High-precision egg cell / early embryo electric activation device and method

Abstract

The invention discloses a high-precision egg cell / early embryo electric activation device. The device comprises a power supply; the electrical activation chip comprises a base and an electrode layer, the base is provided with a plurality of electrical activation grooves arranged at intervals and two opposite electrode pits communicated with the electrical activation grooves, the electrode layer comprises two electrode assemblies, each electrode assembly comprises an electrode part and a conductive part connected to the electrode part, the electrode parts are arranged in the electrical activation grooves, and the conductive parts are arranged in the electrical activation grooves. The two electrode parts and the electric activation groove form an electric activation chamber, and the conductive parts are arranged in the corresponding electrode pits; and the wire assembly is used for connecting the power supply with the two conductive parts of the electrically activated chip. The invention also discloses a high-precision egg cell / early embryo electric activation method. According to the invention, the accuracy and the stability of egg cell / early embryo electric activation are greatly improved, a stable and uniform electric field can be provided under a low-voltage condition, the voltage demand is reduced, the electric damage to cell membranes is reduced, and the cell survival rate and the activation efficiency are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical fields of cell electrostimulation and embryo engineering, and particularly to a high-precision electroactivation device and method for oocytes / early embryos. Background Art

[0002] In embryo engineering technology, the activation of early embryos is a key step in initiating embryo development and is usually performed during the micromanipulation process. Mammalian oocytes are arrested at metaphase II of meiosis before fertilization, and their activation is achieved by the entry of sperm, which induces an increase in intracellular calcium concentration. Compared with chemical activation, electroactivation has become the mainstream technology because of its controllable parameters, high activation rate, simple operation, and no chemical toxicity. It forms temporary pores on the cell membrane through a transient high voltage, enhances the permeability, and realizes the exchange of ions and small molecule substances inside and outside the cell. In the presence of external calcium ions, electroactivation simulates the dynamic changes of calcium oscillations during natural fertilization, optimizes the activation process by adjusting the electro-pulse parameters, enables the oocytes to replicate the natural fertilization process physiologically and morphologically, and promotes normal development. However, the existing technology still has problems such as low survival rate and unstable activation effect.

[0003] Currently, traditional electroactivation systems usually adopt parallel plate electrode devices to generate an approximately uniform electric field in the cell suspension. However, since the time required for each region of the cell membrane to reach the transmembrane threshold potential is different, the distribution of membrane pores is uneven. In addition, the distance between the parallel plate electrodes is 1-10 mm, which is relatively large, and a high voltage of several hundred to several thousand volts needs to be applied, which is likely to cause irreparable damage to the cell membrane. At the same time, such devices usually focus on batch processing and are difficult to meet the needs of personalized and fine electroactivation of single cells. Coupled with the generation of bubbles, uneven electric field distribution, and unstable cell position and posture, the experimental uncertainty is further increased. Even under the same equipment and conditions, the activation effect may still fluctuate, resulting in low activation efficiency and survival rate. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a high-precision electroactivation device and method for oocytes / early embryos.

[0005] To achieve the above purpose, the technical solution provided by an embodiment of the present invention is as follows:

[0006] A high-precision electroactivation device for oocytes / early embryos, comprising:

[0007] A power supply for providing the electric energy required for electrostimulation of oocytes / early embryos;

[0008] The electrically activated chip includes a base and an electrode layer. The base is provided with a plurality of electrically activated grooves arranged at intervals and two electrode pits that communicate with the plurality of electrically activated grooves and are opposite to each other. The electrode layer includes two electrode assemblies. Each electrode assembly includes an electrode part and a conductive part connected to the electrode part. The electrode part is disposed in the electrically activated groove, and the two electrode parts and the electrically activated groove form an electrically activated chamber. The conductive part is disposed in the corresponding electrode pit;

[0009] A wire assembly for connecting the power supply to the two conductive parts of the electrically activated chip.

[0010] As a further improvement of the present invention, a plurality of microstructural pillars are disposed in the electrically activated groove. The plurality of microstructural pillars are distributed on a circumference centered on the center of the electrically activated groove, and the two electrode parts are disposed outside the plurality of microstructural pillars.

[0011] As a further improvement of the present invention, the microstructural pillars are cylindrical.

[0012] As a further improvement of the present invention, the number of the microstructural pillars is six. The six microstructural pillars are divided into a first group and a second group. The first group includes two first microstructural pillars, and the second group includes four second microstructural pillars. The diameter of the first microstructural pillar is greater than the diameter of the second microstructural pillar.

[0013] As a further improvement of the present invention, the depth of the electrically activated groove is greater than the depth of the electrode pit.

[0014] As a further improvement of the present invention, two baffles are disposed in the electrically activated groove, and the two electrode parts are located on both sides of the two baffles.

[0015] As a further improvement of the present invention, the electrode part is arc-shaped.

[0016] As a further improvement of the present invention, the electrode part and the conductive part are integrally formed and connected.

[0017] As a further improvement of the present invention, the electrode layer is fixed to the base by press-fitting or bonding.

[0018] A high-precision method for electrically activating oocytes / early embryos using the electrically activating device includes the following steps:

[0019] (1) Preparatory stage: Place the pre-treated oocytes / early embryos into the electrically activated chamber, and inject the electrically activating solution into the electrically activated chamber;

[0020] (2) Electrical activation stage of cells: Turn on the power supply, set the required electrical activation parameters, and perform electrical activation on the oocytes / early embryos. When the oocytes / early embryos are subjected to the electric field provided by the two electrode parts, the permeability of the cell membrane increases;

[0021] (3) Post-treatment: Take out the electrically activated oocytes / early embryos and place them in cell culture medium for cultivation.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) The present invention can form a uniform and stable transmembrane electric field on the surface of the egg membrane at a relatively low input voltage to achieve uniform expansion of the egg membrane, reduce the extreme excessive voltage in the local area of the egg membrane, and thus avoid damage to cell activity. This device needs to be able to reduce the input voltage and achieve uniform electrical stimulation of the oocyte membrane.

[0024] (2) The present invention can perform personalized and precise electrical activation on individual oocytes / early embryos, ensure uniform distribution of pores on the membrane, and avoid electrical damage caused by local over-concentration.

[0025] (3) The present invention realizes high-precision and high-efficiency electrical activation of oocytes / early embryos, simplifies the operation steps, significantly improves the experimental repeatability, activation efficiency and survival rate of oocytes / early embryos, and provides stable and reliable technical support for the high-efficiency electrical activation of oocytes / early embryos.

[0026] (4) The electrical activation groove and the two electrode parts together form a semi-closed electrical activation chamber, which is convenient for operators to quickly place and take out oocytes / early embryos, reduce their exposure time in the external environment, protect the activity of oocytes / early embryos and reduce environmental interference. Moreover, the semi-closed structure does not require complex sealing or disassembly, simplifies the operation process, improves the experimental efficiency, and at the same time ensures the accurate positioning of oocytes / early embryos in the electric field area, ensuring the safety and reliability of the experiment.

[0027] (5) The present invention has the characteristics of miniaturization and precision, high structural strength and good structural stability, improves the experimental reliability, and at the same time simplifies the manufacturing and assembly processes, which is conducive to high-efficiency mass production. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1Schematic diagram of the overall structure of the high-precision oocyte / early embryo electroactivation device according to the preferred embodiment of the present invention;

[0030] Figure 2 Pulse diagram required to be provided by the power supply of the high-precision oocyte / early embryo electroactivation device according to the preferred embodiment of the present invention;

[0031] Figure 3 Schematic diagram of the principle of the high-precision oocyte / early embryo electroactivation device according to the preferred embodiment of the present invention;

[0032] Figure 4 Three-dimensional view of the electroactivation chip according to the preferred embodiment of the present invention;

[0033] Figure 5 is Figure 4 Enlarged schematic diagram of A in

[0034] Figure 6 Exploded structure schematic diagram of the electroactivation chip according to the preferred embodiment of the present invention;

[0035] Figure 7 Top view of the electroactivation chip according to the preferred embodiment of the present invention;

[0036] Figure 8 Front view of the electroactivation chip according to the preferred embodiment of the present invention;

[0037] Figure 9 Side view of the electroactivation chip according to the preferred embodiment of the present invention;

[0038] Figure 10 Schematic diagram of the structure with electroactivation grooves and electrode pits provided on the base of the electroactivation chip according to the preferred embodiment of the present invention;

[0039] Figure 11 Partial enlarged view of the electroactivation groove and the electrode pit according to the preferred embodiment of the present invention;

[0040] Figure 12 Schematic diagram of the structure of the electrode layer of the electroactivation chip according to the preferred embodiment of the present invention;

[0041] Figure 13 Partial enlarged view of the electrode part and the conductive part according to the preferred embodiment of the present invention;

[0042] Figure 14 Schematic diagram of the arrangement of different microstructural struts;

[0043] Figure 15 Effect diagram of cell electroactivation by different arrangements of microstructural struts;

[0044] Figure 16Comparison diagram of the maximum transmembrane voltage and the maximum pore density on the cell membrane under different electrode shapes;

[0045] Figure 17 Schematic diagram of the transmembrane voltage distribution on the egg membrane of different electrode models;

[0046] Figure 18 Schematic diagram of the pore density distribution on the egg membrane of different electrode models;

[0047] Figure 19 Total transmembrane voltage and pore density on the egg membrane under different electrode shapes;

[0048] In the figure: 1. Power supply, 2. Electro-activation chip, 21. Base, 211. Electro-activation groove, 212. Electrode pit, 213. First microstructural pillar, 214. Second microstructural pillar, 215. Baffle, 22. Electrode layer, 221. Electrode assembly, 222. Electrode part, 223. Conductive part, 23. Electro-activation chamber, 3. Wire assembly, 31. Wire, 4. Oocyte / early embryo. Detailed implementation manners

[0049] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0050] Please refer to Figure 1 、 Figures 3 - 13, an embodiment of the present application discloses a high-precision electroactivation device for oocytes / early embryos, which includes a power supply 1, an electroactivation chip 2, and a wire assembly 3. The power supply 1 is used to provide the required electrical energy for the electrostimulation of oocytes / early embryos. The electroactivation chip 2 includes a base 21 and an electrode layer 22. The base 21 and the electrode layer 22 are arranged from bottom to top. The base 21 is used to provide structural support, and the electrode layer 22 is used to generate a uniform electric field required for electroactivation. The base 21 is provided with a plurality of electroactivation grooves 211 arranged at intervals and two opposite electrode pits 212 communicating with the plurality of electroactivation grooves 211. The electrode layer 22 includes two electrode assemblies 221. Each electrode assembly 221 includes an electrode part 222 and a conductive part 223 connected to the electrode part 222. The electrode part 222 is disposed in the electroactivation groove 211. The two electrode parts 222 and the electroactivation groove 211 form an electroactivation chamber 23, which is used to provide a uniform electric field during the electroactivation process to achieve electroactivation. The conductive part 223 is disposed in the corresponding electrode pit 212. The wire assembly 3 is used to connect the power supply 1 to the two conductive parts 223 of the electroactivation chip 2 to transmit the electrical energy of the power supply 1 to the electrode layer 22.

[0051] This device has the characteristics of miniaturization and precision, high structural strength and good structural stability, which improves the experimental reliability. At the same time, it simplifies the manufacturing and assembly processes, which is conducive to high-efficiency mass production. The compact structure reduces the support requirements, ensures the precise positioning of the electrode layer 22, realizes uniform electric field distribution, further optimizes the electroactivation effect, and reflects the high efficiency and practicality of the device. A uniform and stable electric field can be formed on the surface of the egg membrane at a relatively low input voltage, realizing uniform expansion of the cell membrane, avoiding extreme excessive voltage in local areas, and thus effectively reducing the risk of electrical damage to the cell membrane. In addition, the device can perform personalized fine electroactivation on individual oocytes / early embryos, significantly improving the experimental repeatability, activation efficiency, and survival rate of oocytes / early embryos, providing stable and reliable technical support for the high-efficiency electroactivation of oocytes / early embryos. The electroactivation groove 211 and the two electrode parts 222 together form a semi-closed electroactivation chamber 23, which is convenient for operators to quickly place and take out oocytes / early embryos, reducing their exposure time in the external environment, protecting the activity of oocytes / early embryos and reducing environmental interference. Compared with the fully enclosed design, the semi-closed structure does not require complex sealing or disassembly, simplifies the operation process, improves the experimental efficiency, and at the same time ensures the precise positioning of oocytes / early embryos in the electric field area, ensuring the safety and reliability of the experiment.

[0052] As Figure 1As shown, power supply 1 is a standard laboratory power supply, but it is not limited to a standard laboratory power supply. A commercially available or self-made power supply can also be used, as long as the output pulse of the power supply can be adjusted to provide electrical energy for electrically activating oocytes / early embryos. The wire assembly 3 includes two wires 31, and the two wires 31 are respectively connected to two conductive parts 223. The wire 31 is a supporting wire for the power supply 1. One end of the wire 31 is inserted into the pulse interface of the power supply 1, and the other end is connected to the electro-activation chip 2. Specifically, the other end of the wire 31 connected to the electro-activation chip 2 is a bare wire and is fixed to the conductive part 223 of the electro-activation chip 2 through an insulating tape.

[0053] As Figure 2 shown, it is a schematic diagram of the pulse signal required to be provided by the power supply 1 during the electro-activation of oocytes / early embryos. During the electro-activation process, a single-pulse stimulation method is adopted. The pulse amplitude can be adjusted within the range of 1 - 100V to adapt to different experimental conditions and the personalized needs of oocytes / early embryos. At the same time, the pulse width is between 50 μs and 200 μs to ensure the accuracy of the electrical stimulation and the effective action on the cell membrane. This setting of the pulse signal can ensure the activation efficiency while minimizing the damage to oocytes / early embryos, thereby improving the cell survival rate of electro-activation.

[0054] Preferably, the base 21 is made of a flexible insulating material, such as a biocompatible resin material, PDMS, etc. The base 21 is a flexible structure that can adapt to different experimental environments, reducing the mechanical damage to the electro-activation chip 2 caused by external forces; excellent biocompatibility and non-toxicity ensure that oocytes / early embryos are not affected by the released substances of the material during electro-activation; good electrical insulation performance can effectively isolate the electric field, avoiding problems such as short circuits or electric shocks; strong processability enables the fine manufacturing of complex microstructures; in addition, the flexible base 21 also significantly reduces the overall weight and volume of the electro-activation chip 2, improving the operation convenience and portability, being suitable for high-efficiency mass production, and meeting diverse application requirements.

[0055] Preferably, the base 21 and the electrode layer 22 are fixed by press-fitting or gluing. Specifically, the two conductive parts 223 are respectively fixed to the two electrode pits 212 by press-fitting or gluing, and the two electrode parts 222 are fixed to the electro-activation grooves 211 by press-fitting or gluing. Preferably, the size of the electro-activation chip 2 is 10 cm × 6 cm × 0.2 cm, but it is not limited to this size and can be adjusted according to requirements.

[0056] Please refer to Figure 5 、 Figure 11 、 Figure 13, preferably, a plurality of microstructural pillars are provided in the electroactivation groove 211. The plurality of microstructural pillars are distributed on a circumference centered on the center of the electroactivation groove 211, and the two electrode portions 222 are disposed around the plurality of microstructural pillars. By providing a plurality of microstructural pillars, it is possible to effectively restrict the irregular movement of the oocyte / early embryo during the experiment due to its activity, achieve stable cell position, avoid the cell being adsorbed to the surface of the electrode portion 222 due to liquid flow and operation error, ensure that the cell is within the observation field of view, and at the same time avoid the inconsistency of the electroporation effect caused by uneven electric field distribution, avoid too high local electric field intensity, avoid damage or apoptosis to the cell, and further uniform the number and distribution of pores on the cell membrane, improving the accuracy of the experimental results.

[0057] Preferably, the microstructural pillar is cylindrical. Due to its symmetry and uniform geometric structure, the cylindrical microstructural pillar can concentrate and guide the electric field lines, making the electric field intensity distribution on the cell membrane more uniform. This characteristic can not only form more uniformly distributed pores during the electroporation process, but also reduce the damage to the cell caused by too high local electric field intensity. In addition, the cylindrical microstructural pillar exhibits lower liquid resistance in the liquid environment, which is more conducive to stabilizing the position of the cell and reducing the influence of external interference on the experimental process. At the same time, the design of the cylindrical microstructural pillar is simple and is easy to be manufactured with high precision by photolithography and 3D printing technologies, having the advantages of low cost and high reliability.

[0058] Please refer to Figure 11 , in this embodiment, the number of microstructural pillars is six. The six microstructural pillars are divided into a first group and a second group. The first group includes two first microstructural pillars 213, and the second group includes four second microstructural pillars 214. The diameter of the first microstructural pillar 213 is larger than the diameter of the second microstructural pillar 214. By setting like this, it is possible to significantly improve the efficiency and uniformity of electroporation while restricting the free movement of the oocyte / early embryo, and it is possible to take into account the electroporation efficiency, cell stability and operation feasibility. Further preferably, the diameter of the first microstructural pillar 213 is 30 μm, and the diameter of the second microstructural pillar 214 is 10 μm. Considering that the diameter range of mouse oocytes / early embryos is 60 μm to 100 μm, it is preferred that the diameter of the circumference where the two first microstructural pillars 213 and the four second microstructural pillars 214 are located is 130 μm, which is convenient for placing the oocyte / early embryo.

[0059] Preferably, the two electrode pits 212 are symmetrically arranged with respect to the plurality of electroactivation grooves 211. Please refer to Figure 10, in this embodiment, the number of the electro-activation grooves 211 is ten, but is not limited to ten, and can be set to eleven, twelve or even more according to needs. In this embodiment, ten oocytes / early embryos can be subjected to personalized and delicate electro-stimulation simultaneously, greatly improving the experimental efficiency and effect. Preferably, the distance between adjacent electro-activation grooves 211 is 8900 μm. The distance between adjacent electro-activation grooves 211 is far enough to avoid mutual electric field interference during the electro-activation process.

[0060] Please refer to Figure 11 , preferably, the depth of the electro-activation groove 211 is greater than the depth of the electrode pit 212, so as to prevent liquid leakage caused by the splicing of the base 21 and the electrode layer 22 at the edge of the electro-activation groove 211, and avoid the risk of oocytes / early embryos being sucked into the bottom of the electrode part 222, resulting in cell loss, experimental failure, or even short circuit. This structure effectively avoids such potential hazards and significantly improves the experimental safety and stability. Preferably, the size of the electro-activation groove 211 can be 900 μm × 900 μm × 300 μm. At this time, the depth of the electro-activation groove 211 is 300 μm. Preferably, the depth of the electrode pit 212 is 100 μm.

[0061] Please refer to Figure 5 , Figure 11 , Figure 13 , two baffles 215 are arranged in the electro-activation groove 211, and the two electrode parts 222 are located on both sides of the two baffles 215. On the one hand, the baffle 215 can effectively control the distance between the two electrode parts 222 and reduce the risk of electrode burnout caused by excessive electric field concentration; on the other hand, the baffle 215 also plays a reference role during the assembly process of the conductive part 223 and the electrode pit 212, and the electrode part 222 and the electro-activation groove 211, making the assembly of the electrode layer 22 and the base 21 more accurate and convenient. Preferably, the two baffles 215 are arranged in a direction perpendicular to the direction opposite to the two electrode pits 212. Preferably, the size of the baffle 215 is 180 μm × 20 μm × 300 μm. The corresponding dimensions are the length L, thickness and height of the baffle 215. At this time, the height of the baffle 215 is the same as the depth of the electro-activation groove 211.

[0062] Preferably, the electrode part 222 is arc-shaped. The arc-shaped electrode can distribute the electric field more evenly, reduce the phenomenon of local overvoltage concentration, and thus optimize the uniformity of pore generation, which is of great significance for improving the electro-poration efficiency and reducing cell damage. Preferably, the distance D between the two electrode parts 222 is 500 μm. Compared with the traditional electro-activation device, in the present invention, by reducing the electrode distance, according to the formula Where E is the electric field strength, U is the voltage, and d is the electrode spacing, such that the required voltage is significantly reduced when applying the same electric field strength. Simulation verification shows that the pulse amplitude in this embodiment only needs to be set to 21 v to form an electric field with a strength of 1.5×10 5 V / m around the oocytes / early embryos, which not only reduces the potential electric shock risk to the operator and the object being operated on, improves the overall operation safety, but also reduces the risk of electrical damage to the cell membrane due to excessive voltage, thereby significantly improving the cell survival rate and activation efficiency. At the same time, the spacing between the two electrode parts 222 takes into account the actual operation requirements, leaving enough operation space in the electroactivation chamber 23 to ensure that the cells can be accurately placed in the electroactivation chamber 23.

[0063] Please refer to Figure 13 , in this embodiment, the electrode part 222 and the conductive part 223 are integrally formed and connected. This setting avoids the problem of poor contact that may be caused by the connection of multiple components, improves the uniformity of electric field transmission, ensures the stability and reliability of the electroactivation process. At the same time, the integrated structure reduces the complexity of manufacturing and assembly, improves the overall integration and mass production efficiency of the chip, and further optimizes the convenience of experimental operation and the repeatability of results. The conductive part 223 perfectly matches the electrode pit 212, and the two electrode parts 222 are matched in the electroactivation groove 211 to ensure stable fixation and precise electrical connection. Preferably, the thickness of the conductive part 223 is 100 μm. Preferably, the electrode layer 22 is made of a highly conductive metal or conductive glass material, with excellent electrical conductivity and mechanical stability. The highly conductive metal includes gold, silver, or copper.

[0064] Please refer to Figure 14 , which are three different arrangements of the microstructural struts. Among them, Figure 14 -(a) The structure is Method 1, with four microstructural struts, and the diameters of the four microstructural struts are all the same; Figure 14 -(b) The structure is Method 2, with six microstructural struts, and the diameters of the six microstructural struts are all the same; Figure 14 -(c) The structure is Method 3, with six microstructural struts, including two first microstructural struts 213 and four second microstructural struts 214. Please refer to Figure 15 , Figure 15 -(a) shows the distribution of the corresponding pores on the cell membrane in the three methods, Figure 15 -(b) shows the total number of pores that appear on the cell membrane in the three methods. The simulation results show that this arrangement of Method 3 can significantly improve the uniformity and efficiency of electroporation while restricting the free movement of oocytes / early embryos, optimize the local electric field distribution, and increase the pore density and evenly distribute them.

[0065] As Figure 16As shown, multiple electrode models are established through simulation software, including interdigital electrodes, facing electrodes, tip cone electrodes, tip flat electrodes, concentric circle electrodes, peripheral electrode 1, peripheral electrode 2, arc-shaped electrodes, and small cylindrical electrodes. When the contact electric field of the cell membrane is 1.5×10 5 V / m, the interaction simulation calculation of the electric field and the cell membrane is carried out. The results show that the influence of the electrode shape on the maximum transmembrane voltage and the amplitude of the maximum pore density is small. Then, on the basis of the same maximum transmembrane voltage and amplitude of the maximum pore density, the transmembrane voltage and pore density uniformity under different electrode shapes are observed through simulation. As Figure 17 , Figure 18 shown, concentric circle electrodes, peripheral electrode 2, etc. generate a strong electric field in local areas, resulting in a more concentrated pore density and increasing the risk of local damage to the cell membrane. While arc-shaped electrodes and small cylindrical electrodes make the pore density more evenly distributed on the cell membrane surface, reducing the concentration of transmembrane voltage and pores, thus improving the overall stability and uniformity of the cell membrane.

[0066] To study the influence of different electrode shapes on the total amount of transmembrane voltage and pore density on the egg membrane, a program is written by the gradient method. The result data graphs calculated by the above-mentioned electrodes with different shapes are imported into MATLAB, and the total amounts of the transmembrane voltage and pore density curves under each electrode shape are calculated. As Figure 19 shown, the influence of different electrode shapes on the total amount of transmembrane voltage is small, but the influence on the total amount of pore density is more significant. Under the same electric field conditions, the total amount of pores generated by the concentric circle electrode is the smallest, while the total amount of pores generated by the arc-shaped electrode is the largest. This shows that the electrode shape significantly affects the generation of pores by influencing the distribution pattern of the electric field on the cell membrane.

[0067] It can be comprehensively verified that the arc-shaped electrode can distribute the electric field more evenly, reduce the phenomenon of local overvoltage concentration, and thus optimize the uniformity of pore generation.

[0068] The embodiment of the present application also discloses a high-precision electroactivation method for oocytes / early embryos, using the above-mentioned electroactivation device, including the following steps:

[0069] S1. Preparatory stage: Put the pre-treated oocytes / early embryos 4 into the electroactivation chamber, and inject the electroactivation solution into the electroactivation chamber 23;

[0070] S2. Electroactivation stage of cells: Turn on the power supply 1, set the required electroactivation parameters, and electroactivate the oocytes / early embryos 4. When the oocytes / early embryos 4 are subjected to the electric field provided by the two electrode parts 222, the permeability of the cell membrane increases;

[0071] S3. Post-treatment: Take out the electroactivated oocytes / early embryos 4 and put them into the cell culture medium for cultivation.

[0072] To further illustrate the electrical stimulation method of the present invention, as a preferred solution, it includes the following steps:

[0073] (1) Preparatory stage: Use a holding needle to accurately place the pre-treated oocyte / early embryo 4 in the space surrounded by two first microstructural struts 213 and four second microstructural struts 214. Subsequently, inject the prepared electroactivation solution into the semi-closed electroactivation chamber 23 to ensure that the liquid evenly covers the electroactivation chamber 23, providing an ideal medium environment for electroactivation. Among them, the outer diameter of the holding needle can be 150 μm. The formulation of the electroactivation solution can be: 54.66 g of mannitol, 9.9 mg of magnesium chloride (MgCl 2 ), 11.5 mg of calcium chloride (CaCl 2 ), 2.383 g of Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1 mg / ml of bovine serum albumin (BSA), made up to 50 ml with deionized water, then filtered and dispensed with a 0.22 μm filter, sealed, and stored at 4°C to obtain. However, it is not limited to this formulation of electroactivation solution, and any electroactivation solution that can achieve electroactivation when an electric field is applied can be used, which is not defined here.

[0074] (2) Electroactivation stage of cells: Turn on the power supply 1, set the electroactivation parameters according to experimental requirements, set the voltage to 21 V and the pulse width to 150 μs. At this time, the electric field strength in the field reaches 1.5×10 5 V / m, and start the electroactivation program. Under the action of the stable and uniform electric field provided by the arc-shaped electrode part 222, the cell membrane permeability of the oocyte / early embryo 4 is improved, thus achieving an efficient electroactivation effect, while reducing the risk of electrical damage and improving the cell survival rate.

[0075] (3) Post-treatment: After electroactivation is completed, quickly take out the oocyte / early embryo from the electroactivation chamber 23 and place it in cell culture medium for subsequent culture to ensure its activity and subsequent growth state.

[0076] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0077] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A high-precision egg cell / early embryo electrical activation device, characterized in that: include: A power source for providing the electrical energy required for electrical stimulation of the egg cell / early embryo; An electro-activated chip comprises a base and an electrode layer, wherein the base is provided with a plurality of electro-activated grooves arranged at intervals and two electrode pits connected to and opposite to the plurality of electro-activated grooves, the electrode layer comprises two electrode assemblies, each of the electrode assemblies comprises an electrode portion and a conductive portion connected to the electrode portion, the electrode portion is arranged in the electro-activated groove, the two electrode portions and the electro-activated groove form an electro-activated chamber, and the conductive portion is arranged in the corresponding electrode pit; A wire assembly is used to connect the power source and the two conductive parts of the electrically activated chip.

2. The high-precision egg cell / early embryo electrical activation device according to claim 1, characterized in that: A plurality of microstructure pillars are arranged in the electro-activation groove, and the plurality of microstructure pillars are distributed on a circumference with the center of the electro-activation groove as the center, and the two electrode parts are arranged outside the plurality of microstructure pillars.

3. The high-precision egg cell / early embryo electrical activation device according to claim 2, characterized in that: The microstructure pillar is cylindrical.

4. The high-precision egg cell / early embryo electrical activation device according to claim 3, characterized in that: The number of the microstructure pillars is six, and the six microstructure pillars are divided into a first group and a second group. The first group includes two first microstructure pillars, and the second group includes four second microstructure pillars. The diameter of the first microstructure pillar is greater than the diameter of the second microstructure pillar.

5. The high-precision egg cell / early embryo electrical activation device according to claim 1, characterized in that: The depth of the electrically activated groove is greater than the depth of the electrode pit.

6. The high-precision egg cell / early embryo electrical activation device according to claim 1, characterized in that: Two baffles are arranged in the electrically activated groove, and the two electrode parts are located on both sides of the two baffles.

7. The high-precision egg cell / early embryo electrical activation device according to claim 1, characterized in that: The electrode portion is in an arc shape.

8. The high-precision egg cell / early embryo electrical activation device according to claim 1, characterized in that: The electrode portion is integrally formed and connected with the conductive portion.

9. The high-precision egg cell / early embryo electrical activation device according to claim 1, characterized in that: The electrode layer is fixed to the base by press-fitting or bonding.

10. A high-precision egg cell / early embryo electrical activation method, characterized in that: Using the electro-activation device according to any one of claims 1 to 9 comprises the following steps: (1) Preliminary preparation stage: placing the pretreated oocytes / early embryos into the electro-activation chamber and injecting the electro-activation liquid into the electro-activation chamber; (2) Cell electrical activation stage: Turn on the power supply, set the required electrical activation parameters, and electrically activate the egg cell / early embryo. When the egg cell / early embryo is subjected to the electric field provided by the two electrode parts, the permeability of the cell membrane increases; (3) Post-processing: The electrically activated oocytes / early embryos are removed and placed in cell culture medium for culture.

Images