Robotized in-situ operation method for reconstructed embryo electrofusion

Abstract

The invention discloses a robotized in-situ operation method for reconstructed embryo electrofusion, and belongs to the technical field of cell-level micromanipulation. According to the method, somatic cell nuclear transplantation operation and reconstructed embryo electrofusion operation are integrated on the same operation platform, and an electrode microneedle with suction and electrical stimulation functions is introduced into a left mechanical arm; the right mechanical arm introduces parallel double needles for nuclear transplantation and electrical stimulation respectively, and the method comprises the following key steps: cell positioning, cell holding, cell enucleation, cell nuclear injection, conversion of an injection needle and an electrode microneedle, electrical stimulation fusion, and finally release of operated cells. According to a traditional reconstructed embryo electrofusion method, a reconstructed embryo subjected to nuclear transfer is transferred to a specially-made parallel electrode plate, electric pulse fusion is applied, and experimental results show that compared with a traditional operation process, the method provided by the invention has the advantages that about 45.3% of time can be saved, and the efficiency and repeatability of reconstructed embryo electrofusion are effectively improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cell-level micromanipulation technology, and particularly relates to a robotic in-situ operation method for reconstituted embryo electric fusion. BACKGROUND

[0002] Reconstituted embryo electric fusion is a key step of cloning technology and is a next step after somatic cell nuclear transfer. Current reconstituted embryo electric fusion operation needs to put the embryo after nuclear transfer into a cell electric fusion device. Existing commercial cell electric fusion devices usually apply a fusion electric field on two parallel electrode plates with a distance of 1-10 mm to realize electric fusion. Since the reconstituted embryo needs to be transferred between different devices and the positions and postures of the embryo between the parallel electrode plates are difficult to unify, the process will cause a certain degree of damage to the cells and is low in efficiency. From the perspective of cell operation, the two cells required for cell fusion are similar to single cell operation, and it is expected to realize in-situ expansion from single cell operation to embryo operation. Therefore, it is necessary to design a robotic in-situ operation method for reconstituted embryo electric fusion, which does not need to transfer the embryo between different devices and can realize precise electric stimulation fusion of the reconstituted embryo to improve the operation efficiency. SUMMARY

[0003] In order to overcome the problems of low operation efficiency, damage to cells in the operation process and difficulty in precise electric stimulation in current reconstituted embryo electric fusion operation, the present application aims to provide a robotic in-situ operation method for reconstituted embryo electric fusion to optimize the reconstituted embryo electric fusion process and improve the efficiency of robotic reconstituted embryo electric fusion.

[0004] The present application adopts the following technical solution to solve the above problems:

[0005] A robotic in-situ operation method for reconstituted embryo electric fusion, the method comprising the following steps:

[0006] S1: Cell positioning: an improved robotic in-situ operation platform is adopted, the operation platform comprising a group of electrode microneedles with suction and electric stimulation functions and a group of parallel injection needles and electrode microneedles, and the operation platform is automatically positioned at a cell to be operated in the center of a liquid drop in a culture dish during movement of the operation platform;

[0007] S2: Cell suction: the suction pressure of the operation platform is adjusted so that the electrode microneedles with suction and electric stimulation functions just hold the cell at the current position, the zona pellucida of the oocyte just contacts the electrode wire and does not deform, and other cells to be operated and operated cells are not affected;

[0008] S3: Cell enucleation: the injection needle is used to enucleate the suctioned oocyte;

[0009] S4: Cell injection: using the injection needle to suck a somatic cell and inject it into the perivitelline space of the oocyte;

[0010] S5: Injection needle and electrode microneedle conversion: moving the parallel injection needle and electrode microneedle at the same time, moving the injection needle out of the microscopic field of view, and moving the electrode microneedle into the microscopic field of view;

[0011] S6: Electric stimulation fusion: using a cell fusion instrument to provide a direct current rectangular pulse signal to cause cell membrane perforation and promote oocyte and somatic cell fusion

[0012] S7: Release the operated cell for the next cell operation.

[0013] Further, in S1, the preparation steps of the electrode microneedle with holding function and electric stimulation function are as follows:

[0014] (1) Draw the glass tube to form the capillary microneedle;

[0015] (2) Needle breaking: breaking the needle at the outer diameter of 80 μm of the capillary microneedle using a needle breaking instrument;

[0016] (3) Inserting a metal electrode: inserting a platinum wire with a diameter of 30 μm so that the platinum wire is basically flush with the end needle port;

[0017] (4) End bending: in order to ensure that the end of the electrode microneedle is horizontal to the culture dish during the experiment, bending is performed at a distance of 3-5 mm from the end using an alcohol lamp, and the bending angle is about 30°;

[0018] (5) Fixing the electrode microneedle: inserting the prepared electrode microneedle into the patch clamp amplifier glass electrode holder.

[0019] Further, the glass tube is heated and drawn to a set shape and size by a capillary glass microneedle needle drawing instrument to form a capillary microneedle.

[0020] Further, the end diameter of the electrode microneedle is 80 μm, and the platinum wire inside the electrode microneedle has a diameter of 80 μm.

[0021] Further, the model of the patch clamp amplifier glass electrode holder is AXON-700B.

[0022] Further, in S1, the robotic in-situ operation platform includes a culture dish, the electrode microneedle with holding function and electric stimulation function, and the parallel injection needle and electrode microneedle are respectively arranged on both sides of the culture dish, the upper ends of the electrode microneedle with holding function and electric stimulation function and the parallel injection needle and electrode microneedle are provided with needle holders, and the electrode microneedle with holding function and electric stimulation function is connected with the needle holder through the patch clamp amplifier glass electrode holder.

[0023] Furthermore, in S1, the center of the cell is located by calculating the ratio of the radius to the perimeter of the cell to be operated on.

[0024] Furthermore, in S2, the minimum pressure required to hold the cell is 1550 Pa.

[0025] Furthermore, in S5, the injection microneedle is a commonly used injection microneedle in somatic cell nuclear transfer technology, with a needle radius ranging from 15 to 20 μm. The electrode microneedle is a tungsten steel needle with a tip diameter of 30 μm. The conversion between the injection needle and the electrode microneedle requires calibrating the distance between the injection needle and the electrode microneedle under low magnification. When operating under high magnification, the parallel injection needle and electrode microneedle are moved simultaneously to the calibrated distance.

[0026] Furthermore, in S6, the cell fusion instrument is model CF-150B, and the DC rectangular pulse signal provided by the cell fusion instrument has an amplitude of 5.5V and a duration of 30μs.

[0027] Compared with the prior art, the present invention has the following advantages and effects:

[0028] This invention introduces a specially designed microneedle electrode that combines cell holding and electrical stimulation functions; it also incorporates parallel double needles for nuclear transfer and electrical stimulation fusion, respectively, eliminating the time required to transfer embryos to different devices and the time spent on objective lens switching and focusing, enabling in-situ manipulation of reconstructed embryo electrofusion. Experimental results show that, compared with previous reconstructed embryo electrofusion procedures, this method increases the operation speed by 45.3% while maintaining essentially the same success rate and cell viability, effectively improving the efficiency and reproducibility of reconstructed embryo electrofusion. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 This is a flowchart of the experimental operation method;

[0031] Figure 2 A schematic diagram of a robotic in-situ manipulation platform for reconstructive embryo electrofusion.

[0032] Figure 3 A physical image of the electrode microneedles used in robotic in-situ manipulation for reconstructive embryo electrofusion;

[0033] inFigure 3 (a) shows the left electrode microneedle; Figure 3 (b) is the right-side electrode microneedle;

[0034] Figure 4 This is a schematic diagram of the experimental procedure;

[0035] in Figure 4 (a) Cell localization; Figure 4 (b) Cell adsorption; Figure 4 (c) Enucleation of the cell; Figure 4 (d) Cell enema; Figure 4 (e) Switching between injection needle and electrode microneedle; Figure 4 (f) Electrical stimulation fusion;

[0036] Figure 5 This is a schematic diagram illustrating the distance calibration between the injection needle and the electrode microneedle.

[0037] in Figure 5 (a) is a schematic diagram of the field of view under a 10x scope; Figure 5 (b) is a schematic diagram of the field of view under a 4x scope;

[0038] Figure 6 Images of the distance calibration experiment between the injection needle and the electrode microneedle under a 4x microscope.

[0039] In the figure, 1-first needle holder; 2-patch clamp AXON-700B amplifier glass electrode holder; 3-left electrode microneedle; 4-culture dish; 5-injection needle; 6-right electrode microneedle; 7-second needle holder; 8-third needle holder. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] like Figure 1 As shown in the flowchart, this invention provides a robotic in-situ manipulation method for reconstructed embryo electrofusion, comprising the following steps:

[0042] S1: Cell localization: An improved robotic in-situ manipulation platform is used. The platform includes a set of electrode microneedles that have both suction and electrical stimulation functions, as well as a set of parallel injection needles and electroelectrode microneedles. The cell center position is located by calculating the radius-to-circumference ratio of the cell to be manipulated. During the movement of the manipulation platform, the cell to be manipulated is automatically located in the center of the droplet in the culture dish.

[0043] S2: Cell Adsorption: Adjust the adsorption pressure of the operating platform so that the left electrode microneedle just adsorbs the cell at the current position, so that the zona pellucida of the oocyte just contacts the electrode wire without deformation, and at the same time does not affect other cells to be operated or already operated. The minimum pressure for adsorbing cells is 1550 Pa.

[0044] S3: Enucleation: Enucleation of the oocytes held in aspirate is performed by injection;

[0045] S4: Cell infusion: A somatic cell is aspirated using an injection needle and injected into the perivitelline space of the oocyte;

[0046] S5: Switching between injection needle and electrode microneedle: Move the parallel injection needle and electrode microneedle simultaneously, so that the injection needle moves out of the microscopic field of view and the electrode microneedle moves into the microscopic field of view. The injection microneedle is the commonly used injection microneedle in somatic cell nuclear transfer technology, with a needle radius of approximately 15 to 20 μm. The electrode microneedle is a tungsten steel needle with a tip diameter of 30 μm. Switching between the injection needle and electrode microneedle requires calibrating the distance between the injection needle and electrode microneedle under low magnification. When operating under high magnification, simply move the parallel injection needle and electrode microneedle simultaneously to the calibrated distance.

[0047] S6: Electrical stimulation fusion: A DC rectangular pulse signal is provided using a cell fusion instrument to perforate the cell membrane and promote the fusion of oocytes and somatic cells. The cell fusion instrument model is CF-150B. The DC rectangular pulse signal provided by the cell fusion instrument has an amplitude of 5.5V and a duration of 30μs.

[0048] S7: Release the operated cell and proceed to the next cell operation.

[0049] The method will be described in detail below with reference to specific embodiments. In this embodiment, the target cells are porcine oocytes and porcine embryonic fibroblasts.

[0050] like Figure 2 As shown, the robotic in-situ manipulation platform used in this method includes a culture dish 4. For ease of understanding, the electrode microneedle prepared in this application, which has both suction and electrical stimulation functions, is referred to as the left electrode microneedle 3. The parallel injection needle and electrode microneedle are referred to as the injection needle 5 and the right electrode microneedle 6, respectively. The left electrode microneedle 3 and the parallel injection needle 5 and right electrode microneedle 6 are respectively located on both sides of the culture dish. The left electrode microneedle 3 is connected to a patch clamp AXON-700B amplifier glass electrode holder 2. The patch clamp AXON-700B amplifier glass electrode holder 2 is connected to a first needle holder 1. The injection needle 5 is connected to a second needle holder 7. The right electrode microneedle 6 is connected to a third needle holder 8.

[0051] To prepare the left-side electrode microneedle 3, such as Figure 3 As shown in (a), the process steps are as follows:

[0052] (1) Drawing a glass tube to form a capillary needle; as a preferred option, a capillary glass needle drawing instrument is used to heat and draw the glass tube to a set shape and size to form a capillary needle.

[0053] (2) Needle breakage: The needle is broken at 80 μm outer diameter of the capillary needle using a needle-burning instrument;

[0054] (3) Inserting metal electrodes: Inserting a platinum wire with a diameter of 30 μm, so that the platinum wire is basically flush with the end of the needle, and the diameter of the platinum wire inserted into the electrode microneedle is 80 μm.

[0055] (4) End bending: To ensure that the end of the left electrode microneedle 3 is horizontal with the culture dish 4 during the experiment, use an alcohol lamp to bend the end of the left electrode microneedle 3 at a distance of 3-5 mm from the end of the left electrode microneedle 3, with a bending angle of about 30°.

[0056] (5) Fixing the electrode microneedles: Insert the prepared left electrode microneedles 3 into the AXON-700B amplifier glass electrode holder 2.

[0057] like Figure 3 As shown in (b), in this embodiment, the injection needle 5 used is a commonly used injection microneedle in somatic cell nuclear transfer technology, with a needle radius ranging from approximately 15 to 20 μm; the right electrode microneedle 6 is a tungsten steel needle with a tip diameter of 30 μm.

[0058] After completing the improvements to the robotic in-situ operation platform, the specific operation method includes the following steps:

[0059] S1: Cell localization, such as Figure 4 As shown in (a), the process of moving the XY operation platform allows the oocyte to appear in the microscopic field of view and automatically positions the cell to be operated in the center of the droplet in the culture dish.

[0060] S2: Cell adsorption, such as Figure 4 As shown in (b), the holding pressure is adjusted so that the left electrode microneedle 3 just holds the cell at the current position, so that the zona pellucida of the oocyte just contacts the electrode wire without deformation, and at the same time does not affect other cells to be operated or already operated; to ensure successful holding, we set the minimum pressure applied in the embodiment to 1600 Pa.

[0061] S3: Enucleation of the cell, such as... Figure 4 As shown in (c), the oocyte was enucleated using injection needle 5;

[0062] S4: Cell infusion, such as Figure 4 As shown in (d), a somatic cell was aspirated using injection needle 5 and injected into the perivitelline space of the oocyte;

[0063] S5: Switching between injection needle and electrode microneedle, such as Figure 4 As shown in (e), under low magnification, as Figure 5 (a) Figure 6 As shown, the distance ΔD between the calibrated injection needle 5 and the right-side electrode microneedle 6 is determined under high magnification, as follows: Figure 5 As shown in (b), the parallel injection needle 5 and the right electrode microneedle 6 are moved simultaneously by a calibrated distance ΔD, so that the injection needle 5 moves out of the microscopic field of view and the right electrode microneedle 6 moves into the microscopic field of view.

[0064] S6: Electrical stimulation fusion, such as Figure 4 As shown in (f), a DC rectangular pulse signal was provided by a CF-150B cell fusion instrument. The pulse amplitude was 5.5V and the pulse duration was 30μs, which caused perforation of the cell membrane and promoted the fusion of oocytes and somatic cells.

[0065] S7: Release the operated cell and proceed to the next cell operation.

[0066] Experimental results show that, compared with previous reconstructed embryo electrofusion procedures, this method increases the operation speed by 45.3% while maintaining essentially the same success rate and cell survival rate, effectively improving the efficiency and reproducibility of reconstructed embryo electrofusion. Specifically, of 20 reconstructed embryo electrofusions performed using this robotic method, 17 were successful, with all 17 surviving, and the average operation time was 82 seconds. In contrast, of 20 somatic cell nuclear transfers performed manually by professional researchers, 17 were successful, with all 17 surviving, and the average operation time was 150 seconds.

[0067] Furthermore, it should be noted that the shapes and names of the parts and components described in the specific embodiments described in this specification may differ. All equivalent or simple variations made to the structure, features, and principles of this invention are included within the scope of protection of this invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the structure of this invention or exceed the scope defined in these claims, all of which should fall within the scope of protection of this invention.

Claims

1. A robotic in-situ manipulation method for electrofusion of reconstructed embryos, characterized in that: The method includes the following steps: S1: Cell localization: An improved robotic in-situ manipulation platform is used, which includes a set of electrode microneedles that have both suction and electrical stimulation functions, as well as a set of parallel injection needles and electrode microneedles. During the movement of the manipulation platform, the cells to be manipulated are automatically located in the center of the culture droplet. S2: Cell Adsorption: Adjust the adsorption pressure of the operating platform so that the electrode microneedles, which have both adsorption and electrical stimulation functions, can just adsorb the cell at the current position, so that the zona pellucida of the oocyte just contacts the electrode wire without deformation, and at the same time, it does not affect other cells to be operated or already operated. S3: Enucleation: Enucleation of the oocyte held in aspirate is performed by injection; S4: Cell infusion: A somatic cell is aspirated using an injection needle and injected into the perivitelline space of the oocyte; S5: Switching between injection needle and electrode microneedle: Move the parallel injection needle and electrode microneedle simultaneously, so that the injection needle moves out of the microscopic field of view and the electrode microneedle moves into the microscopic field of view. S6: Electrical stimulation fusion: A DC rectangular pulse signal is provided using a cell fusion instrument to perforate the cell membrane, promoting the fusion of oocytes and somatic cells. S7: Release the operated cell and proceed to the next cell operation.

2. The robotic in-situ manipulation method for electrofusion of reconstructed embryos according to claim 1, characterized in that: In S1, the preparation steps of the electrode microneedle that combines the functions of suction and electrical stimulation are as follows: (1) Drawing glass tubes to form capillary needles; (2) Needle breakage: The needle is broken at 80 μm outer diameter of the capillary needle using a needle-burning instrument; (3) Inserting metal electrodes: Inserting a platinum wire with a diameter of 30 μm, so that the platinum wire is basically flush with the end of the needle; (4) End bending: To ensure that the end of the electrode microneedle remains horizontal with the culture dish during the experiment, use an alcohol lamp to bend it 3-5 mm away from the end, with a bending angle of about 30°. (5) Fixing the electrode microneedles: Insert the prepared electrode microneedles into the patch clamp amplifier glass electrode holder.

3. The robotic in-situ manipulation method for electrofusion of reconstructed embryos according to claim 2, characterized in that: The glass tube is heated and drawn to a set shape and size using a capillary glass microneedle drawing instrument to form capillary microneedles.

4. The robotic in-situ manipulation method for electrofusion of reconstructed embryos according to claim 2, characterized in that: The electrode microneedle has a terminal diameter of 80 μm, and the platinum wire inserted inside the electrode microneedle has a diameter of 80 μm.

5. A robotic in-situ manipulation method for electrofusion of reconstructed embryos according to claim 2, characterized in that: The diaphragm clamp amplifier glass electrode holder is model AXON-700B.

6. The robotic in-situ manipulation method for electrofusion of reconstructed embryos according to claim 2, characterized in that: In S1, the robotic in-situ operation platform includes a culture dish. The electrode microneedles that combine suction and electrical stimulation functions, as well as the parallel injection needles and electroelectrode microneedles, are respectively disposed on both sides of the culture dish. The electrode microneedles that combine suction and electrical stimulation functions, as well as the parallel injection needles and electroelectrode microneedles, are all provided with needle holders at their upper ends. The electrode microneedles that combine suction and electrical stimulation functions are connected to the needle holders through a patch clamp amplifier glass electrode holder.

7. A robotic in-situ manipulation method for electrofusion of reconstructed embryos according to any one of claims 1-6, characterized in that: In S1, the center of the cell is located by calculating the ratio of the radius to the perimeter of the cell to be operated on.

8. A robotic in-situ manipulation method for electrofusion of reconstructed embryos according to any one of claims 1-6, characterized in that: In S2, the minimum pressure required to hold the cell is 1550 Pa.

9. A robotic in-situ manipulation method for electrofusion of reconstructed embryos according to any one of claims 1-6, characterized in that: In S5, the injection microneedle is a commonly used injection microneedle in somatic cell nuclear transfer technology, with a needle radius ranging from 15 to 20 μm. The electrode microneedle is a tungsten steel needle with a tip diameter of 30 μm. The conversion between the injection needle and the electrode microneedle requires calibrating the distance between the injection needle and the electrode microneedle under low magnification. When operating under high magnification, the parallel injection needle and electrode microneedle are moved simultaneously to the calibrated distance.

10. A robotic in-situ manipulation method for electrofusion of reconstructed embryos according to any one of claims 1-6, characterized in that: In S6, the cell fusion instrument is model CF-150B, and the DC rectangular pulse signal provided by the cell fusion instrument has an amplitude of 5.5V and a duration of 30μs.

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