Sperm sorting device and sperm sorting method

Abstract

A sperm sorting device and a sperm sorting method are provided. The sperm sorting device comprises a collection tank, a waste liquid tank, a sorting flow channel, and a material layer. The waste liquid tank is located on a side of the collection tank. The sorting flow channel extends between the collection tank and the waste liquid tank. The sorting flow channel is in communication with the collection tank and the waste liquid tank. The sorting flow channel comprises a lower surface, an upper surface, and a sidewall. The sidewall is located between the lower surface and the upper surface. The sidewall is connected to the lower surface and the upper surface. The material layer is located on the lower surface and the upper surface of the sorting flow channel. The material layer has a negative potential or a positive potential. The range of the negative potential is -1 millivolt to -30 millivolts. The range of the positive potential is 1 millivolt to 30 millivolts. The described sperm sorting device can effectively separate X sperm from Y sperm, and the separated X sperm and Y sperm can have relatively high activity.

Description

Sperm sorting device and sperm sorting method Technical Field

[0001] The present invention relates to a sorting device and a sorting method, and in particular to a sperm sorting device and a sperm sorting method. Background Art

[0002] Currently, flow cytometry is primarily used to separate X chromosome-bearing sperm (i.e., X sperm) from Y chromosome-bearing sperm (i.e., Y sperm) in semen. While flow cytometry can effectively separate X chromosome-bearing sperm from Y chromosome-bearing sperm, it can also result in poor sperm motility (approximately less than 50%).

[0003] Summary of the Invention

[0004] The present invention provides a sperm sorting device and a sperm sorting method, which can effectively separate X sperm from Y sperm, and the separated X sperm and Y sperm can have higher activity.

[0005] The present invention provides a sperm sorting device, comprising a collection tank, a waste liquid tank, a sorting channel, and a material layer. The waste liquid tank is located on one side of the collection tank. The sorting channel extends between the collection tank and the waste liquid tank. The sorting channel is connected to the collection tank and the waste liquid tank. The sorting channel includes a lower surface, an upper surface, and side walls. The side walls are located between the lower surface and the upper surface. The side walls are connected to the lower surface and the upper surface. The material layer is located on the lower surface and the upper surface of the sorting channel. The material layer has a negative potential or a positive potential. The negative potential ranges from -1 millivolt (mV) to -30 mV. The positive potential ranges from 1 mV to 30 mV.

[0006] According to an embodiment of the present invention, in the sperm sorting device, the material of the material layer may include polystyrene (PS), poly-L-lysine (poly-L-lysine) or F-127( F-127) (trade name, manufactured by Bio Basic Inc.) polymer material.

[0007] According to one embodiment of the present invention, the sperm sorting device may include a first substrate, a second substrate, and an adhesive layer. The second substrate is positioned on the first substrate. The second substrate may include a first opening, a second opening, a specimen injection slot, and a third opening. The specimen injection slot is positioned between the first and second openings. The third opening may be connected to the bottom of the specimen injection slot. The adhesive layer is positioned between the first and second substrates. The adhesive layer may include a fourth opening, a fifth opening, and a sixth opening. The fifth opening is positioned between the fourth and sixth openings. The fifth opening may be connected to the fourth and sixth openings. The fourth opening may be connected to the first opening. The sixth opening may be connected to the second opening. The fifth opening may be connected to the third opening.

[0008] According to an embodiment of the present invention, in the sperm sorting device, the material of the first substrate may include polycarbonate (PC).

[0009] According to an embodiment of the present invention, in the sperm sorting device, the material of the second substrate may include polycarbonate.

[0010] According to an embodiment of the present invention, in the sperm sorting device, the material of the adhesive layer may include light-curing adhesive, heat-curing adhesive, resin, or double-sided tape, but the present invention is not limited thereto.

[0011] According to one embodiment of the present invention, in the sperm sorting device, the first substrate may have a first surface. The second substrate may have a second surface. The second surface may face the first surface. The material layer may be located on the first surface and the second surface.

[0012] According to one embodiment of the present invention, in the sperm sorting device, the sorting channel may include a fifth opening. The lower surface of the sorting channel may be the portion of the first surface overlapping with the fifth opening. The upper surface of the sorting channel may be the portion of the second surface overlapping with the fifth opening.

[0013] According to an embodiment of the present invention, in the sperm sorting device, the fourth opening may be aligned with the first opening. The collecting tank may include the first opening and the fourth opening.

[0014] According to an embodiment of the present invention, in the sperm sorting device, the sixth opening may be aligned with the second opening. The waste liquid tank may include the second opening and the sixth opening.

[0015] According to an embodiment of the present invention, in the sperm sorting device, the pattern of the third opening in top view may be a stripe.

[0016] According to an embodiment of the present invention, in the sperm sorting device, an extending direction of the third opening may intersect with an extending direction of the sorting channel.

[0017] According to an embodiment of the present invention, in the sperm sorting device, the sorting channel may include a first portion and a second portion, wherein the first portion is located between the collecting tank and the second portion.

[0018] According to an embodiment of the present invention, in the sperm sorting device, the first portion may taper toward the collecting tank.

[0019] According to an embodiment of the present invention, in the sperm sorting device, the second portion may taper toward the waste liquid tank.

[0020] According to one embodiment of the present invention, in the sperm sorting device, the end of the sorting channel connected to the waste liquid tank may be a narrow channel, and the width of the sorting channel may be a minimum at the narrow channel.

[0021] The present invention provides a sperm sorting method, which may include the following steps. A sperm sorting device is provided. The sperm sorting device includes a collection tank, a waste liquid tank, a sorting channel, and a material layer. The waste liquid tank is located on one side of the collection tank. The sorting channel extends between the collection tank and the waste liquid tank. The sorting channel connects the collection tank and the waste liquid tank. The sorting channel includes a lower surface, an upper surface, and sidewalls. The sidewalls are located between the lower surface and the upper surface. The sidewalls are connected to the lower surface and the upper surface. The material layer is located on the lower surface and the upper surface of the sorting channel. The material layer has a negative potential or a positive potential. The negative potential ranges from -1 millivolt to -30 millivolts. The positive potential ranges from 1 millivolt to 30 millivolts. A medium solution is provided to the collection tank, the waste liquid tank, and the sorting channel, wherein the liquid levels of the medium solution in the collection tank, the waste liquid tank, and the sorting channel are consistent. A sperm sample is injected into the sorting channel. The sperm sample includes at least one X sperm and at least one Y sperm. X sperm are sperm carrying an X chromosome. Y sperm are sperm carrying a Y chromosome. A culture medium is injected into the collection tank, allowing the culture medium to flow from the collection tank into the sorting channel, forming a culture medium flow. In the sorting channel, the X and Y sperm in the sperm sample are sorted by the flow field of the culture medium flow and the electric field of the material layer.

[0022] According to one embodiment of the present invention, in the sperm sorting method, the material layer may have a negative potential. During the sorting process, due to electrostatic repulsion, Y sperm may be closer to the upper and lower surfaces than X sperm.

[0023] According to one embodiment of the present invention, in the sperm sorting method, the material layer may have a positive potential. During the sorting process, due to electrostatic absorption, X sperm may be closer to the upper and lower surfaces than Y sperm.

[0024] According to one embodiment of the present invention, the sperm sorting method may further include the following steps: removing the solution containing Y sperm from the collection tank; removing the waste liquid from the waste liquid tank; after removing the solution containing Y sperm from the collection tank and the waste liquid from the waste liquid tank, injecting culture medium into the waste liquid tank to flush the solution in the sorting flow channel into the collection tank; and removing the solution containing X sperm from the collection tank.

[0025] Based on the above, in the sperm sorting device and sperm sorting method proposed in the present invention, a material layer is located on the lower and upper surfaces of the sorting channel. The material layer has a negative or positive potential. The negative potential ranges from -1 millivolt to -30 millivolts, while the positive potential ranges from 1 millivolt to 30 millivolts. Therefore, X sperm and Y sperm in the sperm sample are sorted in the sorting channel based on the flow field of the culture medium flow and the electric field of the material layer. This effectively separates X sperm from Y sperm, and the separated X and Y sperm have higher motility.

[0026] In order to make the above features and advantages of the present invention more clearly understood, embodiments are given below with reference to the accompanying drawings for detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 is a perspective view of a sperm sorting device according to some embodiments of the present invention;

[0028] FIG2 is an exploded view of a sperm sorting device according to some embodiments of the present invention;

[0029] FIG3 is a top view of a sperm sorting apparatus according to some embodiments of the present invention;

[0030] FIG4 is a cross-sectional schematic diagram of a sorting flow channel when a sperm sorting device is used to sort a sperm sample according to some embodiments of the present invention;

[0031] FIG5 is a schematic cross-sectional view of a sorting flow channel when a sperm sorting device is used to sort a sperm sample according to other embodiments of the present invention;

[0032] FIG6 is a schematic diagram of combining a micronized magnetic ball with a Y sperm according to some embodiments of the present invention;

[0033] FIG7 is a flow chart of a sperm sorting method according to some embodiments of the present invention;

[0034] FIG8 is a graph showing the relationship between the ratio of Y sperm and sorting time according to an experimental example of the present invention;

[0035] FIG9 is a graph showing the relationship between sperm activity and sorting time according to an experimental example of the present invention;

[0036] FIG10 is a graph showing the relationship between the cumulative number of sperm collected and the sorting time according to an experimental example of the present invention.

[0037] Explanation of the accompanying figures: 10: Sperm sorting device 100: Material layer 102, 104: Substrate 106: Adhesive layer 200: X sperm 202: Y sperm AB1: Y sperm antibody AG1: Surface antigen AR: Culture medium flow BS1, BS2: Bottom surface BT1: Biotin C1: Collection tank C2: Waste liquid tank C3: Sorting flow channel C4: Sample injection tank D1, D2: Extension direction M1: Micron magnetic ball P1: First part P2: Second part P3: Third part NC: Narrow channel Lanes OP1, OP2, OP3, OP4, OP5, OP6: openings OS1: lower outer surface OS2: upper outer surface S1: lower surface S2: upper surface S3, S4, S5, S6, S7, S8, S9: sidewalls SA1: streptavidin SL: starting lines SS1, SS2: surfaces S100, S102, S104, S106, S108, S110, S112, S114: steps W1, W2, W3: width DETAILED DESCRIPTION

[0038] The following examples are illustrated in detail with accompanying figures. However, these examples are not intended to limit the scope of the present invention. For ease of understanding, identical components will be designated by the same reference numerals throughout the following description. Furthermore, the figures are for illustrative purposes only and are not drawn to scale. The dimensions of various features may be arbitrarily increased or decreased for clarity.

[0039] Figure 1 is a perspective view of a sperm sorting device according to some embodiments of the present invention. Figure 2 is an exploded view of a sperm sorting device according to some embodiments of the present invention. In Figure 2, the dashed lines on the substrate 102 represent the outlines of the collection tank C1, waste liquid tank C2, and sorting channel C3 projected onto the substrate 102. Figure 3 is a top view of a sperm sorting device according to some embodiments of the present invention. Figure 4 is a schematic cross-sectional view of the sorting channel when the sperm sorting device is used to sort a sperm sample according to some embodiments of the present invention. Figure 5 is a schematic cross-sectional view of the sorting channel when the sperm sorting device is used to sort a sperm sample according to other embodiments of the present invention. Figures 4 and 5 are cross-sectional views taken along the II' section line in Figure 1. Furthermore, the components in the cross-sectional views of Figures 4 and 5 are not drawn to the same scale as the components in the perspective view of Figure 1. Figure 6 is a schematic diagram of combining a micronized magnetic ball with Y sperm according to some embodiments of the present invention.

[0040] Referring to Figures 1 to 3 , a sperm sorting device 10 includes a collection tank C1, a waste liquid tank C2, a sorting channel C3, and a material layer 100. In some embodiments, the collection tank C1 is used to collect sorted sperm. The waste liquid tank C2 is located to one side of the collection tank C1. In some embodiments, the waste liquid tank C2 is used to collect impurities from a solution containing sperm with low or no activity, or from seminal plasma.

[0041] The sorting channel C3 extends between the collection tank C1 and the waste liquid tank C2. The sorting channel C3 connects the collection tank C1 and the waste liquid tank C2. The sorting channel C3 includes a lower surface S1, an upper surface S2, and a sidewall S3. The sidewall S3 is located between the lower surface S1 and the upper surface S2. The sidewall S3 connects the lower surface S1 and the upper surface S2. In some embodiments, the sorting channel C3 can be used for sperm sorting.

[0042] The material layer 100 is located on the lower surface S1 and the upper surface S2 of the sorting channel C3. The material layer 100 has a negative potential or a positive potential. The negative potential ranges from -1 mV to -30 mV. In some embodiments, the negative potential ranges from -1 mV to -10 mV. In some embodiments, the negative potential ranges from -10 mV to -20 mV. In some embodiments, the negative potential ranges from -20 mV to -30 mV. The positive potential ranges from 1 mV to 30 mV. In some embodiments, the positive potential ranges from 1 mV to 10 mV. In some embodiments, the positive potential ranges from 10 mV to 20 mV. In some embodiments, the positive potential ranges from 20 mV to 30 mV. In some embodiments, the material of the material layer 100 may include polystyrene, poly-L-lysine or F-127 (trade name, manufactured by Bio Basic) is a polymer material or a ceramic material. In some embodiments, the material layer 100 can be formed on the lower surface S1 and the upper surface S2 by coating, evaporation deposition, or sputtering.

[0043] In some embodiments, X sperm and Y sperm in a sperm sample can be sorted in the sorting channel C3 by the flow field of the culture medium flow and the electric field of the material layer 100. X sperm are sperm carrying the X chromosome. Y sperm are sperm carrying the Y chromosome. In some embodiments, X sperm and Y sperm may have different electrical potentials. In some embodiments, the surface potential of X sperm may be -20 mV, and the surface potential of Y sperm may be -16 mV. In some embodiments, the swimming speed of X sperm may be 20 μm / s, and the swimming speed of Y sperm may be 22 μm / s.

[0044] As shown in Figure 4, when the material layer 100 has a negative potential, an electrostatic repulsion effect is generated between the material layer 100 and the sperm (including X sperm 200 and Y sperm 202). The electrostatic force between the X sperm 200 and the material layer 100 is greater than the electrostatic force between the Y sperm 202 and the material layer 100. Therefore, after the sperm sample is injected into the sorting channel C3, the Y sperm 202 may be closer to the upper surface S2 and lower surface S1 of the sorting channel C3 than the X sperm 200. Furthermore, after the culture medium is injected into the collection tank C1, the culture medium flowing into the sorting channel C3 forms a culture medium flow AR. The culture medium flow AR can flow toward the waste liquid tank C2. Because the culture medium flow AR in the sorting channel C3 has a flow field gradient, the flow rate of the culture medium flow AR near the middle region of the sorting channel C3 is faster than the flow rate of the culture medium flow AR near the boundaries of the sorting channel C3 (e.g., the lower surface S1 and the upper surface S2). As a result, the upstream swimming resistance of the X sperm 200 repelled into the middle region of the sorting channel C3 is greater than the upstream swimming resistance of the Y sperm 202 near the boundary of the sorting channel C3. Furthermore, the swimming speed of the X sperm 200 itself is slower than that of the Y sperm 202. As the sorting time increases and the combined effects of the flow field of the culture medium AR and the electric field of the material layer 100 increase, the distance between the Y sperm 202 and the X sperm 200 increases. Therefore, the X sperm 200 and Y sperm 202 in the sperm sample are sorted in the sorting channel C3 in response to the flow field of the culture medium AR and the electric field of the material layer 100. Consequently, a high proportion of Y sperm 202 can be collected in the collection tank C1.

[0045] As shown in Figure 5, when the material layer 100 has a positive potential, an electrostatic attraction effect is generated between the material layer 100 and sperm (including X sperm 200 and Y sperm 202). The electrostatic force between X sperm 200 and the material layer 100 is greater than the electrostatic force between Y sperm 202 and the material layer 100. Therefore, after the sperm sample is injected into the sorting channel C3, X sperm 200 may be closer to the upper surface S2 and lower surface S1 of the sorting channel C3 than Y sperm 202. Furthermore, after the culture medium is injected into the collection tank C1, the culture medium flowing into the sorting channel C3 forms a culture medium flow AR. The culture medium flow AR can flow toward the waste liquid tank C2. Due to the stronger electrostatic attraction, X sperm 200 gather near the upper surface S2 and lower surface S1 of the sorting channel C3, thereby displacing Y sperm 202 to the central region of the sorting channel C3. Furthermore, because the flow rate of the culture solution AR near the middle region of the sorting channel C3 is faster than that near the boundary of the sorting channel C3, the swimming speed of the Y sperm 202 in the culture solution AR is reduced. Consequently, the upstream resistance faced by the Y sperm 202 near the middle region of the sorting channel C3 is greater than the upstream resistance faced by the X sperm 200 near the boundary of the sorting channel C3. As the sorting time increases and the combined effects of the flow field of the culture solution AR and the electric field of the material layer 100 increase, the distance between the Y sperm 202 and the X sperm 200 increases. Consequently, the X sperm 200 and Y sperm 202 in the sperm sample are sorted in the sorting channel C3 in response to the flow field of the culture solution AR and the electric field of the material layer 100. Consequently, a high proportion of X sperm 200 can be collected in the collection tank C1.

[0046] As shown in FIG6 , when the surface of the magnetic microbeads M1 modified with Y sperm antibody AB1 is bound to the surface antigen AG1 of Y sperm 202, an additional weight burden is imposed on the Y sperm 202. For example, the Y sperm antibody AB1 can be bound to the magnetic microbeads M1 via biotin BT1 and streptavidin SA1. Due to the weight of the magnetic microbeads M1 (approximately 2.0×10 -9 grams) is much larger than the weight of Y sperm 202 (about 1.0×10 -19Therefore, regardless of the potential of the material layer 100, after the sperm sample is injected into the sorting channel C3, the X sperm 200 can swim faster than the Y sperm 202. Furthermore, the mobility of the Y sperm 202, whose surfaces are modified with micronized magnetic spheres M1, is susceptible to interference from external magnetic fields and forces. If one or more magnets (not shown) (with a magnetic force ranging from 200 gauss to 8000 gauss) are placed near the lower outer surface OS1 and / or upper outer surface OS2 of the sperm sorting device 10, after the sperm sample is injected into the sorting channel C3, the Y sperm 202, whose surfaces are modified with micronized magnetic spheres M1, will be attracted and fixed to the lower surface S1 and / or upper surface S2 by the magnetic force. Within the sorting channel C3, only the X sperm 200 have the freedom to swim toward the collection tank C1.

[0047] Conversely, although not shown in the figure, when the micronized magnetic spheres modified with X sperm antibodies are pre-bound to the surface antigens of X sperm 200, an additional weight burden is imposed on the X sperm 200. After the sperm sample is injected into the sorting channel C3, the Y sperm 202 can swim faster than the X sperm 200. Furthermore, the mobility of the X sperm 200 modified with the micronized magnetic spheres is easily disturbed by external magnetic fields and forces. If a single or multiple magnets are placed near the lower outer surface OS1 and / or upper outer surface OS2 of the sperm sorting device 10, after the sperm sample is injected into the sorting channel C3, the X sperm 200 modified with the micronized magnetic spheres will be attracted and fixed to the lower surface S1 and / or upper surface S2 by the magnetic force. Within the sorting channel C3, only the Y sperm 202 remain free to swim toward the collection tank C1.

[0048] In some embodiments, as the sorting time increases and the flow field effect of the culture medium flow AR and the external magnetic force are exerted, the distance between the Y sperm 202 and the X sperm 200 will be increased. Therefore, the X sperm 200 and the Y sperm 202 in the sperm sample can produce a sorting effect in the sorting flow channel C3 due to the magnetic field effect corresponding to the flow field of the culture medium flow AR and the external magnetic force.

[0049] In some embodiments, as shown in FIG3 , the sorting channel C3 may include a first portion P1 and a second portion P2. The first portion P1 is located between the collecting tank C1 and the second portion P2. The first portion P1 may serve as the upstream region of the sorting channel C3, and the second portion P2 may serve as the downstream region of the sorting channel C3.

[0050] In some embodiments, the first portion P1 may taper toward the collection tank C1. That is, the width W1 of the first portion P1 may gradually decrease toward the collection tank C1. This creates a gentle flow field in the upstream region of the sorting channel C3 (i.e., the first portion P1) after the culture fluid flows into the sorting channel C3, reducing the effort required for sperm to swim backward.

[0051] In some embodiments, the second portion P2 may taper toward the waste trough C2. That is, the width W2 of the second portion P2 may gradually decrease toward the waste trough C2. This allows the culture fluid AR to flow faster in the downstream region of the sorting channel C3 (i.e., the first portion P2) after it flows into the sorting channel C3, thereby rapidly flushing low-motility or inactive sperm into the waste trough C2. In some embodiments, the end of the sorting channel C3 connecting to the waste trough C2 may be a narrow channel NC. In some embodiments, the width of the sorting channel C3 may be at its minimum at the narrow channel NC.

[0052] In some embodiments, the sorting channel C3 may further include a third portion P3 between the first portion P1 and the second portion P2. The width W3 of the third portion P3 may be approximately equal to the maximum width W1 of the first portion P1 and the maximum width W2 of the second portion P2.

[0053] The sperm sorting device 10 may include a substrate 102, a substrate 104, and an adhesive layer 106. The substrate 102 may have a surface SS1. The material layer 100 may be located on the surface SS1. In some embodiments, the material of the substrate 102 may include polycarbonate.

[0054] Substrate 104 is positioned on substrate 102. Substrate 104 may have a surface SS2. Surface SS2 may face surface SS1. Material layer 100 may be positioned on surface SS2. Substrate 104 may include an opening OP1, an opening OP2, a sample injection slot C4, and an opening OP3. Sample injection slot C4 is positioned between opening OP1 and opening OP2. In some embodiments, sample injection slot C4 may be a depression in substrate 104. Opening OP3 may be connected to the bottom of sample injection slot C4. In some embodiments, a sperm sample may be injected into sample injection slot C4, and the sperm sample in sample injection slot C4 may be injected into sorting channel C3 via opening OP3. In some embodiments, the top view pattern of opening OP3 may be strip-shaped, so that sperm injected into sorting channel C3 (e.g., X sperm 200 and Y sperm 202 in Figures 4 and 5) begin swimming on the same starting line SL. In some embodiments, the extension direction D1 of opening OP3 may intersect with the extension direction D2 of sorting channel C3. In some embodiments, the extension direction D1 of the opening OP3 may be perpendicular to the extension direction D2 of the sorting channel C3. In some embodiments, the material of the substrate 104 may include polycarbonate.

[0055] Adhesive layer 106 is positioned between substrates 102 and 104. Adhesive layer 106 may include opening OP4, opening OP5, and opening OP6. Opening OP5 is positioned between opening OP4 and opening OP6. Opening OP5 may communicate with opening OP4 and opening OP6. Adhesive layer 106 may be made of, but is not limited to, a light-curing adhesive, a heat-curing adhesive, a resin, or double-sided tape.

[0056] Opening OP4 may be connected to opening OP1. Opening OP4 may be aligned with opening OP1. Collection tank C1 may include opening OP1 and opening OP4. Sidewall S4 of collection tank C1 may include sidewall S5 of opening OP1 and sidewall S6 of opening OP4. Bottom surface BS1 of collection tank C1 may be the portion of surface SS1 that overlaps with opening OP4.

[0057] Opening OP6 may be connected to opening OP2. Opening OP6 may be aligned with opening OP2. Waste liquid tank C2 may include opening OP2 and opening OP6. Sidewall S7 of waste liquid tank C2 may include sidewall S8 of opening OP2 and sidewall S9 of opening OP6. Bottom surface BS2 of waste liquid tank C2 may be the portion of surface SS1 that overlaps with opening OP6.

[0058] Opening OP5 may be connected to opening OP3. Sorting channel C3 may include opening OP5. Sorting channel C3 and opening OP5 may have the same sidewall S3. Lower surface S1 of sorting channel C3 may be the portion of surface SS1 that overlaps with opening OP5. Upper surface S2 of sorting channel C3 may be the portion of surface SS2 that overlaps with opening OP5.

[0059] Hereinafter, a sperm sorting method using the sperm sorting apparatus in the above-described embodiment will be described with reference to FIG. 7 .

[0060] FIG. 7 is a flow chart of a sperm sorting method according to some embodiments of the present invention.

[0061] 1 to 7 , step S100 is performed to provide a sperm sorting device 10. The details of the sperm sorting device 10 can be found in the description of the above embodiment and will not be repeated here.

[0062] Next, step S102 is performed, where the culture fluid is provided to the collection tank C1, waste tank C2, and sorting channel C3, wherein the culture fluid levels in the collection tank C1, waste tank C2, and sorting channel C3 are aligned. In some embodiments, the method for providing the culture fluid to the collection tank C1, waste tank C2, and sorting channel C3 may include the following steps, but the present invention is not limited thereto. First, the culture fluid can be injected into the sample injection tank C4 using a pipette, and then injected into the sorting channel C3 through opening OP3 to wet the sorting channel C3. Next, approximately 1000 μl of culture fluid can be injected into each of the collection tank C1 and waste tank C2 using a pipette, and the culture fluid levels in the collection tank C1, waste tank C2, and sorting channel C3 can be aligned for approximately 5 minutes. In some embodiments, the culture fluid can include a phosphate solution, a citrate solution, a tris (hydroxymethyl)aminomethane) solution, skim milk powder, egg yolk, glucose, fructose, lactose, or lecithin.

[0063] Next, step S104 is performed to inject the sperm sample into the sorting channel C3. In some embodiments, approximately 1 ml of sperm sample can be injected into the sample injection slot C4 using a pipette. The sperm sample in the sample injection slot C4 is then injected into the sorting channel C3 through the strip-shaped opening OP3, allowing the sperm sample to spread throughout the sorting channel C3. As shown in Figures 4 and 5, the sperm sample includes at least one X sperm 200 and at least one Y sperm 202. X sperm 200 is sperm carrying an X chromosome. Y sperm 202 is sperm carrying a Y chromosome.

[0064] Next, step S106 is performed, where culture fluid is injected into the collection tank C1, allowing the culture fluid to flow from the collection tank C1 into the sorting channel C3, forming a culture fluid flow AR. In the sorting channel C3, the X sperm 200 and Y sperm 202 in the sperm sample are sorted by the flow field of the culture fluid flow AR and the electric field of the material layer 100. In some embodiments, in step S106, the culture fluid level in the collection tank C1 can be higher than the culture fluid level in the waste tank C2. Therefore, the culture fluid can be driven by the difference in liquid levels between the collection tank C1 and the waste tank C2, generating a flow rate and thus forming the culture fluid flow AR. In some embodiments, a pipette can be used to inject approximately 70 μl of culture fluid into the collection tank C1 every minute to generate a net pressure differential, thereby driving the culture fluid until the sorting is complete.

[0065] As shown in Figures 4 and 5 , X sperm 200 and Y sperm 202 in the sperm sample are sorted in the sorting channel C3 by the flow field of the culture medium flow AR and the electric field of the material layer 100. In some embodiments, as shown in Figure 4 , the material layer 100 may have a negative potential, and during the sorting process, due to electrostatic repulsion, the Y sperm 202 may be closer to the upper surface S2 and lower surface S1 than the X sperm 200. In some embodiments, as shown in Figure 5 , the material layer 100 may have a positive potential, and during the sorting process, due to electrostatic attraction, the X sperm 200 may be closer to the upper surface S2 and lower surface S1 of the sorting channel C3 than the Y sperm 202. The details of the sorting effect have been fully described in the above embodiments (Figures 4 and 5) and will not be repeated here. Furthermore, the following embodiments illustrate the case where the material layer 100 has a negative potential, but the present invention is not limited to this.

[0066] Next, step S108 can be performed to remove the solution containing Y sperm 202 from collection tank C1. In some embodiments, a pipette can be used to extract all of the solution from collection tank C1. Because Y sperm 202 swim out of sorting channel C3 more quickly and enter collection tank C1, the proportion of Y sperm 202 in the solution removed from collection tank C1 will be higher than that of X sperm 200. Furthermore, because this sperm sorting method utilizes the retrograde swimming characteristics of highly active sperm, the Y sperm 202 collected in collection tank C1 are likely to have higher motility.

[0067] Furthermore, step S110 may be performed to remove waste liquid from waste liquid tank C2. The waste liquid may contain sperm with low or no activity. In some embodiments, a pipette may be used to remove all of the solution in waste liquid tank C2. In some embodiments, steps S108 and S110 may be performed simultaneously, but the present invention is not limited thereto. In other embodiments, step S108 may be performed first, followed by step S110. In still other embodiments, step S110 may be performed first, followed by step S108.

[0068] Subsequently, step S112 can be performed. After the solution containing Y sperm 202 is removed from the collection tank C1 and the waste liquid is removed from the waste liquid tank C2, culture medium is injected into the waste liquid tank C2, and the solution in the sorting channel C3 is flushed into the collection tank C1. In some embodiments, approximately 2000 μl of culture medium can be injected into the waste liquid tank C2 using a pipette, and the solution in the sorting channel C3 is flushed into the collection tank C1.

[0069] Next, step S114 is performed to remove the solution containing X sperm 200 from collection tank C1. In the solution removed from collection tank C1, the proportion of X sperm 200 is higher than the proportion of Y sperm 202. In some embodiments, the solution containing X sperm 200 can be removed using a pipette. Furthermore, because this sperm sorting method utilizes the retrograde swimming characteristics of highly active sperm, the X sperm 200 collected in collection tank C1 are likely to have a higher motility.

[0070] Based on the above embodiments, it can be seen that in the sperm sorting device 10 and sperm sorting method, the material layer 100 is located on the lower surface S1 and upper surface S2 of the sorting channel C3. The material layer 100 has a negative or positive potential. The negative potential ranges from -1 millivolts to -30 millivolts, while the positive potential ranges from 1 millivolt to 30 millivolts. Therefore, the X sperm 200 and Y sperm 202 in the sperm sample are sorted in the sorting channel C3 by the flow field of the culture medium flow AR and the electric field of the material layer 100. This effectively separates the X sperm 200 and Y sperm 202, and the separated X sperm 200 and Y sperm 202 have higher activity.

[0071] FIG8 is a graph showing the relationship between the ratio of Y sperm and sorting time according to an experimental example of the present invention.

[0072] As shown in FIG8 , after sperm sorting using the sperm sorting method of the above embodiment, the optimal time point is 20 minutes. At this time, the proportion of Y sperm reaches approximately 80%. Similarly, the proportion of X sperm in the downstream area also increases by the same proportion.

[0073] Figure 9 shows the relationship between sperm motility and sorting time, according to an experimental example of the present invention. As shown in Figure 9, after recording the changes in sperm motility at various sorting times, the following observations can be made: After a prolonged sorting period, sperm motility only gradually decreased from an initial level of approximately 82%, and remained at approximately 78% activity at 20 minutes.

[0074] FIG10 is a graph showing the relationship between the cumulative number of sperm collected and the sorting time according to an experimental example of the present invention. As shown in FIG8 to FIG10 , based on the proportion of Y sperm, sperm motility, and the cumulative number of sperm collected, 20 minutes is the optimal collection point, and a cumulative total of approximately 40 million sperm with an activity of approximately 78% can be collected. If four sperm sorting devices 10 are used simultaneously for sperm sorting, a cumulative total of approximately 160 million sperm can be collected within the same operating time (20 minutes), maintaining a good Y sperm ratio (approximately 80%) and excellent sperm motility (approximately 78%). This level has surpassed the quality of sperm sorted by flow cytometry, as shown in Table 1 below.

[0075] Table 1

[0076] In summary, in the sperm sorting device and sperm sorting method of the above-described embodiments, the material layer is located on the lower and upper surfaces of the sorting channel. The material layer has a negative or positive potential. The negative potential ranges from -1 mV to -30 mV, while the positive potential ranges from 1 mV to 30 mV. Therefore, X sperm and Y sperm in the sperm sample are sorted in the sorting channel by the flow field of the culture medium and the electric field of the material layer. This effectively separates X sperm from Y sperm, and the separated X and Y sperm have higher motility.

[0077] Although the present invention has been disclosed above by way of embodiments, they are not intended to limit the present invention. Any person skilled in the art may make slight changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A sperm sorting device comprising: Collection tank; a waste liquid tank, located on one side of the collecting tank; A sorting channel extending between the collecting tank and the waste liquid tank and communicating with the collecting tank and the waste liquid tank, wherein the sorting channel comprises a lower surface, an upper surface, and a side wall, wherein the side wall is located between the lower surface and the upper surface and connected to the lower surface and the upper surface; as well as The material layer is located on the lower surface and the upper surface of the sorting channel and has a negative potential or a positive potential, wherein the negative potential ranges from -1 mV to -30 mV, and the positive potential ranges from 1 mV to 30 mV.

2. The sperm sorting device according to claim 1, wherein the material layer comprises polystyrene, poly-L-lysine or F-127 polymer material.

3. The sperm sorting device according to claim 1, comprising: a first substrate; a second substrate, located on the first substrate, wherein the second substrate comprises a first opening, a second opening, a sample injection slot, and a third opening, wherein the sample injection slot is located between the first opening and the second opening, and the third opening is connected to the bottom of the sample injection slot; as well as An adhesive layer is located between the first substrate and the second substrate, wherein the adhesive layer includes a fourth opening, a fifth opening and a sixth opening, the fifth opening is located between the fourth opening and the sixth opening and is connected to the fourth opening and the sixth opening, the fourth opening is connected to the first opening, the sixth opening is connected to the second opening, and the fifth opening is connected to the third opening. The sperm sorting device according to claim 3 , wherein the material of the first substrate comprises polycarbonate. The sperm sorting device according to claim 3 , wherein the material of the second substrate comprises polycarbonate.

6. The sperm sorting device according to claim 3, wherein the material of the adhesive layer comprises light-curing glue, heat-curing glue, resin or double-sided tape.

7. The sperm sorting device according to claim 3, wherein The first substrate has a first surface, The second substrate has a second surface, The second surface faces the first surface, and The material layer is located on the first surface and the second surface.

8. The sperm sorting device according to claim 7, wherein The sorting channel includes the fifth opening, The lower surface of the sorting channel is a portion of the first surface that overlaps with the fifth opening, and The upper surface of the sorting channel is a portion of the second surface overlapping with the fifth opening.

9. The sperm sorting device according to claim 3, wherein The fourth opening is aligned with the first opening, and The collecting tank includes the first opening and the fourth opening.

10. The sperm sorting device according to claim 3, wherein The sixth opening is aligned with the second opening, and The waste liquid tank includes the second opening and the sixth opening. The sperm sorting device according to claim 3 , wherein the pattern of the third opening in a top view comprises a stripe shape. 12 . The sperm sorting device according to claim 3 , wherein an extending direction of the third opening intersects with an extending direction of the sorting channel. 13 . The sperm sorting device according to claim 1 , wherein the sorting channel comprises a first portion and a second portion, and the first portion is located between the collecting tank and the second portion.

14. The sperm sorting device according to claim 13, wherein the first portion tapers toward the collecting tank. The sperm sorting device according to claim 13 , wherein the second portion tapers toward the waste fluid tank. 16 . The sperm sorting device according to claim 1 , wherein one end of the sorting channel connected to the waste liquid tank is a narrow channel, and the width of the sorting channel is minimum at the narrow channel.

17. A sperm sorting method comprising: A sperm sorting device is provided, wherein the sperm sorting device comprises: Collection tank; a waste liquid tank, located on one side of the collecting tank; The sorting channel extends between the collecting tank and the waste liquid tank and is connected to the collecting tank and the waste liquid tank. A waste liquid tank, wherein the separation channel comprises a lower surface, an upper surface, and a side wall, the side wall being located between the lower surface and the upper surface and connected to the lower surface and the upper surface; and a material layer located on the lower surface and the upper surface of the sorting channel and having a negative potential or a positive potential, wherein the negative potential ranges from -1 mV to -30 mV, and the positive potential ranges from 1 mV to 30 mV; Providing culture fluid to the collecting tank, the waste fluid tank, and the separation channel, wherein the liquid levels of the culture fluid in the collecting tank, the waste fluid tank, and the separation channel are consistent; injecting a sperm sample into the sorting flow channel, wherein the sperm sample includes at least one X sperm and at least one Y sperm, wherein the X sperm is a sperm carrying an X chromosome, and the Y sperm is a sperm carrying a Y chromosome; and The culture solution is injected into a collection tank so that the culture solution flows from the collection tank into the separation channel to form a culture solution flow, wherein the X sperm and the Y sperm in the sperm sample are separated in the separation channel according to the flow field of the culture solution flow and the electric field of the material layer.

18. The sperm sorting method according to claim 17, wherein The material layer has the negative potential, and During the sorting process, due to the effect of electrostatic repulsion, the Y sperms are closer to the upper surface and the lower surface of the sorting channel than the X sperms.

19. The sperm sorting method according to claim 17, wherein The material layer has the positive potential, and During the sorting process, due to the influence of electrostatic adsorption, the X sperms are closer to the upper surface and the lower surface of the sorting channel than the Y sperms.

20. The sperm sorting method according to claim 17, further comprising: Taking out the solution containing the Y sperm from the collection tank; Taking out waste liquid from the waste liquid tank; After the solution containing the Y sperm is taken out from the collection tank and the waste liquid is taken out from the waste liquid tank, the culture liquid is injected into the waste liquid tank to flush the solution in the sorting flow channel into the collection tank; as well as The solution containing the X sperm is taken out from the collection tank.

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