A sperm handling chip and methods of use thereof

Abstract

This invention relates to the field of sperm sorting technology, specifically to a sperm processing chip and its usage method. The sperm processing chip includes: a second tank disposed within a first tank, forming a sample loading chamber between the first and second tanks for placing sperm samples to be tested; the outer wall of the second tank faces the sample loading chamber, and the top of the outer wall is sloped, with one end of the slope pointing towards the sample loading chamber and the other end connected to the interior of the second tank; microchannels extending along the slope's inclination direction are formed on the slope; and a collection chamber is formed on the inner wall of the second tank for collecting sperm that swim from the sample loading chamber to the slope microchannels and fall into the collection chamber; the height of the first tank wall is higher than that of the second tank wall. This sperm processing chip can achieve active sorting without chemical drugs and sperm screening under drug action, and can effectively maintain sperm activity. It has the advantages of high versatility, high recovery rate, high sorting accuracy, high motility, and ease of operation.

Description

Technical Field

[0001] This invention relates to the field of sperm sorting technology, specifically to a sperm processing chip and its usage method. Background Technology

[0002] Assisted reproductive technology is an effective way to solve infertility, and obtaining sperm with high fertilization potential in a clinically significant way is a prerequisite for successful conception. Therefore, how to screen high-quality sperm is a crucial step in the process of assisted reproductive technology.

[0003] Currently, the main methods used clinically for sperm sorting are the swim-up method and density gradient centrifugation. The swim-up method utilizes the autonomous motility of motile sperm, allowing them to swim upwards from the bottom of the test tube to the surface of the culture medium. Density gradient centrifugation leverages the differences between normal sperm and inactive sperm, or other components of semen, including differences in their motility trajectories and abilities. In a density gradient column, these different components will remain in their respective density gradient columns, thus allowing for separation. After centrifugation, these different components will remain in columns with different density gradients, thereby selecting the more normal sperm for artificial insemination.

[0004] However, both upstream methods and density gradient centrifugation rely on a single standard for sperm sorting. In density gradient centrifugation, the centrifugation step increases reactive oxygen species, damaging DNA double strands and affecting embryo quality. Sperm quality evaluation criteria include multiple aspects such as morphology, motility, DNA fragmentation rate, and sperm characteristics (including chemotaxis, thermotaxis, mesotaxis, and thallotaxis). Correspondingly, sperm sorting needs to be performed from multiple dimensions.

[0005] Furthermore, clinical practice currently employs two completely different techniques and instruments for sperm testing and drug testing. After drug testing, routine sperm testing is still required to determine the sperm's response to the drug. This process is prone to sampling errors, time-consuming, and requires various consumables, making it cumbersome and unable to provide a direct and rapid assessment of the drug's effect on sperm. Summary of the Invention

[0006] One of the objectives of this invention is to provide a sperm processing chip that overcomes the shortcomings of existing technologies. This sperm processing chip can achieve active sorting without the action of chemical drugs and sperm screening under the action of drugs, and can effectively maintain sperm activity. It has the advantages of high versatility, high recovery rate, high sorting accuracy, high motility and easy operation.

[0007] The second objective of this invention is to provide a method for using a sperm processing chip.

[0008] To achieve one of the above objectives, the present invention provides the following technical solution:

[0009] A sperm processing chip is provided, comprising:

[0010] First tank,

[0011] The second tank is disposed within the first tank.

[0012] A sample loading chamber for placing the sperm sample to be tested is formed between the first tank and the second tank.

[0013] The outer wall of the second tank faces the sample loading pool, and the top of the outer wall is sloped. One end of the slope points to the sample loading pool, and the other end connects to the interior of the second tank. The slope has microchannels extending along its inclination direction.

[0014] The inner wall of the second tank forms a collection pool for collecting sperm that swim from the sample loading pool to the slope microchannel and fall into the collection pool.

[0015] The wall height of the first tank is higher than that of the second tank.

[0016] In some embodiments, the bottom surface of the sample addition tank is lower than the bottom surface of the collection tank.

[0017] In some embodiments, the outer wall of the second tank is configured as a slope.

[0018] The slope includes a connecting plate, which is connected to a plate. The plate has several aligned microchannels. The connecting plate is detachably connected to the slope.

[0019] In some embodiments, the angle between the slope and the horizontal line is 20° to 60°.

[0020] The beneficial effects of the sperm processing chip of the present invention are as follows:

[0021] (1) The sperm processing chip of the present invention, through the arrangement of the first tank and the second tank, makes the sample loading pool and the collection pool present a U-shape, and a slope is set on the path from the sample loading pool to the collection pool. The slope is provided with microchannels, which can use the characteristic of sperm tending to move by adhering to the wall to screen the sperm with good activity to the collection pool. Sperm with poor activity and poor linear movement ability cannot swim to the collection pool and remain in the sample loading pool. Sperm sorting can be achieved without damage, and it has the advantages of easy operation and low sorting cost.

[0022] (2) The sperm processing chip of the present invention, since the collection pool is located in the sample loading tank, can add chemicals that simulate the microenvironment of sperm-egg binding in the reproductive system or drugs for drug evaluation in the collection pool. The chemicals can diffuse from the collection pool to the sample loading tank so that the solution of the entire processing chip gradually forms a gradient, realizing the simulation of the human reproductive system environment. Then, in the sperm processing chip, it can sort out sperm with good activity and forward motility, as well as sperm that are sensitive to hormones in the reproductive tract. The drugs can diffuse from the collection pool to the sample loading tank so that the solution of the entire processing chip gradually forms a gradient, realizing the setting of drug concentrations, and then evaluating the effect of drugs on sperm, improving the comprehensiveness of sperm sorting, and having the advantage of easy operation.

[0023] (3) The sperm processing chip of the present invention can control the sample volume by changing the size of the first tank and the second tank, which overcomes the problem of small sample volume in traditional sperm sorting and processing, and can greatly improve the sorting efficiency.

[0024] To achieve the second objective mentioned above, the present invention provides the following technical solution:

[0025] A sperm sorting method is provided, comprising the following steps: using the aforementioned sperm processing chip,

[0026] The sperm to be sorted is placed in the sample loading tank, and sperm culture medium is added to the tank so that the level of the sperm culture medium is higher than the highest point of the slope. The opening of the first tank is sealed, and the tank is incubated for a certain period of time for sorting. The sperm in the collection tank is then aspirated.

[0027] The sperm culture medium is pre-equilibrated at 35℃~40℃ before being added to the sample addition tank.

[0028] The method for sealing the opening of the first tank is as follows: place the sperm processing chip in a water bath container and cover the water bath container, or directly seal the opening of the first tank, and then place the sperm processing chip in a carbon dioxide incubator for 5 min to 60 min, at a temperature of 34℃ to 40℃ and a carbon dioxide concentration of 3% to 7%.

[0029] A sperm sorting method simulating the human body environment is provided, comprising the following steps: using the aforementioned sperm processing chip,

[0030] A chemical that simulates the microenvironment of sperm-egg fusion in the female reproductive system is added to the collection pool. Sperm culture medium is added to the sample loading pool, ensuring that the sperm culture medium covers the highest point of the slope and fills the collection pool. The chemical diffuses in the sperm culture medium, forming a concentration gradient that gradually decreases from the collection pool to the sample loading pool.

[0031] The sperm to be sorted is placed at the bottom of the sample loading tank, the opening of the first tank is sealed, and after a certain period of incubation, the sperm is aspirated from the collection tank.

[0032] The sperm culture medium is pre-equilibrated at 35℃~40℃ before being added to the sample addition tank.

[0033] The method for sealing the opening of the first tank is as follows: place the sperm processing chip in a water bath container and cover the water bath container, or directly seal the opening of the first tank. Then place the sperm processing chip in a carbon dioxide incubator for 5 min to 60 min at a temperature of 34℃ to 40℃ and a carbon dioxide concentration of 3% to 7%, and wait for the chemical to form a concentration gradient that gradually decreases from the collection pool to the sample addition pool.

[0034] A drug-based sperm testing method is provided, comprising the following steps: using the sperm processing chip described above, adding a drug to the collection pool, adding a liquid medium to the sample loading pool, such that the liquid medium covers the highest point of the slope and fills the collection pool, the drug diffuses in the liquid medium and forms a concentration gradient, the concentration gradient gradually decreasing from the collection pool to the sample loading pool;

[0035] The sperm to be tested is placed at the bottom of the sample loading tank, the opening of the first tank is sealed, and after a certain period of incubation, the sperm in the collection tank is aspirated, and the performance of the aspirated sperm is tested.

[0036] The liquid medium is pre-equilibrated at 35℃~40℃ before being added to the sample addition tank.

[0037] The method for sealing the opening of the first tank is as follows: place the sperm processing chip in a water bath container and cover the water bath container, or directly seal the opening of the first tank. Set the time, temperature and carbon dioxide concentration of the sperm processing chip in the carbon dioxide incubator according to the experimental needs, and wait for the drug to form a concentration gradient that gradually decreases from the collection pool to the sample addition pool.

[0038] A release-controlled sperm testing method is provided, comprising the following steps:

[0039] Using the aforementioned sperm processing chip,

[0040] The slope is prepared using a releaseable material. The sperm to be tested is placed in the sample loading tank and culture medium is added so that the liquid level of the culture medium reaches at least the highest point of the slope. After a certain period of culture, the sperm is collected from the collection tank. The culture medium is pre-equilibrated at 35℃~40℃ before being added to the sample loading tank.

[0041] In some implementations...

[0042] The releaseable controlled material includes a sustained-release substrate and a chemical drug, wherein the chemical drug is encapsulated by the sustained-release substrate.

[0043] In some embodiments, the slope is divided into several regions, and the composition of the release-controlled materials corresponding to these regions is adjusted to form a composite drug microenvironment.

[0044] In some embodiments, the sustained-release substrate is one or a combination of two or more of the following: hydrogel, hyaluronic acid, chitosan, nanofibers, polyorthoester, polyphospholipid, and polyemulsion.

[0045] The beneficial effects of using the sperm processing chip of the present invention are as follows:

[0046] The sperm sorting method of the present invention utilizes the characteristics of sperm swimming along the wall and the forward movement of high-quality sperm, so that the sperm to be sorted can swim along the slope and fall into the collection pool. Since the slope has a certain inclination, the sperm with good activity can swim in the collection pool within a specified time, while the sperm with poor activity cannot swim across the slope. Finally, the sperm in the collection pool are collected as the sperm with good activity, thus realizing sperm activity sorting. It has the advantages of being easy to operate and will not damage the sperm to be sorted.

[0047] The sperm sorting method simulating the human body environment of the present invention first adds chemical drugs simulating the human body environment to the collection pool. With the intervention of sperm culture medium, the chemical drugs in the collection pool can gradually diffuse to the sample loading pool, forming an environment in which the concentration of secretions from the female reproductive system gradually decreases from the outside to the inside, thereby improving the accuracy of sperm sorting.

[0048] The present invention relates to a drug-based sperm testing method, in which a drug is first added to a collection pool. This drug can be a variety of different drug components. With the intervention of a culture medium, the drug in the collection pool can gradually diffuse into the sample loading pool. Utilizing the chemotaxis of sperm, the sperm can swim along the direction of the high-concentration drug culture medium. Since the drug concentration is the highest in the collection pool, the sperm swimming to the collection pool have good activity and the drug's treatment effect on sperm can be further tested in the collection pool. In addition, by changing the type of drug, the sperm can be subjected to different drug tests.

[0049] The release-controlled sperm testing method of the present invention sets the slope as a time-degradable release-controlled material, which enables control over the sperm climbing time. Once the slope degrades, subsequent sperm cannot continue to swim to the collection pool, thus achieving precise sorting of sperm with the desired activity. At the same time, since the slope is a release-controlled material, different drugs can be mixed on the slope to achieve corresponding drug release-controlled detection, further improving the accuracy of the detection.

[0050] (5) The method of using the sperm processing chip of the present invention allows for free combination and flexible replacement of the component structure. It enables flexible control and adjustment of different structures, sizes, materials, types of chemicals, and concentrations to meet different purposes such as sperm sorting, detection, and drug testing. It proposes a sperm sorting method; a high-throughput sperm processing method; and a method for simultaneously achieving sperm sorting and sperm therapy efficacy evaluation on the same device. It also proposes a microenvironment construction method and a testing method for studying the effects of different components on sperm. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the structure of the sperm processing chip according to a specific embodiment of the present invention.

[0052] Figure 2 This is an exploded view of the structure of the sperm processing chip according to a specific embodiment of the present invention.

[0053] Figure 3 This is a schematic diagram of the slope structure according to a specific embodiment of the present invention.

[0054] Figure 4 This is a cross-sectional view of the sperm processing chip according to a specific embodiment of the present invention.

[0055] Figure 5 This is a physical image of the sperm processing chip according to a specific embodiment of the present invention.

[0056] Figure 6 This is a flowchart of the addition of chemical drugs in Example 7 of the present invention.

[0057] Figure label:

[0058] 1. First tank; 2. Second tank; 3. Sample addition tank; 4. Slope; 5. Microchannel; 6. Collection tank; 7. Inclined slope; 8. Connecting plate; 9. Plate. Detailed Implementation

[0059] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0060] The terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the invention. The singular forms "a", "the", and "the" used in this invention and the appended claims are also intended to include the plural forms unless the context clearly dictates otherwise. It should also be understood that the term "and / or" used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0061] It should be understood that although the terms "first", "second", "third", etc. may be used in this invention to describe various information, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of this invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of this invention, "a plurality of" means two or more unless otherwise specifically defined.

[0062] Example 1

[0063] Please refer to Figures 1-4 , the sperm processing chip disclosed in this embodiment includes:

[0064] The first trough

[0065] The second trough 2, and the second trough 2 is arranged in the first trough

[0066] A sample loading pool 3 for placing the sperm sample to be detected is formed between the first trough and the second trough 2

[0067] The outer wall surface of the second trough 2 faces the sample loading pool 3 and is configured as a slope 4. One end of the slope 4 points to the sample loading pool 3, and the other end is connected to the inside of the second trough 2. A microchannel 5 extending along the inclined direction of the slope 4 is provided on the slope 4. This slope 4 facilitates screening out the sperm that can swim upwards and forwards. In practical applications, due to the characteristic that sperm has a tendency to move towards the wall surface, setting this slope 4 can also ensure that sperm crawls along this slope 4. In this embodiment, the angle between the slope 4 and the horizontal line is 20° to 60°.

[0068] A collection pool 6 is formed on the inner wall surface of the second trough 2 for collecting the sperm that swim from the sample loading pool 3 to the slope 4 and fall into the collection pool 6. Preferably, both the first trough and the second trough 2 are set as rectangles, and the first trough and the second trough 2 are configured as a "hui" character. In practical applications, other configurations can also be set as long as the second trough 2 can be kept arranged in the first trough.

[0069] The height of the wall of the first tank is higher than that of the wall of the second tank 2, which ensures that the culture medium or sperm sample is not easily overflowed from the first tank, thus ensuring the accuracy of the detection.

[0070] In this embodiment, the bottom surface of the sample loading chamber 3 is lower than the bottom surface of the collection chamber 6. This lower bottom surface of the sample loading chamber 3 allows for the placement of a larger sperm sample, increasing the detection volume and thus improving the detection accuracy.

[0071] In this embodiment, the outer wall surface of the second tank 2 is configured as a slope 7.

[0072] The slope 4 includes a connecting plate 8, which is connected to a plate 9. The plate 9 has several aligned microchannels 5. The connecting plate 8 is detachably connected to the slope 7.

[0073] Since the slope 4 can be detachably connected to the ramp 7, when microchannels 5 of different sizes are needed, the slope 4 can be directly replaced. The connection methods between the slope 4 and the ramp 7 include, but are not limited to, bonding, welding, and magnetic connection, with the aim of fixing the two relatively and preventing displacement during use. The plurality of microchannels 5 are configured as more than three microchannels 5, arranged equidistantly in parallel or radial patterns. The width of the microchannels 5 is between 10μm and 500μm, and the cross-sectional shape of the microchannels 5 can be rectangular, arc-shaped, polygonal, or other shapes.

[0074] The material of the sperm processing chip needs to be non-toxic to sperm, such as glass, PDMS, acrylic resin, biosafe resin, or other biocompatible materials that are non-toxic to sperm, cells, and the human body.

[0075] The slope is divided into several regions. By adjusting the composition of the release control material corresponding to these regions, a composite drug microenvironment can be formed. Specifically, since the release control material consists of a matrix material and a drug contained within the matrix material, by adjusting the drug composition and / or matrix material in each region of the slope, a preset composite drug microenvironment can be effectively formed, making the test controllable.

[0076] The material of the slope surface 4 can be the same as or different from the material of the first tank and the second tank 2, and each slope surface 4 can also be made of a different material. For example, the slope surface 4 may contain chemicals or may not contain chemicals. For example, the material of the slope surface 4 can be a water-soluble material or a water-insoluble material.

[0077] Exemplarily, the slope 4 material containing chemicals can be made of a chemical drug to be tested and a matrix material with a sustained-release function (such as hydrogel, hyaluronic acid, chitosan, nanofibers, polyorthoesters, polyphospholipids, and polylactide, etc.), and the degradation time of the matrix material is at least greater than 30 minutes. The side length range of the first tank is 5 - 100 mm, and the overall height range is 2 - 30 mm. The sample addition pool 3 is in a "hui" - shaped structure, the outer side length should be at least 0.5 mm less than the side length of the sperm processing chip, the outer side length range of the sample addition pool 3 is 4.5 - 99.5 mm, the depth of the sample addition pool 3 should be at least 0.5 mm less than the height of the sperm processing chip, the depth range of the sample addition pool 3 is 1.5 - 29.5 mm, the width range of the sample addition pool 3 is 1 - 5 mm, and the inner side length range of the sample addition pool 3 is 2 - 98.5 mm. The depth range of the collection pool 6 is 1.5 - 29.5 mm, the height of the collection pool 6 should be lower than the height of the sperm processing chip and the highest point of the processing structure in the vertical height, and the side length range of the collection pool 6 is 1 - 98 mm.

[0078] Example 2

[0079] The sperm sorting method disclosed in this example includes the following steps: using the sperm processing chip described in Example 1,

[0080] Placing the sperm to be sorted in the sample addition pool 3, adding sperm culture medium to the sample addition pool 3 so that the liquid level of the sperm culture medium is higher than the highest point of the slope 4, covering the opening of the first tank, culturing for a certain period of time for sorting, and sucking the sperm in the collection pool 6.

[0081] Among them, the sperm culture medium is pre - equilibrated at 35°C - 40°C, preferably 37°C, and then added to the sample addition pool 3.

[0082] To prevent liquid evaporation during the sorting process, the treatment method for covering the opening of the first tank is: placing the sperm processing chip in a water bath container and covering the water bath container, or directly covering the opening of the first tank, and then placing the sperm processing chip in a carbon dioxide incubator, culturing for 5 min - 60 min, at a temperature of 34°C - 40°C, and a carbon dioxide concentration of 3% - 7%.

[0083] Example 3

[0084] The sperm sorting method simulating the human body environment disclosed in this example includes the following steps: using the sperm processing chip described in Example 1.

[0085] A chemical substance simulating the microenvironment of sperm-egg fusion in the female reproductive system is added to the collection pool 6. Sperm culture medium is added to the sample loading pool 3, ensuring that the sperm culture medium covers the highest point of the slope 4 and fills the collection pool 6. The chemical substance diffuses in the sperm culture medium, forming a concentration gradient that gradually decreases from the collection pool 6 to the sample loading pool 3.

[0086] To maintain a concentration gradient environment in the sperm sample from the outset, the sperm to be sorted is placed at the bottom of the sample loading chamber 3. Preferably, the sperm sample is slowly added to the bottom of the sample loading chamber 3 through the sperm culture medium using a pipette or micropipette. Preferably, the sample is added in equal amounts to all four sides of the rectangular sample loading chamber 3. The opening of the first tank is sealed, and after a certain period of incubation, the sperm in the collection chamber 6 is aspirated. The aspirated sperm are those with good motility.

[0087] The sperm culture medium is pre-equilibrated at 35℃~40℃, preferably 37℃, before being added to the sample addition tank 3.

[0088] To prevent liquid evaporation during the sorting process, the method for sealing the opening of the first tank is as follows: place the sperm processing chip in a water bath container and cover the water bath container, or directly seal the opening of the first tank. Then place the sperm processing chip in a carbon dioxide incubator for 5 min to 60 min at a temperature of 34℃ to 40℃ and a carbon dioxide concentration of 3% to 7%, and wait for the chemical to form a concentration gradient that gradually decreases from the collection pool 6 to the sample addition pool 3.

[0089] Example 4

[0090] The drug-based sperm testing method disclosed in this embodiment includes the following steps: using the sperm processing chip described in Example 1,

[0091] Add the drug to the collection tank 6 and add liquid medium to the sample addition tank 3 so that the sperm culture medium covers the highest point of the slope 4 and fills the collection tank 6. The drug diffuses in the sperm culture medium and forms a concentration gradient, which gradually decreases from the collection tank 6 to the sample addition tank 3.

[0092] To ensure the sperm sample is initially in a concentration gradient environment, the sperm to be tested is placed at the bottom of the sample loading chamber 3. Preferably, the sperm sample is slowly added to the bottom of the sample loading chamber 3 through the sperm culture medium using a pipette or micropipette. The opening of the first tank is sealed, and after a certain period of incubation, the sperm in the collection chamber 6 is aspirated to test the sperm aspiration performance.

[0093] The culture medium is pre-equilibrated at 35℃~40℃, preferably 37℃, before being added to the sample addition tank 3.

[0094] The method for sealing the opening of the first tank is as follows: place the sperm processing chip in a water bath container and cover the water bath container, or directly seal the opening of the first tank. Set the time, temperature and carbon dioxide concentration of the sperm processing chip in the carbon dioxide incubator according to the experimental needs, and wait for the drug to form a concentration gradient that gradually decreases from the collection pool 6 to the sample addition pool 3.

[0095] Example 5

[0096] The release-controlled sperm testing method disclosed in this embodiment includes the following steps:

[0097] Using the sperm processing chip described in Example 1,

[0098] The slope 4 is prepared using a releaseable material. The sperm to be tested is placed in the sample loading tank 3 and culture medium is added so that the liquid level of the culture medium reaches at least the highest point of the slope 4. After a certain period of culture, the sperm is extracted from the collection tank 6. The sperm culture medium is pre-equilibrated at 35℃~40℃, preferably 37℃, before being added to the sample loading tank 3.

[0099] In this embodiment, the releaseable controlled material includes a sustained-release substrate and a chemical drug, wherein the chemical drug is coated by the sustained-release substrate.

[0100] In this embodiment, the sustained-release substrate is one or a combination of two or more of the following: hydrogel, hyaluronic acid, chitosan, nanofibers, polyorthoester, polyphospholipid, and polyemulsion.

[0101] The method for preparing the slope surface 4 using the releaseable and controllable material includes the following steps:

[0102] A digital model of the required processing structure is established using 3D modeling and fabricated using methods such as 3D printing, laser processing, or machining. A negative mold of the processing structure is prepared using materials such as silicone or polydimethylsiloxane through a molding process. The target chemical and a matrix material with sustained-release function (such as hydrogel, hyaluronic acid, chitosan, nanofibers, polyorthoesters, polyphospholipids, and polyemulsions) are thoroughly mixed and added to the negative mold obtained in the previous step. After degassing, the mixture is cured. Preferably, the four processing structures of the chip can each be composed of N (1≤N≤4) kinds of chemicals. After complete curing, the mold is removed, and the processing structure containing the target chemical is attached and fixed to the surface of the processing area of ​​the chip.

[0103] Further explanation of specific embodiments of the present invention is provided in Examples 5 to 7.

[0104] Example 6

[0105] Sperm sorting using sperm processing chips

[0106] A digital model of the sperm processing chip was created using 3D design software. The sperm processing chip is a square with a side length of 20 mm and an overall height of 6.5 mm. The sample loading chamber 3 has a side length of 18.2 mm, a depth of 5.5 mm, a groove width of 3.25 mm, and a height of 2.6 mm from the bottom of the sample loading chamber 3 to the lowest point of the processing structure. The angle between the processing area and the bottom of the sperm processing chip is set at 45°. The base of the processing structure is trapezoidal, with a long side of 12 mm, a short side of 9 mm, a width of 2.3 mm, and a height of 0.65 mm. The surface of the processing structure has 12 equidistant, balanced microchannels with a height of 0.65 mm and a width of 100 micrometers. The processing structure and the sperm processing chip are integrally molded.

[0107] Biocompatible resin was selected as the material, and the sperm processing chip designed in step 1 was prepared using ultra-precision 3D printing equipment. The surface residual resin was washed with anhydrous ethanol, then rinsed with deionized water and physiological saline in sequence, and finally sterilized with ultraviolet light for later use.

[0108] Human fallopian tube fluid was used as the sorting medium. It was placed in a carbon dioxide incubator overnight for pre-equilibration at 37°C and a carbon dioxide concentration of 5%.

[0109] Shake the liquefied semen sample thoroughly, then use a pipette to extract 500 μL of the semen sample and add it to sample cell 3. The semen level should be lower than the lowest point of the treatment structure.

[0110] Slowly add pre-balanced human fallopian tube fluid to collection pool 6 until the fluid level is higher than the highest point of the processing area and level with the highest point of the inner wall of the sperm processing chip.

[0111] The chips were placed in a covered plastic petri dish and then placed in a carbon dioxide incubator at a temperature of 37°C and a carbon dioxide concentration of 5% for 25 minutes.

[0112] After the sorting time is complete, remove the sperm processing chip from the CO2 incubator. Use a pipette to extract the sorted sperm from collection pool 6 for subsequent testing or assisted reproductive technology.

[0113] Example 7

[0114] Using progesterone to induce sperm sorting

[0115] A stock solution with a concentration of 1 mmol / L was prepared by mixing dimethyl sulfoxide and progesterone, and then the progesterone stock solution was diluted with human fallopian tube fluid to a concentration of 10 μmol / L.

[0116] A digital model of the sperm processing chip was created using 3D design software. The sperm processing chip is a square with a side length of 30mm and an overall height of 9mm. The sample loading chamber 3 has a side length of 26mm, a depth of 7.5mm, a groove width of 3mm, and a height of 4mm from the bottom of the sample loading chamber 3 to the lowest point of the processing structure. The angle between the processing area and the bottom of the sperm processing chip is set at 45°. The base of the processing structure is trapezoidal, with a long side of 20mm, a short side of 14.5mm, a width of 3.8mm, and a height of 1mm. The surface of the processing structure has 12 equidistant, balanced microchannels, each 1mm high and 200μm wide. The processing structure and the sperm processing chip are integrally molded.

[0117] Biocompatible resin was selected as the material, and the sperm processing chip designed in step 1 was prepared using ultra-precision 3D printing equipment. The surface residual resin was washed with anhydrous ethanol, then rinsed with deionized water and physiological saline in sequence, and finally sterilized with ultraviolet light for later use.

[0118] Human fallopian tube fluid was used as the sorting medium. It was placed in a carbon dioxide incubator overnight for pre-equilibration at 37°C and a carbon dioxide concentration of 5%.

[0119] Please see Figure 6 Add 3 μL of progesterone stock solution to the collection pool 6 of the sperm processing chip, and slowly add 1300 μL of pre-equilibrated human fallopian tube fluid to the sample loading pool 3. At this time, the level of human fallopian tube fluid is higher than the lowest point of the processing structure. The sample loading pool 3, the processing area and the collection pool 6 inside the sperm processing chip have formed a continuous space with liquid as the medium.

[0120] The chip was placed in a sealed container with a water bath and then placed in a carbon dioxide incubator for 15 minutes at a temperature of 37°C and a carbon dioxide concentration of 5%, allowing progesterone to diffuse and form a concentration gradient.

[0121] Take 500 μL of liquefied semen sample, mix thoroughly, and then use a pipette to slowly add the semen sample to the bottom of the sample loading chamber 3 along the four sides of the loading chamber 3 until the liquid level in the chip is level with the highest point of the chip.

[0122] The chip was placed in a sealed container with a water bath and then placed in a carbon dioxide incubator for 30 minutes at a temperature of 37°C and a carbon dioxide concentration of 5%.

[0123] After processing, the sperm samples are removed from the incubator and collected in collection pool 6 for subsequent testing or assisted reproductive technology.

[0124] Example 8

[0125] Constructing a multi-chemical component microenvironment using sperm processing chips for sperm research

[0126] A digital model of the sperm processing chip was created using 3D design software. The sperm processing chip was set as a square with a side length of 30mm and an overall height of 9mm. The sample loading chamber 3 had a side length of 26mm, a depth of 7.5mm, a groove width of 3mm, and a height of 4mm from the bottom of the sample loading chamber 3 to the lowest point of the processing structure. The angle between the processing area and the bottom of the sperm processing chip was set at 45°, and the depth of the connection point of the processing area was 1mm to facilitate connection with the processing structure.

[0127] The base of the processing structure is trapezoidal, with a long side of 20mm, a short side of 14.5mm, a width of 3.8mm, and a height of 1mm. The surface of the processing structure has 24 equidistant, balanced microchannels. The processing structure itself is 1mm high, and the microchannels are 100 micrometers wide. The overall height of the processing structure is 2mm.

[0128] Biocompatible resin was selected as the material, and the sperm processing chip designed in step 1 was prepared using ultra-precision 3D printing equipment. The surface residual resin was washed with anhydrous ethanol, then rinsed with deionized water and physiological saline in sequence, and finally sterilized with ultraviolet light for later use.

[0129] The structure designed in step 2 was prepared using 3D printing technology, and a negative mold of the structure was prepared using polydimethylsiloxane through a molding process.

[0130] Drugs A, B, C, and D to be tested were prepared to the target concentration and mixed with hyaluronic acid respectively. After defoaming under negative pressure, they were placed in an oven and heated to 35°C for curing before being demolded.

[0131] Using the same photosensitive resin as the sperm processing chip as an adhesive, the drug-containing processing structure is bonded and fixed to the sperm processing chip.

[0132] Take 1 ml of liquefied semen sample, mix thoroughly, and slowly add it to sample loading chamber 3. The height of the semen sample should not exceed the lowest point of the processing structure.

[0133] When using Hanks solution as a test buffer, it needs to be pre-equilibrated at 37°C. When using it, slowly add Hanks solution until the liquid level is flush with the highest point of the sperm processing chip.

[0134] The chip was placed in a sealed petri dish and incubated in a carbon dioxide incubator for 30 minutes at a temperature of 37°C and a carbon dioxide concentration of 5% before testing.

[0135] After the experiment, the sperm processing chip was removed from the incubator, and the test was completed. The sample could then be observed, recorded, and analyzed under a microscope, or a sperm sample could be aspirated from collection chamber 6 for further characterization.

[0136] The sperm processing chip and its usage method proposed in this invention can overcome the above-mentioned problems, construct a suitable microenvironment for sperm sorting, and allow for adjustment of the structure and chemical concentrations according to different needs to achieve sperm sorting. Furthermore, different types of chemicals can be replaced to test the effects of different drugs on sperm, enabling the evaluation of sperm treatment efficacy.

[0137] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A sperm processing chip, characterized in that, include: First tank, The second tank is disposed within the first tank. A sample loading chamber for placing the sperm sample to be tested is formed between the first tank and the second tank. The outer wall of the second tank faces the sample loading pool, and the top of the outer wall is sloped. One end of the slope points to the sample loading pool, and the other end connects to the interior of the second tank. The slope has microchannels extending along its inclination direction. The inner wall of the second tank forms a collection pool for collecting sperm that swim from the sample loading pool to the slope microchannel and fall into the collection pool. The wall height of the first tank is higher than that of the second tank.

2. The sperm processing chip according to claim 1, characterized in that, The bottom surface of the sample addition tank is lower than the bottom surface of the collection tank.

3. The sperm processing chip according to claim 2, characterized in that, The slope includes a connecting plate, which is connected to a plate. The plate has several aligned microchannels, and the connecting plate is detachably connected to the slope.

4. The sperm processing chip according to claim 1, characterized in that, The angle between the slope and the horizontal line is 20° to 60°.

5. A sperm sorting method, characterized in that, The process includes the following steps: using the sperm processing chip described in any one of claims 1 to 4. The sperm to be sorted is placed in the sample loading tank, and sperm culture medium is added to the tank so that the level of the sperm culture medium is higher than the highest point of the slope. The opening of the first tank is sealed, and the tank is incubated for a certain period of time for sorting. The sperm in the collection tank is then aspirated. The method for sealing the opening of the first tank is as follows: place the sperm processing chip in a water bath container and cover the water bath container, or directly seal the opening of the first tank, and then place the sperm processing chip in a carbon dioxide incubator for 5 min to 60 min, at a temperature of 34℃ to 40℃ and a carbon dioxide concentration of 3% to 7%.

6. A sperm sorting method simulating the human body environment, characterized in that, The process includes the following steps: using the sperm processing chip described in any one of claims 1 to 4. A chemical that simulates the microenvironment of sperm-egg fusion in the female reproductive system is added to the collection pool. Sperm culture medium is added to the sample loading pool, ensuring that the sperm culture medium covers the highest point of the slope and fills the collection pool. The chemical diffuses in the sperm culture medium, forming a concentration gradient that gradually decreases from the collection pool to the sample loading pool. The sperm to be sorted is placed at the bottom of the sample loading tank, the opening of the first tank is sealed, and after a certain period of incubation, the sperm is aspirated from the collection tank. The method for sealing the opening of the first tank is as follows: place the sperm processing chip in a water bath container and cover the water bath container, or directly seal the opening of the first tank. Then place the sperm processing chip in a carbon dioxide incubator for 5 min to 60 min at a temperature of 34℃ to 40℃ and a carbon dioxide concentration of 3% to 7%, and wait for the chemical to form a concentration gradient that gradually decreases from the collection pool to the sample addition pool.

7. A drug-based sperm testing method, characterized in that, The process includes the following steps: using the sperm processing chip described in any one of claims 1 to 4. Drug is added to the collection pool, and liquid medium is added to the sample addition pool, so that the liquid medium covers the highest point of the slope and fills the collection pool. The drug diffuses in the liquid medium and forms a concentration gradient, which gradually decreases from the collection pool to the sample addition pool. The sperm to be tested is placed at the bottom of the sample loading tank, the opening of the first tank is sealed, and after a certain period of incubation, the sperm in the collection tank is aspirated, and the performance of the aspirated sperm is tested. The method for sealing the opening of the first tank is as follows: place the sperm processing chip in a water bath container and cover the water bath container, or directly seal the opening of the first tank. Set the time, temperature and carbon dioxide concentration of the sperm processing chip in the carbon dioxide incubator according to the experimental needs, and wait for the drug to form a concentration gradient that gradually decreases from the collection pool to the sample addition pool.

8. A sperm testing method based on release control, characterized in that, Includes the following steps: Using the sperm processing chip according to any one of claims 1 to 4, The slope is prepared using a releaseable material. The sperm to be tested is placed in the sample loading tank and culture medium is added so that the liquid level of the culture medium reaches at least the highest point of the slope. After a certain period of culture, the sperm is collected from the collection tank. The culture medium is pre-equilibrated at 35℃~40℃ before being added to the sample loading tank.

9. The sperm testing method based on release control according to claim 8, characterized in that, The releaseable controlled material includes a sustained-release substrate and a chemical drug, wherein the chemical drug is encapsulated by the sustained-release substrate.

10. The sperm testing method based on release control according to claim 9, characterized in that, The slope is divided into several regions, and the composition of the release-controlled materials in these regions is adjusted to create a composite drug microenvironment.

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