CN111849768A - Egg cell multi-efficiency accurate electrical stimulation device and method - Google Patents
Egg cell multi-efficiency accurate electrical stimulation device and method Download PDFInfo
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- CN111849768A CN111849768A CN202010673131.1A CN202010673131A CN111849768A CN 111849768 A CN111849768 A CN 111849768A CN 202010673131 A CN202010673131 A CN 202010673131A CN 111849768 A CN111849768 A CN 111849768A
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Abstract
The invention discloses an egg cell multi-efficiency accurate electrical stimulation device, which comprises: a base; the electric stimulation mechanism comprises two first electric stimulation components and two second electric stimulation components, wherein each first electric stimulation component comprises a liquid metal capillary microneedle electrode and a first needle holding instrument connected with the liquid metal capillary microneedle electrode, and each second electric stimulation component comprises a metal parallel plate microelectrode and a second needle holding instrument connected with the metal parallel plate microelectrode; the cell pose adjusting chip is arranged on the base and used for adjusting the pose of the cell; and the rotating mechanism is arranged on the base, is connected with the cell pose adjusting chip and is used for driving the cell pose adjusting chip to rotate. The invention also discloses an egg cell multi-efficiency accurate electrical stimulation method. The invention can carry out accurate electrical stimulation on specific parts of the egg cells at different stages in the whole cloning process, realize multiple effects on the targeted electrical stimulation at each stage of the somatic cell cloning process, and improve the success rate of the whole somatic cell cloning process.
Description
Technical Field
The invention relates to the technical field of biology related to cloning, in particular to an egg cell multi-efficiency accurate electrical stimulation device and method for a cloning process.
Background
Cloning is the process of producing the progeny of the same genome as the original individuals by asexual propagation by using biotechnology. Since birth, cloning technology has always occupied the most important place in life science research and related medical technology. The egg cells contain important biogenetic information and are the main operation objects of the cloning process. However, the success rate of cloning experiments is very low, and one of the main reasons is that the activity of in vitro egg cells is greatly reduced, the egg cells are easy to die in the experimental process, even if the egg cells are not killed during reconstruction, development retardation is easy to occur in subsequent cultivation, and at this time, the egg cells need to be stimulated manually to activate the cells. Among the various stimulation methods, the electro-stimulation egg cells become the best scheme due to the advantages of convenient operation, accurate parameter control, high activation rate, high biocompatibility, no pollution, small damage and the like. Electrical stimulation of egg cells is largely classified into electrical activation and electrical fusion. The electrical activation is the increase of cell membrane permeability of the oocyte membrane under the excitation of DC pulse voltage, and the extracellular Ca2+Influx into the cell to make intracytoplasmic Ca2+At elevated concentrations, the egg cells are activated. If the DC pulse voltage continues to rise, irreversible cell membrane perforation occurs, and at the moment, if two cells are attached to each other, the two cells are fused into one cell, and the cell is electrofusion. The basic principle is that the stability of the phosphodiester bimolecular structure of the cell membrane is changed under the stimulation of electric pulses.
The traditional egg cell electric stimulation device basically arranges a batch of egg cells between macroscopic parallel electrode plates, leads in electric stimulation signals to generate a uniform electric field, and finds out the optimal electric stimulation parameters through accurate adjustment to obtain higher electric stimulation success rate and lower cell death rate. However, the distance between the electrodes of the macroscopic parallel plate is large, so that the voltage required by applying electric stimulation is also large, and the cells are extremely easy to damage and are unsafe; and the traditional electric stimulator cannot perform accurate electric stimulation on specific parts of the egg cells, and cannot perform various electric stimulations (electric activation, electric fusion, mitochondrial membrane potential lifting and the like) required by cloning the egg cells at each stage by using one set of electric stimulation device.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an egg cell multi-efficiency accurate electrical stimulation device and method oriented to a cloning process.
In order to achieve the above object, an embodiment of the present invention provides the following technical solutions:
an accurate electrostimulation device of oocyte multipotency, comprising:
a base;
the electric stimulation mechanism comprises two first electric stimulation components and two second electric stimulation components, each first electric stimulation component comprises a liquid metal capillary microneedle electrode and a first needle holding instrument connected with the liquid metal capillary microneedle electrode, and each second electric stimulation component comprises a metal parallel plate microelectrode and a second needle holding instrument connected with the metal parallel plate microelectrode;
The cell pose adjusting chip is arranged on the base and used for adjusting the pose of the cell;
and the rotating mechanism is arranged on the base, is connected with the cell pose adjusting chip and is used for driving the cell pose adjusting chip to rotate.
As a further improvement of the present invention, the liquid metal capillary microneedle electrode is manufactured by the following steps: firstly, drawing a glass tube to form a capillary microneedle, then injecting liquid metal from the tail of the capillary microneedle, then throwing the liquid metal to the tip end of the capillary microneedle, finally trimming the tip end of the capillary microneedle, and leading out a lead from the tail of the capillary microneedle.
As a further improvement of the invention, the diameter of the tip of the liquid metal capillary microneedle electrode is 10 μm.
As a further improvement of the present invention, the metal parallel plate microelectrode comprises a rod-like electrode body and parallel plate electrodes, the rod-like electrode body and the parallel plate electrodes being integrally formed.
As a further improvement of the invention, the head diameter of the rod-shaped electrode body is 0.5mm, and the size of the parallel plate electrode is 2mm multiplied by 1 mm.
As a further improvement of the present invention, the cell pose adjustment chip includes an upper substrate, a lower substrate, and an intermediate layer disposed between the upper substrate and the lower substrate, the upper substrate is provided with a micro-hole therein, the intermediate layer is provided with an air chamber, and the lower substrate is provided with an air guide column.
As a further improvement of the invention, the diameter of the micropores is 15 μm.
As a further improvement of the present invention, the first needle holder includes a first screw cap, a second screw cap, a connecting seat, a third screw cap, and a fixing rod, the first screw cap is in threaded connection with the second screw cap, both the second screw cap and the third screw cap are in threaded connection with the connecting seat, and the fixing rod is in threaded connection with the third screw cap.
As a further improvement of the invention, the rotating mechanism comprises a motor, a transmission gear connected with the motor and a bearing seat connected with the transmission gear, and the bearing seat is connected with the cell pose adjusting chip.
An accurate electrical stimulation method for egg cell multi-potency, which uses the device and comprises the following steps:
(1) egg cell activation phase: moving the egg cells onto a cell pose adjusting chip and fixing the egg cells, controlling two liquid metal capillary microneedle electrodes to be tightly attached to an egg cell transparent belt in an included angle of 50-70 degrees, continuously introducing high-voltage direct current pulses, and then rotating the cell pose adjusting chip through a rotating mechanism to drive the egg cells fixed on the cell pose adjusting chip to horizontally rotate until all cell membranes are fully stimulated;
(2) Egg cell enucleation stage: enucleating the activated egg cells;
(3) and (3) reconstructing embryo electrofusion stage: after a nuclear donor is injected into an enucleated oocyte to form a reconstructed embryo, the reconstructed embryo is fixed on a cell pose adjusting chip, two liquid metal capillary microneedle electrodes are moved to be tightly attached to the reconstructed embryo at an included angle of 180 degrees, the contact surface of the nuclear donor and the enucleated oocyte is perpendicular to the direction of an electric field, and high-voltage direct current pulse is introduced to enable the nuclear donor and the enucleated oocyte to be electrically fused to form a reconstructed embryo;
(4) and (3) a recombinant embryo culture stage: and (3) placing the recombined embryo after electrofusion between two metal parallel plate microelectrodes, and introducing a low-frequency alternating-current micro-current signal to the metal parallel plate microelectrodes.
The invention has the beneficial effects that:
(1) the liquid metal capillary microneedle electrode and the metal parallel plate microelectrode provided by the invention can be used for accurately electrically stimulating single egg cells and specific parts of the egg cells, such as cell membranes and mitochondria, so that the electrical damage to other parts of the egg cells is reduced as much as possible, and the success rate and the survival rate of each stage of the somatic cell cloning process are improved.
(2) The method can complete the electrical stimulation required by the egg cells at each stage of the somatic cell cloning, and comprises the steps of performing electrical stimulation on the cell membranes of the egg cells before denucleation to activate the egg cells, completing the electrical fusion of a nuclear donor and the denucleated egg cells at the reconstructed embryo electrical fusion stage, completing the improvement of the mitochondrial membrane potential of the reconstructed embryo at the reconstructed embryo development stage, and realizing multiple effects through the targeted electrical stimulation on each stage of the somatic cell cloning process, thereby helping an operation object break through each stage to continue to develop.
(3) Because the liquid metal capillary micro-needle electrode and the metal parallel plate microelectrode are adopted, the electrode distance d is smaller according to a formula
When the stimulation of the same size electric field is applied, the required voltage is smaller, and the safety of operators and operation objects is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a schematic structural view of an electrostimulation device according to a preferred embodiment of the present invention;
FIG. 2 is a top view of an electrical stimulation apparatus of a preferred embodiment of the present invention;
FIG. 3 is an enlarged partial view of an electrical stimulation apparatus in accordance with a preferred embodiment of the present invention;
FIG. 4 is a partial top view of an electrical stimulation apparatus in accordance with a preferred embodiment of the present invention;
fig. 5 is a schematic structural view of a cell pose adjustment chip according to a preferred embodiment of the present invention;
fig. 6 is an exploded schematic view of a cell pose adjustment chip according to a preferred embodiment of the present invention;
FIG. 7 is a schematic structural diagram of a first electrostimulation assembly according to a preferred embodiment of the invention;
FIG. 8 is an enlarged view of A in FIG. 7;
FIG. 9 is a schematic structural diagram of a second electrostimulation assembly according to a preferred embodiment of the invention;
FIG. 10 is an enlarged view of B in FIG. 9;
fig. 11 is a flow chart of the preparation of a liquid metal capillary microneedle electrode according to a preferred embodiment of the present invention;
FIG. 12 is a schematic illustration of the application of electrical stimulation to cells at various stages of cloning in accordance with a preferred embodiment of the present invention;
FIG. 13 is a flow chart of the application of electrical stimulation during somatic cell cloning in accordance with a preferred embodiment of the present invention;
in the figure: 10. the device comprises a base, 12, a first electrical stimulation component, 14, a second electrical stimulation component, 16, a liquid metal capillary microneedle electrode, 18, a first needle holder, 20, a metal parallel plate microelectrode, 24, a second needle holder, 26, a cell pose adjusting chip, 28, a tip, 30, a rod-shaped electrode body, 31, a parallel plate electrode, 32, a head, 33, an upper substrate, 34, a lower substrate, 35, an intermediate layer, 36, a micropore, 38, an air chamber, 40, an air guide column, 42, a groove, 44, a first spiral cover, 46, a second spiral cover, 48, a connecting seat, 50, a third spiral cover, 52, a fixing rod, 54, a first annular gasket, 56, a second annular gasket, 58, a channel, 59, a lead wire, 60, a motor, 62, a bearing seat, 63, a supporting plate, 64, an egg cell, 66, a nuclear donor, 68, an enucleated egg cell, 70, a reconstructed embryo, 72 and a reconstructed embryo.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1 to 4, an egg cell multi-potency precise electrical stimulation device includes:
a base 10;
the electrostimulation mechanism comprises two first electrostimulation components 12 and two second electrostimulation components 14, wherein each first electrostimulation component 12 comprises a liquid metal capillary microneedle electrode 16 and a first needle holding instrument 18 connected with the liquid metal capillary microneedle electrode 16, and each second electrostimulation component 14 comprises a metal parallel plate microelectrode 20 and a second needle holding instrument 24 connected with the metal parallel plate microelectrode 20;
a cell pose adjusting chip 26 mounted on the base 10 for adjusting the pose of the cell;
and a rotating mechanism mounted on the base 10 and connected to the cell pose adjustment chip 26 for driving the cell pose adjustment chip 26 to rotate.
In order to more precisely realize the electrical stimulation to single cells and specific parts of cells, such as cell membranes and mitochondria, the liquid metal capillary microneedle electrode 16 is preferably prepared by the following process, as shown in fig. 11, the process steps are as follows:
(1) the drawn glass tube is formed into capillary microneedles. As a preferred scheme, a capillary microneedle drawing instrument is used for heating and drawing the glass tube to a set shape and size to form the capillary microneedle, and the capillary microneedle drawing instrument adopts a common structure in a biological laboratory.
(2) Liquid metal is injected from the tail of the capillary microneedle. Preferably, the liquid metal is injected from the tail of the capillary micro-needle by a high-precision injector, and the liquid metal is gallium-based liquid metal.
(3) The liquid metal was thrown towards the capillary microneedle tip. Preferably, the liquid metal is thrown to the tip of the capillary microneedle by using centrifugal force generated by a centrifugal machine until the liquid metal is full of the tip and has no bubbles when observed under a microscope, the rotating speed of the centrifugal machine is set to 6500r/min before the centrifugal machine works, the duration is 1-2 minutes, and the rotating speed and the time can be adjusted according to the diameter of the capillary microneedle.
(4) And (4) trimming the tip of the capillary micro-needle, and leading out a lead from the tail of the capillary micro-needle glass tube to form the liquid metal capillary micro-needle electrode. And leading out a lead from the tail part of the capillary micro-needle glass tube for connecting a power supply before the liquid metal is oxidized and solidified. The surface of the liquid metal exposed in the air is quickly oxidized to form a thin solid oxide film with the thickness of about 1nm, which is beneficial to stabilizing the shape of the liquid metal capillary microneedle electrode, preventing the liquid metal from flowing out from the tip end of the capillary microneedle and avoiding affecting the performance of the capillary microneedle electrode.
As shown in fig. 8, the tip 28 of the preferred liquid metal capillary microneedle electrode 16 of the present invention has a diameter of 10 μm.
The metal parallel plate microelectrode 20 is made of a metal which does not chemically react with the cell culture solution when energized. In the embodiment of the present invention, the metal parallel plate micro-electrode 20 is made of platinum, but is not limited to platinum, and may be gold, silver, or the like. The preferred metal parallel plate microelectrode 20 of the present invention comprises a rod electrode body 30 and a parallel plate electrode 31, wherein the rod electrode body 30 and the parallel plate electrode 31 are integrally formed. It is further preferable that the diameter of the head 32 of the rod-like electrode body 30 is 0.5mm and the size of the parallel plate electrode 31 is 2mm × 1 mm.
As shown in fig. 5 and 6, in order to fix cells, the cell pose adjusting chip 26 according to the present invention preferably includes an upper substrate 33, a lower substrate 34, and an intermediate layer 35 disposed between the upper substrate 33 and the lower substrate 34, the upper substrate 33 has micropores 36, the intermediate layer 35 has an air chamber 38, the lower substrate 34 has an air guide pillar 40, the micropores 36, the air chamber 38, and the air guide pillar 40 are sequentially communicated, the micropores 36 are used for holding cells, the air chamber 38 is used for stabilizing air pressure and reducing fluctuation, and the air guide pillar 40 is connected to an air pump (not shown) for generating negative pressure for fixing ova.
It is further preferable that the diameter of the micropores 36 is 15 μm. Further, it is preferable that the upper substrate 33, the lower substrate 34, and the intermediate layer 35 have a rectangular shape for easy mounting. Preferably, the upper substrate 33 is centrally provided with a recess 42, and the micro-holes 36 are provided on the bottom wall of the recess 42. It is further preferred that the recess 42 is circular and that the micro-holes 6 are provided in the central bottom wall of the recess 42. Preferably, the upper substrate 33 and the lower substrate 34 are made of organic glass, and the intermediate layer 35 is made of PDMS.
As shown in fig. 7 and 8, the first needle-holding instrument 18 of the present invention preferably includes a first screw cap 44, a second screw cap 46, a connecting base 48, a third screw cap 50 and a fixing rod 52, wherein the first screw cap 44 is screwed with the second screw cap 46, the second screw cap 46 and the third screw cap 50 are both screwed with the connecting base 48, and the fixing rod 52 is screwed with the third screw cap 50. In order to improve the stability of the connection between the first screw cap 44 and the second screw cap 46, and between the second screw cap 46 and the connecting seat 48, it is preferable that the second screw cap 46 is padded with a first annular gasket 54, and the connecting seat 48 is padded with a second annular gasket 56. As shown in fig. 9 and 10, the first needle holder 18 and the second needle holder 24 preferably have the same structure and size. According to the invention, the first screw cap 44, the second screw cap 46 and the connecting seat 48 are preferably provided with a channel 58, the liquid metal capillary microneedle electrode 16 is partially clamped in the channel 58, and the channel 58 can facilitate the leading-out of a lead 59 of the liquid metal capillary microneedle electrode 16.
In order to facilitate the position adjustment of the liquid metal capillary microneedle electrode 16 and the metal parallel plate microelectrode 20, it is preferable that each of the first needle holder 18 and the second needle holder 24 is respectively fixed on a corresponding four-degree-of-freedom mechanical arm (not shown in the figure), and the four-degree-of-freedom mechanical arm can realize translation along X, Y, Z three directions and rotation around the X direction, and the Y direction refers to the direction parallel to the tip 28 or the head 32.
The preferred rotating mechanism of the present invention includes a motor 60, a transmission gear (not shown in the figure) connected to the motor 60, and a carrying base 62 connected to the transmission gear, and the cell pose adjustment chip 26 is mounted on the carrying base 62. It is further preferable that the carrying seat 62 is installed in the base 10, the carrying seat 62 is circular, the carrying seat 62 extends inward to form at least one supporting plate 63, the cell pose adjusting chip 26 is fixed on the supporting plate 63, a gear (not shown) is sleeved on the outer peripheral surface of the carrying seat 62, the transmission gear is connected with the gear on the outer peripheral surface of the carrying seat 62, the motor 60 is started, and the carrying seat 62 is driven to rotate through the transmission gear, so as to drive the cell pose adjusting chip 26 to rotate.
Referring to fig. 12, the method for accurately electrically stimulating egg cells with multiple efficiencies of the present invention is described, and the method using the above-mentioned apparatus comprises the following steps:
(1) Egg cell activation phase: moving the egg cells 64 to the cell pose adjusting chip 26 and fixing the egg cells, as shown in fig. 12- (a), controlling the two liquid metal capillary microneedle electrodes 16 to be tightly attached to the transparent belts of the egg cells 64 at an included angle of 50-70 degrees, continuously introducing high-voltage direct current pulses, and then rotating the cell pose adjusting chip 26 through a rotating mechanism to drive the egg cells 64 fixed on the cell pose adjusting chip 26 to horizontally rotate until all cell membranes are fully stimulated;
(2) egg cell enucleation stage: enucleating the activated egg cells 64;
(3) and (3) reconstructing embryo electrofusion stage: injecting a nuclear donor 66 into an enucleated oocyte 68 to form a reconstructed embryo 70, as shown in fig. 12- (b), fixing the reconstructed embryo 70 on a cell pose adjusting chip 26, moving two liquid metal capillary microneedle electrodes 16 to be tightly attached to the reconstructed embryo 70 at an included angle of 180 degrees, enabling the contact surface of the nuclear donor 66 and the enucleated oocyte 68 to be vertical to the direction of an electric field, representing the direction of the electric field by the arrow in the figure, and introducing high-voltage direct-current pulses to enable the nuclear donor 66 and the enucleated oocyte 68 to be electrically fused to form a reconstructed embryo 72;
(4) and (3) a recombinant embryo culture stage: the recombined embryo 72 after electrofusion is placed between two parallel plate electrodes 31, as shown in FIG. 12- (c), and a low-frequency AC micro-current signal is applied to the metal parallel plate microelectrode 20.
As shown in fig. 13, in order to further explain the electrical stimulation method of the present invention, it preferably includes the steps of:
(1) egg cell activation phase: moving the egg cells 64 to the cell pose adjusting chip 26, starting a negative pressure pump to fix the egg cells 64, controlling the two liquid metal capillary microneedle electrodes 16 to be tightly attached to the transparent belts of the egg cells 64 at an included angle of 60 degrees, keeping the distance between the needle points of the two liquid metal capillary microneedle electrodes 16 between 8 and 12 mu m, realizing that the electric field between the two liquid metal capillary microneedle electrodes 16 is focused on the cell membrane part of the egg cells 64, continuously introducing high-voltage direct current pulses, ensuring that the electric field strength is 0.5 to 1.0kv/cm and the pulse width is 50 to 100 mu s, starting the motor 60 to drive the rotating mechanism to rotate the cell pose adjusting chip 26, driving the egg cells 64 fixed at the center of the cell pose adjusting chip 26 to horizontally rotate until all the cell membranes are fully stimulated, increasing the permeability of the cell membranes after electrical stimulation, and being beneficial to Ca in the2+Enters the egg cell 64 and completes the activation of the egg cell 64. It is further preferred that the tips of the two liquid metal capillary microneedle electrodes 16 are spaced 10 μm apart. It is further preferred that the egg 64 is an ovarian oocyte.
(2) Egg cell enucleation stage: activated egg cells 64 were enucleated with the aid of a microscope using blind aspiration.
(3) And (3) reconstructing embryo electrofusion stage: after a nuclear donor 66 is injected into an enucleated oocyte 68 to form a reconstructed embryo 70, the reconstructed embryo 70 is fixed on a cell pose adjusting chip 26, two liquid metal capillary microneedle electrodes 16 are moved to be tightly attached to the reconstructed embryo 70 at an included angle of 180 degrees, the contact surface of the nuclear donor 66 and the enucleated oocyte 68 is perpendicular to the direction of an electric field, high-voltage direct current pulses are introduced, the electric field strength is 1-2 kv/cm, the pulse width is 50-100 microseconds, and the nuclear donor 66 and the enucleated oocyte 68 are electrically fused to form a recombinant embryo 72. It is further preferred that the nuclear donor 66 be injected into the perivitelline space of the enucleated oocyte 68.
(4) And (3) a recombinant embryo culture stage: the recombined embryo 72 after electrofusion is placed between two parallel plate electrodes 31, a low-frequency alternating current micro-current signal is introduced into the metal parallel plate microelectrode 20, the voltage is 40-50V, the pulse width is 20-30 ms, and the frequency is 30-50 Hz, so that the mitochondrial membrane potential of the recombined embryo 72 is improved, and the activity of the recombined embryo 72 is further improved to promote the division of the recombined embryo 72.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. An accurate electrical stimulation device of egg cell multipotency, characterized by, includes:
a base;
the electric stimulation mechanism comprises two first electric stimulation components and two second electric stimulation components, each first electric stimulation component comprises a liquid metal capillary microneedle electrode and a first needle holding instrument connected with the liquid metal capillary microneedle electrode, and each second electric stimulation component comprises a metal parallel plate microelectrode and a second needle holding instrument connected with the metal parallel plate microelectrode;
the cell pose adjusting chip is arranged on the base and used for adjusting the pose of the cell;
and the rotating mechanism is arranged on the base, is connected with the cell pose adjusting chip and is used for driving the cell pose adjusting chip to rotate.
2. The egg cell multipotency precise electrical stimulation device according to claim 1, wherein the liquid metal capillary microneedle electrode is manufactured by the following steps: firstly, drawing a glass tube to form a capillary microneedle, then injecting liquid metal from the tail of the capillary microneedle, then throwing the liquid metal to the tip end of the capillary microneedle, finally trimming the tip end of the capillary microneedle, and leading out a lead from the tail of the capillary microneedle.
3. The egg cell multipotency precise electrical stimulation device according to claim 2, wherein the diameter of the tip of the liquid metal capillary microneedle electrode is 10 μm.
4. The egg cell multiprocessing accurate electrical stimulation device according to claim 1, wherein the metal parallel plate microelectrode comprises a rod electrode body and a parallel plate electrode, the rod electrode body and the parallel plate electrode are integrally formed.
5. The egg cell multipotency precise electrical stimulation device according to claim 4, wherein the diameter of the head of the rod-shaped electrode body is 0.5mm, and the size of the parallel plate electrode is 2mm x 1 mm.
6. The egg cell multi-potency precise electrical stimulation device according to claim 1, wherein the cell pose adjustment chip comprises an upper substrate, a lower substrate and an intermediate layer arranged between the upper substrate and the lower substrate, the upper substrate is internally provided with micropores, the intermediate layer is provided with an air chamber, and the lower substrate is provided with an air guide column.
7. The egg cell multipotency precise electrical stimulation device according to claim 6, wherein the diameter of the micropores is 15 μm.
8. The precise electrical stimulation device for egg cell multipotency according to claim 1, wherein the first needle holding instrument comprises a first screw cap, a second screw cap, a connecting seat, a third screw cap and a fixing rod, the first screw cap is in threaded connection with the second screw cap, the second screw cap and the third screw cap are both in threaded connection with the connecting seat, and the fixing rod is in threaded connection with the third screw cap.
9. The egg cell multi-efficacy precise electrical stimulation device as claimed in claim 1, wherein the rotating mechanism comprises a motor, a transmission gear connected with the motor and a bearing seat connected with the transmission gear, and the bearing seat is connected with the cell pose adjusting chip.
10. A method of precise electrical stimulation of egg cell multipotency using the device of any one of claims 1-9, comprising the steps of:
(1) egg cell activation phase: moving the egg cells onto a cell pose adjusting chip and fixing the egg cells, controlling two liquid metal capillary microneedle electrodes to be tightly attached to an egg cell transparent belt in an included angle of 50-70 degrees, continuously introducing high-voltage direct current pulses, and then rotating the cell pose adjusting chip through a rotating mechanism to drive the egg cells fixed on the cell pose adjusting chip to horizontally rotate until all cell membranes are fully stimulated;
(2) Egg cell enucleation stage: enucleating the activated egg cells;
(3) and (3) reconstructing embryo electrofusion stage: after a nuclear donor is injected into an enucleated oocyte to form a reconstructed embryo, the reconstructed embryo is fixed on a cell pose adjusting chip, two liquid metal capillary microneedle electrodes are moved to be tightly attached to the reconstructed embryo at an included angle of 180 degrees, the contact surface of the nuclear donor and the enucleated oocyte is perpendicular to the direction of an electric field, and high-voltage direct current pulse is introduced to enable the nuclear donor and the enucleated oocyte to be electrically fused to form a reconstructed embryo;
(4) and (3) a recombinant embryo culture stage: and (3) placing the recombined embryo after electrofusion between two metal parallel plate microelectrodes, and introducing a low-frequency alternating-current micro-current signal to the metal parallel plate microelectrodes.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010673131.1A CN111849768B (en) | 2020-07-14 | 2020-07-14 | Egg cell multi-efficiency accurate electrical stimulation device and method |
| PCT/CN2020/103160 WO2022011729A1 (en) | 2020-07-14 | 2020-07-21 | Multi-efficiency accurate electrical stimulation device and method for egg cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010673131.1A CN111849768B (en) | 2020-07-14 | 2020-07-14 | Egg cell multi-efficiency accurate electrical stimulation device and method |
Publications (2)
| Publication Number | Publication Date |
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| CN111849768A true CN111849768A (en) | 2020-10-30 |
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| WO2024045482A1 (en) * | 2022-08-30 | 2024-03-07 | 苏州大学 | Combined activation apparatus and method for early embryos |
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| CN111849768B (en) | 2022-07-19 |
| WO2022011729A1 (en) | 2022-01-20 |
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