An embryo freezing dish and a method of embryo vitrification

Abstract

The application discloses an embryo freezing dish and an embryo vitrification method, and belongs to the field of medical devices. The embryo freezing dish is provided with a receiving hole and at least two liquid storage pools. The receiving hole is used for accommodating embryos, and the liquid storage pools are used for containing vitrification solutions. The at least two liquid storage pools are respectively communicated with the receiving hole. A gland is installed on a dish body. A stand column extends into the dish body and is located at the upper end of the receiving hole. A micro flow channel is formed between the stand column and the receiving hole. The micro flow channel is communicated with the at least two liquid storage pools. Through the above design, different volumes of reagents are injected into the two liquid storage pools. The reagents are communicated through the micro flow channel but do not flow. The reagent on the side with a small volume is sucked. Under the liquid tension, the liquid on the other side will flow to the other side through the micro flow channel and be sucked. In the process, the vitrification reagent will pass through the receiving hole and can fully soak the embryo, realizing the washing effect on the embryo and achieving the vitrification treatment effect on the embryo. In the embryo vitrification and freezing operation process, the embryo does not need to be transferred from the freezing dish, so that the damage is reduced.

Description

Technical Field

[0001] This invention relates to the field of medical devices, and in particular to an embryo freezing dish and a method for vitrifying embryos. Background Technology

[0002] In assisted reproductive technology (IVF) cycles, cryopreservation technology has been successfully applied to oocytes or fertilized eggs. Embryo cryopreservation allows for the reasonable limitation of the number of embryos transferred, effectively reducing the rate of multiple pregnancies. It also provides a second chance for patients who have experienced failed fresh embryo transfer cycles or miscarriages, reducing costs and alleviating patient suffering. Vitrification, a technique that has emerged in recent years, offers advantages over traditional programmed freezing. Programmed freezing requires cooling from room temperature to the freezing point, then slowly lowering to the temperature before immersion in liquid nitrogen (-80℃ to -100℃), a more cumbersome process. Vitrification, on the other hand, allows for direct and rapid immersion in liquid nitrogen. Therefore, vitrification is simple, fast, and economical, offering significant advantages and clinical application value compared to programmed freezing, which requires sophisticated cryopreservation equipment, is complex to operate, time-consuming, and consumes large amounts of liquid nitrogen. Vitrification is a freezing method in which a high-concentration cryoprotectant solution solidifies upon freezing, resulting in a dramatic increase in viscosity and a transformation from a liquid to a structureless glassy state. This method effectively preserves the molecular and ionic distribution of cells, maintains relatively low transmembrane solute concentration and osmotic pressure difference, and minimizes damage to cell membranes and organelles. This technique uses a high-concentration cryoprotectant solution to displace intracellular water, followed by rapid cooling (15,000–30,000 °C / min) to transform the liquid state into a glassy, ​​amorphous solid. Therefore, it is a result of combining rapid freezing with a high-concentration cryoprotectant.

[0003] Current artificial vitrification methods involve exposing embryos to two or three vitrification solutions using conventional petri dishes or multiwell plates. In a typical protocol, the embryo is first transferred to the first solution, then to the second solution using a pipette, and so on, repeating the washing process in multiple solutions over a predetermined time period until it is ready for cryopreservation. Finally, the droplet containing the embryo is aspirated onto a cryopreservation apparatus (such as a cryopreservation carrier), transferred, and directly immersed in liquid nitrogen for long-term cryopreservation.

[0004] In the existing embryo vitrification and freezing process, the embryos need to be physically transferred multiple times, which can cause potential damage, and the process cannot be automated. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide an embryo freezing dish in which the embryo does not need to be transferred from the freezing dish during the vitrification and freezing process.

[0006] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide an embryo vitrification method in which the embryo does not need to be transferred from the freezing dish during the embryo vitrification and freezing process.

[0007] One of the objectives of this invention is achieved through the following technical solution:

[0008] An embryo freezing dish includes a dish body with a receiving hole and at least two liquid reservoirs. The receiving hole is used to receive embryos, and the liquid reservoirs are used to contain vitrification solution. The at least two liquid reservoirs are respectively connected to the receiving hole. The embryo freezing dish also includes a cap with a column. The cap is installed on the dish body, and the column extends into the dish body and is located at the upper end of the receiving hole. A microchannel is formed between the column and the receiving hole, and the microchannel is connected to the at least two liquid reservoirs.

[0009] Furthermore, the receiving hole is located at the intersection of at least two of the liquid storage pools, and the width of each liquid storage pool narrows near the receiving hole, forming a slit area at the intersection of the at least two liquid storage pools.

[0010] Furthermore, the width of the slit area is 1-1.4mm, the gap between the side wall of the column and the side wall of the slit area is 0.01-0.03mm, the inner diameter of the storage hole is 0.3-0.5mm, and the distance between the end of the column and the top of the storage hole is 0.08-0.1mm.

[0011] Furthermore, the vessel body includes two surrounding plates and a bottom plate, and the two surrounding plates and the bottom plate are fixedly connected to form two liquid storage pools.

[0012] Furthermore, the enclosure is in the shape of a reverse bow, the storage hole is provided on the bottom plate, the two ends of the bottom plate are inclined, and the lowest point of the bottom plate is near the storage hole.

[0013] Furthermore, each of the enclosure panels includes a panel body and two limiting portions. The limiting portions extend from the outer wall of the panel body and are spaced apart. The cover also includes a snap-on plate. The snap-on plate has a contact surface on each of its two end faces. The snap-on plate is snapped between the two limiting portions, and the contact surface abuts against the limiting portions.

[0014] Furthermore, the vessel also includes an outer wall, with the two surrounding plates and the bottom plate located inside the outer wall and fixedly connected to it, forming a clamping space between the plates and the outer wall.

[0015] Furthermore, the outer wall includes a main body and a boss, the boss being located on top of the main body, and the width of the boss being greater than the width of the main body to facilitate sealing of the freezing dish.

[0016] Furthermore, the pressure cap also includes a top plate, the column and the two buckle plates extend from the top plate, the column is located between the two buckle plates, the top plate has an opening, and the position of the opening corresponds to the liquid storage tank.

[0017] Furthermore, the top plate is X-shaped.

[0018] Furthermore, the dish body is integrally formed.

[0019] Furthermore, the bottom of the storage hole is made of a transparent material.

[0020] The second objective of this invention is achieved by the following technical solution:

[0021] An embryo vitrification method, performed using any of the above-mentioned embryo cryopreservation dishes, includes the following steps:

[0022] S1: Place the embryonic cells into the receiving hole;

[0023] S2: Install the cap onto the dish body;

[0024] S3: Extend the pipette to the bottom of one side of the reservoir and inject vitrification reagent 1. The volume of reagent 1 is A.

[0025] S4: The pipette is extended to the bottom of the other reservoir and vitrification reagent 1 is injected. The volume of reagent 1 is B, which is less than A. The vitrification reagent 1 in the two reservoirs is connected through the microchannel, but due to the surface tension of the micro liquid, the liquids on both sides will not flow freely and there is still a height difference.

[0026] S5: The pipette slowly draws up the vitrification reagent 1 from the smaller side. Under the liquid tension, the liquid in the larger side reservoir will slowly flow through the microchannel between the dish body and the cap to the other reservoir and be drawn away. During this process, the vitrification reagent will pass through the receiving hole and fully wet the embryo, achieving the washing effect on the embryo and achieving the vitrification treatment effect.

[0027] S6: Repeat steps S4 and S5, using different vitrification reagents to treat the embryos.

[0028] Compared to existing technologies, the embryo freezing dish of this invention has a receiving hole and at least two liquid reservoirs. The receiving hole is used to hold embryos, and the liquid reservoirs are used to contain vitrification solution. The at least two liquid reservoirs are respectively connected to the receiving hole. The embryo freezing dish also includes a cap, which includes a column. The cap is installed on the dish body, and the column extends into the dish body and is located at the upper end of the receiving hole. A microchannel is formed between the column and the receiving hole. The microchannel connects to the at least two liquid reservoirs. Through the above design, reagents of different volumes are injected into the two liquid reservoirs. The reagents are connected through the microchannels but do not flow. The reagent on the side with the smaller volume is drawn in. Under the liquid tension, the liquid on the side with the larger volume will slowly flow through the microchannel to the other liquid reservoir and be drawn away. During this process, the vitrification reagent will pass through the receiving hole and can fully wet the embryo, achieving a washing effect on the embryo and achieving the vitrification treatment effect. During the vitrification and freezing operation, the embryo does not need to be transferred from the freezing dish, reducing damage. Attached Figure Description

[0029] Figure 1 This is a perspective view of the embryo freezing dish of the present invention;

[0030] Figure 2 for Figure 1 A three-dimensional view of the body of an embryo cryopreservation dish;

[0031] Figure 3 for Figure 2 Another three-dimensional view of the dish;

[0032] Figure 4 for Figure 1 A three-dimensional view of the capping of an embryo freezing dish;

[0033] Figure 5 for Figure 4 Another perspective view of the cap;

[0034] Figure 6 for Figure 1 A three-dimensional cross-sectional view of an embryo freezing dish;

[0035] Figure 7 for Figure 1 A cross-sectional view of an embryo freezing dish;

[0036] Figure 8 for Figure 7 A magnified view of point A in the embryo cryopreservation dish.

[0037] In the diagram: 220, dish body; 2201, outer wall; 22010, main body; 22011, boss; 2202, partition; 2203, surrounding plate; 22031, plate body; 22030, limiting part; 2204, clamping space; 2205, liquid storage tank; 2206, storage hole; 2207, bottom plate; 2208, microchannel; 221, pressure cap; 2210, top plate; 22101, opening; 2211, column; 2212, reinforcing block; 2213, snap plate; 22130, contact surface; 22131, guiding surface; 200, embryo. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or it can be fixed through another intermediate component. When a component is said to be "connected to" another component, it can be directly connected to the other component or it may be fixed through another intermediate component. When a component is said to be "set on" another component, it can be set directly on the other component or it may be set through another intermediate component. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0041] like Figure 1 As shown, the embryo freezing dish of the present invention includes a dish body 220 and a cap 221.

[0042] Please continue reading. Figure 2 as well as Figure 3 The vessel body 220 is integrally formed. The vessel body 220 includes an outer wall 2201, a partition 2202, a surrounding plate 2203, and a bottom plate 2207.

[0043] The outer wall 2201 has an annular hollow structure; in this embodiment, the outer wall 2201 is rectangular. The outer wall 2201 includes a main body 22010 and a boss 22011. The boss 22011 extends from the top of the main body 22010, and its width is greater than the width of the main body 22010. When the embryo freezing dish is sealed, the boss 22011 is pressed against the sealing film. Therefore, appropriately designing a slightly larger width for the boss 22011 can ensure the reliability of the heat seal. Preferably, the width of the boss 22011 is 1.3 mm.

[0044] The partition 2202 is located inside the outer wall 2201 and extends from the middle of the outer wall 2201 to the enclosure 2203, thereby strengthening the structural strength of the vessel body 220.

[0045] The enclosure 2203 is used to form the liquid storage tank 2205. Specifically, there are two enclosures 2203, which are symmetrically arranged. Each enclosure 2203 extends from one end of the outer wall 2201 to the opposite end. The height of the enclosure 2203 is lower than the height of the outer wall 2201, so that there is space above the enclosure 2203 for installing the pressure cap 221. A clamping space 2204 is formed between each enclosure 2203 and the outer wall 2201, and the liquid storage tank 2205 is formed between the two enclosures 2203. Each enclosure 2203 includes a plate body 22031 and a limiting part 22030. The plate body 22031 is curved, which divides the liquid storage tank 2205 into two. The two liquid storage tanks 2205 are connected and form a narrow area. The narrow area facilitates the flow of fluid from one side to the other. Specifically, the plate 22031 is in the shape of an inverted bow, and there are two limiting parts 22030. The two limiting parts 22030 extend from the outer wall of the plate 22031 and are located within the clamping space 2204. The two limiting parts 22030 are spaced apart, and a snap-fit ​​plate 2213 is used between the two limiting parts 22030 to snap the cap 221, so that the cap 221 is installed on the dish body 220. The distance between the two plates 2203 near the outer wall 2201 is 6 mm, and the width of the slit area in the middle is 1.2 mm.

[0046] A base plate 2207 extends from one end of the outer wall 2201 to the other, and its edge connects to the two surrounding plates 2203, forming two liquid storage pools 2205. The liquid storage pools 2205 gradually narrow in width from the outer wall 2201 towards the center of the vessel. The base plate 2207 is inclined at both ends to facilitate fluid flow towards the central slit area under gravity; a typical slope angle is 30°. The slopes of the two liquid storage pools 2205 extend symmetrically downwards, leaving a flat area at the bottom. Preferably, each liquid storage pool 2205 can hold 50 μL of liquid.

[0047] A receiving hole 2206 is provided on the bottom plate 2207 at the center bottom of the slit area. The inner diameter of the receiving hole 2206 is 0.3-0.5 mm, preferably 0.4 mm. The depth of the receiving hole 2206 is 0.2-0.4 mm, preferably 0.3 mm. The receiving hole 2206 is used to accommodate the embryo to be processed. The local wall thickness of the bottom and sides of the receiving hole 2206 is 0.1-0.2 mm. To ensure the feasibility of the injection molding process, a thin-walled transition zone is designed in the receiving hole 2206 and the flat bottom to avoid abrupt changes in wall thickness that would prevent molding. The purpose of the local thin-wall treatment is to minimize thermal resistance and improve the heat conduction efficiency inside and outside the dish, so that the embryo freezing dish can be quickly frozen when placed in a liquid nitrogen environment.

[0048] The bottom wall of the storage well 2206 is made of transparent material, which makes it easy to observe the changes in embryo morphology caused by the introduction of reagents at each step of the vitrification process.

[0049] Please continue reading. Figure 4 as well as Figure 5 The cover 221 includes a top plate 2210, a column 2211, and a snap plate 2213.

[0050] The top plate 2210 is used to connect the column 2211 and the snap-fit ​​plate 2213. The top plate 2210 has an opening 22101 to facilitate the vertical insertion of a pipette into the flat bottom of the dish body 220 for liquid injection and aspiration operations. The two sides of the top plate 2210 are arc-shaped, which creates more space between the outer wall 2201 and the top plate 2210, facilitating the gripping operation of the cap 221. Specifically, the top plate 2210 is X-shaped. The thickness of the top plate 2210 is equal to the height difference between the surface of the main body 22010 and the boss 22011, so that after the embryo cryopreservation dish is sealed, the sealing film adheres to the boss 22011, which can just press down on the cap 221 and prevent the cap 221 from falling out in the vertical direction.

[0051] The column 2211 extends from the center of the top plate 2210 and is perpendicular to the top plate 2210. The column 2211 is cylindrical, and its diameter is slightly smaller than the width of the slit area of ​​the dish body 220. The height of the column 2211 is less than the height from the upper surface of the surrounding plate 2203 to the flat bottom, so as to facilitate the formation of the microchannel 2208.

[0052] There are two snap-fit ​​plates 2213, which extend from both sides of the top plate 2210 and are perpendicular to the top plate 2210. Each snap-fit ​​plate 2213 is arc-shaped, with its convex surface facing the column 2211. Four contact surfaces 22130 are provided at the four ends of each snap-fit ​​plate 2213, and these contact surfaces 22130 are parallel to the line connecting the centers of the two snap-fit ​​plates 2213. A guide surface 22131 is provided at the end of each contact surface 22130; the guide surface 22131 is beveled to facilitate the installation of the pressure cap 221.

[0053] Please continue reading. Figures 6 to 8 When using an embryo freezing dish, place the embryo 200 into the receiving hole 2206 and install the cap 221 onto the dish body 220. At this time, the latch plate 2213 moves along the guide surface 22131 to between the two limiting parts 22030. The column 2211 extends into the slit area. The gap between the side wall of the column 2211 and the side wall of the slit is 0.02 mm, and a small gap, between 0.08-0.1 mm, is left between the end face and the bottom surface of the slit. A microchannel 2208 is formed between the column 2211 and the receiving hole 2206. The microchannel 2208 connects the two liquid storage pools 2205 and is located at the upper end of the receiving hole 2206. Since the diameter of the embryo is greater than 0.1 mm, the small gap allows liquid to flow from one side of the liquid storage pool 2205 to the other side, while preventing the embryo from escaping from the receiving hole 2206 with the liquid.

[0054] At this time, the translational freedom of the cap 221 relative to the dish body 220 in the direction of the two reservoirs 205 is constrained by the contact surface 22130 and the limiting part 22030. After assembly, the column 2211 can be kept in the center of the slit area without moving left or right. The pipette is extended to the bottom of one reservoir 2205 and vitrification reagent 1 is injected, with a volume of 40 μL; the pipette is extended to the bottom of the other reservoir 2205 and vitrification reagent 1 is injected, with a volume of 10 μL. The vitrification reagent 1 in the two reservoirs 2205 is connected through the microchannel 2208, but due to the surface tension of the micro-liquid, the liquids on both sides will not flow freely and there is still a height difference; the pipette is slowly drawn from the smaller volume of vitrification reagent 1 on the side with the smaller volume, and the liquid surface tension is maintained. Under pressure, the liquid in the larger storage tank 2205 will slowly flow through the microchannel 2208 between the dish body 220 and the cap 221 to the other storage tank 2205 and be absorbed. During this process, the vitrifying agent will pass through the receiving hole 2206 and fully wet the embryo 200, achieving the washing effect on the embryo 200 and achieving the vitrification treatment effect on the embryo 200. Repeat the above process and use different vitrifying agents (vitrifying agent 2 and vitrifying agent 3) to treat the embryo 200.

[0055] After vitrification of the embryos, most of the reagents are removed, leaving only a small amount (approximately 0.2-0.5 μL) in the storage hole 2206 of the dish 220, encapsulating the embryos. In this state, heat sealing can be performed, for example, by using a hot-pressing process to seal an aluminum film onto the protrusion 22011 of the dish 220. This completely isolates the inside and outside of the dish 220 before immersing it in liquid nitrogen for cryopreservation. The thin-walled treatment of the storage hole 2206 at the bottom of the dish 220 allows for rapid cooling of the embryos in liquid nitrogen, ensuring effective freezing. To further improve liquid flow, the embryo cryopreservation dish proposed in this invention can undergo surface hydrophilic treatment before use. To ensure the material's resistance under liquid nitrogen, PP (polypropylene) is the preferred material for the embryo cryopreservation dish.

[0056] The present invention also relates to a method for vitrifying embryos, implemented using the above-mentioned embryo cryopreservation dish, comprising the following steps:

[0057] S1: Place the embryonic cells into the receiving well and wrap them with 2-3 μL of liquid droplets;

[0058] S2: Install the cap onto the dish body;

[0059] S3: Extend the pipette to the bottom of one side of the reservoir and inject vitrification reagent 1. The volume of reagent 1 is A.

[0060] S4: The pipette is extended to the bottom of the other reservoir and vitrification reagent 1 is injected. The volume of reagent 1 is B, which is less than A. The vitrification reagent 1 in the two reservoirs is connected through the microchannel, but due to the surface tension of the micro liquid, the liquids on both sides will not flow freely and there is still a height difference.

[0061] S5: Use a pipette to slowly aspirate the vitrification reagent 1 from the smaller side at a rate (typically 0.5-5 μL / s). Under liquid tension, the liquid in the larger reservoir will slowly flow through the microchannel between the dish body and the cap to the other reservoir and be aspirated. During this process, the vitrification reagent will pass through the receiving hole and fully wet the embryo, achieving a washing effect on the embryo and achieving the vitrification treatment effect.

[0062] S6: Repeat steps S4 and S5, using different vitrification reagents to treat the embryos.

[0063] Through the above design, reagents of different volumes are injected into two reservoirs 2205. The reagents are connected but not flowing through microchannels 2208. The reagent from the smaller volume side is drawn in, and under liquid tension, the liquid from the larger volume side slowly flows through microchannels 2208 to the other reservoir 2205 and is drawn away. During this process, the vitrification reagent passes through the receiving hole 2206 and fully wets the embryo 200, achieving a washing effect on the embryo 200 and achieving the vitrification treatment effect. During the vitrification and freezing operations, the embryo 200 does not need to be transferred from the freezing dish, reducing damage. The receiving hole 2206 allows for observation of the embryos. After each vitrification reagent treatment, the embryos can be observed through an inverted microscope to evaluate the operation effect.

[0064] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These are all equivalent modifications and improvements made to the above embodiments based on the essential technology of the present invention, and all of these fall within the protection scope of the present invention.

Claims

1. An embryo freezing dish, comprising a dish body, characterized in that: The dish body is provided with a receiving hole and at least two liquid storage pools. The receiving hole is used to receive embryos, and the liquid storage pools are used to contain vitrification solution. The at least two liquid storage pools are respectively connected to the receiving hole. The embryo freezing dish also includes a cap. The cap includes a column. The cap is installed on the dish body. The column extends into the dish body and is located at the upper end of the receiving hole. A microchannel is formed between the column and the receiving hole. The microchannel is connected to the at least two liquid storage pools. The receiving hole is located at the intersection of at least two of the liquid storage tanks, and the width of each liquid storage tank narrows near the receiving hole. At least two liquid storage tanks form a slit area at the intersection. The width of the slit area is 1-1.4 mm, the gap between the side wall of the column and the side wall of the slit area is 0.01-0.03 mm, the inner diameter of the receiving hole is 0.3-0.5 mm, and the distance between the end of the column and the top of the receiving hole is 0.08-0.1 mm. The vessel body includes two surrounding plates and a bottom plate. The two surrounding plates and the bottom plate are fixedly connected and form two liquid storage tanks. The surrounding plates are in the shape of an inverted bow. The receiving hole is provided on the bottom plate. The two ends of the bottom plate are inclined. The lowest point of the bottom plate is near the receiving hole. Each surrounding plate includes a plate body and two limiting parts. The limiting parts extend from the outer wall of the plate body. The two limiting parts are spaced apart. The pressure cap also includes a buckle plate. The two ends of the buckle plate are respectively provided with abutting surfaces. The buckle plate is buckled between the two limiting parts. The abutting surfaces abut against the limiting parts. The dish also includes an outer wall, with the two surrounding plates and the bottom plate located inside the outer wall and fixedly connected to it, forming a clamping space between the plates and the outer wall; the outer wall includes a main body and a boss, with the boss located on top of the main body, and the width of the boss being greater than the width of the main body to facilitate sealing of the freezing dish.

2. The embryo freezing dish according to claim 1, characterized in that: The pressure cap also includes a top plate, the column and the two buckle plates extend from the top plate, the column is located between the two buckle plates, the top plate has an opening, and the position of the opening corresponds to the liquid storage tank.

3. The embryo freezing dish according to claim 2, characterized in that: The top plate is X-shaped.

4. The embryo freezing dish according to any one of claims 1-3, characterized in that: The dish body is integrally molded.

5. The embryo freezing dish according to any one of claims 1-3, characterized in that: The bottom of the storage hole is made of transparent material.

6. A method for vitrifying embryos, implemented using an embryo cryopreservation dish as described in any one of claims 1-3, characterized in that, Includes the following steps: S1: Place the embryonic cells into the receiving hole; S2: Install the cap onto the dish body; S3: Extend the pipette to the bottom of one side of the reservoir and inject vitrification reagent 1. The volume of reagent 1 is A. S4: The pipette is extended to the bottom of the other reservoir and vitrification reagent 1 is injected. The volume of reagent 1 is B, which is less than A. The vitrification reagent 1 in the two reservoirs is connected through the microchannel, but due to the surface tension of the micro liquid, the liquids on both sides will not flow freely and there is still a height difference. S5: Use a pipette to slowly draw up the vitrification reagent 1 from the smaller side. Under the liquid tension, the liquid in the larger side reservoir will slowly flow through the microchannel between the dish body and the cap to the other reservoir and be drawn away. During this process, the vitrification reagent will pass through the receiving hole and fully wet the embryo, achieving the washing effect on the embryo and achieving the vitrification treatment effect. S6: Repeat steps S4 and S5, using different vitrification reagents to treat the embryos.

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