A method and device for selecting microsecond pulse electric field parameters for killing senescent cells

Abstract

A method and apparatus for selecting microsecond pulsed electric field parameters for killing senescent cells. The method includes: 1) obtaining biological tissue cells and culturing them to obtain n groups of cell reagents containing normal cells and n groups of cell reagents containing senescent cells; 2) setting n groups of pulsed electric field parameters for killing senescent cells; 3) placing the i-th group of cell reagents containing normal cells and the i-th group of cell reagents containing senescent cells between an electrode pair, and electrically stimulating the i-th group of cell reagents containing normal cells and the i-th group of cell reagents containing senescent cells using the i-th group of pulsed electric field parameters; 4) performing cell activity detection, PI positivity rate detection, and electrosensitivity detection on each group of cell reagents after electrical stimulation to determine the optimal pulsed electric field parameters. The apparatus includes an electrode pair and a pulse generator. Compared with traditional treatment methods for chronic wounds, this invention only requires an electric field to efficiently and accurately kill senescent cells, without thermal effects or side effects.

Description

Technical Field

[0001] This invention relates to the field of wound treatment technology, specifically to a method and device for selecting microsecond pulse electric field parameters for killing senescent cells. Background Technology

[0002] Diabetes is a common chronic disease. Statistics show that as of 2022, China had 140 million diabetic patients, ranking first in the world. It is a difficult-to-cure disease with a low control rate, only 36.7%. Diabetic patients often suffer from numerous complications, the most common being diabetic chronic wounds or foot ulcers. Chronic wounds in diabetic patients often have long treatment cycles, slow healing, and high recurrence rates, and in severe cases, can even lead to amputation. Typical characteristics of chronic wounds include poor blood circulation, abnormal inflammation, and the inability to form an outermost layer of epidermis. Diabetic wounds differ from ordinary wounds; their wound microenvironment is complex, influenced by numerous factors such as hyperglycemia, excessive ROS production, and persistent inflammation. Cellular senescence in the wound is one of the important pathophysiological factors contributing to wound non-healing in diabetic patients. Hyperglycemia, oxidative stress, and mitochondrial and DNA damage in the diabetic wound environment are potential factors inducing cellular senescence; the number of senescent cells in diabetic wounds is far greater than that in normal wounds. Senescent cells are cells that have permanently stopped proliferating, undergone morphological changes, secrete complex pro-inflammatory factors, exhibit increased autophagy, and possess strong resistance to apoptosis. Compared to normal cells, senescent cells have a wrinkled cell membrane, resulting in increased cell volume. Simultaneously, the cell and nucleus enlarge, while the distance between the nucleus and cell membrane decreases. The prolonged presence of senescent cells keeps wounds in an inflammatory state, hindering healing. Cellular senescence impedes tissue regeneration and triggers persistent low-level inflammation, thereby accelerating the senescence of other cells.

[0003] The wound site contains a variety of cell types, including fibroblasts, monocytes, vascular endothelial cells, and epidermal cells. Currently, there is still no ideal solution for eliminating senescent cells without damaging normal cells. Because senescent and normal cells in the wound site differ in morphology, changes in their cell membranes and nuclear membranes alter the electroporation threshold, leading to differences in their impedance values. There are currently gaps in the measurement of electrical impedance to senescent and normal cells, as well as in the measurement of cell morphology and nuclear membrane size. Similarly, research on how to selectively kill senescent cells in the wound to promote wound healing is somewhat insufficient. Summary of the Invention

[0004] The purpose of this invention is to provide a method and apparatus for selecting microsecond pulse electric field parameters for killing senescent cells.

[0005] The technical solution adopted to achieve the technical objective of this invention is as follows: a method for selecting microsecond pulse electric field parameters for killing senescent cells, comprising the following steps:

[0006] 1) Obtain biological tissue cells and perform cell culture to obtain n sets of cell reagents containing normal cells and n sets of cell reagents containing senescent cells, where n is a positive integer.

[0007] 2) Set n sets of pulse electric field parameters as follows:

[0008]

[0009] In the formula, I is the pulse electric field parameter matrix. i Let I be the pulse electric field parameters for the i-th group, i = 1, 2, ..., n. ij Let j be the j-th parameter of the i-th group of pulse electric field parameters, where j = 1, 2, ..., m, and m is a positive integer.

[0010] 3) Place the cell reagent containing normal cells in the i-th group between the electrode pairs, and set the parameters of the pulse generator to the pulse electric field parameters I of the i-th group. i .

[0011] The pulse generator transmits a pulse signal to the electrode pair, causing the electrode pair to generate electrical stimulation on the i-th group of cellular reagents containing normal cells.

[0012] The cellular reagent containing senescent cells in the i-th group is placed between the electrode pairs, and the parameters of the pulse generator are set to the pulse electric field parameters I of the i-th group. i .

[0013] The pulse generator transmits a pulse signal to the electrode pair, causing the electrode pair to generate electrical stimulation on the i-th group of cellular reagents containing senescent cells, with i initially valued at 1.

[0014] 4) Let i = i + 1, and repeat step 3) until all cell reagents are electrically stimulated.

[0015] 5) Perform cell viability, PI positivity rate, and electrosensitivity tests on each group of cells after electrical stimulation to determine the optimal pulsed electric field parameters.

[0016] Furthermore, the pulse electric field parameters include pulse amplitude, electric field strength, pulse width, and pulse count.

[0017] Furthermore, the pulse amplitude ranges from 100V to 400V.

[0018] The electric field strength ranges from 500V / cm to 2000V / cm.

[0019] Furthermore, the pulse width ranges from 50µs to 200µs.

[0020] Furthermore, the steps for detecting cell viability are as follows:

[0021] 5.1.1) Add the electrically stimulated cell reagent to a 96-well plate and place the 96-well plate in a cell incubator for a period of time.

[0022] 5.1.2) Add CCK-8 reagent to the 96-well plate containing the cell reagent, and then place the 96-well plate in a cell incubator for a period of time.

[0023] 5.1.3) Measure the absorbance of the cultured cell reagents using an enzyme-linked immunosorbent assay (ELISA) reader.

[0024] 5.1.4) By comparing the absorbance values ​​of normal cells and senescent cells under the same set of pulsed electric field parameters, the pulsed electric field parameters that yield the maximum difference in cell activity between normal cells and senescent cells are obtained.

[0025] Furthermore, the steps for detecting the PI positivity rate are as follows:

[0026] 5.2.1) Add PI staining working solution to the unstimulated cell reagent to resuspend the cells, and obtain cell suspensions containing normal cells and cell suspensions containing senescent cells.

[0027] 5.2.2) Using the i-th group of pulse electric field parameters I i Electrical stimulation was applied to the cell suspension containing normal cells in group i and the cell suspension containing senescent cells in group i.

[0028] 5.2.3) The cell suspension after electrical stimulation was incubated in the dark for a period of time, and the cell suspension after incubation in the dark was detected by flow cytometry to obtain the PI positivity rate of the cells.

[0029] 5.2.4) Compare the PI positivity rates of normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that maximize the difference in PI positivity rates between normal cells and senescent cells.

[0030] Furthermore, the steps for the electrosensitivity detection are as follows:

[0031] 5.3.1) Add PBS to the unstimulated cell reagent for washing.

[0032] 5.3.2) Add Calcein AM / PI detection working solution to the washed cell reagent and incubate in the dark for a period of time to obtain detection solutions containing normal cells and detection solutions containing senescent cells.

[0033] 5.3.3) Using the i-th group of pulse electric field parameters I i Electrical stimulation was applied to the test solution containing normal cells in group i and the test solution containing senescent cells in group i, and the fluorescence changes of the cells were recorded in real time using a fluorescence microscope.

[0034] 5.3.4) Compare the fluorescence dissipation of normal cells and senescent cells and the PI content in cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that cause rapid fluorescence dissipation in senescent cells and high PI content in normal cells.

[0035] An apparatus for applying the above method includes an electrode pair and a pulse generator.

[0036] The electrodes are used to electrically stimulate the reagents used to treat the cells.

[0037] The pulse generator is used to generate microsecond pulse electric fields and transmit them to the electrode pair.

[0038] Furthermore, the electrode cup includes a positive electrode and a negative electrode.

[0039] The positive and negative electrodes are placed opposite each other.

[0040] The reagent for the cells to be treated is placed between the positive and negative electrodes.

[0041] Furthermore, there is a gap between the positive electrode and the negative electrode.

[0042] The technical effects of this invention are beyond doubt. This invention proposes a new method for selectively killing senescent cells using microsecond pulsed electric fields. Compared with traditional treatments for chronic wounds, this invention can efficiently and accurately kill senescent cells using only an electric field, without thermal effects or side effects.

[0043] This invention combines the volume difference and different cell impedance characteristics between senescent cells and normal cells, and uses a pulsed electric field to achieve different degrees of electroporation of the two types of cells, thereby achieving the effect of causing the death of senescent cells while maintaining a high survival rate of normal cells.

[0044] This invention proposes to efficiently and selectively eliminate senescent cells by regulating the pulsed electric field parameters (amplitude, pulse width, frequency, and duration) acting on cells based on the morphological and electrical impedance differences between senescent and normal cells.

[0045] Compared to traditional chemotherapy or radiotherapy, electroporation is safer. Because electroporation only physically perforates the cell membrane, it avoids the side effects of chemotherapy or radiation damage, resulting in less damage to the wound and a shorter recovery period.

[0046] This invention selectively kills senescent cells, offering superior results compared to other traditional treatments during wound repair. Electroporation technology enables precise killing of senescent cells. By precisely controlling the electric field strength, pulse count, and pulse width, senescent cells can be selectively perforated while causing minimal damage to normal cells. This selective killing maximizes the protection of normal cells and reduces treatment side effects. Attached Figure Description

[0047] Figure 1 A flowchart of a method for selecting microsecond pulse electric field parameters for killing senescent cells;

[0048] Figure 2 Schematic diagram of the morphology of normal and senescent 3T3 adherent cells; Figure 2 (a) is a schematic diagram of the morphology of normal adherent 3T3 cells; Figure 2 (b) is a schematic diagram of the morphology of senescent adherent cells;

[0049] Figure 3 Schematic diagram of the morphology of normal and senescent 3T3 suspension cells; Figure 3 (a) is a schematic diagram of the morphology of normal 3T3 suspension cells; Figure 3 (b) is a schematic diagram of the morphology of senescent suspended cells;

[0050] Figure 4 A schematic diagram showing the real and imaginary parts of impedance in normal and senescent 3T3 cells.

[0051] Figure 5 This is a schematic diagram of the cell activity of normal 3T3 cells and senescent cells. Figure 5 (a) is a schematic diagram of cell activity under different electrical parameters with a pulse width of 50 μs; Figure 5 (b) is a schematic diagram of cell activity with different electrical parameters at a pulse width of 100 μs; Figure 5 (c) is a schematic diagram of cell activity under different electrical parameters at a pulse width of 200 μs;

[0052] Figure 6 Schematic diagram of flow cytometry PI single staining of normal 3T3 cells and senescent cells;

[0053] Figure 7 This is a schematic diagram showing the fluorescence dissipation of normal 3T3 cells and senescent cells. Figure 7 (a) is a schematic diagram of fluorescence in normal 3T3 cells before electrical stimulation; Figure 7 (b) is a schematic diagram of fluorescence after electrical stimulation of normal 3T3 cells; Figure 7 (c) is a schematic diagram of PI content after electrical stimulation of normal 3T3 cells; Figure 7 (d) is a schematic diagram of fluorescence in senescent cells before electrical stimulation; Figure 7 (e) is a schematic diagram of fluorescence after electrical stimulation of senescent cells; Figure 7 (f) is a schematic diagram of PI content after electrical stimulation of senescent cells;

[0054] Figure 8 This is a schematic diagram of the measurement system. Detailed Implementation

[0055] The present invention will be further described below with reference to embodiments, but it should not be construed that the scope of the present invention is limited to the following embodiments. Various substitutions and modifications made based on ordinary technical knowledge and common practices in the art without departing from the above-described technical concept of the present invention should be included within the scope of protection of the present invention.

[0056] Example 1:

[0057] See Figures 1 to 8 A method for selecting microsecond pulse electric field parameters for killing senescent cells includes the following steps:

[0058] 1) Obtain biological tissue cells and perform cell culture to obtain n sets of cell reagents containing normal cells and n sets of cell reagents containing senescent cells, where n is a positive integer.

[0059] 2) Set n sets of pulse electric field parameters as follows:

[0060]

[0061] In the formula, I is the pulse electric field parameter matrix. i Let I be the pulse electric field parameters for the i-th group, i = 1, 2, ..., n. ij Let j be the j-th parameter of the i-th group of pulse electric field parameters, where j = 1, 2, ..., m, and m is a positive integer.

[0062] 3) Place the cell reagent containing normal cells in the i-th group between the electrode pairs, and set the parameters of the pulse generator to the pulse electric field parameters I of the i-th group. i .

[0063] The pulse generator transmits a pulse signal to the electrode pair, causing the electrode pair to generate electrical stimulation on the i-th group of cellular reagents containing normal cells.

[0064] The cellular reagent containing senescent cells in the i-th group is placed between the electrode pairs, and the parameters of the pulse generator are set to the pulse electric field parameters I of the i-th group. i .

[0065] The pulse generator transmits a pulse signal to the electrode pair, causing the electrode pair to generate electrical stimulation on the i-th group of cellular reagents containing senescent cells, with i initially valued at 1.

[0066] 4) Let i = i + 1, and repeat step 3) until all cell reagents are electrically stimulated.

[0067] 5) Perform cell viability, PI positivity rate, and electrosensitivity tests on each group of cells after electrical stimulation to determine the optimal pulsed electric field parameters.

[0068] Example 2:

[0069] A method for selecting microsecond pulse electric field parameters for killing senescent cells is described in Example 1. Further, the pulse electric field parameters include pulse amplitude, electric field strength, pulse width, and pulse count.

[0070] Example 3:

[0071] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 1 to 2, wherein the pulse amplitude range is 100V-400V.

[0072] The electric field strength ranges from 500V / cm to 2000V / cm.

[0073] Example 4:

[0074] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 1 to 3, wherein the pulse width is in the range of 50µs-200µs.

[0075] Example 5:

[0076] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 1 to 4, and further, the steps for detecting cell activity are as follows:

[0077] 5.1.1) Add the electrically stimulated cell reagent to a 96-well plate and place the 96-well plate in a cell incubator for a period of time.

[0078] 5.1.2) Add CCK-8 reagent to the 96-well plate containing the cell reagent, and then place the 96-well plate in a cell incubator for a period of time.

[0079] 5.1.3) Measure the absorbance of the cultured cell reagents using an enzyme-linked immunosorbent assay (ELISA) reader.

[0080] 5.1.4) By comparing the absorbance values ​​of normal cells and senescent cells under the same set of pulsed electric field parameters, the pulsed electric field parameters that yield the maximum difference in cell activity between normal cells and senescent cells are obtained.

[0081] Example 6:

[0082] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 1 to 5, and further, the steps for PI positivity rate detection are as follows:

[0083] 5.2.1) Add PI staining working solution to the unstimulated cell reagent to resuspend the cells, and obtain cell suspensions containing normal cells and cell suspensions containing senescent cells.

[0084] 5.2.2) Using the i-th group of pulse electric field parameters I i Electrical stimulation was applied to the cell suspension containing normal cells in group i and the cell suspension containing senescent cells in group i.

[0085] 5.2.3) The cell suspension after electrical stimulation was incubated in the dark for a period of time, and the cell suspension after incubation in the dark was detected by flow cytometry to obtain the PI positivity rate of the cells.

[0086] 5.2.4) Compare the PI positivity rates of normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that maximize the difference in PI positivity rates between normal cells and senescent cells.

[0087] Example 7:

[0088] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 1 to 6, and further, the steps of the electrosensitivity detection are as follows:

[0089] 5.3.1) Add PBS to the unstimulated cell reagent for washing.

[0090] 5.3.2) Add Calcein AM / PI detection working solution to the washed cell reagent and incubate in the dark for a period of time to obtain detection solutions containing normal cells and detection solutions containing senescent cells.

[0091] 5.3.3) Using the i-th group of pulse electric field parameters I i Electrical stimulation was applied to the test solution containing normal cells in group i and the test solution containing senescent cells in group i, and the fluorescence changes of the cells were recorded in real time using a fluorescence microscope.

[0092] 5.3.4) Compare the fluorescence dissipation of normal cells and senescent cells and the PI content in cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that cause rapid fluorescence dissipation in senescent cells and high PI content in normal cells.

[0093] Example 8:

[0094] An apparatus for applying the method of any one of embodiments 1 to 7 includes an electrode pair and a pulse generator.

[0095] The electrodes are used to electrically stimulate the reagents used to treat the cells.

[0096] The pulse generator is used to generate microsecond pulse electric fields and transmit them to the electrode pair.

[0097] Example 9:

[0098] A microsecond pulse electric field parameter selection device for killing senescent cells, the main technical contents of which are described in Example 8, further wherein the electrode cup includes a positive electrode and a negative electrode.

[0099] The positive and negative electrodes are placed opposite each other.

[0100] The reagent for the cells to be treated is placed between the positive and negative electrodes.

[0101] Example 10:

[0102] A microsecond pulse electric field parameter selection device for killing senescent cells, the main technical contents of which are described in any one of Embodiments 8 to 9, further wherein there is a gap between the positive electrode and the negative electrode.

[0103] Example 11:

[0104] See Figures 1 to 8 A method for selecting microsecond pulse electric field parameters for killing senescent cells includes the following steps:

[0105] 1) Obtain biological tissue cells and perform cell culture to obtain n sets of cell reagents containing normal cells and n sets of cell reagents containing senescent cells, where n is a positive integer.

[0106] The mouse fibroblast cell line 3T3-L1 was used and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 1% penicillin and streptomycin (100 μg / mL) and 10% inactivated fetal bovine serum (FBS) in a constant temperature and humidity incubator at 37°C and 5% CO2. The cells were passaged using 0.25% trypsin (0.25% Trypsin-EDTA) throughout the culture process to obtain cell reagents containing normal cells.

[0107] 3T3-L1: Take cells with a density of 80%, discard the original culture medium, add culture medium supplemented with H2O2 (300uM / mL) to stimulate the cells for 4h, then replace with fresh culture medium and culture for 24h to obtain cell reagent containing senescent cells.

[0108] 2) Set n sets of pulse electric field parameters as follows:

[0109]

[0110] In the formula, I is the pulse electric field parameter matrix. i Let I be the pulse electric field parameters for the i-th group, i = 1, 2, ..., n. ij Let j be the j-th parameter of the i-th group of pulse electric field parameters, where j = 1, 2, ..., m, and m is a positive integer.

[0111] 3) Place the cell reagent containing normal cells in the i-th group between the electrode pairs, and set the parameters of the pulse generator to the pulse electric field parameters I of the i-th group. i .

[0112] The pulse generator transmits a pulse signal to the electrode pair, causing the electrode pair to generate electrical stimulation on the i-th group of cellular reagents containing normal cells.

[0113] The cellular reagent containing senescent cells in the i-th group is placed between the electrode pairs, and the parameters of the pulse generator are set to the pulse electric field parameters I of the i-th group. i .

[0114] The pulse generator transmits a pulse signal to the electrode pair, causing the electrode pair to generate electrical stimulation on the i-th group of cellular reagents containing senescent cells, with i initially valued at 1.

[0115] 4) Let i = i + 1, and repeat step 3) until all cell reagents are electrically stimulated.

[0116] 5) Perform cell viability, PI positivity rate, and electrosensitivity tests on each group of cells after electrical stimulation to determine the optimal pulsed electric field parameters.

[0117] Example 12:

[0118] A method for selecting microsecond pulse electric field parameters for killing senescent cells is described in Example 11. Further, the pulse electric field parameters include pulse amplitude, electric field strength, pulse width, and pulse count.

[0119] Example 13:

[0120] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 11 to 12, wherein the pulse amplitude range is 100V-400V.

[0121] The electric field strength ranges from 500V / cm to 2000V / cm.

[0122] Example 14:

[0123] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 11 to 13, wherein the pulse width is in the range of 50µs-200µs.

[0124] Example 15:

[0125] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 11 to 14, further wherein the steps for detecting cell activity are as follows:

[0126] 5.1.1) Add the electrically stimulated cell reagent to a 96-well plate and place the 96-well plate in a cell incubator for a period of time.

[0127] 5.1.2) Add CCK-8 reagent to the 96-well plate containing the cell reagent, and then place the 96-well plate in a cell incubator for a period of time.

[0128] 5.1.3) Measure the absorbance of the cultured cell reagents using an enzyme-linked immunosorbent assay (ELISA) reader.

[0129] 5.1.4) By comparing the absorbance values ​​of normal cells and senescent cells under the same set of pulsed electric field parameters, the pulsed electric field parameters that yield the maximum difference in cell activity between normal cells and senescent cells are obtained.

[0130] Collect cells in good growth condition with a density of 70-80%, discard the original culture medium, rinse once with phloase buffer solution (PBS), add trypsin and incubate in a cell culture incubator for 1 min to allow cell detachment. Add fresh culture medium to stop digestion, centrifuge the cell suspension at 800 rpm for 5 min, discard the supernatant, and resuspend in culture medium (3 × 10⁻⁶). 6 Cells / mL). Cell suspensions were electrically stimulated using a self-developed pulse generator and electrode cups (2mm distance between positive and negative electrodes). 100µL of cell suspension was added to the electrode cups each time, and the entire unit was placed on the electrode base. Different pulse electric field parameters were adjusted for varying electric field strength and pulse width. The cell suspensions were treated with a pulsed electric field at a frequency of 1Hz for 8 seconds. After treatment, the cells were transferred to 96-well plates and incubated for 3 hours. Then, 10µL of cell proliferation / toxicity assay kit (Cell Counting Kit-8, CCK-8) was added to each well, and the cells were incubated for another 2 hours. The absorbance at 450nm was then measured using a microplate reader. Data processing yielded... Figure 5The cell activity graph shown shows that, through experiments with different pulsed electric field parameters, it was found that under an electric field strength of 1000 V / cm and a pulse width of 100 μs for 8 s, 3T3 normal cells and senescent cells could achieve the greatest difference in cell activity.

[0131] Example 16:

[0132] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 11 to 15, further wherein the steps for PI positivity rate detection are as follows:

[0133] 5.2.1) Add PI staining working solution to the unstimulated cell reagent to resuspend the cells, and obtain cell suspensions containing normal cells and cell suspensions containing senescent cells.

[0134] 5.2.2) Using the i-th group of pulse electric field parameters I i Electrical stimulation was applied to the cell suspension containing normal cells in group i and the cell suspension containing senescent cells in group i.

[0135] 5.2.3) The cell suspension after electrical stimulation was incubated in the dark for a period of time, and the cell suspension after incubation in the dark was detected by flow cytometry to obtain the PI positivity rate of the cells.

[0136] 5.2.4) Compare the PI positivity rates of normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that maximize the difference in PI positivity rates between normal cells and senescent cells.

[0137] Collect cells in good growth condition with a density of 70-80%, digest and centrifuge them, discard the supernatant, and resuspend the cells in propidium iodide (PI) staining working solution (1×10⁻⁶). 7 (cells / mL) 100 μL of cell suspension was added to a 2 mm electroporation cuvette each time, and the entire cuvette was placed in the electrode holder. Pulsed electric field treatment was performed according to different electric field parameters. The treated cell suspension was transferred to a 1.5 mL EP tube and incubated at 37°C in the dark for 30 min before flow cytometry analysis. The PI positivity rate of normal cells and senescent cells was statistically analyzed. The results are shown below. Figure 6 As shown, under an electric field strength of 1000 V / cm and a pulse width of 100 μs for 8 s, the difference in PI positivity rate between normal 3T3 cells and senescent cells was maximized.

[0138] Example 17:

[0139] A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are described in any one of Examples 11 to 16, further wherein the steps of the electrosensitivity detection are as follows:

[0140] 5.3.1) Add PBS to the unstimulated cell reagent for washing.

[0141] 5.3.2) Add Calcein AM / PI detection working solution to the washed cell reagent and incubate in the dark for a period of time to obtain detection solutions containing normal cells and detection solutions containing senescent cells.

[0142] 5.3.3) Using the i-th group of pulse electric field parameters I i Electrical stimulation was applied to the test solution containing normal cells in group i and the test solution containing senescent cells in group i, and the fluorescence changes of the cells were recorded in real time using a fluorescence microscope.

[0143] 5.3.4) Compare the fluorescence dissipation of normal cells and senescent cells and the PI content in cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that cause rapid fluorescence dissipation in senescent cells and high PI content in normal cells.

[0144] Cells with good growth and a density of 70-80% were collected and prepared into a cell suspension (5×10⁻⁶). 6 cells / mL), at 5×10 5 Cells / wells were seeded into 35 mm diameter laser confocal dishes and cultured for 24 h. The original culture medium was removed, and the cells were slowly washed twice with PBS. 1 mL of Calcein AM / PI detection working solution was added to each dish, and the cells were incubated at 37°C in the dark for 30 min. The laser confocal dishes were then placed on the stage of a laser confocal microscope. Using the optimal parameters from the previous experiment, an electric field strength of 1000 V / cm, a pulse width of 100 μs, and 8 pulses were selected for pulsed electrotherapy of the cells. The fluorescence changes of the cells were observed and recorded in real time under a fluorescence microscope during the stimulation process. Figure 7 As shown, observations revealed that senescent cells were more sensitive to pulsed electric fields and their fluorescence dissipated too quickly, while the fluorescence of normal cells was concentrated inside the cell. After reversible electroporation, normal cells showed stronger activity and a large amount of PI entered the cell.

[0145] Example 18:

[0146] An apparatus for applying the method of any one of embodiments 11 to 17 includes an electrode pair and a pulse generator.

[0147] The electrodes are used to electrically stimulate the reagents used to treat the cells.

[0148] The pulse generator is used to generate microsecond pulse electric fields and transmit them to the electrode pair.

[0149] Example 19:

[0150] A microsecond pulse electric field parameter selection device for killing senescent cells, the main technical contents of which are described in Example 18, further wherein the electrode cup includes a positive electrode and a negative electrode.

[0151] The positive and negative electrodes are placed opposite each other.

[0152] The reagent for the cells to be treated is placed between the positive and negative electrodes.

[0153] The electrodes used in this shoot are 35mm long × 5.4mm wide × 1.6mm high.

[0154] Example 20:

[0155] A microsecond pulse electric field parameter selection device for killing senescent cells, the main technical contents of which are described in any one of Embodiments 18 to 19, further wherein there is a gap between the positive electrode and the negative electrode.

[0156] Example 21:

[0157] See Figures 1 to 8 A method for selecting microsecond pulse electric field parameters for killing senescent cells, the main technical contents of which are as follows:

[0158] Cell culture: The mouse fibroblast cell line 3T3-L1 was used and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 1% penicillin and streptomycin (100 μg / mL) and 10% inactivated fetal bovine serum (FBS) in a constant temperature and humidity incubator at 37°C and 5% CO2. The cells were passaged by digestion with 0.25% trypsin (0.25% Trypsin-EDTA) throughout the culture process.

[0159] Cellular senescence model construction: 3T3-L1: Take cells with a density of 80%, discard the original culture medium, add culture medium supplemented with H2O2 (300uM / mL) to stimulate the cells for 4h, and then replace with fresh culture medium and culture for 24h.

[0160] Experiment on selective killing of senescent cells by pulsed electric field:

[0161] 1. Size measurement of normal and senescent 3T3 cells

[0162] Collect cells in good growth condition, centrifuge and resuspend them, then administer at 1×10⁻⁶. 6Cells / wells were seeded into 6-well plates and cultured for 24 hours. Cells from each well were then digested and collected into 1.5 mL EP tubes. After centrifugation and discarding the supernatant, 500 μL / tube of DiI (Cell Plasma Membrane Staining Kit with DiI) and Hoechst (Hoechst 33258) (blue fluorescent dye for nuclei) were added to stain the cell membrane and nucleus, respectively. The cells were incubated at 37°C in the dark for 20 min, centrifuged at 500 rpm for 5 min, and the staining solution was discarded. The cells were washed three times with PBS and then dropped onto a glass slide. A coverslip was placed on the slide, and the images were observed using a laser confocal microscope. Statistical analysis of the cell and nuclear dimensions of senescent and normal cells was performed using Image processing software. Figure 2 and Figure 3 The test results showed that the morphology and nuclear membrane size of normal 3T3 cells were smaller than those of senescent cells.

[0163] 2. Impedance testing of normal and senescent cells using 3T3.

[0164] Impedance spectroscopy of cell suspension was measured using a precision impedance analyzer and testing fixtures. Before measurement, the impedance analyzer underwent open-circuit calibration, short-circuit calibration, and load compensation. During measurement, 20 μL of cell suspension was placed in a self-made electrode holder, measuring 35 mm (L × W × H) and 1.6 mm (H). The central gold electrode measured 5 mm × 1.6 mm with a spacing of 2 mm. This holder was fixed to the bottom of a standard 35 mm culture dish. The impedance spectrum was measured in the frequency range of 20 Hz to 20 MHz using the impedance analyzer. A schematic diagram of the measurement system is shown below. Figure 8 As shown, data were read three times for each sample. The measured impedance spectroscopy data were then fitted using EISSpectrum Analyzer 1.0 software. Cell membrane electrical parameters were calculated using equivalent circuit inversion. The fitting results are shown below. Figure 4 As shown, there is a certain difference in the real part of the impedance values ​​between normal 3T3 cells and senescent cells.

[0165] 3. CCK8 cell viability assay

[0166] Collect cells in good growth condition with a density of 70-80%, discard the original culture medium, wash once with PBS, add trypsin and incubate in a cell culture incubator for 1 min to allow cell detachment. Add fresh culture medium to stop digestion, centrifuge the cell suspension at 800 rpm for 5 min, discard the supernatant, and resuspend in culture medium (3 × 10⁻⁶). 6Cells / mL). Cell suspensions were electrically stimulated using a self-developed pulse generator and electrode cups (2mm distance between positive and negative electrodes). 100µL of cell suspension was added to the electrode cups each time, and the entire unit was placed on the electrode base. Different electric field intensities and pulse widths were adjusted according to the pulse electric field parameters in Table 1. The cell suspensions were treated with a pulsed electric field at a frequency of 1Hz for 8 seconds. After treatment, the cells were transferred to 96-well plates and incubated for 3 hours. Then, 10µL of LCK-8 (Cell Counting Kit-8) reagent was added to each well, and the cells were incubated for another 2 hours. The absorbance at 450nm was then measured using a microplate reader. Data processing yielded... Figure 5 The cell activity graph shown shows that, through experiments with different pulsed electric field parameters, it was found that under an electric field strength of 1000 V / cm and a pulse width of 100 μs for 8 s, 3T3 normal cells and senescent cells could achieve the greatest difference in cell activity.

[0167] Table 1. Experimental electrophysiological parameters for selective killing of 3T3 senescent cells.

[0168]

[0169] Flow PI single staining after 4-pulse electric field treatment

[0170] Collect cells in good growth condition with a density of 70-80%, digest and centrifuge them, discard the supernatant, and resuspend the cells in propidium iodide (PI) staining working solution (1×10⁻⁶). 7 100 μL of cell suspension was added to a 2 mm electroporation cuvette each time, and the entire cuvette was placed in the electrode holder. Pulsed electric field treatment was performed according to the electric field parameters in Table 2. The treated cell suspension was then transferred to a 1.5 mL EP tube and incubated at 37°C in the dark for 30 min before flow cytometry analysis. The PI positivity rate of normal cells and senescent cells was statistically analyzed, and the results are as follows: Figure 6 As shown, under an electric field strength of 1000 V / cm and a pulse width of 100 μs for 8 s, the difference in PI positivity rate between normal 3T3 cells and senescent cells was maximized.

[0171] Table 2. Flow cytometry parameters of normal and senescent 3T3 cells.

[0172]

[0173] 5. Real-time imaging with PI fluorescence staining (to investigate the electrosensitivity of senescent 3T3 cells compared to normal cells)

[0174] Cells with good growth and a density of 70-80% were collected and prepared into a cell suspension (5×10⁻⁶). 6 cells / mL), at 5×10 5Cells / wells were seeded into 35 mm diameter laser confocal dishes and cultured for 24 h. The original culture medium was removed, and the cells were slowly washed twice with PBS. 1 mL of Calcein AM / PI detection working solution was added to each dish, and the cells were incubated at 37°C in the dark for 30 min. The laser confocal dishes were then placed on the stage of a laser confocal microscope. Using the optimal parameters from the previous experiment, a pulsed electrotherapy was performed with an electric field strength of 1000 V / cm, a pulse width of 100 μs, and 8 pulses. The fluorescence changes of the cells were observed and recorded in real time under a fluorescence microscope during the stimulation process. The electrodes used in this imaging were 35 mm long × 5.4 mm wide × 1.6 mm high, with a central gold electrode measuring 5 × 1.6 mm and a spacing of 2 mm. Figure 7 As shown, observations revealed that senescent cells were more sensitive to pulsed electric fields and their fluorescence dissipated too quickly, while the fluorescence of normal cells was concentrated inside the cell. After reversible electroporation, normal cells showed stronger activity and a large amount of PI entered the cell.

Claims

1. A method for selecting microsecond pulse electric field parameters for killing senescent cells, characterized in that, Includes the following steps: 1) Obtain biological tissue cells and perform cell culture to obtain n sets of cell reagents containing normal cells and n sets of cell reagents containing senescent cells, where n is a positive integer; 2) Set n sets of pulse electric field parameters as follows: (1) In the formula, is the pulse electric field parameter matrix; Let be the i-th group of pulse electric field parameters, i=1,2,...,n, and let be the j-th parameter of the i-th group of pulse electric field parameters, j=1,2,...,m, where m is a positive integer; The pulse electric field parameters include pulse amplitude, electric field strength, pulse width, and pulse count; The pulse amplitude range is 100V-400V; The electric field strength ranges from 500 V / cm to 2000 V / cm; The pulse width ranges from 50µs to 200µs; 3) Place the cell reagent containing normal cells in the i-th group between the electrode pairs, and set the parameters of the pulse generator to the pulse electric field parameters of the i-th group; The pulse generator transmits a pulse signal to the electrode pair, causing the electrode pair to generate electrical stimulation on the i-th group of cellular reagents containing normal cells; Place the cellular reagent containing senescent cells in the i-th group between the electrode pairs, and set the parameters of the pulse generator to the pulse electric field parameters of the i-th group; The pulse generator transmits a pulse signal to the electrode pair, causing the electrode pair to generate electrical stimulation on the i-th group of cellular reagents containing senescent cells, with i initially valued at 1. 4) Let i = i + 1, and repeat step 3) until all cell reagents have been electrically stimulated; 5) Perform cell activity detection, PI positivity detection, and electrosensitivity detection on each group of cells after electrical stimulation to determine the optimal pulsed electric field parameters.

2. The method for selecting microsecond pulse electric field parameters for killing senescent cells according to claim 1, characterized in that, The steps for detecting cell viability are as follows: 5.1.1) Add the electrically stimulated cell reagent to a 96-well plate and place the 96-well plate in a cell incubator for a period of time; 5.1.2) Add CCK-8 reagent to the 96-well plate containing cell reagents, and then place the 96-well plate in a cell incubator for a period of time; 5.1.3) Measure the absorbance of the cultured cells using an enzyme-linked immunosorbent assay (ELISA) reader; 5.1.4) By comparing the absorbance values ​​of normal cells and senescent cells under the same set of pulsed electric field parameters, the pulsed electric field parameters that yield the maximum difference in cell activity between normal cells and senescent cells are obtained.

3. The method for selecting microsecond pulse electric field parameters for killing senescent cells according to claim 1, characterized in that the step of detecting the PI positivity rate is as follows: 5.2.1) Add PI staining working solution to the cell reagent that has not been electrically stimulated to resuspend the cells, and obtain cell suspensions containing normal cells and cell suspensions containing senescent cells; 5.2.2) The cell suspension containing normal cells in group i and the cell suspension containing senescent cells in group i are electrically stimulated using the pulse electric field parameters of group i. 5.2.3) The cell suspension after electrical stimulation was incubated in the dark for a period of time, and the cell suspension after incubation in the dark was detected by flow cytometry to obtain the PI positivity rate of the cells; 5.2.4) Compare the PI positivity rates of normal cells and senescent cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that maximize the difference in PI positivity rates between normal cells and senescent cells.

4. The method for selecting microsecond pulse electric field parameters for killing senescent cells according to claim 1, characterized in that, The steps for the electrosensitivity detection are as follows: 5.3.1) Wash the cells with PBS that have not been electrically stimulated; 5.3.2) Add Calcein AM / PI detection working solution to the washed cell reagent and incubate in the dark for a period of time to obtain detection solutions containing normal cells and detection solutions containing senescent cells; 5.3.3) The i-th group of pulsed electric field parameters were used to electrically stimulate the i-th group of detection solutions containing normal cells and the i-th group of detection solutions containing senescent cells, and the fluorescence change process of the cells was recorded in real time by fluorescence microscopy. 5.3.4) Compare the fluorescence dissipation of normal cells and senescent cells and the PI content in cells under the same set of pulsed electric field parameters to obtain the pulsed electric field parameters that cause senescent cells to dissipate fluorescence quickly and normal cells to have a high PI content.

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