Embryo automatic medium exchange culture dish

Abstract

The present invention discloses an embryo automatic liquid exchange culture dish, comprising at least one group of culture units, wherein the culture unit comprises an injection pool, a culture pool and a waste liquid cavity connected in sequence; wherein the upper part of the injection pool is connected with a liquid inlet channel, and its bottom is connected with the bottom of the culture pool through an overflow channel; the bottom of the culture pool is provided with at least one embryo microhole, and the bottom of the embryo microhole is connected to the waste liquid cavity through a waste liquid channel. The embryo automatic liquid exchange culture dish of the present invention can achieve complete liquid exchange and effectively meet the material requirements required for each development stage of the embryo; the present invention avoids the damage to the embryo culture environment and the damage to the embryo itself caused by repeated manual intervention operations through automatic liquid exchange, can improve the stability of embryo culture and the quality of blastocysts, and effectively increase the clinical pregnancy rate and live birth rate.

Description

Technical Field

[0001] The invention relates to the field of cell culture, in particular to an embryo automatic liquid replacement culture dish. Background Art

[0002] Maintaining a suitable and stable environment for the embryo culture system is one of the key factors affecting the results of in vitro fertilization and embryo transfer (IVF-ET). Embryo culture medium is an essential and important substance in the process of in vitro fertilization. During the fertilization stage, the sperm and egg combine in vitro to form a fertilized egg, which is then placed in a culture medium for culture. This simulates the uterine environment and allows the embryo to grow and develop well. Embryo culture medium not only plays an important role in embryo development, but also has a certain impact on the development of offspring after embryo implantation. It has become one of the hot spots of attention and research in the field of assisted reproductive technology (ART) around the world.

[0003] The common components of human embryo culture medium mainly include: (1) water, which accounts for 99% of the total components of the culture medium. The culture medium is strictly sterilized purified water; (2) inorganic ions, such as calcium ions and magnesium ions, which are conducive to embryo compaction; (3) energy substrates, which are mainly designed according to the environment of energy substrates in the fallopian tube and uterine cavity, such as glucose, pyruvate, amino acids, etc.; (4) hormones and growth factors, such as luteinizing hormone, follicle stimulating hormone, epidermal growth factor and insulin-like growth factor, which can improve the in vitro maturation rate and developmental potential of oocytes; (5) protein, the most added of which is human serum albumin (HSA), which has the functions of stabilizing and protecting embryo cell membranes, maintaining stable colloidal osmotic pressure and preventing embryos from attaching to embryo culture dishes; (6) vitamins, which regulate cell metabolism and prevent the influence of adverse culture conditions on embryo development.

[0004] During the in vitro embryo culture process, embryos at different stages need to be replaced with different culture media. In the sequential culture media system currently used in clinical practice, the fertilization fluid, cleavage fluid and blastocyst culture media are designed based on the natural changes in the environment in the fallopian tube, so the cleavage fluid and blastocyst fluid need to be replaced on the first and third days after the oocyte is fertilized. The components of the cleavage fluid can meet the nutritional needs of the embryo from the first to the third day, while the various components of the blastocyst culture media can better meet the material needs of blastocyst development.

[0005] Currently, when doctors change the culture medium, they need to take the embryo culture dish out of the incubator each time, suck out the culture medium with a manual pipette on the workbench, and then add new culture medium. This operation not only separates the embryo from the original relatively stable culture environment, but the manual intervention of pipetting can easily cause damage to the embryo itself. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an automatic embryo culture dish for changing liquid in view of the above-mentioned deficiencies in the prior art, so as to solve the problems of the destruction of the embryo culture environment and the damage to the embryo itself caused by the current manual liquid change operation, so as to improve the stability of embryo culture and the quality of blastocysts, and increase the clinical pregnancy rate and live birth rate.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: an embryo automatic liquid replacement culture dish, comprising at least one group of culture units, wherein the culture unit comprises a liquid injection pool, a culture pool and a waste liquid chamber connected in sequence;

[0008] The upper part of the injection pool is connected with a liquid inlet channel, and the bottom of the injection pool is connected with the bottom of the culture pool through an overflow channel;

[0009] At least one embryo microwell is arranged at the bottom of the culture pool, and the bottom of the embryo microwell is connected to the waste liquid chamber through a waste liquid flow channel.

[0010] Preferably, a vent hole is provided on the top of the liquid injection pool.

[0011] Preferably, an air suction port is provided at the place where the waste liquid chamber communicates with the outside.

[0012] Preferably, the culture unit is arranged inside the culture dish body, the inlet end of the liquid inlet channel forms a liquid inlet on the side of the culture dish body, and the end of the liquid inlet channel is connected to the side wall of the upper part of the liquid injection pool.

[0013] Preferably, all the liquid inlet channels are in the same plane and are arranged horizontally, and through the bending layout, the lengths of all the liquid inlet channels from the liquid inlet to the liquid injection tank are the same.

[0014] Preferably, the diameter and depth of the embryo microwell are both 0.4-0.5 mm, and each of the embryo microwell can only accommodate a single embryo.

[0015] Preferably, the waste liquid flow channel is arranged in a horizontal plane close to the bottom of the embryo microwell, and the inner diameter of the waste liquid flow channel is not greater than 0.1 mm.

[0016] Preferably, the waste liquid flow channel includes a main flow channel and two branch flow channels, the inlets of the two branch flow channels are connected to the bottom of the embryo microwell, the ends of the two branch flow channels converge to the main flow channel, and the end of the main flow channel is connected to the waste liquid chamber;

[0017] The end of the main flow channel is higher than the bottom surface of the waste liquid chamber.

[0018] Preferably, the layout and length of the waste fluid flow channels from all the embryo microholes to the waste fluid chamber are consistent.

[0019] Preferably, the two branch channels are symmetrically distributed on both sides of the embryo microwell.

[0020] The beneficial effects of the present invention are as follows: the embryo automatic fluid exchange culture dish of the present invention can achieve thorough fluid exchange and effectively meet the material requirements of the embryo at each developmental stage; the present invention avoids the destruction of the embryo culture environment and the damage to the embryo itself caused by repeated manual intervention operations through automatic fluid exchange, can improve the stability of embryo culture and the quality of blastocysts, and effectively increase the clinical pregnancy rate and live birth rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a perspective structural schematic diagram of the embryo automatic medium replacement culture dish of the present invention;

[0022] Figure 2 It is a perspective view of the embryo automatic fluid replacement culture dish of the present invention when viewed from above;

[0023] Figure 3 This is a schematic diagram of the external structure of the embryo automatic medium replacement culture dish of the present invention;

[0024] Figure 4 It is a cross-sectional view of the embryo automatic medium replacement culture dish of the present invention from the side;

[0025] Figure 5 It is a schematic structural diagram of the waste liquid flow channel of the present invention.

[0026] Description of reference numerals:

[0027] 1—culture dish body; 2—culture unit; 3—liquid injection pool; 4—culture pool; 5—waste liquid chamber; 6—liquid inlet channel; 7—overflow channel; 8—embryo micropore; 9—waste liquid channel; 30—air vent; 50—air extraction port; 60—liquid inlet; 90—main channel; 91—branch channel; 10—culture liquid; 11—oil film. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0029] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0030] like Figure 1-5 As shown, an automatic embryo liquid exchange culture dish of this embodiment includes a culture dish body 1, at least one group of culture units 2 is arranged inside the culture dish body 1, and each group of culture units 2 includes a liquid injection pool 3, a culture pool 4 and a waste liquid chamber 5 which are connected in sequence;

[0031] The top of the injection tank 3 is connected with a liquid inlet channel 6, and the bottom thereof is connected with the bottom of the culture tank 4 through an overflow channel 7;

[0032] At least one embryo microwell 8 is provided at the bottom of the culture pool 4 , and the bottom of the embryo microwell 8 is connected to the waste liquid chamber 5 through a waste liquid flow channel 9 .

[0033] A vent hole 30 is provided on the top of the liquid injection pool 3, which can discharge the gas in the liquid inlet channel 6 during the liquid injection process and balance the surface air pressure of the liquid injection pool 3 and the culture pool 4.

[0034] The inlet end of the liquid inlet channel 6 forms a liquid inlet port 60 on the side of the culture dish body 1, and the end of the liquid inlet channel 6 is connected to the side wall of the upper part of the liquid injection pool 3. The culture liquid passes through the liquid inlet channel 6 to reach the inner side wall of the upper part of the liquid injection pool 3. Through slow supply, the culture liquid will adhere to the inner wall of the liquid injection pool 3 and slowly flow down to the liquid surface without causing the appearance of bubbles.

[0035] All the liquid inlet channels 6 are arranged horizontally in the same plane, and the lengths of all the liquid inlet channels 6 from the liquid inlet 60 to the liquid injection pool 3 are the same through the bending layout. The new culture fluid of the culture dish is supplied through the liquid inlet 60 by a multi-channel micro pump or a drip pump and enters the liquid inlet channel 6. Since the liquid inlet channels 6 have the same length, the supply flow rate and volume of the culture fluid in each channel can be kept consistent.

[0036] An overflow channel 7 is provided at the bottom of the injection pool 3 and is connected to the bottom of the culture pool 4. When new culture fluid is injected into the injection pool 3, the liquid level of the injection pool 3 will gradually rise, and there will be a certain height difference with the liquid level of the culture pool 4. Under the action of gravity and external air pressure, the new culture fluid in the injection pool 3 will flow into the culture pool 4 through the overflow channel 7.

[0037] The culture pool 4 contains culture fluid. The embryo culture cycle is relatively long. To prevent the culture fluid from volatilizing, a mineral oil film 11 is required to be covered on the upper layer during use.

[0038] The diameter and depth of the embryo microwell 8 are both 0.4-0.5 mm, and each embryo microwell 8 can only accommodate a single embryo.

[0039] The waste liquid flow channel 9 is arranged in a horizontal plane near the bottom of the embryo micropore 8, and the inner diameter of the waste liquid flow channel 9 is not greater than 0.1 mm. In contrast, the diameter of a general embryo is about 0.2 mm to 0.3 mm, which can ensure that the embryo will not flow into the waste liquid flow channel 9.

[0040] The bottom surface of the waste liquid chamber 5 is lower than the outlet of the waste liquid channel 9, and the total volume within the height range from the outlet of the waste liquid channel 9 to the bottom surface of the waste liquid chamber 5 is used as the maximum allowable waste liquid storage capacity of the culture dish.

[0041] The waste liquid flow channel 9 includes a main channel 90 and two branch channels 91, the entrances of the two branch channels 91 are connected to the bottom of the embryo micropore 8, and the two branch channels 91 are distributed at a certain angle, preferably 180° (i.e. symmetrically distributed on both sides of the embryo micropore 8). The ends of the two branch channels 91 converge to the main channel 90, and the ends of the main channel 90 are connected to the waste liquid chamber 5. The layout and length of the waste liquid flow channels 9 from all embryo micropores 8 to the waste liquid chamber 5 are consistent. The entrances of the two branch channels 91 can effectively avoid the problem that a single entrance is easily blocked by the embryo, causing difficulty in discharging waste liquid, and can also avoid the problem of increased negative pressure caused by blockage in more serious cases that the embryo itself is damaged.

[0042] The waste liquid chamber 5 is provided with an air suction port 50 at the place where it communicates with the outside world. It is used to connect a negative pressure pump. Under the air suction effect of the negative pressure pump, the air pressure in the waste liquid chamber 5 is significantly lower than the outside air pressure. Under the effect of the pressure difference, the culture liquid in the culture pool 4 is discharged from the branch channel 91 at the bottom of the micropores to the waste liquid chamber 5 through the waste liquid flow channel 9. Since the air pressure in the waste liquid chamber 5 is uniform, the layout and length of each waste liquid flow channel 9 are the same. Therefore, the difference in the waste liquid flow rate discharged from each embryo micropore 8 is small, and the consistency of waste liquid discharge is good.

[0043] Among them, the material of the culture dish can be glass, quartz, high molecular polymer, etc., or a combination of these materials. The manufacturing process of the microchannel part (especially the waste liquid channel 9) adopts conventional methods, such as a bonding method similar to a microfluidic chip. Other structural parts can be 3D printed or injection molded, and the present invention does not make any improvements here.

[0044] The embryo automatic liquid exchange culture dish of the present invention has a similar preparation process to conventional culture dishes before use, requiring manual addition of culture solution, removal of visible bubbles in the culture solution, covering with an anti-volatile mineral oil film 11, and introduction of embryos, wherein the air extraction port 50 needs to be blocked when adding culture solution to prevent excessive flow of culture solution into the waste liquid flow channel 9, or gas in the waste liquid flow channel 9 from overflowing from the micropores to cause bubbles. Finally, the prepared culture dish is placed in a matching time-lapse imaging incubator or a special embryo incubator with a real-time monitoring system, and then the liquid inlet 60 and the air extraction port 50 of the culture dish are respectively connected to the corresponding external gas-liquid pipeline ports to complete the operation.

[0045] In one embodiment, the automatic fluid replacement process of the embryo automatic fluid replacement culture dish of the present invention is as follows: a negative pressure pump is connected to the air suction port 50, and the negative pressure pump is first controlled to form a uniform and suitable negative pressure space in the waste liquid chamber 5, forcing part of the culture fluid in the culture pool 4 to be discharged from the bottom of the embryo micropore 8 through the waste liquid flow channel 9 into the waste liquid chamber 5, and then the multi-channel micro pump or drip pump is controlled to supply new culture fluid, and the culture fluid reaches the injection pool 3 through the liquid inlet flow channel 6, and slowly flows down to the liquid surface along the inner wall of the injection pool 3. As the liquid level rises, under the action of gravity, the new culture fluid flows into the culture pool 4 through the overflow channel 7, and the concentration of the new culture fluid will gradually increase. After several rounds of repetition, a relatively thorough fluid replacement effect can be achieved.

[0046] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. An automatic embryo culture dish, characterized in that: It comprises at least one group of culture units, wherein the culture unit comprises a liquid injection pool, a culture pool and a waste liquid chamber which are connected in sequence; The upper part of the injection pool is connected with a liquid inlet channel, and the bottom of the injection pool is connected with the bottom of the culture pool through an overflow channel; At least one embryo microwell is provided at the bottom of the culture tank, and the bottom of the embryo microwell is connected to the waste liquid cavity through a waste liquid flow channel; A vent hole is provided on the top of the liquid injection pool, and a suction port is provided at the place where the waste liquid chamber communicates with the outside world; The waste liquid flow channel includes a main flow channel and two branch flow channels, the inlets of the two branch flow channels are connected to the bottom of the embryo micropore, the ends of the two branch flow channels converge to the main flow channel, and the end of the main flow channel is connected to the waste liquid chamber; The end of the main channel is higher than the bottom surface of the waste liquid chamber; The two branch channels are symmetrically distributed on both sides of the embryo micropore; When new culture fluid is injected into the injection pool, the liquid level of the injection pool will gradually rise, and there will be a certain height difference with the liquid level of the culture pool. Under the action of gravity and external air pressure, the new culture fluid in the injection pool will flow into the culture pool through the overflow channel; The culture tank contains culture fluid and is covered with a mineral oil film on the upper layer when in use.

2. The embryo automatic medium replacement culture dish according to claim 1, characterized in that: The culture unit is arranged inside the culture dish body, the inlet end of the liquid inlet channel forms a liquid inlet on the side of the culture dish body, and the end of the liquid inlet channel is connected to the side wall of the upper part of the injection pool.

3. The embryo automatic medium replacement culture dish according to claim 2, characterized in that: All of the liquid inlet flow channels are in the same plane and are arranged horizontally. Through the bending layout, the lengths of all of the liquid inlet flow channels from the liquid inlet to the liquid injection pool are the same.

4. The embryo automatic medium replacement culture dish according to claim 1, characterized in that: The diameter and depth of the embryo microwell are both 0.4-0.5 mm, and each embryo microwell can only accommodate a single embryo.

5. The embryo automatic medium replacement culture dish according to claim 1, characterized in that: The waste liquid flow channel is arranged in a horizontal plane close to the bottom of the embryo micropore, and the inner diameter of the waste liquid flow channel is no more than 0.1 mm.

6. The embryo automatic medium replacement culture dish according to claim 1, characterized in that: The layout and length of the waste fluid flow channels from all the embryo microholes to the waste fluid chamber are consistent.

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