JP2022188912A - Microfluidic device, method of manufacturing microfluidic device, and method of using microfluidic device - Google Patents

Abstract

To provide microfluidic devices capable of efficiently separating and culturing a cell body and a projection-like cell component extending from the cell body, to provide methods of manufacturing a microfluidic device, and to provide methods of using the microfluidic device.SOLUTION: Provided is a microfluidic device capable of culturing in a state in which a cell body and a projection-like cell component extending from the cell body are separated, the device comprising: a first culture space capable of culturing a cell body; and a second culture space extending from the first culture space and capable of culturing the cell component, the second culture space having a flat shape when viewed in the extension direction, and having a width that allows culturing of the cell component extending from the cell body and a height that does not allow the cell body to enter.SELECTED DRAWING: Figure 4

Description

The present invention relates to microfluidic devices, methods of manufacturing microfluidic devices, and methods of using microfluidic devices.

As a conventional method for evaluating neurotoxicity, there is an observational test to observe reflex movements in a method using experimental animals. Also, in culture tests using cultured cells, there is an electrophysiological measurement method using a multi-electrode array.

However, in the method using laboratory animals, since animals have species differences from humans, the reliability of verification for humans is low. In addition, the cell electrophysiology method requires a very high degree of expertise for measurement and analysis. Another problem is that the measuring instrument is expensive and cannot be used for many tests.

Furthermore, as a method of evaluating neurotoxicity, a method of forming neurites from nerve cells using a microfluidic device and observing drug stimulation and its response is known (for example, Patent Documents 1 and 2 below). Patent Document 1 discloses a microfluidic device having channels in which neurites (also called axons) extending from cell bodies can be cultured. Patent Literature 2 discloses a microfluidic device having channels in which neurites extending from cell bodies can be cultured.

Japanese Patent No. 6430680 Japanese Patent No. 5904789

However, the channel of Patent Document 1 has a width of 100 to 150 μm and a height of 100 to 200 μm. Protrusions assemble to form bundles. Channel structures that generate bundles of neurites do not allow observation and analysis of individual growth of neurites.

The channel of Patent Literature 2 has an inner diameter of 10 μm, and neurites must pass through a very small channel, which makes it difficult for neurites to grow and reduces the efficiency of the test. In addition, the growth and function of neurites are imperfect because the cells that make up myelin, which covers and protects neurites, cannot pass through the channels. As a result, a drug test that reproduces a living body cannot be obtained.

In view of the above problems, the present invention provides a microfluidic device, a method for manufacturing a microfluidic device, and a microfluidic device that can efficiently separate and culture a cell body and a protruding cell component part extending from the cell body. The purpose is to provide a method of using

A microfluidic device according to the present invention is a microfluidic device that can be cultured in a state in which a cell body and a protruding cell component part extending from the cell body are separated,
A first culture space capable of culturing the cell bodies, and a second culture space extending from the first culture space and capable of culturing the cell constituent parts, wherein the second culture space extends in the extending direction. When viewed from above, it has a flat shape and has a width that allows the cell constituent parts extending from the cell bodies to be cultured and a height that does not allow the cell bodies to enter.
In addition, the second culture space has a flat shape and is configured to have a width that is seven times or more the diameter of the cell-constituting portion extending from the cell body.
Furthermore, the second culture space is configured such that the width is ten times or more the height.

According to this configuration, the first culture space can culture cell bodies, and the second culture space can culture cell constituent parts extending from the cell bodies. At this time, the second culture space has a flat shape and a width that is 7 times or more the diameter of the cell constituent part extending from the cell body. Furthermore, it is configured in a flat cross-sectional shape so that the width is 10 times or more the height. For this reason, multiple neurites do not form a fascicle-like structure, and are cultured so that the neurites are aligned in the width direction. As a result, according to the microfluidic device of the present invention, it is possible to efficiently separate and culture the cell body and the protrusion-like cell constituent parts extending from the cell body.

In the microfluidic device according to the present invention, the height of the second culture space may be 5 μm or more and 80 μm or less. According to this configuration, the cell constituent parts can be appropriately cultured in the second culture space.

In the microfluidic device according to the present invention, the first culture space and/or the second culture space may be configured to be observable from the outside. According to this configuration, a series of operations from culturing to observation and evaluation can be performed within one microfluidic device.

The microfluidic device according to the present invention may be constructed of a thermoplastic resin. According to this configuration, the microfluidic device can be easily molded by injection molding.

In the microfluidic device according to the present invention, the thermoplastic resin may be a cycloolefin polymer (COP). Since COP has low drug adsorption, it is possible to perform accurate drug evaluation with little drug adsorption. In addition, since COPs have high transparency, cells can be easily observed from the outside.

The method for manufacturing a microfluidic device according to the present invention comprises
a step of forming in a first substrate a through hole extending from a first main surface of the first substrate toward a second main surface, and a recess provided in the second main surface and communicating with the through hole; ,
a step of bringing a second substrate into contact with the second main surface of the first substrate to cover the through hole and the recess;
and bonding the first substrate and the second substrate by optical bonding.

According to this configuration, by joining the first substrate and the second substrate, the through holes function as the first culture space, and the recess functions as the second culture space. Further, by bonding the first substrate and the second substrate by optical bonding, it is possible to obtain a microfluidic device that does not generate eluates that adversely affect drug evaluation or damage cells.

A method of using the microfluidic device according to the present invention comprises:
culturing the cell body in the first culture space and culturing the cell constituent part extending from the cell body in the second culture space;
administering a drug into the second culture space and evaluating efficacy and/or toxicity of the drug on the cell-constituting site.

According to the method of using the microfluidic device of the present invention, the cell body and the protrusion-like cell constituent part extending from the cell body can be efficiently separated and cultured, so that drug evaluation that reproduces a state close to the biological environment can be performed. It can be performed.

FIG. 2 is a perspective view of the microfluidic device according to the present embodiment before manufacturing; Top view of microfluidic device III-III line sectional view of the microfluidic device shown in FIG. A plan view schematically showing how neurons are cultured in a microfluidic device. VV line cross-sectional view of the microfluidic device 1 shown in FIG. Plan view of a microfluidic device according to another embodiment Cross-sectional view of a microfluidic device according to another embodiment Plan view of a microfluidic device according to another embodiment Cross-sectional view of a microfluidic device according to another embodiment Plan view of a microfluidic device according to another embodiment Cross-sectional view of a microfluidic device according to another embodiment Plan view of a microfluidic device according to another embodiment Cross-sectional view of a microfluidic device according to another embodiment

[Structure of microfluidic device]
A microfluidic device according to the present invention will be described with reference to the drawings. It should be noted that each drawing disclosed in this specification is only schematically illustrated. That is, the dimensional ratios on the drawings and the actual dimensional ratios do not necessarily match, and the dimensional ratios do not necessarily match between the drawings.

FIG. 1 is a perspective view of a microfluidic device 1 according to this embodiment before manufacturing. The microfluidic device 1 has a first substrate 10 and a second substrate 20 and is manufactured by bonding these substrates. FIG. 1 corresponds to a perspective view showing the first substrate 10 and the second substrate 20 just prior to bonding.

The microfluidic device 1 is laminated on one main surface 20a of the second substrate 20 so that one main surface 10b (corresponding to the second main surface of the present invention) of the first substrate 10 is partially in contact with the main surface 20a. are joined together to form the The main surface refers to a surface of the substrates 10 and 20 that has a much larger area than the other surfaces. The substrates 10, 20 each have two main surfaces, which are arranged opposite each other. The main surface 10b of the first substrate 10, which is partially in contact with the second substrate 20, has a recess (described later). Another main surface 10a (corresponding to the first main surface of the present invention) of the first substrate 10 is located on the opposite side of the second substrate 20 and has ports 11 and 12 (described later).

In the following description, in a state in which the first substrate 10 and the second substrate 20 are bonded, a plane parallel to the main surfaces 10a and 10b of the first substrate 10 and the main surfaces 20a and 20b of the second substrate 20 is defined as the XY plane. , and the XYZ coordinate system, in which the direction perpendicular to the XY plane is the Z direction, is appropriately referred to.

In addition, in this specification, when distinguishing between positive and negative directions when expressing directions, positive and negative signs are added, such as “+X direction” and “−X direction”. Moreover, when expressing a direction without distinguishing between positive and negative directions, it is simply described as “X direction”. That is, in the present specification, the term “X direction” includes both “+X direction” and “−X direction”. The same applies to the Y direction and Z direction. The microfluidic device 1 is normally used with the Z direction as the vertical direction, and the -Z direction corresponds to the upward direction.

FIG. 2 is a plan view of the microfluidic device 1. FIG. FIG. 3 shows a schematic cross-sectional view of the microfluidic device 1 in which the first substrate 10 is bonded onto the second substrate 20, and the schematic cross-sectional view is a cross-sectional view taken along line III-III of the microfluidic device 1 shown in FIG. is. The schematic cross-sectional view of FIG. 3 shows only the lines appearing on the cross section among the contour lines of both substrates in order to facilitate understanding of the drawing. The same applies to FIG. 5, which will be described later.

The first substrate 10 and the second substrate 20 are rectangular substrates having principal surfaces of the same shape. The length and width of the main surfaces of the first substrate 10 and the second substrate 20 are, for example, 10 mm and 20 mm. Also, the thickness of the first substrate 10 is greater than the thickness of the second substrate 20 . The thickness of the first substrate 10 is, for example, 0.2 to 10 mm, and is 3 mm in this embodiment. Also, the thickness of the second substrate 20 is, for example, 0.1 to 2 mm, and is 1 mm in this embodiment.

As shown in FIG. 1, the first substrate 10 is formed with a through hole 11a and a through hole 12a extending from the first main surface 10a toward the second main surface 10b. The through holes 11a and the through holes 12a are arranged side by side in the X direction. The port 11 is the opening of the through hole 11a on the first main surface 10a, and the port 12 is the opening of the through hole 12a on the first main surface 10a.

The ports 11 , 12 have at least one purpose of injecting liquid into the microfluidic device 1 and/or discharging liquid from the microfluidic device 1 . For example, port 11 may be used as a liquid inlet and port 12 as a liquid outlet.

Through hole 11 a functions as first culture space 31 by bonding first substrate 10 and second substrate 20 . However, although an example in which the through holes 11 a function as the first culture space 31 is shown in this embodiment, the through holes 12 a may function as the first culture space 31 . Moreover, both the through-holes 11a and the through-holes 12a may function as the first culture spaces 31, respectively.

The through holes 11a and 12a of the present embodiment are both circular holes extending in the Z direction. The diameter d (see FIG. 2) of the through holes 11a and 12a is, for example, 0.05 to 5 mm, and is 2 mm in this embodiment. Also, the distance L (see FIG. 2) between the through holes 11a and 12a is, for example, 1 to 100 mm, and is 7 mm in this embodiment.

The second main surface 10b of the first substrate 10 has a recess 13a, as shown in FIG. The concave portion 13a is formed in the Y-direction central portion of the second main surface 10b so as to extend in the X-direction. A cross section (a cross section parallel to the YZ plane) perpendicular to the extending direction (X direction) of the recess 13a has a flat rectangular shape.

The recess 13a communicates with the through-hole 11a and the through-hole 12a. The concave portion 13 a functions as a hollow second culture space 32 sandwiched between the substrates 10 and 20 by joining the first substrate 10 and the second substrate 20 .

The recess 13a has a slit shape extending in the X direction with a constant width and depth. The width of the concave portion 13a is ten times or more the depth. That is, the second culture space 32 is configured such that the width W (see FIG. 2) is ten times or more the height H (see FIG. 3) when viewed in the extending direction. The second culture space 32 is configured such that the width W is preferably 10 times or more, more preferably 20 times or more as large as the height H. Moreover, it is preferable that the width W is 100 times or less the height H. In a conventional microfluidic device made of silicone rubber, it was difficult to maintain the height of a flat channel whose width is 10 times or more as large as its height due to the effects of gravity and the like.

As described above, the microfluidic device 1 of the present embodiment includes the first culture space 31 and the second culture space 32 extending from the first culture space 31, and the second culture space 32 extends The width W is 10 times or more the height H when viewed in the direction. The first culture space 31 can culture cell bodies, and the second culture space 32 can culture projecting cell constituent parts extending from the cell bodies. As a result, the microfluidic device 1 can be cultured in a state in which the cell bodies and the cell constituent parts are separated.

4 and 5 are a plan view and a cross-sectional view schematically showing how nerve cells are cultured in the microfluidic device 1. FIG. FIG. 5 is a cross-sectional view of the microfluidic device 1 shown in FIG. 4 taken along line VV.

A nerve cell C1 (an example of a cell body) is cultured in the first culture space 31, while a protruding neurite C2 (an example of a cell component part) extending from the nerve cell C1 is cultured in the second culture space 32. It is At this time, as shown in FIGS. 4 and 5, the second culture space 32 has a flat shape in which the nerve cells C1 do not enter the second culture space 32 and the neurites C2 can be cultured.
Nerve cells C1 are classified according to the presence or absence of myelin, diameter, conduction velocity, etc. Type A, which has myelin, has diameters of Aα (13-22 μm), Aβ (8-13 μm), and Aγ (4 μm), respectively. ~8 μm) and Aδ (1 to 4 μm). Autonomic nerves are called type B even when they have a myelin sheath, and their diameter is 1 to 3 μm. Furthermore, C-type fibers without myelin sheath (C-fibers) have a diameter of 0.2-1.0 μm.
Furthermore, since the second culture space 32 is configured to have a flat cross-sectional shape such that the width W is ten times or more the height H, as shown in FIG. It is cultured so that each neurite C2 is aligned in the width direction (Y direction) without taking a structure.

When neurite C2 is composed only of axons (without myelin), the diameter of neurite C2 is 0.2-1.5 μm, and neurite C2 is composed of axons covered with myelin. In this case, the diameter of the neurite C2 is 1-22 μm. By giving the second culture space 32 a width of 7 times or more the diameter of the neurite C2, the neurite C2 can grow while being divided into a plurality of pieces without contacting the side wall of the second culture space 32. can. If the width of the second culture space 32 is 7 times or less with respect to the diameter of the neurite C2, the adjacent neurite C2 itself and the side wall of the second culture space 32 are contacted during culture of the neurite C2, and growth is suppressed. It will be done.

In the second culture space 32, the width W is preferably 50 μm or more and 8000 μm or less, more preferably 500 μm or more and 1000 μm or less. If the width W is narrower than 50 μm, it becomes difficult to obtain a sufficient observation range. On the other hand, if the width W is larger than 8000 μm, both the channel height and the channel width that can be separated cannot be satisfied. In this embodiment, the width W is 800 μm.

In the second culture space 32, the height H is preferably 5 μm or more and 80 μm or less, more preferably 20 μm or more and 80 μm or less. If the height H is less than 5 μm, it is difficult for the neurites C2 to invade, and even if the neurites C2 invade, their growth is inhibited. On the other hand, if the height H is higher than 80 μm, the nerve cells C1 also enter the second culture space 32, and the nerve cells C1 and neurites C2 cannot be separated. In this embodiment, the height H is 40 μm.

When culturing neurites (unmyelinated nerves) without myelin (myelin sheath) or when using other cells, the height H is preferably 5 μm or more, which is larger than the diameter of the unmyelinated nerves. When culturing neurites (myelinated nerves) having myelin (myelin sheath), the height H is preferably 20 μm or more, which is the thickness of the myelinated nerves.

When the individual cells are separated and the dispersed cells are seeded, it is necessary to prevent floating cells from flowing into the second culture space 32 . The size of the cells when dispersed into single cells is 10 to 30 μm in diameter, depending on the cell type, so the height H is preferably 20 μm or less. Moreover, when the cells are seeded in the state of cell aggregates, the height H is preferably 80 μm or less, which is smaller than the cell aggregates.

[Method for producing microfluidic device]
Next, a method for manufacturing the microfluidic device 1 will be described in detail.

(Substrate preparation process)
A first substrate 10 and a second substrate 20 that constitute the microfluidic device 1 are prepared.

The material forming the substrates 10 and 20 is a transparent thermoplastic resin. Examples of thermoplastics include polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), cycloolefin polymer (COP), polystyrene (PS). Medical grade COP is particularly preferable as the thermoplastic resin that constitutes the substrates 10 and 20 . COP is a thermoplastic resin with high transparency, low autofluorescence, and low drug adsorption.

The shapes of the substrates 10 and 20 in this embodiment will be described. The first substrate 10 and the second substrate 20 are rectangular substrates having the same main surface. Also, the thickness of the first substrate 10 is greater than the thickness of the second substrate 20 . However, the main surfaces of the first substrate 10 and the second substrate 20 may not have the same shape. For example, the vertical and horizontal dimensions of the main surface of the first substrate 10 may be larger than the vertical and horizontal dimensions of the main surface of the second substrate 20, and the vertical and horizontal dimensions of the main surface of the second substrate 20 may be , may be greater than the longitudinal and lateral dimensions of the main surface of the first substrate 10 . Also, the thickness of the first substrate 10 may be the same as the thickness of the second substrate 20 , or the thickness of the first substrate 10 may be smaller than the thickness of the second substrate 20 .

Both of the two main surfaces 20a and 20b of the second substrate 20 in this embodiment are flat. A first major surface 10 a of the first substrate 10 has ports 11 and 12 . The ports 11 and 12 are openings of through holes 11 a and 12 a formed in the first substrate 10 . In addition, the second main surface 10b of the first substrate 10 has a concave portion 13a for forming a hollow second culture space 32 after being joined to the second substrate 20. As shown in FIG.

In order to provide the through holes 11a and 12a and the recessed portion 13a in the first substrate 10, for example, injection molding, a combination of a photolithography process and an etching process, casting, cutting, etc. are available. The most appropriate means should be selected accordingly. As an example, by forming the first substrate 10 from the above-described thermoplastic resin, the through holes 11a and 12a and the recess 13a can be easily formed by injection molding.

(Substrate bonding process)
The principal surface 10b of the manufactured first substrate 10 and the principal surface 20a of the second substrate 20 are joined. The bonding method described below is a method that does not require the formation of a thin film as an adhesive on the substrate, and is carried out in the following procedure.

First, the bonding surfaces (10b, 20a) of both substrates are subjected to surface activation treatment. As a method of surface activation treatment, a method of irradiating ultraviolet rays can be used.

For the method of irradiating ultraviolet rays, for example, from an ultraviolet light source such as a xenon excimer lamp that emits light with a wavelength of 172 nm, the main surface 10b of the first substrate 10 and the main surface 20a of the second substrate 20 are irradiated with a wavelength of 200 nm. It is performed by irradiating the following vacuum ultraviolet (VUV). As other examples of ultraviolet light sources, a low-pressure mercury lamp having a bright line at 185 nm and a deuterium lamp having a bright line in the wavelength range of 120 to 200 nm can be preferably used. The illuminance of vacuum ultraviolet rays is, for example, 10 to 500 mW/cm ² , and the irradiation time is appropriately set depending on the resin, and is, for example, 5 to 6 seconds.

Next, the first substrate 10 and the second substrate 20 are overlapped so that the bonding surfaces (10b, 20a) of both substrates subjected to the surface activation treatment are in contact, and both substrates are pressed using a press machine. Then, a joining step of joining is performed. In order to maintain the surface activation state, the bonding process is preferably performed within a predetermined time period, for example, within 10 minutes after the ultraviolet irradiation process is completed.

This bonding step is performed in an optionally heated environment to strengthen the bond. In the bonding step, bonding conditions such as heating temperature and pressing force are set according to the constituent material of the first substrate 10 and the constituent material of the second substrate 20 . Specifically, the pressing temperature is, for example, 40 to 130° C., and the pressing force for joining is, for example, 0.1 to 10 MPa.

After pressurizing the substrate in which the first substrate 10 and the second substrate 20 are bonded (hereinafter sometimes referred to as “bonded substrate”) for a predetermined period of time, the substrate may be further heated for a predetermined period of time as necessary. By heating the bonded substrate after pressurizing it, a portion where a sufficient bonded state is obtained and a portion where a sufficient bonded state is not obtained coexist at the bonding interface of the laminated substrate after pressure is applied. Even in the case where the bonding state is not sufficient, heating can bring the bonding state to the desired state.

A pressurized state of the bonded substrates is maintained for a predetermined time, and then the pressurized state is released, the temperature of the bonded substrates is increased to a predetermined temperature, and the temperature is maintained until a desired bonding state is obtained. can be Here, the predetermined temperature is a temperature at which the bonded substrates are not deformed. For example, the heating temperature is, for example, 40 to 130° C., and the heating time is, for example, 60 to 600 seconds.

After that, through a cooling step, the microfluidic device 1 in which the first substrate 10 is bonded onto the main surface 20a of the second substrate 20 is produced.

[How to use the microfluidic device]
Next, how to use the microfluidic device 1 will be described. Specifically, it is a method of evaluating a drug using the microfluidic device 1 .

First, nerve cells C1 are seeded from the port 11 to the first culture space 31 . The first culture space 31 and the second culture space 32 are filled with a suitable medium. By culturing the nerve cell C1 in the first culture space 31 for several days, the neurite C2 extends from the nerve cell C1 and invades the second culture space 32 . As a result, the nerve cell C1 and the neurite C2 can be cultured separately. The neurite C2 cultured in the second culture space 32 can be observed from the outside. The neurite C2 is observed, for example, from the main surface 20b side of the second substrate 20 using a fluorescence microscope or the like. In order to properly observe the neurites C2 from the outside, the first substrate 10 and the second substrate 20 are made of a transparent thermoplastic resin, and the main surfaces of the first substrate 10 and the second substrate 20 are flat. is preferred.

A drug is then administered into the second culture space 32 to assess efficacy and/or toxicity of the drug to neurite C2. As described above, according to the microfluidic device 1 of the present invention, a series of operations from culturing to drug testing and observation evaluation can be performed within one device.

In addition, as the timing of administering the drug, a method of administering the drug into the second culture space 32 after the neurite C2 has sufficiently elongated in the second culture space 32, and evaluating efficacy and / or toxicity; There is a method in which neurites C2 are cultured in a state in which a drug is preliminarily administered to the culture medium in the culture space 32 (also the first culture space 31), and efficacy and/or toxicity are evaluated.

Although the embodiments of the present invention have been described above with reference to the drawings, it should be considered that the specific configuration is not limited to these embodiments. The scope of the present invention is indicated not only by the description of the above embodiments but also by the scope of claims, and includes all modifications within the scope and meaning equivalent to the scope of claims.

It is possible to adopt the structure adopted in each of the above embodiments in any other embodiment. The specific configuration of each part is not limited to the above-described embodiment, and various modifications are possible without departing from the scope of the present invention.

(1) The microfluidic device 1 may further include a reservoir for storing medium. 6A and 6B are a plan view and a cross-sectional view showing an example in which the microfluidic device 1 has a reservoir 33. FIG. A reservoir 33 is connected to the first culture space 31 . The volume ratio of the reservoir 33 and the first culture space 31 to the second culture space 32 is, for example, 2000:1.

In addition, in FIGS. 6A and 6B, the reservoir 33 is shown as a configuration separate from the first culture space 31, but it may be integrated. That is, the first culture space 31 may be configured as part of the reservoir 33 .

(2) FIGS. 7A and 7B show another example in which the configuration of the second culture space 32 is different from the above embodiment. In this example, the second culture space 32 is partly exposed to the outside.

(3) FIGS. 8A and 8B show another example in which the configuration of the second culture space 32 is different from the above embodiment. In this example, the second culture space 32 is configured by a recess formed in the second substrate 20 . A part of the second culture space 32 is exposed to the outside.

(4) FIGS. 9A and 9B show an example in which a chemical introduction port 14 communicating with the second culture space 32 is provided. A drug can be administered from the drug solution introduction port 14 to the second culture space 32 .

(5) In the above embodiments, nerve cells were used as cell bodies, but the cells are not limited to this. Examples of cell bodies include follicular dendritic cells, which are antigen-presenting cells that constitute the immune system in lymphoid tissue, and melanocytes, which are cells that produce melanin in the skin and the like. .

Reference Signs List 1: microfluidic device 10: first substrate 10a: first main surface 10b: second main surface 11: port 11a: through hole 12: port 12a: through hole 13a: concave portion 14: chemical solution introduction port 20: second substrate 20a : Main surface 20b : Main surface 31 : First culture space 32 : Second culture space 33 : Reservoir C1 : Neuron C2 : Neurite W : Width of second culture space H : Height of second culture space

Claims (10)

A microfluidic device capable of culturing in a state in which a cell body and a projecting cell component part extending from the cell body are separated,
a first culture space capable of culturing the cell bodies, and a second culture space extending from the first culture space and capable of culturing the cell constituent parts,
The second culture space has a flat shape when viewed in the extension direction, and has a width that allows culturing of the cell constituent part extending from the cell body and a height that does not allow the cell body to enter. A microfluidic device characterized by: 2. The microfluidic device according to claim 1, wherein the second culture space has a flat shape and has a width seven times or more as large as the diameter of the cell component portion extending from the cell body. 2. The microfluidic device according to claim 1, wherein the width of the second culture space is 10 times or more as large as the height. 2. The microfluidic device according to claim 1, wherein the second culture space has a height of 5 [mu]m or more and 80 [mu]m or less. 2. The microfluidic device according to claim 1, wherein the first culture space and/or the second culture space are configured to be observable from the outside. 2. The microfluidic device according to claim 1, wherein said microfluidic device is made of a thermoplastic resin. 7. The microfluidic device of claim 6, wherein the thermoplastic resin is cycloolefin polymer (COP). 2. The microfluidic device according to claim 1, wherein the cell body is a nerve cell, and the cell constituent part is a neurite extending from the nerve cell. A method of manufacturing the microfluidic device of claim 1, comprising:
a step of forming in a first substrate a through hole extending from a first main surface of the first substrate toward a second main surface, and a recess provided in the second main surface and communicating with the through hole; ,
a step of bringing a second substrate into contact with the second main surface of the first substrate to cover the through hole and the recess;
and bonding the first substrate and the second substrate by optical bonding. A method of using the microfluidic device of claim 1, comprising:
culturing the cell body in the first culture space and culturing the cell constituent part extending from the cell body in the second culture space;
A method of using a microfluidic device, comprising administering a drug into the second culture space and evaluating efficacy and/or toxicity of the drug on the cell-constituting site.

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