KR20090041962A - Fertilized egg or ovum culture system which mimics physical and biological features of oviduct based on microfluid mechanics - Google Patents

Abstract

A culture system for fertilized egg or ovum using micro-fluid mechanics is provided to improve culture of fertilized egg and ovum, thereby being used for artificial insemination and external fertilization for infertility couples. A culture system for fertilized egg or ovum comprises: an inlet(1) for fertilized egg or ovum; an ovum movement micro-fluidic channel(10), linked with the inlet, in which ovum can move; a plurality of ovum compression micro-fluidic channels(20) formed by reducing diameter of the ovum movement micro-fluidic channel; and an outlet(60) for fertilized egg or ovum connected to the ovum movement micro-fluidic channel.

Description

Fertilized egg or ovum culture system which mimics physical and biological features of oviduct based on microfluid mechanics}

The present invention relates to a fertilized egg and egg culture system that mimics the physical and biological characteristics of the fallopian tube on the basis of microfluidic control technology, and more specifically, to a device that can mimic the compression, peristalsis and ciliary motion in the fallopian tube. In combination with microfluidic control technology, the present invention provides a method for efficiently culturing and developing fertilized eggs and eggs.

The development of stem cells and the development of adjuvant reproduction have led to the development of in vitro culture (IVC) and fertilization (IVF) techniques for fertilized eggs and eggs. One of the most important processes in in vitro fertilization and in vitro procedures to address the development and infertility of stem cells, which emerge as the only alternative to the treatment of intractable disease, is the culture of fertilized eggs and eggs. Existing culture techniques for fertilized eggs and eggs have relied on the same Petri dish culture as other cell culture methods, and techniques for individually handling embryos consisted of glass pipettes or holding pipettes.

In order to improve the high development failure rate of the existing culture method, multiple birth and ethical and economic problems, devices for cultivating fertilized eggs and eggs with microfluidic system have been developed (Domestic Patent Publication No. 10-2005-0008731, Korean Patent Publication 10-2007-0033946). This system has helped to overcome the limitations of conventional culture dishes and glass pipettes, thereby increasing the success rate. However, the devices still have a limitation in their efficiency, showing a lot of difference from the shape of the oviduct through which the actual embryo moves within the organism.

Human oviduct is an elongated tube that connects the uterus to the ovary, where fertilization takes place and serves to move the fertilized egg into the uterus. The total length is about 10 cm and consists of four parts, nipples, fallopian tubes, flat and narrow. The fundibulum is a funnel-shaped part of the ovary, about 10 mm in diameter. The fimbriae is the edge of the fallopian tube, with fingers like extensions of its margins. The ampulla is one-third of the end of the fallopian tube and is an important part of the fertilization. Lastly, the isthmus is the part of the uterine cavity that has the narrowest diameter of 30 to 100 μm of the fallopian tubes. If the diameter of the fertilized egg is about 100 μm it can be seen that the fertilized egg is compressed and deformed under slight pressure while passing through the isthmus while moving the fallopian tube toward the uterus. The fallopian epithelium is ciliated simple columnar epithelium. The fertilized egg that meets sperm in the flat part is sent to the uterus by ciliary movement by ciliary epithelial cells in the fallopian tube and peristalsis of the fallopian tube itself.

Thus, the present inventors have fabricated a culture apparatus that mimics the movement of fertilized eggs in the fallopian tubes in living organisms by combining the above-described physical and biological characteristics of the fallopian tubes and microfluidic control techniques, and the healthy differentiation of the fertilized eggs in the culture apparatus. The present invention was completed by confirming.

The purpose of the present invention is to improve the culture efficiency and stem cell and embryology research by imitating the physical and biological characteristics of the oviduct in order to increase the low efficiency of fertilized egg and egg culture system based on the existing microfluidics (microfluidics) Is to provide convenience.

In order to achieve the above object, the present invention is a fertilized egg and medium inlet (1);

An egg moving microfluidic channel part 10 connected to the fertilized egg and the medium inlet 1 and movable in the egg;

An oocyte-compressed microfluidic channel part 20 which reduces the diameter of the oocyte-moving microfluidic channel part 10 formed in at least one number in the oocyte-moving microfluidic channel part 10; And,

The egg transfer microfluidic channel unit 10 provides a fertilized egg or an egg culture system in which a fertilized egg and a medium outlet 60 are connected.

In addition, the present invention 1) injecting the medium into the inlet (1) of the fertilized egg or egg culture system;

2) injecting a fertilized egg or an egg into the inlet (1) and controlling the speed of the microfluidic flow;

3) culturing the fertilized egg or egg while putting the system of step 2) in a 37 ° C. incubator to maintain the proper temperature; And,

4) It provides a method for cultivating fertilized eggs or eggs using the system comprising the step of discharging the cultured eggs or eggs of step 3) from the fertilized eggs and the medium outlet (51).

In addition, the present invention is a fertilized egg and medium inlet (1);

An egg moving microfluidic channel part 10 connected to the fertilized egg and the medium inlet 1 and movable in an egg and provided with a valve (pump) channel part 25 in an upstream region;

An oocyte-compressed microfluidic channel part 20 which reduces the diameter of the oocyte-moving microfluidic channel part 10 formed in at least one number in the oocyte-moving microfluidic channel part 10; And,

The egg transfer microfluidic channel unit 10 provides a fertilized egg or an egg culture system in which a fertilized egg and a medium outlet 60 are connected.

In addition, the present invention 1) injecting the medium into the inlet (1) of the fertilized egg or egg culture system;

2) injecting a fertilized egg or an egg into the inlet (1) and controlling the speed of the microfluidic flow;

3) injecting microfluid into the valve (pump) channel part 25 when the fertilized egg or egg of step 2) passes the valve (pump) channel part 25 for peristaltic contraction;

4) When the fertilized egg or egg of step 3) has passed the valve (pump) channel part 25, culturing the fertilized egg or egg while putting the system of step 3) in a 37 ° C. incubator to maintain an appropriate temperature; And,

5) It provides a fertilized egg or egg culture method using the system comprising the step of discharging the cultured fertilized egg or egg of step 4) from the fertilized egg and medium outlet (51).

Hereinafter, the present invention will be described in detail.

The present invention is a fertilized egg and medium inlet (1);

An egg moving microfluidic channel part 10 connected to the fertilized egg and the medium inlet 1 and movable in the egg;

An oocyte-compressed microfluidic channel part 20 which reduces the diameter of the oocyte-moving microfluidic channel part 10 formed in at least one number in the oocyte-moving microfluidic channel part 10; And,

The egg transfer microfluidic channel unit 10 provides a fertilized egg or an egg culture system in which a fertilized egg and a medium outlet 60 are connected.

The egg transfer microfluidic channel 10 is preferably 10% to 100% wide and 5% to 50% high in blastocyst diameter, in which the fertilized egg is developed. It is preferable that the egg movement microfluidic channel part 10 includes two to 300 egg compression microfluidic channel parts 20. The egg compression microfluidic channel part 20 may be included, and thus, the number of compression stimuli may be adjusted and mimic the length of the oviduct. The egg compressed microfluidic channel portion 20 is preferably 60% to 90% of the width and height of the blastocyst diameter, and the width and height of the egg compressed microfluidic channel portion 20 are adjusted to control the intensity of the compression stimulus. Can be. Preferably, the egg compressed microfluidic channel part 20 has a length of 50 μm to 150 μm, and the width and height of the egg compressed microfluidic channel part 20 are adjusted to control the strength of the compression stimulus. . As a result of using the system, the cultured fertilized eggs were confirmed to be healthy differentiation up to the blastocyst form (see Fig. 6a).

In addition, the present invention 1) injecting the medium into the inlet (1) of the fertilized egg or egg culture system;

2) injecting a fertilized egg or an egg into the inlet (1) and controlling the speed of the microfluidic flow;

3) culturing the fertilized egg or egg while putting the system of step 2) in a 37 ° C. incubator to maintain the proper temperature;

4) It provides a method for cultivating fertilized eggs or eggs using the system comprising the step of discharging the cultured eggs or eggs of step 3) from the fertilized eggs and the medium outlet (51).

In addition, the egg transfer microfluidic channel unit 10 is connected to the fertilized egg and the medium inlet (1) and the egg is movable;

An egg moving microfluidic channel part 10 connected to the fertilized egg and the medium inlet 1 and movable in an egg and provided with a valve (pump) channel part 25 in an upstream region;

An oocyte-compressed microfluidic channel part 20 which reduces the diameter of the oocyte-moving microfluidic channel part 10 formed in at least one number in the oocyte-moving microfluidic channel part 10; And,

The egg transfer microfluidic channel unit 10 provides a fertilized egg or an egg culture system in which a fertilized egg and a medium outlet 60 are connected.

The valve (pump) channel portion 25 is located in an upstream region of the lower microfluidic channel plate 50 through which the fertilized egg or egg can move, and the valve (pump) channel portion 25 and the lower microfluidic channel plate 50 are formed. A membrane may exist between the fertilized eggs or eggs that move the channel portion of the lower microfluidic channel plate 50. Preferably the membrane is polydimethylsiloxane. A peristaltic pump is additionally connected to the valve (pump) channel portion 25 to apply pressure to the lower channel plate 50 so that the fluid in the lower portion can flow in a constant direction and speed.

In addition, the present invention 1) injecting the medium into the inlet (1) of the fertilized egg or egg culture system;

2) injecting a fertilized egg or an egg into the inlet (1) and controlling the speed of the microfluidic flow;

3) injecting microfluid into the valve (pump) channel part 25 when the fertilized egg or egg of step 2) passes the valve (pump) channel part 25 for peristaltic contraction;

4) When the fertilized egg or egg of step 3) has passed the valve (pump) channel part 25, culturing the fertilized egg or egg while putting the system of step 3) in a 37 ° C. incubator to maintain an appropriate temperature;

5) It provides a fertilized egg or egg culture method using the system comprising the step of discharging the cultured fertilized egg or egg of step 4) from the fertilized egg and medium outlet (51).

It provides a culture system characterized in that the ciliated cells of the fallopian tube surface is further attached to the inner wall of the culture system of the present invention.

Chemicals may be treated on the inner wall of the culture system prior to the attachment of the ciliated cells, such as collagen, fibronectin, laminin, poly -L-lysine, natural extracellular matrix, and the like, and most preferred is the extracellular matrix. In addition, the present invention may be replaced by MEF (mouse embryonic fibroblast) in place of the ciliated cells.

The oocyte moving microfluidic channel part 10, the oocyte compressed microfluidic channel part 20 and the valve (pump) channel part 25 of the culture system of the present invention are polydimethylsiloxane (polydimethylsiloxane, PDMS), polymethyl methacrylate Selected from the group consisting of polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polystyrene, polycyclic olefins, polyimides and polyurethanes It may be either, PDMS was used in a preferred embodiment of the present invention.

Also in a preferred embodiment of the present invention the culture system may be bonded to any one selected from the group consisting of slide glass, crystal, glass glass and PDMS, and may be fixed on the device to form a slope capable of generating microfluidic flow. Can be. In addition, the microfluidic flow may be generated by adjusting the medium injection amount with a syringe pump.

In the preferred embodiment of the present invention, as shown in Figure 2c, fertilized egg and medium inlet (1), egg transfer microfluidic channel section 10, 14 oocyte compressed microfluidic channel section 20 and fertilized egg and medium outlet (60) Provided is a fertilized egg or egg culture system.

The system can be adjusted to allow fluid and fertilized eggs to move inside the microchannel by immobilizing the culture system on a device that forms a slope for the flow of the microfluid, causing a height difference on the left and right of the system (see FIG. 5). As a result of using the system, the cultured fertilized eggs were confirmed to be healthy differentiation up to the blastocyst form (see Fig. 6b).

In addition, in a preferred embodiment of the present invention, the culture system shown in Figure 2c, shown in Figure 3a is a top, and the culture system (Fig. 3b) that is in phase with the system is a bottom, the top and bottom are joined Provided is a fertilized egg or egg culture system open from the fertilized egg and medium inlet 1 to the outside of the system in the opposite direction to the egg transfer channel portion. In addition, oocytes may be attached to the inner wall of the culture system.

In addition, in a preferred embodiment of the present invention, a valve (pump) channel portion 25 is further included at the upper end of the egg moving microfluidic channel portion 10 of the culture system shown in FIG. Provided is a fertilized egg or egg culture system. The ciliated cells of the fallopian tube surface may be further attached to the inner wall of the culture system.

 In a preferred embodiment of the present invention as a result of culturing the fertilized egg in the culture system shown in Figure 4 it was confirmed that the cultured fertilized egg is healthy differentiation up to blastocyst form.

Since the present invention mimics the physical and biological characteristics of the oviduct in order to increase the efficiency of the microfluidic system-based fertilized egg and egg culture system, other culture systems using microfluidic control technology as well as the conventional petri dish type culture method. Is expected to be different in the degree of physical and biological stimulation given to the fertilized egg, it was confirmed through the preferred embodiment of the present invention that the cultured fertilized eggs are healthy differentiation to blastocyst form. That is, the culture system of the present invention will be more effective than the conventional culture system, such as culture rate, growth and fertilized egg after fertilization. In addition, the microfluidic channel part of the present invention can be adjusted in the width and height of the channel portion, there is no limit to the type of ciliocytes that can be attached to the channel portion, so it can be applied as a culture system for fertilized eggs or eggs derived from various species Could be.

The oviduct biomimetic fertilized egg culture system based on the microfluidic control technology according to the present invention can help efficient culture and development of fertilized eggs and eggs, thus providing convenience and high efficiency in stem cell and embryology research, and effectively in general gynecology. It can be applied to artificial insemination and in vitro fertilization for infertile women.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1> Preparation of basic unit of fertilized egg or egg culture system

<1-1> Imitation of Physical Characteristics (Compression)

The basic unit of the fertilized egg or egg culture system that mimics the constriction among the physical characteristics of the oviduct based on microfluidics according to the present invention is as follows. As shown in FIG. 1A, the system has a structure in which a structure layer 41 having a microchannel pattern and a transparent plate layer 70 are stacked. The structure layer 41 having the microchannel pattern was manufactured by a molding method using transparent and elastic PDMS (polydimethylsiloxane). As a master for molding, a method of patterning a photoresist on a silicon wafer substrate was used. As the photosensitive material, an egg transfer microfluidic channel part 10 and an egg compressed microfluidic channel part 20 were manufactured using SU-8 (FIG. 2B). At this time, in order to produce a microfluidic channel similar to the size of an actual oviduct, the egg moving microfluidic channel part 10 was set to a width of 200 μm and a height of 200 μm, which is more than 10% wider than the actual blastocyst diameter. In addition, the channel width and height of the oocyte-compressed microfluidic channel 20 for simulating the compression that may occur when the fertilized egg moves in the fallopian tube are 90% or less of the blastocyst diameter (FIG. 1A), and the width and height of the passage are It is smaller than the size of the fertilized egg and the egg can give a constriction effect on the surface of the fertilized egg and the egg. The mold was poured with pre-PDMS (Sylgard 184, Dow Corning) mixed with a curing agent in a ratio of 10: 1, and cured at 60 ° C. for 3 hours. After the hardening process, a hole was formed in the PDMS substrate to prepare an inlet (1) and an outlet (60) through which cells and various fluid materials can flow. The structure layer 41 and the transparent plate layer 70 having the microchannel pattern thus formed are oxidized and stacked by using an air plasma to complete a fertilized egg or egg culture system consisting of two layers.

<1-2> Imitation of Physical Characteristics (Interlocking Motion)

Among the physical characteristics of the oviduct based on microfluidics according to the present invention, the basic unit of a fertilized egg or egg culture system that mimics a peristaltic contraction is as follows. As shown in FIG. 1B, the system has a valve (pump) channel portion 25 for a peristaltic contraction. The lower microfluidic channel plate 50 through which the fertilized egg can flow can be placed thereon, and the channel portion of the lower microfluidic channel plate 50 can be pressed by generating a fluid flow in which the direction of the fluid flow is perpendicular to the system. A valve (pump) channel section 25 was placed on top. Connecting a pipe capable of introducing and discharging fluid into the valve (pump) channel part 25 to the wall surface of the upper microfluidic channel plate (FIG. 4), and the upper microfluidic channel plate 40 and the lower microfluidic channel. Between the structure layers of the plate 50, a membrane was made of a soft material such as PDMS. The valve (pump) channel portion 25 contacts the channel portion of the lower panel plate 50 so as to press the channel portion to move not only the fluid of the channel portion of the lower microfluidic channel plate 50 but also the fertilized egg. In addition, by connecting a peristaltic pump to the valve (pump) channel part 25 to apply pressure to the lower microfluidic channel plate 50, the lower fluid was controlled to flow in a constant direction and speed.

In the basic manufacturing process, a silicon mold having a round microfluidic channel was manufactured, and a lower microfluidic channel plate 50 was manufactured through PDMS curing. The upper microfluidic channel plate 40 including the upper valve (pump) channel portion 25 was bonded through the alignment. After completion of the bonding, the valve (pump) channel part 25 and the lower microfluidic channel plate 50 are removed from the lower wafer and laminated on the transparent plate 70 to incubate the fertilized egg or egg having the valve (pump) channel part 25. Complete the system.

<1-3> Mimicking Biological Features

Embryos or egg culture systems that mimic the biological characteristics of the oviducts based on microfluidics according to the present invention are as follows. After flowing the DMEM medium into the fertilized egg and the medium inlet (1) in the culture system of the present invention, ciliated cells (Amy LW, Adv ) on the surface of the oviduct Physiol Educ 30: 237-241, 2006) were injected through the fertilized egg and medium inlet (1). At this time, if necessary, the inside of the channel was pre-coated with a chemical (collagen) to help the ciliated cells adhere well. After the fluid was waited for the ciliated cells to adhere to the channel part, the images were analyzed under a microscope and cultured for 2 hours or more until the density of the ciliated cells became high.

< Example  2> Fabrication of Embryo or Oocyte Culture System

<2-1> culture system 1

The fertilized egg or egg culture system based on the microfluidics according to the present invention has an egg moving microfluidic channel part 10 and an ovum compressing microfluidic channel part 20 to mimic repetitive compression in an oviduct. While repeated repeatedly at regular intervals, the length of the system could be applied to the basic channel plate 40 of the adjusted form. As shown in FIG. 2A, the system may be composed of a fertilized egg and a medium inlet 1, an egg moving microfluidic channel part 10, an egg compressed microfluidic channel part 20, a fertilized egg and a medium outlet 60, and the egg. The moving microfluidic channel part 10 and the ovum compressed microfluidic channel part 20 (FIG. 2B) were repeatedly connected at regular intervals (500 µm).

The culture system was fixed on the device for forming the slope for the flow of the microfluid, thereby adjusting the fluid and the fertilized egg to move inside the microchannel by causing a height difference on the left and right of the system (FIG. 5). At this time, in order to control the moving speed of the fertilized egg or egg close to the actual moving speed in the oviduct (oviduct) to give a gradient repetition every 30 minutes to give a flow of fluid to form a flow.

<2-2> culture system 2

The fertilized egg or egg culture system based on the microfluidics according to the present invention is an egg movement microfluidic channel part 10 and egg compression of the present system in order to mimic the repetitive compression in the oviduct and the length of the oviduct. When the microfluidic channel part 20 is repeatedly connected at regular intervals, the length of the system is extended by the length of the actual oviduct (13 cm) while bending the oocyte moving microfluidic channel part 10 in a U shape (FIG. 2C). gave.

In order to finely control the moving speed of the fertilized egg, the fluid is filled in the inlet 1 and flows in a constant direction, and a large port is attached to the outlet 60 to induce evaporation so that the fluid flows in a constant direction. In order to control the fluid velocity in the microchannel by attaching ports having different sizes to the inlet 1 and the outlet 60, a method of injecting the fluid at a constant speed using a syringe pump was used.

<2-3> culture system 3

In order to facilitate the fertilization of eggs and eggs, two culture systems 1 of Example <2-1> may be overlapped to face each other, and then an inlet may be applied to the side of the culture system. As shown in FIG. 3A, the upper microfluidic channel plate 40 is the upper microfluidic channel plate 40, and the reverse phase thereof is the lower microfluidic channel plate 50 (FIG. 3B). 40 and the lower microfluidic channel plate 50 were combined. In addition, as shown in Figure 3a, the inlet port (1) on the side of the culture system of the upper microfluidic channel plate 40 and the lower microfluidic channel plate 50 is combined to be used in the actual embryology and stem cell laboratory Pipette 31 was used to easily inject the fertilized egg 30.

In addition, in order to allow the fertilized egg or egg entering the egg moving microfluidic channel part 10 through the injection hole 1 by imitation of physical characteristics (interlocking motion) to perform a peristaltic motion in the actual oviduct. A valve (pump) channel section 25 was installed at the beginning of the microfluidic channel of the system (Fig. 4), to improve the performance of the culture system by imitation of biological characteristics (ciliary movement). The ciliated cells were attached to the culture system by the method of Example <1-3> to the inner walls of the egg moving microfluidic channel part 10 and the egg compression microfluidic channel part 20.

In order to finely control the moving speed of the fertilized egg, the valve (pump) channel part 25 uses a peristaltic pump to apply a pressure to the channel part of the lower microfluidic channel plate 50 to supply the fluid in a constant direction and speed. Injecting was used.

< Example  3> Cell culture using fertilized egg or egg culture system

<3-1> culture system 1

Fertilized eggs were cultured using the culture system 1 of FIG. 2A prepared in Example 2-1 of the present invention.

First, CR1aa medium was flowed into the fertilized egg and the medium inlet (1) to allow sufficient medium inside the system, and the fertilized egg and the medium to be cultured were placed in the inlet (1). The culture system 1 was placed in a 37 ° C. incubator and incubated while maintaining the appropriate temperature. After one week to ten days, the fertilized egg was discharged into the fertilized egg and the medium outlet (60). Thereafter, the cultured fertilized egg was observed through a microscope (Olympus, Japan).

As a result, the fertilized egg slowly moved while repeatedly passing through the ovum compacted microfluidic channel part 20 and the ovule moving microfluidic channel part 10 (FIG. 7). Observation was possible (FIG. 6A).

<3-2> culture system 2

Embryos were cultured using the culture system 2 of FIG. 2C prepared in Example 2-2 of the present invention.

First, CR1aa medium was flowed into the fertilized egg and the medium inlet (1) to allow sufficient medium inside the system, and the fertilized egg and the medium to be cultured were placed in the inlet (1). The rate of movement of the fertilized egg or egg was controlled to be close to 0.157 μm / s, which is the rate of movement in the actual oviduct. The culture system 2 was placed in a 37 ° C. incubator and incubated while maintaining the appropriate temperature. After one week to ten days, the fertilized egg was discharged into the fertilized egg and the medium outlet (60). Thereafter, the cultured fertilized egg was observed through a microscope (Olympus, Japan).

As a result, it was observed that the fertilized eggs cultured well up to blastocyst form (FIG. 6B).

<3-3> culture system 3

Embryos were cultured using the culture system 3 of FIG. 4 prepared in Example 2-3 of the present invention.

First, CR1aa medium was flowed into the fertilized egg and the medium inlet (1) to allow sufficient medium inside the system, and then the fertilized egg and the medium to be cultured were put in the inlet (1). The rate of movement of the fertilized egg or egg was controlled to be close to 0.157 μm / s, which is the rate of movement in the actual oviduct. Culture system 3 was placed in a 37 ° C. incubator and incubated while maintaining the proper temperature. After one week to ten days, the fertilized egg was discharged to the fertilized egg and the medium outlet (60). Thereafter, the cultured fertilized egg was observed through a microscope (Olympus, Japan).

As a result, it was observed that the fertilized eggs cultured well up to the blastocyst stage.

1 is a schematic diagram of the basic unit of the fertilized egg or egg culture system of the present invention:

1A: basic unit of culture system mimics physical feature compression; And,

1b: Basic unit of a culture system that mimics physical feature peristalsis.

2 is a schematic diagram of a culture system that mimics the characteristics of the compression and length of the fallopian tube of the present invention:

2A: System repeating physical feature compression imitation;

2b: an enlarged view of a base unit that mimics physical feature compression; And,

Fig. 2c: Base channel plate repeating physical feature compression mimics similar to the length of the oviduct.

3 is a schematic diagram of a fertilized egg or egg culture system adapted to facilitate infusion of fertilized eggs and eggs:

3a: a system combining the base channel plate of FIG. 2c and the lower channel plate of FIG. 3b; And,

Figure 3b: the lower channel plate of the reverse phase of Figure 2c.

4 is a schematic diagram of a fertilized egg or egg culture system that mimics the peristaltic features of the present invention.

5 is an actual picture of the culture system according to the present invention.

Figure 6 is a diagram showing that the fertilized egg cultured in the culture system of the present invention is healthyly differentiated up to blastocyst form:

6A: Porcine fertilized eggs cultured in culture system 1; And,

6B: Porcine fertilized eggs cultured in culture system 2.

Figure 7 is a view showing a pig fertilized egg passing through the ovum compressed microfluidic channel portion 20 of the culture system according to the present invention.

<Explanation of symbols for the main parts of the drawings>

1: fertilized egg and medium inlet

10: Oocyte movement microfluidic channel part

20: Oocyte compression microfluidic channel part

25: valve (pump) channel section for peristaltic contractions

30: fertilized egg

31: micro-pipette for injecting fertilized eggs

40: upper channel plate

41: structure layer with a microchannel pattern

50: lower channel plate

60: fertilized egg and medium outlet

70: transparent plate layer

Claims (17)

Fertilized egg and medium inlet (1); An egg moving microfluidic channel part 10 connected to the fertilized egg and the medium inlet 1 and movable in the egg; An oocyte-compressed microfluidic channel part 20 which reduces the diameter of the oocyte-moving microfluidic channel part 10 formed in the egg moving microfluidic channel part 10 by at least one number; And, A fertilized egg or egg culture system in which the fertilized egg and the medium outlet (60) are connected to the egg moving microfluidic channel (10). The culture system according to claim 1, wherein the ovum microfluidic channel part (10) is 10% to 100% wide and 5% to 50% high in blastocyst diameter in the form of a fertilized egg. [Claim 2] The culture system according to claim 1, wherein two to 300 ovum compressed microfluidic channel parts are included. The culture system according to claim 1, wherein the oocyte compressed microfluidic channel portion (20) is 60% to 90% of the width and height of the blastocyst diameter. The culture system according to claim 1, wherein the oocyte compressed microfluidic channel portion (20) is adjusted in width and height to adjust the intensity of the compression stimulus. Fertilized egg and medium inlet (1); An egg moving microfluidic channel part 10 connected to the fertilized egg and the medium inlet 1 and movable in an egg and provided with a valve (pump) channel part 25 in an upstream region; An oocyte-compressed microfluidic channel part 20 which reduces the diameter of the oocyte-moving microfluidic channel part 10 formed in at least one number in the oocyte-moving microfluidic channel part 10; And, A fertilized egg or egg culture system in which the fertilized egg and the medium outlet (60) are connected to the egg moving microfluidic channel part (10). 7. The valve (pump) channel portion 25 is located above the lower microfluidic channel plate 50, through which the fertilized egg or egg can move, and the valve (pump) channel portion 25 and the lower microfluidic channel. Cultivation system, characterized in that the membrane between the plate 50 is able to press the fertilized egg or egg to move the channel portion of the lower microfluidic channel plate (50). 8. The culture system of claim 7, wherein the membrane is polydimethylsiloxane. 7. A culture system according to claim 6, further comprising a peristaltic pump connected to the valve (pump) channel section (25). 7. The culture system according to claim 1 or 6, wherein the ciliated cells of the fallopian tube surface are further attached to the inner wall of the culture system. The method of claim 10, wherein the collagen, fibronectin, laminin, poly-L-lysine and natural extracellular matrix can aid in the attachment of the ciliocytes prior to the attachment of the ciliocytes. A culture system, characterized in that any one selected from the group consisting of is treated on the inner wall of the culture system. The method according to claim 1 to 11, wherein the oocyte moving microfluidic channel portion 10, the oocyte compressed microfluidic channel portion 20 and the valve (pump) channel portion 25 of the culture system is polydimethylsiloxane (PDMS) , Polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polystylene, polycyclic olefins, polyimides and polyurethanes Culture system, characterized in that any one selected from the group consisting of. The culture system according to any one of claims 1 to 11, wherein the culture system is bonded to any one selected from the group consisting of slide glass, crystal, glass glass and PDMS. 12. The culture system according to any one of the preceding claims, wherein the culture system is fixed on an apparatus to form a slope capable of generating microfluidic flow. 1) injecting the medium into the inlet (1) of the fertilized egg or egg culture system of claim 1; 2) injecting a fertilized egg or an egg into the inlet (1) and controlling the speed of the microfluidic flow; 3) culturing the fertilized egg or egg while putting the system of step 2) in a 37 ° C. incubator to maintain the proper temperature; And, 4) A fertilized egg or egg culture method using the system of claim 1, comprising the step of discharging the cultured egg or egg of step 3) from the fertilized egg and the medium outlet (51).  1) injecting the medium into the inlet (1) of the fertilized egg or egg culture system of claim 6; 2) injecting a fertilized egg or an egg into the inlet (1) and controlling the speed of the microfluidic flow; 3) injecting microfluid into the valve (pump) channel part 25 when the fertilized egg or egg of step 2) passes the valve (pump) channel part 25 for peristaltic contraction; 4) when the fertilized egg or egg of step 3) has passed the valve (pump) channel part 25, culturing the fertilized egg or egg while putting the system of step 3) in a 37 ° C. incubator to maintain a suitable temperature; And, 5) A fertilized egg or egg culture method using the system of claim 6, comprising the step of discharging the cultured fertilized egg or egg of step 4) from the fertilized egg and the medium outlet (51). The method of claim 15, wherein the fertilized egg or egg culture system is the culture system of claim 10.

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