Sperm processing chip and use method therefor

Abstract

The present invention relates to the technical field of sperm sorting, and provides a sperm processing chip and a use method therefor. The sperm processing chip comprises: a second tank arranged in a first tank, wherein a sample adding pool for placement of a sperm sample to be tested is formed between the first tank and the second tank, an outer wall surface of the second tank faces the sample adding pool, the top of the outer wall surface is constructed as a slope, one end of the slope points to the sample adding pool and the other end is connected to the interior of the second tank, the slope is provided with a microchannel extending in an inclined direction of the slope, an inner wall surface of the second tank forms a collection pool for collecting sperm that flows from the sample adding pool to the microchannel of the slope and falls into the collection pool, and the tank wall height of the first tank is higher than the tank wall height of the second tank. The sperm processing chip can achieve active sorting without the use of chemicals, and sperm screening under the action of chemicals, can effectively maintain sperm vitality, and has the advantages of high versatility, high recovery rate, high sorting accuracy, high motility rate, and ease of operation.

Description

A sperm processing chip and a method of using the same TECHNICAL FIELD

[0001] The present application relates to the technical field of sperm sorting, in particular to a sperm processing chip and a method of using the same. BACKGROUND

[0002] The incidence of infertility in China has rapidly climbed from 3% to 12.5%, and by 2018, there were about 50 million couples of childbearing age facing the problem of infertility in China, and there was a trend of youth. Assisted reproductive technology is an effective method to solve infertility, and obtaining sperm with high clinical fertilization potential is the premise of successful pregnancy, so how to screen high-quality sperm is a crucial step in the process of assisted reproductive technology.

[0003] Currently, the methods for sorting sperm in clinical use are mainly the upstream method and the density gradient centrifugation method. The upstream method mainly utilizes the self-movement ability of motile sperm, which can move upwards from the bottom of the test tube to the liquid surface layer of the culture medium. The density gradient centrifugation method utilizes the differences between normal sperm and non-motile sperm or other components in semen in various aspects, including the trajectory of movement and the ability to move, so that they can be distinguished and separated in the density gradient solution column. After centrifugation, these different components will stay in different density gradient solution columns, thereby screening relatively normal sperm for artificial insemination.

[0004] However, both the upstream method and the density gradient centrifugation method rely on a single standard for sorting sperm. In the density gradient centrifugation method, the centrifugation step can increase reactive oxygen species and damage DNA double strands, affecting embryo quality. Sperm quality evaluation criteria include its morphology, movement, DNA fragmentation rate, and sperm characteristics (including chemotaxis, thermotaxis, rheotaxis, and thigmotaxis), and accordingly, sperm needs to be sorted from multiple dimensions.

[0005] In addition, current clinical sperm detection and drug testing usually use two completely different technologies and instruments. After drug testing, routine sperm detection is still needed to determine the response of sperm to drugs, which can cause sampling errors, take a long time, and consume multiple consumables, making the process complicated and unable to intuitively and quickly determine the effect of drugs on sperm. SUMMARY

[0006] One of the purposes of the present application is to avoid the shortcomings in the prior art and provide a sperm processing chip that can achieve active sorting without the action of chemical drugs, sperm screening under the action of drugs, and effectively maintain sperm activity, with the advantages of high universality, high recovery rate, high sorting accuracy, high motility rate, and easy operation.

[0007] The second object of the present application is to provide a method for using the sperm processing chip.

[0008] To achieve the above-mentioned one of the objects, the present application provides the following technical solutions.

[0009] The sperm processing chip comprises:

[0010] a first groove,

[0011] a second groove arranged in the first groove,

[0012] a sample pool for placing a sperm sample to be detected is formed between the first groove and the second groove,

[0013] an outer wall surface of the second groove faces the sample pool, and a top of the outer wall surface is configured as a slope, one end of the slope points to the sample pool, and the other end is connected to the inside of the second groove, and the slope is provided with a microchannel extending along the slope,

[0014] an inner wall surface of the second groove forms a collection pool for collecting sperm swimming from the sample pool to the slope microchannel and falling into the collection pool,

[0015] a groove wall height of the first groove is higher than a groove wall height of the second groove.

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

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

[0018] the slope comprises a connecting plate, the connecting plate is connected with a plate block, the plate block is provided with a plurality of aligned microchannels, and the connecting plate is detachably connected to the slope.

[0019] In some embodiments, an included angle between the slope and a horizontal line is 20°-60°.

[0020] The sperm processing chip has the following advantages:

[0021] (1) The sperm processing chip is provided with the first groove and the second groove, so that the sample pool and the collection pool present a character-shaped structure, and the slope provided on the path from the sample pool to the collection pool is provided with the microchannel, which can utilize the characteristics of sperm adhesion and directional movement to screen the sperm with good activity to the collection pool, and the sperm with poor activity and poor straight-line movement ability cannot swim to the collection pool and stay in the sample pool, so that the sperm sorting can be realized without damage, and the sperm processing chip has the advantages of easy operation and low sorting cost.

[0022] (2) The sperm processing chip of the present application can add chemicals simulating the microenvironment of sperm-egg combination in the reproductive system or drugs for drug evaluation into the collection pool, and the chemicals can diffuse from the collection pool to the sample groove, so that the solution of the whole processing chip gradually forms a gradient, the environment of the human reproductive system is simulated, and then sperm with good activity and good forward motility and sperm sensitive to hormones in the reproductive tract can be sorted out in the sperm processing chip; the drugs can diffuse from the collection pool to the sample groove, so that the solution of the whole processing chip gradually forms a gradient, the setting of various concentrations of the drugs is realized, and then the effect of the drugs on sperm can be evaluated, the comprehensiveness of sperm sorting is improved, and the sperm processing chip has the advantages of easy operation.

[0023] (3) The sperm processing chip of the present application can control the sample amount by changing the size of the first groove body and the second groove body, overcome the problem of small sample amount in traditional sperm sorting and processing, and greatly improve the efficiency of sorting.

[0024] To achieve the above-mentioned second purpose, the present application provides the following technical solutions:

[0025] A sperm sorting method is provided, which comprises the following steps:

[0026] Placing the sperm to be sorted into the sample pool, adding sperm culture solution to the sample pool, so that the liquid level of the sperm culture solution is higher than the highest point of the slope, covering the notch of the first groove body, culturing for a certain period of time for sorting, and sucking the sperm in the collection pool,

[0027] Pre-equilibrating the sperm culture solution at 35 DEG C to 40 DEG C and then adding it to the sample pool,

[0028] The processing mode of covering the notch of the first groove body is: placing the sperm processing chip in a water bath container and covering the water bath container, or directly covering the notch of the first groove body, and then placing the sperm processing chip in a carbon dioxide incubator, culturing for 5 min to 60 min, the temperature is 34 DEG C to 40 DEG C, and the carbon dioxide concentration is 3% to 7%.

[0029] A sperm sorting method simulating the human environment is provided, which comprises the following steps:

[0030] Adding chemicals simulating the microenvironment of sperm-egg combination in the female reproductive system into the collection pool, adding sperm culture solution to the sample pool, so that the sperm culture solution covers the highest point of the slope and fills the collection pool, the chemicals diffuse in the sperm culture solution and form a concentration gradient, and the concentration gradient gradually decreases from the collection pool to the sample pool,

[0031] Placing the sperm to be sorted at the bottom of the sample pool, covering the notch of the first groove body, and after a certain period of culture, sucking the sperm in the collection pool,

[0032] Pre-equilibrating the sperm culture solution at 35-40°C and then adding it to the sample pool,

[0033] The covering method for the notch of the first groove body is to place the sperm treatment chip in a water bath container and cover the water bath container, or directly cover the notch of the first groove body, and then place the sperm treatment chip in a carbon dioxide incubator for 5-60 minutes at a temperature of 34-40°C and a carbon dioxide concentration of 3-7%, and wait for the chemical to form a concentration gradient gradually decreasing from the collection pool to the sample pool.

[0034] A drug-based sperm testing method is provided, including the following steps: using the sperm treatment chip, adding a drug to the collection pool, and adding a liquid medium to the sample 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, and the concentration gradient gradually decreases from the collection pool to the sample pool.

[0035] Placing the sperm to be tested at the bottom of the sample pool, covering the notch of the first groove body, and after a certain period of culture, sucking the sperm in the collection pool, and detecting the performance of the sucked sperm,

[0036] Pre-equilibrating the liquid medium at 35-40°C and then adding it to the sample pool,

[0037] The covering method for the notch of the first groove body is to place the sperm treatment chip in a water bath container and cover the water bath container, or directly cover the notch of the first groove body, and according to the experimental requirements, set the time, temperature, and carbon dioxide concentration of the sperm treatment chip in the carbon dioxide incubator, and wait for the drug to form a concentration gradient gradually decreasing from the collection pool to the sample pool.

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

[0039] Using the sperm treatment chip,

[0040] The slope is made of a releaseable material, the sperm to be tested is placed in the sample pool and a culture solution is added, so that the liquid level of the culture solution reaches at least the highest point of the slope, after a certain period of culture, the sperm in the collection pool is extracted, and the culture solution is pre-equilibrated at 35-40°C before being added to the sample pool.

[0041] In some embodiments,

[0042] The controllable release material comprises a slow-release base material and a chemical drug, and the chemical drug is coated by the slow-release base material.

[0043] In some embodiments, the slope surface is divided into several regions, and a composite drug microenvironment is formed by adjusting the corresponding release control material composition of the several regions.

[0044] In some embodiments, the slow-release base material is one or a combination of more than two of a hydrogel, hyaluronic acid, chitosan, nanofiber, polyorthoester, polyphospholipid, and polylactide.

[0045] The sperm processing chip has the following advantages:

[0046] (1) The sperm sorting method utilizes the characteristics of sperm adhesion and movement and the forward movement force of high-quality sperm, so that the sperm to be sorted can move along the slope surface and fall into the collection pool.

[0047] (2) The sperm sorting method simulates the human body environment by adding chemical drugs in the collection pool, which can gradually diffuse into the sample pool under the intervention of the sperm culture solution, forming an environment where the concentration of female reproductive system secretions gradually decreases from the outside to the inside, thereby improving the accuracy of sperm sorting.

[0048] (3) The sperm testing method based on drugs adds drugs in the collection pool, which can be a variety of different drug components, and the drugs in the collection pool can gradually diffuse into the sample pool under the intervention of the culture solution.

[0049] (4) The sperm testing method based on release control sets the slope surface as a timed degradation release control material, so that the time for sperm to climb the slope can be controlled.

[0050] (5) The sperm processing chip of the application can be freely combined, and the sperm processing chip structure of the spare parts can be flexibly replaced. Different structures, different sizes, different materials, different types of chemicals, and different concentrations can be flexibly controlled and adjusted to meet different purposes such as sperm sorting, detection, and drug testing, and a sperm sorting method is provided. A large-flux sperm processing method is provided. A method for simultaneously realizing sperm sorting and sperm treatment drug efficacy evaluation on the same device is provided. A microenvironment construction method is provided. A test method for studying the influence of different components on sperm is provided. BRIEF DESCRIPTION OF DRAWINGS

[0051] Fig. 1 is a structural schematic diagram of the sperm processing chip of the embodiment of the application.

[0052] Fig. 2 is an exploded view of the sperm processing chip of the embodiment of the application.

[0053] Fig. 3 is a structural schematic diagram of the slope surface of the embodiment of the application.

[0054] Fig. 4 is a sectional view of the sperm processing chip of the embodiment of the application.

[0055] Fig. 5 is a physical diagram of the sperm processing chip of the embodiment of the application.

[0056] Fig. 6 is a flowchart of adding a chemical drug according to the embodiment 7 of the application.

[0057] Reference signs:

[0058] 1, first groove; 2, second groove; 3, sample pool; 4, slope surface; 5, microchannel; 6, collection pool; 7, slope; 8, connecting plate; 9, plate. DETAILED DESCRIPTION

[0059] The preferred embodiments of the application will be described in more detail with reference to the drawings. Although the preferred embodiments of the application are shown in the drawings, it should be understood that the application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the application more thorough and complete, and to fully convey the scope of the application to those skilled in the art.

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

[0061] It should be understood that although the terms "first", "second", "third", etc. may be used in the present 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 the present 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 the present invention, "a plurality of" means two or more unless otherwise specifically defined.

[0062] Embodiment 1

[0063] Referring to FIGS. 1-4, the sperm processing chip disclosed in this embodiment includes:

[0064] A first tank

[0065] A second tank 2, the second tank 2 is disposed inside the first tank

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

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

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

[0069] The height of the tank wall of the first tank is higher than the height of the tank wall of the second tank 2, which can ensure that the culture solution or sperm sample is not easily spilled out of the first tank and ensure the detection accuracy.

[0070] In this embodiment, the bottom surface of the sample addition pool 3 is lower than the bottom surface of the collection pool 6. The bottom surface of the sample addition pool 3 being lower than the bottom surface of the collection pool 6 can hold more sperm samples, increase the detection amount, and then improve the detection accuracy.

[0071] In the embodiment, the outer wall surface of the second groove body 2 is formed as a slope 7,

[0072] The slope surface 4 comprises a connecting plate 8, the connecting plate 8 is connected with a plate block 9, the plate block 9 is provided with a plurality of aligned microchannels 5, and the connecting plate 8 is detachably connected to the slope 7.

[0073] Since the slope surface 4 can be detachably connected to the slope 7, when microchannels 5 of different sizes are needed, the slope surface 4 can be directly replaced. The connection mode of the slope surface 4 and the slope 7 includes but is not limited to adhesion, welding, magnetic attraction connection, etc., and the purpose is to relatively fix the two, and no displacement occurs during use. The plurality of microchannels 5 are arranged as more than 3 microchannels 5, equidistantly parallel or equidistantly radially, the width of the microchannel 5 is between 10 μm and 500 μm, and the cross-sectional shape of the microchannel 5 can be rectangular, arc-shaped, polyline or other shapes.

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

[0075] The slope surface is divided into a plurality of regions, and a composite drug microenvironment is formed by adjusting the corresponding release control material composition of the plurality of regions. Specifically, since the release control material is composed of a base material and a drug contained in the base material, by adjusting the drug composition and / or the base material on each region of the slope surface, a preset composite drug microenvironment can be effectively formed, so that the test has controllability.

[0076] The material of the slope surface 4 can be the same as or different from the material of the first groove body and the second groove body 2, and the material of each slope surface 4 can also be different. Illustratively, the slope surface 4 can contain chemicals or not. Illustratively, the slope surface 4 material can be water-soluble material or non-water-soluble 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 has 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. 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. 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 time for sorting, and aspirating the sperm in the collection pool 6.

[0081] Among them, the sperm culture medium is pre - balanced 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 for simulating the human body environment disclosed in this example includes the following steps: using the sperm processing chip described in Example 1,

[0085] The chemical substance simulating the microenvironment of sperm-egg binding in female reproductive system is added to the collection pool 6, and the sperm culture solution is added to the sample pool 3, so that the sperm culture solution covers the highest point of the slope 4 and fills the collection pool 6, the chemical substance diffuses in the sperm culture solution and forms a concentration gradient gradually decreasing from the collection pool 6 to the sample pool 3,

[0086] To keep the sperm sample in the concentration gradient environment from the beginning, the sperm to be sorted is placed at the bottom of the sample pool 3, preferably, the sperm sample is slowly added to the bottom of the sample pool 3 through the sperm culture solution of the sample pool 3 by using a pipette or a micro-sampler. Preferably, the sample is added in equal amounts at the four edges of the rectangular sample pool 3 of the chip. After covering the notch of the first groove body, the sperm in the collection pool 6 is aspirated after a certain incubation time, and the aspirated sperm is good in activity.

[0087] Preferably, the sperm culture solution is pre-equilibrated at 35-40°C, preferably at 37°C, and then added to the sample pool 3,

[0088] To prevent liquid evaporation during the sorting process, the first groove body is covered in the following manner: the sperm treatment chip is placed in a water bath container and the water bath container is covered, or the notch of the first groove body is directly covered, and then the sperm treatment chip is placed in a carbon dioxide incubator for 5-60 min at a temperature of 34-40°C and a carbon dioxide concentration of 3-7%, and the chemical substance is allowed to form a concentration gradient gradually decreasing from the collection pool 6 to the sample pool 3.

[0089] Example 4

[0090] The drug-based sperm test method disclosed in this embodiment comprises the following steps: using the sperm treatment chip of Example 1,

[0091] The drug is added to the collection pool 6, and the liquid medium is added to the sample pool 3, so that the sperm culture solution covers the highest point of the slope 4 and fills the collection pool 6, the drug diffuses in the sperm culture solution and forms a concentration gradient gradually decreasing from the collection pool 6 to the sample pool 3;

[0092] To keep the sperm sample in the concentration gradient environment from the beginning, the sperm to be tested is placed at the bottom of the sample pool 3, preferably, the sperm sample is slowly added to the bottom of the sample pool 3 through the sperm culture solution of the sample pool 3 by using a pipette or a micro-sampler. The notch of the first groove body is covered, and the sperm in the collection pool 6 is aspirated after a certain incubation time, and the performance of the aspirated sperm is detected.

[0093] Preferably, the sperm culture solution is pre-equilibrated at 35-40°C, preferably at 37°C, and then added to the sample pool 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 demolded, and the processing structure containing the target chemical is attached and fixed to the surface of the processing area of ​​the chip.

[0103] Further, specific embodiments of the present invention will be described in Examples 5 to 7.

[0104] Example 6

[0105] Sperm sorting using sperm processing chips

[0106] A digital model of the sperm handling chip was established using three-dimensional design software. The sperm handling chip was set as a square with a side length of 20 mm. The overall height of the sperm handling chip was 6.5 mm. The length of the side of the loading pool 3 was 18.2 mm. The depth of the loading pool 3 was 5.5 mm. The width of the groove of the loading pool 3 was 3.25 mm. The height from the bottom of the loading pool 3 to the lowest point of the handling structure was 2.6 mm. The angle between the handling area and the bottom of the sperm handling chip was set as 45°. The base of the handling structure was 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. Twelve equidistant and balanced microchannels were provided on the surface of the handling structure, with a height of 0.65 mm and a width of 100 microns. The handling structure and the sperm handling chip were integrally formed.

[0107] The biocompatible resin was selected as the material, and the sperm handling chip designed in step 1 was prepared using an ultra-precision 3D printing device. The surface residual resin was washed with anhydrous ethanol, and then sequentially washed with deionized water and physiological saline. Finally, it was sterilized by ultraviolet and ready for use.

[0108] Human oviduct fluid was selected as the sorting liquid medium, which was pre-equilibrated in a carbon dioxide incubator for one day and overnight at a temperature of 37°C and a carbon dioxide concentration of 5%.

[0109] The liquefied semen sample was shaken thoroughly, and 500 μL of the semen sample was extracted using a pipette gun and added to the loading pool 3. The height of the semen was lower than the lowest point of the handling structure.

[0110] The pre-equilibrated human oviduct fluid was slowly added to the collection pool 6 until the liquid surface was higher than the highest point of the handling area and flush with the highest point of the inner wall of the sperm handling chip.

[0111] The chip was placed in a plastic culture dish with a cover and placed in a carbon dioxide incubator for processing at a temperature of 37°C and a carbon dioxide concentration of 5% for 25 minutes.

[0112] After the sorting time was over, the sperm handling chip was taken out of the carbon dioxide incubator. The sorted sperm was extracted from the collection pool 6 using a pipette gun for subsequent testing or assisted reproductive technology.

[0113] Example 7

[0114] Sperm sorting induced by progesterone

[0115] Dimethyl sulfoxide was used to prepare a progesterone mother liquor with a concentration of 1 mmol / L. The progesterone mother liquor was diluted with human oviduct fluid to a concentration of 10 μmol / L.

[0116] A digital model of the sperm handling chip is established using three-dimensional design software. The sperm handling chip is set as a square with a side length of 30 mm. The overall height of the sperm handling chip is 9 mm. The length of the side of the sample pool 3 is 26 mm. The depth of the sample pool 3 is 7.5 mm. The width of the groove of the sample pool 3 is 3 mm. The height from the bottom of the sample pool 3 to the lowest part of the handling structure is 4 mm. The angle between the handling area and the bottom of the sperm handling chip is set as 45°. The base of the handling structure is trapezoidal. The long side of the base is 20 mm. The short side of the base is 14.5 mm. The width of the base is 3.8 mm. The height of the base is 1 mm. The surface of the handling structure is provided with 12 equidistant and balanced micro-channels. The height of the micro-channels is 1 mm. The width of the micro-channels is 200 microns. The handling structure and the sperm handling chip are integrally formed.

[0117] The biocompatible resin is selected as the material. The sperm handling chip designed in step 1 is prepared by using an ultra-precision 3D printing device. The surface residual resin is washed with anhydrous ethanol. Then, the sperm handling chip is sequentially washed with deionized water and physiological saline. Finally, the sperm handling chip is sterilized by ultraviolet and is ready for use.

[0118] Human oviduct fluid is selected as the sorting liquid medium. The human oviduct fluid is pre-equilibrated in a carbon dioxide incubator for one day and is left overnight. The temperature is 37°C. The carbon dioxide concentration is 5%.

[0119] Referring to FIG. 6, 3 μL of a progesterone solution is added to the collection pool 6 of the sperm handling chip. 1300 μL of the human oviduct fluid which has been pre-equilibrated is slowly added to the sample pool 3. At this time, the liquid level of the human oviduct fluid is higher than the lowest part of the handling structure. The sample pool 3, the handling area and the collection pool 6 in the sperm handling chip form a continuous space with the liquid as the medium.

[0120] The chip is placed in a capped container provided with a water bath and is placed in a carbon dioxide incubator for 15 minutes. The temperature is 37°C. The carbon dioxide concentration is 5%. The progesterone diffuses to form a concentration gradient.

[0121] 500 μL of the liquefied semen sample is taken. After being mixed thoroughly, the semen sample is slowly added to the bottom of the sample pool 3 through the culture solution in the chip by using a pipette. The liquid level in the chip is flush with the highest point of the chip.

[0122] The chip is placed in a capped container provided with a water bath and is placed in a carbon dioxide incubator for 30 minutes. The temperature is 37°C. The carbon dioxide concentration is 5%.

[0123] After the treatment is completed, the chip is taken out of the incubator. The treated sperm sample in the collection pool 6 is taken for subsequent testing or assisted reproductive technology.

[0124] Example 8

[0125] Constructing a multi-chemical component microenvironment for sperm research using a sperm handling chip

[0126] A digital model of the sperm handling chip was established using three-dimensional design software. The sperm handling chip was set as a square with a side length of 30 mm. The overall height of the sperm handling chip was 9 mm. The length of the side of the sample pool 3 was 26 mm. The depth of the sample pool 3 was 7.5 mm. The width of the groove of the sample pool 3 was 3 mm. The height from the bottom of the sample pool 3 to the lowest part of the processing structure was 4 mm. The angle between the processing area and the bottom of the sperm handling chip was set to 45°. The depth of the connection of the processing area was 1 mm, which facilitated the connection with the processing structure.

[0127] The base of the processing structure was trapezoidal, with a long side of 20 mm, a short side of 14.5 mm, a width of 3.8 mm, and a base height of 1 mm. The surface of the processing structure was provided with 24 equidistant balanced microchannels, with a processing structure height of 1 mm and a microchannel width of 100 microns. The overall height of the processing structure was 2 mm.

[0128] Biocompatible resin was selected as the material, and the sperm handling chip designed in step 1 was prepared using ultra-precision 3D printing equipment. The surface residual resin was washed with anhydrous ethanol, and then sequentially washed with deionized water and physiological saline. Finally, it was sterilized by ultraviolet and ready for use.

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

[0130] The target concentrations of the test drugs A, B, C, and D were prepared, mixed with hyaluronic acid, degassed under negative pressure, and then placed in an oven to heat to 35°C for solidification. After solidification, the sample was demolded.

[0131] The processing structure containing the drug was bonded and fixed to the sperm handling chip using the same photosensitive resin as the adhesive.

[0132] 1 ml of liquefied semen sample was taken and mixed thoroughly before being slowly added to the sample pool 3. The maximum height of the semen sample should not exceed the lowest part of the processing structure.

[0133] Hanks solution was used as the test buffer, which was pre-equilibrated at 37°C. When used, Hanks solution was slowly added until the liquid surface was level with the highest point of the sperm handling chip.

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

[0135] After the experimental time was over, the sperm handling chip was taken out of the incubator, and the test was completed. The sperm sample could be observed, recorded, and analyzed under a microscope or collected from the collection pool 6 for subsequent characterization.

[0136] The sperm treatment chip and the use method thereof can overcome the above problems, build a microenvironment suitable for sperm sorting, and adjust the structure and the concentration of chemicals according to different requirements to realize sperm sorting. In addition, different kinds of chemicals can be replaced to test the effects of different drugs on sperm, and the efficacy of sperm treatment is evaluated.

[0137] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A sperm handling chip, characterized in that, The chip comprises: a first groove, a second groove arranged in the first groove, a sample pool formed between the first groove and the second groove for placing a sperm sample to be detected, an outer wall surface of the second groove faces the sample pool, and a top of the outer wall surface is configured as a slope, one end of the slope points to the sample pool, and the other end is connected to the inside of the second groove, and the slope is provided with micro-channels extending along the slope, an inner wall surface of the second groove forms a collection pool for collecting sperm swimming from the sample pool to the slope micro-channels and falling into the collection pool, the height of the groove wall of the first groove is higher than the height of the groove wall of the second groove.

2. The sperm handling chip of claim 1, wherein, The bottom surface of the sample pool is lower than the bottom surface of the collection pool.

3. The sperm handling chip of claim 2, wherein, The outer wall surface of the second groove is configured as a slope, the slope comprises a connecting plate connected with a plate, the plate is provided with a plurality of aligned micro-channels, and the connecting plate is detachably connected to the slope.

4. The sperm handling chip of claim 1, wherein, The included angle between the slope and the horizontal line is 20°-60°.

5. A method of sperm sorting, characterized in that, The chip comprises: placing sperm to be sorted in the sample pool, adding a sperm culture solution to the sample pool, making the liquid level of the sperm culture solution higher than the highest point of the slope, covering the groove opening of the first groove, culturing for a certain period of time for sorting, and sucking the sperm in the collection pool, the covering method of the groove opening of the first groove is that the sperm treatment chip is placed in a water bath container and the water bath container is covered, or the groove opening of the first groove is directly covered, and then the sperm treatment chip is placed in a carbon dioxide incubator for 5-60 min, the temperature is 34-40°C, and the carbon dioxide concentration is 3-7%.

6. A method of sperm sorting which simulates the environment of the human body, characterized by, The chip comprises: adding chemicals simulating the micro-environment of sperm-egg combination in the female reproductive system to the collection pool, adding a sperm culture solution to the sample pool, making the sperm culture solution cover the highest point of the slope and fill the collection pool, the chemicals diffuse in the sperm culture solution and form a concentration gradient gradually decreasing from the collection pool to the sample pool, placing sperm to be sorted at the bottom of the sample pool, covering the groove opening of the first groove, and sucking the sperm in the collection pool after culturing for a certain period of time, the covering method of the groove opening of the first groove is that the sperm treatment chip is placed in a water bath container and the water bath container is covered, or the groove opening of the first groove is directly covered, and then the sperm treatment chip is placed in a carbon dioxide incubator for 5-60 min, the temperature is 34-40°C, and the carbon dioxide concentration is 3-7%, and the chemicals form a concentration gradient gradually decreasing from the collection pool to the sample pool.

7. A drug-based sperm test method, characterized by, The chip comprises: The chip comprises: adding a drug to the collection pool and adding a liquid medium to the sample pool, making the liquid medium cover the highest point of the slope and fill the collection pool, the drug diffuses in the liquid medium and forms a concentration gradient gradually decreasing from the collection pool to the sample pool. The sperm to be tested is placed at the bottom of the sample pool, the notch of the first groove body is covered, the sperm in the collection pool is extracted after a certain period of culture, and the performance of the extracted sperm is detected, The processing mode of covering the notch of the first groove body is that the sperm processing chip is placed in a water bath container and the water bath container is covered, or the notch of the first groove body is directly covered, and the time, temperature and carbon dioxide concentration of placing the sperm processing chip in a carbon dioxide incubator are set according to the experimental requirements, and a concentration gradient gradually decreasing from the collection pool to the sample pool is formed by waiting for the drug.

8. A method for testing sperm based on release control, characterized by, The method comprises the following steps: The sperm processing chip according to any one of claims 1-5 is used, The slope surface is prepared by using a controllable release material, the sperm to be tested is placed in the sample pool and culture solution is added, and the liquid level of the culture solution reaches at least the highest point of the slope surface, the sperm in the collection pool is extracted after a certain period of culture, and the culture solution is pre-equilibrated at 35-40 DEG C before being added to the sample pool.

9. The sperm test method based on a controllable release according to claim 8, characterized in that, The controllable release material comprises a slow-release base material and a chemical drug, and the chemical drug is coated by the slow-release base material.

10. The release-based sperm test method of claim 9, wherein, The slope surface is divided into several regions, and a composite drug microenvironment is formed by adjusting the corresponding controllable release material composition of the several regions.

Images