阅读说明：本技术 一种斑马鱼原代心肌细胞分离与培养的新方法 (Novel method for separating and culturing primary cardiomyocytes of zebra fish ) 是由 陈桢玥 王秋云 席其良 陆国平 于 2018-08-29 设计创作，主要内容包括：本发明公开了一种斑马鱼原代心肌细胞分离与培养的新方法,其包含以下步骤：步骤1：将斑马鱼用A1溶液进行清洗以去除杂物,然后用乙醇洗涤；步骤2：处死斑马鱼,并快速取出斑马鱼心脏；步骤3：将取出的心脏用A2溶液进行洗涤除菌；步骤4：用剪刀精细切碎心脏,用细胞培养液A3吹打成单细胞悬液；步骤5：将单细胞悬液离心后弃去细胞培养液A3,加入细胞培养液A4进行培养。本发明平均一个斑马鱼心脏能够得到约9.2×10<Sup>5</Sup>个原代心肌细胞。可进一步用于研究心脏发育与再生功能、疾病机制研究,也可以应用于筛选小分子药物。本方法效率高、经济、方便；分离的斑马鱼原代细胞具有功能性；经免疫荧光鉴定,纯度达到95％以上。(The invention discloses a novel method for separating and culturing primary cardiomyocytes of zebra fish, which comprises the following steps: step 1: washing zebra fish with A1 solution to remove impurities, and washing with ethanol; step 2: the zebra fish is sacrificed and the heart of the zebra fish is taken out quickly; and step 3: washing the removed heart with A2 solution for sterilization; and 4, step 4: finely cutting the heart with scissors, and blowing and beating the heart into single cell suspension by using cell culture solution A3; and 5: the single cell suspension was centrifuged, the cell culture solution A3 was discarded, and the cell culture solution A4 was added for culture. The invention can obtain about 9.2 multiplied by 10 on average one zebra fish heart 5 And (3) obtaining primary myocardial cells. Can be further used for researching the development and regeneration functions of the heart and the mechanism of diseases, and can also be applied to screening small molecular drugs. The method has high efficiency, economy and convenience; the isolated zebrafish primary cells are functional; through immunofluorescence identification, the purity reaches more than 95%.)

1. A novel method for separating and culturing primary cardiomyocytes of zebra fish is characterized by comprising the following steps:

step 1: washing zebra fish with A1 solution to remove impurities, and washing with ethanol;

step 2: the zebra fish is sacrificed and the heart of the zebra fish is taken out quickly;

and step 3: washing the removed heart with A2 solution for sterilization;

and 4, step 4: finely cutting the heart with scissors, and blowing and beating the heart into single cell suspension by using cell culture solution A3;

and 5: centrifuging the single cell suspension, removing a cell culture solution A3, and adding a cell culture solution A4 for culture;

the preparation method of the A1 solution comprises the following steps: taking 0.17-0.37g of potassium dihydrogen phosphate, 1.32-1.52g of disodium hydrogen phosphate, 7-9g of sodium chloride and 0.1-0.3g of potassium chloride, mixing, adding 700 mL of deionized water, stirring and dissolving, adding concentrated hydrochloric acid to adjust the pH value to 7-8, then fixing the volume to 1L, and finally adding 80-120 units/mL of penicillin and 80-120 mu g/mL of streptomycin;

the formula of the A2 solution is an A1 solution with double concentrations of penicillin and streptomycin;

the formula of the cell culture solution A3 is as follows: DMEM complete culture medium, fetal calf serum with volume fraction of 8% -12%, 80-120 units/mL penicillin and 80-120 mug/mL streptomycin;

the formula of the cell culture solution A4 is as follows: DMEM complete culture medium, horse serum with volume fraction of 2% -8% and 8-12 mu mol/L of cytarabine; 0.05-0.15mmol/L brdu, 1-3. mu.g/mL mitomycin C, 80-120 units/mL penicillin and 80-120. mu.g/mL streptomycin.

2. The novel method for separating and culturing zebrafish primary cardiomyocytes according to claim 1, wherein the A1 solution is prepared by the following steps: 0.27g of potassium dihydrogen phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride are taken, mixed and dissolved in 800mL of deionized water under stirring, concentrated hydrochloric acid is added to adjust the pH value to 7.4, then the volume is adjusted to 1L, and finally 100 units/mL of penicillin and 100 micrograms/mL of streptomycin are added.

3. The novel method for separating and culturing zebrafish primary cardiomyocytes of claim 1, wherein the formula of the cell culture solution A3 is as follows: DMEM complete medium, fetal bovine serum with a volume fraction of 10%, 100 units/mL penicillin and 100. mu.g/mL streptomycin.

4. The novel method for separating and culturing zebrafish primary cardiomyocytes of claim 1, wherein the formula of the cell culture solution A4 is as follows: DMEM complete culture medium, horse serum with volume fraction of 5% and 10 mu mol/L of cytarabine; 0.1mmol/L brdu, 2. mu.g/mL mitomycin C, 100 units/mL penicillin and 100. mu.g/mL streptomycin.

5. The novel method for isolation and culture of zebrafish primary cardiomyocytes according to claim 1 or4, wherein brdu and mitomycin C are added to the cell culture broth A4 only during the first 24 hours of the primary cardiomyocyte culture process.

6. The novel method for isolation and culture of zebrafish primary cardiomyocytes according to claim 1, wherein in step 1, said zebrafish are adult fish of 15-18 months of age, which are bred in an aqueous environment at a temperature of 28.5 ℃ under 14 hours of light and 10 hours of dark light cycle, and randomly selected among different breeding groups.

7. The novel method for isolation and culture of zebrafish primary cardiomyocytes according to claim 1, wherein in step 1 the zebrafish is washed at least 3 times with a solution a 1; the heart was washed at least 5 times with a solution of a2 in step 3.

8. The novel method for isolation and culture of zebrafish primary cardiomyocytes according to claim 1, wherein in step 2, zebrafish is killed by placing it in ice water.

9. The novel method for isolation and culture of zebrafish primary cardiomyocytes according to claim 1, wherein in step 5, the cell culture fluid A3 is washed again and centrifuged again before the cell culture fluid a4 is added, and the cell culture fluid A3 is discarded.

10. The novel method for isolating and culturing zebrafish primary cardiomyocytes of claim 1, wherein the A1 solution, the A2 solution, the cell culture solution A3, and the cell culture solution A4 are prepared and then sterilized by filtration.

Technical Field

The invention relates to the technical field of cell separation and culture, in particular to a novel method for separating and culturing primary cardiomyocytes of zebra fish.

Background

Cardiovascular disease is one of the leading causes of human death and is on an increasing trend year by year. Apoptosis or growth inhibition of myocardial cells can lead to myocardial dysfunction and even death of the body. Myocardial regeneration is a hot problem in current cardiovascular disease research. The heart tissue engineering research solves the problems of limited self-renewal and regeneration capacity of the myocardial cells and the like by immortalizing the myocardial cells, cell models of embryos or fetuses and newborns and inducing the directional differentiation of stem cells from different sources into the myocardial cells, and provides a new treatment strategy for myocardial regeneration; however, the cardiomyocytes produced by various research methods do not have the full function of adult cells. The cardiomyocytes are permanently differentiated cells, the in vitro culture is extremely difficult, and the conventional simulated cardiomyocytes (such as H9C2 cells) have partial activity of the cardiomyocytes but are not true cardiomyocytes; therefore, the invention provides a novel efficient method for culturing adult cardiomyocytes in vitro, which is helpful for solving the great problem that the real cardiomyocytes are difficult to culture in vitro in the current cardiovascular research field.

Zebrafish (Danio rerio) embryos develop in vitro and are transparent in whole bodies, have a genetic background highly homologous to humans, and are widely used for developmental studies. However, most of human cardiovascular diseases are developed in adults, so in recent years, adult models of zebra fish are gradually rising. At the present stage, the research of embryo zebra fish as a model mainly focuses on the aspects of heart development and regeneration. In the aspect of adult models, the application of adult zebra fish cardiovascular disease models and the research of related mechanisms are limited due to the small size of zebra fish, the small amount of heart tissues and the difficulty in primary culture of myocardial cells. The existing separation method of the primary cardiomyocytes of the zebra fish is low in efficiency, and the number of the obtained primary cardiomyocytes is very limited; meanwhile, the number of passages of the primary cells is limited, so that the application prospect of the zebra fish myocardial cell line is limited to a great extent. The difficulty in constructing an in-vitro myocardial cell culture methodology causes great limitation on the in-vitro research of adult zebra fish myocardial cells, and therefore, the method becomes one of important reasons that the establishment and research of adult models of zebra fish cardiovascular diseases are not widely developed. The method greatly improves the primary culture efficiency and quality of the myocardium of the adult heart of the zebra fish, and breaks through the bottleneck of zebra fish model research.

Disclosure of Invention

The invention aims to provide a novel method for separating and culturing primary zebra fish cardiomyocytes, which aims to overcome the defects and shortcomings of the existing method for separating and culturing the primary zebra fish cardiomyocytes and provide tool cells for research and application of the primary zebra fish cardiomyocytes.

In order to achieve the purpose, the invention provides a novel method for separating and culturing primary cardiomyocytes of zebra fish, which comprises the following steps:

step 1: washing zebra fish with A1 solution to remove impurities, and washing with ethanol;

step 2: the zebra fish is sacrificed and the heart of the zebra fish is taken out quickly;

and step 3: washing the removed heart with A2 solution for sterilization;

and 4, step 4: finely cutting the heart with scissors, and blowing and beating the heart into single cell suspension by using cell culture solution A3;

and 5: centrifuging the single cell suspension, removing a cell culture solution A3, and adding a cell culture solution A4 for culture;

the preparation method of the A1 solution comprises the following steps: taking 0.17-0.37g of potassium dihydrogen phosphate, 1.32-1.52g of disodium hydrogen phosphate, 7-9g of sodium chloride and 0.1-0.3g of potassium chloride, mixing, adding 700 mL of deionized water, stirring and dissolving, adding concentrated hydrochloric acid to adjust the pH value to 7-8, then fixing the volume to 1L, and finally adding 80-120 units/mL of penicillin and 80-120 mu g/mL of streptomycin;

the formula of the A2 solution is an A1 solution with double concentrations of penicillin and streptomycin;

the formula of the cell culture solution A3 is as follows: DMEM complete culture medium, fetal calf serum with volume fraction of 8% -12%, 80-120 units/mL penicillin and 80-120 mug/mL streptomycin;

the formula of the cell culture solution A4 is as follows: DMEM complete culture medium, horse serum with volume fraction of 2% -8% and 8-12 mu mol/L of cytarabine; 0.05-0.15mmol/L brdu, 1-3. mu.g/mL mitomycin C, 80-120 units/mL penicillin and 80-120. mu.g/mL streptomycin.

The novel method for separating and culturing the zebra fish primary myocardial cells comprises the following steps: 0.27g of potassium dihydrogen phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride are taken, mixed and dissolved in 800mL of deionized water under stirring, concentrated hydrochloric acid is added to adjust the pH value to 7.4, then the volume is adjusted to 1L, and finally 100 units/mL of penicillin and 100 micrograms/mL of streptomycin are added.

The novel method for separating and culturing the zebra fish primary myocardial cells comprises the following steps of: DMEM complete medium, fetal bovine serum with a volume fraction of 10%, 100 units/mL penicillin and 100. mu.g/mL streptomycin.

The novel method for separating and culturing the zebra fish primary myocardial cells comprises the following steps of: DMEM complete culture medium, horse serum with volume fraction of 5% and 10 mu mol/L of cytarabine; 0.1mmol/L brdu, 2. mu.g/mL mitomycin C, 100 units/mL penicillin and 100. mu.g/mL streptomycin.

The method for separating and culturing the zebra fish primary cardiomyocytes comprises the step of adding brdu and mitomycin C into the cell culture solution A4 only in the first 24 hours of the primary cardiomyocyte culture process.

In the step 1, the zebra fish is grown in a water environment with the temperature of 28.5 ℃ under 14-hour light and 10-hour dark light circulation, and is randomly selected from different breeding groups to be 15-18 months old.

The novel method for separating and culturing the primary cardiomyocytes of the zebra fish is characterized in that the zebra fish is washed for 3 times by using the A1 solution in the step 1; the heart was washed at least 5 times with a solution of a2 in step 3.

The novel method for separating and culturing the zebra fish primary myocardial cells comprises the step 2 of putting the zebra fish in ice water for killing.

In the step 5, before the cell culture solution A4 is added, the cell culture solution A3 is used for washing again and repeatedly centrifuging once, and then the cell culture solution A3 is discarded.

The novel method for separating and culturing the zebra fish primary cardiomyocytes comprises the steps of preparing the A1 solution, the A2 solution, the cell culture solution A3 and the cell culture solution A4, and then performing filtration sterilization.

Compared with the prior art, the invention has the following beneficial effects:

the method is to obtain the primary zebra fish myocardial cells by separating and culturing the zebra fish primary myocardial cells. On average, a zebrafish heart can be obtained at about 9.2 × 10⁵And (3) obtaining primary myocardial cells. Can be further used for researching the development and regeneration functions of the heart and can also be applied to screening small molecular drugs. The method has high efficiency, economy and convenience; the isolated zebrafish primary cells are functional; through immunofluorescence identification, the purity reaches more than 95%.

Drawings

FIG. 1 is a photograph of zebrafish on the third day after primary cardiomyocytes have been isolated;

FIG. 2 is a zebra fish primary myocardial cell immunofluorescence assay.

Detailed Description

The invention will be further described by the following specific examples in conjunction with the drawings, which are provided for illustration only and are not intended to limit the scope of the invention.

The invention provides a novel method for separating and culturing primary cardiomyocytes of zebra fish, which comprises the following steps:

step 1: washing zebra fish with A1 solution for at least 3 times to remove impurities, and washing with ethanol; the zebra fish is bred in a water environment with the temperature of 28.5 ℃ under the condition of 14-hour illumination and 10-hour dark light circulation, and 15-18 months old adult fishes are randomly selected from different breeding groups.

The preparation method of the A1 solution comprises the following steps: taking 0.17-0.37g of potassium dihydrogen phosphate, 1.32-1.52g of disodium hydrogen phosphate, 7-9g of sodium chloride and 0.1-0.3g of potassium chloride, mixing, adding 700 mL of deionized water, stirring and dissolving, adding concentrated hydrochloric acid to adjust the pH value to 7-8, then fixing the volume to 1L, and finally adding 80-120 units/mL of penicillin and 80-120 mu g/mL of streptomycin; and (5) filtering and sterilizing.

Preferably, the preparation method of the A1 solution comprises the following steps: taking 0.27g of monopotassium phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride, mixing, adding 800mL of deionized water, stirring for dissolving, adding concentrated hydrochloric acid to adjust the pH value to 7.4, then fixing the volume to 1L, and finally adding 100 units/mL of penicillin and 100 micrograms/mL of streptomycin; and (5) filtering and sterilizing.

Step 2: putting the zebra fish into ice water, killing the zebra fish by adopting a low-temperature method, and quickly taking out the heart of the zebra fish;

and step 3: washing the removed heart with A2 solution for at least 5 times for sterilization; the formula of the A2 solution is an A1 solution with double concentrations of penicillin and streptomycin, so that a better sterilization effect is realized;

and 4, step 4: finely cutting the heart with scissors, and blowing and beating the heart into single cell suspension by using cell culture solution A3;

the formula of the cell culture solution A3 is as follows: DMEM complete medium (cell general basic culture solution), fetal calf serum (cell culture nutrient substance) with volume fraction of 8% -12%, 80-120 units/mL penicillin and 80-120 mug/mL streptomycin (antibiotic, bacteriostasis); and (5) filtering and sterilizing.

Preferably, the formula of the cell culture solution A3 is as follows: DMEM complete medium, fetal calf serum with volume fraction of 10%, 100 units/mL penicillin and 100 mug/mL streptomycin; and (5) filtering and sterilizing.

And 5: centrifuging the single cell suspension, removing the cell culture solution A3, washing with the cell culture solution A3 again, centrifuging again, removing the cell culture solution A3 again, and adding the cell culture solution A4 for culturing;

the formula of the cell culture solution A4 is as follows: DMEM complete medium (cell universal basic culture solution), horse serum (nutrient substances for myocardial cell culture) with volume fraction of 2% -8%, and 8-12 mu mol/L of cytarabine (inhibiting the growth of hybrid cells); 0.05-0.15mmol/L of 5-bromodeoxyuridine (brdu, which inhibits the growth of the heterocytes by adding only the first 24 hours of the primary culture), 1-3 mug/mL of mitomycin C (which inhibits the growth of the heterocytes by adding only the first 24 hours of the primary culture), 80-120 units/mL of penicillin and 80-120 mug/mL of streptomycin (antibiotics, bacteriostasis); and (5) filtering and sterilizing.

Preferably, the formula of the cell culture solution A4 is as follows: DMEM complete culture medium, horse serum with volume fraction of 5% and 10 mu mol/L of cytarabine; 0.1mmol/L brdu, 2. mu.g/mL mitomycin C, 100 units/mL penicillin and 100. mu.g/mL streptomycin; and (5) filtering and sterilizing.

Example (c):

isolation, culture and identification of zebra fish primary cardiomyocytes

Animals:

adult zebrafish AB (Danio rerio) were obtained from commercial suppliers and maintained and bred at zebrafish house breeding center belonging to rekins hospital at shanghai university of transportation. Zebra fish is bred in water environment at 28.5 deg.C under 14 hr light and 10 hr dark light cycle, and adult fish of 15-18 months age are randomly selected from different breeding groups.

Reagent:

potassium dihydrogen phosphate (KH)₂PO₄) Disodium hydrogen phosphate (Na)₂HPO₄) Sodium chloride (NaCl), potassium chloride (KCl) and concentrated hydrochloric acid were purchased from the national drug group, penicillin, streptomycin, cytarabine, 5-bromodeoxyuridine (brdu) and mitomycin C were purchased from Sigma, 70% ethanol, horse serum, fetal bovine serum, DMEM were purchased from Hyclone, cardiac Myosin Heavy Chain (MHC), α smooth muscle actin (α -SMA), troponin T (TnT), troponin I (TnI) were purchased from Abcam, Vimentin (Vimentin) was purchased from Santa cruz, anti-rabbit Alexa Fluor488, anti-rabbit Alexa Fluor594, anti-mouse Alexa Fluor488, anti-mouse Alexa Fluor594 from Shanghai Bay Biotech Ltd.

The test method comprises the following steps:

(1) zebrafish were washed 3 times with a solution of a1 for 30 seconds each time, and then 3 times with 70% ethanol for 30 seconds each time. The formulation of solution a1 was as follows: pH 7.4; potassium dihydrogen phosphate (KH)₂PO₄): 0.27 g; disodium hydrogen phosphate (Na)₂HPO₄): 1.42 g; sodium chloride (NaCl): 8g of the total weight of the mixture; potassium chloride (KCl): 0.2 g; adding deionized water about 800mL, stirring thoroughly to dissolve, adding concentrated hydrochloric acid to adjust pH to 7.4, diluting to 1L, and adding penicillin 100 units/mL and streptomycin 100 μ g/mL. And (5) filtering and sterilizing.

(2) The zebrafish were placed in ice water, sacrificed cryogenically, and the zebrafish heart was quickly removed under a dissecting scope and placed in a solution a 2. The A2 solution is prepared into A1 solution with double concentration of penicillin and streptomycin to realize better sterilization effect. And (5) filtering and sterilizing.

(3) Washing the removed heart with A2 solution for 5 times, each for 30 seconds;

(4) the heart was finely minced with scissors and blown into a single cell suspension with zebrafish cardiomyocyte medium a 3. The formula of the cell culture solution A3 is as follows: DMEM complete medium; fetal Bovine Serum (FBS) with a volume fraction of 10%; 100 units/mL penicillin and 100. mu.g/mL streptomycin. And (5) filtering and sterilizing.

(5) The cells were centrifuged at 2000 rpm for 5 minutes, the cell culture solution A3 was discarded after centrifugation, and the cells were washed again with the cell culture solution A3, centrifuged again and the cell culture solution A3 was discarded. Adding zebra fish myocardial cell culture solution A4. 5% CO at 28 ℃₂Cultured in an incubator.

The beating function of the zebrafish cardiomyocytes after 24 hours of culture is shown in figure 1 (original magnification of 20-fold observed in bright field). The formula of the A4 solution is as follows: DMEM complete medium; horse serum with volume fraction of 5%; 10 μmol/L of an cytarabine; 0.1mmol/L brdu (plus primary first 24 hours only); 2. mu.g/ml mitomycin C (plus primary only for the first 24 hours); 100 units/mL penicillin and 100. mu.g/mL streptomycin. And (5) filtering and sterilizing.

(6) After 72 hours, cells are passaged, and the zebrafish myocardial cells are identified by an immunofluorescence method, the result is shown in figure 2 (the original magnification is 20 times), the myocardial cell identification is that the staining of Vimentin (Vimentin) is negative, the staining of myocardial Myosin Heavy Chain (MHC), α smooth muscle actin (α -SMA), troponin T (TnT) and troponin I (TnI) are positive, and the purity of the separated zebrafish primary cells is over 95 percent through the immunofluorescence identification.

In summary, the method has the following advantages:

(1) the efficiency is high: according to the previous method: in vitro culture of zebrafish cardiomyocytes and cardiac progenitors and three-dimensional culture of adult zebrafish cardiac tissue as disclosed by Zeng WR et al (Zeng WR, Beh SJ, Bryson-Richardson RJ, Doran PM. production of zebrafish cardiospheres and cardiac pro)genetic cell vitro and three-dimensional culture of adult Zebraf fish tissue antigens, Biotechnology and bioengineering 2017,114:2142-⁵And (3) obtaining primary myocardial cells. The method can obtain 9.2 × 10 heart of each zebra fish⁵And (3) obtaining primary myocardial cells. The efficiency is improved by 2.3 times.

(2) Economy: according to the previous method: in vitro culture of zebrafish cardiomyocytes and cardiac progenitors and three-dimensional culture of adult zebrafish cardiac tissue as disclosed by Zeng WR et al (Zeng WR, Beh SJ, Bryson-Richardson RJ, Donan PM.Production of zebrafish heart proteins and heart promoter cells in vitro and three-dimensional culture of gene-dimensional culture of adult zebrafish heart tissue in biotechnology and biotechnology 2017,114: 2142-. Because the efficiency of the method is 2.4 times that of the former old method, the method only needs 25-30 zebra fishes and is more economical.

(3) The purity is high: the purity of the zebra fish primary cells separated by the method is over 95 percent through immunofluorescence identification.

(4) The application is wide: the zebra fish primary cells separated by the method have the characteristics of high efficiency, economy and high purity, so that the zebra fish primary cells can be applied to basic research and can also be used as cell models for screening new clinical drugs.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.