Oocyte / embryo automatic vitrification and rewarming device and method

Abstract

The invention relates to an oocyte / embryo automatic vitrification and rewarming device and method. The device comprises a microfluidic mixing mechanism, a mechanical sliding table mechanism, a water absorbing material, a micro electric push rod, an electric clamping jaw and a receiving part, the micro-fluidic mixing mechanism comprises a micro-injection pump, a micro-injection needle, a silicone tube, a micro-fluidic chip, a steel needle and a micro-grid capillary tube; the micro-grid capillary tube is used as a freezing carrier; the mechanical sliding table mechanism comprises an X-axis assembly and a Z-axis assembly connected with the X-axis assembly. And the receiving piece is a liquid nitrogen tank. Compared with the prior art, the oocyte / embryo automatic vitrification and rewarming device provided by the invention optimizes a protective agent loading and removing mode through the microfluidic system so as to reduce permeation damage to cells. Meanwhile, cell transfer is reduced, the whole process of loading the protective agent, freezing the cells and unloading the protective agent is automatic, and the system can realize automation and standardization of the vitrification process of the oocytes / embryos.

Description

Technical Field

[0001] The present invention relates to the technical field of biological cell detection, processing and operation, and in particular to an oocyte / embryo automatic vitrification and rewarming device and method. Background Art

[0002] At present, human fertility is on a downward trend, and fertility preservation can help alleviate the problem of declining fertility. Some women who want to postpone their childbearing plans for personal reasons will have the need to preserve their fertility when they are young. In addition, radiotherapy, chemotherapy, surgery, etc. are also factors that affect patients' fertility. In order to preserve fertility and the quality of life after recovery, these women also have the need to preserve their fertility.

[0003] Common methods for preserving female fertility include cryopreservation of female oocytes, embryos, ovarian tissues, etc. Vitrification has the advantages of fast cooling rate, sufficient vitrification, and high recovery rate, and is a common method in the current oocyte / embryo cryopreservation process. However, artificial vitrification of oocytes / embryos is one of the most time-consuming and labor-intensive procedures in IVF laboratories. This process requires manual operation by highly skilled embryologists. It takes 15-20 minutes to process an oocyte / embryo, and requires high concentration. There may be differences in the repeatability and consistency of operators. Therefore, consistency, reliability, and safety are crucial for operators, patients, oocytes, and embryos.

[0004] In order to improve the success rate and efficiency of vitrification, the introduction of automation technology will greatly improve efficiency and save costs. At present, some progress has been made in automatic vitrification devices. For example, Arav et al. developed an automatic vitrification preservation system (FertileSafe Ltd, Israel), the carrier used in this device is a carrier that combines a special grid and a 0.25mL straw. The robotic arm moves the straw in different protective agent containers to change the protective agent concentration around the cells, and finally enters the liquid nitrogen for freezing. The device still transfers oocytes / embryos at different concentrations of protective agents, and a sudden change in the protective agent concentration will cause osmotic damage. Roy et al. developed a semi-automatic closed "Gavi" system for embryo vitrification, which first injects a lower concentration of CPA into the cryocarrier Pod, then absorbs the CPA and injects a higher concentration of CPA for the second stage of equilibrium after a period of balancing, and then absorbs the CPA again. The whole process takes about 13 to 18 minutes. After loading, the device can automatically seal the Pod, and then manually remove the Pod and vertically put it into the liquid nitrogen bucket placed next to it to complete the freezing. The heat transfer performance of the freezing carrier Pod of this device is poor, and it cannot guarantee complete vitrification. In addition, Miao et al. designed an embryo vitrification robotic system. They proposed a new open microfluidic chip. The equilibrium solution and vitrification solution were loaded / removed in the solution exchange chamber by a syringe pump, and then the microvalve was opened and the embryo entered the transplantation channel. The embryo was transferred from the open microfluidic chip to the Cryotop using positive pressure, and then transferred and immersed in liquid nitrogen using a gripper. This system currently only realizes the processing of single embryos, and the loading speed of its protective agent is too fast, which is easy to damage cells. Automatic rewarming in this system is also a time-consuming process, and the degree of automation is low.

[0005] Therefore, in view of the above problems, the present invention aims to develop an automatic vitrification device with high automation, simple operation and short time consumption. Summary of the invention

[0006] The purpose of the present invention is to provide an automatic vitrification and rewarming device and method for oocytes / embryos in order to overcome the defects of the above-mentioned prior art. Through the microfluidic system, the loading and removal of the protective agent are optimized to reduce the osmotic damage to the cells. At the same time, the cell transfer is reduced, and the entire process of protective agent loading-cell freezing-protective agent unloading is automated. The system can realize the automation and standardization of the vitrification process of oocytes / embryos. The concentration gradient of the protective agent is realized by a microinjection pump and a microfluidic chip; an oocyte / embryo protective agent loading and freezing carrier that can be combined with a microfluidic chip is designed, and a medical absorbent dressing is placed under the carrier. Through the cooperation of an electric clamp and a micro electric push rod, excess liquid is sucked out after loading is completed to minimize the remaining sample volume; a programmable mechanical slide is used to control the descent of the carrier and the movement of the liquid nitrogen tank. In the preset process, the oocyte / embryo loading and freezing carrier can be quickly descended to the liquid nitrogen tank for vitrification in a very short time. Thereby, the loading and freezing of the oocyte / embryo protective agent are completed in the same carrier, reducing the osmotic damage and cell loss caused by cell movement. Use mechanized operations to standardize the entire process and reduce problems such as accidental deviations and errors caused by human operations.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The first object of the present invention is to provide an oocyte / embryo automatic vitrification and rewarming device for loading / removing and freezing an oocyte / embryo protective agent, wherein the device comprises a microfluidic mixing mechanism, a mechanical slide mechanism, a water-absorbing material, a micro electric push rod, an electric clamp, and a receiving member; the microfluidic mixing mechanism comprises a microinjection pump, a microinjection needle, a silicone tube, a microfluidic chip, a steel needle (steel needle) at the outlet, and a microgrid capillary; the microinjection pump is connected to the microinjection needle; the silicone tube is connected to the microinjection needle; the silicone tube is connected to the microfluidic chip; the microfluidic chip is connected to the steel needle (steel needle) at the outlet; the steel needle (steel needle) at the outlet is connected to the microgrid capillary; The capillaries are arranged relatively to each other; the microgrid capillary is used as a freezing carrier; the water-absorbing material is arranged below the microgrid capillary, and is used to absorb the protective agent flowing out of the microgrid capillary during the loading process and absorb the excess protective agent after the loading is completed to achieve the minimum amount of residual liquid; the mechanical slide mechanism includes an X-axis component and a Z-axis component connected to the X-axis component; the receiving part is connected to the X-axis component; a micro electric push rod is arranged on the Z-axis component; the water-absorbing material is connected to the micro electric push rod; the electric clamp is connected to the Z-axis component; the electric clamp includes electric clamp fingers; the electric clamp fingers are used to clamp and put down the microgrid capillary; the receiving part is a liquid nitrogen tank.

[0009] Furthermore, the microgrid capillary comprises a tube body and a grid; the inner diameter of the tube body is 0.56 mm, the outer diameter is 0.8 mm, the wall thickness is 0.12 mm, and the length is 30 mm; the bottom of the tube body is wrapped with a grid with a pore size of 0.048 mm.

[0010] Furthermore, the grid is a polyamide grid.

[0011] Furthermore, the holding force of the electric clamp is 15N, which can smoothly clamp the cryocarrier microgrid capillary tube away from the steel needle without causing obvious deformation of the cryocarrier microgrid capillary tube. The overlap of the cryocarrier microgrid capillary tube and the steel needle at the connection point is 3mm to ensure that no overflow occurs at the gap at the connection point during the loading process. The electric clamp clamps the cryocarrier microgrid capillary tube. The first stage of the descending distance is 6mm.

[0012] Furthermore, the mechanical slide mechanism includes an X-axis assembly and a Z-axis assembly connected to the X-axis assembly; the X-axis assembly includes an X-axis motor, an X-axis guide rail, and an X-axis slider; the X-axis slider is slidably connected to the X-axis guide rail; the X-axis motor is connected to the X-axis slider to drive the X-axis slider to slide on the X-axis guide rail; the receiving member is connected to the X-axis slider; the Z-axis assembly includes a Z-axis motor, a Z-axis guide rail, and a Z-axis slider; the Z-axis slider is slidably connected to the Z-axis guide rail; the Z-axis motor is connected to the Z-axis slider to drive the Z-axis slider to slide on the Z-axis guide rail; a structure-chip bracket is provided at the top of the Z-axis guide rail, and the structure-chip bracket is used to place a microfluidic chip for mixing a protective agent; a micro electric push rod is provided on the Z-axis guide rail; a placement slot is provided on the head of the micro electric push rod, and the placement slot is used to place a water-absorbing material; the electric clamp is connected to the Z-axis slider (fixed connection).

[0013] Furthermore, the device also includes an electric control mechanism; the electric control mechanism includes a relay control box, an LED light, and a controller; the controller is respectively connected to the microinjection pump, the relay control box, and the LED light; the motor driver is respectively connected to the X-axis motor and the Z-axis motor; the LED light includes LED No. 1, LED No. 2, and LED No. 3, all of which are used to prompt the stage process.

[0014] Furthermore, the electric control mechanism also includes a transformer and a motor driver; the controller is connected to the transformer and the motor driver respectively; the transformer is used for voltage transformation; and the motor driver is used for driving the X-axis motor and the Z-axis motor.

[0015] Furthermore, two microinjection pumps are provided, and the two microinjection pumps are connected via a linkage switch; the relay control box is used to control the start and stop of the linkage switch by powering on and off, thereby controlling the working state of the microinjection pump, and operating at preset time intervals through a program on the control panel of the controller.

[0016] Furthermore, the linkage switch is made according to the working principle of the Footswitch Input interface, using a 3.5mm direct-insert audio plug and a foot switch joint welded together to simultaneously control the start and stop of two microinjection pumps.

[0017] Furthermore, the micro electric push rod is equipped with two medical absorbent dressings. The head of the micro electric push rod is provided with two placement slots, namely slot No. 1 and slot No. 2 adjacent to slot No. 1. Slot No. 1 absorbs excess liquid, shrinks 3mm after the injection procedure, and absorbs the remaining liquid in the carrier in slot No. 2. Finally, the micro electric push rod shrinks 2mm again to provide descending space for the cryocarrier microgrid capillary.

[0018] Furthermore, the water-absorbing material is a medical water-absorbing dressing, specifically a medical gelatin sponge; two pieces of water-absorbing material are provided.

[0019] Furthermore, the microfluidic chip was used to load and remove the protective agent, and the loading and removal time was 8 min. 2 )x 2 The loading-removal curve was optimized to obtain the highest oocyte / embryo survival rate.

[0020] Furthermore, the X-axis slider can realize the movement of the receiving part, which is translated 100mm to the left after the micro electric push rod is retracted, directly under the cryocarrier microgrid capillary. The Z-axis slider can realize the lowering of the electric gripper to the receiving part, down to 1mm from the liquid nitrogen tank, so that the electric gripper can release the cryocarrier microgrid capillary.

[0021] A second object of the present invention is to provide a method for loading an oocyte / embryo cryoprotectant, the loading method using an oocyte / embryo automatic vitrification and rewarming device, the loading method comprising the following steps:

[0022] a) firstly, a buffer solution and a cryoprotectant are loaded into a microinjection needle respectively, a silicone tube is used to connect the outlet of the microinjection needle and the injection port of the microfluidic chip, and the microinjection needle of the buffer solution is pushed until the buffer solution fills the microfluidic chip;

[0023] b) after the oocyte is moved into the cryocarrier microgrid capillary by using the egg transfer needle, the microgrid capillary is connected to the steel needle at the outlet of the microfluidic chip, the microfluidic chip is fixed on the structure-chip bracket, a hole is reserved at a corresponding position on the structure-chip bracket for the microgrid capillary to extend out of the structure-chip bracket, and the microgrid capillary is just in the slot below after extending out of the small hole;

[0024] c) The two micro-injection pumps work simultaneously and the program runs for 8 minutes before the micro-injection pumps stop injecting.

[0025] The third object of the present invention is to provide an oocyte / embryo cryopreservation method, wherein the cryopreservation method uses an oocyte / embryo automatic vitrification and rewarming device, and the cryopreservation method comprises the following steps:

[0026] a) The electric gripper fixed on the Z-axis slider moves downward by 3 mm under the movement of the Z-axis slider, the top of the microgrid capillary is separated from the steel needle at the outlet of the microfluidic chip, and the bottom is close to the water-absorbing material and stays for 2 seconds, and the remaining sample in the microgrid capillary is sucked out;

[0027] b) After the aspiration step is completed, the electric gripper holds the microgrid capillary and descends. At the same time, the liquid nitrogen tank connected to the X-axis moves to the bottom of the microgrid capillary. The electric gripper fingers extend and the microgrid capillary falls freely into the liquid nitrogen tank, completing the vitrification procedure.

[0028] A fourth object of the present invention is to provide a method for removing an oocyte / embryo cryoprotectant, the method using an oocyte / embryo automatic vitrification and rewarming device, the method comprising the following steps:

[0029] a) Preheat the rewarming reagent to 37°C, make droplets in a culture dish, take out the microgrid capillary from the liquid nitrogen tank, place the microgrid capillary in the rewarming reagent at 37°C for 1 min, load the buffer solution and the rewarming solution into a microinjection needle respectively, and connect the microinjection needle and the inlet of the microfluidic chip with a silicone tube;

[0030] b) After 1 minute, the microgrid capillary is connected to the steel needle at the outlet of the microfluidic chip for injection. After 8 minutes, the injection procedure ends and the microinjection pump stops injecting;

[0031] c) transferring, the electric gripper descends to hold the microgrid capillary away from the microfluidic chip, and the electric gripper fingers of the electric gripper are released to hold the microgrid capillary;

[0032] d) Connect a pipette to the top opening of the microgrid capillary, immerse the bottom grid of the microgrid capillary in the BS solution, aspirate twice to make the liquid into droplets, observe the droplets and find the cells in the droplets;

[0033] e) Use an egg transfer needle to transfer the cells from the BS droplet to the M2 culture medium for culture.

[0034] Further, the vitrification process (vitrification procedure):

[0035] Step 1: Turn on the power, the device is powered on, and LED light 1 lights up, indicating that the device is ready.

[0036] Step 2: Turn on the foot switch, the microinjection pump starts, and the injection procedure is carried out with a delay of 480s.

[0037] Step 3: The foot switch is turned off, the microinjection pump stops injecting, and LED light 2 lights up, indicating that the injection is completed and the next step can be carried out.

[0038] Step 4: The electric gripper is powered on and starts to move, and the electric gripper fingers 10 contract and clamp the carrier microgrid capillary.

[0039] Step 5: The micro electric push rod is powered on and performs a contraction movement for 0.15 s. It is known that the running speed of the micro electric push rod is 30 mm / s. At this time, the micro electric push rod moves 4.5 mm, and the carrier micro grid capillary is facing the water absorbent material in the No. 2 card slot.

[0040] Step 6: Move the Z-axis slider downward by 6mm to make the bottom grid of the carrier microgrid capillary close to the absorbent material in the No. 2 card slot, and move the X-axis slider to the left by 100mm to make the liquid nitrogen tank directly under the carrier microgrid capillary. Delay for 3s, of which 2s is the waiting time to ensure that the remaining liquid in the carrier microgrid capillary is sucked out and the remaining sample volume is minimized. 1s is the contraction movement of the micro electric push rod for 1s.

[0041] Step 7: The Z-axis slide moves down 117mm, and the electric gripper stays just above the liquid nitrogen tank.

[0042] Step 8: The motorized gripper fingers extend and the carrier microgrid capillary falls into the liquid nitrogen tank.

[0043] Step 9: The X-axis slider moves 100mm to the right, the Z-axis slider moves 123mm upward, the micro electric push rod passes positive current and extends 10mm. LED light 3 lights up, indicating that the whole program is completed.

[0044] Further, the rewarming and protective agent removal process:

[0045] Preheat the rewarming reagent to 37°C, make droplets in a culture dish, take the microgrid capillary out of the liquid nitrogen tank, and place the microgrid capillary in the rewarming reagent at 37°C for 1 min.

[0046] Connect the microgrid capillary to the steel needle at the outlet of the microfluidic chip and start the following protective agent removal process.

[0047] Step 1: Turn on the power, the device is powered on, and LED light 1 lights up, indicating that the device is ready.

[0048] Step 2: Turn on the foot switch, the microinjection pump starts, and the injection procedure is carried out with a delay of 480s.

[0049] Step 3: The foot switch is turned off, the microinjection pump stops injecting, and LED light 2 lights up, indicating that the injection is completed and the next step can be carried out.

[0050] Step 4: The electric gripper is powered on and starts to move, and the two fingers contract to clamp the carrier microgrid capillary.

[0051] Step 5: The micro electric push rod is powered on and performs a contraction movement for 0.4s. It is known that the running speed of the micro electric push rod is 30mm / s. At this time, the micro electric push rod moves 10mm and is completely retracted.

[0052] Step 6: Move the Z-axis slider down 123mm.

[0053] Step 7: The motorized gripper fingers extend to hold the carrier microgrid capillary.

[0054] Step 8: The Z-axis slider moves upward 123mm, the micro electric push rod is connected to the positive current and extends 10mm. LED light 3 lights up, indicating that the whole program is completed.

[0055] The fifth object of the present invention is to provide a loading / removal scheme of the protective agent in the microfluidic chip, that is, a loading / removal curve of a quadratic function, so that the survival rate of the oocyte is maximized. The segmented loading curve is a quadratic equation y=ax 2 (x is time, y is rate). Given a total flow rate of 30 μL / min and a total duration of 8 min, we know that this function passes through points (0,0) and (8,30), so we can get a=30 / 8 2 , so the quadratic function is y = (30 / 8 2 )x 2 The value is taken every 1 minute to make the loading-removal curve closer to the function curve.

[0056] The sixth object of the present invention is to provide a loading and freezing carrier suitable for the automatic vitrification and rewarming storage device of oocytes / embryos, which integrates the protective agent loading / vitrification freezing / protective agent removal / cell rewarming process into the same carrier, so as to realize the integration of automatic vitrification and rewarming storage of oocytes / embryos. After the protective agent loading / removal process is completed, the excess liquid is sucked out, and the liquid is sucked out within 2s, and the carrier can directly carry the cells and put them into liquid nitrogen.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 1) This technical solution provides an automatic vitrification and rewarming device for oocytes / embryos, which realizes the concentration gradient change of the protective agent through a microinjection pump and a microfluidic chip; designs an oocyte / embryo protective agent loading and freezing carrier that can be combined with a microfluidic chip, and places a medical absorbent dressing under the carrier. After loading is completed, the excess liquid is sucked out by the cooperation of electric grippers and micro-electric push rods to minimize the remaining sample volume; a programmable mechanical slide is used to control the descent of the carrier and the movement of the liquid nitrogen tank. In the preset process, the oocyte / embryo loading and freezing carrier can be quickly lowered into the liquid nitrogen tank for vitrification in a very short time. Thereby, the loading and freezing of oocyte / embryo protective agents are completed in the same carrier, reducing the osmotic damage and cell loss caused by cell movement.

[0059] 2) The technical solution provides an automatic vitrification and rewarming device for oocytes / embryos, which uses mechanized operation to standardize the entire process and reduce the problems of accidental deviations and errors caused by human operation. Through the microfluidic system, the way of loading and removing the protective agent is optimized to reduce the osmotic damage to the cells. At the same time, cell transfer is reduced, and the entire process of protective agent loading-cell freezing-protective agent unloading is automated. The system can realize the automation and standardization of the vitrification process of oocytes / embryos.

[0060] 3) The technical solution provides an automatic vitrification and thawing device for oocytes / embryos, which is a set of automatic vitrification devices with high automation, simple operation and short time consumption. Designing a microfluidic chip that can continuously load and remove protective agents can reduce the penetration damage during the loading and removal of protective agents. The cryocarrier adopts a microgrid capillary to ensure the smooth flow of the protective agent while retaining the cells. After loading is completed, the excess protective agent can be quickly absorbed by the absorbent material to minimize the amount of remaining liquid. The assembly of each component is realized according to the functional requirements, so that the components can achieve precise cooperation to complete the automatic vitrification process, so as to improve the efficiency and survival rate of oocyte / embryo vitrification. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Schematic diagram of the overall device of the oocyte / embryo automatic vitrification and thawing device in an embodiment of the present invention.

[0062] Figure 2 Schematic diagram of liquid absorption of the oocyte / embryo automatic vitrification and thawing device in an embodiment of the present invention, wherein: (a) initial state of protective agent loading / unloading, (b) protective agent loading / unloading process, (c) bottom close to the water-absorbing material of tank No. 2, and (d) remaining liquid is absorbed.

[0063] Figure 3It is a 8-min quadratic function loading-removal curve diagram of the oocyte / embryo automatic vitrification and thawing device in an embodiment of the present invention.

[0064] Figure 4 Schematic diagram of the microfluidic mixing mechanism of the oocyte / embryo automatic vitrification and thawing device in an embodiment of the present invention.

[0065] Figure 5 for Figure 1 Partial schematic diagram of part A in the middle (the viewing angle is slightly biased, but it does not affect the judgment of the structure).

[0066] in:

[0067] 1. Microinjection pump; 2. Relay control box; 3. LED light; 4. Controller; 5. Transformer; 6. Motor driver; 7. Microfluidic chip; 8. Steel needle; 9. Microgrid capillary; 10. Electric gripper finger; 11. Water-absorbing material; 12. Micro electric push rod; 13. Electric gripper; 14. Z-axis guide; 15. Z-axis motor; 16. X-axis motor; 17. X-axis guide; 18. Microinjection needle; 19. Silicone tube; 20. Receiver; 21. Placement slot; 22. X-axis slider; 23. Z-axis slider; 24. Structure-chip holder; 25. Oocyte / embryo. DETAILED DESCRIPTION

[0068] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The features such as component models, material names, connection structures, etc. that are not clearly described in this technical solution are all regarded as common technical features disclosed in the prior art.

[0069] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral one; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements; "up", "down", "left", "right" and the like are only used to indicate relative positional relationships, and when the absolute position of the described object changes, the relative positional relationship may change. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0070] It should be noted that, in the present invention, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0071] One of the purposes of the present invention is to maximize the survival rate of oocytes by the loading / removal scheme of the protective agent in the microfluidic chip 7, that is, the loading / removal curve of the quadratic function. The segmented loading curve is a quadratic equation y=ax 2 (x is time, y is rate). Given a total flow rate of 30 μL / min and a total duration of 8 min, we know that this function passes through points (0,0) and (8,30), so we can get a=30 / 8 2 , so the quadratic function is y = (30 / 8 2 )x 2 The value is taken every 1 minute to make the loading-removal curve closer to the function curve.

[0072] The second purpose of the present invention is to provide an oocyte / embryo automatic vitrification and rewarming device, which consists of a programmable mechanical slide (mechanical slide mechanism), a micro-injection pump 1, a foot switch, a 3.5mm head straight plug audio plug, a modular electric clamp 13, a micro electric push rod 12, an LED indicator light (LED light 3), a relay module (relay control box 2), a fixing component, a liquid nitrogen tank, a medical absorbent dressing, etc.

[0073] The third object of the present invention is to provide a loading and freezing carrier suitable for an oocyte / embryo automatic vitrification and rewarming storage device, which integrates the protective agent loading / vitrification freezing / protective agent removal / cell rewarming process into the same carrier, thereby realizing the integration of oocyte / embryo automatic vitrification and rewarming storage. After the protective agent loading / removal process is completed, the excess liquid is sucked out, and the liquid is completely sucked out within 2 seconds, and the carrier can directly carry the cells and put them into liquid nitrogen.

[0074] The fourth purpose of the present invention is to provide a method for combining a microinjection pump with a programmable mechanical slide, control the start and stop of the linkage switch by the up and down power control of the relay (relay control box 2), thereby controlling the working state of the microinjection pump 1, and realize the operation according to the preset time interval through the program of the control panel.

[0075] The fifth object of the present invention is to provide a method for the electric clamp 13 and the micro electric push rod 12 to cooperate in sucking out the excess liquid in the carrier. A double-slot container is placed at the head of the micro electric push rod 12. During the loading process, the carrier faces the center of the medical absorbent dressing in the No. 1 slot. During the protective agent loading process, the dripping reagent is sucked out. After the loading is completed, the micro electric push rod 12 retracts 3mm. At this time, the carrier faces the medical absorbent dressing in the No. 2 slot to suck out the remaining sample in the tube body to minimize the amount of remaining sample.

[0076] The sixth object of the present invention is to provide a method for removing excess liquid from a carrier by using an electric gripper and a micro electric push rod. After the removal, the mechanical slider carrying the liquid nitrogen tank moves 100 mm to the left, and is now directly below the carrier. The slider carrying the electric gripper 13 descends to the top of the liquid nitrogen tank, and the carrier that has been clamped and processed descends to the edge of the liquid nitrogen tank. At this time, the electric gripper fingers 10 open, and the carrier freely falls into the liquid nitrogen tank.

[0077] The present invention is further described in detail below in conjunction with specific embodiments.

[0078] Example 1

[0079] like Figure 1 , 4 As shown in Figure 5, an automatic vitrification and rewarming device for oocytes / embryos in this embodiment includes a microfluidic mixing mechanism, a mechanical slide mechanism, a water-absorbing material 11, an electric clamp 13, and a receiving part 20, wherein the receiving part 20 is a liquid nitrogen tank. The microfluidic mixing mechanism includes a microinjection pump 1, a microinjection needle 18, a silicone tube 19, a microfluidic chip 7, a steel needle (steel needle 8) at the outlet, and a microgrid capillary 9; the microinjection pump 1 is connected to the microinjection needle 18; the silicone tube 19 is connected to the microinjection needle 18; the silicone tube 19 is connected to the microfluidic chip 7; the microfluidic chip 7 is connected to the steel needle (steel needle 8) at the outlet; the steel needle (steel needle 8) at the outlet is arranged opposite to the microgrid capillary 9; the microgrid capillary 9 serves as a freezing carrier.

[0080] The water-absorbing material 11 is disposed below the microgrid capillary 9 and is used to absorb the protective agent flowing out of the microgrid capillary 9 during the loading process and to absorb the excess protective agent after the loading is completed to minimize the amount of residual liquid.

[0081] The mechanical slide mechanism includes an X-axis assembly and a Z-axis assembly connected to the X-axis assembly; the X-axis assembly includes an X-axis motor 16, an X-axis guide rail 17, and an X-axis slider 22; the X-axis slider 22 is slidably connected to the X-axis guide rail 17; the X-axis motor 16 is connected to the X-axis slider 22 to drive the X-axis slider 22 to slide on the X-axis guide rail 17; the receiving member 20 is fixedly connected to the X-axis slider 22; the Z-axis assembly includes a Z-axis motor 15, a Z-axis guide rail 14, and a Z-axis slider 23; the Z-axis slider 23 is connected to the Z-axis guide rail 14 Sliding connection; the Z-axis motor 15 is connected to the Z-axis slider 23 to drive the Z-axis slider 23 to slide on the Z-axis guide rail 14; a structure-chip holder 24 is provided at the top of the Z-axis guide rail 14, and the structure-chip holder 24 is used to place the microfluidic chip 7 for protective agent mixing; a micro electric push rod 12 is provided on the Z-axis guide rail 14; a placement slot 21 is provided at the head of the micro electric push rod 12, and the placement slot 21 is used to place the water-absorbing material 11; the electric clamp 13 is connected to the Z-axis slider 23 (fixed connection).

[0082] The mechanical slide mechanism is composed of two axes, X and Z, and a structure-chip holder 24 is designed at the top of the Z-axis guide rail 14 to place the microfluidic chip 7 for protective agent mixing. A micro electric push rod 12 with a telescopic distance of 10 mm is set at 36 mm from the bottom of the structure-chip holder 24, and a card slot 21 is placed at the head of the micro electric push rod 12 for placing medical absorbent dressings.

[0083] The electric gripper 13 includes electric gripper fingers 10 ; the electric gripper fingers 10 are used to grip and put down the micro-grid capillary 9 .

[0084] The device also includes an electric control mechanism; the electric control mechanism includes a relay control box 2, an LED lamp 3, a controller 4, a transformer 5, and a motor driver 6; the controller 4 is respectively connected to the microinjection pump 1, the relay control box 2, the LED lamp 3, the transformer 5, and the motor driver 6; the motor driver 6 is respectively connected to the X-axis motor 16 and the Z-axis motor 15; the LED lamp 3 includes LED No. 1, LED No. 2, and LED No. 3, all of which are used to prompt the stage process; the transformer 5 is used for voltage transformation; the motor driver 6 is used to drive the X-axis motor 16 and the Z-axis motor 15; the relay control box 2 is used to control the start and stop of the linkage switch by the upper and lower power control to control the working state of the microinjection pump 1, and realize the operation according to the preset time interval through the program of the control panel.

[0085] Two microinjection pumps 1 are provided, and the two microinjection pumps 1 are connected by a linkage switch; the relay control box 2 is used to control the start and stop of the linkage switch by turning on and off electricity to control the working state of the microinjection pump 1, and the operation is realized according to the preset time interval through the program of the control panel of the controller 4. The linkage switch is a linkage switch that can control the start and stop of two microinjection pumps 1 at the same time, which is made by welding a 3.5mm head straight-insertion audio plug and a joint of a foot switch according to the working principle of the FootswitchInput interface.

[0086] like Figure 2 As shown, the micro electric push rod 12 carries two medical absorbent dressings. The head of the micro electric push rod 12 is provided with two placement slots 21, namely slot 1 and slot 2 adjacent to slot 1. Slot 1 absorbs excess liquid, shrinks 3mm after the injection procedure, and absorbs the remaining liquid in the carrier in slot 2. Finally, the micro electric push rod 12 shrinks 2mm again to provide a descending space for the cryocarrier microgrid capillary 9. Figure 2 The steel needle 8 is the steel needle 8 at the outlet of the microfluidic chip 7, the steel needle 8 is connected to the microgrid capillary 9, the electric clamp finger 10 is used to clamp the microgrid capillary 9, the oocyte / embryo 25 is placed in the microgrid capillary 9, and the absorbent material 5 is an absorbent material 5 that absorbs liquid.

[0087] The water-absorbing material 11 is a medical water-absorbing dressing, and specifically can be a medical gelatin sponge.

[0088] The microfluidic chip 7 was used to load and remove the protective agent. The loading and removal time were both 8 min. y = (30 / 8 2 )x 2 The loading-removal curve was optimized to obtain the highest oocyte / embryo survival rate.

[0089] The X-axis slider 22 can realize the movement of the receiving part 20, and after the micro-electric push rod 12 is retracted, it is translated to the left by 100 mm, directly below the cryocarrier microgrid capillary 9. The Z-axis slider 23 can realize the lowering of the electric gripper 13 to the receiving part 20, and lower it to 1 mm away from the liquid nitrogen tank, so that the electric gripper 13 can release the cryocarrier microgrid capillary 9.

[0090] The vitrification solution / thawing solution and the basic solution are loaded into the microinjection needle respectively, and the microinjection needle 18 is connected to the inlet of the microfluidic chip 7 through a 0.5 mm silicone tube 19. Then the microgrid capillary 9 is connected to the outlet of the microfluidic chip 7, and the microfluidic mixing mechanism is completed. Figure 4 As shown, the injection speed of one micro-injection pump 1 is set to 0-30 μL / min, and the injection speed of another micro-injection pump 1 is set to 30 μL / min-0, and the total flow rate is 30 μL / min.

[0091] The mixed protective agent at the outlet of the microfluidic chip 7 will flow into the microgrid capillary 9 where the cells are placed. The slowly rising concentration of the protective agent will reduce the damage to the cells caused by the sudden change in osmotic pressure, and the cells do not need to be moved again after being loaded into the carrier, thus avoiding mechanical damage caused during the movement.

[0092] The microgrid capillary 9 includes a tube body and a grid; the inner diameter of the tube body is 0.56 mm, the outer diameter is 0.8 mm, the wall thickness is 0.12 mm, the length is 30 mm, and both ends are open; the bottom of the tube body is wrapped with a polyamide grid with a pore size of 0.048 mm, and the grid is fixed by heating at the lower end of the polypropylene tube carrier body using a heatable plastic sealing machine.

[0093] The holding force of the electric clamp 13 is 15N, which can smoothly clamp the cryocarrier microgrid capillary tube 9 away from the steel needle 8 without causing obvious deformation of the cryocarrier microgrid capillary tube 9. The overlap of the connection between the cryocarrier microgrid capillary tube 9 and the steel needle 8 by 3mm can ensure that no overflow occurs at the gap of the connection during the loading process. The electric clamp 13 clamps the cryocarrier microgrid capillary tube 9. The first stage of the descending distance is 6mm.

[0094] In this embodiment, the microinjection pump 1 may be a Harvard microinjection pump Pump 11 Pico Plus Elite (Harvard, USA).

[0095] In this embodiment, the electric gripper 13 may be a JODELL EPG-M series modular electric gripper.

[0096] In this embodiment, the micro electric linear actuator is a commercially available or purchased micro electric linear actuator.

[0097] In this embodiment, the relay control box 2 may use an 8-way 24V relay module.

[0098] In this embodiment, the liquid nitrogen tank is a 300 mL liquid nitrogen tank.

[0099] The microfluidic chip 7 is an S-shaped channel mixing system made of a polymethyl methacrylate (PMMA) plate, with a size of 33mm×33mm×1.75mm, a total length of a mixing channel of 55mm, and a channel width of 0.2mm. In order to reduce the resistance during the flow of liquid and simplify the solution of fixing the chip to the Z axis, the outlet direction is designed to be parallel to the microfluidic chip 7 during the manufacturing process. In order to prevent the steel needle 8 from being parallel to the microfluidic chip 7 and causing overflow, the specifications of the steel needle 8 at the outlet part are selected to be consistent with the thickness of the PMMA plate, both of which are 0.5mm. The solution outlet of the top layer of the microfluidic chip 7 is connected with a steel needle 8 with an outer diameter of 0.6mm and the inlet is connected with a silicone tube 19 with an inner diameter of 0.5mm. The inlet and outlet are fixed and sealed with UV light curing glue to prevent solution leakage.

[0100] Example 2

[0101] This embodiment provides an application method of an oocyte / embryo automatic vitrification and thawing device in loading and removing an oocyte / embryo cryoprotectant, including a cryoprotectant loading method.

[0102] This embodiment is a method for loading an oocyte / embryo cryoprotectant, wherein the cryopreservation method uses an oocyte / embryo automatic vitrification and rewarming device, and the cryopreservation method comprises the following steps:

[0103] a) First, the buffer solution and the cryoprotectant are each loaded into a 1 mL microinjection needle 18, and a 0.5 mm silicone tube 19 is used to connect the outlet of the microinjection needle 18 and the injection port of the microfluidic chip 7, and the microinjection needle 18 of the buffer solution is pushed until the buffer solution fills the microfluidic chip 7;

[0104] b) after the oocyte is moved to the cryocarrier microgrid capillary 9 by using the egg transfer needle, the microgrid capillary 9 is connected to the steel needle 8 at the outlet of the microfluidic chip 7, the microfluidic chip 7 is fixed on the structure-chip bracket 24, a hole is reserved at a corresponding position on the structure-chip bracket 24 for the microgrid capillary 9 to extend out of the structure-chip bracket 24, and the microgrid capillary 9 is just in the slot below after extending out of the small hole;

[0105] c) Click the program loading switch and then turn on the foot switch. The two micro-injection pumps 1 will work simultaneously. At the same time, LED light 1 will light up to indicate that the injection program is in progress. After the program runs for 8 minutes, micro-injection pump 1 will automatically stop injecting.

[0106] Example 3

[0107] This embodiment provides an application method of an oocyte / embryo automatic vitrification and thawing device in loading and removing an oocyte / embryo cryoprotectant, including a cryoprotectant loading method, a cryopreservation method, and a thawing and cryoprotectant removal method.

[0108] This embodiment provides an oocyte / embryo cryopreservation method, wherein the cryopreservation method uses an oocyte / embryo automatic vitrification and thawing device, and the cryopreservation method comprises the following steps:

[0109] a) The electric gripper 13 fixed on the Z-axis slider 23 moves downward by 3 mm under the movement of the Z-axis slider 23, the top of the microgrid capillary 9 is separated from the steel needle 8 at the outlet of the microfluidic chip 7, and the bottom is close to the water-absorbing material 11 for 2 seconds, and the remaining sample in the microgrid capillary 9 is sucked out;

[0110] b) After the suction step is completed, the electric clamp 13 holding the microgrid capillary 9 is rapidly lowered, and at the same time, the liquid nitrogen tank connected to the X-axis is rapidly moved to the left to just below the microgrid capillary 9, the electric gripping fingers 10 are extended, and the microgrid capillary 9 freely falls into the liquid nitrogen tank, completing the vitrification procedure.

[0111] Example 4

[0112] This embodiment provides an application method of an oocyte / embryo automatic vitrification and thawing device in loading and removing an oocyte / embryo cryoprotectant, including a method for removing the cryoprotectant.

[0113] This embodiment provides a method for removing an oocyte / embryo cryoprotectant, the method using an oocyte / embryo automatic vitrification and rewarming device, and the method comprising the following steps:

[0114] a) preheating the rewarming reagent to 37° C., making droplets in a culture dish, taking out the microgrid capillary 9 from the liquid nitrogen tank, placing the microgrid capillary 9 in the rewarming reagent at 37° C. for 1 min, loading the buffer solution and the rewarming solution into a 1 ml microinjection needle 18, and connecting the microinjection needle and the inlet of the microfluidic chip 7 with a 0.5 mm silicone tube 19;

[0115] b) After 1 minute, the microgrid capillary 9 is connected to the steel needle 8 at the outlet of the microfluidic chip 7, and the rewarming program switch is clicked. The No. 1 LED light is on, indicating that the injection program is in progress, and the injection is performed. After 8 minutes, the injection program ends and the microinjection pump 1 stops injecting;

[0116] c) LED light No. 2 lights up, indicating that the transfer procedure is in progress, and the transfer is carried out. The electric gripper 13 descends to hold the microgrid capillary 9 away from the microfluidic chip 7, and the electric gripper finger 10 of the electric gripper 13 is released, and the microgrid capillary 9 is held in hand;

[0117] d) connecting a pipette to the top opening of the microgrid capillary 9, immersing the bottom grid of the microgrid capillary 9 in the BS solution, aspirating twice to make the liquid into droplets, observing the droplets and finding cells in the droplets;

[0118] e) Use an egg transfer needle to transfer the cells from the BS droplet to the M2 culture medium for culture.

[0119] Example 5

[0120] This embodiment provides an application method of an oocyte / embryo automatic vitrification and thawing device in loading and removing an oocyte / embryo cryoprotectant, including a vitrification process.

[0121] The vitrification process (vitrification procedure) includes the following steps:

[0122] Step 1: Turn on the power, the device is powered on, and LED light 1 lights up, indicating that the device is ready.

[0123] Step 2: Turn on the foot switch, start the microinjection pump 1, and perform the injection procedure with a delay of 480 seconds.

[0124] Step 3: The foot switch is turned off, microinjection pump 1 stops injecting, and LED light No. 2 lights up, indicating that the injection is completed and the next step can be carried out.

[0125] Step 4: The electric gripper 13 is powered on and starts to move, and two fingers are contracted to clamp the carrier microgrid capillary 9.

[0126] Step 5: The micro electric push rod 12 is powered on and performs a contraction movement for 0.15 s. It is known that the running speed of the micro electric push rod 12 is 30 mm / s. At this time, the micro electric push rod 12 moves 4.5 mm, and the carrier micro grid capillary 9 is facing the water absorbent material 11 in the No. 2 card slot.

[0127] Step 6: The Z-axis slider 23 moves downward by 6 mm, so that the bottom grid of the carrier microgrid capillary 9 is close to the water-absorbing material 11 in the No. 2 card slot, and the X-axis slider 22 moves leftward by 100 mm, so that the liquid nitrogen tank is directly under the carrier microgrid capillary 9. The delay is 3 seconds, of which 2 seconds is the waiting time to ensure that the remaining liquid in the carrier microgrid capillary 9 is sucked out and the remaining sample volume is minimized, and 1 second is the contraction movement of the micro electric push rod 12 for 1 second.

[0128] Step 7: The Z-axis slider 23 moves downward by 117 mm, and the electric gripper 13 stays just above the liquid nitrogen tank.

[0129] Step 8: The electric gripper fingers 10 are extended, and the carrier microgrid capillary 9 falls into the liquid nitrogen tank.

[0130] Step 9: The X-axis slider 22 moves 100mm to the right, the Z-axis slider 23 moves 123mm upward, and the micro electric push rod 12 passes positive current and extends 10mm. LED light No. 3 lights up, indicating that the whole program is completed.

[0131] Example 5

[0132] This embodiment provides an application method of an oocyte / embryo automatic vitrification and rewarming device in loading and removing an oocyte / embryo cryoprotectant, including a rewarming process.

[0133] Rewarming and protective agent removal process (protective agent removal procedure):

[0134] Preheat the rewarming reagent to 37° C., make droplets in a culture dish, take out the microgrid capillary 9 from the liquid nitrogen tank, and place the microgrid capillary 9 in the rewarming reagent at 37° C. and let it stand for 1 minute.

[0135] The microgrid capillary 9 is connected to the steel needle 8 at the outlet of the microfluidic chip 7 to start the protective agent removal process.

[0136] Step 1: Turn on the power, the device is powered on, and LED light 1 lights up, indicating that the device is ready.

[0137] Step 2: Turn on the foot switch, start the microinjection pump 1, and perform the injection procedure with a delay of 480 seconds.

[0138] Step 3: The foot switch is turned off, microinjection pump 1 stops injecting, and LED light No. 2 lights up, indicating that the injection is completed and the next step can be carried out.

[0139] Step 4: The electric gripper 13 is powered on and starts to move, and two fingers are contracted to clamp the carrier microgrid capillary 9.

[0140] Step 5: The micro electric push rod 12 is powered on and performs a contraction movement for 0.4 seconds. It is known that the running speed of the micro electric push rod 12 is 30 mm / s. At this time, the micro electric push rod 12 moves 10 mm and is completely retracted.

[0141] Step 6: The Z-axis slider 23 moves downward 123 mm.

[0142] Step 7: The electric gripper fingers 10 are extended to hold the carrier microgrid capillary 9.

[0143] Step 8: The Z-axis slider 23 moves upward by 123 mm, and the micro electric push rod 12 passes positive current and extends by 10 mm. LED light No. 3 lights up, indicating that the whole program is finished.

[0144] Example 6

[0145] This embodiment provides an application method of an oocyte / embryo automatic vitrification and thawing device in oocyte / embryo cryoprotectant loading and removal, including a protectant loading-removal scheme.

[0146] The specific implementation method of the loading-removal scheme of the protective agent 8min quadratic function is as follows:

[0147] Regarding the loading method of the protective agent, it is considered that gradually increasing the concentration of the protective agent may reduce the osmotic damage to the cells. Under the premise of controlling the total flow rate to 30μL, the injection curve was tested with an 8-min loading time. The results showed that the oocyte survival rate, cleavage rate, and blastocyst rate all reached the highest. The total loading flow rate of VS and BS was 30μL / min, the number of segments was 4, and the duration was 8min. The segmented loading curve was given by the quadratic equation y=ax 2 (x is time, y is rate). Given a total flow rate of 30 μL / min and a total duration of 8 min, we know that this function passes through points (0,0) and (8,30), so we can get a=30 / 8 2 , so the quadratic function is y = (30 / 8 2 )x 2 . The microinjection pump can realize piecewise linear function. In order to make the loading curve closer to the function curve, the value is taken every 1 minute. The injection speed of one microinjection pump 1 is set to 0-30μL / min, and the injection speed of the other microinjection pump 1 is set to 30μL / min-0, and the total flow rate is 30μL / min. During the protective agent loading process, BS=30-VS, and during the protective agent removal process, BS=30-TS. The loading-removal curve of the quadratic function for 8 minutes can be obtained as shown Figure 3 shown.

[0148] Example 7

[0149] This embodiment provides an application method of an oocyte / embryo automatic vitrification and thawing device in loading and removing oocyte / embryo cryoprotectants, including a cryoprotectant loading method, an oocyte / embryo vitrification method, a cryoprotectant removal method, and an oocyte / embryo thawing method.

[0150] Among them, the specific steps of loading the cryoprotectant are:

[0151] a) Use a microinjection needle 18 to draw 0.5 ml of basic solution (BS) and vitrification solution (VS), respectively, and fix the 1 ml microinjection needle 18 on the corresponding microinjection pump 1. Use two 1 ml microinjection needles 18 to draw 0.5 ml of BS and VS, respectively, and fix the microinjection needles 18 on the corresponding microinjection pumps 1, and use a 0.5 mm silicone tube 19 to connect the outlet of the microinjection needle 18 to the inlet of the microfluidic chip 7.

[0152] b Place the connected microfluidic chip 7 on the structure-chip holder 24 at the top of the Z-axis guide rail 14. The steel needle 8 at the outlet of the microfluidic chip 7 extends out of the structure-chip holder 24 through the hole reserved at the lower end of the structure-chip holder 24, so as to facilitate better connection with the carrier (microgrid capillary 9) later.

[0153] c) Push the 1mL microinjection needle 18 loaded with BS to fill the inside of the microfluidic chip 7 with BS. Set the flow rate range of BS to 30-0μL / min and the flow rate range of VS to 0-30μL / min on the microinjection pump 1, and keep the total flow rate of the reagent to 30μL / min.

[0154] d) 200 μL of BS is drawn into a culture dish to form a droplet, and the bottom end of the carrier microgrid capillary 9 is immersed in the droplet. The BS is automatically drawn by the capillary stress of the carrier itself until the carrier is filled.

[0155] e) Using an egg transfer needle, the oocytes / embryos are transferred to a carrier, and the liquid environment around the cells is maintained to prevent the cells from losing water and dying. Under a stereomicroscope, it is observed that the cells exist in the liquid column and are in good condition, which confirms that the vitrification process can be carried out.

[0156] f) Connect the loaded carrier to the steel needle 8 at the outlet of the microfluidic chip 7, with the two overlapping by 3mm to ensure that there is no leakage during the liquid loading process. At this time, the carrier is exactly located in the center of the two electric gripping fingers 10 of the electric gripping clamp 13.

[0157] g) Select the program to be executed on the control panel and click Start. The loading program starts, the foot switch is powered on, and the two micro-injection pumps 1 start working at the same time according to the preset injection program. The reagent will be injected at the pre-set flow rate, and the medical absorbent dressing (absorbent material 11) will absorb the dripping reagent in time.

[0158] Among them, oocyte / embryo vitrification method:

[0159] a) After the injection procedure lasts for 8 minutes, the microinjection pump 1 automatically stops the injection. The electric gripper finger 10 contracts to clamp the carrier. When the electric gripper 13 grabs the carrier and stops contracting, it ensures that the carrier will not be squeezed and deformed. The carrier can be separated from the outlet of the microfluidic chip 7 and then dropped 6mm. At this time, the carrier is separated from the steel needle 8 at the outlet of the microfluidic chip 7, and the top of the tube body contacts the air, and the pressure is the same as the atmospheric pressure. After dropping 1mm, the grid at the lower end of the carrier is tightly attached to the medical absorbent dressing, and the liquid in the carrier is sucked out. At this time, try to minimize the amount of liquid in the carrier.

[0160] b) After the liquid is removed, the carrier carrying the cells descends 100 mm in 1 second on the Z axis. At the same time, the liquid nitrogen tank moves 100 mm to the left to the bottom of the carrier, and the carrier enters the liquid nitrogen tank for vitrification.

[0161] Among them, the specific steps of removing the cryoprotectant are:

[0162] a) Preheat the thawing solution (TS) to 37°C in an incubator, make droplets in a culture dish, take out the carrier from the cryotube, quickly put it into the TS droplet, and wait for the white frost on the surface of the microgrid capillary 9 to disappear before connecting it to the outlet of the microfluidic chip 7.

[0163] b) TS and BS are loaded into the microinjection needle respectively, and the microinjection needle 18 is fixed on the corresponding microinjection pump 1. The microinjection needle 18 is connected to the inlet of the microfluidic chip 7 using a 0.5 mm silicone tube 19, and the microinjection needle 18 containing TS is pushed to fill the microfluidic chip 7.

[0164] c) Click the rewarming program switch, the device starts working, and LED light 1 lights up, indicating that the injection program is currently in progress. The foot switch is powered on, and the two micro-injection pumps 1 start working at the same time. The flow rate range of TS is 30-0μL / min, and the flow rate range of BS is 0-30μL / min. The total flow rate is maintained at 30μL / min.

[0165] d) After 8 minutes, the injection procedure ends and the microinjection pump 1 stops injecting. LED light No. 2 lights up, indicating the next step of operation. The carrier is separated from the outlet of the microfluidic chip 7, the bottom grid is immersed in the M2 solution, the upper opening is connected to the pipette and blown and sucked twice, and then the M2 solution containing the oocytes is blown into the culture dish.

[0166] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An oocyte / embryo automatic vitrification and rewarming device, used for loading / removing and freezing oocyte / embryo protective agents, characterized in that: The device comprises a microfluidic mixing mechanism, a mechanical slide mechanism, a water absorbing material (11), a micro electric push rod (12), an electric clamp (13), and a receiving member (20); The microfluidic mixing mechanism comprises a microinjection pump (1), a microinjection needle (18), a silicone tube (19), a microfluidic chip (7), a steel needle (8), and a microgrid capillary (9); The microinjection pump (1) is connected to a microinjection needle (18); The silicone tube (19) is connected to the microinjection needle (18); The silicone tube (19) is connected to the microfluidic chip (7); The microfluidic chip (7) is connected to the steel needle (8); The steel needle (8) and the microgrid capillary (9) are arranged opposite to each other; The microgrid capillary (9) serves as a freezing carrier; The water-absorbing material (11) is arranged below the microgrid capillary (9) and is used to absorb the protective agent flowing out of the microgrid capillary (9) during the loading process and to absorb the excess protective agent after the loading is completed to minimize the amount of residual liquid; The mechanical slide mechanism includes an X-axis assembly and a Z-axis assembly connected to the X-axis assembly; The receiving member (20) is connected to the X-axis assembly; The Z-axis assembly is provided with a micro electric push rod (12); The water absorbing material (11) is connected to the micro electric push rod (12); The electric clamp (13) is connected to the Z-axis assembly; The electric gripper (13) comprises an electric gripper finger (10); The electric gripper fingers (10) are used to grip and place down the microgrid capillary (9); The receiving component (20) is a liquid nitrogen tank.

2. An oocyte / embryo automatic vitrification and rewarming device according to claim 1, characterized in that: The microgrid capillary (9) comprises a tube body and a grid; The bottom of the tube body is wrapped with a mesh.

3. The oocyte / embryo automatic vitrification and rewarming device according to claim 1, characterized in that: The mechanical slide mechanism includes an X-axis assembly and a Z-axis assembly connected to the X-axis assembly; The X-axis assembly comprises an X-axis motor (16), an X-axis guide rail (17), and an X-axis slide block (22); The X-axis slider (22) is slidably connected to the X-axis guide rail (17); The X-axis motor (16) is connected to the X-axis slider (22) to drive the X-axis slider (22) to slide on the X-axis guide rail (17); The receiving member (20) is connected to the X-axis slider (22); The Z-axis assembly comprises a Z-axis motor (15), a Z-axis guide rail (14), and a Z-axis slide block (23); The Z-axis slider (23) is slidably connected to the Z-axis guide rail (14); The Z-axis motor (15) is connected to the Z-axis slider (23) to drive the Z-axis slider (23) to slide on the Z-axis guide rail (14); A structure-chip support (24) is provided at the top end of the Z-axis guide rail (14), and the structure-chip support (24) is used to place a microfluidic chip (7) for mixing the protective agent; A micro electric push rod (12) is provided on the Z-axis guide rail (14); The head of the micro electric push rod (12) is provided with a placement slot (21), and the placement slot (21) is used to place the water absorbing material (11); The electric clamp (13) is connected to the Z-axis slider (23).

4. The oocyte / embryo automatic vitrification and rewarming device according to claim 1, characterized in that: The device also includes an electric control mechanism; The electric control mechanism comprises a relay control box (2), an LED lamp (3), and a controller (4); The controller (4) is connected to the microinjection pump (1), the relay control box (2), and the LED lamp (3) respectively; The motor driver (6) is connected to the X-axis motor (16) and the Z-axis motor (15) respectively; The LED lights (3) include LED No. 1, LED No. 2, and LED No. 3, all of which are used to indicate the stage process.

5. An oocyte / embryo automatic vitrification and rewarming device according to claim 4, characterized in that: Two microinjection pumps (1) are provided. Two microinjection pumps (1) are connected via a linkage switch; The relay control box (2) is used to control the start and stop of the linkage switch by powering on and off, thereby controlling the working state of the microinjection pump (1), and to operate at preset time intervals through a program on the control panel.

6. The oocyte / embryo automatic vitrification and rewarming device according to claim 1, characterized in that: The water-absorbing material (11) is a medical water-absorbing dressing; Two pieces of water-absorbing material (11) are provided.

7. An oocyte / embryo automatic vitrification and rewarming device according to claim 1, characterized in that: The microfluidic chip (7) was used to load and remove the protective agent. The loading and removal time was 8 min. y = (30 / 8 2 )x 2 The loading-removal curve was optimized to obtain the highest oocyte / embryo survival rate.

8. A method for loading an oocyte / embryo cryoprotectant, the method using the oocyte / embryo automatic vitrification and rewarming device according to any one of claims 1 to 7, characterized in that: The loading method comprises the following steps: a) firstly, a buffer solution and a cryoprotectant are respectively loaded into a microinjection needle (18), a silicone tube (19) is used to connect the outlet of the microinjection needle (18) and the injection port of the microfluidic chip (7), and the microinjection needle (18) of the buffer solution is pushed until the buffer solution fills the microfluidic chip (7); b) after the oocyte is transferred into the cryocarrier microgrid capillary (9) by using an egg transfer needle, the microgrid capillary (9) is connected to the steel needle (8) at the outlet of the microfluidic chip (7), the microfluidic chip (7) is fixed on the structure-chip support (24), a hole is reserved at a corresponding position on the structure-chip support (24) for the microgrid capillary (9) to extend out of the structure-chip support (24), and the microgrid capillary (9) is just in the slot below after extending out of the small hole; c) Two micro-injection pumps (1) work simultaneously, and the micro-injection pumps (1) stop injecting after the program runs for 8 minutes.

9. A method for cryopreservation of oocytes / embryos, the method using the oocyte / embryo automatic vitrification and thawing device according to any one of claims 1 to 7, characterized in that: The cryopreservation method comprises the following steps: a) the electric gripper (13) fixed on the Z-axis slider (23) moves downward by 3 mm under the movement of the Z-axis slider (23), the top of the microgrid capillary (9) is separated from the steel needle (8) at the outlet of the microfluidic chip (7), and the bottom is closely attached to the water-absorbing material (11) and stays for 2 seconds, and the remaining sample in the microgrid capillary (9) is sucked out; b) After the suction step is completed, the electric clamp (13) clamps the microgrid capillary (9) and descends, and at the same time, the liquid nitrogen tank connected to the X-axis moves to the bottom of the microgrid capillary (9), the electric gripping fingers (10) extend, and the microgrid capillary (9) falls freely into the liquid nitrogen tank, completing the vitrification process.

10. A method for removing an oocyte / embryo cryoprotectant, the method using the oocyte / embryo automatic vitrification and rewarming device according to any one of claims 1 to 7, characterized in that: The removal method comprises the following steps: a) preheating the rewarming reagent to 37° C., making droplets in a culture dish, taking out the microgrid capillary (9) from the liquid nitrogen tank, placing the microgrid capillary (9) in the rewarming reagent at 37° C. and letting it stand for 1 minute, respectively loading the buffer solution and the rewarming solution into a microinjection needle (18), and connecting the microinjection needle and the entrance of the microfluidic chip (7) with a silicone tube (19); b) After 1 minute, the microgrid capillary (9) is connected to the steel needle (8) at the outlet of the microfluidic chip (7) to perform injection. After 8 minutes, the injection procedure is completed and the microinjection pump (1) stops injecting; c) transferring, the electric gripper (13) descends to clamp the microgrid capillary (9) away from the microfluidic chip (7), the electric gripper finger (10) of the electric gripper (13) is released, and the microgrid capillary (9) is held; d) connecting a pipette to the top opening of the microgrid capillary (9), immersing the bottom grid of the microgrid capillary (9) in the BS solution, aspirating twice to make the liquid into droplets, observing the droplets and finding cells in the droplets; e) Use an egg transfer needle to transfer the cells from the BS droplet to the M2 culture medium for culture.

Images