KR20210062976A - Curved microfluidic chip, Preparation method thereof, and Sperm screening method using the same - Google Patents

Abstract

The present invention relates to a microfluidic chip for segregating sperm by imitation of a female reproductive system, a method for manufacturing the same, and a method for selecting sperm using the same, wherein the microfluidic chip can select excellent sperm under conditions similar to those of a body without a separate pump and chemical inducing materials, thereby increasing the possibility of pregnancy of infertile couples.

Description

Curved microfluidic chip, manufacturing method thereof, and method of screening sperm using the same {Curved microfluidic chip, Preparation method thereof, and Sperm screening method using the same}

The present invention relates to a microfluidic chip for separating sperm by imitation of a female reproductive system, a method for manufacturing the same, and a method for selecting sperm using the same.

As the number of infertile couples is increasing worldwide, it is reported that in Korea, 1 to 2 couples out of 10 married couples are infertile couples. To overcome infertility, infertile couples use reproductive technologies such as in vitro fertilization (IVF), Intra Cytoplasmic Sperm Injection ICSI, and intrauterine insemination (IUI) to obtain a child. are making efforts for Five million babies worldwide are being born with assisted reproductive technology, and the number of babies born with this technology is expected to gradually increase.

Males account for 30-50% of all infertility cases, and 40-88% of male infertility cases are caused by low sperm count, decreased sperm motility, abnormal morphology, and DNA damage in sperm. to be. Even if the ovum and fertilization are inhibited or fertilized due to the above causes, it affects the low embryonic development and fertility rate and causes infertility, and it is very important to isolate good sperm from infertile men to increase the fertility.

However, in most fertility clinics, density gradient centrifugation (DGC) and swim-up methods are used to separate good sperm by morphology and motility, but these methods are time consuming and use good sperm. There is a limit to separating them. Existing methods for isolating sperm as described above do not take into account environments that mimic the female reproductive system at all, and despite advances in reproductive technology, the pregnancy rate of infertile couples is still at a standstill.

Therefore, there is a need for research and development on a chip capable of separating good sperm by mimicking the female reproductive system.

Republic of Korea Patent Publication No. 10-2015-0130280

It is an object of the present invention to provide a microfluidic chip comprising a curved microchannel and a water-soluble polymer having a viscosity in the microchannel, a method for manufacturing the same, and a method for screening sperm using the same.

The present invention provides an injection port for providing sperm;

A channel through which sperm provided from the inlet moves, the microchannel formed in a curved shape from the inlet; And

and a separation hole in which the sperm that have moved through the microchannel are collected,

The microchannel provides a microfluidic chip comprising 1% to 5% of a water-soluble polymer.

In addition, the present invention comprises the steps of forming an injection hole, a microchannel and a separation hole in the polymer substrate constituting the upper plate and the lower plate;

laminating the prepared upper and lower plates; And

It provides a method of manufacturing a microfluidic chip comprising adding 1% to 5% of a water-soluble polymer to the medium of the microchannel.

In addition, the present invention provides a method for screening sperm using the above-described microfluidic chip.

The present invention is a microfluidic chip that mimics a female reproductive system, and it is possible to select excellent sperm under conditions similar to those of the body without a separate pump and chemical inducing substances, thereby increasing the possibility of pregnancy of infertile couples.

1 is a schematic chemical image of a microfluidic chip according to the present invention and an image of an actual microfluidic chip.
FIG. 2 is an image obtained by using a microfluidic chip according to the present invention to capture sperm motility according to a polymer concentration in a microchannel using a phase-contrast microscope.
3 is a graph showing sperm motility according to time for each polymer concentration in a microchannel using the microfluidic chip according to the present invention.
4 is an image taken with a phase contrast microscope of sperm according to time for each concentration of polymer in a microchannel using the microfluidic chip according to the present invention.
5 is an image and graph showing the distribution of the number of vacuoles according to the polymer concentration of the microchannel using the microfluidic chip according to the present invention.

In the present invention, various modifications may be made and various embodiments may be provided, and specific embodiments will be illustrated in the drawings and described in detail in the detailed description.

However, this is not intended to limit the present invention to a specific embodiment, it should be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention.

Existing sperm sorting chips use a separate pump in the sperm sorting chip to select sperm with excellent motility to form a flow opposite to the sperm movement and induce sperm by adding a chemical inducing substance to the collecting port where the sperm arrives. On the other hand, the microfluidic chip according to the present invention can select excellent sperm without a separate pump and chemical inducing material, so that excellent sperm can be obtained at low cost.

The present invention provides an injection port for providing sperm;

A channel through which sperm provided from the inlet moves, the microchannel formed in a curved shape from the inlet; And

and a separation hole in which the sperm that have moved through the microchannel are collected,

The microchannel provides a microfluidic chip comprising 1% to 5% of a water-soluble polymer.

As shown in Fig. 1, the microfluidic chip according to the present invention was manufactured by mimicking the female reproductive system. Specifically, an injection hole, a microchannel, and a separation hole are formed in a polymer plastic substrate, and the microchannel has a curved shape and contains a certain amount of water-soluble polymer to realize a physical viscous environment similar to that of a female reproductive organ. In addition, the microchannel may have a narrower diameter as it approaches the separation hole, thereby making it easier to separate sperm.

For example, the injection hole, the microchannel, and the separation hole are continuously connected, and the injection hole and the separation hole may be positioned in a straight line on the x-axis or the y-axis line based on the xy plane. In addition, the microchannel connecting the inlet and the separation port may have a curved shape. By having the structure as described above, it is possible to select sperm having excellent motility under conditions similar to those of female reproductive organs.

Specifically, the microfluidic chip may have a structure in which an upper plate and a lower plate are laminated. An injection hole, a micro panel, and a separation hole are formed on the upper plate and the lower plate, respectively, and the micro channel may be sealed in a laminated structure.

For example, the upper plate and the lower plate may independently be a polymer substrate including at least one selected from polyacrylate, polydimethylsiloxane, and polystyrene polymer. Specifically, the upper plate and the lower plate may independently be a polymer substrate including polyacrylate, polydimethylsiloxane, or polystyrene polymer. By including the polymer substrate as described above, the microfluidic chip can visually observe the movement of the sample and, if necessary, observe the sperm with a microscope.

As an example, the microchannel may contain 1% to 5% of a water-soluble polymer. Specifically, the microchannel may contain 1% to 4%, 1% to 3%, 2% to 5%, 2% to 4%, 2% to 3%, or 2.5% to 3.5% of a water-soluble polymer. By including the water-soluble polymer in the above content, the microfluidic chip according to the present invention realizes a viscous environment similar to that of a female reproductive organ, thereby selecting sperm with excellent motility. The content of the water-soluble polymer may be volume %.

The water-soluble polymer is not particularly limited as long as it can form a viscous environment in the microchannel. For example, it may include at least one selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinyl alcohol, and polyethylene oxide. Specifically, the water-soluble polymer may be polyvinylpyrrolidone (PVP), polyvinyl alcohol or polyethylene oxide.

Also, the microchannels may have an average diameter of less than 1000 μm. Specifically, the average diameter of the microchannel is less than 900 μm, less than 800 μm, 600 μm to 1000 μm, 600 μm to 900 μm, 600 μm to 800 μm, 700 μm to 1000 μm, 700 μm to 900 μm, 700 μm to It may be 800 μm or 700 μm to 900 μm.

For example, the diameter of the microchannel may decrease from the injection hole to the separation hole. Specifically, the diameter of the microchannel connected to the separation hole may be 100㎛ to 600㎛. More specifically, the diameter of the microchannel in the region 0.5mm to 5mm, 0.5mm to 3mm, or 1mm to 2mm away from the separation hole is 100 μm to 600 μm, 100 μm to 500 μm, 100 μm to 400 μm, 200 μm to 600 μm, 200 μm to 500 μm, 200 μm to 400 μm, 300 μm to 600 μm, 300 μm to 500 μm, or 300 μm to 400 μm. By having a narrow diameter as described above, in the microfluidic chip according to the present invention, the sperm can move freely without a separate pump, and the sperm can be separated efficiently.

In addition, independently forming the injection hole, the microchannel and the separation hole in the polymer substrate constituting the upper plate and the lower plate;

laminating the prepared upper and lower plates; And

It provides a method of manufacturing a microfluidic chip comprising adding 1% to 5% of a water-soluble polymer to the medium of the microchannel.

Specifically, in the step of adding the water-soluble polymer to the medium of the microchannel, 1% to 5% of the water-soluble polymer may be added. Specifically, the step of adding the water-soluble polymer to the medium of the microchannel is 1% to 4%, 1% to 3%, 2% to 5%, 2% to 4%, 2% to 3%, or 2.5% to 3.5% of water-soluble polymer may be added. By adding the water-soluble polymer in the same amount as described above, the microfluidic chip according to the present invention realizes a viscous environment similar to that of a female reproductive organ, thereby selecting sperm with excellent motility. The amount of the water-soluble polymer added may be volume %.

The water-soluble polymer is not particularly limited as long as it can form a viscous environment in the microchannel. For example, it may include one or more selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinyl alcohol, and polyethylene oxide. Specifically, the water-soluble polymer may be polyvinylpyrrolidone (PVP), polyvinyl alcohol or polyethylene oxide.

As an example, in the step of forming the injection hole, the microchannel, and the separation hole in the polymer substrate constituting the upper plate and the lower plate, the injection hole, the microchannel and the separation hole are sequentially formed in the upper plate and the lower plate of the polymer substrate by using a laser. have. Specifically, the polymer substrate forming the upper and lower plates uses a laser to form an injection hole for injecting a solution containing sperm and a separation hole through a microchannel, but when forming microchannels on the upper and lower plates, the laser It can be manufactured by forming a groove with

In addition, the diameter of the microchannel may be formed to an average of 1000 μm or less. Specifically, the average diameter of the microchannel is less than 900 μm, less than 800 μm, 600 μm to 1000 μm, 600 μm to 900 μm, 600 μm to 800 μm, 700 μm to 1000 μm, 700 μm to 900 μm, 700 μm to It can be formed in 800㎛ or 700㎛ to 900㎛.

In addition, the microchannel may be formed such that the diameter of the microchannel decreases from the injection hole to the separation hole. Specifically, the diameter of the microchannel connected to the separation hole may be formed in a range of 100 μm to 600 μm. More specifically, the diameter of the microchannel in the region 0.5mm to 5mm, 0.5mm to 3mm, or 1mm to 2mm away from the separation hole is 100 μm to 600 μm, 100 μm to 500 μm, 100 μm to 400 μm, 200 μm to 600 μm, 200 μm to 500 μm, 200 μm to 400 μm, 300 μm to 600 μm, 300 μm to 500 μm, or 300 μm to 400 μm.

By forming the narrow diameter as described above, in the microfluidic chip according to the present invention, the sperm can move freely without a separate pump, and the sperm can be separated efficiently.

As an example, in the step of laminating the upper plate and the lower plate, a closed microchannel may be formed by laminating a polymer substrate having an injection hole, a microchannel, and a separation hole formed thereon. The polymer substrate of the upper plate and the lower plate may be independently formed of at least one selected from polyacrylate, polydimethylsiloxane, and polystyrene polymer. Specifically, the polymer substrate of the upper plate and the lower plate may be independently formed of a polymer substrate including polyacrylate, polydimethylsiloxane, or polystyrene polymer. By using the polymer substrate as described above, the microfluidic chip can see the movement of the sample with the naked eye and, if necessary, observe the sperm with a microscope.

Specifically, the step of laminating the upper plate and the lower plate may be laminated by applying an adhesive to the polymer substrate of the upper plate and the lower plate to the portion in contact with the upper plate and the lower plate. The adhesive may have adhesive properties, for example, polydimethylsiloxane (PDMS), polyimide, polyester, polyurethane or polyethylene may be used as the adhesive.

The method of manufacturing a microfluidic chip according to the present invention may include curing the adhesive by applying heat after laminating the upper and lower plates. Specifically, after laminating the upper and lower plates, the adhesive may be cured by maintaining it at a temperature of 50° C. to 100° C. for 1 hour to 10 hours. More specifically, after laminating the upper plate and the lower plate at a temperature of 50 ℃ to 90 ℃, 50 ℃ to 80 ℃, 50 ℃ to 70 ℃ or 60 ℃ to 70 ℃ 1 hour to 8 hours, 2 hours to 8 hours, 3 The adhesive can be cured by holding for hours to 7 hours or 4 hours to 6 hours.

In addition, the present invention provides a method for screening sperm using the above-described microfluidic chip.

As an example, injecting a sperm sample into the injection hole of the microfluidic chip; And it may include the step of collecting the sperm in the separation port. Specifically, in the step of injecting the sperm sample into the injection port of the microfluidic chip, the sperm sample may be raw semen obtained from a male, and the sperm fluid may include sperm with vacuoles and/or sperm without vacuoles. can When the sperm sample is injected into the injection hole, the sperm in the sample moves along the microchannel of the microfluidic chip, and only the sperm with excellent motility can pass through the microchannel according to the viscosity of the microchannel.

The method for screening sperm according to the present invention may be performed for 10 to 60 minutes after injecting a sperm sample into a microfluidic chip. Specifically, after injecting the sperm sample into the microfluidic chip, 10 minutes to 50 minutes, 10 minutes to 40 minutes, 10 minutes to 30 minutes, 20 minutes to 60 minutes, 20 minutes to 50 minutes, 20 minutes to 40 minutes, 20 minutes to 30 minutes, 30 minutes to 50 minutes, or 30 minutes to 40 minutes.

As an example, the step of collecting the sperm in the separator may be collected by suctioning the sperm collected in the separator with a pipette. The sperm in the separation port may be a sperm with excellent motility and no vacuole among the sperm included in the sperm sample.

In addition, it may further include the step of observing the collected sperm under a microscope after the step of collecting the sperm in the separation sphere. The step of observing the collected sperm under a microscope may be a step of determining whether the collected sperm has excellent motility and does not have vacuoles.

Hereinafter, the present invention will be described in more detail through Examples and the like according to the present invention, but the scope of the present invention is not limited by the Examples presented below.

Example 1

The microfluidic chip according to the present invention used 2D Auto Cad, and the upper and lower plates of the microfluidic chip were made of 1.3 mm thick 18 mm Х 16 mm poly(methyl methacrylate) (PMMA) with a laser cutter. (laser cutter, MYL-0705 Model, MYCNC company) was manufactured using. Specifically, an inlet (inlet) and an outlet (outlet) were cut to have diameters of 2.2 mm and 1 mm, respectively, to form a hole in the PMMA upper plate. Then, to create a microchannel through which the sperm pass, a microchannel with a 1mm width semi-elliptical structure was formed on the upper and lower PMMA plates using a laser cutter, and the microchannel directly connected to the separation hole was formed to have a width of 350㎛. did. After laminating the upper and lower plates of PMMA with microchannels formed by using polydimethyl siloxane (PDMS) adhesive, put them in a 60 mm petri dish, and hold them at a temperature of 65° C. for 5 hours to prepare a microfluidic chip. did.

1% polyvinylpyrrolidone was injected into the culture medium in the microchannel of the microfluidic chip prepared above, and 10 μl of sperm was put into the injection hole, and then the sperm collected in the separation hole was collected 10 minutes later.

Example 2

A microfluidic chip was prepared under the same conditions as in Example 1, except that 3% of polyvinylpyrrolidone was injected into the microchannel.

Comparative Example 1

A microfluidic chip was prepared under the same conditions as in Example 1, except that polyvinylpyrrolidone was not injected into the microchannel.

Experimental Example 1

In order to confirm the sperm selectivity of the microfluidic chip according to the present invention, the motility of the separated sperm and the number of vacuoles according to the polymer viscosity of the microchannel were observed for the microfluidic chips of Examples 1 and 2 and Comparative Example 1. , the results are shown in FIGS. 2 to 4 .

2 is a phase-contrast microscope image of the sperm passing through the microchannel in order to examine the motility of the sperm according to the polymer concentration of the microchannel in the microfluidic chips of Examples 1, 2, and Comparative Example 1. As shown in FIG. Referring to FIG. 2 , as the concentration of polyvinylpyrrolidone in the micropanel increased, the number of sperm finally obtained through the microchannel decreased, while it was confirmed that the sperm activity and rectilinear motility were very good. In addition, since various contaminants and blood cells present in the seminal fluid were completely removed, only purely highly motile sperm were isolated.

3 is a graph showing the motility of sperm according to time for each polymer concentration, and FIG. 4 is an image taken with a phase contrast microscope of raw sperm and sperm according to time according to polymer concentration of the microfluidic chip. 3 and 4, as a result of confirming the motility of sperm over time, the number of sperm passing through microchannels decreased as the polyvinylpyrrolidone concentration increased, but it was schematically shown that excellent motility was exhibited even after about 40 minutes had elapsed. will show it

5 is an image and graph showing the distribution of the number of vacuole heads of sperm according to the polymer concentration of the microchannel using the initial sperm and the microfluidic chip. Referring to FIG. 5 , it was confirmed that the number of vacuole heads of sperm was decreased when sperm were separated by adding polyvinylpyrrolidone. In the sperm head, the vacuole plays a role in interfering with embryo development and pregnancy when fertilized with the egg.

Through this, it is to prove that the microfluidic chip according to the present invention is a technology that dramatically reduces time and cost compared to the conventional method of separating sperm with vacuole heads.

Claims (12)

an inlet for providing sperm;
A channel through which sperm provided from the inlet moves, the microchannel formed in a curved shape from the inlet; and
and a separation hole in which the sperm that have moved through the microchannel are collected,
Microfluidic chip, characterized in that the microchannel contains 1% to 5% of a water-soluble polymer. The method of claim 1,
The water-soluble polymer is a microfluidic chip, characterized in that at least one selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinyl alcohol and polyethylene oxide. The method of claim 1,
The microchannel has a curved shape,
The microfluidic chip, characterized in that the average diameter of the microchannel is less than 1000㎛. The method of claim 1,
The microfluidic chip, characterized in that the diameter of the microchannel connected to the separation hole is 100㎛ to 600㎛. The method of claim 1,
The microfluidic chip has a structure in which an upper plate and a lower plate are laminated,
The microfluidic chip, characterized in that the upper plate and the lower plate are independently selected from polyacrylate, polydimethylsiloxane, and polystyrene polymer. The method of claim 1,
The microfluidic chip, characterized in that the microchannel is in a sealed form. Forming an injection hole, a microchannel, and a separation hole independently of the polymer substrate constituting the upper plate and the lower plate;
laminating the prepared upper and lower plates; and
A method of manufacturing a microfluidic chip comprising adding 1% to 5% of a water-soluble polymer to the medium of the microchannel. The method of claim 7,
The step of forming the injection hole, the microchannel and the separation hole in the polymer substrate is a method of manufacturing a microfluidic chip using a laser. The method of claim 7,
The step of forming the injection hole, the microchannel and the separation hole in the polymer substrate forms the microchannel in the form of a curve,
The method of manufacturing a microfluidic chip to form the diameter of the microchannel to an average of 1000㎛ or less. The method of claim 8,
A method of manufacturing a microfluidic chip in which the diameter of the microchannel connected to the separation hole is 100 μm to 600 μm. The method of claim 7,
A method of manufacturing a microfluidic chip wherein the polymer substrate of the upper plate and the lower plate is independently selected from polyacrylate, polydimethylsiloxane, and polystyrene polymer. A method for screening sperm using the microfluidic chip according to any one of claims 1 to 6.

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