组合物2”)。
在一个特别优选的实施方式中，所述用于分离的组合物中包含如下成分：3-15μM的(-)-Blebbistatin，20-25mM的葡萄糖，3-8mM的丙酮酸钠，3-8mM的肌酸，15-25mM的β-氨基乙磺酸，3-6mM的4-羟乙基哌嗪乙磺酸(HEPES)，50-300U/ml的青霉素，50-300μg/ml的链霉素，3-8mM的氯化镁，3-8mM的氯化钾，100-150mM的氯化钠，3-15mM的磷酸二氢钠，以及适量的NaOH，使得组合物的pH值为7.3-7.5(下称“分离组合物3”)。
在一个优选的实施方式中，所述用于分离的组合物中包含如下成分：1-100μM的para-aminoblebbistatin，5-50mM的葡萄糖，0.1-20mM的丙酮酸钠，0.1-20mM的肌酸，5-50mM的β-氨基乙磺酸，0.5-10mM的4-羟乙基哌嗪乙磺酸(HEPES)，10-500U/ml的青霉素，10-500μg/ml的链霉素，0.1-20mM的氯化镁，0.1-20mM的氯化钾，10-500mM的氯化钠，0.5-30mM的磷酸二氢钠，以及适量的NaOH，使得组合物的pH值为7.0-7.8(下称“分离组合物4”)。
在一个更优选的实施方式中，所述用于分离的组合物中包含如下成分：5-50μM的para-aminoblebbistatin，10-30mM的葡萄糖，1-10mM的丙酮酸钠，1-10mM的肌酸，10-30 mM的β-氨基乙磺酸，1-8mM的4-羟乙基哌嗪乙磺酸(HEPES)，20-400U/ml的青霉素，20-400μg/ml的链霉素，1-10mM的氯化镁，1-10mM的氯化钾，50-200mM的氯化钠，1-20mM的磷酸二氢钠，以及适量的NaOH，使得组合物的pH值为7.2-7.6(下称“分离组合物5”)。
在一个特别优选的实施方式中，所述用于分离的组合物中包含如下成分：8-25μM的para-aminoblebbistatin，20-25mM的葡萄糖，3-8mM的丙酮酸钠，3-8mM的肌酸，15-25mM的β-氨基乙磺酸，3-6mM的4-羟乙基哌嗪乙磺酸(HEPES)，50-300U/ml的青霉素，50-300μg/ml的链霉素，3-8mM的氯化镁，3-8mM的氯化钾，100-150mM的氯化钠，3-15mM的磷酸二氢钠，以及适量的NaOH，使得组合物的pH值为7.3-7.5(下称“分离组合物6”)。
(二)用于心肌细胞分离的方法
在所述心肌细胞分离方法中包括脱钙、消化和细胞收集步骤。在脱钙步骤中，可使用本发明所述的任一种用于心肌细胞分离的组合物作为无钙液；在消化步骤中，可向所述无钙液中加入本领域公知用于细胞消化的酶液，从而获得用于消化的组合物，以及向所述无钙液中加入血清，从而获得用于细胞转运的组合物。除此以外，其余操作、试剂和条件都与现有技术中一般的细胞分离方法所用操作、试剂和条件相同。
在一个实施方式中，在所述消化步骤用作消化液的组合物，是向无钙液(即本发明所述的任一种用于心肌细胞分离的组合物)中加入酶液和氯化钙而获得的，所述酶液例如为蛋白酶(例如蛋白酶XXIV)和/或胶原酶(例如胶原酶I、胶原酶II)。在一个实施方式中，在单独或组合使用的情况下，所述消化液中蛋白酶(例如蛋白酶XXIV)的浓度为0.1-5U/ml，优选0.5-3U/ml，更优选1-1.5U/ml，特别优选约1.2U/ml；胶原酶(例如胶原酶I、胶原酶II)的浓度例如为50-800U/ml，优选100-600U/ml，更优选200-400U/ml，特别优选约300U/ml。在一个实施方式中，所述消化液中氯化钙的浓度为0.002-0.1mM，优选0.005-0.05mM，更优选0.01-0.02mM，特别优选约0.015mM。
在一个优选的实施方式中，所述用作消化液的组合物(其pH为7.0-7.8)中除了对于前述分离组合物1或4所描述的成分以外，还含有如下成分：50-800U/ml的胶原酶II和0.002-0.1mM的氯化钙；在另一个具体实施方式中，其还另外含有0.1-5U/ml的蛋白酶XXIV。
在一个更优选的实施方式中，所述用作消化液的组合物(其pH为7.2-7.6)中除了对于前述分离组合物2或5所描述的成分以外，还含有如下成分：100-600U/ml的胶原酶II和0.005-0.05mM的氯化钙；在另一个具体实施方式中，其还另外含有0.5-3U/ml的蛋白酶XXIV。
在一个特别优选的实施方式中，所述用作消化液的组合物(其pH为7.3-7.5)中除了前述分离组合物3或6所描述的成分以外，还含有如下成分：200-400U/ml的胶原酶II和0.01-0.02mM的氯化钙；在另一个具体实施方式中，其还另外含有1-1.5U/ml的蛋白酶XXIV。
本发明所述的消化步骤可分阶段进行，且各阶段中可使用相同或不同的上述各实施方式中所述的组合物作为消化液。
在另一个实施方式中，在所述细胞转运步骤中用作细胞转运液的组合物，是向无钙液(即本发明所述的任一种用于心肌细胞分离的组合物)中加入血清而获得的。
在本文中，术语“血清”可以指任何常用于细胞培养领域的血清、或其中的主要成分(例如血清白蛋白等)。在一个实施方式中，血清为胎牛血清(FBS)。在另一个实施方式中，血清为牛血清白蛋白(BSA)。在一个优选的实施方式中，血清为胎牛血清，且胎牛血清占所述用作细胞转运液的组合物的体积分数为1％-20％，优选2％-15％，更优选3％-12％，特别优选约5％-10％；和/或，血清为牛血清白蛋白，且牛血清白蛋白在所述用作细胞转运液的组合物中的浓度为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。
在一个优选的实施方式中，所述用作细胞转运液的组合物(其pH为7.0-7.8)是向前述分离组合物1或4中加入胎牛血清和/或牛血清白蛋白而获得的，其中胎牛血清占所述心肌细胞培养组合物的体积分数为1％-20％，牛血清白蛋白在所述心肌细胞培养组合物中的浓度为0.1-10g/ml。
在一个更优选的实施方式中，所述用作细胞转运液的组合物(其pH为7.2-7.6)是向前述分离组合物2或5中加入胎牛血清和/或牛血清白蛋白而获得的，其中胎牛血清占所述心肌细胞培养组合物的体积分数为2％-15％，牛血清白蛋白在所述心肌细胞培养组合物中的浓度为0.2-5g/ml。
在一个特别优选的实施方式中，所述用作细胞转运液的组合物(其pH为7.3-7.5)是向前述分离组合物3或6中加入胎牛血清和/或牛血清白蛋白而获得的，其中胎牛血清占所述心肌细胞培养组合物的体积分数为3％-12％，牛血清白蛋白在所述心肌细胞培养组合物中的浓度为0.3-1g/ml。
(三)用于心肌细胞培养的组合物
本发明的用于心肌细胞培养的组合物，是向本领域常规用于细胞培养的培养基中加入(-)-Blebbistatin和/或para-aminoblebbistatin、血清和抗生素所得到的。其中，培养基例如为选自M199系列、MEM系列、DMEM系列的一种或多种培养基；优选地，该培养基可选自M199培养基、M199-无谷氨酰胺培养基、M199-4-羟乙基哌嗪乙磺酸-无谷氨酰胺培养基、MEM培养基、MEM-GlutaMAX培养基、MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX培养基、DMEM-高糖培养基、DMEM-低糖培养基；更优选地，该培养基可为M199培养基、MEM-GlutaMAX培养基或MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX培养基。另外，(-)-Blebbistatin和/或para-aminoblebbistatin的含量、以及血清和抗生素的种类和含量与前文第(一)部分针对“用于心肌细胞分离的组合物”和第(二)部分针对“用于心肌细胞分离的方法”所定义的内容相同。
在一个优选的实施方式中，所述用于心肌细胞培养的组合物是向上述任一系列或任一种培养基(例如M199、MEM-GlutaMAX或MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX培养基) 中加入以下成分获得的(以下统称“培养组合物1”)：
·(-)-Blebbistatin和/或para-aminoblebbistatin，其中所述组合物中(-)-Blebbistatin的浓度为1-50μM，para-aminoblebbistatin的浓度为1-100μM；
·青霉素、和/或链霉素、和/或Primocin，其中青霉素在所述组合物中的浓度为10-500U/ml，链霉素在所述组合物中的浓度为10-500μg/ml，Primocin在所述组合物中的浓度为10-500μg/ml；
·胎牛血清和/或牛血清白蛋白，其中胎牛血清占所述组合物的体积分数为1％-20％，牛血清白蛋白在所述组合物中的浓度为0.1-10g/ml。
在一个更优选的实施方式中，所述用于心肌细胞培养的组合物是向上述任一系列或任一种培养基(例如M199、MEM-GlutaMAX或MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX培养基)中加入以下成分获得的(以下统称“培养组合物2”)：
·(-)-Blebbistatin和/或para-aminoblebbistatin，其中所述组合物中(-)-Blebbistatin的浓度为2.5-20μM，para-aminoblebbistatin的浓度为5-50μM；
·青霉素、和/或链霉素、和/或Primocin，其中青霉素在所述组合物中的浓度为20-400U/ml，链霉素在所述组合物中的浓度为20-400μg/ml，Primocin在所述组合物中的浓度为20-400μg/ml；
·胎牛血清和/或牛血清白蛋白，其中胎牛血清占所述组合物的体积分数为2％-15％，牛血清白蛋白在所述组合物中的浓度为0.2-5g/ml。
在一个特别优选的实施方式中，所述用于心肌细胞培养的组合物是向上述任一系列或任一种培养基(例如M199、MEM-GlutaMAX或MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX培养基)中加入以下成分获得的(以下统称“培养组合物3”)：
·(-)-Blebbistatin和/或para-aminoblebbistatin，其中所述组合物中(-)-Blebbistatin的浓度为3-15μM(例如约10μM)，para-aminoblebbistatin的浓度为8-25μM(例如约10μM或20μM)；
·青霉素、和/或链霉素、和/或Primocin，其中青霉素在所述组合物中的浓度为50-300U/ml(例如约100U g/ml或200U/ml)，链霉素在所述组合物中的浓度为50-300μg/ml(例如约100μg/ml或200μg/ml)，Primocin在所述组合物中的浓度为50-300μg/ml(例如约100μg/ml或200μg/ml)；
·胎牛血清和/或牛血清白蛋白，其中胎牛血清占所述组合物的体积分数为5％-10％(例如约5％)，牛血清白蛋白在所述组合物中的浓度为0.3-1g/ml(例如约0.5g/ml)。
(四)用于心肌细胞培养的试剂盒
本发明的用于心肌培养的试剂盒包含：
(1)前述任一种用于心肌培养的组合物；和
(2)包被组合物，所述包被组合物包含基质胶(Matrigel)和/或层粘连蛋白(Laminin)，基质胶和/或层粘连蛋白的作用是促进细胞粘附于壁上生长。例如，在使用层粘连蛋白时， 其浓度为10-500μg/ml，优选20-300μg/ml，更优选50-200μg/ml，特别优选约50μg/ml或约200μg/ml。
在一个优选的实施方式中，所述试剂盒包含：
(1)前述培养组合物1中的任一种组合物；和
(2)包被组合物1，所述包被组合物1包含基质胶，和/或10-500μg/ml的层粘连蛋白。
在一个更优选的实施方式中，所述试剂盒包含：
(1)前述培养组合物2中的任一种组合物；和
(2)包被组合物2，所述包被组合物2包含基质胶，和/或20-300μg/ml的层粘连蛋白。
在一个特别优选的实施方式中，所述试剂盒包含：
(1)前述培养组合物3中的任一种组合物；和
(2)包被组合物3，所述包被组合物3包含基质胶，和/或50-200μg/ml的层粘连蛋白。
另外，在包被组合物中含有基质胶时，基质胶的用量与孔的实际容积相适应即可，例如可为150-200μl/孔。
(五)用于心肌细胞培养的方法
本发明的心肌细胞培养方法中，使用前文第(三)部分所述的组合物或第(四)部分所述的试剂盒，对细胞进行培养。
更优选地，在本发明的细胞培养方法中，先使用前文第(四)部分所述的包被组合物对培养板进行包被，然后再使用前文第(三)部分所述的组合物作为培养基对细胞进行培养。
(六)制备心肌细胞分离试剂或心肌细胞培养试剂的用途
本发明还涉及(-)-Blebbistatin和/或para-aminoblebbistatin在用于制备心肌细胞分离试剂或心肌细胞培养试剂中的用途，其使用方式如前文第(一)至(五)部分所述。
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：人心肌细胞分离试验
心肌细胞分离过程一般包括组织转运及切片、脱钙、酶液消化和细胞收集。其中，无钙台式液(无钙液)为分离过程中使用的主要试剂，用于脱钙、酶液消化和细胞收集。本发明中参照文献(Liang Chen,Guang-ran Guo,Man Rao,Kai Chen,Jiang-ping Song and Sheng-shou Hu,“A Modified Method for Isolation of Human Cardiomyocytes to Model Cardiac Diseases.”,Journal of Translational Medicine(2018).)中记载的心肌细胞分离实验方案进行操作。具体步骤如下。
将离体的左心耳立即置于器官保存液(威斯康星大学制造，美国)(约4℃)中，迅速转移至超净台内的60mm细胞培养皿中，加入器官保存液(约4℃)，并将其放在提前预冷(-20℃冰箱)的冰盒上，用剪刀去除多余的脂肪并对其进行修剪，将组织粘在样本台 上，使用切片机(莱卡VT1200S，德国)对组织进行切片(300μm)，然后于超净台内手动剪碎。将剪碎的心耳分为三组，分别使用本发明无钙液对组织进行脱钙处理，共三次。在锥形瓶内加入15-20ml无钙液，脱钙三次，室温摇晃锥形瓶，分别在2min、3min后更换新鲜无钙液，第三次脱钙时间为4min，脱钙结束后将无钙液弃掉，分别加入由对应的无钙液配制的酶液1(37℃预热)，锡箔纸包好瓶口后，在37℃的水浴摇床(Bibby Stuart SBS40，英国)中摇晃，随时观察酶液颜色的变化，由清澈逐渐变得浑浊。将酶液收集到15ml离心管内，锥形瓶中加入新鲜酶液1，继续在水浴锅中消化。将收集的酶液置于预冷的4℃离心机(Eppendorf centrifuge 5804R，德国)中离心(100×g，1min)，去上清，观察管底沉淀是否有心肌细胞，并判断杆状细胞所占比例。第二次消化同第一次。当沉淀中含有的心肌细胞杆状细胞率在70％以上时，将酶液1更换为由酶液1中使用的无钙液配制的酶液2，继续消化，此次开始收集浑浊酶液中的心肌细胞，采用的收集方法为，在离心(4℃，100×g，1min)后用3ml转运液重悬细胞。
其中，无钙液使用分别含有5μM(-)-Blebbistatin、10μM(-)-Blebbistatin和20mM BDM的无钙液。无钙液及酶液1、2的组成如下表1-3所示。
[表1]
Figure PCTCN2020082949-appb-000003
NaOH调整PH值，pH＝7.4
[表2]
组分 浓度
蛋白酶XXIV 1.2U/ml
胶原酶Ⅱ 300U/ml
CaCl 2 0.015mM
溶剂：无钙台式液
[表3]
组分 浓度
胶原酶Ⅱ 300U/ml
CaCl 2 0.015mM
溶剂：无钙台式液
结果如图1所示。由图1A可知，将20mM BDM组分离获得的细胞杆状率标准化为1.0，5μM和10μM(-)-Blebbistatin组获得的标准化杆状细胞率达到1.2-1.5(p<0.05)，显著提高了人心肌细胞存活率。由图1B和图1C可知，5μM和10μM(-)-Blebbistatin组分离得到的人心肌细胞的长度也明显长于20mM BDM组(p<0.05)。图1D是分离后的人心肌细胞的显微照片(莱卡，DMI4000B，10X)，可知20mM BDM组的杆状细胞形态并没有很好地得到维持，视野中可见到大量球形细胞；而5μM和10μM(-)-Blebbistatin组均可良好地维持人心肌细胞长杆状形态，10μM(-)-Blebbistatin组的效果更优。上述结果表明，含有5-10μM Blebbistatin的分离试剂的组均能够良好地在细胞分离中维持人心肌细胞形态并提高其杆状率。
此外，为了观察两种成分无钙液(20mM BDM和10μM(-)-Blebbistatin)分离得到的人心肌细胞在体外培养时细胞状态是否有所不同，我们对分离得到的人心肌细胞进行复钙。具体而言，人心肌细胞在无钙离子的环境中很难存活，本发明采用5步复钙法梯度恢复人心肌细胞的钙离子浓度。在细胞收集液中加入的CaCl 2(储备浓度30mM)的浓度梯度依次为0.08mM(5min)、0.25mM(5min)、0.5mM(5min)、1mM(5min)、1.8mM(15min)。20mM BDM组分离得到的人心肌细胞使用M199培养基，加入10％胎牛血清(FBS)(注：实施例及相应附图中胎牛血清的浓度百分比为体积百分比)、100U/ml青霉素和100μg/ml链霉素及20mM BDM进行培养。10μM(-)-Blebbistatin组分离得到的人心肌细胞使用M199培养基，加入10％胎牛血清、100U/ml青霉素和100μg/ml链霉素及10μM(-)-Blebbistatin进行培养。具体而言，将细胞置于37℃培养箱(5％CO 2，相对饱和湿度为95％，Thermo Scientific Heracell VIOS CO 2 160i，美国)中，分别培养6天。
其中，细胞收集液(表1-3)的组成如下表4所示。
结果如图1E所示，10μM(-)-Blebbistatin组的心肌细胞杆状率为80％以上，明显高于20mM BDM组(p<0.05)。这表明(-)-Blebbistatin不仅在人心肌细胞分离中对细胞起保护作用，还有利于人心肌细胞的体外培养。
[表4]
组分 含量
无钙台式液 36ml
胎牛血清 4ml
实施例2：人心肌细胞培养试验
实施例2.1：不同浓度的(-)-Blebbistatin和para-aminoblebbistatin对人心肌细胞杆状率的影响
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9组，在基础培养基中分别加入0、2.5、5、10、20μM的(-)-Blebbistatin和5μM、10μM、20μM、50μM的para-aminoblebbistatin。将分离得到的人心肌细胞分为9组，使用9种培养基将细胞铺于提前使用200μg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2所示。
由图2A可知，(-)-Blebbistatin使用浓度与细胞杆状率显著相关，加入(-)-Blebbistatin的各组杆状细胞率明显高于无(-)-Blebbistatin组，有统计学差异(p<0.001)。由此可以确认2.5-20μM的(-)-Blebbistatin可以显著提高心肌细胞存活率。另外，(-)-Blebbistatin组的心肌细胞杆状率随浓度上升而提高。
由图2B所示的心肌细胞培养图可知，培养1天后5μM和10μM(-)-Blebbistatin组的心肌细胞状态明显好于0μM组。与此同时，我们发现与5μM(-)-Blebbistatin组相比，10μM(-)-Blebbistatin组更好地维持了心肌细胞长杆状形态。培养7天后结果更为明显，10μM(-)-Blebbistatin组细胞边缘也逐渐变圆，但其形态及杆状细胞数明显好于0μM及5μM(-)-Blebbistatin组。综合考虑(-)-Blebbistatin对细胞活性的影响以及其水溶性和光敏性，可以确定10μM为培养心肌细胞的更合适的(-)-Blebbistatin浓度。
此外，由图2C可知，10μM的para-aminoblebbistatin组与10μM的(-)-Blebbistatin组无统计学差异，也即，两者对人心肌细胞存活能力的影响大致为同等程度。而且，可以确认5-50μM的para-aminoblebbistatin(简称PAB)可以显著提高心肌细胞存活率。
实施例2.2：包被组合物对人心肌细胞贴壁数量的影响
分别用以下试剂包被48孔培养板：层粘连蛋白(Gibco公司，商品编号：23017015；50μg/ml或200μg/ml，37℃培养箱孵育过夜，晾干)，纤维结合蛋白(fibronectin，Sigma Aldrich公司，商品编号：F0895-2MG；5μg/ml或50μg/ml，晾干)，多聚-L-赖氨酸(poly-L-lysine，Sigma Aldrich公司，商品编号：P04832-50ML；0.01％，室温孵育5min，晾干)和基质胶(康宁公司，商品编号：354234；37℃，30min)。在M199培养基中加入20mM的BDM、10％胎牛血清、100U/ml青霉素和100μg/ml链霉素而得到的本实验所用的培养基。将分离得到的心肌细胞铺于已包被的48孔培养板，培养72小时(h)后显微镜下拍照，每组取4个视野，求平均杆状细胞数，结果表示为均数±标准差。
结果如图3所示，培养72小时后，观察到多聚-L-赖氨酸和纤维结合蛋白(5μg/ml， 50μg/ml)组杆状细胞数量明显低于基质胶组、50μg/ml层粘连蛋白组及200μg/ml层粘连蛋白组，其中200μg/ml层粘连蛋白组杆状细胞数最高。这表明使用基质胶和层粘连蛋白可以让心肌细胞粘附生长。现有技术中作为包被组合物使用的层粘连蛋白的浓度通常为约10μg/ml，而本申请发明人发现在本发明中层粘连蛋白的浓度为50-200μg/ml时可使心肌细胞有效贴壁。
实施例2.3：血清浓度对人心肌细胞平均杆状细胞率的影响
在M199培养基中加入20mM的BDM、100U/ml青霉素和100μg/ml链霉素，然后分别加入不同浓度血清(0％、5％、10％和20％的胎牛血清)，人心肌细胞铺于提前用200μg/ml的层粘连蛋白包被的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％)，培养5天。
由图4可知，实验组平均杆状细胞率(杆状细胞占总细胞比例)明显高于对照组，结果有统计学差异(p<0.05)。但是，三种不同血清浓度实验组之间并无统计学差异(p>0.05)，10％胎牛血清组的平均杆状细胞率略高于5％胎牛血清组和20％胎牛血清组。由此可以确定心肌细胞培养过程中适合的胎牛血清浓度为5-20％，优选为10％。
实施例2.4：不同类型的成品培养基对人心肌细胞存活率的影响
选择了下述8种常用成品培养基，分别为MEM(购自Thermo公司)、MEM-GlutaMAX(购自Thermo公司)、MEM-GlutaMAX-4-羟乙基哌嗪乙磺酸(购自Thermo公司)、M199(购自Sigma公司)、M199-无谷氨酰胺(购自Thermo公司)、M199-4-羟乙基哌嗪乙磺酸-无谷氨酰胺(购自Thermo公司)、DMEM-高糖(购自Thermo公司)以及DMEM-低糖-丙酮酸钠(购自Thermo公司)。
8种不同成品培养基中分别加入20mM的BDM、100U/ml青霉素和100μg/ml链霉素。将分离得到的人心肌细胞铺于提前用200μg的层粘连蛋白包被的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％)，培养5天。
结果如图5所示，8种培养基均达到了约50％-70％以上的杆状细胞率，因此均可以用于人心肌细胞培养。特别是，与实施例2.1中所用的M199培养基相比，其它几种培养基(M199-无谷氨酰胺培养基、M199-4-羟乙基哌嗪乙磺酸-无谷氨酰胺培养基、MEM培养基、MEM-GlutaMAX培养基、MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX培养基、DMEM-高糖培养基、DMEM-低糖培养基)均在培养过程中取得了接近、持平或甚至更高的杆状细胞率，表明培养基的选择对人心肌细胞存活率的影响不大。
实施例2.5：肌球蛋白抑制剂N-苄基-对甲苯磺酰胺(BTS)和MYK-461对人心肌细胞存活率的影响
为了探索除了BDM和(-)-Blebbistatin以外是否存在其他对心肌细胞的杆状率有改善作用的肌球蛋白ATP酶抑制剂，将(-)-Blebbistatin、BTS、MYK-461对心肌细胞活性的影响进行了对比。针对分离的人心肌细胞，使用分别含有10μM(-)-Blebbistatin、BTS(浓度分别为5μM、10μM、20μM、30μM、40μM、50μM)、MYK-461(浓度分别为0.5μM、1μM、2μM、5μM、10μM、20μM)的MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX培养基， 并向所述培养基中加入10％胎牛血清、100U/ml青霉素和100μg/ml链霉素。
结果如图6所示，不同浓度的BTS和MYK-461组中，杆状细胞百分比无明显差异，但均明显低于(-)-Blebbistatin组(p<0.05)。这进一步说明了并不是所有肌球蛋白ATP酶抑制剂都可以在人心肌细胞的培养中取得良好效果。
实施例2.6：代谢调节剂对人心肌细胞存活率的影响
将代谢调节剂丙酮酸钠(sodium pyruvate，2mM)、胰岛素(insulin，10m g/L)、肌酸(creatine，5mM)、β-氨基乙磺酸(taurine，5mM)和L-左旋肉碱(L-carnitine，2mM)混合加入培养基中，上述代谢调节剂的混合物简称为“PICCT”。实验组根据基础培养基(M199培养基，其中添加有10％胎牛血清、100U/ml青霉素和100μg/ml链霉素)中加入不同浓度(-)-Blebbistatin(0μM、5μM、10μM)分为三组，每组包括有无PICCT两个条件，用以探讨生长因子及代谢底物对人心肌细胞存活率的影响。于37℃细胞培养箱(5％CO 2，相对饱和湿度为95％)中培养7天后，计算校准心肌细胞存活率(第7天细胞存活率/第0天细胞存活率×100％)。在心肌细胞培养过程中，部分细胞失去原有的长杆状细胞形态而贴壁生长，这部分细胞被视为死细胞，不计入活细胞数量。
如图7所示，与对应浓度的(-)-Blebbistatin组相比，加入PICCT对存活率无明显影响。以上结果说明，在细胞培养过程中，对细胞存活起更关键作用的成分是(-)-Blebbistatin，而不是PICCT中的代谢调节剂成分。
实施例2.7：本发明培养基与ACCIT培养基的对比
使用文献(V.Bistola,M.Nikolopoulou,A.Derventzi,A.Kataki,N.Sfyras,N.Nikou,M.Toutouza,P.Toutouzas,C.Stefanadis,and M.M.Konstadoulakis,“Long-Term Primary Cultures of Human Adult Atrial Cardiac Myocytes:Cell Viability,Structural Properties and Bnp Secretion in Vitro”,Int J Cardiol,131(2008),113-22.)中记载的ACCIT培养基(组分及含量如下述表5所示)和本发明培养基(在MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX成品培养基中加入10μM(-)-Blebbistatin、10％胎牛血清、100U/ml青霉素和100μg/ml链霉素而得到的培养基)分别对人心肌细胞培养6天，计算杆状细胞在全部细胞中所占的比例。
结果如图8所示，本发明完全培养基组的杆状心肌细胞比例明显高于ACCIT组，结果有统计学差异(p<0.05)。这进一步表明本发明培养基能够取得提高人心肌细胞存活率的优异效果。
[表5]
Figure PCTCN2020082949-appb-000004
综上所述，本发明优选的人心肌细胞的培养条件为：使用基质胶(37℃，30min)或50-200μg/ml层粘连蛋白(37℃培养箱孵育过夜，晾干)提前包被培养板，使用在选自MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX、M199-无谷氨酰胺、M199-4-羟乙基哌嗪乙磺酸-无谷氨酰胺、MEM、MEM-GlutaMAX、DMEM-高糖中的任一种成品培养基中加入10％-20％的胎牛血清、2.5-20μM(-)-Blebbistatin、及100U/ml青霉素和100μg/ml链霉素而得到的培养基。
以上结果表明，含有(-)-Blebbistatin的本发明培养基不仅能够显著提高分离人心肌细胞时的存活率，还能显著提高人心肌细胞培养过程中的存活率。
实施例2.8：培养时间对细胞形态的影响
在MEM-GlutaMAX中加入100μg/ml青霉素、100μg/ml链霉素、100μg/ml Primocin和10μM(-)Blebbistatin，得到本实验所用的基础培养基。实验共分2组，在基础培养基中分别加入BSA和FBS，获得分别含1g/ml BSA和10％FBS的基础培养基(注：为简便起见，对于实施例及相应附图中的BSA浓度单位，有时以近似的质量分数“％”来代替“g/ml”，例如，以“1％BSA”表示“1g/ml BSA”)。左心耳组织来源于45-65岁的病人，将分离得到的人原代心肌细胞分为2组，分别用含1％BSA和10％FBS的基础培养基进行重悬，使用200μg/ml的层连蛋白包被的96孔细胞培养板培养，每孔种入6000个细胞，置于37℃细胞培养箱(5％CO 2，相对饱和湿度为95％)中培养7天。培养过程中，每两天换液一次，用Incucyte(S3)仪器每隔4个小时进行自动拍照。数据处理时，测量每个细胞的长度和宽度，将长度和宽度比例大于2的细胞算作杆状细胞。计算每个培养条件下的细胞杆状率，实验重复进行6次，结果如图9所示。
虽然图9A表明两种培养基培养的心肌细胞杆状率均随着时间的延长而下降，但图9B表明10％FBS组的细胞长度在所试验的时间段内保持得相对较好，图9C表明1％BSA组在第3天后的杆状细胞的存活率保持得相对较好。
此外，本发明人进一步设计了将BSA和FBS同时用于细胞培养的方案，检测培养3天和6天时细胞杆状率、细胞长度、细胞宽度的变化，以期BSA和FBS同时应用会减小对细胞形态的影响。具体分组为在基础培养基中分别加入以下成分：0％BSA+0％FBS、1％BSA、10％FBS、1％BSA+10％FBS、0.5％BSA+10％FBS、1％BSA+5％FBS、0.5％BSA+5％FBS，共7组，重复进行3次。其他实验条件不变。结果如图10所示，表明各组均在约3天时和6天时维持了细胞的形态。
实施例2.9：连续培养对细胞转录水平的影响
人原代心肌细胞分离后分别用如实施例2.8所述的含1％BSA和10％FBS的基础培养基培养，并于培养后一天和三天后分别与分离当天进行比较。具体地，首先将由此分别获得的细胞样品离心，然后将弃掉上清浆的细胞沉淀储存于-80℃冰箱。试剂盒GeneJET RNA Purification Kit(Thermo Scientific,K0731)进行提取RNA，用NanoDrop 2000(Thermo Scientific)测定RNA浓度和质量。随后用KAPA mRNA HyperPrep Kit(Illumina,KK8581)将500ng的RNA构建cDNA文库。cDNA文库的大小和浓度分别用2100 Bioanalyzer (Agilent)和Qubit(Thermo Fisher Scientific)进行测定。将文库稀释到2-3pM后用Illumina NextSeq 500测序平台测序，相应的试剂盒为75cycles of NextSeq 500/550 High Output Kit v2.5kit(Illumina,20024906)。根据测序结果制作热图、统计上调和下调基因数及上下调基因变化量(|log2FoldChange|)。log2FoldChange≥0.58为上调，log2FoldChange≤-0.58为下调。使用t-检验比较了D1上调、D1下调、D3上调、D3下调中，1％BSA和10％FBS变化。由图11A可知，1％BSA和10％FBS在培养1天和3天的基因上下调变化的趋势基本相同，表明二者都能够保持心肌细胞原有的细胞状态。图11B中显示培养1天和3天的上下调基因数可见1％BSA组变化的基因数甚至比10％FBS组更少；图11C可知，在上调基因中，培养1天和培养3天1％BSA组的变化程度也比10％FBS组更少，这表明1％BSA组的效果甚至更优于10％FBS组。

Claims (10)

  1. 一种组合物，其含有(-)-Blebbistatin和/或para-aminoblebbistatin，以及任选的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、抗生素、等渗剂，其中
    (-)-Blebbistatin是由下述式(1)表示的化合物，
    Figure PCTCN2020082949-appb-100001
    para-aminoblebbistatin是由下述式(2)表示的化合物，
    Figure PCTCN2020082949-appb-100002
  2. 如权利要求1所述的组合物，其含有以下成分：1-50μM的(-)-Blebbistatin，5-50mM的葡萄糖，0.1-20mM的丙酮酸钠，0.1-20mM的肌酸，5-50mM的β-氨基乙磺酸，0.5-10mM的4-羟乙基哌嗪乙磺酸(HEPES)，10-500U/ml的青霉素，10-500μg/ml的链霉素，0.1-20mM的氯化镁，0.1-20mM的氯化钾，10-500mM的氯化钠，0.5-30mM的磷酸二氢钠，以及适量的NaOH，使得组合物的pH值为7.0-7.8。
  3. 一种心肌细胞分离方法，其包括脱钙、消化和细胞收集步骤，其中，在脱钙、消化和细胞收集步骤中使用权利要求1或2所述的组合物。
  4. 一种组合物，其通过如下获得：向M199系列、MEM系列、DMEM系列培养基中添加(-)-Blebbistatin和/或para-aminoblebbistatin、抗生素和血清。
  5. 如权利要求4所述的组合物，其通过向M199培养基、MEM-GlutaMAX培养基或MEM-4-羟乙基哌嗪乙磺酸-GlutaMAX培养基中加入以下成分而获得：
    ·(-)-Blebbistatin和/或para-aminoblebbistatin，其中所述组合物中(-)-Blebbistatin的浓度为1-50μM，para-aminoblebbistatin的浓度为1-100μM；
    ·青霉素、和/或链霉素、和/或Primocin，其中青霉素在所述组合物中的浓度为10-500U/ml，链霉素在所述组合物中的浓度为10-500μg/ml，Primocin在所述组合物中的浓度为10-500μg/ml；
    ·胎牛血清和/或牛血清白蛋白，其中胎牛血清占所述组合物的体积分数为1％-20％，牛血清白蛋白在所述组合物中的浓度为0.1-10g/ml。
  6. 一种试剂盒，其包含：
    (1)如权利要求4或5所述的组合物；和
    (2)包被组合物，所述包被组合物包含基质胶或层粘连蛋白。
  7. 一种心肌细胞培养方法，其中，使用权利要求4或5所述的组合物作为培养基。
  8. 如权利要求7所述的心肌细胞培养方法，其中，在培养细胞之前使用包被组合物对培养板进行包被。
  9. 如权利要求6所述的试剂盒或如权利要求8所述的方法，其中所述包被组合物包含基质胶和/或10-500μg/ml的层粘连蛋白。
  10. (-)-Blebbistatin和/或para-aminoblebbistatin在用于制备心肌细胞分离试剂或心肌细胞培养试剂中的用途。
PCT/CN2020/082949 2019-04-04 2020-04-02 人心肌细胞分离试剂、培养基、分离方法和培养方法 WO2020200272A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/594,072 US20220169989A1 (en) 2019-04-04 2020-04-02 Human cardiomyocyte separation reagent, culture medium, separation method, and culture method
EP20781916.0A EP3967751A4 (en) 2019-04-04 2020-04-02 HUMAN CARDIOMYOCYTE SEPARATION REAGENT, CULTURE MEDIUM, SEPARATION PROCEDURE AND CULTURE PROCEDURE

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910275447.2A CN111778205B (zh) 2019-04-04 2019-04-04 人心肌细胞分离试剂、培养基、分离方法和培养方法
CN201910275447.2 2019-04-04

Publications (1)

Publication Number Publication Date
WO2020200272A1 true WO2020200272A1 (zh) 2020-10-08

Family

ID=72664961

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/082949 WO2020200272A1 (zh) 2019-04-04 2020-04-02 人心肌细胞分离试剂、培养基、分离方法和培养方法

Country Status (4)

Country Link
US (1) US20220169989A1 (zh)
EP (1) EP3967751A4 (zh)
CN (1) CN111778205B (zh)
WO (1) WO2020200272A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115698263A (zh) * 2020-04-09 2023-02-03 青岛百洋智心科技有限公司 用于心肌细胞冻存的组合物
CN113424816A (zh) * 2021-04-07 2021-09-24 上海慧灯医疗科技有限公司 一种心肌细胞冻存液及其制备方法
WO2024050139A1 (en) * 2022-09-02 2024-03-07 AnaBios Corporation Therapeutic uses of cardiac myosin inhibitors

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101481415A (zh) * 2009-02-04 2009-07-15 山东农业大学 一种新型prrs病毒受体及该受体的阻断抑制剂

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110212480A1 (en) * 2010-02-26 2011-09-01 Kyle Louis Kolaja Use of primary human cardiomyocytes
WO2013063305A2 (en) * 2011-10-25 2013-05-02 Mount Sinai School Of Medicine Directed cardiomyocyte differentiation of stem cells
JP2017108705A (ja) * 2015-12-18 2017-06-22 国立大学法人京都大学 心筋細胞の製造方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101481415A (zh) * 2009-02-04 2009-07-15 山东农业大学 一种新型prrs病毒受体及该受体的阻断抑制剂

Non-Patent Citations (13)

* 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
E. J. SHARPEJ. R. ST CLAIRC. PROENZA: "Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice", J VIS EXP, 2016
FARMAN, GP ET AL.: "Blebbistatin: use as inhibitor of muscle contraction", PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, vol. volume 455, no. 6, 10 November 2007 (2007-11-10), XP019590131, ISSN: 0031-6768, DOI: 20200508113351A *
JR. WILLIAM A. CLARKRICHARD A. CHIZZONITEALAN W. EVERETTMURRAY RABINOWITZRADOVAN ZAK: "Species Correlations between Cardiac Isomyosins. A Comparison of Electrophoretic and Immunological Properties", THE JOURNAL OF BIOLOGICACL CHEMISTRY, 1981
K. W. LINZR. MEYERR. SURGESS. MEINARDUSJ. VEESA. HOFFMANN0. WINDHOLZC. GROHE: "Rapid Modulation of L-Type Calcium Current by Acutely Applied Oestrogens in Isolated Cardiac Myocytes from Human, Guinea-Pig and Rat", EXPERIMENTAL PHYSIOLOGY, 1998
KABAEVA, Z ET AL.: "Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression", AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, vol. 294, no. 4,, 30 April 2008 (2008-04-30), XP055739361, ISSN: 0002-9440, DOI: 20200508113736X *
LIANG CHENGUANG-RAN GUOMAN RAOKAI CHENJIANG-PING SONGSHENG-SHOU HU: "A Modified Method for Isolation of Human Cardiomyocytes to Model Cardiac Diseases", JOURNAL OF TRANSLATIONAL MEDICINE, 2018
M. ACKERS-JOHNSONP. Y. LIA. P. HOLMESS. M. O'BRIEND. PAVLOVICR. S. FOO: "A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes from the Adult Mouse Heart", CIRC RES, vol. 119, 2016, pages 909 - 20
S. WERFELS. NOTHJUNGET. SCHWARZMAYRT. M. STROMT. MEITINGERS. ENGELHARDT: "Characterization of Circular Rnas in Human, Mouse and Rat Hearts", JMOL CELL CARDIOL, vol. 98, 2016, pages 103 - 7, XP029724140, DOI: 10.1016/j.yjmcc.2016.07.007
STEPHANE N. HATEMMARTIN MORADJAMES S. K. SHAM: "Species Differences in the Activity of the Na(+)-Ca2+ Exchanger in Mammalian Cardiac Myocytes", JOURNAL OF PHYSIOLOGY, 1995
V. BISTOLAM. NIKOLOPOULOUA. DERVENTZIA. KATAKIN. SFYRASN. NIKOUM. TOUTOUZAP. TOUTOUZASC. STEFANADISM. M. KONSTADOULAKIS: "Long-Term Primary Cultures of Human Adult Atrial Cardiac Myocytes: Cell Viability, Structural Properties and Bnp Secretion in Vitro", INT J CARDIOL, vol. 131, 2008, pages 113 - 22
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
ZHAO MEI , QI QI-XUE , HE RONG : "The Effect of Blebbistatin on Primary Adult Mouse Cardiac Myocytes Culture and GFP Gene Transfer", CHINESE JOURNAL OF HEMORHEOLOGY, vol. 17, no. 4, 15 December 2007 (2007-12-15), CN, pages 529 - 532, XP055860194, ISSN: 1009-881X *

Also Published As

Publication number Publication date
EP3967751A1 (en) 2022-03-16
CN111778205B (zh) 2022-09-30
EP3967751A4 (en) 2022-12-21
US20220169989A1 (en) 2022-06-02
CN111778205A (zh) 2020-10-16

Similar Documents

Publication Publication Date Title
WO2020200272A1 (zh) 人心肌细胞分离试剂、培养基、分离方法和培养方法
Hamad et al. Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations
Rupert et al. IGF1 and NRG1 enhance proliferation, metabolic maturity, and the force‐frequency response in hESC‐derived engineered cardiac tissues
Zhang et al. Universal cardiac induction of human pluripotent stem cells in two and three-dimensional formats: implications for in vitro maturation
Sreejit et al. An improved protocol for primary culture of cardiomyocyte from neonatal mice
Liu et al. Metabolic reconfiguration supports reacquisition of primitive phenotype in human mesenchymal stem cell aggregates
CN105229145B (zh) 片状细胞培养物的制造方法
EP3008172B1 (en) Culture medium compositions for maturating cardiomyocytes derived from pluripotent mammalian stem cells
Malandraki-Miller et al. Changing metabolism in differentiating cardiac progenitor cells—can stem cells become metabolically flexible cardiomyocytes?
TW200521234A (en) Method of single cell culture of undifferentiated human embryonic stem cells
US20080254003A1 (en) Differentiation of Human Embryonic Stem Cells and Cardiomyocytes and Cardiomyocyte Progenitors Derived Therefrom
JP2016536975A (ja) 多能性幹細胞から機能的心筋へと直接分化させる方法
Pennarossa et al. Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of low-stiffness substrate
Apdik et al. Dose-dependent effect of boric acid on myogenic differentiation of human adipose-derived stem cells (hADSCs)
US20240189363A1 (en) Cardiomyocytes and compositions and methods for producing the same
US11913020B2 (en) Method for improving stem cell migration using ethionamide
Zhu et al. Disruption of histamine/H 1 R signaling pathway represses cardiac differentiation and maturation of human induced pluripotent stem cells
Wang et al. Neuregulin-1 enhances differentiation of cardiomyocytes from embryonic stem cells
Song et al. N-acetylcysteine promotes the proliferation of porcine adipose-derived stem cells during in vitro long-term expansion for cultured meat production
US11395863B2 (en) Modification method for sheet-shaped cell culture
Yamanaka et al. Enhanced proliferation of monolayer cultures of embryonic stem (ES) cell-derived cardiomyocytes following acute loss of retinoblastoma
Saadeldin et al. Generation of porcine endometrial organoids and their use as a model for enhancing embryonic attachment and elongation
Buikema et al. Expanding Mouse Ventricular Cardiomyocytes Through GSK‐3 Inhibition
Loos et al. Human model of primary carnitine deficiency cardiomyopathy reveals ferroptosis as a novel mechanism
Nakajima et al. Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method

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: 20781916

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: 2020781916

Country of ref document: EP

Effective date: 20211104