JP2012075391A - Apparatus and method for culturing fertilized egg - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fertilized egg culture device for arraying fertilized eggs and successfully retaining the arrayed fertilized eggs, and to provide and a fertilized egg culture apparatus and a fertilized egg culture method using the device.SOLUTION: The fertilized egg culture device includes a micro channel structure where a mammalian fertilized egg and a culture solution are distributed. The micro channel structure includes: a main channel; and a meandering channel sequentially crossing the main channel. The main channel includes a narrowed part having a width small enough not to allow the fertilized egg to pass therethrough. An area between the narrowed part and the meandering channel crossing the main channel upstream thereof serves as a cage area for capturing the fertilized egg. The sidewall of the meandering channel on a downstream side is cut open so that the cage area is expanded toward the downstream of the meandering channel. Moreover, the cage area is provided at the bottom surface thereof with a recessed part.

Description

  The present invention relates to a culture apparatus used for culturing fertilized eggs and a method for culturing fertilized eggs using the culture apparatus.

Mammal fertilized egg culturing is a fundamental technology that is indispensable in various fields of life science such as medical and livestock fields, and recently, establishment of embryonic stem cells (ES cells). It has been following. In the process of transplanting fertilized eggs into the womb for infertility treatment or livestock breeding, in order to ensure reliable implantation and subsequent growth, cultivate once to a stage called blastocyst outside the body, and only fertilized eggs in good condition Are selected and transplanted.
Conventionally, when such fertilized eggs are transplanted, operations such as washing, culturing and selection have been performed.
In vitro culture of fertilized eggs is usually performed by the microdroplet method. This method is a method in which a culture liquid droplet is formed on a petri dish, and a plurality of fertilized eggs are placed in the droplet to perform culture. However, the microdroplet method requires advanced techniques, and there is a problem that the quality and incidence of fertilized eggs are likely to vary depending on the operator's technique. For example, when placing a fertilized egg in a droplet, connect a micropipette or mouthpiece to the glass capillary, collect the fertilized egg in the capillary by the balance between intake and exhaust, Operations such as moving to another environment are generally performed. However, fertilized eggs are vulnerable to disturbances, and the incidence of blastocysts tends to decrease due to poor operation. Therefore, the method as described above has a problem of low quantitativeness and reproducibility, such as variations in the quality of fertilized eggs and the incidence of blastocysts depending on the skill level of the person performing the procedure. Furthermore, in the conventional technique, it is difficult to individually manage and cultivate fertilized eggs, so it is difficult to evaluate the development process of each fertilized egg.
Therefore, automation is expected in order to culture fertilized eggs with good quantification and reproducibility regardless of skill level. In order to automate the culture of fertilized eggs, both fertilized eggs can be cultured with quality control individually, and only the fertilized eggs that have developed into blastocysts can be selectively recovered. A system is required, but such a system has not yet been realized.

On the other hand, in recent years, in the field of μTAS (Micro Total Analysis System) to which microfabrication technology is applied, research dealing with minute bioparticles such as cells, bacteria, and biomolecules has been conducted. In μTAS, a micro system involving a micro fluid operation is realized in a micro flow path formed by a microfabrication technique, and various bio particle operation methods have been proposed so far. For example, the inventors of the present invention can arrange a large amount of fine particles at a specific site only by a simple microfluidic operation, exchange a solution in the arranged state, and give a drug stimulus. We are researching microfluidic devices with microchannels (dynamic microarrays) that allow microparticle manipulation, such as moving to a site. For example, Patent Document 1 discloses, as an apparatus for arranging microbeads, a linear flow channel having a narrowed region while microbeads are flown, a square wave channel that sequentially intersects the linear flow channel, and microbeads. If there is an empty stenosis area in which the microbead is not captured, the microbead is captured in the empty stenosis area. If there is a stenosis area in which the microbead is captured, the stenosis area is bypassed and the microbead is moved downstream. Disclosed is an array device comprising microbead moving means for movement.
In this dynamic microarray, when the liquid is flowed in the absence of particles, the flow in the linear flow path becomes the main stream, and the flow in the square wave flow path becomes the substream. Yes. Therefore, when particles are caused to flow along with the liquid to the dynamic microarray, the particles flow linearly at a position where the linear flow channel, which is the main flow channel, and the rectangular wave flow channel intersect (hereinafter also referred to as a crossing route). It flows into the path and is dammed by a narrowed portion formed with a width that prevents particles from passing through. When the particles are dammed up, the next flowing particles flow into the square wave channel, which is a detour, and move downstream, and flow into the straight channel at the next intersection, downstream It is dammed to the stenosis. In this way, particles are sequentially captured between the crossing path and the stenosis portion downstream thereof (cage region) and arrayed (arranged). Therefore, by designing the dynamic microarray so that one particle is captured in one cage region, the particles captured in each cage region can be individually managed.

JP 2007-186456 A

The inventors of the present invention applied a microfluidic device provided with a dynamic microarray having a main flow path and a meandering flow path that sequentially intersects the main flow path, as described in Patent Document 1, to fertilized egg culture. As a result, it has been found that the microfluidic device has several problems in the culture of fertilized eggs.
One of the problems is that a plurality of fertilized eggs are trapped in one cage region and cannot be arrayed well. In other words, in order to culture fertilized eggs to the state of blastocysts, it is necessary to set the size of the cage region according to the size of the blastocysts. However, it is about 1.3 to 2 times the initial fertilized egg. Therefore, since the cage area is too wide compared to the initial fertilized egg, another fertilized egg enters the cage area where the fertilized egg has already been captured. If a plurality of fertilized eggs are captured in one cage region, individual management cannot be performed. Further, when fertilized eggs flow in series, a plurality of fertilized eggs may be caught at the intersection of the main flow path and the meandering flow path, and the fertilized egg may block the flow path itself.
Another problem is that the position of the captured fertilized egg may not be maintained during culture. For example, the position of the fertilized egg in the cage region may change during culture, or in the worst case, the fertilized egg may be released from the cage region before it develops to the blastocyst. If the position in the cage region changes, it becomes difficult to individually observe the fertilized egg.
When cultivating a fertilized egg, the fertilized egg is vulnerable to disturbance, so it is desirable to reduce the flow rate of the liquid in the flow channel as compared with the case where microbeads are arranged as described in Patent Document 1. If the flow rate is lowered, problems such as the above release tend to occur.
Since these problems hinder the automation of fertilized egg culture, improvements are required.
The present invention has been made in view of the above circumstances, and provides a fertilized egg culturing device, a fertilized egg culturing apparatus, and the device that can satisfactorily hold an array of fertilized eggs or an arrayed fertilized egg. It aims at providing the culture method of the used fertilized egg.

The present invention for solving the above problems has the following aspects.
[1] A microchannel structure through which a fertilized egg and a culture solution of a mammal circulate is provided.
The microchannel structure has a main channel and a meandering channel that sequentially intersects the main channel,
The main flow path is provided with a narrowed portion having a width through which the fertilized egg does not pass, and a cage region that captures the fertilized egg between the narrowed portion and a meandering flow channel that intersects the main flow channel upstream of the narrowed portion. And
The cage region is a fertilized egg culturing device in which a side wall on the downstream side of the meandering channel is notched and expanded in the downstream direction of the meandering channel.
[2] A microchannel structure through which a fertilized egg and a culture solution of a mammal circulate,
The microchannel structure has a main channel and a meandering channel that sequentially intersects the main channel,
The main flow path is provided with a narrowed portion having a width through which the fertilized egg does not pass, and a cage region that captures the fertilized egg between the narrowed portion and a meandering flow channel that intersects the main flow channel upstream of the narrowed portion. And
A fertilized egg culturing device in which a recess is provided on the bottom surface of the cage region.
[3] The fertilized egg culture device according to [1], wherein a recess is provided on a bottom surface of the cage region.
[4] The fertilized egg culturing device according to [1], wherein the side wall of the cage region on the upstream side of the meandering flow path and the side wall of the meandering flow path merge substantially vertically.
[5] The side wall on the upstream side of the meandering flow path and the side wall of the meandering flow path of the cage region are joined substantially vertically, [2] or [3] Fertilized egg culture device.
[6] A fertilized egg culturing apparatus comprising the fertilized egg culturing device according to any one of [1] to [5].
[7] A method of culturing a fertilized egg of a mammal using the fertilized egg culturing device according to any one of [1] to [5],
Disposing the fertilized egg in the cage region by circulating the fertilized egg together with a culture solution in the microchannel structure; and culturing the fertilized egg while circulating the culture solution in the microchannel structure; A culture method comprising:
[8] A method of culturing a mammalian fertilized egg using the fertilized egg device according to [2], [3] or [5],
Arranging the fertilized egg in the cage region by circulating the fertilized egg together with a culture solution in the microchannel structure, and culturing the fertilized egg without circulating the culture solution in the microchannel structure And a culture method.

  According to the present invention, it is possible to provide a fertilized egg culturing device, a fertilized egg culturing apparatus, and a method for culturing a fertilized egg using the device, which can satisfactorily hold an array of fertilized eggs or an arrayed fertilized egg. .

It is a top view of the microchannel structure 10 with which the fertilized egg culture device of 1st embodiment is provided. 1 is a partially enlarged view of a microchannel structure 10. It is a top view of the microchannel structure 20 with which the fertilized egg culture device of 2nd embodiment is provided. (A) is the elements on larger scale of the microchannel structure 20, (b) is a longitudinal cross-sectional view in position X-X 'in (a). It is a top view of the microchannel structure 30 with which the fertilized egg culture device of 3rd embodiment is provided. 2 is a photograph of the microfluidic device obtained in Example 1. 4 is a photograph of the microchannel structure of the microfluidic device obtained in Example 2. 4 is a photograph of the microchannel structure of the microfluidic device obtained in Example 3. 2 is a photograph of a microchannel structure of a microfluidic device obtained in Comparative Example 1. It is a photograph which shows the result of having performed the arrangement | positioning operation of a fertilized egg using the microfluidic device obtained by the comparative example 1 in the test example 1. FIG. It is a photograph which shows the result of having performed the arrangement operation of a fertilized egg using the microfluidic device obtained in Example 1 in Test Example 1. It is a photograph which shows the result of having performed the placement operation of a fertilized egg using the microfluidic device obtained in Example 3 in Test Example 1. It is a photograph which shows the result of having performed the arrangement operation of a fertilized egg using the microfluidic device obtained in Example 2 in Test Example 1.

A fertilized egg culturing device of this embodiment will be described with reference to the accompanying drawings.
<First embodiment>
1 and 2 show a microchannel structure 10 provided in the fertilized egg culture device of the first embodiment of the present invention. FIG. 1 is a top view of a microchannel structure 10 included in a fertilized egg culturing device of this embodiment, and FIG. 2 is a partially enlarged view thereof. The arrow in FIGS. 1-2 shows the distribution direction of a culture solution.
The microchannel structure 10 includes a linear main channel 11 and a square-wave meandering channel 12 that sequentially intersects the main channel 11.
The main channel 11 is provided with a plurality of narrowed portions 11a having a width that does not allow a fertilized egg to be cultured to pass between each narrowed portion 11a and a meandering channel 12 that intersects the main channel 11 upstream thereof (in FIG. 2). Is a cage region 11b that captures a fertilized egg.
The cage region 11 b extends in the downstream direction of the meandering flow path 12 by providing a notch 11 c by notching the downstream side wall of the meandering flow path 12. Hereinafter, the area | region where the width | variety is expanded by the notch 11c among the cage areas 11b is called the expansion part 11d.
A region in the cage region 11b on the downstream side of the expanded portion 11d has a semicircular cross section (semicircular portion 11e).
Here, the “main flow channel” means a flow channel having the highest flow rate when the culture solution is introduced into the micro flow channel structure in a state where the fertilized egg is not captured at all. That is, in embryo culture device 10, the volume flow rate to _{Q 1} the main channel 11, the ratio of the volume flow rate _{Q 2} of the serpentine channel 12 _(Q 1 / Q ₂₎ has a greater than 1. When the main flow path 11 and the meandering flow path 12 are designed to satisfy Q ₁ / Q ₂ > 1, a cage region that is vacant when the fertilized egg is circulated through the fertilized egg culture device 10 together with the culture solution. A fertilized egg is introduced into 11b.
Q ₁ / Q ₂ is preferably more than 1 and 5 or less, and more preferably 2 or more and 4 or less. When the value of Q ₁ / Q ₂ is more than 1, particularly 2 or more, it is easy to place a fertilized egg in the cage region 11b, and when the value of Q ₁ / Q ₂ is 5 or less, one cage A plurality of fertilized eggs are not easily captured in the region 11b, and the array can be favorably performed.

Width W ₁ and height of the main flow path 11, width W ₂ and height of the meandering flow path 12, width W ₃ and length W _{5 of} the constricted portion 11 a, cage region 11 b in the culture medium flow direction, expansion portion 11 d, The lengths W ₄ , W _a , and W _b of the semicircular portions 11e are set according to the value of Q ₁ / Q ₂ and the fertilized egg to be cultured, respectively.
The value of Q ₁ / _{Q 2} has a width _{W 3,} the length _{W 5} and the height of the constriction 11a, the width _{W 2,} the length of the serpentine channel 12, defined by the balance of the height and the like.
Here, the fertilized egg cultured by the fertilized egg culturing device of the present invention is a fertilized egg of a mammal, and examples of the mammal include human, mouse, cow, and pig. After fertilization, the fertilized egg of a mammal increases in the number of cells from the 2 cell stage, the 4 cell stage, and the 8 cell stage by cleavage, and develops into a blastocyst through a morula. A blastocyst is composed of a trophectoderm and an inner cell mass inside it. In in vitro fertilization, transplantation into the uterus is usually performed from the 4-8 cell stage to the blastocyst stage. Is called.
The size of a fertilized egg immediately after fertilization is usually about 130 μm for humans, about 80 μm for mice, and about 120 to 130 μm for cows and pigs. The size of the fertilized egg at the blastocyst stage is about 1.3 to 2 times the size of the fertilized egg immediately after fertilization. This magnification varies depending on the animal species, for example, about 1.3 times for mice and about 2 times for cows.
Therefore, the width W ₁ and a height of the main channel 11, the width W ₂ and a height of the serpentine channel 12, respectively set, the introduction of the embryo, in order to perform the collection, etc., based on the size of the blastocyst It is preferable to do. Specifically, the width and height of the blastocyst can be moved, but the size of the blastocyst is preferably +5 μm to +50 μm.
Width W ₃ of the constriction 11a in order to trap the introduced fertilized egg should be smaller than the size of the embryo at the minimum size. Here, the “size of the fertilized egg at the minimum size” is the same size as the fertilized egg immediately after fertilization, and from the time of fertilization to the initial blastocyst is the “size of the fertilized egg at the minimum size”. is there. On the other hand, if the width W ₃ is too narrow, the resistance to flow of the constriction direction is increased from the intersection, the flow rate of the serpentine channel 12 becomes larger than the flow rate of the main flow path, may not be caught successfully fertilized is there. However even by decreasing the width W _3, by increasing the length) of the serpentine channel 12, increasing the resistance to flow in the downstream direction of the serpentine channel 12 from the intersection, the value of Q 1 _/ Q ₂ Therefore, the lower limit of the width W ₃ is not particularly limited. Here, the length of the meandering channel 12 is the length of the center of the channel between one of the positions where the meandering channel 12 intersects the main channel 11 and the next position where it intersects the main channel 11. .
But not limited length W _5, especially stenosis 11a, is preferably short in terms of Shoryuro space. However the length W ₅ is correlated with the length of the serpentine channel 12, also increases the length of the serpentine channel 12 length W ₅ becomes longer. Therefore, it is preferable to consider the value of Q ₁ / Q ₂ when setting the width W ₃ and the length W ₅ of the narrowed portion 11a.
The length W ₄ of the cage area 11b in the flow direction of the culture, in order to generate the fertilized egg to the blastocyst stage, it is preferable to set the size of the blastocyst as a reference. Specifically, a fertilized egg may be any until the blastocyst length of the whole fertilized egg is accommodated in the cage area 11b, but for example, the length W _4, as long as the magnitude of the blastocyst or when slightly smaller design than is, it is possible to blastocysts reach the same size as the length W ₄ is automatically pop out from the cage area 11b by the flow, to be released. From these viewpoints, the size of the blastocyst is preferably −10 μm to +50 μm. The length _{W a} of the extension portion 11d in the flow direction of the culture, it is preferable that the radius -5μm~ + 25μm blastocyst. The length _{W b} of semicircular portion 11e in the flow direction of the culture, it is preferable that the radius -5μm~ + 25μm blastocyst.

The cage region 11c has a semicircular portion 11e having a semicircular cross section on the downstream side, and the notch 11c has a shape cut from one end of the arc in the downstream direction of the meandering channel 12. .
And the side wall of the notch 11c, the angle theta _b formed by the side wall of the serpentine channel 12 is preferably 110 to 160 °, more preferably 120 to 150 °.
The side wall of the cage region 11c opposite to the side where the notch 11c is provided, that is, the upstream side wall of the meandering channel 12 and the side wall of the meandering channel 12 are joined substantially vertically. Here, “substantially perpendicular” means within a range of 80 to 100 °. A cutout side wall opposite to the 11c of the cage region 11c, the angle theta _a formed by the side wall of the serpentine channel 12, preferably 80 to 150 °, more preferably 90 to 120 °.

When fertilized eggs are introduced into the microchannel structure 10 together with the culture solution, the fertilized eggs are sequentially captured in the cage region 11b and arrayed. That is, when the cage region 11b is vacant (the fertilized egg is not captured), the resistance to the flow from the intersection of the main flow path 11 and the meandering flow path 12 toward the narrowed portion 11a is downstream of the meandering flow path 12. Since it is lower than the resistance to the flow in the direction, the flow rate of the main flow channel 11 is larger than the flow rate of the meandering flow channel 12. Therefore, the fertilized egg introduced together with the culture solution moves in the direction of the narrowed portion 11a from the intersection. Since the stenosis part 11a is formed with a width that allows the culture solution to pass but not the fertilized egg, the fertilized egg is trapped by the stenosis part 11a, and the fertilized egg is trapped in the cage region 11b. When the fertilized egg is captured in the cage region 11b, the resistance to the flow from the upstream crossing path toward the narrowed portion 11a is significantly increased, so that the flow rate of the meandering flow channel 12 is higher than the flow rate of the main flow channel 11. Will also increase. Therefore, the fertilized egg introduced next moves along the meandering flow path 12, bypasses the cage region 11b where the fertilized egg is captured, and is captured by the next empty cage region 11b.
In Patent Document 1, both the side walls of the cage region 11b are shaped to merge perpendicularly to the side walls of the meandering channel 12 (see FIG. 5 of Patent Document 1). However, in the case of the cage region having such a shape, as described above, a plurality of fertilized eggs are easily captured in one cage region. In the present embodiment, by providing the notches 11c, it is possible to form a good array in which one fertilized egg is captured in one cage region 11b. This is because even if another fertilized egg enters the cage region 11b in which the fertilized egg has been captured, it is likely to be pushed away along the notch 11c when receiving the flow of the meandering channel 12, so that the inside of the cage region 11b This is considered to flow to the meandering flow path 12 without stagnation.
As described above, according to the fertilized egg culturing device of this aspect, the cage regions 11b provided on the main flow path 11 are formed from the upstream side only by sequentially introducing the fertilized eggs together with the culture solution into the micro flow path structure 10. The fertilized eggs are sequentially blocked, and as a result, the fertilized eggs can be well arrayed.
According to the fertilized egg culturing device of this aspect, since a good array in which one fertilized egg is captured in one cage region can be formed, a plurality of fertilized eggs can be cultured individually while quality control is performed. This is useful for automating culture operations.

The microchannel structure 10 is usually provided inside a plate-like support.
The material constituting the support is not particularly limited, and can be appropriately selected from known materials conventionally used for forming a microchannel structure according to the purpose. For example, when the inside of the microchannel structure 10 is visually observed from the outside, a transparent material that transmits visible light is preferable. Examples of the transparent material include silicone rubber such as polydimethylsiloxane (hereinafter referred to as PDMS), various resin materials such as acrylic and polystyrene, glass, and gel. Among these, polystyrene resin is preferable because it is inexpensive and easily mass-produced.
Other than transparent materials, metals such as silicon and platinum can be used.
In the present invention, since the fertilized egg is cultured, the material constituting the support is preferably a material that is compatible with the fertilized egg, and in particular, oxygen in the atmosphere outside the device is micro-flowed. A material having oxygen permeability (oxygen permeable material) is preferable because it can be supplied into the path structure 10. As the oxygen permeable material, any known oxygen permeable material can be used, and examples thereof include biocompatible oxygen permeable materials used for oxygen permeable contact lenses. In particular, since the cultured cells in the microchannel structure 10 can be observed from the outside of the device, the material having oxygen permeability is preferably a transparent material.
Specific examples of the biocompatible oxygen permeable material include silicone rubber and gel. In particular, PDMS is preferable because it is biocompatible, has transparency and oxygen permeability, and is an inexpensive material.

A device in which the microchannel structure 10 is formed inside the support can be manufactured, for example, by the following procedure.
First, a structure in which the microchannel structure 10 is formed on the surface of the support is manufactured. The structure can be manufactured by using a known microfabrication method such as a method conventionally used for manufacturing a microdevice having a minute channel structure. Examples of the fine processing method include a lithography method, an etching method, cutting, and injection molding.
If an example of the manufacturing method using the lithography method is given, a structure in which the microchannel structure 10 is formed on the surface of the support can be produced by performing the following steps (1) to (5).
(1) First, a photoresist is applied on a substrate by spin coating to form a photoresist layer.
(2) The photoresist layer is patterned by exposing and developing the photoresist layer through a mask having a pattern corresponding to the microchannel structure 10.
(3) A prepolymer layer of UV curable or thermosetting polymer is applied on the patterned photoresist layer (template) to form a prepolymer layer.
(4) The prepolymer layer is cured by irradiating or heating UV to form a polymer layer.
(5) The polymer layer is peeled off.
The surface of the polymer layer (support) thus obtained is a pattern obtained by inverting the pattern of the photoresist layer (for example, a space pattern when the pattern of the photoresist layer is a line pattern). A microchannel structure 10 having the same height (depth) as the thickness is formed.
A through hole (a port for introducing a culture solution and / or a fertilized egg upstream of the microchannel structure 10) into the structure having the microchannel structure 10 formed on the surface of the support thus obtained. A through-hole that becomes a port for leading a culture solution and / or a fertilized egg from the downstream of the micro-channel structure 10) is performed.
The device in which the microchannel structure 10 is formed inside the support includes, for example, a plate-like support (such as a flat plate or glass plate of PDMS) on the surface of the structure on which the microchannel structure 10 is formed. ). Further, in the same procedure as the structure in which the microchannel structure 10 is formed on the surface of the support, a microchannel structure having a pattern in which the pattern of the microchannel structure 10 is reversed is formed on the surface of the support. Can also be produced by aligning and bonding the surfaces of the two structures on which the microchannel structure is formed.

<Second embodiment>
The microchannel structure 20 with which the fertilized egg culture device of 2nd embodiment of this invention is provided in FIGS. 3-4 is shown. FIG. 3 is a top view of the microchannel structure 20 provided in the fertilized egg culture device of this embodiment, FIG. 4 (a) is a partially enlarged view thereof, and FIG. 4 (b) is a diagram in FIG. 4 (a). It is a longitudinal cross-sectional view in position XX '. The arrows in FIGS. 3 to 4 indicate the flow direction of the culture solution.
The microchannel structure 20 includes a linear main channel 21 and a rectangular wave meandering channel 22 that sequentially intersects the main channel 21.
The main flow path 21 is provided with a plurality of narrow portions 21a having a width that does not allow a fertilized egg to be cultured to pass between each narrow portion 21a and a meandering flow path 22 that intersects the main flow path 21 upstream thereof (FIG. 4 ( A hatched portion in a) is a cage region 21b for capturing a fertilized egg.
A cylindrical cup structure (concave portion) 22c is provided on the bottom surface of the cage region 21b.

The width and height of the main channel 21, the width and height of the meandering channel 22, the width and length of the constricted portion 21 a, and the length of the cage region 21 b in the culture medium flow direction are the same as in the first embodiment. The width W ₁ and height of the main flow path 11, the width W ₂ and height of the meandering flow path 12, the width W ₃ and length W _{5 of} the constricted portion 11a, and the length W ₄ of the cage region 11b in the culture medium flow direction It is set in the same way.
The cage region 21c has a semicircular cross section on the downstream side, and the cup structure 21c is provided at the downstream end of the cage region 21b, that is, at a position in contact with the narrowed portion 21a.
The diameter _{D 1} of the cup-shaped structure 21c is 1.3 to 2 times the size of preferred at the minimum size of the embryo to be cultured. The depth _{D 2} of the cup-shaped structure 21c is 1 / 3-1 / 2 times the size of preferred at the minimum size of the embryo to be cultured. When the diameter D ₁ is too small, there is a possibility that interfere with the generation of the embryo. Further, when the depth D ₂ is too small, the effect of providing a cup-shaped structure 21c might not be sufficiently obtained. If the diameter D ₁ or the depth D ₂ is too large, a plurality of fertilized eggs will enter the cage region at the time of placement, and the blastocyst will be trapped in the cup structure 21c after culture, making recovery difficult. . However, the diameter D ₁ must be W ₁ less in size.

When a fertilized egg is introduced into the microchannel structure 20 together with a culture solution, the fertilized egg is sequentially captured and arrayed in the cage region 21b as in the first embodiment.
In Patent Document 1, the bottom surface of the cage region is smooth. However, in the case of such a cage portion, as described above, the position of the captured fertilized egg cannot be maintained during the culture, and the position of the fertilized egg within the cage region changes during the culture, It may be released from the cage region before it develops to the blastocyst.
An increase in the incubator internal pressure can be cited as a cause of the change in the position of the fertilized egg. In other words, when culturing fertilized eggs, the microfluidic device is housed in an incubator and carbon dioxide gas or nitrogen gas is supplied in order to make the carbon dioxide concentration and oxygen concentration in the environment suitable for culturing fertilized eggs. Yes. As a result, the incubator internal pressure increases. This increase in internal pressure causes a back flow of the culture solution in the main channel, and this flow is thought to move the fertilized egg in the upstream direction.
The change in position due to the backflow is not a problem when the flow rate of the culture solution is large, but becomes a serious problem as the flow rate is lowered. When fertilized eggs are cultivated, the fertilized eggs are vulnerable to disturbances. Therefore, in order to improve the incidence, the liquid in the flow path is less than when microbeads are arranged as described in Patent Document 1. It is desirable to reduce the flow rate. In the present embodiment, the cup structure 21c is provided on the bottom surface of the cage region 21b, so that a low flow rate (for example, 0.1 μL / min or less in the case of a channel size for mouse fertilized eggs, Even in the case of a flow path size (approximately 0.4 μL / min or less in the case of a flow path size for mouse fertilized eggs), fertilized eggs captured in the cage region 21b can be stably placed at the position of the cup structure 21c. Can hold. Therefore, the fertilized egg culturing device of the present embodiment is useful for automating the culturing operation of fertilized eggs since it can perform quality control by holding the fertilized eggs in a predetermined position until they develop in blastocysts. is there.

Furthermore, the following effects are also acquired by providing the cup structure 21c.
(1) Autocline / paracline effect.
(2) Maintaining dissolved oxygen concentration.
(3) Easy co-culture.
Regarding (1) among the above, the fertilized egg has been developed under the influence of various growth factors before reaching the post-fertilization implantation. By providing the cup structure 21c, for example, the growth factor released from the fertilized egg remains in the cup structure 21c as it is, and the concentration of the growth factor in the cage region 21b increases. Therefore, it can culture | cultivate in the environment nearer to a living body, and generation | occurrence | production efficiency improves.
For (2), for example, a substance whose fluorescence intensity varies depending on the amount of oxygen consumption is attached to the bottom surface of the cup structure 21c and cultured. At that time, the dissolved oxygen concentration in the cage region 21b can be measured with high sensitivity by measuring the fluorescence intensity of the cup structure 21c. By adjusting the dissolved oxygen concentration of the culture solution supplied to the microchannel structure 20 according to the measurement result, the dissolved oxygen concentration in the cage region 21b can be maintained at a desired concentration. At the same time, the respiratory activity of the fertilized egg can be measured, and the result can be used as an index of the quality of the fertilized egg.
With regard to (3), by providing the cup structure 21c, a feeder cell for co-culture can be attached to the bottom surface thereof. When supporting cells such as endometrial cells are attached to the bottom surface of the cup structure 21c and the fertilized egg is cultured (co-culture), it can be cultured in an environment closer to the living body and the generation efficiency is improved.
Here, “co-culture” means a method of simultaneously culturing cultured cells (fertilized eggs in the case of the present invention) and somatic cells of the same or different species.
The feeder cells used for co-culture with a mammalian fertilized egg are preferably mammalian somatic cells or tissues of the same species. Specifically, fibroblasts, reproductive organ-derived cells (endometrial cells, Oviduct epithelial cells, etc.). Moreover, the tissue which consists of these cells may be sufficient. Of these, oviduct epithelial cells are preferred.
The attachment of the feeder cells to the bottom surface of the cup structure 21c is performed, for example, by introducing a culture solution containing the feeder cells into the microchannel structure 20 in advance and then culturing it in a closed state before introducing the fertilized egg. it can. Supporting cells seeded on the bottom surface of the cup structure can be cultured well without being adversely affected by flow, such as shear stress.

The microchannel structure 20 is usually provided inside a plate-like support, similarly to the microchannel structure 10 in the first embodiment.
A device in which the microchannel structure 20 is provided inside the support can be manufactured in the same procedure as the device of the first embodiment.

<Third embodiment>
FIG. 5 shows a microchannel structure 30 provided in the fertilized egg culture device of the third embodiment of the present invention. FIG. 5 is a top view of the microchannel structure 30 provided in the fertilized egg culture device of this embodiment. The arrows in FIG. 5 indicate the flow direction of the culture solution.
The microchannel structure 30 includes a linear main channel 31 and a square-wave meandering channel 32 that sequentially intersects the main channel 31.
The main flow path 31 is provided with a plurality of narrowed portions 31a each having a width that does not allow a fertilized egg to be cultured to pass. Is a cage region 31b.
The cage region 31 b extends in the downstream direction of the meandering flow path 32 by providing a notch 31 c by notching the downstream side wall of the meandering flow path 32. Hereinafter, in the cage region 31b, a region whose width is expanded by the notch 31c is referred to as an expanded portion.
A region in the cage region 31b on the downstream side of the expanded portion has a semicircular cross section.
In addition, a cylindrical cup structure (concave portion) 31d is provided on the bottom surface of the cage region 31b.

The microchannel structure 30 in the present embodiment is the same as the microchannel structure 10 of the first embodiment except that a cup structure 31c is further provided.
The cup structure 31d is the same as the cup structure 21c described in the second embodiment.
Therefore, according to the fertilized egg culturing device of this aspect, the plurality of cage regions 31b provided on the main flow path 31 are fertilized sequentially from the upstream side only by sequentially introducing the fertilized eggs together with the culture solution into the micro flow path structure 30. As a result, the fertilized eggs can be well arrayed. In addition, the fertilized eggs arranged in an array can be stably held at the position of the cup structure 31d in the cage region 31b even under a low flow rate. Therefore, the fertilized egg culturing device of this aspect is useful for automating the culturing operation of the fertilized egg.
Furthermore, the following effects described above can also be obtained.
(1) Autocline / paracline effect.
(2) Maintaining dissolved oxygen concentration.
(3) Easy co-culture.

The microchannel structure 30 is usually provided inside a plate-like support, similarly to the microchannel structure 10 in the first embodiment.
A device in which the microchannel structure 30 is provided inside the support can be manufactured in the same procedure as the device of the first embodiment.

The fertilized egg culture device of the present invention is not limited to the above embodiment.
For example, in the first embodiment to the third embodiment, the main flow paths 11, 21, and 31 are linear, and the meandering flow paths 12, 22, and 32 are square wave shapes that perpendicularly intersect the main flow path. Although shown, the present invention is not limited to this, and an effect can be obtained if the flow channel structure has a shape in which the flow rate of the main flow channel is larger than the flow rate of the meandering flow channel. Therefore, as long as the flow rate of the main flow path is larger than the flow rate of the meandering flow path, for example, the main flow path may be curved, bent, or the like, and the meandering flow path is curved, bent, It is good also as a sawtooth wave shape, a triangular wave shape, a sine wave shape. In order to design a flow channel structure in which the flow rate of the main flow channel is larger than the flow rate of the meandering flow channel, the flow resistance determined by the height, width, and length of the flow channel is determined from the main flow channel. Should be set to be higher.
In the first embodiment and the third embodiment, an example in which the notches 11c and 31c are linear is shown, but the notches may be curved.
In the second embodiment and the third embodiment, the cup structures 21c and 31d are cylindrical, but may be hemispherical.
In the first embodiment to the third embodiment, the cage regions 11b, 21b, and 31b have been shown to have a semicircular cross section on the downstream side. The taper shape etc. which become narrow toward the side may be sufficient.

As described above, the fertilized egg culturing device of the present invention is useful for automating the culturing operation of the fertilized egg.
The fertilized egg culturing apparatus of the present invention comprises the fertilized egg culturing device of the present invention, a liquid feeding means, a fertilized egg observing means, and an incubator.
As an example of a fertilized egg automatic culture apparatus for automatically culturing a fertilized egg, an apparatus including a fertilized egg culturing device, a syringe pump, a time lapse device, a multi-gas incubator, and a waste liquid tank can be cited. In the fertilized egg automatic culture apparatus, the fertilized egg culture device is installed on a time-lapse apparatus. The inlet-side port of the micro-channel structure of the fertilized egg culture device is connected to a syringe pump for feeding liquid, and the outlet-side port is connected to a waste liquid tank by a silicone tube.
In such a fertilized egg automatic culture apparatus, the placement, culture and recovery of the fertilized egg are performed as follows.
First, a fertilized egg is introduced into a microchannel structure together with a culture solution using a syringe pump and distributed. Thereby, as above-mentioned, a fertilized egg is capture | acquired by the vacant cage area | region in a microchannel structure. By repeating this operation, fertilized eggs are arranged (arrayed) in a plurality of cage regions in the microchannel structure.
The fertilized egg may be a fertilized egg of a mammal, and examples thereof include fertilized eggs such as humans, mice, cows, and pigs.
Before introducing a fertilized egg, support cells for co-culture may be introduced in advance and cultured to attach the support cells to the bottom surface of the cage region or the like.

Next, the fertilized egg arranged in the microchannel structure is cultured.
It is preferable to culture the fertilized egg while circulating the culture solution through the microchannel structure. In this case, the composition of the cage region can be adjusted by adjusting the composition of the supplied culture solution (type and concentration of nutrient components, dissolved oxygen concentration, etc.). However, this invention is not limited to this, You may culture | cultivate, without distribute | circulating a culture solution. When fertilized eggs are cultured using a conventional microchannel structure that does not have an extension or recess, if the culture is performed without circulating the culture solution, backflow occurs in the main channel, and the fertilized eggs are placed in the cage region. It cannot be stably held in place, and the fertilized egg is released from the cage region when it has not developed in the blastocyst. Although the release can be prevented by flowing the culture solution at a certain high flow rate, there is a concern that the development efficiency to the blastocyst may be adversely affected. On the other hand, in the case of a fertilized egg culturing device in which a recess is provided on the bottom surface of the cage region as shown in the second embodiment, the fertilized egg is placed in a predetermined position in the cage region without circulating the culture solution. Can be held stably.
When culturing fertilized eggs while circulating the culture medium through the microchannel structure, the flow rate of the culture solution supplied to the microchannel structure is determined from the point of efficiency of the fertilized egg blastocyst development. In the case of a use channel, 0.5 μL / min or less is preferable, and 0.2 μL / min or less is more preferable. The lower limit of the flow rate is not particularly limited, but is preferably 0.2 μL / min or more in the case of a mouse fertilized egg flow path from the viewpoint that the fertilized egg can be stably held at a predetermined position in the cage region. More than minutes are more preferable. The flow rate can be controlled by a syringe pump or the like.
The composition of the culture solution to be used and other culture conditions (culture temperature, culture time, etc.) can be appropriately set according to the fertilized egg to be cultured.
Fertilized eggs are preferably cultured until they develop into blastocysts.
The state of the fertilized egg during development (development stage, etc.) can be individually observed for each cage region by a time lapse apparatus. The stage of the time lapse apparatus can automatically control the XYZ axes by a computer, and can observe a fertilized egg in each cage region in culture over time. Therefore, not only the morphological evaluation of blastocysts but also the way of development can be observed and evaluated, and only high-quality fertilized eggs can be recovered from the microdevice. Improving the conception rate can be expected by transplanting high-quality fertilized eggs into the womb.
Here, an example in which observation for each cage region is performed using a time lapse device has been shown, but the present invention is not limited to this, and is performed using a general optical microscope, a moving image photographing device, or the like. Also good.

  For collection of fertilized eggs in the microchannel structure, for example, bubble generating means for generating bubbles is provided in the narrowed portion downstream of the cage region where the fertilized eggs to be collected are captured, and the bubble generating means provides This can be done by generating bubbles. Examples of the bubble generating means include an electrode and a laser. For example, when an electrode (platinum electrode or the like) is installed in a narrowed portion on the downstream side of the cage region and a voltage is applied to the electrode, bubbles are generated and grow. As a result, pressure is applied from the constriction to the cage region. This pressure pushes the fertilized egg out of the cage area and releases it into the serpentine channel. The released fertilized egg is collected from the outlet port of the device.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to this embodiment.
<Example 1>
A PDMS microfluidic device having the microchannel structure 10 having the configuration shown in FIGS. 1 and 2 on the surface was manufactured by the following procedure.
(1) The silicon substrate was washed with a pyrana solution (sulfuric acid: hydrogen peroxide solution = 2: 1) and buffered hydrofluoric acid (BHF).
(2) A negative photoresist SU-8 2075 (manufactured by MicroChem) was spin-coated on a cleaned silicon substrate so as to have a film thickness of 120 μm, and heated to be cured (prebaked).
(3) The SU-8 surface was masked with a glass photomask on which the channel shape was drawn, and UV was irradiated.
(4) After the post-baking, the silicon substrate was immersed in the SU-8 developer and developed. At this time, SU-8 which was not irradiated with UV by masking was removed. As a result, the convex shape of the microchannel structure could be manufactured on the silicon substrate.
(5) After development, the silicon substrate (hereinafter, mold master) was washed with isopropanol and pure water.
(6) In order to easily release the PDMS from the mold master, the surface of the mold master was coated with trifluorocarbon using a reactive ion etching (RIE) apparatus.
(7) Unpolymerized PDMS and a curing agent were stirred at a weight ratio of 10: 1, poured into a mold master, and cured by heating at 75 degrees for 2 and a half hours.
(8) The cured PDMS was released from the mold master. A concave structure was transferred on the surface of the released PDMS (hereinafter referred to as PDMS chip).
(9) Using a trepan, a through hole was made in a portion corresponding to the upstream (inlet) port, the downstream (exit) port, and the fertilized egg introduction port of the PDMS chip channel.
(10) A microfluidic device having the microchannel structure 10 is obtained by bonding the PDMS chip channel structure surface and a PDMS flat plate or glass plate by an oxygen plasma bonding method using an RIE apparatus. It was.
The dimensions of the microchannel structure 10 in the obtained PDMS microfluidic device were as follows.
The width W ₁ of the main flow path 11 is 120 μm, and the height is 120 μm.
The meandering channel 12 has a width W ₂ = 120 μm, a height = 120 μm, and a length of 1060 μm. The length of the meandering channel 12 is the center of the channel between one of the positions where the meandering channel 12 intersects the main channel 11 and the next position where it intersects the main channel 11 as described above. Length.
The width W ₃ = 40 μm, height = 120 μm, and length W ₅ = 30 μm of the constricted portion 11a.
The length W ₄ = 110 μm of the cage region 11b in the flow direction of the culture solution, the length W _a = 50 μm of the expansion portion 11d, and the length W _b = 60 μm of the semicircular portion 11e.
An angle θ _b = 135 ° formed by the side wall of the notch 11 c and the side wall of the meandering flow path 12.
An angle θ _a = 90 ° formed by the side wall of the cage region 11 c opposite to the notch 11 c and the side wall of the meandering channel 12.
The microfluidic device was photographed with a digital camera. The photograph is shown in FIG.

<Example 2>
A PDMS microfluidic device having the microchannel structure 20 having the configuration shown in FIGS. 3 to 4 on the surface was produced by performing the basic procedures (2) to (5) twice in Example 1. The specific procedure is as follows.
(1 ′) A convex shape having a micro-channel structure was manufactured by the procedures (1) to (5) in Example 1.
(2 ′) In order to fabricate the cup structure, the procedure was returned to the procedure (2) of Example 1, and SU-8 2050 was spin-coated so that the film thickness was 40 μm. The surface of SU-8 was masked with the glass photomask on which the cup structure was drawn in the procedure (3) of Example 1 and irradiated with UV. At this time, alignment (positioning) was performed so as to produce a cup structure in the cage region of the flow path.
After (3 ′), a microfluidic device was manufactured in the same procedure as (4) to (10) in Example 1.
The dimensions of the microchannel structure 20 in the obtained PDMS microfluidic device were as follows.
The width W ₁ of the main channel 21 is 120 μm and the height is 120 μm.
The width W ₂ of the meandering channel 22 is 120 μm, the height is 120 μm, and the length is 1060 μm.
The width W ₃ = 40 μm, height = 120 μm, and length W ₅ = 30 μm of the constricted portion 21a.
The length W ₄ of the cage region 21b is 110 μm.
Angle formed by the side wall of the cage region 21b and the side wall of the meandering channel 12 = 90 °.
The diameter D ₁ = 100 μm and the depth D ₂ = 40 μm of the cup structure 21c.
A part of the microchannel structure 20 of the PDMS microfluidic device was photographed using an inverted microscope and a digital camera. The photograph is shown in FIG.

<Example 3>
5 except that the PDMS microfluidic device having the microchannel structure 30 having the structure shown in FIG. 5 on the surface is changed to a glass photomask having a notch in the channel structure in Example 2. It was manufactured according to the procedure.
The dimensions of the microchannel structure 30 in the obtained PDMS microfluidic device were as follows.
The width W ₁ of the main flow path 31 is 120 μm and the height is 120 μm.
The width W ₂ of the meandering channel 32 is 120 μm, the height is 120 μm, and the length is 1060 μm.
The width W ₃ = 40 μm, the height = 120 μm, and the length W ₅ = 30 μm of the narrowed portion 31a.
The length W ₄ of the cage region 31b is 110 μm.
The length W ₄ = 110 μm of the cage region 31 b in the flow direction of the culture solution, the length W _a = 50 μm of the extended portion, and the length W _b = 60 μm of the semicircular portion.
An angle θ _b = 135 ° formed by the side wall of the notch 31c and the side wall of the meandering flow path 32.
An angle θ _a = 90 ° formed by the side wall of the cage region 31c opposite to the notch 31c and the side wall of the meandering channel 32.
The diameter D ₁ = 100 μm and the depth D ₂ = 40 μm of the cup structure 31d.
A part of the microchannel structure 30 of the PDMS microfluidic device was photographed using an inverted microscope and a digital camera. The photograph is shown in FIG.

<Comparative Example 1>
A PDMS microfluidic device having a microchannel structure having the same configuration as that of the microchannel structure 10 except that the notch 11c is not provided, and the same as in Production Example 1 except that the glass photomask used is changed. It was manufactured according to the procedure.
The dimensions of the microchannel structure in the obtained PDMS microfluidic device were as follows.
The width W ₁ of the main flow path 11 is 120 μm, and the height is 120 μm.
The meandering channel 12 has a width W ₂ = 120 μm, a height = 120 μm, and a length of 1060 μm.
The width W ₃ = 40 μm, height = 120 μm, and length W ₅ = 30 μm of the constricted portion 11a.
The length W ₄ of the cage region 11b is 110 μm.
Angle formed by the side wall of the cage region 11b and the side wall of the meandering flow path 12 = 90 °.
Further, the microchannel structure portion of the PDMS microfluidic device was photographed using an inverted microscope and a digital camera. The photograph is shown in FIG.

<Test Example 1>
The inlet port of the microchannel structure of the microfluidic device manufactured in Examples 1 to 3 and Comparative Example 1 was connected to a syringe pump for liquid feeding, and the outlet port was connected to a waste liquid tank by a silicone tube. Thereafter, the microfluidic device was placed on a time lapse apparatus.
First, using a syringe pump, the culture solution was introduced from the inlet side port side of the microchannel structure, and the inside of the microchannel structure was filled with the culture solution. Next, a mouse fertilized egg was introduced from a fertilized egg introduction port (a port installed immediately downstream of the inlet side port; upstream of the microchannel structure). At this time, the placement operation of the fertilized egg in the microchannel structure was observed over time using an inverted microscope and a digital video camera. Mouse fertilized eggs were prepared by in vitro fertilization method proposed by Toyoda et al. Using 13-week-old female BDF1 and male ICR, and the diameter at the time of introduction was about 80 μm. The culture solution was fed at a flow rate of 1.0 μL / min.
In each device, the volume flow ratio Q ₁ / Q ₂ of the main flow path and the meandering flow path was about 2.5.

As a result, in the example using the microfluidic device obtained in Comparative Example 1, many cage regions where a plurality of fertilized eggs were captured were confirmed as shown in FIG. Moreover, as shown in FIG.10 (b), the case where the fertilized egg which was caught in the cage area obstruct | occluded the flow path was also confirmed.
In contrast, in the example using the microfluidic device obtained in Examples 1 and 3, fertilized eggs were sequentially captured in the cage region of the microchannel structure. Each fertilized egg was captured in each cage region. FIGS. 11 and 12 show the state where the fertilized egg is captured in the cage region in the example using the microfluidic device obtained in Examples 1 and 3, respectively.
In the example using the microfluidic device obtained in Example 2, the fertilized eggs were sequentially captured in the cage region of the microchannel structure. However, as shown in FIG. 13, there was a slight cage region in which 2 to 3 fertilized eggs were captured.

<Test Example 2>
The effect of stabilizing the fertilized egg holding position by the cup structure was evaluated by the following procedure.
In Test Example 1 above, from the time when introduction of the fertilized egg was started to 72 hours later, a specific cage region was observed at a fixed point by a type lapse apparatus, and the stability of the position of the captured fertilized egg was evaluated.
As a result, in the example using the microfluidic device of Comparative Example 1, after fertilized eggs were introduced (culture medium was fed at a flow rate of 1.0 μL / min) and arrayed, the culture solution was flowed at a flow rate of 0.1 μL / min. However, most of the fertilized eggs disappeared from the cage area in the end until 72 hours after the start of the experiment. It was.
On the other hand, in the example using the microfluidic device obtained in Examples 2 and 3, after fertilized eggs were introduced (culture solution was fed at a flow rate of 1.0 μL / min) and arrayed, the flow rate was 0.1 μL. Of course, the fertilized egg was held at the position of the cup structure 72 hours after the start even when the culture solution was continuously fed at a rate of 1 min / min, even without feeding the culture solution (flow rate 0). .

  DESCRIPTION OF SYMBOLS 10 ... Microchannel structure, 11 ... Main channel, 11a ... Narrow part, 11b ... Cage area | region, 11c ... Notch, 11d ... Expansion part, 11e ... Semicircle part, 12 ... Serpentine channel, 20 ... Microchannel structure 21 ... main flow path, 21a ... constriction, 21b ... cage region, 21c ... cup structure (concave), 22 ... serpentine flow path, 30 ... micro flow path structure, 31 ... main flow path, 31a ... constriction, 31b ... cage Area, 31c ... notch, 31d ... cup structure (recessed part), 32 ... meandering channel

Claims (8)

Provided with a micro-channel structure through which mammalian fertilized eggs and culture fluid circulate,
The microchannel structure has a main channel and a meandering channel that sequentially intersects the main channel,
The main flow path is provided with a narrowed portion having a width through which the fertilized egg does not pass, and a cage region that captures the fertilized egg between the narrowed portion and a meandering flow channel that intersects the main flow channel upstream of the narrowed portion. And
The cage region is a fertilized egg culturing device in which a side wall on the downstream side of the meandering channel is notched and expanded in the downstream direction of the meandering channel. Provided with a micro-channel structure through which mammalian fertilized eggs and culture fluid circulate,
The microchannel structure has a main channel and a meandering channel that sequentially intersects the main channel,
The main flow path is provided with a narrowed portion having a width through which the fertilized egg does not pass, and a cage region that captures the fertilized egg between the narrowed portion and a meandering flow channel that intersects the main flow channel upstream of the narrowed portion. And
A fertilized egg culturing device in which a recess is provided on the bottom surface of the cage region.   The fertilized egg culture device according to claim 1, wherein a concave portion is provided on a bottom surface of the cage region.   The fertilized egg culturing device according to claim 1, wherein a side wall of the cage region on the upstream side of the meandering flow path and a side wall of the meandering flow path merge substantially vertically.   The fertilized egg culturing device according to claim 2 or 3, wherein a side wall of the cage region on the upstream side of the meandering flow path and a side wall of the meandering flow path merge substantially vertically.   A fertilized egg culture apparatus comprising the fertilized egg culture device according to any one of claims 1 to 5. A method for culturing a mammalian fertilized egg using the fertilized egg culturing device according to any one of claims 1 to 5,
Disposing the fertilized egg in the cage region by circulating the fertilized egg together with a culture solution in the microchannel structure; and culturing the fertilized egg while circulating the culture solution in the microchannel structure; A culture method comprising: A method for culturing a mammalian fertilized egg using the fertilized egg culturing device according to claim 2, 3 or 5,
Arranging the fertilized egg in the cage region by circulating the fertilized egg together with a culture solution in the microchannel structure, and culturing the fertilized egg without circulating the culture solution in the microchannel structure And a culture method.