Electro-transfection instrument special for low-damage electro-transfection suitable for NK cells and T cells and preparation method of electro-transfection instrument

Abstract

The invention provides a low-damage electrotransfection special electrotransfection instrument suitable for NK cells and T cells, an electrotransfection instrument body is provided with an electrotransfection box, and an inner cavity of the electrotransfection box is provided with a fixing groove; an electric revolving cup is arranged on the fixing groove, the electric revolving cup comprises a cup cover, a cup body and an electrode, and the cup body is of a cavity structure; the electrode comprises an upper arc-shaped electrode and a lower arc-shaped electrode, the upper arc-shaped electrode is arranged on the cup cover, and the lower arc-shaped electrode is arranged in the bottom end of the electric revolving cup body; the cup cover of the electric revolving cup is also provided with a knob adjusting device which is elastically connected with the middle part of the upper arc-shaped electrode; and the knob adjusting device is used for adjusting the curvature of the upper arc-shaped electrode. The problem that the electrotransfection rate and the cell survival rate are reduced due to uneven electric field distribution is solved, the apoptosis rate of immune cells after electrotransfection can be controlled to be smaller than or equal to 10%, the cell activity is effectively reserved, and the method can be widely applied to the fields of immune cell gene modification and tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of cell biology experimental equipment, specifically to a low-damage electroporation instrument for NK cells and T cells and its preparation method. Background Technology

[0002] Traditional cell electroporators suffer from uneven electric field distribution, affecting the effectiveness of cell electrotransfection. The electrode cup design of traditional cell electroporators is flawed; the cell suspension exhibits a concave surface (higher at the edges, lower in the center) due to surface tension, resulting in uneven contact between the electrode edges and the liquid surface. The lower resistance at the edges leads to insufficient voltage, hindering cell electrotransfection, while the higher resistance at the center results in excessive voltage, potentially killing cells. When setting the voltage for the electroporator, increasing the voltage to allow cells at the edges to perforate increases cell death at the concave center. Conversely, decreasing the voltage to reduce cell death prevents cells at the edges from perforating, reducing electrotransfection efficiency. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a low-damage electroporation device for NK cells and T cells and its preparation method.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a low-damage electroporation device for NK cells and T cells, wherein the main body of the electroporation device is provided with an electroporation box, and the inner cavity of the electroporation box is provided with a fixing groove;

[0005] An electric rotating cup is configured on the fixed groove. The electric rotating cup includes a cup lid, a cup body and an electrode. The cup body has a cavity structure. The electrode includes an upper arc-shaped electrode and a lower arc-shaped electrode. The upper arc-shaped electrode is disposed on the cup lid, and the lower arc-shaped electrode is disposed inside the bottom end of the electric rotating cup body. The electric rotary cup has electrode connection parts at both ends along the diameter direction of the cup lid. The electrode connection parts have built-in slots. The two ends of the upper arc-shaped electrode are slidably connected to the electrode connection parts of the cup lid. The length of the upper arc-shaped electrode exceeds the distance between the two electrode connection parts of the cup lid. The ends of the upper arc-shaped electrode have a spare length to accommodate in the slots. The lid of the electric rotating cup is also equipped with a knob adjustment device that is elastically connected to the middle of the upper arc-shaped electrode. The knob adjustment device adjusts the curvature of the upper arc-shaped electrode.

[0006] The electrodes of the electroporation cup in the aforementioned low-damage electroporation device for NK and T cells are configured as an upper arc-shaped electrode and a lower arc-shaped electrode. The two ends of the upper arc-shaped electrode are slidably connected to the electrode connection parts of the cup lid. The length of the upper arc-shaped electrode exceeds the distance between the two electrode connection parts of the cup lid, and the ends of the upper arc-shaped electrode have a margin to accommodate in the slots. This length, exceeding the distance between the two electrode connection parts of the cup lid and having a margin to accommodate in the slots, allows for adjustment of the curvature of the upper arc-shaped electrode. When the curvature is increased, the width of the electroporation cup becomes uniform, the length of the upper arc-shaped electrode protruding from the cup increases, and the length of the upper arc-shaped electrode with margins accommodated in the slots decreases.

[0007] The curvature of the upper arc-shaped electrode is adjusted via a knob to match the concave curvature of the cells to be tested. This better avoids the problem of the cell suspension exhibiting a "concave liquid surface" (higher at the edges and lower at the center) due to surface tension. This ensures that the electrode distance between the edges and the center of the cell suspension undergoing electroporation is more uniform, resulting in a more even electric field distribution. This overcomes the technical problem that when setting the voltage for the electroporator, increasing the voltage to allow edge cells to perforate leads to increased cell death at the concave center, while decreasing the voltage to reduce cell death prevents edge cells from perforating, thus reducing electroporation efficiency. This invention achieves a better electroporation rate and reduces cell death. It can control the apoptosis rate of immune cells after electroporation to ≤10%, effectively preserving cell viability and can be widely applied in the fields of immune cell gene modification and tumor immunotherapy.

[0008] Preferably, the upper arc-shaped electrode has clamps at both ends that are perpendicular to the ends of the arc-shaped electrode, and the slot has a slot opening, with the length of the clamps being greater than the width of the slot opening.

[0009] The upper arc-shaped electrode has clips at both ends that are perpendicular to the ends of the arc-shaped electrode to prevent slippage during the adjustment of the arc (curvature) of the upper arc-shaped electrode.

[0010] Preferably, the knob adjustment device includes an adjustment knob, a knob scale, and the adjustment knob has a screw and a hand-tightening part fixed on the screw. The screw of the adjustment knob is threadedly installed with the lid of the electric rotating cup, and the bottom of the screw of the adjustment knob is exposed and abuts against the midpoint of the upper arc-shaped electrode. The upper arc-shaped electrode is made of an elastic conductive material.

[0011] The electroporation cup of the aforementioned low-damage electroporator for NK and T cells uses a screw and thread to adjust the exposed length of the screw bottom. When the exposed length of the screw extends, it compresses the upper arc-shaped electrode, causing deformation and increasing the curvature of the concave surface of the upper arc-shaped electrode. When the exposed length of the screw decreases, the force of the screw compressing the arc-shaped electrode decreases, and due to elastic recovery, the two ends of the upper arc-shaped electrode retract into the slot, reducing the curvature.

[0012] Preferably, the inner cavity of the electro-transfer box has n×n fixed slots arranged in a matrix, where n is a natural number not less than 2.

[0013] The aforementioned low-damage electroporation instrument, suitable for NK cells and T cells, can achieve multi-channel electroporation.

[0014] Preferably, the width of the electrospinning cup is 2-4 mm, the cup body is cubic, the electrode is an aluminum sheet electrode, and the material of the electrospinning cup is polycarbonate or quartz.

[0015] Preferably, the electrostatic oscillator includes a housing and a sealing cover. The housing has a square box structure with an opening at the top. The sealing cover is fitted to the opening at the top of the housing, and a sealing sleeve is installed on the bottom surface of the sealing cover.

[0016] This invention also provides a method of using any of the above-mentioned low-damage electroporation devices suitable for NK cells and T cells, the method of use including the following steps: (1) Prepare the cell solution to be treated, take the cell solution to test the surface tension of the cell solution, and calculate the curvature or radius of curvature of the cell solution according to the Yang-Laplace formula; (2) Calculate the height of the concave surface of the cell fluid in the electric rotating cup based on the curvature or radius of curvature of the cell fluid and the width of the electric rotating cup in step (1); (3) Add the prepared cell solution to the electroporation cup, cover the electroporation cup with the cup lid, and adjust the adjustment knob on the cup lid until the height of the concave surface of the upper arc electrode is consistent with the calculated height of the concave surface of the cell solution in the electroporation cup. (4) Turn on the electroporation apparatus to perform electroporation.

[0017] Preferably, the method for measuring the surface tension of cell fluid is the capillary rise method, which measures the surface tension, liquid density, capillary inner diameter, liquid level rise height, and contact angle of the cell fluid.

[0018] The present invention also provides an electro-rotating cup, which includes a cup lid, a cup body and electrodes, wherein the cup body has a cavity structure; The electrode includes an upper arc-shaped electrode and a lower arc-shaped electrode. The upper arc-shaped electrode is disposed on the cup lid, and the lower arc-shaped electrode is disposed inside the bottom end of the electric rotating cup body. The electric rotary cup has electrode connection parts at both ends along the diameter direction of the cup lid. The electrode connection parts have built-in slots. The two ends of the upper arc-shaped electrode are slidably connected to the electrode connection parts of the cup lid. The length of the upper arc-shaped electrode exceeds the distance between the two electrode connection parts of the cup lid. The ends of the upper arc-shaped electrode have a spare length to accommodate in the slots. The lid of the electric rotating cup is also equipped with a knob adjustment device that is elastically connected to the middle of the upper arc-shaped electrode. The knob adjustment device adjusts the curvature of the upper arc-shaped electrode.

[0019] Preferably, the upper arc-shaped electrode has clamps at both ends that are perpendicular to the ends of the arc-shaped electrode, and the slot has a slot opening, with the length of the clamps being greater than the width of the slot opening.

[0020] Preferably, the knob adjustment device includes an adjustment knob, a knob scale, and the adjustment knob has a screw and a hand-tightening part fixed on the screw. The screw of the adjustment knob is threadedly installed with the lid of the electric rotating cup, and the bottom of the screw of the adjustment knob is exposed and abuts against the midpoint of the upper arc-shaped electrode. The upper arc-shaped electrode is made of an elastic conductive material.

[0021] Preferably, the width of the electrospinning cup is 2-4 mm, the cup body is cubic, the electrode is an aluminum sheet electrode, and the material of the electrospinning cup is polycarbonate or quartz.

[0022] The beneficial effects of this invention are as follows: This invention provides a low-damage electroporation instrument for NK cells and T cells, and its preparation method. The electrodes of the electroporation cup in this low-damage electroporation instrument for NK cells and T cells are configured as an upper arc-shaped electrode and a lower arc-shaped electrode. The two ends of the upper arc-shaped electrode are slidably connected to the electrode connection parts of the cup lid. The length of the upper arc-shaped electrode exceeds the distance between the two electrode connection parts of the cup lid, and the ends of the upper arc-shaped electrode have a margin to accommodate in the slot. The fact that the length of the upper arc-shaped electrode exceeds the distance between the two electrode connection parts of the cup lid and that the ends of the upper arc-shaped electrode have a margin to accommodate in the slot allows for adjustment of the curvature of the upper arc-shaped electrode. When the curvature is increased, the width of the electroporation cup becomes uniform, the length of the upper arc-shaped electrode protruding from the cup increases, and the length of the upper arc-shaped electrode with a margin to accommodate in the slot becomes shorter.

[0023] The curvature of the upper arc-shaped electrode is adjusted via a knob to match the concave curvature of the cells to be tested. This better avoids the problem of the cell suspension exhibiting a "concave liquid surface" (higher at the edges and lower at the center) due to surface tension. This ensures that the electrode distance between the edges and the center of the cell suspension undergoing electroporation is more uniform, resulting in a more even electric field distribution. This overcomes the technical problem that when setting the voltage for the electroporator, increasing the voltage to allow edge cells to perforate leads to increased cell death at the concave center, while decreasing the voltage to reduce cell death prevents edge cells from perforating, thus reducing electroporation efficiency. This invention achieves a better electroporation rate and reduces cell death. It can control the apoptosis rate of immune cells after electroporation to ≤10%, effectively preserving cell viability and can be widely applied in the fields of immune cell gene modification and tumor immunotherapy. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of a low-damage electroporation device for NK cells and T cells according to an embodiment of the present invention.

[0025] Figure 2 This is a schematic diagram of the electroporation cup of a low-damage electroporation device for NK cells and T cells according to an embodiment of the present invention.

[0026] Figure 3 This is a schematic diagram of the cup lid and upper arc-shaped electrode of an electroporation cup for a low-damage electroporation device suitable for NK cells and T cells, according to an embodiment of the present invention.

[0027] The components include: 1. Electro-polarizer body; 2. Electro-polarizer box; 3. Fixing groove; 4. Electro-polarizer sealing cover; 5. Electro-polarizer outer shell; 6. Electro-polarizer cup; 7. Cup body; 8. Cup lid; 9. Upper arc-shaped electrode; 10. Lower arc-shaped electrode; 11. Knob adjustment device; 12. Hand-tightening part of the adjustment knob fixed on the screw; 13. Screw of the adjustment knob. Detailed Implementation

[0028] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0029] Example 1 As an embodiment of the present invention, a low-damage electroporation device suitable for NK cells and T cells is provided, such as... Figure 1 , such as 2, Figure 3 As shown, the main body (1) of the electro-spinning instrument is provided with an electro-spinning box (2), and the inner cavity of the electro-spinning box is provided with a fixing groove (3). An electric rotating cup (6) is disposed on the fixed groove (3). The electric rotating cup (6) includes a cup lid (8), a cup body (7) and electrodes (9, 10). The cup body (7) has a cavity structure. The electrodes (9, 10) include an upper arc-shaped electrode (9) and a lower arc-shaped electrode (10). The upper arc-shaped electrode (9) is disposed on the cup lid, and the lower arc-shaped electrode (10) is disposed inside the bottom end of the electric rotating cup body. The lid (8) of the electric rotating cup (6) is provided with electrode connection parts at both ends along the diameter direction of the lid. The electrode connection parts have built-in slots. The two ends of the upper arc electrode (9) are slidably connected to the electrode connection parts of the lid (8). The length of the upper arc electrode (9) exceeds the distance between the two electrode connection parts of the lid (8). The ends of the upper arc electrode (9) have a spare length to accommodate in the slots. The lid (8) of the electric rotating cup (6) is also provided with a knob adjustment device (11) that is elastically connected to the middle of the upper arc electrode. The knob adjustment device (11) adjusts the curvature of the upper arc electrode (9).

[0030] Furthermore, the upper arc-shaped electrode (9) is provided with clips perpendicular to the ends of the arc-shaped electrode at both ends, and the slot has a slot opening, the length of the clip being greater than the width of the slot opening of the slot.

[0031] The upper arc-shaped electrode has clips at both ends that are perpendicular to the ends of the arc-shaped electrode to prevent slippage during the adjustment of the arc (curvature) of the upper arc-shaped electrode.

[0032] Furthermore, the knob adjustment device (11) includes an adjustment knob (12, 13), a knob scale, and the adjustment knob has a screw (13) and a hand-tightening part (12) fixed on the screw. The screw (13) of the adjustment knob is threadedly installed with the cup lid (8) of the electric rotating cup, and the bottom of the screw of the adjustment knob is exposed and abuts against the midpoint of the upper arc electrode. The upper arc electrode is made of an elastic conductive material.

[0033] The electroporation cup of the aforementioned low-damage electroporator for NK and T cells uses a screw and thread to adjust the exposed length of the screw bottom. When the exposed length of the screw extends, it compresses the upper arc-shaped electrode, causing deformation and increasing the curvature of the concave surface of the upper arc-shaped electrode. When the exposed length of the screw decreases, the force of the screw compressing the arc-shaped electrode decreases, and due to elastic recovery, the two ends of the upper arc-shaped electrode retract into the slot, reducing the curvature.

[0034] Furthermore, the inner cavity of the electric transfer box (2) is provided with n×n fixed slots arranged in a matrix, where n is a natural number not less than 2.

[0035] The aforementioned low-damage electroporation instrument, suitable for NK cells and T cells, can achieve multi-channel electroporation.

[0036] Furthermore, the width of the electro-spinning cup (6) is 2~4mm, the cup body of the electro-spinning cup is cubic, the electrode is an aluminum sheet electrode, and the material of the electro-spinning cup is polycarbonate or quartz.

[0037] Furthermore, the electrostatic generator includes a housing (5) and a sealing cover (4). The housing adopts a square box structure and has an opening at the top. The sealing cover is matched with the opening at the top of the housing and a sealing sleeve is installed on the bottom surface of the sealing cover.

[0038] This embodiment describes the method of using a low-damage electroporation device specifically designed for NK cells and T cells. The method includes the following steps: (1) Prepare the cell solution to be treated, take the cell solution to test the surface tension of the cell solution, and calculate the curvature or radius of curvature of the cell solution according to the Yang-Laplace method; the method for taking the cell solution to test the surface tension of the cell solution is the capillary rise method, and the surface tension, liquid density, capillary inner diameter, liquid rise height and contact angle of the cell solution are measured. (2) Calculate the height of the concave surface of the cell fluid in the electric rotating cup based on the curvature or radius of curvature of the cell fluid and the width of the electric rotating cup in step (1); (3) Add the prepared cell solution to the electroporation cup, cover the electroporation cup with the cup lid, and adjust the adjustment knob on the cup lid until the height of the concave surface of the upper arc electrode is consistent with the calculated height of the concave surface of the cell solution in the electroporation cup. (4) Turn on the electroporation apparatus to perform electroporation.

[0039] Example 2 As an embodiment of the present invention, it is applicable to an electric rotary cup, such as Figure 2 , Figure 3 As shown, the electro-rotating cup (6) includes a cup lid (8), a cup body (7), and electrodes (9, 10), with the cup body (7) having a cavity structure; The electrode includes an upper arc-shaped electrode (9) and a lower arc-shaped electrode (10). The upper arc-shaped electrode (9) is disposed on the cup lid (8), and the lower arc-shaped electrode (10) is disposed inside the bottom end of the electric rotating cup body (7). The electric rotary cup has electrode connection parts at both ends along the diameter direction of the cup lid. The electrode connection parts have built-in slots. The two ends of the upper arc-shaped electrode are slidably connected to the electrode connection parts of the cup lid. The length of the upper arc-shaped electrode exceeds the distance between the two electrode connection parts of the cup lid. The ends of the upper arc-shaped electrode have a spare length to accommodate in the slots. The lid of the electric rotating cup is also provided with a knob adjustment device (11) that is elastically connected to the middle of the upper arc-shaped electrode. The knob adjustment device adjusts the curvature of the upper arc-shaped electrode.

[0040] Furthermore, the upper arc-shaped electrode has clips at both ends that are perpendicular to the ends of the arc-shaped electrode, and the slot has a slot opening, with the length of the clips being greater than the width of the slot opening.

[0041] Furthermore, the knob adjustment device (11) includes an adjustment knob (12, 13), a knob scale, and the adjustment knob has a screw (13) and a hand-tightening part (12) fixed on the screw. The screw of the adjustment knob is threadedly installed with the lid of the electric rotating cup, and the bottom of the screw of the adjustment knob is exposed and abuts against the midpoint of the upper arc-shaped electrode. The material of the upper arc-shaped electrode is an elastic conductive material.

[0042] Furthermore, the width of the electro-rotating cup is 2-4 mm, the cup body is cubic, the electrode is an aluminum sheet electrode, and the material of the electro-rotating cup is polycarbonate or quartz.

[0043] Experimental methods: (I) Main experimental equipment 1. Based on the size specifications of the electroporation cup of the Thermo Fisher Neon NxT electroporation system, an electroporation cup as described in Example 2 was custom-made for electroporation experiments.

[0044] (II) Electroporation Experiment 1. Primary T cells: Purified from human peripheral blood mononuclear cells (PBMCs) using CD3 / CD28 magnetic beads; (providing a channel for cell acquisition) 2. Activate the cultivation system: Basic culture medium: RPMI-1640 (containing 2mM L-glutamine, 100U / mL penicillin, and 100μg / mL streptomycin) + 10%-15% fetal bovine serum (FBS, which must be mycoplasma-free); Activator: Add CD3 / CD28 activation magnetic beads to each well (magnetic beads: cells = 1:1); Cytokines: Add IL-2 (final concentration 300-500 IU / mL, to maintain T cell survival and proliferation). Culture conditions: 37℃, 5% CO2 constant temperature incubator, activation for 48h (under the microscope, the cells show clustered growth, and the viability is >90%, which is sufficient for electroporation).

[0045] 3. Electroporation reagents Table 1 Electroporation reagents

[0046] 4. Electroporation experiment (1) Resuspend the cells in 100 μL of electroporation buffer, mix 10 μL of the cell suspension with 10 μL of trypan blue, and count the cells using a hemocytometer to ensure a cell concentration of 1 × 10⁻⁶. 6 -5×10 6 cells / 100μL (too low a concentration will reduce transfection efficiency, too high a concentration will easily lead to cell accumulation and damage), viability must be >90%.

[0047] (2) Prepare a mixed system of "cell-nucleic acid-electroporation buffer", with 90 μL of electroporation buffer and 1×10 6 Cells, 5 μg of exogenous nucleic acid, adjusted to 100 μL.

[0048] (3) Electro-polarizer operation. The electro-polarizer operation is performed in three parallel operations, and the average value is taken in subsequent tests.

[0049] The experimental group pre-sampled cell sap to detect its surface tension and calculated the curvature or radius of curvature of the cell sap using the Yang-Laplace method. The method for detecting the surface tension of the cell sap was the capillary rise method, which measured the surface tension, liquid density, capillary inner diameter, liquid level rise height, and contact angle. The concave height of the cell sap in the electro-rotating cup was calculated based on the curvature or radius of curvature of the cell sap and the width of the electro-rotating cup. The adjustment knob on the cup lid was adjusted until the concave height of the upper arc electrode was consistent with the calculated concave height of the cell sap in the electro-rotating cup.

[0050] In the control group, the height of the concave surface of the upper arc electrode was not adjusted, and the adjustment knob and screw were removed by rotation.

[0051] (iii) Detection of electroporation rate and cell viability.

[0052] The test samples are cell sap from the experimental group and the control group after electroporation.

[0053] 1. Resume culture: Add 1 mL of preheated T cell culture medium (containing IL-2300 IU / mL, without activating magnetic beads) to each well and incubate at 37°C in a 5% CO2 incubator (avoid shaking the culture plate for the first 24 hours to allow the cells to fully repair themselves). After 24 hours of incubation, replace 50% of the culture medium (to remove any possible residual nucleic acid toxicity substances) and continue incubation for another 48 hours.

[0054] 2. Transfection efficiency testing: siRNA or gene editing components: Transfection rate was calculated by detecting the mRNA expression level of the target gene using qPCR; 3. Cell viability retest: Viability was determined by trypan blue staining 48 hours after transfection.

[0055] The experimental results are shown in Table 2: Table 2 Electroporation rate and cell viability The above verification experiments show that adjusting the curvature of the upper arc electrode using the knob adjustment device to match the concave curvature of the cells to be tested better avoids the problem of the cell suspension forming a "concave liquid surface" (higher at the edges and lower at the center) due to surface tension. This makes the electrode distance between the edges and the center of the cell suspension to be electrotransfected more consistent, resulting in a more uniform electric field distribution. This overcomes the technical problem that when setting the voltage for the electrotransfer instrument, increasing the voltage to allow edge cells to perforate leads to increased cell death at the concave center, while decreasing the voltage to reduce cell death results in edge cells not being able to perforate, thus reducing the efficiency of electrotransfection. This invention achieves a better electrotransfection rate and reduces cell death. The invention can control the apoptosis rate of immune cells after electrotransfection to ≤10%, effectively preserving cell viability, and can be widely applied in the fields of immune cell gene modification and tumor immunotherapy.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A low-damage electroporation instrument specifically designed for NK cells and T cells, characterized in that: The main body of the electro-polarizer is provided with an electro-polarizer box, and the inner cavity of the electro-polarizer box is provided with a fixing groove; An electric rotating cup is configured on the fixed groove. The electric rotating cup includes a cup lid, a cup body and an electrode. The cup body has a cavity structure. The electrode includes an upper arc-shaped electrode and a lower arc-shaped electrode. The upper arc-shaped electrode is disposed on the cup lid, and the lower arc-shaped electrode is disposed inside the bottom end of the electric rotating cup body. The electric rotary cup has electrode connection parts at both ends along the diameter direction of the cup lid. The electrode connection parts have built-in slots. The two ends of the upper arc-shaped electrode are slidably connected to the electrode connection parts of the cup lid. The length of the upper arc-shaped electrode exceeds the distance between the two electrode connection parts of the cup lid. The ends of the upper arc-shaped electrode have a spare length to accommodate in the slots. The lid of the electric rotating cup is also equipped with a knob adjustment device that is elastically connected to the middle of the upper arc-shaped electrode. The knob adjustment device adjusts the curvature of the upper arc-shaped electrode.

2. The low-damage electroporation apparatus for NK cells and T cells according to claim 1, characterized in that, The upper arc-shaped electrode has clips at both ends that are perpendicular to the ends of the arc-shaped electrode. The slot has a slot opening, and the length of the clip is greater than the width of the slot opening.

3. The low-damage electroporation apparatus for NK cells and T cells according to claim 1, characterized in that, The knob adjustment device includes an adjustment knob, a knob scale, and the adjustment knob has a screw and a hand-tightening part fixed on the screw. The screw of the adjustment knob is threadedly installed with the lid of the electric rotating cup, and the bottom of the screw of the adjustment knob is exposed and abuts against the midpoint of the upper arc-shaped electrode. The upper arc-shaped electrode is made of an elastic conductive material.

4. The low-damage electroporation apparatus for NK cells and T cells according to claim 1, characterized in that, The inner cavity of the electro-rotator box has n×n fixed slots arranged in a matrix, where n is a natural number not less than 2.

5. The low-damage electroporation apparatus for NK cells and T cells according to claim 1, characterized in that, The width of the electro-rotating cup is 2~4mm, the cup body is cubic, the electrode is an aluminum sheet electrode, and the material of the electro-rotating cup is polycarbonate or quartz.

6. The low-damage electroporation apparatus for NK cells and T cells according to claim 1, characterized in that, The electrostatic precipitator includes an outer casing and a sealing cover. The outer casing adopts a square box structure with an opening at the top. The sealing cover is fitted to the opening at the top of the outer casing, and a sealing sleeve is installed on the bottom surface of the sealing cover.

7. The method of using the low-damage electroporation apparatus for NK cells and T cells as described in any one of claims 1-6, characterized in that, The usage method includes the following steps: (1) Prepare the cell solution to be treated, take the cell solution to test the surface tension of the cell solution, and calculate the curvature or radius of curvature of the cell solution according to the Yang-Laplace formula; (2) Calculate the height of the concave surface of the cell fluid in the electric rotating cup based on the curvature or radius of curvature of the cell fluid and the width of the electric rotating cup in step (1); (3) Add the prepared cell solution to the electroporation cup, cover the electroporation cup with the cup lid, and adjust the adjustment knob on the cup lid until the height of the concave surface of the upper arc electrode is consistent with the calculated height of the concave surface of the cell solution in the electroporation cup. (4) Turn on the electroporation apparatus to perform electroporation.

8. The method of using the low-damage electroporation apparatus for NK cells and T cells according to claim 7, characterized in that, The method for measuring the surface tension of cell sap is the capillary rise method, which measures the surface tension, liquid density, capillary inner diameter, liquid level rise height, and contact angle of the cell sap.

9. An electric rotary cup, characterized in that, The electro-rotating cup includes a cup lid, a cup body, and electrodes, with the cup body having a cavity structure. The electrode includes an upper arc-shaped electrode and a lower arc-shaped electrode. The upper arc-shaped electrode is disposed on the cup lid, and the lower arc-shaped electrode is disposed inside the bottom end of the electric rotating cup body. The electric rotary cup has electrode connection parts at both ends along the diameter direction of the cup lid. The electrode connection parts have built-in slots. The two ends of the upper arc-shaped electrode are slidably connected to the electrode connection parts of the cup lid. The length of the upper arc-shaped electrode exceeds the distance between the two electrode connection parts of the cup lid. The ends of the upper arc-shaped electrode have a spare length to accommodate in the slots. The lid of the electric rotating cup is also equipped with a knob adjustment device that is elastically connected to the middle of the upper arc-shaped electrode. The knob adjustment device adjusts the curvature of the upper arc-shaped electrode.

10. The electro-rotator according to claim 9, characterized in that, The upper arc-shaped electrode has clips at both ends that are perpendicular to the ends of the arc-shaped electrode, and the slot has a slot opening. The length of the clip is greater than the width of the slot opening. The knob adjustment device includes an adjustment knob, a knob scale, and the adjustment knob has a screw and a hand-tightening part fixed on the screw. The screw of the adjustment knob is threadedly installed with the lid of the electric rotary cup, and the bottom of the screw of the adjustment knob is exposed and abuts against the midpoint of the upper arc electrode. The width of the electro-rotating cup is 2-4 mm, the cup body is cubic, the electrode is an aluminum sheet electrode, and the material of the electro-rotating cup is polycarbonate or quartz.

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