Method for extracting characterization and metabolism information of cell mechanical response behavior

Abstract

The invention discloses a cell mechanical response behavior characterization and metabolism information extraction method. The method comprises the following steps: 1, constructing a device integrated with an ultrasonic mechanical stimulation device and electrochemical detection; step 2, adding a cell culture solution containing cells into the device in the step 1, then applying voltage by a signal generator in the device, and then observing the arrangement condition of the cells on the electrode by using a microscope; and 3, changing the quantity of the cells in the cell culture fluid, and then observing the arrangement condition of the cells on the electrode by using the microscope again. According to the characterization and metabolism information extraction method of the cell mechanical response behavior, an ultrasonic mechanical stimulation device and an electrochemical detection technology are integrated, and real-time in-situ monitoring on the change of an electrical signal of the cell is realized while the cell is subjected to mechanical stimulation.

Description

Technical Field

[0001] The present invention relates to the field of cell mechanics, and more particularly to a method for characterizing cell mechanical response behavior and extracting metabolic information. Background Art

[0002] Currently, the characterization of cell mechanical response behavior and the extraction of metabolic information are mainly achieved through optical technologies, such as optical microscopy (Nature Nanotechnology, 2011, 6 (12), 809–814.), optical tweezers (Journal of the Royal Society Interface, 2008, 5 (24), 671–690.), and photohydrazide stretching (Journal of Biomechanical Engineering, 2010, 132 (4), 044504.). These technologies have made significant achievements in the study of cell mechanical response behavior and greatly promoted the rapid development of cell mechanical property measurement technology. However, in order to gain a deeper understanding of cell signal transduction coupling behavior and the oxidative stress response it triggers, and to explore its deep biological mechanisms, it is necessary to obtain more extensive metabolic information. The capture of electrical signals can expand the dimension of cell metabolic information extraction, so that the acquisition of cell mechanical response characteristics is not limited to the morphological distribution changes revealed by optics, but can also delve into the electrophysiological activities inside the cell. Furthermore, existing technologies primarily characterize and detect cellular responses to mechanical loading, leaving room for improvement in detection timeliness, data continuity, and sensitivity to subtle phenomena. Therefore, by enabling real-time monitoring of cellular responses to dynamic mechanical stimulation and extracting information about their dynamic changes, we can provide immediate insight into subtle fluctuations in cellular metabolic activity.

[0003] However, existing technologies primarily characterize and detect cellular response characteristics after mechanical loading, leaving room for improvement in detection timeliness, data continuity, and sensitivity to subtle phenomena. Furthermore, existing cellular mechanical response information is primarily extracted through mechanical characterization, leaving room for improvement in this area. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for characterizing the mechanical response behavior of cells and extracting metabolic information by integrating ultrasonic mechanical loading technology and electrochemical detection technology.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for characterizing cell mechanical response behavior and extracting metabolic information, comprising the following steps: Step 1: Build a device that integrates ultrasonic mechanical stimulation and electrochemical detection; Step 2: Add a cell culture medium containing cells to the device in step 1, then apply voltage via a signal generator in the device, and then observe the arrangement of cells on the electrodes using a microscope; Step 3: Change the amount of cells in the cell culture medium and then observe the arrangement of cells on the electrode again using a microscope; Among them, if the cells are observed to be clearly arranged in one dimension at the bottom of the electrode, it means that the cell culture fluid of this amount of cells can be successfully stimulated by ultrasonic mechanics and arranged in a directional manner. Otherwise, it means that the cell culture fluid of this amount of cells cannot be successfully stimulated by ultrasonic mechanics.

[0006] As a further improvement of the present invention, the step three further includes the following steps: Select a cell culture medium that can be successfully stimulated by ultrasonic mechanical stimulation and oriented, then change the applied voltage of the signal generator. If it is observed that the cells do not show obvious arrangement at the bottom of the electrode, it means that the degree of mechanical stimulation applied at this voltage is low and the cells cannot be effectively arranged. Conversely, it means that the degree of mechanical stimulation applied at this voltage is high, which allows the cells to be effectively arranged.

[0007] As a further improvement of the present invention, the step three further includes the following steps: Select cell culture medium that can successfully be stimulated by ultrasonic mechanics and oriented in a certain direction, and then perform live-dead staining experiments on the cells after 1 hour, 2 hours, 3 hours, and 5 hours, respectively. Then, observe the survival rate of the cells under a fluorescence microscope after being placed in the device for several hours.

[0008] As a further improvement of the present invention, the step three further includes the following steps: Cell culture fluid that can be successfully stimulated by ultrasonic mechanical stimulation and oriented in a directional manner was selected. A signal generator was used to apply gradient mechanical stimulation of 8V, 12V, 16V, and 20V, respectively. The changes in the impedance electrical signal of HeLa cells were then monitored in real time for 5 minutes using a CHI660 electrochemical workstation. The impedance value of the cells was then monitored at a test frequency of 10,000 Hz and a sampling interval of 20 s.

[0009] As a further improvement of the present invention, the specific steps of constructing a device integrating ultrasonic mechanical stimulation device and electrochemical detection in step 1 are as follows: Step 1: Customize a rectangular acrylic container with the following specifications: 4 mm internal diameter, square cross-section with 4 mm side length, 2 mm wall thickness, and 1 cm overall height. The top and bottom of the container should be open, not closed. In steps 1 and 2, a gold microelectrode with a glass substrate and dimensions of 10 x 6 x 0.75 mm was used, which contained a working electrode, a reference electrode, and an auxiliary electrode; In step 13, purchase piezoelectric ceramics with dimensions of 10 x 8 x 0.65 mm and glue them symmetrically on both sides of the rectangular container in step 1 using 502 glue; Step 14: Use sealing glue to fix the container with piezoelectric ceramics in step 13 to the center of the gold microelectrode in step 12; In step 15, insert the device completed in step 14 into the pre-purchased electrochemical interface, ensuring that the pins in the interface can accurately mate with the three electrode points of the gold electrode. Then, connect the other end of the interface to the electrochemical workstation. Step 16: Connect the positive and negative wires on the piezoelectric ceramic in step 13 to the positive and negative electrodes of the signal generator respectively. After applying voltage, a device integrating ultrasonic mechanical stimulation and electrochemical detection is obtained.

[0010] The beneficial effect of the present invention is that, through the setting of step one, a device integrating ultrasonic mechanical stimulation device and electrochemical detection can be effectively constructed. Then, based on the device, through the effects of steps two and three, the device can be used to realize the extraction of cell mechanical response behavior and metabolic information, and the ultrasonic mechanical stimulation device is integrated with electrochemical detection technology to realize real-time in situ monitoring of the changes in the electrical signals of cells while they are mechanically stimulated. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A schematic diagram of the rectangular container used for the device; Figure 2 Schematic diagram of the gold microelectrode used; Figure 3 A schematic diagram showing how the pin can precisely dock with the three electrode points of the gold electrode; Figure 4 Schematic diagram of HeLa cells showing a clear one-dimensional arrangement at the bottom of the electrode; Figure 5 a, b, and c are schematic diagrams of adding different numbers of HeLa cells; Figure 6 Schematic diagram of providing different voltage stimulations for a, b, c, d, e, and f; Figure 7 Schematic diagram of live-dead staining experiment; Figure 8 Schematic diagram of real-time monitoring of impedance electrical signals of cells a and b; Figure 9 Schematic diagram of a macrophage as an example. DETAILED DESCRIPTION

[0012] The present invention will be further described below with reference to the embodiments shown in the accompanying drawings.

[0013] A method for characterizing cell mechanical response behavior and extracting metabolic information in this embodiment specifically includes the following steps: Step 1: Customize a rectangular acrylic container with the following specifications: internal diameter of 4 mm (square cross section, 4 mm side length), wall thickness of 2 mm, and overall height of 1 cm. The top and bottom of the container are open, not closed ( Figure 1 ).

[0014] Step 2: Purchase a gold microelectrode with a glass substrate and a size of 10 x 6 x 0.75 mm, which contains a working electrode, a reference electrode and an auxiliary electrode ( Figure 2 ) Step 3: Purchase piezoelectric ceramics with dimensions of 10 x 8 x 0.65 mm and glue them symmetrically on both sides of the rectangular container in step 1 using 502 glue.

[0015] Step 4: Use sealing glue to fix the container with piezoelectric ceramics in step 3 at the center of the gold microelectrode in step 2.

[0016] Step 5: Insert the device completed in step 4 into the pre-purchased electrochemical interface, ensuring that the pins in the interface can accurately connect with the three electrode points of the gold electrode ( Figure 3 ), and then connect the other end of the interface to the electrochemical workstation.

[0017] Step six, connect the positive and negative wires on the piezoelectric ceramic in step three to the positive and negative electrodes of the signal generator respectively, and after applying voltage, the device integrating ultrasonic mechanical stimulation device and electrochemical detection is obtained.

[0018] In the device integrating ultrasonic mechanical stimulation and electrochemical detection prepared based on the above steps, the mechanical stimulation of different numbers of HeLa cells in the device is specifically described: 150 μL of cell culture medium containing 50 w HeLa cells was added to the rectangular container of the above device. After applying a voltage of 20 V using a signal generator, the arrangement of cells on the electrodes was observed using a microscope. Figure 4 It can be seen that the cells are clearly arranged in one dimension at the bottom of the electrode, indicating that HeLa cells can be successfully stimulated by ultrasonic mechanics and arranged in a directional manner in this device.

[0019] To further explore the effect of cell number on the arrangement, we added 100 w ( Figure 5 a); 200w ( Figure 5 b) and 300w ( Figure 5 c) HeLa cells were applied with a 20 V voltage using a signal generator, and the cell alignment on the electrode was observed using a microscope. When the cell count was 100 W, good directional alignment was still achieved. However, when the cell count increased to 200 W, the alignment became increasingly blurred. At 300 W, no clear one-dimensional arrangement of the HeLa cells could be observed. This may be because the increased cell count resulted in close contact between cells, leaving insufficient space for directional alignment. This indicates that the optimal cell count for this integrated device is between 50 and 200 W.

[0020] In the device of integrated ultrasonic mechanical stimulation and electrochemical detection prepared based on the above steps, the conditions of HeLa cells being stimulated by different gradient mechanical stimuli in the device are specifically described: 150 μL of cell culture medium containing 100 w HeLa cells was added to the rectangular container of the above device, and a voltage of 5 V was applied using a signal generator. The arrangement of cells on the electrodes was observed using a microscope. Figure 6 As can be seen from a, the cells did not show obvious arrangement at the bottom of the electrode, indicating that at 5 V, the applied mechanical stimulation was too low to effectively arrange the cells.

[0021] To further explore the effect of applied voltage on cell arrangement, we applied voltages of 6V, 7V, 8V, 9V, and 10V, and observed the arrangement of cells on the electrodes using a microscope. Figure 6 bf shows that when the voltage reaches 8V ( Figure 6 d), the cells can be clearly arranged at the bottom of the electrode.

[0022] The survival of HeLa cells in the device integrating ultrasonic mechanical stimulation and electrochemical detection prepared based on the above steps is described: 150 μL of cell culture medium containing 100 w HeLa cells was added to the rectangular container of the above device. Live-dead staining experiments were performed on the cells after 1 h, 2 h, 3 h, and 5 h, respectively. The survival rate of the cells after being placed in the device for several hours was then observed under a fluorescence microscope. Figure 7 As can be seen, no obvious large-scale cell death occurred during the 5-hour observation period, indicating that the device does not cause cell death in the short term and does not affect the cell stress and subsequent electrical signal testing.

[0023] The device integrating ultrasonic mechanical stimulation and electrochemical detection prepared based on the above steps is used to describe the real-time monitoring of the impedance electrical signal of HeLa cells under mechanical stimulation in the device. 150 μL of cell culture medium containing 100 w HeLa cells was added to the rectangular container of the above device. After applying gradient mechanical stimulation of 8V, 12V, 16V, and 20V respectively using a signal generator, the changes in the HeLa cell impedance electrical signal were monitored in real time for 5 minutes using a CHI660 electrochemical workstation. Figure 8 As shown in a, when the test frequency is 10000 Hz and the sampling interval is 20s, the impedance signal of HeLa cells becomes larger after mechanical stimulation, and the impedance value gradually increases with the increase of mechanical stimulation. Under the same mechanical stimulation, the impedance value does not change significantly within 5 minutes. The average of the three impedance values in each minute is taken as the average impedance of this minute, which is obtained by Figure 8 As shown in Figure b, after averaging, the impedance value still shows a slow upward trend within 5 minutes under the same mechanical stimulation. These results demonstrate that the device can monitor the impedance electrical response of cells in real time while applying mechanical stimulation to them.

[0024] Furthermore, this embodiment provides the following examples: In the device integrating ultrasonic mechanical stimulation and electrochemical detection prepared based on the above steps, the real-time monitoring of the impedance electrical signal of RAW264.7 macrophages under mechanical stimulation in the device is described: 150 μL of cell culture medium containing 100 w RAW 264.7 macrophages was added to the rectangular container of the above device. After applying a 20V gradient mechanical stimulation using a signal generator, the RAW 264.7 macrophage impedance electrical signal changes were monitored in real time for 30 minutes using a CHI660 electrochemical workstation. The test frequency was 10000 Hz, the sampling interval was 20 seconds, and the average of every three values was taken as the impedance value within this minute. Figure 9 As can be seen, after mechanical stimulation of RAW 264.7 macrophages, their impedance signal first increased and then decreased within 30 minutes. These results demonstrate that the device can monitor the impedance electrical response of cells in real time while applying mechanical stimulation to them for a long period of time.

[0025] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for characterizing cell mechanical response behavior and extracting metabolic information, characterized by: The steps include: Step 1: Build a device that integrates ultrasonic mechanical stimulation and electrochemical detection; Step 2: Add a cell culture medium containing cells to the device in step 1, then apply voltage using a signal generator in the device, and then observe the arrangement of cells on the electrodes using a microscope; Step 3: Change the amount of cells in the cell culture medium and then observe the arrangement of cells on the electrode again using a microscope; Among them, if the cells are observed to be clearly arranged in one dimension at the bottom of the electrode, it means that the cell culture fluid of this amount of cells can be successfully stimulated by ultrasonic mechanics and arranged in a directional manner. Otherwise, it means that the cell culture fluid of this amount of cells cannot be successfully stimulated by ultrasonic mechanics.

2. The method for characterizing cell mechanical response behavior and extracting metabolic information according to claim 1, characterized in that: The step three also includes the following steps: Select a cell culture medium that can be successfully stimulated by ultrasonic mechanical stimulation and oriented, then change the applied voltage of the signal generator. If it is observed that the cells do not show obvious arrangement at the bottom of the electrode, it means that the degree of mechanical stimulation applied at this voltage is low and the cells cannot be effectively arranged. Conversely, it means that the degree of mechanical stimulation applied at this voltage is high, which allows the cells to be effectively arranged.

3. The method for characterizing cell mechanical response behavior and extracting metabolic information according to claim 1 or 2, characterized in that: The step three also includes the following steps: Select cell culture medium that can be successfully stimulated by ultrasonic mechanics and oriented in a certain direction, and then perform live-dead staining experiments on the cells after 1 hour, 2 hours, 3 hours, and 5 hours, respectively. Then, observe the survival rate of the cells under a fluorescence microscope after being placed in the device for several hours.

4. The method for characterizing cell mechanical response behavior and extracting metabolic information according to claim 1 or 2, characterized in that: The step three also includes the following steps: Cell culture fluid that can be successfully stimulated by ultrasonic mechanical stimulation and oriented in a directional manner was selected. A signal generator was used to apply gradient mechanical stimulation of 8V, 12V, 16V, and 20V, respectively. The changes in the impedance electrical signal of HeLa cells were then monitored in real time for 5 minutes using a CHI660 electrochemical workstation. The impedance value of the cells was then monitored at a test frequency of 10,000 Hz and a sampling interval of 20 s.

5. The method for characterizing cell mechanical response behavior and extracting metabolic information according to claim 1 or 2, characterized in that: The specific steps of constructing a device integrating ultrasonic mechanical stimulation device and electrochemical detection in step 1 are as follows: Step 1: Customize a rectangular acrylic container with the following specifications: 4 mm internal diameter, square cross-section with 4 mm side length, 2 mm wall thickness, and 1 cm overall height. The top and bottom of the container should be open, not closed. In steps 1 and 2, a gold microelectrode with a glass substrate and dimensions of 10 x 6 x 0.75 mm was used, which contained a working electrode, a reference electrode, and an auxiliary electrode; In step 13, purchase piezoelectric ceramics with dimensions of 10 x 8 x 0.65 mm and glue them symmetrically on both sides of the rectangular container in step 1 using 502 glue; Step 14: Use sealing glue to fix the container with piezoelectric ceramics in step 13 to the center of the gold microelectrode in step 12; In step 15, insert the device completed in step 14 into the pre-purchased electrochemical interface, ensuring that the pins in the interface can accurately mate with the three electrode points of the gold electrode. Then, connect the other end of the interface to the electrochemical workstation. Step 16: Connect the positive and negative wires on the piezoelectric ceramic in step 13 to the positive and negative electrodes of the signal generator respectively. After applying voltage, a device integrating ultrasonic mechanical stimulation and electrochemical detection is obtained.

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