Methods for Isolating Primary Neonatal Rat Cardiomyocytes

Abstract

The present invention relates to the field of biotechnology, and more particularly to a method for isolating primary neonatal rat cardiomyocytes (NRCMs), wherein the method uses two digestion enzymes for stepwise digestion and does not include a centrifugation step.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for isolating primary neonatal rat cardiomyocytes (NRCMs). Background Art

[0002] NRCM cultured in vitro is the basis of many studies in the field of cardiovascular disease and is crucial for studying cardiac function and drug discovery. It is widely used in the study of cardiovascular disease mechanisms, cardiac drug activity screening, and the clarification of drug myocardial toxicity or drug action mechanism. Isolating and culturing high-quality, high-purity, and high-activity cardiomyocytes is the key to the experiment.

[0003] The existing methods for isolating primary cardiomyocytes are similar, and the main differences lie in the selection, ratio and digestion time of digestive enzymes.

[0004] Centrifugation is a common method for removing broken cells and digestion fluid. The process of separating and culturing NRCM in the prior art all includes the step of centrifugation.

[0005] There remains a need in the art for improved methods of isolating primary neonatal rat cardiomyocytes. Summary of the invention

[0006] The object of the present application is to provide an improved method for isolating primary neonatal rat cardiomyocytes.

[0007] To achieve the above objectives, the present disclosure provides a method for isolating primary neonatal rat cardiomyocytes, which uses two digestive enzymes for stepwise digestion and does not include a centrifugation step.

[0008] The present disclosure uses two digestive enzymes for stepwise digestion instead of the traditional single enzyme method or mixed enzyme method, and for the first time uses the stepwise digestion method combined with the terminated digestion direct culture method (IV) to separate and culture primary cardiomyocytes, and verifies the simplicity, high efficiency and reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Embodiments of the present application will be described below by way of example only and with reference to the accompanying drawings, in which:

[0010] Figure 1A Comparison of the total number of cells in each group is shown (n=5).

[0011] Figure 1B Comparison of cell survival rates among groups is shown (n=5).

[0012] Figure 2 The morphological changes of NRCMs of each group at 48 hours (×200), 96 hours (×100) and 96 hours (×40) are shown.

[0013] Figure 3 FITC confocal immunofluorescence (green) staining of cardiomyocytes in each group is shown (×200).

[0014] Figure 4A The results of cytotoxicity of Curcuma aromatica extract in NRCM isolated and cultured by Method IV are shown.

[0015] Figure 4B The viability responses of NRCM and H9c2 cardiomyocytes in each group to pretreatment with Curcuma aromatica extract in H / R are shown. DETAILED DESCRIPTION

[0016] In the following description, certain specific details are included to provide a comprehensive understanding of each disclosed embodiment. However, one skilled in the relevant art will recognize that the embodiments can be implemented without one or more of these specific details, but with other methods, components, materials, etc.

[0017] Unless otherwise required in this application, throughout the specification and the claims that follow, the word "comprising" should be interpreted in its open, inclusive sense, ie, "including, but not limited to."

[0018] References to "one embodiment" or "an embodiment" or "in another embodiment" or "in certain embodiments" throughout this specification are intended to include in at least one embodiment the specific referenced elements, structures or features described in connection with that embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, the specific elements, structures or features may be combined in any appropriate manner in one or more embodiments.

[0019] It should also be understood that the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise.

[0020] The present disclosure provides a method for isolating primary neonatal rat cardiomyocytes, which uses two digestion enzymes for stepwise digestion and does not include a centrifugation step.

[0021] In a specific embodiment, stepwise digestion is performed with 0.02-0.10% by weight trypsin and 0.04-0.12% by weight collagenase II.

[0022] In a more specific embodiment, stepwise digestion is performed with 0.03-0.09% by weight trypsin and 0.05-0.11% by weight collagenase II.

[0023] In a more specific embodiment, stepwise digestion is performed with 0.04-0.08% by weight trypsin and 0.06-0.10% by weight collagenase II.

[0024] In a more specific embodiment, stepwise digestion is performed with 0.05-0.07% by weight trypsin and 0.07-0.09% by weight collagenase II.

[0025] In an exemplary embodiment of the present invention, stepwise digestion was performed using 0.06% by weight of trypsin and 0.08% by weight of collagenase II.

[0026] The present disclosure uses two digestive enzymes for stepwise digestion instead of the traditional single enzyme method or mixed enzyme method, and for the first time uses the stepwise digestion method combined with the terminated digestion direct culture method to separate and culture primary cardiomyocytes, and verifies the simplicity, high efficiency and reliability.

[0027] The present disclosure firstly digests with trypsin for the first time and discards the supernatant, and then further digests and collects the obtained cell suspension. The present disclosure adopts a step-by-step digestion method with a digestion solution, and firstly digests the myocardial tissue with trypsin having a strong digestion ability for the first time. On the one hand, blood cells and dead myocardial cells can be removed as quickly as possible, and on the other hand, the myocardial tissue can be made soft and fluffy, which can shorten the next digestion time and avoid trypsin from damaging the cell membrane, thereby potentially increasing cell survival rate and cell viability.

[0028] In the present disclosure, the inventors first attempted to terminate the digestion and direct culture method (IV), that is, the cell suspension obtained by step-by-step digestion of the digestive solution was passed through a BD plug to remove residual tissue, and then directly subjected to differential adherence culture without centrifugation. After the digestion was terminated by the complete culture medium of the digestive solution, its digestive effect was lost, and a small amount of small cell fragments could be removed by replacing the cell culture medium for the first time after 48 hours. From the results analyzed by the inventors, it can be seen that the cell survival rate, spontaneous beating rate, and cell purity obtained by the terminated digestion and direct culture method (IV) are superior to those using traditional trypsin.

[0029] In summary, this application is the first to use the step-by-step digestion followed by terminated digestion and direct culture method (IV) to isolate primary cardiomyocytes, which can shorten the separation and digestion time, ensure the number and survival rate of the obtained cells, and ensure the purity and vitality of the cells.

[0030] Example

[0031] Hereinafter, the present invention will be explained in detail by the following examples so as to better understand the various aspects of the present invention and its advantages. However, it should be understood that the following examples are non-limiting and are only used to illustrate certain embodiments of the present invention.

[0032] Methods and Materials

[0033] animal

[0034] Postnatal day 2 SPF Sprague-Dawley (SD) rats [certificate number SCXK-(Gui)2014-0002] were purchased from the Experimental Animal Center of Guangxi Medical University. The animals were approved by the Animal Care and Use Committee of Guangxi Medical University (Guangxi, China).

[0035] Chemicals and reagents

[0036] High-glucose DMEM medium (Gibco, USA): low-glucose DMEM medium (Gibco, USA); fetal bovine serum (FBS) (Gibco, USA); 10,000 U / ml penicillin / streptomycin (Gibco); phosphate buffered saline (PBS, pH 7.4, 1X) (Gibco, USA); trypan blue solution (Solarbio, Beijing), dimethyl sulfoxide (DMSO) (Solarbio); MTT (Amresco). Trypsin and collagenase II (Gibco Life Technologies, USA).

[0037] Rabbit anti-Sarcomeric Alpha Actinin (α-SA) antibody (Abcam, UK), fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit secondary antibody (Sigma, USA), 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, USA), rat H9c2 cardiomyocyte cell line (Cell Bank of Chinese Academy of Sciences, Beijing), Curcuma aromatica extract (extracted in this experiment) and stored at 4°C for future use.

[0038] Statistical analysis

[0039] The results of confocal immunofluorescence were analyzed by Image Proplus 6.0 software. Data were expressed as mean ± SD and analyzed by SPSS 22.0 statistical software. One-way analysis of variance (ANOVA) statistical method was used to analyze the significance, and P < 0.05 was considered statistically significant.

[0040] Example 1 NRCM separation

[0041] Place 8-10 2-day-old SD rats in a beaker filled with 75% alcohol and soak for 10 seconds. Wipe the chest and abdominal skin with a 75% alcohol cotton ball. Use straight scissors to cut the skin from the abdomen to the neck along the linea alba. Use another pair of straight scissors to open the chest along the xiphoid process. Squeeze the back skin to expose the heart. Use curved forceps to quickly remove the heart and immediately transfer it to a sterile culture dish containing 4°C precooled phosphate buffered saline solution (PBS). Use curved scissors to carefully remove non-ventricular muscle tissues such as the auricle and atrium, replace the PBS in the culture dish, and wash the ventricular muscle three times. Transfer the ventricular muscle to a sterile penicillin bottle, add 1 mL of precooled PBS, and use another pair of curved scissors to cut it into pieces of about 1-3 mm. 3 The tissue fragments were then transferred to a 15 mL sterile centrifuge tube containing 6 ml of 0.08% trypsin for primary digestion. The tissue fragments were stirred intermittently for 2 min and then allowed to stand at 37°C for 1 min. This step was repeated 5 times for 15 min (n=5), and then the supernatant was carefully removed. The main components of the supernatant were non-cardiac cells such as blood cells.

[0042] The tissue precipitate obtained above was digested step by step with different digestion solutions: (1) Digestion solution A was added for further digestion until the tissue became soft and fluffy for 15 min (n=5), the cell supernatant was carefully collected in another 15 ml centrifuge tube, and an equal amount of complete medium (high-glucose DMEM containing 20% ​​inactivated FBS and 1% penicillin / streptomycin) was added to terminate the digestion, and then stored at 37°C. (2) The remaining tissue pieces were resuspended with digestion solution B and digestion was continued until the tissue pieces were nearly completely digested (25-30 min), and then complete medium was added to stop digestion. These two digestion processes were repeated, and the tissue pieces were stirred intermittently for 2 min, and then left to stand at 37°C for 1 min. The cell suspension obtained in the above two steps was collected and then passed through a 100 μm BD plug to provide cell suspension without tissue fragments.

[0043] The tissue suspension was divided into four groups: (I) digestion solution A and digestion solution B both contained 0.06% by weight trypsin; (II) digestion solution A and digestion solution B both contained 0.08% by weight collagenase II; (III) digestion solution A contained 0.06% by weight trypsin and digestion solution B contained 0.08% by weight collagenase II; (IV) digestion solution A contained 0.06% by weight trypsin and digestion solution B contained 0.08% by weight collagenase II. The cell suspensions obtained by methods I, II and III were centrifuged (400 g, 10 min) respectively, and the obtained cell pellets were resuspended in complete medium; the cell suspension of method IV was not centrifuged.

[0044] In order to selectively obtain highly pure primary cardiomyocytes, the cell suspensions obtained by the above-mentioned stepwise digestion centrifugation method (I, II and III) and the stepwise digestion combined with the terminated digestion direct culture method (IV) were transferred to 25 cm 2 In cell culture, the cells were placed in a carbon dioxide incubator (37°C, 5% CO2, 95% O2) for 2 hours to separate non-cardiomyocytes by differential attachment (mainly to remove a large number of fibroblasts). Then, each group of non-attached cells (mainly containing cardiomyocytes) were re-seeded in a new culture dish, 6-well plate or 96-well plate, and 5-BrudU was added at a final concentration of 100 μM to inhibit the growth of fibroblasts during the culture process, so as to obtain cardiomyocytes with higher purity.

[0045] Example 2 NRCM and H9c2 cardiomyocyte culture

[0046] The medium of the above four groups of NRCMs was changed for the first time after 48 h, and the medium was changed every day thereafter.

[0047] After H9c2 cardiomyocytes were revived, they were cultured in complete medium. When the third subculture reached 70-80% confluence, 6 × 10 5 Each well was seeded in a 96-well plate.

[0048] Example 3 Trypan blue staining method for separating cell counting and cell viability detection

[0049] Take 100 μL of cell suspension after differential attachment for 2 hours and 900 μL of 0.04% trypan blue solution in a 1.5 ml sterile tube and mix them evenly. Stain for 3 minutes respectively. Observe and record under an inverted microscope. Dead cells are stained blue, while living cells are colorless and transparent. Therefore, the total number of NRCMs and living cells can be counted with a white blood cell counter. Each experiment was repeated 5 times (n=5), and the results are expressed as mean ± SD.

[0050] Cell viability is closely related to experimental results and is usually expressed as a percentage, namely:

[0051] Cell survival rate (%) = number of unstained cells / total number of cells × 100

[0052] The total number of cells and viability of NRCMs obtained by the four isolation methods are shown in Figure 2. Figure 1A and 1B There was no significant difference in the total number of cells obtained by method IV and the other three methods (I-III) (P>0.05); however, in terms of cell survival rate, method IV was significantly higher than method I (P<0.05).

[0053] Example 4 Observation of cell morphology and spontaneous beating rate

[0054] From the second day to the fifteenth day after isolation and culture, the morphological changes and growth status of each group of primary cardiomyocytes were continuously observed by inverted microscope, and 5 cell clusters were randomly selected to record their spontaneous beating rates. The spontaneous beating rates were expressed as mean ± SD.

[0055] After 12 hours of cell culture, cardiomyocytes began to grow on the wall. After 24 hours, cardiomyocytes grew polygonal pseudopodia, and some cardiomyocytes began to contract spontaneously. After 48 hours, the pseudopodia of the cells were cross-linked to form a network, which was pulsating in a sheet-like manner. After 72 hours, the cells grew in clusters and connected to each other to form a dense network, which was pulsating synchronously and contracting vigorously, and after 96 hours, it was pulsating in an island-like manner ( Figure 2 The cardiomyocyte beating rate was continuously recorded from day 3 to day 15, with a synchronous rate of 60-100 beats / min (Table 1). There was no significant difference in morphology and spontaneous beating rate between method IV and the other three isolation methods.

[0056] Table 1 Time variation of spontaneous beating rate of myocardial cells in each group (times / minute)

[0057]

[0058] Example 5 Identification and Purity Observation of Cardiomyocytes

[0059] Confocal immunofluorescence was used to identify cardiomyocytes and determine their purity. After the cells in each group were cultured on the coverslips of 6-well plates for 72 hours, confocal immunofluorescence staining was performed. In short, the culture medium was aspirated, dried naturally, and fixed in 4% paraformaldehyde for 30 minutes, washed 3 times with PBS (5 minutes each time), permeabilized with 0.2% TritonX-100 for 30 minutes, washed 3 times with PBS (5 minutes each time), and incubated with rabbit anti-mouse primary antibody (1:50) at 4°C overnight. After being taken out the next day, washed 3 times with PBS (5 minutes each time), incubated with FITC-labeled goat anti-rabbit secondary antibody (1:200) for 60 minutes, washed 3 times with PBS (5 minutes each time), added DAPI working solution and incubated at room temperature in the dark for 10 minutes, washed 3 times with PBS (5 minutes each time), added anti-fluorescence quencher, and immediately observed the immunofluorescence staining of each group using a laser confocal microscope (Nikon A1, Japan).

[0060] Immunofluorescence staining of cardiomyocytes in each group Figure 3As shown. α-SA is a cardiac actin marker. In the confocal immunofluorescence staining results, primary cardiomyocyte α-SA was stained green by FITC, and other non-cardiomyocytes such as fibroblasts and vascular epithelial cells were not stained by FITC, but all cell nuclei were stained blue by DIPI. The purity of the four groups of cardiomyocytes was 85.70% (I), 90.09% (II), 97.39% (III) and 97.70% (IV), respectively.

[0061] Example 6 MTT method to measure cell viability

[0062] The MTT method was used to evaluate the reliability of NRCM isolated and cultured by method IV and the three methods. First, the cytotoxicity of Curcuma aromatica extract was determined using NRCM isolated and cultured by method IV, and then the cell viability of NRCM isolated and cultured by the four methods and H9c2 cardiomyocytes was determined.

[0063] Method IV When the NRCM inoculated in a 96-well culture plate reached 70-80% confluence, the culture was discarded and pretreated with a high glucose DMEM medium containing Curcuma aromatica extract (2000 μg / ml, 1000 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.25 μg / ml) at 37°C under normoxic conditions for 12 h. The Curcuma aromatica extract was dissolved in DMSO, and the concentration of DMSO in the final culture medium did not exceed 0.1%. The experimental concentration of the Curcuma aromatica extract can be determined by a cytotoxicity test.

[0064] When the NRCMs of the four extraction methods and H9c2 cardiomyocytes were seeded in 96-well culture plates and reached 70-80% confluence, the culture was discarded and the culture medium was replaced with high-glucose DMEM medium containing Curcuma aromatica extract. The cardiomyocytes of each group were cultured under normal oxygen conditions for 12 hours, and then the in vitro H / R model was established. In short, the culture medium in the cardiomyocyte culture plate of each group was sucked clean as much as possible with a pipette, and then replaced with sugar-free DMEM medium without FBS and hypoxic in the hypoxia chamber for 4 hours. Subsequently, the hypoxic solution in the culture plate was removed and replaced with high-glucose DMEM, and reoxygenated in a carbon dioxide incubator (37°C, 5% CO2, 95% O2) for 4 hours. Each well was repeated three times (n=3). The cells of the control group were cultured under normoxic conditions with high-glucose DMEM culture medium without FBS. After the H / R of the cardiomyocytes of each group was completed, 20 μL of MTT solution (5 mg / ml) was added to each well, and the 96-well plate was wrapped with tin foil and placed in a carbon dioxide box to avoid light and cultured for 4 hours. After the culture medium in each well was removed by a pipette, 150 μL DMSO was added and the cells were shaken at a low speed for 10 min to fully dissolve the crystals. The OD value of each well was measured at 570 nm using an enzyme-linked immunosorbent assay (Thermo Scientific, MA, USA). The results were expressed as mean ± SD.) The experimental results were expressed as cell viability:

[0065] Cell viability (%) = (OD 实验孔 -OD 空白孔 ) / (OD 正常孔 -OD 空白孔 )×100%.

[0066] like Figure 4A As shown, the concentration of Curcuma aromatica extract <125 μg / ml was considered to be non-cytotoxic. Therefore, the concentrations of Curcuma aromatica extract were 125, 62.5, 31.25, 15.625, 7.8125, and 0 μg / ml for each group and H9c2 cardiomyocytes to verify the reliability of the NRCM viability response of each group.

[0067] like Figure 4B As shown, the results showed that there was no significant difference in the viability response of NRCM and H9c2 cardiomyocytes in each group to H / R of Curcuma aromatica extract, and this trend was consistent with the preliminary study of the inventors in AMI, and showed a good dose relationship. However, at the same dose of Curcuma aromatica extract, NRCM responded better than H9c2 cardiomyocytes. Compared with the control group, *P < 0.05. This means that method IV digestion and separation of NRCM and the other three methods can meet the screening of cardiovascular disease drug activity and mechanism of action research.

[0068] It can be understood from the foregoing that although the specific embodiments of the present application are described for the purpose of illustrative illustration, various modifications or improvements can be made by those skilled in the art without departing from the spirit and scope of the present application. These modifications or improvements should all fall within the scope of the claims attached to the present application.

Claims

1. A method for isolating primary neonatal rat cardiomyocytes, the method comprising stepwise digestion using two digestive enzymes and excluding a centrifugation step, wherein the stepwise digestion is performed using 0.05-0.07% by weight of trypsin and 0.07-0.09% by weight of collagenase II, The cells were first digested with trypsin and the supernatant was discarded, and then further digested and the resulting cell suspension was collected. The cell suspension obtained by step-by-step digestion with the digestion solution was passed through a BD plug to remove residual tissue, and then directly subjected to differential adherence culture without centrifugation.

2. The method of claim 1, wherein the stepwise digestion is performed using 0.06% by weight trypsin and 0.08% by weight collagenase II.

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