Method for culturing myocardial cells

Abstract

The invention relates to the technical field of cell culture, and particularly discloses a myocardial cell culture method which comprises the following steps: 1) selecting an experimental subject capable of carrying out myocardial cell culture; 2) separating the cardiac muscle tissue of the experimental subject; 3) separating cardiac muscle cells from the separated cardiac muscle tissue; and (4) adding the separated myocardial cells into a high-glucose DMEM culture solution, then adding 5-bromodeoxyuridine, uniformly mixing, inoculating into a culture dish which is coated with polyglutamic acid overnight at 37 DEG C in advance according to the density of (1.0-6.0) * 10 < 5 > cells / cm < 2 >, and culturing in a 5% CO2 cell culture box at 37 DEG C, after 48h, replacing a high-glucose DMEM culture solution which does not contain 5-bromodeoxyuridine and contains 15% of fetal calf serum, and subsequently replacing the high-glucose DMEM culture solution which does not contain 5-bromodeoxyuridine and contains 15% of fetal calf serum according to the growth state of the cells for 1d-2d for continuous culture. The myocardial cell culture method provided by the invention can efficiently and stably obtain a large number of myocardial cells with good activity, high survival rate and high purity.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, in particular to a multifunctional traditional Chinese medicine extraction system. Background Art

[0002] In vitro culture of cardiomyocytes can provide homogeneous colonies of single cells and is not affected by neural or humoral factors. It has become one of the basic methods for studying myocardial signal transduction and drug screening. As an in vitro experimental research model, cardiomyocytes are irreplaceable. They can eliminate the interference of neural, humoral, and endocrine factors and observe and study the development of the heart and the occurrence and development mechanism of cardiovascular diseases alone. At the same time, they have spontaneous pulsation and maintain many of their structures and functions in the body.

[0003] Existing methods for culturing cardiomyocytes mainly include tissue block adhesion method and enzyme digestion separation method. Although tissue block adhesion is simple and economical to operate, it also has obvious disadvantages, such as poor adhesion of tissue blocks, and the cells cultured on the wall also contain a large number of non-cardiomyocytes such as fibroblasts, and the screening and separation of cardiomyocytes is repeated. In addition, not every piece of tissue can grow cells, and a large amount of tissue needs to be cultured and screened, which leads to a low success rate of culturing cardiomyocytes through the tissue block adhesion method, a large workload, and low culturing efficiency. The enzymes mainly used in the enzyme digestion separation method are pancreatin and collagenase, which decompose the intercellular matrix in the myocardial tissue, thereby dispersing the cells into single cells, which is convenient for subsequent culture and use. However, pancreatin not only decomposes the inter-tissue protein components, but also has a strong destructive effect on cell membrane proteins and intracellular microtubules and microfilaments. Therefore, long-term digestion with pancreatin alone causes a large number of cardiomyocytes to lose the ability to adhere to the wall and spontaneously beat, greatly affecting the vitality and survival rate of the cells. In addition, the tissue treated with pancreatin digestion can release a large amount of flocculent collagen, which is difficult to separate the supernatant, and the number of cardiomyocytes obtained is small and the vitality is poor. Collagenase mainly decomposes the collagen fibers between cells. The appropriate application of collagenase can fully disperse the clumped cardiomyocytes to increase the number of cardiomyocyte digestion. Its action is moderate, and the damage to cardiomyocytes is small, but it is difficult to completely digest. At present, most of them use traditional mixed enzymes, that is, the method of mixed digestion of pancreatin and collagenase to separate cardiomyocytes. Although the number of cells separated and digested is large, there are still problems such as poor cell adhesion, poor activity and low purity.

[0004] In addition, since cardiomyocytes are highly differentiated near-terminal cells with the characteristics of losing the ability to proliferate, divide and differentiate, the culture process is easily affected by various processing factors and operation levels. There have always been many shortcomings such as low adhesion rate, low survival rate, low beating rate and low purity of cardiomyocytes, which will have a great impact on the subsequent related experimental results. Therefore, how to make the separated cardiomyocytes achieve the ideal survival rate and purity is a bottleneck problem that needs to be solved urgently. Summary of the invention

[0005] The object of the present invention is to provide a method for culturing cardiomyocytes that can efficiently and stably obtain cardiomyocytes with good vitality, high survival rate and high purity in large quantities.

[0006] The present invention is achieved through the following technical solution: A method for culturing cardiomyocytes comprises the following steps:

[0007] (1) Select experimental subjects that can culture cardiomyocytes;

[0008] (2) Isolating the myocardial tissue of the experimental subjects;

[0009] (3) isolating cardiomyocytes from isolated myocardial tissue;

[0010] (4) After adding the isolated cardiomyocytes to high-glucose DMEM culture medium, 5-bromodeoxyuridine was added and mixed, and then the cells were seeded at a density of (1.0-6.0)×105 cells / cm2 in a culture dish pre-coated with polyglutamic acid at 37°C overnight, and cultured in a cell culture incubator at 37°C and 5% CO2. After 48 hours, the high-glucose DMEM culture medium containing no 5-bromodeoxyuridine and 15% fetal bovine serum was replaced. Subsequently, the high-glucose DMEM culture medium containing no 5-bromodeoxyuridine and 15% fetal bovine serum was replaced for 1 day to 2 days according to the growth status of the cells and continued to be cultured.

[0011] In order to better implement the method of the present invention, further, the experimental subjects selected in step (1) that can be used for cardiomyocyte culture include at least one of KM rat suckling mice, Wistar rat suckling mice, C57 mouse suckling mice, suckling pigs, and suckling rabbits, and the experimental subjects are all SPF grade.

[0012] In order to better implement the method of the present invention, further, the experimental subjects for separation in step (2) are suckling mice, and the specific process of isolating the myocardial tissue is as follows:

[0013] (2.1) Soak the suckling mice in 75% alcohol for 8-10 seconds for disinfection, then quickly transfer them to the clean bench and fix them on a sterile foam board;

[0014] (2.2) Cut the skin just to the left of the middle of the sternum of the suckling mouse, separate the skin with forceps, fully expose the chest, and make a longitudinal incision to open the chest cavity;

[0015] (2.3) According to the beating of the heart, squeeze out the heart and remove it with curved forceps;

[0016] (2.4) Place the removed heart in ice-cold phosphate buffer and wash it by blowing to clean the red blood cells;

[0017] (2.5) After removing the atria and fibrous tissue, move the heart to another clean culture dish and cut the heart 2 to 3 times, washing it 3 times in total.

[0018] In order to better implement the method of the present invention, further, the operation processes of step (2.2) and step (2.5) are both completed by ophthalmic scissors and assisted by ophthalmic forceps.

[0019] In order to better implement the method of the present invention, further, the operation process of step (2.5) is completed on ice.

[0020] In order to better implement the method of the present invention, further, the process of isolating myocardial cells from the isolated myocardial tissue in step (3) is:

[0021] (3.1) The heart tissue was aspirated into a glass culture bottle, 10 mL of 0.25% trypsin digestion solution containing ethylenediaminetetraacetic acid was added, and the solution was incubated at 4°C for 12 to 16 hours.

[0022] (3.2) Take the heart tissue after resting, add 10 mL of high-glucose DMEM culture medium, and keep in a 37°C constant temperature water bath for 5 min to stop the digestion of pancreatic enzymes;

[0023] (3.2) Discard the supernatant and keep the heart tissue. Add 10 mL of collagen digestion solution and place in a 37°C constant temperature water bath. Shake gently for 1 min on a shaker with the speed controlled at 15 r / min.

[0024] (3.3) Discard the supernatant, transfer the heart tissue to another culture bottle, add 12 mL of type II collagenase, place in a 37°C constant temperature water bath, and shake gently on a shaker for 30 minutes at a speed of 15 r / min;

[0025] (3.4) Transfer the supernatant to a new centrifuge tube and repeat the digestion 3 to 4 times. When only flocculent material is seen, the myocardial tissue is completely digested.

[0026] (3.5) The collected supernatant was centrifuged for 5 min at a speed of 1000 r / min, the supernatant was discarded, 30 mL of high-glucose DMEM culture medium was added, the tube wall was gently tapped to suspend the cells, and then inoculated into a 100 mm culture dish and incubated in a 37°C, 5% CO2 incubator for 70 min. Non-cardiac cells such as fibroblasts and endothelial cells were removed by the differential adhesion method; the resulting cell suspension contained the isolated cardiomyocytes.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] (1) The present invention uses poly-lysine pre-coating to treat the culture dish. Glutamine can upregulate the expression of heat shock protein 70 and metallothionein, which are protective proteins of myocardial cells, thereby stabilizing the cell structure, improving the energy metabolism of cells, and helping to promote myocardial cell adhesion and maintain longer spontaneous beating.

[0029] (2) The present invention treats the heart tissue by adding ethylenediaminetetraacetic acid to the trypsin digestion solution, wherein ethylenediaminetetraacetic acid is a chelating agent for calcium ions, a non-enzymatic digestion product, and its main function is to destroy the connection between cells. Therefore, adding ethylenediaminetetraacetic acid to the trypsin digestion solution can enhance the digestion effect of trypsin, reduce damage to cells, and have a better digestion effect;

[0030] (3) The present invention uses a high-glucose DMEM culture medium that does not contain 5-bromodeoxyuridine and contains 15% fetal bovine serum to culture cardiomyocytes. Although 5-bromodeoxyuridine is beneficial to the further purification of cardiomyocytes, 5-bromodeoxyuridine also has a certain effect on the vitality of cardiomyocytes while inhibiting the proliferation of fibroblasts. Therefore, the high-glucose DMEM culture medium that does not contain 5-bromodeoxyuridine and contains 15% fetal bovine serum is used. The use of a high-glucose DMEM culture medium that effectively contains 15% fetal bovine serum is helpful to promote the proliferation of cardiomyocytes, maintain the function and vitality of cells, increase cell density, and reduce the interference of non-cardiomyocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments with reference to the following drawings:

[0032] Figure 1 It is a schematic diagram of the process of the present invention;

[0033] Figure 2 The morphology of myocardial cells under an optical microscope after 48 hours of culture using the method of the present invention;

[0034] Figure 3 The invention relates to immunofluorescence identification of cardiomyocytes cultured by the culture method of the present invention. DETAILED DESCRIPTION

[0035] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0036] In order to make the purpose, process conditions and advantages of the present invention more clearly understood, the present invention is further described in detail in conjunction with the following embodiments, but the embodiments of the present invention are not limited thereto. Without departing from the above-mentioned technical ideas of the present invention, various substitutions and changes are made according to the common technical knowledge and customary means in the field, which should all be included in the scope of the present invention. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] Embodiment 1:

[0038] This embodiment provides a method for culturing cardiomyocytes. The specific process is as follows: Figure 1 As shown, the following steps are included:

[0039] (1) Select suckling mice as the experimental subjects for separation;

[0040] (2) The specific process of isolating myocardial tissue from neonatal mice is as follows:

[0041] (2.1) Soak the suckling mice in 75% alcohol for 8-10 seconds for disinfection, then quickly transfer them to the clean bench and fix them on a sterile foam board;

[0042] (2.2) Use ophthalmic scissors to cut the skin on the left side of the middle of the sternum of the suckling mouse, separate the skin with forceps, fully expose the chest, make a longitudinal incision to open the chest cavity, try not to exceed the range of the first skin cut, and avoid opening the abdominal cavity to avoid contamination;

[0043] (2.3) According to the beating of the heart, squeeze out the heart and remove it with curved forceps;

[0044] (2.4) Place the removed heart in ice-cold phosphate buffer and wash it by blowing to clean the red blood cells;

[0045] (2.5) Perform the lower part of the operation on ice: Use ophthalmic forceps and ophthalmic scissors to remove excess atrium and surrounding lung tissue, then move the heart to another clean culture dish and cut the heart 2 to 3 times (just enough to make sure it is not broken), and wash it 3 times in total

[0046] (3) isolating cardiomyocytes from isolated myocardial tissue;

[0047] (3.1) The heart tissue was aspirated into a glass culture bottle, 10 mL of 0.25% trypsin digestion solution containing ethylenediaminetetraacetic acid was added, and the solution was incubated at 4°C for 12 to 16 hours.

[0048] (3.2) Take the heart tissue after resting, add 10 mL of high-glucose DMEM culture medium, and keep in a 37°C constant temperature water bath for 5 min to stop the digestion of pancreatic enzymes;

[0049] (3.2) Discard the supernatant and keep the heart tissue. Add 10 mL of collagen digestion solution and place in a 37°C constant temperature water bath. Shake gently for 1 min on a shaker with the speed controlled at 15 r / min.

[0050] (3.3) Discard the supernatant, transfer the heart tissue to another culture bottle, add 12 mL of type II collagenase, place in a 37°C constant temperature water bath, and shake gently on a shaker for 30 minutes at a speed of 15 r / min;

[0051] (3.4) Transfer the supernatant to a new centrifuge tube and repeat the digestion 3 to 4 times. When only flocculent material is seen, the myocardial tissue is completely digested.

[0052] (3.5) The collected supernatant was centrifuged for 5 min at a speed of 1000 r / min, the supernatant was discarded, 30 mL of high-glucose DMEM culture medium was added, the tube wall was gently tapped to suspend the cells, and then inoculated into a 100 mm culture dish and incubated in a 37°C, 5% CO2 incubator for 70 min. Non-cardiac cells such as fibroblasts and endothelial cells were removed by the differential adhesion method; the resulting cell suspension contained the isolated cardiomyocytes.

[0053] (4) After adding the isolated cardiomyocytes to high-glucose DMEM culture medium, 5-bromodeoxyuridine was added and mixed, and then the cells were seeded at a density of (1.0-6.0)×105 cells / cm2 in a culture dish pre-coated with polyglutamic acid at 37°C overnight, and cultured in a cell culture incubator at 37°C and 5% CO2. After 48 hours, the high-glucose DMEM culture medium containing no 5-bromodeoxyuridine and 15% fetal bovine serum was replaced. Subsequently, the high-glucose DMEM culture medium containing no 5-bromodeoxyuridine and 15% fetal bovine serum was replaced for 1 day to 2 days according to the growth status of the cells and continued to be cultured.

[0054] Embodiment 2:

[0055] This embodiment tests the survival rate of cultured cardiomyocytes on the basis of the above embodiment. The specific process is as follows:

[0056] Take 90 μL of myocardial cell suspension and add 10 μL of 0.4% trypan blue dye solution to mix evenly. After standing for about 3 minutes, take 1 to 2 drops of the mixed cell suspension and drip along the edge of the cover glass. Place the counting plate under an inverted phase contrast microscope for observation and counting. The round cells with strong refractive index that are not stained are living myocardial cells, and the cells stained blue are dead cells. Randomly take 4 fields of view for cell counting. The survival rate (%) of the counted myocardial cells = number of living cells / (number of living cells + number of dead cells) × 100%, and repeat the counting calculation 5 times.

[0057] Experiments show that the cardiomyocyte culture method provided by the present invention can obtain a large number of cardiomyocytes, and the cell survival rate detected by trypan blue staining can reach more than 99%. This method is simple, economical, and has good cell adhesion culture. The differential adhesion method is used to remove non-cardiomyocytes such as fibroblasts, and the chemical reagent BrdU is added to inhibit the growth and proliferation of residual fibroblasts. After culturing for 2 to 5 days, the vast majority of cells in the field of view are beating cardiomyocytes or connected cardiomyocyte clusters, and no obvious growth and proliferation of non-cardiomyocytes such as fibroblasts is observed.

[0058] Embodiment 3:

[0059] This embodiment, based on the above embodiment, performs beating and morphological observation on cultured cardiomyocytes, and the specific process is as follows:

[0060] The cardiomyocytes were inoculated into 100 mm culture dishes pre-coated with poly-lysine and cultured to adhere to the wall. The beating frequency, rhythm, and intensity of the cardiomyocytes were observed under an inverted phase contrast microscope to determine the vitality of the cardiomyocytes. The growth status and morphological changes of the cardiomyocytes were observed and photographed.

[0061] The experimental results are as follows Figure 2 As shown, the cardiomyocyte culture method provided by the present invention is that the newly separated and digested cardiomyocytes are suspended in the cell fluid and are round or elliptical. After 2 to 4 hours of culture, the cells begin to grow attached to the wall. At the beginning, the cells are single and round. After 6 to 8 hours, a single cardiomyocyte occasionally beats, and the beating frequency is slow and spindle-shaped. After 48 hours, the pseudopodia extended by the cells form cross-links with each other, clustering, interweaving densely and forming a network, and contracting synchronously at about 120 to 140 times / min.

[0062] Embodiment 4:

[0063] Based on the above embodiment, this embodiment performs myocardial cell purity identification on cultured myocardial cells, and the specific process is as follows:

[0064] Cardiomyocytes were cultured at a rate of 1-2 × 10 5The cells were inoculated at a density of 100 μg / L in a 24-well plate with a sterile glass slide pre-coated with polyglutamic acid to prepare a cell slide; after 48 h of adherence culture, the slide was taken out and rinsed 3 times with 0.01 mmol / L PBS buffer (2 min each time); fixed with 40 g / L paraformaldehyde fixative for 5-10 min, 0.5 mL was added to each well, the fixative was removed, and the cells were rinsed 3 times with 0.01 mmol / L PBS buffer (2 min each time); perforated with 3% TritonX-100 at room temperature for 30 min, the permeabilization solution was removed, and the cells were rinsed 3 times with 0.01 mmol / L PBS buffer (2 min each time) (avoid shaking); 200 μL 5% albumin was added and incubated at 37 degrees for 60 min n, discard excess liquid, do not wash; take 200 μL of α-actinin antibody diluted 1:100 and drop it on the coverslip inoculated with cardiomyocytes, keep it in a refrigerator at 4℃ overnight, rinse it 3 times with 0.01mmol / L PBS buffer, 2min each time; add 1:100 diluted Dylight488 labeled anti-mouse IgG and incubate it at room temperature in the dark for 60min, rinse it 3 times with 0.01mmol / L PBS buffer, 2min each time; use DAPI dye solution to stain the nucleus, incubate it at room temperature in the dark for 3-5min, rinse it 3 times with 0.01mmol / L PBS buffer, 2min each time; use absorbent paper to dry the liquid on the slide, and drop the sealing liquid containing anti-fluorescence quencher to seal the slide. Observe and collect images under a fluorescence microscope. Immunofluorescence staining is positive for cardiomyocytes. Randomly take 10 high-power fields to count the number of positive cells and the total number of cells, and repeat 5 times (positive cell rate = number of positive cells / total number of cells × 100%).

[0065] The experimental results are as follows Figure 3 As shown, under an inverted fluorescence microscope, it can be seen that: in the cytoplasm, actin (α-actinin) is specifically stained green, which is positive for immunofluorescence staining, and the nucleus stained with DAPI is blue, which confirms that it is a cardiomyocyte. The cell cytoplasm is not stained, and the nucleus is blue, which is a non-cardiomyocyte. The results show that more than 99% of the cells are positive for α-actinin staining, and the purity is obviously very high. It can be seen that the cardiomyocytes isolated and cultured by this method are of high purity, and the growth of other non-cardiomyocytes is rare.

[0066] Embodiment 5:

[0067] This embodiment is further limited on the basis of the above embodiment, and the experimental subjects selected in the step (1) for cardiomyocyte culture include at least one of KM rat suckling mice, Wistar rat suckling mice, C57 mouse suckling mice, suckling pigs, and suckling rabbits, and the experimental subjects are all SPF grade.

[0068] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for culturing cardiomyocytes, characterized in that: The following steps are involved: (1) Select experimental subjects that can culture cardiomyocytes; (2) Isolating the myocardial tissue of the experimental subjects; (3) isolating cardiomyocytes from isolated myocardial tissue; (4) After the isolated cardiomyocytes were added to the high-glucose DMEM culture medium, 5-bromodeoxyuridine was added and mixed, and the mixture was diluted to (1.0-6.0)×10 5 Cells / cm 2 The cells were inoculated at a density of 1.50 μg / cm2 in a culture dish pre-coated with polyglutamic acid overnight at 37°C and cultured in a cell culture incubator at 37°C and 5% CO2. After 48 h, the high-glucose DMEM culture medium containing 15% fetal bovine serum and without 5-bromodeoxyuridine was replaced. Subsequently, the high-glucose DMEM culture medium containing 15% fetal bovine serum and without 5-bromodeoxyuridine was replaced for 1 to 2 days according to the growth status of the cells and continued to be cultured.

2. The method for culturing cardiomyocytes according to claim 1, characterized in that: The experimental subjects selected in step (1) for culturing cardiomyocytes include at least one of KM rat suckling mice, Wistar rat suckling mice, C57 mouse suckling mice, suckling piglets, and suckling rabbits, and the experimental subjects are all SPF grade.

3. A method for culturing cardiomyocytes according to claim 2, characterized in that: In the step (2), the experimental subjects for separation are suckling mice, and the specific process of isolating the myocardial tissue is as follows: (2.1) Soak the suckling mice in 75% alcohol for 8-10 seconds for disinfection, then quickly transfer them to the clean bench and fix them on a sterile foam board; (2.2) Cut the skin just to the left of the middle of the sternum of the suckling mouse, separate the skin with forceps, fully expose the chest, and make a longitudinal incision to open the chest cavity; (2.3) According to the beating of the heart, squeeze out the heart and remove it with curved forceps; (2.4) Place the removed heart in ice-cold phosphate buffer and wash it by blowing to clean the red blood cells; (2.5) After removing the atria and fibrous tissue, move the heart to another clean culture dish and cut the heart 2 to 3 times, washing it 3 times in total.

4. The method for culturing cardiomyocytes according to claim 3, characterized in that: The operation processes of step (2.2) and step (2.5) are both completed by using ophthalmic scissors and auxiliary ophthalmic forceps.

5. A method for culturing cardiomyocytes according to claim 3 or 4, characterized in that: The operation process of step (2.5) is completed on ice.

6. A method for culturing cardiomyocytes according to claim 3 or 4, characterized in that: The process of isolating myocardial cells from the isolated myocardial tissue in step (3) is as follows: (3.1) The heart tissue was aspirated into a glass culture bottle, 10 mL of 0.25% trypsin digestion solution containing ethylenediaminetetraacetic acid was added, and the solution was incubated at 4°C for 12 to 16 hours. (3.2) Take the heart tissue after resting, add 10 mL of high-glucose DMEM culture medium, and keep in a 37°C constant temperature water bath for 5 min to stop the digestion of pancreatic enzymes; (3.2) Discard the supernatant and keep the heart tissue. Add 10 mL of collagen digestion solution and place in a 37°C constant temperature water bath. Shake gently for 1 min on a shaker with the speed controlled at 15 r / min. (3.3) Discard the supernatant, transfer the heart tissue to another culture bottle, add 12 mL of type II collagenase, place in a 37°C constant temperature water bath, and shake gently on a shaker for 30 minutes at a speed of 15 r / min; (3.4) Transfer the supernatant to a new centrifuge tube and repeat the digestion 3 to 4 times. When only flocculent material is seen, the myocardial tissue is completely digested. (3.5) The collected supernatant was centrifuged for 5 min at a speed of 1000 r / min, the supernatant was discarded, 30 mL of high-glucose DMEM culture medium was added, the tube wall was gently tapped to suspend the cells, and then inoculated into a 100 mm culture dish and incubated in a 37°C, 5% CO2 incubator for 70 min. Non-cardiac cells such as fibroblasts and endothelial cells were removed by the differential adhesion method; the resulting cell suspension contained the isolated cardiomyocytes.

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