A closed microfluidic culture dish for dynamic embryo exchange and its culture method

Abstract

This invention relates to the field of culture equipment technology, and more particularly to a closed-type embryo dynamic fluid exchange microfluidic culture dish and its culture method. The culture dish includes a main body with a culture pool in the middle. The side of the culture pool is connected to a storage pool and a waste pool via inlet and outlet channels, respectively. The storage pool and waste pool are connected to a dynamic fluid exchange device. The invention also discloses a culture method based on the aforementioned closed-type embryo dynamic fluid exchange microfluidic culture dish. Using this closed-type embryo dynamic fluid exchange microfluidic culture dish and its culture method, the characteristics of microfluidics are utilized to simulate the dynamic microenvironment of an in vivo embryo within the fallopian tube, where it is subjected to smooth muscle compression and friction, the impact of fallopian tube fluid flow, and the continuous renewal of fallopian tube fluid carrying away accumulated metabolic waste. This stimulates embryo growth and development, overcoming the shortcomings of traditional static in vitro embryo culture in culture dishes, effectively preventing the embryo from being swept into the flow channels and effectively reducing the shear force exerted on the embryo by the fluid flowing into the culture pool.

Description

Technical Field

[0001] This invention relates to the field of culture equipment technology, and in particular to a closed microfluidic culture dish for dynamic embryo exchange and its culture method. Background Technology

[0002] Embryo culture dishes are the core in vitro culture medium for assisted reproductive technology. A search revealed patent CN102876573B, which discloses an in vitro culture device and method for mammalian embryos. This method indirectly drives the movement of membranes via a square wave generator, thereby moving the embryo culture medium and mineral oil. It simulates the smooth muscle compression, friction, and laminar shear force of fallopian tube fluid experienced by the embryo within the fallopian tube, creating a dynamic growth environment that stimulates embryonic growth and development, thus overcoming the shortcomings of traditional static in vitro culture dishes or plates. This in vitro embryo culture device achieves dynamic embryo culture by causing the culture medium level to fluctuate regularly. However, the magnetic plates and membranes in the device are prone to separation and detachment, causing the culture device to malfunction. The fixed and non-adjustable displacement of the membranes also prevents the device from adapting to the developmental requirements of different animal embryos. Most importantly, this device only addresses the shortcomings of traditional static, two-dimensional culture dishes and fails to remove accumulated metabolic waste and replenish new nutrients during embryo culture. Patent CN103421682B discloses an automatic embryo culture dish that automatically and quantitatively provides embryos with the necessary nutrients and replaces waste liquid in conjunction with an automatic liquid dispensing pump, reducing the damage to embryos caused by repeated moving and changing of the medium during embryo culture. While this dish solves the problem of constantly replenishing nutrient solution and removing waste liquid during embryo culture, it suffers from poor operability in embryo observation. When using this dish, the through-hole in the lid penetrates the cylinder into the embryo culture chamber, obstructing observation and evaluation of the embryos even with the lid on. Furthermore, the overflow channel is located between the embryo culture chamber and the waste liquid chamber. When fresh liquid is continuously added to the embryo culture chamber, the liquid in the culture chamber naturally flows into the waste liquid chamber, achieving the replenishment of fresh liquid and removal of waste liquid. However, embryo waste generally settles at the bottom of the culture chamber. This dish can only remove the upper layer of waste liquid in the culture chamber and cannot remove the large amount of embryo metabolic waste settled at the bottom, resulting in poor embryo culture results. Patent CN101947124A discloses an integrated microfluidic chip device and its usage method. With the aid of culture medium, oocytes are transported to the top of micropits. Due to gravity, the oocytes fall naturally into the micropits and are positioned. Sperm are added at the entrance of the microchannel and swim to the oocyte to complete in vitro fertilization. After fertilization, new culture medium is added from the entrance of the channel to remove sperm waste liquid and embryo culture is carried out. When it is necessary to retrieve the embryo, the upper microchannel is opened to expose the micropit array and the embryo is retrieved. The whole process realizes the functions of oocyte positioning, in vitro fertilization, rapid medium change, embryo culture and embryo retrieval.This microfluidic chip, after in vitro fertilization, adds nutrient solution to the microchannel inlet to remove excess sperm and cell debris, allowing new nutrient solution to flow from the inlet to the outlet. However, after the medium change, waste liquid at the outlet may flow back to the culture pit or even the inlet, due to the principle of communicating vessels. This results in the inlet, pit, and outlet becoming interconnected, and the same applies to rapid medium changes during embryo culture, failing to achieve the true effect of removing old solution and replenishing new solution. Furthermore, since fertilization occurs within microdroplets, mineral oil or other oil-phase liquids are introduced into the channels to prevent evaporation and ensure sealing. Different liquids (nutrient solution, mineral oil, semen) are introduced into the entire microfluidic chip channel. These liquids, due to their different viscosities and inertia, can clog the microfluidic channels, altering fluid flow characteristics, and the mixing of different liquids can negatively impact normal embryo growth and development.

[0003] To address the problem of insufficient medium exchange or backflow of liquid after medium exchange leading to low embryo development capacity in existing embryo culture dishes, there is an urgent need for a closed microfluidic culture dish with dynamic medium exchange for embryos and its culture method. Summary of the Invention

[0004] The purpose of this invention is to provide a closed microfluidic culture dish for dynamic embryo exchange and its culture method, thereby solving the above-mentioned technical problems.

[0005] To achieve the above objectives, the present invention provides a closed microfluidic culture dish for dynamic embryo exchange, comprising a culture dish body, a culture pool with a culture cover in the middle of the culture dish body, the side of the culture pool being connected to a storage pool and a waste pool through an inlet channel and an outlet channel respectively, and the bottom plane of the culture pool having an inlet section, a culture section and an outlet section; the culture section is located between the inlet section and the outlet section, and the culture section has a plurality of culture micropores for placing embryos, and both the inlet section and the outlet section have connecting holes, two connecting holes being respectively arranged opposite to the inlet channel and the outlet channel, and fences are provided between the inlet section and the culture section and between the culture section and the outlet section, the fences including two symmetrically arranged straight fences and an arc-shaped fence located between the two straight fences, with a gap provided between the straight fences and the arc-shaped fences, and the storage pool and the waste pool being connected to a dynamic exchange device.

[0006] Preferably, a protective trough is eccentrically provided at the bottom of the storage tank. The protective trough is elongated and funnel-shaped, with the top of the protective trough being a funnel opening that is connected to the bottom of the storage tank. The bottom diameter of the protective trough is smaller than the top diameter. The bottom of the protective trough is connected to the liquid inlet channel, and the outlet of the liquid inlet channel is lower than the bottom of the culture tank.

[0007] Preferably, the culture micropores are segmented frustum-shaped, increasing in size from bottom to top, with the diameter of the lowest end of the culture micropores being 400μm-600μm.

[0008] Preferably, the gap width is 0.1mm-0.12mm, and the gap width gradually widens from the culture section to the liquid inlet and liquid outlet sections.

[0009] Preferably, the bottom of the culture cover fits into the top of the fence, and the outer wall of the culture cover, the fence, the bottom of the culture tank and the inner wall of the culture tank form a gas storage cavity for storing the inlet channel, the outlet channel and the air bubbles discharged from the culture tank.

[0010] Preferably, a hollow backflow column is provided in the middle of the waste liquid tank, and the bottom of the hollow backflow column is connected to the liquid outlet channel.

[0011] Preferably, at least two centrally symmetrically arranged protruding fasteners are provided on the outside of the storage tank, the outside of the culture tank, and the outside of the waste liquid tank. The protruding fasteners are L-shaped, with one end fixed to the outside of the storage tank and the bottom and inner side of the other end of the protruding fastener being provided with slopes. The storage tank and waste tank are respectively equipped with a storage cover and a waste cover. The outer side of the culture cover, storage cover and waste cover is equipped with a latch and an anti-slip texture. The inner side of the culture cover, storage cover and waste cover is equipped with a sealing ring. The bottom of the latch is equipped with a limiting part, which cooperates with the bottom slope of the other end of the protruding buckle. The side of the latch cooperates with the inner slope of the other end of the protruding buckle.

[0012] Preferably, both the liquid storage cap and the waste liquid cap are provided with hollow Luer connectors at the top, which are used to connect to the dynamic liquid exchange device.

[0013] The specific steps of the culture method based on the above-mentioned closed-system dynamic medium-change microfluidic culture dish for embryos are as follows: Step S1: Preparation phase; The closed embryo dynamic medium exchange microfluidic culture dish, dynamic medium exchange device, culture medium and mineral oil were placed in the same incubator for preheating and equilibration. Step S2: Sample addition stage; Take out the preheated and balanced closed embryo dynamic medium exchange microfluidic culture dish and open the culture cap. Use a pipette to inject the preheated and balanced culture medium into the culture tank until the liquid level is higher than the height of the grid. Then transfer the embryo to be cultured from the outer culture dish into the culture micropores of the culture tank and screw on the culture cap to seal it. Preheated and balanced culture medium is injected into the storage tank and covered with mineral oil. The storage tank and waste tank are sealed and connected to the dynamic liquid exchange device and placed in the incubator. Step S2: Embryo culture and dynamic medium change stage; The dynamic liquid exchange device performs dynamic liquid exchange according to the set parameters. When the dynamic liquid exchange time point is reached, the dynamic liquid exchange device drives the culture medium in the storage tank to flow into the culture tank, and the waste liquid in the culture tank flows into the waste liquid tank at the same time, realizing the dynamic liquid exchange of embryos in the culture dish.

[0014] Preferably, the dynamic liquid exchange device liquid exchange module includes an intermittent dynamic liquid exchange mode, a continuous liquid exchange mode, a positive pressure liquid exchange mode in the storage tank, a negative pressure liquid exchange mode in the waste liquid tank, and a liquid exchange mode that is pumped and injected simultaneously. The dynamic fluid exchange drive can be electric, pneumatic, or hydraulic, and the fluid exchange speed and opening / closing can be adjusted by electric, pneumatic, or hydraulic drive devices.

[0015] Therefore, the present invention employs a closed-type dynamic embryo culture dish and its culture method, which has the following advantages: the culture dish is connected to the storage tank and the waste tank via an inlet channel and an outlet channel on its side. The bottom plane of the culture dish is provided with an inlet section, a culture section, and an outlet section. The culture section is located between the inlet section and the outlet section and has several culture micropores for placing embryos. Both the inlet section and the outlet section have connecting holes, which are respectively arranged opposite to the inlet channel and the outlet channel. Barriers are provided between the inlet section and the culture section and between the culture section and the outlet section. The microfluidic characteristics are used to simulate the dynamic microenvironment of embryos in the fallopian tube, which is subjected to the compression and friction of smooth muscle, the impact of fallopian tube fluid flow, and the continuous renewal of fallopian tube fluid carrying away accumulated metabolic waste. This stimulates embryo growth and development, making up for the shortcomings of traditional static embryo culture in vitro in culture dishes. It effectively prevents embryos from being swept into the flow channel and effectively reduces the shear force caused by the liquid flowing into the culture dish on the embryo.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the three-dimensional structure of a closed-type dynamic embryo fluid exchange microfluidic culture dish according to the present invention; Figure 2 This is a side view of a closed microfluidic culture dish for dynamic embryo exchange according to the present invention. Figure 3 This is a cross-sectional view of a closed-type microfluidic culture dish for dynamic embryo exchange according to the present invention. Figure 4 This is a top view of the culture tank of the present invention; Figure 5 This is a schematic diagram of the microporous structure of the present invention; Figure 6 This is a schematic diagram of the three-dimensional structure of the liquid storage cap of the present invention; Figure 7 This is a cross-sectional view of the liquid storage cap structure of the present invention; Figure 8 This is a schematic diagram of the culture cap structure of the present invention; Figure 9 This is a waste liquid discharge curve diagram of the present invention; Figure 10 This is a simulation diagram of the waste liquid discharge of the present invention; Figure 11 This is a simulation diagram of a shearing process with one input and one output. Figure 12 This is a simulation diagram of a two-input, two-output shearing process. Figure 13 A flow field streamline distribution diagram for one inlet and one outlet; Figure 14 This is a flow field streamline distribution diagram for a two-inlet, two-outlet system.

[0018] Figure Labels 1. Petri dish body; 101. Liquid inlet channel; 102. Liquid outlet channel; 2. Culture tank; 201. Liquid inlet section; 202. Culture section; 203. Liquid outlet section; 204. Culture micropores; 205. Connecting hole; 206. Fence; 2061. Gap; 3. Storage tank; 301. Protective tank; 4. Waste liquid tank; 401. Hollow backflow column; 5. Culture cover; 6. Gas storage cavity; 7. Outward protruding fastener; 701. Sloping surface; 8. Storage cover; 9. Waste liquid cover; 10. Tongue; 1001. Limiting part; 11. Sealing ring; 12. Luer connector. Detailed Implementation

[0019] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product is in use. They are used only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," and "connect" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0020] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0021] like Figure 1 , Figure 2 as well as Figure 3 As shown, a closed microfluidic culture dish for dynamic embryo exchange includes a culture dish body 1. A culture pool 2 with a culture cover 5 is provided in the middle of the culture dish body 1. The side of the culture pool 2 is connected to a storage pool 3 and a waste liquid pool 4 through an inlet channel 101 and an outlet channel 102, respectively.

[0022] like Figure 4As shown, the culture tank 2 is cylindrical with a total capacity of 1 ml. The bottom plane of the culture tank 2 is provided with an inlet section 201, a culture section 202, and an outlet section 203. The culture section 202 is located between the inlet section 201 and the outlet section 203. The culture section 202 has several culture micropores 204 for placing embryos, such as... Figure 5 As shown, the culture microwell 204 is a segmented frustoconical shape, gradually increasing in size from bottom to top. The diameter of the lowest point of the culture microwell 204 is 400μm-600μm, which is approximately 200-400μm larger than the diameter of the embryo. This design helps prevent embryos from shifting or being lost during medium changes or manual transfer. Both the inlet section 201 and the outlet section 203 have connecting holes 205, which are respectively positioned opposite to the inlet channel 101 and the outlet channel 102, and the channels are connected. Barriers 206 are provided between the inlet section 201 and the culture section 202, and between the culture section 202 and the outlet section 203. Gaps 2061 are provided between adjacent barriers 206, with a width of 0.1mm-0.12mm. The width of the gaps 2061 gradually increases from the culture section 202 towards the inlet section 201 and the outlet section 203. The width of the slit 2061 is slightly smaller than the diameter of the embryo, which effectively prevents the embryo from being accidentally carried out of the micropore by the culture medium during medium change and entering the flow channel, thus preventing embryo loss. It also reduces the impact force of the culture medium entering the culture tank 2 during dynamic medium change, reducing the shear force on the embryo. The height of the fence 206 is matched with the diameter of the culture tank 2. The culture cover 5 is recessed to ensure that the bottom surface of the culture cover 5 fits the top surface of the fence 206 in the culture tank 2 after it is closed. The culture medium volume in the middle micropore is the required 200μL. The bottom of the culture cover 5 fits the top of the fence 206. The fence 206 is set at the bottom of the culture tank 2, which can control the culture medium volume of the culture tank 2 and prevent embryo loss. The outer wall of the culture cover 5, the fence 206, the bottom of the culture tank 2, and the inner wall of the culture tank 2 form a gas storage cavity 6, which is used to store the liquid inlet channel 101, the liquid outlet channel 102, and the air bubbles discharged from the culture tank 2. The gas storage chamber 6 is a triangular annular space that can store the gas in the flow channel and the air bubbles that are discharged from the culture tank 2 after the culture cover 5 is closed, so as to avoid the air bubbles on the surface of the micropores of the culture tank 2 from affecting the observation or the effect of the sealed culture of embryos.

[0023] The storage tank 3 and waste tank 4 are connected to the dynamic fluid exchange device. The storage tank 3 is cylindrical with a total capacity of 2 ml. A protective groove 301 is eccentrically positioned at the bottom, extending from the center of the bottom of the storage tank 3 towards the culture tank 2. The protective groove 301 is elongated and flared, with a flared opening at the top that connects to the bottom of the storage tank 3. The flared opening is used for pipette tip alignment and limiting the pipette tip's descent height during embryo fluid exchange. The flared slope prevents the bottom of the culture medium from adhering to the substrate surface of the bottom of the storage tank 3 when the pipette tip is drawing waste fluid, thus preventing cavitation. The bottom diameter of the protective groove 301 is smaller than its top diameter. The bottom of the protective groove 301 connects to the inlet channel 101, which has a diameter of 0.5 mm and a length of approximately 8.5 mm, connecting the storage tank 3 and the culture tank 2. The outlet of the liquid inlet channel 101 is lower than the bottom of the culture tank 2, which can fully replace the waste liquid in the culture tank 2 during dynamic liquid exchange.

[0024] Waste liquid tank 4 is located on one side of the main body of the culture dish 1. It is cylindrical with a total capacity of 2 ml and has a hollow backflow column 401 in the middle. The bottom of the hollow backflow column 401 is connected to the liquid outlet channel 102. It has a diameter of 0.5 mm and a length of about 17.5 mm and is used to connect the culture dish 2 and the waste liquid tank 4. The liquid enters the waste liquid tank 4 through the hollow backflow column 401, while preventing backflow caused by the liquid level in the waste liquid tank 4 being higher than that in the culture dish 2 or the storage tank 3.

[0025] To achieve a seal on the outer sides of the storage tank 3, the culture tank 2, and the waste tank 4, an outwardly protruding fastener 7 and a latch 10 are provided. For example... Figure 6 , Figure 7 as well as Figure 8 As shown, two centrally symmetrically arranged protruding fasteners 7 are provided on the outside of the storage tank 3, the outside of the culture tank 2, and the outside of the waste liquid tank 4. The protruding fasteners 7 are L-shaped, with one end fixed to the outside of the storage tank 3, and the bottom and inner side of the other end of the protruding fasteners 7 are provided with inclined surfaces 701. The storage tank 3 and the waste liquid tank 4 are respectively equipped with a storage cap 8 and a waste liquid cap 9. The culture cap 5 can also be an inward or outward rotating cap. The outer sides of the culture cap 5, the storage cap 8 and the waste liquid cap 9 are provided with a latch 10 and a textured anti-slip pattern. The inner sides of the culture cap 5, the storage cap 8 and the waste liquid cap 9 are provided with a sealing ring 11 (there is a triangular circular rib at the bottom of the cap for sealing with the culture tank 2). The bottom of the latch 10 is provided with a limiting part 1001, which cooperates with the inclined surface 701 at the bottom of the other end of the protruding buckle 7. The side of the latch 10 cooperates with the inclined surface 701 on the inner side of the other end of the protruding buckle 7.

[0026] When the culture cap 5 is sealed to the culture tank 2, the culture cap 5 and the protruding fastener 7 and latch 10 on the culture tank 2 are staggered. Rotating the culture cap 5 causes the latch 10 to engage with the protruding fastener 7 under the action of the inclined surface 701 on the side. The limiting part 1001 contacts the inclined surface 701 on the bottom of the protruding fastener 7. During rotation, the inclined surface 701 transitions to a flat surface, the height of the culture cap 5 decreases, and the sealing ring 11 inside the culture cap 5 presses down onto the inner edge of the culture tank 2, sealing the culture cap 5 and the culture tank 2. The closing process for the liquid storage cap 8 and the waste liquid cap 9 is the same.

[0027] Both the liquid storage cap 8 and the waste liquid cap 9 are equipped with hollow Luer connectors 12 at their tops. The Luer connectors 12 are used to connect to a dynamic liquid exchange device, which is a syringe or a capillary tube with a pump body to supply culture medium.

[0028] The specific steps of the culture method based on the above-mentioned closed-system dynamic medium-change microfluidic culture dish for embryos are as follows: Step S1: Preparation phase.

[0029] The closed embryo dynamic fluid exchange microfluidic culture dish, dynamic fluid exchange device, culture medium and mineral oil were placed in the same incubator for preheating and equilibration.

[0030] Step S2: Sample addition stage.

[0031] Take out the preheated and balanced closed embryo dynamic fluid exchange microfluidic culture dish and open the culture cap 5. Use a pipette to inject the preheated and balanced culture medium into the culture pool 2 until the liquid level is slightly higher than the height of the fence 206. Then transfer the embryo to be cultured from the outer culture dish into the culture micropore 204 of the culture pool 2 and screw on the culture cap 5 to seal it.

[0032] Preheated and balanced culture medium is injected into storage tank 3 and covered with mineral oil. Storage tank 3 and waste liquid tank 4 are sealed and connected to the dynamic liquid exchange device and placed in the incubator.

[0033] Step S2: Embryo culture and dynamic medium exchange stage.

[0034] The dynamic liquid exchange device performs dynamic liquid exchange according to the set parameters. When the dynamic liquid exchange time point is reached, the dynamic liquid exchange device drives the culture medium in the storage tank 3 to flow into the culture tank 2. The waste liquid in the culture tank 2 flows into the waste liquid tank 4 at the same time, realizing the dynamic liquid exchange of embryos in the culture dish.

[0035] The dynamic liquid exchange device includes an intermittent dynamic liquid exchange mode, a continuous liquid exchange mode, a positive pressure liquid exchange mode in the storage tank 3, a negative pressure liquid exchange mode in the waste liquid tank 4, and a liquid exchange mode that is pumped and injected simultaneously.

[0036] The dynamic medium exchange system can be driven electrically, pneumatically, or hydraulically. The speed and operation of the medium exchange process are adjusted via these actuators, allowing for switching between fast and slow exchange modes. It provides controllability and versatility in medium exchange speed, flow rate, and mode, enabling embryo culture and medium exchange in an environment closely resembling in vivo conditions. The microfluidic design simulates the dynamic microenvironment of embryos within the fallopian tubes, characterized by smooth muscle compression and friction, the impact of fallopian tube fluid flow, and the continuous removal of accumulated metabolic waste by the constantly renewing fallopian tube fluid. This stimulates embryonic growth and development, overcoming the shortcomings of traditional static in vitro embryo culture in petri dishes.

[0037] An inlet velocity of 100 μl / 3 s, a culture medium density of approximately 0.9784 g / ml, a waste liquid density of approximately 0.9804 g / m³, and a VOF two-phase flow model with an embryo diameter of 200 μm were used for simulation. Figure 9 and Figure 10 As shown, the wastewater discharge rates of the two structures are very similar. At the 6th second, the structure with one inlet and one outlet (having one inlet slit 2063 and one outlet slit 2063) has 16% of the wastewater remaining, while the structure with two inlets and two outlets (having two inlets 2063 and two outlet slits 2063) has 18% of the wastewater remaining. Figure 11 and Figure 12 As shown, the two-in-two-out structure, due to the additional inlet and outlet, experiences a 50% reduction in velocity at the inlet and outlet compared to a one-in-one-out structure. Consequently, the shear force is also halved, i.e., the impact force is halved. The maximum shear force is located in the embryo pit area near the inlet, followed by the embryo pit area near the outlet. The maximum shear force for a one-in-one-out structure is 0.019 dyn / cm. 2 The maximum shear force for a two-input, two-output configuration is 0.0075 dyn / cm. 2 The flow field distribution is as follows Figure 13 and Figure 14 As shown, the micropore matrix arrangement is simulated using flow field simulation to model the fluid flow within the micropores, predicting the fluid behavior within the chip, including flow velocity, direction, and pressure distribution. The design is optimized to reduce the impact of fluid shear force on the embryo. Waste liquid integral cloud map distribution simulation allows for a direct observation of the flow and distribution of waste liquid within the chip, ensuring effective removal of waste liquid from the culture area and thus reducing potential adverse effects on the embryo. The two-in, two-out structure scheme provides a more rational flow process.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A closed microfluidic culture dish for dynamic embryo exchange, comprising a main body, characterized in that: The culture dish has a culture tank with a culture lid in the middle. The culture tank is connected to the storage tank and the waste tank through the liquid inlet channel and the liquid outlet channel, respectively. The culture tank is provided with a liquid inlet section, a culture section and a liquid outlet section. The culture section is located between the liquid inlet section and the liquid outlet section. The culture section has several culture micropores for placing embryos. Both the liquid inlet section and the liquid outlet section have connecting holes. The two connecting holes are respectively arranged opposite to the liquid inlet channel and the liquid outlet channel. The culture section is provided with a fence between the liquid inlet section and the liquid outlet section. The fence includes several symmetrically arranged fence plates. There are gaps between adjacent fence plates.

2. The closed-loop microfluidic culture dish for dynamic embryo exchange according to claim 1, characterized in that: The bottom of the storage tank is eccentrically equipped with a protective trough, which is long and flared. The top of the protective trough is a flared opening and is connected to the bottom of the storage tank. The bottom diameter of the protective trough is smaller than the top diameter. The bottom of the protective trough is connected to the liquid inlet channel, and the outlet of the liquid inlet channel is lower than the bottom of the culture tank.

3. The closed-loop microfluidic culture dish for dynamic embryo exchange according to claim 2, characterized in that: The culture microwells are segmented frustoconical in shape, increasing in size from bottom to top, with the diameter of the lowest microwell being 400μm-600μm.

4. The closed-loop microfluidic culture dish for dynamic embryo exchange according to claim 3, characterized in that: The slit width is 0.1mm-0.12mm, and the slit width gradually increases from the culture section to the liquid inlet and liquid outlet sections.

5. A closed microfluidic culture dish for dynamic embryo exchange according to claim 4, characterized in that: The bottom of the culture cap fits into the top of the fence. The outer wall of the culture cap, the fence, the bottom of the culture tank, and the inner wall of the culture tank form a gas storage cavity, which is used to store the inlet flow channel, the outlet flow channel, and the air bubbles discharged from the culture tank.

6. A closed microfluidic culture dish for dynamic embryo exchange according to claim 5, characterized in that: The waste liquid tank is equipped with a hollow backflow column, the bottom of which is connected to the liquid outlet channel.

7. A closed microfluidic culture dish for dynamic embryo exchange according to claim 6, characterized in that: At least two centrally symmetrically arranged protruding fasteners are provided on the outside of the storage tank, the outside of the culture tank, and the outside of the waste liquid tank. The protruding fasteners are L-shaped, with one end fixed to the outside of the storage tank and the bottom and inner side of the other end of the protruding fastener being provided with slopes. The storage tank and waste tank are respectively equipped with a storage cover and a waste cover. The outer side of the culture cover, storage cover and waste cover is equipped with a latch and an anti-slip texture. The inner side of the culture cover, storage cover and waste cover is equipped with a sealing ring. The bottom of the latch is equipped with a limiting part, which cooperates with the bottom slope of the other end of the protruding buckle. The side of the latch cooperates with the inner slope of the other end of the protruding buckle.

8. A closed microfluidic culture dish for dynamic embryo exchange according to claim 7, characterized in that: Both the liquid storage cap and the waste liquid cap are equipped with hollow Luer connectors at the top, which are used to connect to the dynamic liquid exchange device.

9. A method for culturing embryos in a closed-loop dynamic medium-change microfluidic culture dish according to any one of claims 1-8, characterized in that, The specific steps are as follows: Step S1: Preparation phase; The closed embryo dynamic medium exchange microfluidic culture dish, dynamic medium exchange device, culture medium and mineral oil were placed in the same incubator for preheating and equilibration. Step S2: Sample addition stage; Take out the preheated and balanced closed embryo dynamic medium exchange microfluidic culture dish and open the culture cap. Use a pipette to inject the preheated and balanced culture medium into the culture tank until the liquid level is higher than the height of the grid. Then transfer the embryo to be cultured from the outer culture dish into the culture micropores of the culture tank and screw on the culture cap to seal it. Preheated and balanced culture medium is injected into the storage tank and covered with mineral oil. The storage tank and waste tank are sealed and connected to the dynamic liquid exchange device and placed in the incubator. Step S2: Embryo culture and dynamic medium change stage; The dynamic liquid exchange device performs dynamic liquid exchange according to the set parameters. When the dynamic liquid exchange time point is reached, the dynamic liquid exchange device drives the culture medium in the storage tank to flow into the culture tank, and the waste liquid in the culture tank flows into the waste liquid tank at the same time, realizing the dynamic liquid exchange of embryos in the culture dish.

10. The culture method based on a closed-loop dynamic embryo culture dish according to claim 9, characterized in that: The dynamic liquid exchange device includes an intermittent dynamic liquid exchange mode, a continuous liquid exchange mode, a positive pressure liquid exchange mode in the storage tank, a negative pressure liquid exchange mode in the waste liquid tank, and a liquid exchange mode that is pumped and injected simultaneously. The dynamic fluid exchange drive can be electric, pneumatic, or hydraulic, and the fluid exchange speed and opening / closing can be adjusted by electric, pneumatic, or hydraulic drive devices.

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