Experimental method for long-term dynamic detection of myocardial cell energy stress state

Abstract

The invention discloses an experimental method for dynamically detecting the energy stress state of cardiac muscle cells for a long time. The experimental method comprises the following steps: constructing a biosensing system based on a microelectrode array, modifying the surface of a microelectrode array chip, culturing primary cardiac muscle cells, and detecting the energy stress state of the cardiac muscle cells. The method comprises the following steps: firstly, coating the surface of the microelectrode array chip with gelatin; then separating and extracting primary myocardial cells, preparing a primary myocardial cell suspension, and planting the primary myocardial cell suspension in a microelectrode array chip culture cavity; finally, culturing the myocardial cells in culture media with different glucose concentrations, and recording changes of extracellular local field potential signals of the myocardial cells in real time; time domain and frequency domain characteristics of the potential signals are extracted and normalized, and a concentration dependent response curve under the glucose concentration condition is obtained. According to the invention, the physiological characteristic of spontaneous discharge of the myocardial cells is utilized, and the microelectrode array chip is combined, so that the electrophysiological change of the myocardial cells under the glucose deprivation condition is recorded in real time, and the detection and evaluation of the energy stress state of the cells are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of micro-nano sensing and cell detection, and particularly relates to an experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes. Background Art

[0002] In recent years, with the rapid development of micro-nano manufacturing technology and sensing detection technology, as well as the increasing maturity of cell culture technology, the research on biosensors for label-free, real-time, and non-invasive cell detection based on cardiomyocytes has attracted more and more attention. A microelectrode array (MEA) is a device used to detect the electrophysiological activities of cells. It consists of multiple microelectrodes and can simultaneously record or stimulate the electrical signals of multiple cells. These electrodes are usually arranged on a substrate to form an array for studying the electrophysiological characteristics of neural networks, cardiomyocytes, etc. The MEA technology is widely used in the fields of biology, medicine, and bioengineering, especially in neuroscience research and cardiology research.

[0003] The effective detection of the electrophysiological characteristics of cardiomyocytes can help researchers and clinicians better understand the pathogenic mechanisms and the development of the disease course of various heart diseases caused by hypoglycemia. However, the current in vitro cell models cannot accurately reflect the electrophysiological changes of cardiomyocytes under glucose deprivation conditions in real time. Therefore, there is an urgent need to develop a new experimental method to help carry out relevant scientific research. Summary of the Invention

[0004] The purpose of the present invention is to provide an experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes in view of the deficiencies of the prior art.

[0005] The purpose of the present invention is achieved by the following technical solutions: An experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes, comprising the following steps: (1) Construct a biosensing system based on a microelectrode array: Connect a signal collector, a signal amplifier, and a computer; the signal collector is used to collect and record electrophysiological signals, and the signal amplifier is used to amplify electrophysiological signals; Insert the microelectrode array chip into the signal collector and place it in a cell culture incubator; (2) Surface modification of the microelectrode array chip: Coat the surface of the microelectrode array chip with gelatin to enhance the adhesion degree of cardiomyocytes on the surface of the microelectrode array chip; (3) Culture primary cardiomyocytes: Isolate and extract primary cardiomyocytes, prepare a primary cardiomyocyte suspension, and plant the primary cardiomyocyte suspension in the culture cavity of the microelectrode array chip, and the cardiomyocytes are evenly distributed on the surface of the microelectrode array chip; (4)Detect the energy stress state of cardiomyocytes: Culture cardiomyocytes in media with different glucose concentrations, and record the changes in the extracellular local field potential signals of cardiomyocytes on the microelectrode array chip in real time; extract the time-domain and frequency-domain characteristics of the detected potential signals, and perform normalization processing to obtain the concentration-dependent response curve under this glucose concentration condition. Judge the energy stress state caused by different degrees of glucose deprivation according to the attenuation trend of the potential signals reflected by the curve.

[0006] Further, in step (2), the surface of the microelectrode array chip is coated with 1% gelatin, and then placed in an incubator for 2 - 4 h; suck out all the gelatin before inoculating cells.

[0007] Further, in step (3), the seeding density of primary cardiomyocytes is 2.5×10 5 ~4.0×10 5 cells / cm 2 ; The cell culture conditions are: temperature 37 °C, culture atmosphere 5.0% CO2, and the culture medium is changed every 24 h.

[0008] Further, in step (3), after planting cardiomyocytes on the microelectrode array chip, use the live / dead cell staining method to determine the growth activity of cardiomyocytes on the microelectrode array chip; use the immunofluorescence staining method of cardiac troponin T to determine the purity of cardiomyocytes; if the survival rate reaches more than 80% and the purity reaches more than 90%, it meets the requirements of cell sensing detection, otherwise, it needs to be cultured again.

[0009] Further, in step (4), the glucose concentration range is 0 - 0.9 g / L; the culture time is 24 h.

[0010] Further, in step (4), the time-domain and frequency-domain characteristics of the potential signals for detecting the energy stress state of cardiomyocytes include: the amplitude and firing frequency of the electrophysiological signals.

[0011] Advantages of the present invention: The present invention utilizes the physiological characteristics of the spontaneous electrical activity of cardiomyocytes, combines with the microelectrode array chip, and records the electrophysiological changes of cardiomyocytes under glucose deprivation conditions in a simple and efficient manner in real time and quickly, realizes the detection and evaluation of the cell energy stress state, and provides a new tool for understanding the impact of hypoglycemia on the heart, which has important value for drug screening and clinical research of diabetes and hypoglycemia. Description of the Drawings

[0012] Figure 1 is the electrophysiological signal change diagram of cardiomyocytes before and after glucose deprivation in the embodiment of the present invention; Figure 2 is the statistical chart of electrophysiological signal characteristic parameters of cardiomyocytes before and after glucose deprivation in the embodiment of the present invention; among them,Figure 2 In (A) therein is a graph showing the relationship between the cell amplitude and time of the sugar-free group, glucose group, and control group; Figure 2 In (B) therein is a graph showing the relationship between the discharge frequency of cardiomyocytes and time during 24 hours of continuous culture of the sugar-free group, glucose group, and control group; Figure 3 It is a graph of the electrophysiological signal changes of cardiomyocytes with glucose deprivation while inhibiting autophagy in the embodiments of the present invention; Figure 4 It is a statistical graph of the electrophysiological signal characteristic parameters of cardiomyocytes with glucose deprivation while inhibiting autophagy in the embodiments of the present invention; wherein, Figure 4 In (A) therein is a graph of the normalized amplitude change of glucose-starved cardiomyocytes treated with the autophagy inhibitor 3-methyladenine (3-MA) monitored continuously for 24 hours; Figure 4 In (B) therein is a graph of the effect of 3-MA on the firing frequency of cardiomyocytes under glucose-starved conditions. Detailed implementation manners

[0013] The structure, preparation, and usage method of the cell electrophysiological sensing device in the present invention are all based on the previous work results of the applicant. Specifically, reference can be made to the "multi-modal microelectrode biosensing system" in the published patent application document "A sensing detection method and system for cardiomyocyte excitation-contraction coupling" (CN115078466A) and the description of the "nano-microelectrode array device" in the published patent application document "A detection system and method for integrated intracellular delivery and electrical sensing of cardiomyocytes" (CN115236049A). Those skilled in the art can make detailed adjustments according to the specific experimental scheme design when reproducing the technical solution of the present invention, and the present invention will not be elaborated further.

[0014] The implementation manner of the present invention will be described in detail below with reference to the accompanying drawings, but the present invention is not limited thereby.

[0015] I. Experimental device of the present invention: An experimental device for long-term dynamically detecting the energy stress state of cardiomyocytes provided by the present invention includes a cell electrophysiological sensing device, a signal collector, a signal amplifier, a computer, and a cell incubator. Among them, the cell electrophysiological sensing device is a technology recorded in existing published literature, including a hollow glass culture cavity, a microelectrode array chip, and a printed circuit board (PCB) adapter; the structure and processing method of this sensing device can be specifically referred to the patent application published document CN115078466A. The signal collector is connected to the signal amplifier through a long shielded cable, and the signal amplifier is connected to the computer by a data connection line. After the cell electrophysiological sensing device is inserted into the signal collector, it can be placed in the cell incubator for long-term collection of cardiomyocyte electrophysiological signals.

[0016] Furthermore, the signal collector and the signal amplifier are respectively placed in two independent metal boxes to reduce the volume. Thus, the sensing device can be placed in an incubator to maintain the viability of cardiomyocytes and achieve long-term and continuous monitoring of electrophysiological signals. Another metal box is placed outside the incubator for convenient operation.

[0017] Furthermore, a metal cover is installed outside the microelectrode array chip slot to shield external signal interference and improve the signal-to-noise ratio.

[0018] II. Detection method of the present invention: Based on the above experimental device, the present invention realizes an experimental method for long-term dynamically detecting the energy stress state of cardiomyocytes, which specifically includes the following steps: (1) Establishment of a biosensing system based on a microelectrode array (MEA): Connect the components in the experimental device, such as the signal collector, the signal amplifier, and the computer, through data lines. Collect and record electrophysiological signals through the signal collector, and synchronously record the extracellular electrophysiological signals of cardiomyocytes at different sites by multiple working electrodes. The electrophysiological signals are amplified 500 times by the signal amplifier and filtered (<7.5 kHz), with a sampling frequency of 20 kHz, and then transmitted to the computer by the data acquisition card for offline analysis. Insert the MEA chip into the signal collector and place it in the cell incubator, and set the environmental parameters to 37°C, 5% CO2, and 100% humidity.

[0019] (2) Surface modification of the microelectrode array chip: Coat the surface of the microelectrode array chip with gelatin to enhance the adhesion degree of cardiomyocytes on the surface of the microelectrode array chip, so as to improve the signal-to-noise ratio of the sensing system for detecting signals.

[0020] As an example, coat the surface of the microelectrode array chip with 1% (m / v) gelatin, and then place it in the incubator and incubate at 37°C for 2 - 4 h. After sucking out the gelatin, the cells can be directly inoculated on the chip.

[0021] When cardiomyocytes are directly inoculated on the chip surface, the average signal-to-noise ratio is 20.5 dB, and the signal-to-noise ratio after coating with gelatin can be increased to an average of 39.1 dB, an increase of 90.7%.

[0022] (3) Isolation and culture of primary cardiomyocytes: Isolate and extract primary cardiomyocytes, prepare a primary cardiomyocyte suspension, and plant the primary cardiomyocyte suspension in the culture cavity of the microelectrode array chip, and the cardiomyocytes are evenly distributed on the surface of the microelectrode array chip.

[0023] As an example, the seeding density of the cardiomyocyte suspension in the culture chamber is 2.5×10 5 ~4.0×10 5 cells / cm 2 ; after seeding, ensure that the cardiomyocytes are evenly distributed on the surface of the microelectrode array chip; the cell culture conditions are a temperature of 37°C and a culture atmosphere of 5.0% (v / v) CO2, and the culture medium is changed every 24 hours.

[0024] Furthermore, for the isolation and culture of the primary cardiomyocytes, after planting the cardiomyocytes in the microelectrode array chip, a live / dead cell staining method needs to be used to determine the growth activity of the cardiomyocytes on the microelectrode array chip; a cardiac troponin T (cTnT) immunofluorescence staining method is used to determine the purity of the cardiomyocytes; those with a survival rate of more than 80% and a purity of more than 90% can meet the requirements of cell sensing detection, otherwise, re-culture is required.

[0025] (4) Detection of the energy stress state of cardiomyocytes: The cardiomyocytes are cultured in media with different glucose concentrations, and the changes in the extracellular local field potential signals of the cardiomyocytes on the microelectrode array chip are recorded in real time, once every 1 hour, each recording for 90 seconds, and continuously recorded for 24 hours; the time domain and frequency domain characteristics of the detected potential signals, that is, the amplitude and firing frequency of the electrophysiological signals, are extracted; subsequently, the collected data is normalized, that is, the electro-signal value collected before changing to a glucose-reduced or glucose-free medium is denoted as X0, and the electro-signal value collected at time t after changing the corresponding medium is denoted as X t , and after normalization calculation, the electro-signal value X nor = X t / X0, so as to eliminate the differences in electro-signals between different channels or different batches of cells, draw the concentration-dependent response curve under this glucose concentration condition, and judge the energy stress state caused by different degrees of glucose deprivation according to the attenuation trend of the potential signals shown by the curve. Among them, the glucose concentration range is 0~0.9 g / L; the culture time is 24 h.

[0026] Example 1

[0027] 1. Preparation of the MEA chip: A photoresist is coated on a 16-square-inch quartz glass substrate. After baking and exposure, a conductive path is deposited using gold / titanium material. Then, an insulating SU-8 layer is generated through photolithography and post-processing. Finally, the glass substrate is divided into 25 device units, and each unit contains 32 microelectrodes. The assembly of the device includes a printed circuit board adapter, a cell culture glass ring, and a sensor chip. The circuit connection is ensured through polydimethylsiloxane (PDMS), silver conductive adhesive, and gold bonding wires, and pins are soldered on the PCB to adapt to the electrophysiological recording system. Before the experiment, the sensor chip is coated with 1% gelatin in a 37°C incubator for 4 hours to promote adhesion. The extracellular action potential of cardiomyocytes is measured on the 3rd day after seeding.

[0028] 2. Isolation of primary cardiomyocytes: Ventricular tissue of neonatal Sprague Dawley (SD) rats aged 0 - 2 days is taken. After washing away the blood in Dulbecco's Modified Eagle Medium (DMEM), it is placed in 3 ml of Hank's Balanced Salt Solution (HBSS), and the tissue is minced into tissue blocks of about 1 mm 3 . The tissue blocks are repeatedly digested with 0.08% trypsin and 0.1% type II collagenase 10 times, 8 minutes each time. The digestion is terminated using DMEM medium containing 10% fetal bovine serum (FBS), and the tissue blocks are dispersed into single cells. Centrifuge at 100 g for 5 min and discard the supernatant. The cell pellet is resuspended and subjected to differential adhesion twice, 45 minutes each time. Finally, the cardiomyocytes are seeded into the MEA chip, about 3.5×10 5 cells per well.

[0029] 3. Electrophysiological detection of cardiomyocytes after glucose deprivation: Inoculate cardiomyocytes on the MEA chip and grow them in high-glucose DMEM medium (4.5 g / ml glucose) containing 10% FBS for 3 - 4 days. Detect whether they have normal electrophysiological signals or observe regular pulsations under the microscope. At this time, aspirate the original medium, wash the cells once with phosphate buffered saline (PBS) with a pH of 7.2 - 7.6, and add sugar-free DMEM medium containing 10% FBS, which is the sugar-free culture group. The 0.45 g / L glucose group is prepared by mixing 10% high-glucose DMEM and 90% sugar-free DMEM and then adding it to the cells. Similarly, the 0.9 g / L glucose group is prepared by mixing 20% high-glucose DMEM and 80% sugar-free DMEM and then adding it to the cells. Use 3 MEA chips for each treatment group to conduct independent repeated experiments, and 32 sensing electrodes on each MEA chip record the electrophysiological signals of cardiomyocytes at different positions simultaneously. Record the electrophysiological signals of cardiomyocytes in each treatment group once an hour; see Figure 1 , Figure 1 shows the extracellular field potential signals of cardiomyocyte responses at different glucose concentrations at 1 hour, 12 hours, and 18 hours. Extract the characteristic parameters (i.e., the amplitude and firing frequency of the electrophysiological signals), import them into GraphPad Prism software for data analysis to obtain Figure 2 the curve of the relationship between the electrophysiological signal and time. See Figure 2 , where Figure 2 (A) in Figure 2 shows that although the amplitudes of cells in the sugar-free group, 0.45 g / L, and 0.9 g / L glucose groups are significantly different from those of the control group, there is no glucose concentration-dependent relationship among the three groups. Among them, the amplitude of the 0.45 g / L group is lower than that of the sugar-free group at one time.

[0030] 4. Evaluation of the effect of inhibiting autophagy on the electrophysiological activity of cardiomyocytes under energy stress: In step 3, while replacing the complete medium with a sugar-free medium, the autophagy inhibitor 3-methyladenine (3-MA) was added at final concentrations of 5 mM, 10 mM, and 20 mM, respectively. The electrophysiological signals of cardiomyocytes in each treatment group were recorded every hour. Refer to Figure 3 , Figure 3 shows the real-time response diagrams of extracellular field potential signals of cardiomyocytes after adding different concentrations of 3-MA in sugar-free medium at 1 hour, 12 hours, and 24 hours. It can be clearly observed that as the concentration increases, the intensity of the electrical signal gradually decreases. The characteristic parameters (i.e., the amplitude and firing frequency of the electrophysiological signal) were extracted and imported into GraphPad Prism software for data analysis to obtain Figure 4 the curve of the relationship between electrophysiological signal and time. Refer to Figure 4 , Figure 4 in (A) is the normalized amplitude change curve of 3-MA-treated glucose-starved cardiomyocytes monitored continuously for 24 hours; compared with the control group and the glucose-starved group, the rate of amplitude decline is more severe after adding 3-MA, and the decline amplitude increases with the increase of 3-MA concentration. No extracellular electrophysiological signal was detected in the 20 mM group after 5 hours of treatment. Figure 4 in (B) is the curve of the effect of 3-MA on the firing frequency of cardiomyocytes under glucose starvation conditions. The trend of the 5 mM group is almost the same as that of the sugar-starved group only in the early stage, that is, it first increases and then slowly decreases, but tachycardia occurs abnormally at the 23rd hour; the 10 mM group shows a small fluctuating trend during this detection period, and finally tachycardia occurs at the 21st hour, and the firing frequency rises sharply; while the 20 mM group shows abnormalities at the 5th hour and the extracellular signal disappears.

[0031] The above embodiments are used to explain the present invention, rather than limit the present invention. Any modifications and changes made within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. An experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes, characterized in that, It includes the following steps: (1) Build a biosensing system based on a microelectrode array: Connect a signal collector, a signal amplifier, and a computer. The signal collector is used to collect and record electrophysiological signals, and the signal amplifier is used to amplify electrophysiological signals. Insert the microelectrode array chip into the signal collector and place it in an incubator. (2) Surface modification of the microelectrode array chip: Coat the surface of the microelectrode array chip with gelatin to enhance the adhesion of cardiomyocytes to the surface of the microelectrode array chip. (3) Culture primary cardiomyocytes: Isolate and extract primary cardiomyocytes, prepare a primary cardiomyocyte suspension, and seed the primary cardiomyocyte suspension in the culture cavity of the microelectrode array chip. The cardiomyocytes are evenly distributed on the surface of the microelectrode array chip. (4) Detect the energy stress state of cardiomyocytes: Culture the cardiomyocytes in media with different glucose concentrations, and record in real time the changes in the extracellular local field potential signals of the cardiomyocytes on the microelectrode array chip. Extract the time-domain and frequency-domain characteristics of the detected potential signals and perform normalization processing to obtain a concentration-dependent response curve under this glucose concentration condition. Judge the energy stress state caused by different degrees of glucose deprivation based on the decay trend of the potential signals reflected by the curve.

2. The experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes according to claim 1, wherein In step (2), the surface of the microelectrode array chip is coated with 1% gelatin, and then placed in an incubator for incubation for 2 - 4 h. Aspirate the gelatin completely before seeding the cells.

3. The experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes according to claim 1, characterized in that In step (3), the seeding density of primary cardiomyocytes is 2.5×10 5 ~4.0×10 5 cells / cm 2 ; the cell culture conditions are: temperature 37°C, culture atmosphere 5.0% CO2, and the culture medium is changed every 24 h during the period.

4. The experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes according to claim 1, characterized in that, In step (3), after seeding cardiomyocytes on the microelectrode array chip, use a live / dead cell staining method to determine the growth activity of cardiomyocytes on the microelectrode array chip. Use a cardiac troponin T immunofluorescence staining method to determine the purity of cardiomyocytes. If the survival rate reaches more than 80% and the purity reaches more than 90%, it meets the requirements for cell sensing detection; otherwise, re-culture is needed.

5. The experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes according to claim 1, wherein In step (4), the glucose concentration range is 0 - 0.9 g / L; the culture time is 24 h.

6. The experimental method for long-term dynamic detection of the energy stress state of cardiomyocytes according to claim 1, wherein In step (4), the time-domain and frequency-domain characteristics of the potential signals for detecting the energy stress state of cardiomyocytes include: the amplitude and firing frequency of the electrophysiological signals.

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