TWI710630B - Integrated chip and method for sperm sorting, oocyte incubation and in vitro fertilization - Google Patents

Abstract

An integrated chip is provided and includes a sperm sorting system, an oocyte incubation and fertilization system and a liquid perfusion system. The sperm sorting system includes an inlet, an outlet, a main channel and a branch channel. The main channel has a width which increases along a direction from the inlet toward the outlet. The branch channel is connected with the main channel. The oocyte incubation and fertilization system is connected with the branch channel and has a first hole part and a second hole part. The diameter of the first hole is larger than an average diameter of the oocyte to hold the oocyte to be fertilized by the sperm. The second hole part is disposed below the first hole part, and the diameter of the second hole is smaller than the oocyte. The selected sperm is introduced to the oocyte incubation and fertilization system through the branch channel, to fertilize with the oocyte in the first hole. The liquid perfusion system is disposed below the second hole part and a liquid exchange is performed onto the incubation and fertilization system through the second hole part.

Description

Integrated chip and method for sperm sorting, egg cultivation and in vitro fertilization

The present invention relates to a system and method, and more particularly to an integrated chip and method for sperm sorting, egg cultivation, and in vitro fertilization.

According to statistics, the proportion of infertility among couples of childbearing age in Taiwan is about 10-15%. Faced with such a high proportion of infertility, in vitro fertilization (IVF) is commonly known as “test tube baby”. ) The successful pregnancy rate is only 33%. Such a low success rate is mostly attributed to the lack of gentle handling of specimens when implementing traditional IVF technology, or human error, resulting in a substantial decline in the health of the specimens or even death. A system that can handle specimens in a gentler way, I believe that the success rate of in vitro fertilization technology can be significantly improved.

The invention provides an integrated chip used for sperm sorting, egg cultivation and in vitro fertilization.

The present invention also provides a method for sperm sorting, egg cultivation and in vitro fertilization.

The integrated chip for sperm sorting, egg cultivation and in vitro fertilization of the present invention includes a sperm sorting system, an egg cultivation and sperm-egg combination system and a liquid perfusion system. The sperm sorting system includes a sperm inlet, a sperm outlet, a main flow channel and a branch flow channel. The main flow channel communicates with the sperm entrance part and the sperm exit part, and the main flow channel has a width that gradually increases from the sperm entrance part to the sperm exit part. The branch flow channel is located between the sperm inlet and the sperm outlet and communicates with the main flow channel. The egg cultivation and sperm-egg combination system is in communication with the branch channel and includes a first through hole portion and a second through hole portion. The first through-hole portion includes a plurality of first through-holes, and the first through-hole has a diameter larger than the average diameter of the egg, and is used for accommodating the egg to be combined with the sperm. The second through hole portion is located below the first through hole portion and includes a plurality of second through holes. The second through hole has a diameter smaller than that of an egg and is used for liquid exchange. Sperm sorted by the sperm sorting system is introduced into the egg cultivation and sperm-egg combination system through the branch channel to combine with the egg located in the first through hole. The liquid perfusion system includes a liquid inlet, a liquid outlet, and a liquid flow channel. The liquid channel is located between the liquid inlet and the liquid outlet and communicates with the liquid inlet and the liquid outlet. A part of the liquid channel is located below the second through-hole portion and exchanges liquid with the egg cultivation and sperm-egg combination system through the second through-hole portion.

In an embodiment of the present invention, the material of the first through-hole portion and the second through-hole portion includes polydimethylsiloxane (PDMS).

In an embodiment of the present invention, the above-mentioned first through holes are arranged in an array.

In an embodiment of the present invention, the above-mentioned second through holes are arranged in an array.

In an embodiment of the present invention, the diameter of the second through hole is greater than 30um.

In an embodiment of the present invention, the above-mentioned main flow path includes a first section and a second section connected to the first section, the first section is located between the sperm entrance portion and the second section, and the second section The segment is located between the first segment and the sperm exit portion, wherein the first segment has substantially the same width, and the second segment has a gradually increasing width with a minimum width greater than that of the first segment.

In an embodiment of the present invention, the width of the aforementioned branch flow channel is smaller than the width of the second section.

In an embodiment of the present invention, the above-mentioned sperm sorting system further includes a blocking structure, the blocking structure is located in the main flow channel and adjacent to the sperm exit portion, and there is a distance between the blocking structure and the side wall of the main flow channel.

In an embodiment of the present invention, the aforementioned blocking structure is dumbbell-shaped.

In an embodiment of the present invention, the above-mentioned sperm inlet and sperm outlet are troughs.

In an embodiment of the present invention, the above-mentioned liquid flow channel has a liquid exchange area, which is located under the egg cultivation and sperm-egg combination system and has a contour conforming to the second through hole.

In an embodiment of the present invention, it includes a first film, a second film under the first film, a third film under the second film, and a fourth film under the third film. The first film includes a first through hole corresponding to the sperm entrance part, a second through hole corresponding to the sperm exit part, a first groove corresponding to the main flow channel and the branch flow channel, and the first groove corresponding to the egg cultivation and sperm-egg combination system The third through hole, the fourth through hole corresponding to the liquid inlet portion, and the fifth through hole corresponding to the liquid outlet portion. The second film includes a first through hole portion, a sixth through hole corresponding to the fourth through hole, and a seventh through hole corresponding to the fifth through hole, wherein the first through hole, the second through hole, and the first groove are located The lower second film serves as the sperm entrance part, the sperm exit part, and the bottom of the main flow channel and the branch flow channel respectively. The third film includes a second through hole portion, an eighth through hole corresponding to the sixth through hole, and a ninth through hole corresponding to the seventh through hole. The fourth film includes a first groove, a second groove, and a second groove connected to each other, wherein the first groove corresponds to the eighth through hole and serves as the bottom of the liquid inlet portion, and the second groove serves as a liquid flow channel, The second groove corresponds to the ninth through hole and serves as the bottom of the liquid outlet portion.

In an embodiment of the present invention, the material of the above-mentioned first film, second film, third film and fourth film includes polydimethylsiloxane.

In an embodiment of the present invention, adjacent two of the aforementioned first film, second film, third film, and fourth film are bonded to each other.

The method for sperm sorting, egg cultivation and in vitro fertilization of the present invention includes the following steps. Provide the aforementioned integrated chip. The sample liquid containing sperm is dropped into the sperm inlet, so that the sperm sorting system performs sperm sorting on the sample liquid containing sperm. Drop the sample liquid containing the eggs into the egg cultivation and sperm-egg combination system so that the eggs are located in the first through holes respectively. The sorted sperm is introduced into the egg cultivation and sperm-egg combination system through the branch channel to combine with the egg in the first through hole to form a fertilized egg.

In an embodiment of the present invention, it further comprises a step of removing the zona pellucida on the egg after dropping the sample liquid containing the egg into the egg incubation and sperm-egg combination system.

In an embodiment of the present invention, after dropping the sample liquid containing the egg into the egg cultivation and sperm-egg binding system, sealing the egg cultivation and sperm-egg binding system, and performing the sperm sorting system on the sperm-containing sample After the sperm sorting is completed by the liquid, the egg culture and sperm-egg combination system is turned on to make the sorted sperm enter the egg culture and sperm-egg combination system through the branch channel.

In an embodiment of the present invention, it further includes sealing the sperm sorting system after dropping the sample liquid containing the sperm into the sperm entrance part, and dropping the sample liquid containing the egg into the egg cultivation and sperm-egg combination system After that, the sperm sorting system is opened to perform sperm sorting.

In an embodiment of the present invention, it further includes culturing the fertilized egg in an egg cultivation and sperm-egg combination system.

In an embodiment of the present invention, it further includes flowing the liquid in the liquid perfusion system, wherein the liquid exchanges with the liquid in the egg incubation and sperm-egg combination system through the second through hole.

Based on the above, the present invention integrates the sperm sorting system, the egg culture and sperm-egg combination system, and the liquid perfusion system on a single chip, so that the high-performance sperm sorted by the sperm sorting system can be directly introduced into the egg culture and sperm-egg The combination system can combine with the cultivated egg in situ to form a fertilized egg, and then achieve the purpose of in vitro fertilization. Since the chip of the present invention passively sorts sperm, processes eggs, and forms fertilized eggs, the process is gentle and can avoid damage to the sperm and eggs, so that the survival rate and combination rate of sperm and eggs are greatly improved. Therefore, the chip of the present invention can not only shorten the operation time of the traditional artificial reproductive technology, but also avoid the death of the specimen due to the lengthy operation time and human misoperation by simplifying the operation process, thereby greatly improving the success rate of the artificial reproductive technology.

In order to make the above-mentioned features and advantages of the present invention more comprehensible, the following specific embodiments are described in detail in conjunction with the accompanying drawings.

In order to make the above and other objects, features, and advantages of the present invention more obvious and understandable, the following will specifically cite the preferred embodiments of the present invention, together with the accompanying drawings, and describe in detail as follows. Furthermore, the directional terms mentioned in the present invention, such as "up", "down", "front", "rear", "left", "right", "in", "out" or "side", etc., Only refer to the direction of the attached drawings. Therefore, the directional terms used are used to describe and understand the present invention, rather than to limit the present invention.

The present invention utilizes the gradual flow channel with the nature of sperm reverse flow, and gradually reduces the flow rate of the flow channel by gradually increasing the cross-sectional area of a certain part of the flow channel, thereby being able to gradually treat sperm with different mobility Perform segmentation. Specifically, the more viable sperm will be able to resist the fluid (that is, the fluid in the sample fluid) and stay in place or go upstream, while the less viable sperm will be carried by the fluid to the exit. On the other hand, the present invention also blocks highly motile sperm through the use of a blocking structure, and brings the sperm with insufficient mobility or dead sperm to the exit. More importantly, the present invention provides branch flow channels so that the sorted sperm can be introduced into the egg cultivation and sperm-egg combination system through the branch flow channels.

1A is a perspective schematic view of an integrated wafer according to an embodiment of the present invention, FIG. 1B is an exploded schematic view of the integrated wafer of FIG. 1A, FIG. 1C is a bottom view of the sperm sorting system of the integrated wafer of FIG. 1A, and FIG. 1D is a schematic cross-sectional view of the integrated chip of FIG. 1A. Referring to FIGS. 1A to 1D at the same time, the integrated chip 10 includes a sperm sorting system 100, an egg cultivation and sperm-egg combination system 200 and a liquid perfusion system 300.

The sperm sorting system 100 includes a sperm inlet 102, a sperm outlet 104, a main flow channel 106, and a branch flow channel 108. In this embodiment, the sperm inlet 102 is used to receive a sample containing sperm. The sperm outlet 104 is used to receive the liquid flowing out of the main flow channel 106. In this embodiment, the sperm inlet 102 and the sperm outlet 104 are, for example, troughs, respectively. The diameter of the sperm entrance portion 102 and the sperm exit portion 104 is, for example, 1.8 mm to 2.2 mm, and is, for example, 2 mm. The depth of the sperm entrance portion 102 and the sperm exit portion 104 is, for example, 1.8 mm to 2.2 mm, and is, for example, 2 mm. The main flow channel 106 and the branch flow channel 108 are, for example, grooves each having a depth. The main flow channel 106 is located between the sperm entrance part 102 and the sperm exit part 104 and communicates the two.

In application, a sample liquid containing sperm is first placed at the sperm entrance portion 102, and then due to the difference in the height of the liquid in the sperm entrance portion 102 and the main flow channel 106, the sample will enter the main flow channel 106 from the sperm entrance portion 102 . Among them, the more viable sperm in the sample will be able to resist the fluid and remain in place or upstream, while the less viable sperm in the sample will be carried to the sperm outlet 104 by the fluid.

In this embodiment, the main runner 106 is, for example, provided with a first section 106A, a second section 106B, and a third section 106C that are connected to each other, which are sections for classifying and sorting sperm. The first section 106A is located between the sperm entrance portion 102 and the second section 106B, has substantially the same width, and the width is less than or equal to the minimum width of the second section 106B. The second section 106B is located between the first section 106A and the third section 106C, and has a gradually increasing width along the direction from the sperm entrance portion 102 to the sperm exit portion 104, so it can also be called a gradual segment . Since the flow rate of the fluid is inversely proportional to the size of the passage through which it passes, the diverging section has to pass fluids of different speeds to separate a variety of sperm with different motility and make them stay in the corresponding section. More specifically, the area of the vertical tangential area of the divergent section relative to the direction of the sperm inlet 102 is smaller than the area of the divergent section relative to the sperm outlet 104. The fluid velocity produced by this design can be calculated by the formula of fluid mechanics. Among them, the motility sperm will resist the fluid velocity and stay at different fluid velocity positions according to the sperm motility. It should be understood that the aforementioned vertical section area refers to a vertical section perpendicular to the extending length direction of the sperm sorting system 100 of the present invention. More specifically, when necessary, the area of the vertical section can be selectively interpreted as the size of the fluid flow through the vertical section, and the area occupied by the material portion defining the sperm sorting system 100 should be ignored.

Furthermore, in order to block highly motile sperm, a third section 106C is optionally added to the main channel 106 of the sperm sorting system 100 of the present invention, so the third area 106C is also called a blocking area. The third section 106C is located between the second section 106B and the sperm exit portion 104 and is a section of the main flow channel 106 adjacent to the sperm exit portion 104. In this embodiment, the third section 106C has substantially the same width, and the width is greater than or equal to the maximum width of the second section 106B. In the third section 106C, the fluid flows through, for example, a flow rate that is three times less than the specified flow rate, but it is not limited to three times. The flow rate of the third section 106C can be three times the specified flow rate. To twenty times. In this embodiment, the third section 106C is provided with a blocking structure 110, for example. The blocking structure 110 is for example used to reduce the space available for fluid flow in the third section 106C of the sperm sorting system 100 to increase fluid flow. The flow rate when passing through the third section 106C. In this embodiment, the blocking structure 110 is, for example, disposed at the junction of the second section 106B and the third section 106C. In this embodiment, the blocking structure 110 is, for example, a dumbbell shape, and the dumbbell-shaped blocking structure 110, for example, has a head end 110A, a connecting end 110B, and an end 110C. The connecting end 110B is, for example, connected and located at the head end 110A and There is a distance d between the end 110C, the head end 110A and the end 110C and the side wall of the third section 106C, and the distance d is, for example, greater than or equal to 50 μm. The arrangement direction of the head end 110A, the connecting end 110B, and the end 110C is, for example, perpendicular to the flow direction of the fluid, so that the fluid can circulate backward from the opposite ends of the blocking structure 110 relative to the connecting end 110B.

In this embodiment, the head end 110A, the connecting end 110B, and the end 110C of the blocking structure 110 are, for example, surrounded by a sperm collecting groove 110D for storing sperm. The sperm collecting groove 110D has a depth, and the extending direction of the depth is, for example, and The flow direction of the fluid is the same. Through the design of the sperm tank 110D, it can effectively block dead and living sperm. In this embodiment, the average vertical cross-sectional area of the sprue 106 in the third section 106C is, for example, one-third to twentieth of the section where the second section 106B connects with the third section 106C. One, by this, the flow rate of the third section 106C will be increased to between three times to twenty times, for example.

The branch channel 108 communicates with the main channel 106 and is used to connect the sperm sorting system 100 and the egg cultivation and sperm-egg combination system 200. In this embodiment, the high-efficiency sperm sorted by the sperm sorting system 100 will flow backward to the junction of the branch channel 108 and the main channel 106, and will be passively pushed into the branch channel 108, and then introduced into the egg cultivation Combine system with sperm and egg 200. In this embodiment, the branch flow channel 108 is, for example, connected to the front end of the second section 106B, that is, the area where the sample liquid enters the second section 106B from the first section 106A. The extension direction of the branch channel 108 is, for example, perpendicular to the extension direction from the sperm entrance portion 102 to the sperm exit portion 104.

In this embodiment, the branch runners 108 have substantially the same width, and the width is, for example, smaller than the minimum width of the main runner 106, that is, smaller than the width of the first section 106A. In an embodiment, the width of the branch flow channel 108 is, for example, 40 μm to 60 μm, and for example, 50 μm, and the width of the first section 106A is, for example, 80 μm to 120 μm, and for example, is 100 μm. In an embodiment, the length of the branch flow channel 108 is, for example, 1.8 mm to 2.2 mm, and for example 2 mm, and the length of the main flow channel 106 is, for example, 8 mm to 10 mm, and is, for example, 12 mm. In this embodiment, the depth of the main flow channel 106 and the branch flow channel 108 are, for example, the same, for example, 80 μm to 120 μm. In an embodiment, the depth of the main flow channel 106 and the branch flow channel 108 is, for example, 100 μm.

The egg breeding and sperm-egg combination system 200 includes a tank body 202 and a first through hole portion 210 and a second through hole portion 220 arranged in the tank body 202. The tank body 202 communicates with the branch flow channel 108. The diameter of the groove body 202 is, for example, 2.5 mm to 3.5 mm, and for example, 3 mm, and the depth of the groove body 202 is, for example, 1.8 mm to 2.2 mm, and is, for example, 2 mm. The first through hole portion 210 is disposed in the groove body 202. The first through-hole portion 210 includes a plurality of first through-holes 212, the diameter of the first through-holes 212 is larger than the average diameter of the egg, and is used to accommodate the egg to be combined with the sperm. That is, the sperm sorted by the sperm sorting system 100 is introduced into the egg culture and sperm-egg combination system 200 through the branch channel 108 to be combined with the eggs located in the first through hole 212. In this embodiment, the aperture of the first through hole 212 is designed so that the first through hole 212 substantially only accommodates one egg, which facilitates the formation of an effective fertilized egg and facilitates the follow-up tracking of a single target cell. In this embodiment, the first through hole portion 210 is made of polydimethylsiloxane (PDMS), for example, and the first through hole 212 penetrates through the polydimethylsiloxane. The hole diameter of the first through hole 212 is, for example, 180 μm to 220 μm, and for example, 200 μm, and the depth of the first through hole 212 is, for example, 180 μm to 220 μm, and is, for example, 200 μm. The first through holes 212 are, for example, regularly arranged, and the closest packing porosity thereof is, for example, 50% to 60%.

The second through hole portion 220 is located below the first through hole portion 210, and the second through hole portion 220 includes a plurality of second through holes 222. The hole diameter of the second through hole 222 is smaller than that of an egg, so that the egg and the fertilized egg can be fixed in the first through hole 212, so the second through hole 222 can be used as the bottom of the tank 202. In addition, the second through hole 222 can exchange liquid with the liquid infusion system 300 below it through the pressure difference, so it is also called a liquid exchange membrane. In this embodiment, the second through hole portion 220 is made of polydimethylsiloxane, for example, and the second through hole 222 penetrates through the polydimethylsiloxane. The hole diameter of the second through hole 222 is, for example, 25 μm to 35 μm, and is, for example, 30 μm, and the depth of the second through hole 222 is, for example, 50 μm to 100 μm, and is, for example, 75 μm. The second through holes 222 are, for example, regularly arranged, and their closest-packed porosity is, for example, 15% to 30%.

The liquid perfusion system 300 includes a liquid inlet portion 302, a liquid outlet portion 304, and a liquid flow channel 306 located between and communicating with the liquid inlet portion 302 and the liquid outlet portion 304. In this embodiment, the liquid inlet portion 302 is used to receive liquid, and the liquid outlet portion 304 is used to receive liquid flowing out of the liquid flow channel 306. In this embodiment, the liquid inlet portion 302 and the liquid outlet portion 304 are, for example, tanks, respectively. The diameter of the liquid inlet portion 302 and the liquid outlet portion 304 is, for example, 1.8 mm to 2.2 mm, and is, for example, 2 mm. The depth of the liquid inlet portion 302 and the liquid outlet portion 304 is, for example, 1.8 mm to 2.2 mm, and is, for example, 2 mm.

The liquid flow channel 306 is, for example, a groove having a depth. In this embodiment, the liquid flow channel 306 has a liquid exchange area 306A, which is located below the second through hole 220 of the egg cultivation and sperm-egg combination system 200. The liquid exchange area 306A exchanges liquid with the second through hole 220 through diffusion, for example, so that the liquid perfusion system 300 exchanges liquid with the egg cultivation and sperm-egg combination system 200. In this embodiment, the liquid exchange area 306A has, for example, a contour conforming to the second through hole portion 220, such as a circular shape.

In this embodiment, as shown in FIG. 1B, the integrated wafer 10 includes, for example, a first film F1, a second film F2 located under the first film F1, a third film F3 located under the second film F2, and a The fourth film F4 below the third film F3. In this embodiment, the material of the first film F1 to the fourth film F4 is, for example, PDMS, but the present invention is not limited to this. In this embodiment, the first film F1 includes a first through hole OP1 corresponding to the sperm entrance part 102, a second through hole OP2 corresponding to the sperm exit part 104, a first through hole OP2 corresponding to the main flow channel 106 and the branch flow channel 108 The groove T1, the protrusion pattern P of the blocking structure 110, the third through hole OP3 corresponding to the trough 202 of the egg cultivation and sperm-egg combination system 200, the fourth through hole OP4 corresponding to the liquid inlet portion 302, and the liquid outlet The fifth through hole OP5 of the part 304. The first through hole OP1 to the fifth through hole OP5 pass through the first film F1, for example, and the first trench T1 has a depth smaller than the thickness of the first film F1, for example.

The second film F2 includes a first through hole portion 210, a sixth through hole OP6 corresponding to the fourth through hole OP4, and a seventh through hole OP7 corresponding to the fifth through hole OP5, wherein the first through hole OP1 and the second The through hole OP2 and the second film F2 below the first groove T1 serve as the sperm inlet 102, the sperm outlet 104, and the bottom of the main flow channel 106 and the branch flow channel 108, respectively. The third film F3 includes a second through hole portion 220, an eighth through hole OP8 corresponding to the sixth through hole OP6, and a ninth through hole OP9 corresponding to the seventh through hole OP7. The fourth film F4 includes a first groove CV1, a second groove T2, and a second groove CV2 that are connected to each other. The first groove CV1 corresponds to the eighth through hole OP8 and serves as the bottom of the liquid inlet portion 302. The groove T2 serves as a liquid flow channel, and the second groove CV2 corresponds to the ninth through hole OP9 and serves as the bottom of the liquid outlet portion 304. In other words, the fourth through hole OP4, the sixth through hole OP6, the eighth through hole OP8, and the first recess CV1 form the liquid inlet portion 302; the fifth through hole OP5, the seventh through hole OP7, the ninth through hole OP9, and the The two grooves CV2 form the liquid outlet portion 304.

In this embodiment, for example, the first film F1 to the fourth film F4 of the integrated chip 10 are fabricated through mold flipping and drilling processes. The following description will be made by making the second film F2 including the first through hole as an example, but it is understandable that the first film F1, the third film F3, and the fourth film F4 can also be manufactured by a similar method. First, a substrate (not shown) is provided, and a columnar array structure (not shown) as a master mold is formed on the substrate by a yellow light lithography process. The columnar array structure includes a plurality of columnar patterns, and the height of the columnar pattern For example, the depth of the first through hole, and the diameter of the columnar pattern is, for example, the hole diameter of the first through hole. The substrate is, for example, a silicon wafer. The material of the columnar array structure is, for example, a negative photoresist, such as SU-8. Then, a thin film material such as PDMS is applied to the surface of the substrate by spin coating and the like and filled into the gap between the columnar array structures. After the thin film material is cured, a thin film including the first through-hole portion is obtained through mold reversal. Then, the sixth through hole OP6 and the seventh through hole OP7 are formed by a drilling process. Of course, in addition to the above methods, other suitable methods such as etching can also be used to fabricate the layers.

In this embodiment, for example, an oxygen plasma modification method is used to bond adjacent two of the first film F1 to the fourth film F4. For example, the surfaces of the second film F2 to the fourth film F4 can be modified and attached to the bottom surfaces of the first film F1 to the third film F3, respectively, so that the first film F1 to F3 The fourth film F4 is joined together to form an integrated chip 10. In one embodiment, for the convenience of carrying, the integrated chip 10 may be combined with a carrier 20, and the material of the carrier 20 may be glass. Furthermore, the integrated chip 10 may further include a sealing element (not shown) to control the difference in air pressure so that the liquid flow is temporarily stopped. For example, the seal is covered on the sperm sorting system 100 to seal the sperm inlet 102 and the sperm outlet 104 of the sperm sorting system 100. Furthermore, another sealing element is, for example, covered on the egg incubation and sperm-egg combination system 200 to seal the tank 202. In one embodiment, the sealing member may be a sealable material such as tape.

Next, the method of sperm sorting, egg cultivation, and in vitro fertilization through the integrated chip will be explained.

First, an integrated chip 10 is provided. Next, a wetting step is performed on the sperm sorting system 100 to wet the main flow channel 106 and the branch flow channel 108. Specifically, the wetting liquid is dripped into the sperm inlet 102, so that the liquid flows through the main flow channel 106 and the branch flow channel 108 to wet it. In this embodiment, the liquid used in the wetting step is, for example, physiological saline, germ cell culture liquid, or other liquids having isotonic pressure with sperm.

Then, the sample liquid containing the eggs is dropped into the tank 202 of the egg culture and sperm-egg combination system 200 so that the eggs are located in the first through holes 212 respectively. In this embodiment, by controlling the size of the first through hole 212, for example, only one egg is disposed in one first through hole 212. Then, pre-treatment steps are performed on the eggs. In this embodiment, the pre-processing step is, for example, the zona pellucida removal step, and this step is performed, for example, by adding enzymes to the tank 202. In this embodiment, after the pre-processing steps are completed (for example, after the effect of the enzyme is completed), the liquid perfusion system 300 is further turned on. That is, the liquid B is supplied to the liquid inlet portion 302 so that the liquid B flows through the liquid flow channel 306 and enters the liquid outlet portion 304. The liquid B may be a liquid used for exchange with the liquid in the tank 202, such as physiological saline, germ cell culture liquid, or other liquids having isotonic pressure with the cells. In this embodiment, due to the pressure difference between the second through hole 220 and the liquid flow channel 306 (ie, the liquid exchange area 306A) below it, harmful substances such as enzymes in the tank body 202 can pass through the second through hole. The portion 220 enters the liquid flow channel 306 and is discharged. In addition, the liquid B in the liquid flow channel 306 can also enter the tank body 202 through the second through hole portion 220 for liquid exchange. In this way, the tank 202 of the egg cultivation and sperm-egg combination system 200 can maintain an environment suitable for cultivating eggs or fertilized eggs. Then, the tank body 202 can be temporarily sealed through the sealing member. Here, the sealing of the tank body 202 can block the communication between the branch flow channel 108 and the tank body 202 to prevent substances (such as undesired sperm) from entering the tank body 202 from the branch flow channel 108 at this time.

Next, the sample liquid containing sperm is dropped into the sperm inlet 102, so that the sperm sorting system 100 performs sperm sorting on the sample liquid containing sperm. In this embodiment, the sample liquid can be semen with sperm from humans, pigs, cows, horses, etc. Then, after the sperm sorting step is completed, the egg cultivation and sperm-egg combination system 200 is turned on, so that the sorted high-efficiency sperm enters the tank 202 through the branch channel 108 to combine with the egg to form a fertilized egg. Here, the sorted high-potency sperm are sperm that can be held in the vicinity of the branch flow channel 108, and are, for example, sperm with moderate swimming motility. In this embodiment, the sorting time is, for example, about 10 minutes, and the proportion of live sperm at this time is about 85%. In this embodiment, the method of opening the egg culture and sperm-egg combination system 200 is, for example, to remove the seal. In addition, after the fertilized egg is formed, the fertilized egg can be further cultured in situ. The process of culturing the fertilized egg in situ includes replacing the culture medium in the tank body 202. Specifically, it can be performed by draining the culture medium in the tank body 202 and supplementing with fresh culture medium. In addition, generally speaking, the sample liquid containing sperm usually contains impurities. Therefore, in one embodiment, a filter member may be further provided in the sperm inlet portion 102, so that the sample liquid containing sperm enters the main flow channel 106. Filter before sorting to avoid impurities affecting the subsequent sorting process. In an embodiment, the filter member may be a filter membrane similar to the second through-hole portion 220. For example, the material of the filter membrane may be PDMS, and the pore size and depth of the through holes of the filter membrane are, for example, 25 μm to 35 μm. With 50μm to 100μm.

To sum up, the present invention integrates the sperm sorting system, the egg culture and sperm-egg combination system, and the liquid perfusion system on a single chip, so that the high-efficiency sperm sorted by the sperm sorting system can be directly introduced into the egg culture and The sperm-egg binding system combines with the cultivated egg in situ to form a fertilized egg to achieve the purpose of in vitro fertilization. In particular, in the present invention, it is only necessary to drop the sample liquid including sperm into the sperm inlet, and drop the sample liquid including the egg into the tank of the egg cultivation and sperm-egg combination system, without other active processing. It can passively sort sperm, process eggs and form fertilized eggs, so the process is gentle and can avoid damage to the sperm and eggs, which greatly improves the survival rate and combination rate of sperm and eggs. Therefore, the chip of the present invention can not only shorten the operation time of the traditional artificial reproductive technology, but also avoid the death of the specimen due to the lengthy operation time and artificial improper operation by simplifying the operation process, thereby greatly improving the success rate of the artificial reproductive technology.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field can make some changes and modifications without departing from the spirit and scope of the present invention. The scope of protection of the present invention shall be determined by the scope of the attached patent application.

10: Integrated wafer 20: Carrier 100: Sperm sorting system 102: Sperm inlet 104: Sperm outlet 106: Main flow channel 106A, 106B, 106C: Section 108: Branch flow channel 110: Blocking structure 110A: Head end 110B: connecting end 110C: end 110D: sperm collecting tank 200: egg cultivation and sperm-egg combination system 202: tank body 210: first through hole 212: first through hole 220: second through hole 222: second through hole Hole 300: liquid perfusion system 302: liquid inlet 304: liquid outlet 306: liquid flow channel 306A: liquid exchange area d: distance

1A is a perspective schematic view of an integrated wafer according to an embodiment of the present invention, FIG. 1B is an exploded schematic view of the integrated wafer of FIG. 1A, FIG. 1C is a bottom view of the sperm sorting system of the integrated wafer of FIG. 1A, and FIG. 1D is a schematic cross-sectional view of the integrated chip of FIG. 1A.

10: Integrated chip

20: carrier board

100: Sperm sorting system

102: Sperm Entrance

104: Sperm Export Department

106: Mainstream

108: branch runner

110: blocking structure

200: Egg cultivation and sperm-egg combination system

202: tank

300: Liquid perfusion system

302: Liquid inlet

304: Liquid Outlet

306: Liquid flow path

Claims (20)

An integrated chip for sperm sorting, egg cultivation and in vitro fertilization, including: a sperm sorting system, including a sperm entrance part, a sperm exit part, a main flow channel and a branch flow channel, the main flow channel and the sperm entrance part Communicating with the sperm outlet, the main flow channel has a width that gradually increases from the sperm inlet toward the sperm outlet, and the branch flow channel is located between the sperm inlet and the sperm outlet And is connected to the main flow channel; the egg cultivation and sperm-egg combination system, which is connected to the branch flow channel, includes a first through hole portion and a second through hole portion, wherein the first through hole portion includes a plurality of first A through hole, the first through hole has a diameter larger than the average diameter of the egg, and is used to accommodate the egg to be combined with the sperm, and the second through hole portion is located below the first through hole portion and includes a plurality of second through holes Hole, the second through hole has a smaller diameter than the egg, and is used for liquid exchange, wherein the sperm sorted by the sperm sorting system is introduced into the egg cultivation and sperm-egg combination system through the branch channel, To be combined with the egg located in the first through hole; and a liquid perfusion system, including a liquid inlet, a liquid outlet, and a liquid channel, wherein the liquid channel is located at the liquid inlet and the liquid outlet And communicate with the liquid inlet portion and the liquid outlet portion, the second through hole portion is provided between the first through hole portion and a part of the liquid flow channel, and the liquid flow channel The part of the second through-hole part exchanges liquid with the first through-hole part of the egg culture and sperm-egg combination system to maintain the first through-hole part It is suitable for an environment suitable for breeding eggs and fertilized eggs. The integrated chip according to the first item of the scope of patent application, wherein the material of the first through-hole portion and the second through-hole portion includes polydimethylsiloxane. According to the integrated chip described in item 1 of the scope of patent application, the first through holes are regularly arranged. According to the integrated chip described in the first item of the patent application, the second through holes are arranged regularly. According to the integrated chip described in the scope of the patent application, the aperture of the second through hole is greater than 30um. The integrated wafer according to the first item of the patent application, wherein the main flow channel includes a first section and a second section connected to the first section, and the first section is located at the sperm entrance Section and the second section, the second section is located between the first section and the sperm exit section, wherein the first section has substantially the same width, the first section The second section has a gradually increasing width and its minimum width is greater than the width of the first section. The integrated wafer according to claim 6, wherein the width of the branch flow channel is smaller than the width of the second section. The integrated wafer according to claim 1, wherein the sperm sorting system further includes a blocking structure, the blocking structure is located in the main flow channel and adjacent to the sperm exit portion, and the blocking structure and There is a distance between the side walls of the main flow channel. The integrated chip according to item 8 of the scope of patent application, wherein the blocking structure is dumbbell-shaped. The integrated wafer according to the first item of the scope of patent application, wherein the sperm entrance part and the sperm exit part are troughs. The integrated chip according to the first item of the patent application, wherein the liquid channel has a liquid exchange area, which is located under the egg cultivation and sperm-egg combination system and has a contour conforming to the second through hole. The integrated wafer described in item 1 of the scope of the patent application includes: a first film including a first through hole corresponding to the sperm entrance portion, a second through hole corresponding to the sperm exit portion, and The main channel and the first groove of the branch channel, the third through hole corresponding to the egg cultivation and sperm-egg combination system, the fourth through hole corresponding to the liquid inlet, and the fourth through hole corresponding to the liquid The fifth through hole of the outlet portion; the second film located below the first film, including the first through hole portion, the sixth through hole corresponding to the fourth through hole, and the fifth through hole Corresponding to the seventh through hole, wherein the second film located under the first through hole, the second through hole and the first groove respectively serve as the sperm entrance part, the sperm exit part and The main flow channel and the bottom of the branch flow channel; a third film located below the second film, including the second through hole portion, an eighth through hole corresponding to the sixth through hole, and A ninth through hole corresponding to the seventh through hole; and The fourth film located under the third film includes a first groove, a second groove, and a second groove connected to each other, wherein the first groove corresponds to the eighth through hole and serves as the The bottom of the liquid inlet portion, the second groove serves as the liquid flow channel, and the second groove corresponds to the ninth through hole and serves as the bottom of the liquid outlet portion. The integrated wafer according to claim 12, wherein the material of the first film, the second film, the third film, and the fourth film includes polydimethylsiloxane. The integrated chip according to claim 13 of the patent application, wherein adjacent two of the first film, the second film, the third film, and the fourth film are bonded to each other. A method for sperm sorting, egg culture, and in vitro fertilization, comprising: providing the integrated chip as described in the first item of the scope of patent application; dropping a sample liquid containing sperm into the sperm inlet so that the The sperm sorting system performs sperm sorting on the sample liquid containing sperm; dropping the sample liquid containing eggs into the egg cultivation and sperm-egg combination system, so that the eggs are respectively located in the first through holes; And the sorted sperm is introduced into the egg cultivation and sperm-egg combining system through the branch flow channel, so as to combine with the egg in the first through hole to form a fertilized egg. The method described in item 15 of the scope of the patent application further includes the step of removing the zona pellucida on the egg after dropping the sample liquid containing the egg into the egg cultivation and sperm-egg combination system. As the method described in item 15 of the scope of the patent application, further comprising, after dropping the egg-containing sample liquid into the egg cultivation and sperm-egg binding system, sealing the egg cultivation and sperm-egg binding system, and After the sperm sorting system completes the sperm sorting of the sperm-containing sample liquid, by opening the egg culture and sperm-egg combination system, the sorted sperm enters the branch channel through the Egg cultivation and sperm-egg combination system. The method described in item 15 of the scope of the patent application further includes sealing the sperm sorting system after dropping the sperm-containing sample liquid into the sperm inlet, and dropping the egg-containing sample liquid After entering the egg cultivation and sperm-egg combination system, the sperm sorting system is opened to perform the sperm sorting. The method described in item 15 of the scope of patent application further includes culturing the fertilized egg in the egg cultivation and sperm-egg combination system. The method described in item 15 of the scope of the patent application further includes flowing a liquid in the liquid perfusion system, wherein the liquid passes through the second through-hole portion and the egg incubation and sperm-egg combination system The liquid undergoes liquid exchange.

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