JP2022120216A - Microfluidic device and method for manufacturing the same, and method for culturing three-dimensional tissue - Google Patents

Abstract

To provide a device capable of artificially constructing a vascular bed and culturing a three-dimensional tissue in a state closer to living tissue, and a method for culturing a three-dimensional tissue using the device.SOLUTION: Provided is a microfluidic device comprising: a device body; a first flow channel provided in the device body; a vascular bed retention chamber adjacent to the first flow channel in the device body and provided through a first wall; an opening provided in the device body and communicating with the vascular bed retention chamber; and a partition wall provided to close the opening. A plurality of first slits is formed in the first wall. The partition wall is removable.SELECTED DRAWING: Figure 1

Description

The present invention relates to a microfluidic device, a method for manufacturing the same, and a method for culturing a three-dimensional tissue using the microfluidic device.

For long-term in vitro culturing of tissues excised from experimental animals, or three-dimensional tissues artificially made from ES cells, iPS cells, or the like, it is necessary to supply nutrients and oxygen from the outside through blood vessels. Until now, we have artificially constructed a vascular bed for three-dimensional tissue culture, and while culturing the three-dimensional tissue using the vascular bed, we have fused the blood vessels in the three-dimensional tissue with the vessels in the vascular bed. , several methods of constructing a new vascular tissue or vascular network within a three-dimensional tissue have been reported.

In Patent Document 1, after culturing angiogenic cells on the surface of a fluorine-containing polyimide membrane, the membrane is inverted and placed on a gel, or the surface of the polyimide membrane is covered with a gel, and culture is continued. A method for forming a vessel-like tissue is disclosed. Further disclosed is a method of forming a vessel-spheroid fusion by seeding spheroids onto a gel layer in which the vessel-like tissue is embedded. In this method, spheroids were observed to grow so as to cover the periphery of the vascular tissue, and fusion of the spheroids and the vascular tissue was also observed in some areas.

In Patent Document 2, a channel for perfusing a culture medium is provided inside the gel, vascular endothelial cells are induced, a vascular bed is created by constructing a vascular network in the gel, and a cell sheet is laminated on the vascular bed. A method for producing a cell sheet laminate is disclosed, characterized in that a vascular network is constructed in the cell sheet by heating.

Non-Patent Document 1 discloses a microfluidic device for co-cultivating vascular networks and cell tissues. The device, called an open-top device, consists of a microchannel with micropores in the ceiling, providing direct fluid access from the reservoir to the microchannel. Fig. 1 of Non-Patent Document 1 discloses that cell spheroids are formed above micropores and a vascular network extends to the cell spheroids through the micropores.

However, the blood vessel/spheroid fusion produced by the method of Patent Document 1 was incomplete, although fusion of spheroids and vascular tissue was observed in some areas. According to the method of Patent Document 2, even if the cell sheet is layered on the vascular bed constructed in the gel, the vascular network and the cell sheet are not in direct contact, and it is thought that the blood vessels in the gel are unlikely to extend to the cell sheet. be done. Also, when using the open-top device of Non-Patent Document 1, the cell spheroids and the vascular bed cannot be in direct contact, and the vascular network can only extend to the cell spheroids through micropores. Three-dimensional tissue cannot be cultured in a similar state.

On the other hand, a microfluidic device is a device that uses microfabrication technology such as MEMS (Micro Electro Mechanical Systems) technology to create microchannels and reaction vessels and apply it to bioresearch and chemical engineering. are collectively called. It is also called microTAS (micro Total Analysis Systems) or Lab on a Chip.

JP 2009-213716 A WO2012/036225

Soojung Oh et al.,“Open-top”microfluidic device for in vitro three dimensional capillary beds, LabChip,2017,17,3405-3414 Nishimoto Y et al., Integrating perfusable vascular networks with a three-dimensional tissue in a microfluidic device, Integr., Biol., 2017, 9, 506-518

An object of the present invention is to provide a device capable of artificially constructing a vascular bed and culturing a three-dimensional tissue in a state closer to living tissue, and a method of culturing a three-dimensional tissue using the device.

The present inventors have made extensive studies to solve the above-described problems of the present invention. The present inventors previously disclosed a method for constructing three-dimensional cell spheroids with perfusable vascular networks using a microfluidic device (Non-Patent Document 2). Specifically, cellular interactions between human umbilical vein endothelial cells (HUVEC) and human lung fibroblasts (hLF) within the channels of the microfluidic device induced angiogenic sprouts from the device towards the spheroids. , also confirmed that a continuous lumen (vascular network) was formed. However, this method of inducing formation of vascular tissue is limited to the combination of HUVEC and hLF, and other cell types cannot produce sufficient angiogenic factors. Cannot connect to spheroids.

Therefore, the present inventors have further improved the above-mentioned device so that any three-dimensional tissue can be cultured, and have developed a new device that allows the vascular bed and the three-dimensional tissue to be cultured in close proximity to each other, and that all three-dimensional tissues can be easily cultured with a single device. A microfluidic device has been found. The present invention is based on this new finding.

A microfluidic device according to the present invention comprises a device body, a first channel provided in the device body, and a blood vessel adjacent to the first channel in the device body and provided through the first wall. a bed holding chamber; an opening provided in the device main body and communicating with the vascular bed holding chamber; formed and the septum is removable.

Using the microfluidic device according to the present invention, a vascular bed is first constructed in a vascular bed holding chamber closed by a septum. By introducing into and further culturing, the blood vessels in the three-dimensional tissue and the blood vessels in the vascular bed are connected, or the blood vessels extend from the vascular bed to construct a new vascular network in the three-dimensional tissue. , three-dimensional tissue can be cultured in a state closer to living tissue.

In the microfluidic device according to the present invention, a pair of first channels may be provided, and each of the pair of first channels may be adjacent to the vascular bed holding chamber via the first wall. As a result, the vascular bed can extend to both sides of the vascular bed holding chamber, and a vascular bed closer to living tissue can be constructed.

In the microfluidic device according to the invention, a septum may be provided between the vascular bed holding chamber and the opening. As a result, there is no step on the ceiling (opening side) of the vascular bed holding chamber, which makes it easier to construct the vascular bed.

In the microfluidic device according to the present invention, the partition may be a partition that can be removed by a chemical method or a physical method, or the partition may be a membrane-like partition made of a water-soluble material. This makes it easier to remove the partition.

In the microfluidic device according to the present invention, the first slits in the first wall may be formed at equal intervals so that the slit width is 50 μm to 200 μm. This makes it easy to supply the components necessary for constructing the vascular bed to the vascular bed holding chamber, to form blood vessels having an optimum thickness, and to facilitate the formation of the vascular bed at regular intervals.

The microfluidic device according to the present invention may further include a first fluid supply section communicating with the first channel. This facilitates the supply or replacement of fluid containing cells or angiogenic factors to the first channel.

The microfluidic device according to the present invention further includes a second channel provided in the device body, the second channel being adjacent to the first channel via the second wall, A plurality of second slits may be formed in the wall. This makes it possible to supply components and the like necessary for constructing the vascular bed to the first channel and the vascular bed holding chamber.

In the microfluidic device according to the present invention, a pair of first channels is provided, each of the pair of first channels is adjacent to the vascular bed holding chamber via the first wall, and the second A pair of flow paths may be provided, and each of the pair of second flow paths may be adjacent to each of the pair of first flow paths via the second wall.

In the microfluidic device according to the present invention, the second slits in the second wall may be formed at regular intervals so that the slit width is 50 μm to 200 μm. This makes it easier to supply components and the like necessary for constructing the vascular bed to the first channel and the vascular bed holding chamber.

The microfluidic device according to the present invention may further include a second fluid supply unit communicating with the second channel. As a result, it is possible to easily supply or replace the fluid containing cells or components necessary for constructing the vascular bed to the second channel.

A method for manufacturing a microfluidic device according to the present invention comprises: a first substrate having a first groove; a recess adjacent to the first groove and provided via a first wall; The through-hole is opened by providing a second substrate and a removable partition, and installing the partition on the surface of the second substrate in contact with the first substrate so as to close the through-hole. forming the first substrate through the partition wall so that the opening is located at a position corresponding to at least a part of the recess on the surface of the second substrate that contacts the first substrate; to form the recess as a vascular bed retention chamber. Since the microfluidic device according to the present invention has a complicated structure, integral molding is impossible. According to the manufacturing method according to the present invention, the microfluidic device according to the present invention can be manufactured by separately preparing the first substrate, the partition wall and the second substrate and placing them in order.

In the manufacturing method according to the present invention, in the step of placing the first substrate, the surfaces of the first substrate and the second substrate that are in contact with each other are plasma-processed, and the first substrate is the second substrate. It may be installed so as to join. Thereby, the first substrate and the second substrate can be strongly bonded.

The method for culturing a three-dimensional tissue according to the present invention uses the microfluidic device according to the present invention to supply a fluid containing angiogenic factors to the first channel, and supply a medium containing vascular endothelial cells to the vascular bed retention chamber. supplying and forming a vascular bed in a vascular bed holding chamber; removing the septum, placing the three-dimensional tissue on the formed vascular bed, and culturing. As a result, a three-dimensional tissue composed of all kinds of cells can be cultured for a long period of time with a single device in a state closer to that of a living tissue. In addition, since it is also possible to transvascularly administer nutrients or drugs to the interior of the three-dimensional tissue through the first channel and the vascular bed connected thereto, drug discovery research and development can be performed while culturing the three-dimensional tissue on the device. Alternatively, it can be used for regenerative medicine research and the like.

In the culture method according to the present invention, when the microfluidic device further comprises a second channel provided in the device main body, supplying an angiogenic factor to the first channel causes fibroblasts to flow into the second channel. It may be performed by supplying medium containing cells.

In the culture method according to the present invention, the step of forming a vascular bed may further include introducing vascular endothelial cells into the first channel. As a result, the vascular endothelial cells within the vascular bed retention chamber tend to connect with the vascular endothelial cells outside the vascular bed retention chamber, making it easier to construct the vascular bed.

The culture method according to the present invention may further include a step of isolating the obtained three-dimensional tissue. The isolated three-dimensional tissue can be used for in vivo experiments using the three-dimensional tissue, living body transplantation, and the like.

According to the microfluidic device and the method for culturing three-dimensional tissue of the present invention, three-dimensional tissue can be cultured in a state closer to living tissue.

1 is a plan view of a microfluidic device of one embodiment. FIG. 1 is an AA' cross-sectional view of a microfluidic device of one embodiment; FIG. 1 is a BB' cross-sectional view of a microfluidic device of one embodiment; FIG. FIG. 3 is an enlarged view of the rectangular frame of the BB' cross-sectional view of the microfluidic device of one embodiment. 1 is a plan view of a first substrate used in a method of manufacturing a microfluidic device according to one embodiment; FIG. FIG. 2 is a plan view of partition walls used in the method of manufacturing a microfluidic device according to one embodiment. FIG. 4 is a plan view of a second substrate used in the method of manufacturing a microfluidic device according to one embodiment; It is a figure which shows the preparation process of the 1st board|substrate of the microfluidic device of one Embodiment. It is a figure which shows the preparation process of the 2nd board|substrate of the microfluidic device of one Embodiment. FIG. 4 is a diagram showing a step of constructing a vascular bed in the three-dimensional tissue culture method of one embodiment. FIG. 4 is a diagram showing a step of constructing a three-dimensional tissue in the three-dimensional tissue culture method of one embodiment. FIG. 10 is a diagram showing the results of Example 3; FIG. 10 is a diagram showing the results of Example 4; FIG. 10 is a schematic diagram of a partition-less device of Experimental Example 5. FIG. FIG. 10 is a diagram showing the results of Experimental Example 5;

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In each figure, the same or corresponding parts are denoted by the same reference numerals, and redundant explanations are omitted.

[Microfluidic device]
1 to 4 show one embodiment of the microfluidic device according to the present invention, but the microfluidic device of the present invention is not limited to this. 1 is a plan view of one embodiment, FIG. 2 is a cross-sectional view of the embodiment along AA', FIG. 3 is a cross-sectional view of the embodiment along BB', and FIG. Figures are shown respectively.

As shown in FIGS. 1 to 4, a microfluidic device 1 of one embodiment includes a device body 1, a first channel 4 provided in the device body 1, and a first channel 4 in the device body 1. , the vascular bed holding chamber 2 provided via the first wall portion 3, the opening 6 provided in the device body 1 and communicating with the vascular bed holding chamber 2, and the opening 6 so as to block and a partition wall 5 provided.

The microfluidic device 1 is not particularly limited as long as it is made of a material suitable for cell culture. Examples of materials include, but are not limited to, plastic resins such as polydimethylsiloxane (PDMS). The shape of the microfluidic device is also not limited, and the size is not particularly limited as long as it is a normal size for a microfluidic device.

The vascular bed holding chamber 2 is a chamber (space) for accommodating cells and a culture medium for forming a vascular bed, and building and holding the vascular bed. Its shape and size are not particularly limited. The height (depth) of the vascular bed holding chamber 2 can be appropriately set according to the size of the vascular bed to be formed, and may be, for example, 50 μm to 500 μm, 50 μm to 300 μm, 50 μm to 250 μm, or 100 μm. It may be ~250 μm.

The microfluidic device 1 may further comprise a vascular endothelial cell supply section 11 communicating with the vascular bed retention chamber 2, as shown in FIG. 1 or FIG. According to the vascular endothelial cell supply unit 11 , it becomes possible to supply cells such as vascular endothelial cells for forming a vascular bed and a fluid such as a medium containing various components to the vascular bed holding chamber 2 . The shape of the vascular endothelial cell supply unit 11 is not particularly limited, and the diameter (maximum diameter) is not particularly limited either, and can be appropriately set according to the purpose.

The first wall portion 3 is interposed between the vascular bed holding chamber 2 and the first channel 4, and the first wall portion 3 is formed with a plurality of first slits 21. As shown in FIG. The first wall portion 3 is a wall for forming the vascular bed retention chamber 2, and the gel-like extracellular matrix containing cells does not pass between the vascular bed retention chamber 2 and the first channel 4. However, it is also a wall that allows components such as low-molecular-weight compounds or proteins to pass through. Here, the low-molecular-weight compounds include carbon sources (sugars), amino acids, vitamins, inorganic salts, etc. necessary for cell culture, and synthetic chemical substances. Serum-derived components such as nutritional factors, angiogenic factors necessary for vascular bed formation, and the like can be mentioned.

The height of the first wall 3 on the side of the vascular bed holding chamber 2 determines the height of the vascular bed holding chamber 2, and the height of the first wall 3 on the side of the first channel 4 is , determines the height of the first channel 4, preferably both being of the same height. The thickness of the first wall portion 3 is not particularly limited, and may be, for example, 50 μm to 500 μm, 50 μm to 300 μm, 50 μm to 250 μm, or 100 μm to 250 μm. Moreover, the length of the first wall portion 3 is not particularly limited, and may be, for example, 50 μm to 500 μm, 50 μm to 300 μm, 50 μm to 250 μm, or 100 μm to 250 μm.

A plurality of first slits 21 are formed in the first wall portion 3, that is, the first wall portion 3 has a micropillar structure. In the first wall portion 3, the plurality of first slits 21 may be formed only in a partial region, or the plurality of first slits 21 may be formed over the entire length. The first slit 21 is a gap having approximately the same height as the first wall portion 3 provided in the first wall portion 3 . The shape of the first slit 21 is not particularly limited. The slit width may have the same width in the thickness direction of the first wall portion 3 (for example, from the vascular bed holding chamber 2 side toward the first channel 4 side), or may have different widths. may For example, as shown in FIG. 4, the slit may have a trapezoidal cross section. Here, the slit width means the narrowest width. The slit width of the first slit 21 does not allow passage of gel-like extracellular matrix containing cells, but may be a width through which components such as low-molecular-weight compounds or proteins can pass. It may be ˜400 μm, 50 μm to 300 μm, 50 μm to 200 μm, or 50 μm to 150 μm. Moreover, it is preferable that a plurality of first slits 21 are formed at equal intervals in the first wall portion 3 . Thereby, a plurality of blood vessels can be formed at equal intervals and with equal diameters, and uniform flow can be imparted to the three-dimensional tissue. The slit width is such that the liquid gel medium does not leak out due to surface tension when the vascular bed holding chamber is filled with the liquid gel medium.

A first channel 4 is a channel for containing and supplying fluid to the vascular bed retention chamber 2 . The fluid contains components such as angiogenic factors that are required for cells to form a vascular bed to form a vascular bed, which are retained in the vascular bed retention chamber 2 . Also, the first channel 4 may contain cells. The cells may be the same cells retained in the vascular bed retention chamber 2 as the cells for forming the vascular bed. The first channel 4 adjoins the vascular bed holding chamber 2 via the first wall 3 . As a result, the components contained in the fluid contained in the first channel 4 pass through the plurality of first slits 21 on the first wall portion 3 and enter the vascular bed holding chamber 2 due to the concentration gradient. being diffused. The first flow path 4 may include a region adjacent to the vascular bed holding chamber 2 via the first wall portion 3 as well as a region communicating with a first fluid supply, which will be described later. The walls of the area for communicating with this first fluid supply may not have slits.

The shape of the first channel 4 is not particularly limited. Two or more flow paths may be formed so as to surround them either permanently or intermittently. Also, the shape of the bottom surface of the first channel 4 is not limited. In one embodiment, as shown in FIGS. 1-3, a pair of first flow channels 4 are provided, each of the pair of first flow channels 4 being connected to the vascular bed retention chamber 2 by a first wall. 3 are adjacent. The pair of first channels 4 preferably extend in parallel in their respective regions adjacent to the vascular bed retention chamber 2 . That is, a vascular bed holding chamber 2 is sandwiched between a pair of first channels 4 . As a result, a vascular bed having a bilaterally symmetrical structure can be formed, and oxygen and nutrients can be uniformly supplied to the three-dimensional tissue via the formed vascular bed.

The height (depth) of the first channel 4 is not particularly limited, and may be, for example, 30 μm to 500 μm, 40 μm to 400 μm, 50 μm to 300 μm, 50 μm to 200 μm, or 50 μm to 150 μm. Preferably, the height of the first channel 4 is the same as the height of the vascular bed retaining chamber 2 and the first wall 3 in order to form a uniform vascular bed. The width of the first channel 4 may be, for example, 100 μm to 5000 μm, 300 μm to 3000 μm, 500 μm to 2000 μm, or 500 μm to 1500 μm.

The partition wall 5 is a lid provided so as to close an opening 6 which will be described later. Here, closing the opening 6 means that the opening 6 is substantially closed. It means that the opening 6 is closed to such an extent that it does not leak out. As long as the medium containing vascular endothelial cells does not leak out, for example, there may be a gap that allows ventilation. As a result, the opening 6 side (ceiling side) of the vascular bed holding chamber 2 is closed, that is, the partition wall 5 becomes a part of the ceiling of the vascular bed holding chamber 2 . This facilitates the formation of vascular beds. On the other hand, if the septum 5 exists after constructing the vascular bed, the 3D tissue cannot be brought into contact with the vascular bed, and the 3D tissue cannot be cultured. Therefore, the partition wall 5 must be removable. By removing the septum 5 after constructing the vascular bed, the 3D tissue can be brought into contact with the vascular bed, and the 3D tissue can be cultured.

The septum 5 may be placed between the vascular bed retention chamber 2 and the opening 6 or in any region of the opening 6 as long as it can substantially occlude the opening 6 . From the viewpoint of ease of fabrication of the device, it is preferably provided between the vascular bed holding chamber 2 and the opening 6 . In this case, the ceiling of the vascular bed holding chamber 2 is approximately flat, so that the vascular bed holding chamber 2 has no steps in the depth direction, so that the vascular endothelial cells described later can be uniformly seeded, thereby forming a vascular bed. likely to be formed. On the other hand, when the partition wall 5 is installed in the opening 6, the ceiling of the vascular bed holding chamber 2 partially protrudes into the opening 6, so that the vascular bed holding chamber 2 has a step in the depth direction. Although uniform seeding of vascular endothelial cells, which will be described later, may not be possible, a vascular bed can be formed. Therefore, the smaller the step between the septum 5 and the rest of the ceiling of the vascular bed holding chamber 2, the better, and most preferably there is no step. If there is a step, the step is preferably 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, 100 μm or less, or 50 μm or less.

The septum 5 is not particularly limited as long as it can be removed without affecting the vascular bed, and may be removable by, for example, a chemical method or a physical method. Chemical methods include, for example, a method of removing by dissolving with a chelating agent or an enzyme, and physical methods include, for example, methods of removing with heat, ultraviolet rays, and the like.

The shape of the partition wall 5 is not particularly limited as long as it can block the opening 6, and may be, for example, sheet-like, film-like, or plug-like. When the partition wall 5 is sheet-like or film-like, its size may be the same as or larger than the cross section of the opening 6, and its thickness may be, for example, 1 to 100 μm or 10 to 50 μm. The partition 5 may be made of a water-soluble material. Here, a water-soluble material refers to a material that has the property of being dissolved in water or an aqueous solution. The water-soluble material may be, for example, a material that can be dissolved by a chelating agent or an enzyme, or a material that can be naturally dissolved by long-term culture for several days until the vascular bed is established. The partition 5 may be a membrane partition made of these water-soluble materials. Examples of water-soluble materials include alginic acid, hyaluronic acid, cellulose, chitosan, chitin, starch, dextran, heparin, carboxymethylcellulose, polysaccharides such as agarose and salts thereof, proteins such as collagen and fibrin, and polyethylene glycol (PEG). , polyglycol salts (PGA) and the like, and alginates are particularly preferred. Alginates include sodium alginate and the like. Chelating agents capable of dissolving such materials include glycol ether diamine tetraacetic acid (EGTA), ethylenediamine tetraacetic acid (EDTA), citric acid, and the like. Enzymes include cellulase, chitinase, collagenase, plasmin, and the like. is mentioned.

The opening 6 is provided in the device body 1 and communicates with the vascular bed holding chamber 2 . Here, the communication between the opening 6 and the vascular bed holding chamber 2 means, depending on the position of the septum 5, whether the communication is established before use or when the septum 5 is removed during use. includes both cases. That is, when the septum 5 is located inside the opening 6 , the opening 6 and the vascular bed retention chamber 2 communicate with each other before use, but the septum 5 is located between the opening 6 and the vascular bed retention chamber 2 . If located in between, the opening 6 and the vascular bed retention chamber 2 are not in communication prior to use and are in communication upon removal of the septum 5 during use. The opening 6 serves as an inlet for introducing a three-dimensional tissue, and is also a space for containing and culturing the three-dimensional tissue. By removing the septum 5, the three-dimensional tissue introduced through the opening 6 is formed in the vascular bed retention chamber 2 and comes into direct contact with the retained vascular bed. The shape of the opening 6 is not particularly limited, and the diameter (maximum diameter) is not particularly limited either, and can be appropriately set according to the size of the desired three-dimensional structure. The opening 6 may be the same size as or smaller than the upper opening of the vascular bed retention chamber 2 (the opening on the side of the opening 6). The height of the opening 6 is not particularly limited as long as it can accommodate a desired three-dimensional tissue.

The microfluidic device 1 may further include a first fluid supply section 9 communicating with the first channel 4, as shown in FIG. 1 or FIG. According to the first fluid supply unit 9, it becomes possible to supply a fluid such as a culture medium containing not only components such as angiogenic factors but also cells to the first channel 4, thereby supplying angiogenic factors and the like. Components are supplied to the vascular bed retention chamber 2 through a first flow path 4 . The shape of the first fluid supply part 9 is not particularly limited, and the diameter (maximum diameter) is not particularly limited either, and can be appropriately set according to the purpose.

The microfluidic device 1 may further include a second channel 8 provided in the device body, the second channel 8 adjoining the first channel 4 via the second wall 7. However, a plurality of second slits 22 may be formed in the second wall portion 7 . The second channel 8 has the same structure as the first channel 4, and the second wall 7 and the second slit 22 are similar to the first wall 3 and the first slit 21, respectively. may have a structure similar to According to the second channel 8, components necessary for constructing a vascular bed, such as angiogenic factors, can be supplied to the first channel 4. FIG. Like the first channel 4, the second channel 8 can contain a medium containing cells as well as a fluid containing components such as angiogenic factors. Cells accommodated in the second channel 8 may be, for example, cells that produce angiogenic factors.

In the microfluidic device 1 , in one embodiment, a pair of first channels 4 are provided, each of the pair of first channels 4 adjoining the vascular bed holding chamber 2 via the first wall 3 . A pair of second flow paths 8 are provided, and each of the pair of second flow paths 8 is adjacent to each of the pair of first flow paths 4 via the second wall portion 7. may be A pair of first channels 4 extend in parallel in respective regions adjacent to the vascular bed retention chamber 2 and a pair of second channels 8 extend in respective regions adjacent to the first channel 4. are preferably extended in parallel in the regions of As a result, a plurality of blood vessels are formed with equal intervals and diameters, and a uniform flow can be applied to the three-dimensional tissue.

In the microfluidic device 1, the slit width of the second slit 22 in the second wall portion 7 is not particularly limited, and may be, for example, 30 μm to 500 μm, 40 μm to 400 μm, 50 μm to 300 μm, 50 μm to 200 μm, or 50 μm. They may be formed at regular intervals of up to 150 μm. This facilitates the supply of angiogenic factors and the like to the first channel 4 and the vascular bed holding chamber 2 .

In one embodiment, the microfluidic device 1 may further include a second fluid supply section 10 communicating with the second channel 8, as shown in FIG. 1 or FIG. According to the second fluid supply unit 10, it becomes possible to supply a fluid such as a medium containing not only components such as angiogenic factors but also cells to the second channel 8, thereby supplying angiogenic factors and the like. Components are fed through the second channel 8 to the first channel 4 and to the vascular bed retention chamber 2 . The shape of the second fluid supply part 10 is not particularly limited, and the diameter (maximum diameter) is not particularly limited either, and can be appropriately set according to the purpose.

[Method for producing microfluidic device]
Since the microfluidic device according to the present invention has a complicated structure, integral molding is impossible. In the method of manufacturing the microfluidic device according to the present invention, the microfluidic device according to the present invention can be manufactured by preparing the first substrate, the partition walls and the second substrate and placing them in order. An example of a method for manufacturing a microfluidic device will be described below with reference to FIGS.

<First step>
In one embodiment, the first step of the method of manufacturing the microfluidic device 1 includes a first substrate 100 shown in FIG. 5, a second substrate 200 shown in FIG. It is a step of preparing a partition wall 5 having a large thickness.

(first substrate)
A first substrate 100 shown in FIG. 5 includes a first groove portion 104 and a recess portion 102 adjacent to the first groove portion 104 and provided via the first wall portion 3 . The first groove portion 104 is a groove for forming the first channel 4 in the microfluidic device 1 , and the concave portion 102 is a concave portion for forming the vascular bed holding chamber 2 in the microfluidic device 1 . First substrate 100 may further comprise a second groove 108 . The second groove portion 108 is a groove for forming the second channel 8 in the microfluidic device 1 . The first groove portion 104 and the second groove portion 108 are groove portions for forming the first flow path 4 and the second flow path 8, respectively. It has the same structure as the channel 4 and the second channel 8 .

A method for manufacturing the first substrate 100 is not particularly limited. As an example, a manufacturing method by soft lithography will be described below. A specific example of the method for manufacturing the first substrate 100 is shown in Example 1. 8 and 9 are schematic diagrams and may not represent the structure accurately. For the construction of each structure shown in FIGS. 8 and 9, please refer to FIGS. 1-7.

In one embodiment, the first substrate 100 is produced by forming a cured product having a desired pattern using a technique capable of forming a fine three-dimensional structure, such as soft lithography. For example, first a material used for soft lithography is placed on the support member 111, the material is patterned into a pattern 112 according to a pre-designed pattern 112, and then a material for forming the first substrate 100 is applied. It is brought into close contact with the pattern 112 and cured to obtain a cured product 113 (FIG. 8(a)).

Here, the support member 111 is preferably separable from the pattern 112. For example, the support member is made of a material that can be removed by a physical or chemical method without destroying the pattern 112. you can Specific examples include materials such as silicon wafers and glass substrates. When the support member 111 is a silicon wafer or a glass substrate, after the pattern 112 is formed on the support member 111, the support member 111 is treated with trichloro-(1H,1H,2H,2H-perfluorooctyl)silane. Support member 111 can be easily removed.

Materials used in soft lithography may be those commonly used, such as epoxy resins and silicone resins.

The pattern 112 is the reverse pattern of the first substrate 100 , i.e. the recesses on the first substrate 100 , the recesses 102 , the first grooves 104 and the second grooves 108 are projected in the pattern 112 . The first wall portion 3 and the second wall portion 7 , which are convex portions on the first substrate 100 , become concave portions in the pattern 112 .

The material for forming the first substrate 100 is not particularly limited as long as it can adhere to the pattern 112 as a fluid and can be cured. It may be a prepolymer of a flexible resin. Examples of the PDMS prepolymer include those obtained by mixing a PDMS main agent with a curing agent. The ratio of main polymer component to curing agent is not particularly limited, but may be, for example, 5:1 to 15:1.

Spin coating may be used to adhere the prepolymer to the pattern 112 . Also, degassing may be performed in a vacuum chamber to prevent air bubbles from entering. Curing of the prepolymer is not particularly limited as long as it is a normal curing method.

After curing, the first substrate 100 may then be peeled from the pattern 112 and then flipped over and placed on a suitable support 114, as shown in FIG. 8(b). The support 114 is not particularly limited, and examples thereof include a cover glass.

(Partition wall)
The partition 5 is as described above, an example of which is shown in FIG. 6, but not limited to this. The method for producing the partition walls 5 is not particularly limited, and conditions can be appropriately set depending on the dissolvable material. When the partition wall 5 is a membrane partition wall formed of a water-soluble material, it can be formed into a thin film having a desired thickness and size according to a general film-forming method. For example, it can be manufactured by spin coating and curing.

When the partition wall 5 is an alginate film, it can be produced, for example, by a cross-linking reaction with calcium ions. After forming a film by drying an alginate aqueous solution with an appropriate concentration, the film is immersed in a calcium salt solution to undergo a cross-linking reaction, and then, for example, it is sandwiched between two glass plates and pressed to form a thin film having a uniform thickness. It can be produced by cutting to a desired size.

(Second substrate)
The second substrate 200 shown in FIG. 7 has through holes 206 provided in the second substrate body 200 . The through-hole 206 is a through-hole for forming the opening 6 in the microfluidic device 1 . Second substrate 200 may further comprise first fluid feedthrough 209 , second fluid feedthrough 210 , and vascular endothelial cell feedthrough 211 . These through-holes are through-holes for forming the first fluid supply section 9, the second fluid supply section 10, and the vascular endothelial cell supply section 11 in the microfluidic device 1, respectively. These through-holes have the same configuration as the corresponding feeders.

The method of manufacturing the second substrate 200 is not particularly limited as long as through holes such as the through holes 206 are provided at desired positions on a polymer substrate having a desired size. A specific example of the method of manufacturing the second substrate 200 is shown in Example 1.

When the septum 5 is provided between the vascular bed holding chamber 2 and the opening 6 , it is preferable to provide a space for installing the septum 5 in the second substrate 200 . This space can be provided in advance by a mold 302, for example, as shown in FIG. 9(a).

<Second step>
In the second step of the method for manufacturing the microfluidic device 1, the partition wall 5 is provided on the surface of the second substrate 200 in contact with the first substrate 100 so as to block the through hole 206, thereby closing the through hole 206. This is the step of forming the opening 6 . The installation method of the partition 5 is not particularly limited, and for example, it may be installed so as to be sandwiched between the first substrate 100 and the second substrate 200 . Here, the surface of the second substrate 200 in contact with the first substrate 100 is called the bottom surface of the second substrate 200, and the surface of the first substrate 100 in contact with the second substrate 200 is called the first substrate 200. is called the top surface of the substrate.

<Third step>
The third step of the method for manufacturing the microfluidic device 1 is to form an opening on the surface of the second substrate 200 in contact with the first substrate 100 (that is, the bottom surface of the second substrate 200) through the partition wall 5. By installing the first substrate 100 so that the first substrate 100 is installed at a position corresponding to at least a part of the recess 102, the opening (ceiling) of the top surface of the recess 102 is formed by the partition wall 5 and the second substrate 200. , thereby forming the recess 102 as the vascular bed retaining chamber 2 .

By the third step, most of the openings on the top surface of the first substrate 100 are blocked by the septum 5 and the bottom surface of the second substrate 200, thereby providing the vascular bed holding chamber 2 as well as the second substrate. A first channel 4 and a second channel 8 are also formed. On the other hand, the ends of first groove 104 (i.e., first channel 4) and second groove 108 (i.e., second channel 8) are located in a second channel, as shown in FIG. Not blocked by the bottom surface of the substrate 200, the first fluid supply through-hole 209 (ie, the first fluid supply section 9) and the second fluid supply through-hole 210 (ie, the second fluid supply section 10), respectively. are in communication. In addition, the end of the recess 102 (that is, the vascular bed holding chamber 2 ) is not blocked by the bottom surface of the second substrate 200 and communicates with the vascular endothelial cell supply section 11 .

The first substrate 100 and the second substrate 200 may be strongly bonded, for which purpose an adhesive may be used, or the contact surfaces may be plasma-processed. Plasma processing of the surface of materials with (-Si-O-) groups such as PDMS or glass causes siloxane bonds consisting of (-Si-O-Si-) between contact surfaces, resulting in strong bonding. .

In the microfluidic device 1 manufactured according to the microfluidic device manufacturing method of the present invention, a partition wall 5 is provided between the vascular bed holding chamber 2 and the opening 6 . The fabricated microfluidic device 1 may be sterilized by UV irradiation or the like before being subjected to cell culture.

The method for manufacturing the microfluidic device described above is merely an example, and the microfluidic device of the present invention can be manufactured by other methods as well. For example, as a modified method of the above manufacturing method, a PDMS prepolymer is poured into a mold, and each channel (first channel 4, second channel 8) and each supply section (first fluid supply section) shown in FIG. 9, each wall portion (first wall portion 3, second wall portion 7, etc.) forming the second fluid supply portion 10, vascular endothelial cell supply portion 11), and the second wall portion shown in FIG. There is a manufacturing method of manufacturing a substrate in which a polymer substrate 304, which is an example of the second substrate 200, is integrated, then attaching the partition wall 5 to a desired position, and finally joining the substrates that will be the bottoms of the channels. .

[Method for culturing three-dimensional tissue]
The three-dimensional tissue culture method according to the present invention uses the microfluidic device 1 according to the present invention to supply a fluid containing an angiogenic factor to the first flow path 4 and to supply vascular endothelial cells to the vascular bed retention chamber 2 . and a step of removing the septum 5, placing a three-dimensional tissue on the formed vascular bed, and culturing it.

10 and 11 show an example of the process of the three-dimensional tissue culture method. Note that (1a) to (5a) in FIGS. 10 and 11 are schematic diagrams of the inside of the rectangular frame of the disconnection in FIG. 5, and (1b) to (5b) in FIGS. 1 is a schematic diagram of the AA′ cross section of the microfluidic device 1 of No. 1. FIG. Since FIGS. 10 and 11 may not accurately represent the structure of the microfluidic device 1 for the sake of clarity, please refer to FIGS. 1-7 for the structure of each structure shown. sea bream.

Figures 10 (1a) and (1b) show the device before it is used. (2a) and (2b) introduce a medium containing vascular endothelial cells into the vascular bed retention chamber 2, introduce a fluid into the pair of first channels 4, and replace the angiogenic factor with a pair of The introduction of fibroblasts into the second channel 8 is shown. (3a) and (3b) further show that vascular endothelial cells are introduced into the pair of first channels 4 to form a vascular bed.

(4a) and (4b) of FIG. 11 show that the three-dimensional tissue is placed on the vascular bed after removing the septum 5, and (5a) and (6b) show that the blood vessel extends to the inside of the three-dimensional tissue. indicate that

The vascular endothelial cells introduced into the vascular bed retention chamber 2 are vascular endothelial cells that form the vascular bed, and the origin thereof is not particularly limited. It may be an endothelial cell, or a vascular endothelial cell obtained by differentiation induction from a stem cell such as a human iPS cell, or a vein endothelial cell, for example, an umbilical vein endothelial cell. preferred.

The medium containing vascular endothelial cells contains components such as carbon sources, nitrogen sources, vitamins, inorganic salts, synthetic chemicals, serum-derived components, etc. necessary for culturing vascular endothelial cells. This can be arbitrarily selected by the trader. A gelatinous medium is preferred so that the self-organization and tube-forming ability of vascular endothelial cells can be used to facilitate the formation of a vascular bed. The gelatinous medium may be a gelatinous medium having an appropriate hardness, for example, by adding a gelling component to a liquid medium. Gelling components include fibrin, collagen, agar, agarose, and the like. When the gelling component is fibrin, the concentration of the gelling component in the medium may be 2-10 mg/mL. The gel medium containing vascular endothelial cells is introduced into the vascular bed retention chamber 2 by suspending the vascular endothelial cells in the dissolved gel medium to a desired concentration, and then passing the suspension through the vascular endothelial cell supply unit 11. It can be carried out by adding the liquid with a pipette or the like and allowing it to gel.

An angiogenic factor is a factor involved in angiogenesis, and the angiogenic factor used in the culture method of the present invention is not particularly limited. For example, vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF) , hepatocyte growth factor (HGF), monocyte chemoattractant protein-1 (MCP-1), sphingosine-1-phosphate (S1P) and the like, particularly human vascular endothelial growth factor, such as human fibroblasts Preferably, it is human vascular endothelial growth factor produced.

The concentration of the angiogenic factor may be appropriately adjusted depending on the type of angiogenic factor or the concentration of vascular endothelial cells in the vascular bed retention chamber 2, and may be, for example, 0.1 ng/mL to 500 ng/mL, It may be from 5 ng/mL to 300 ng/mL. The concentration of the angiogenic factor here means the concentration when concentration equilibrium is reached between the vascular bed holding chamber 2 and the first channel 4 and then the second channel 8 . When the angiogenic factor is introduced into the first channel 4 or the like, the concentration at the time of introduction includes the desired concentration of the angiogenic factor and the concentration of the vascular bed holding chamber 2, the first channel 4 and the second channel 8. can be calculated based on the capacity of

The angiogenic factor itself may be supplied, or cells that produce the angiogenic factor may be introduced into the microfluidic device 1 to produce the angiogenic factor in situ. Cells that produce angiogenic factors are not particularly limited, and examples thereof include human lung fibroblasts, tumor cells such as glioblastoma, and the like.

As a method of introducing cells that produce angiogenic factors into the microfluidic device 1, they may be introduced into the first channel 4 or the second channel 8 of the microfluidic device 1. Since vascular endothelial cells may be introduced, it is preferable to introduce cells that produce angiogenic factors into the second channel 8 . The concentration of angiogenic factors can be adjusted by the number or concentration of angiogenic factor-producing cells to be introduced. When the cells that produce angiogenic factors are human lung fibroblasts, the concentration of human lung fibroblasts in the first channel 4 or the second channel 8 is 1×10 ³ to 1×10 ⁸ cells. cells/mL, or between 1×10 ⁵ and 1×10 ⁷ cells/mL.

The angiogenic factor-containing fluid may be an aqueous solution such as a buffer solution, or a medium for culturing cells. The fluid may contain other angiogenesis-promoting components in addition to angiogenic factors. The medium may be any liquid medium capable of culturing vascular endothelial cells or cells that produce other angiogenic factors, such as EGM-2 (Lonza).

In the culture method according to the present invention, the step of forming a vascular bed may further include introducing vascular endothelial cells into the first channel 4 . As for the addition method, a suspension of vascular endothelial cells may be added via the first fluid supply unit 9, and the vascular endothelial cells are bonded to the side wall of the first channel 4 on the vascular bed holding chamber 2 side. It is preferable to let As a bonding method, for example, after adding a suspension of vascular endothelial cells, the device is tilted at 90° and the side wall on the side of the vascular bed holding chamber 2 is placed downward for bonding by natural sedimentation of the cells. . A vascular bed is constructed in the vascular bed retention chamber 2 without adding vascular endothelial cells. It tends to connect with cells, and the lumen of the vascular endothelial cells formed in the vascular bed retention chamber 2 easily opens to the first channel 4 side. This allows perfusion of the vascular bed by supplying liquid to the first channel 4 . The vascular endothelial cells to be added are preferably of the same type as the vascular endothelial cells to be added in the vascular bed retention chamber 2 .

After supplying a fluid containing an angiogenic factor to the first channel 4 and supplying a medium containing vascular endothelial cells to the vascular bed holding chamber 2, culture is preferably performed while exchanging the medium. The culture conditions are not particularly limited as long as they are general cell culture conditions. Since the amount of medium and fluid is very small, it is better to control the environmental humidity so that it does not dry out.

It is preferable to further introduce vascular endothelial cells into the first channel 4 2 to 4 days after the start of culture. The vascular endothelial cells introduced into the first flow path 4 can form a vascular network having a lumen from the first flow path 4 toward the vascular bed retention chamber 2, so that the first flow path 4 communicates. A vascular bed can be constructed.

Six to seven days after the start of culture, a vascular bed is formed in the vascular bed holding chamber 2 . The vascular bed formed by the culture method according to the present invention is a vascular bed in which a vascular network having a lumen with a diameter of 30 to 80 μm, in some cases about 50 μm, spreads uniformly.

The culture method according to the present invention includes a step of removing the septum 5 after the formation of the vascular bed, placing a three-dimensional tissue on the formed vascular bed, and culturing.

A three-dimensional tissue (three-dimensional tissue) is a tissue or tissue-like cell aggregate having a three-dimensional structure composed of one or more types of cells. The three-dimensional tissue that can be cultured by the culture method of the present invention is not particularly limited, and may be any three-dimensional tissue composed of cells. For example, it may be a three-dimensional tissue separated from a living body or an artificially constructed three-dimensional tissue. A three-dimensional tissue isolated from a living body may be any three-dimensional tissue isolated from any animal, and may be a normal tissue or an abnormal tissue such as a tumor tissue. Artificially constructed three-dimensional tissues include, for example, spheroids simulating a tumor environment, organoids derived from stem cells such as ES cells or human iPS cells, and the like. A three-dimensional tissue may or may not have blood vessels or a network of blood vessels therein.

When the three-dimensional tissue has blood vessels or a vascular network inside, the blood vessels in the vascular bed and the blood vessels in the three-dimensional tissue communicate (fuse) in about 2 to 7 days after placing the three-dimensional tissue on the vascular bed. , to be able to deliver nutrients or drugs from the vascular bed to the tissue. On the other hand, when the three-dimensional tissue does not have blood vessels or a vascular network inside the three-dimensional tissue, the blood vessels extend from the vascular bed to the three-dimensional tissue in about 2 to 7 days after placing the three-dimensional tissue on the vascular bed. A network of blood vessels is formed within, allowing the supply of nutrients or drugs from the vascular bed to the tissue. This allows the three-dimensional tissue to be cultured for a longer period of time.

A three-dimensional tissue cultured on a vascular bed according to the culture method according to the present invention is capable of transvascularly administering nutrients or drugs into the three-dimensional tissue through channels and the vascular bed connected thereto. While culturing the three-dimensional tissue, it can be used for drug discovery research, developmental or regenerative medicine research, and the like.

The culture method according to the present invention may further include a step of isolating the obtained three-dimensional tissue. The isolated three-dimensional tissue can be used for applications such as in vivo experiments using the three-dimensional tissue and for living body transplantation.

EXAMPLES The present invention will now be described more specifically based on examples. However, the present invention is not limited to the following examples.

<Example 1 Fabrication of microfluidic device>
An example of the microfluidic device shown in FIG. 1 was fabricated. First, the first substrate 100 shown in FIG. 5, the partition wall 5 shown in FIG. 6, and the second substrate 200 shown in FIG. 7 were produced.

As the partition wall 5, an alginate film was used as described in J. Am. Cross-linking reaction with calcium ions according to the method disclosed in Li et al. It was made by First, sodium alginate was dissolved in sterilized water to a concentration of 2% to prepare an alginate solution. After putting 3 mL of the alginate solution into a culture dish with a diameter of 35 mm, it was dried overnight at 35° C. to obtain an alginate film. The alginate membrane was soaked in calcium chloride solution for 1 hour. A calcium chloride solution was prepared by dissolving calcium chloride to 100 mM in deionized water containing 30% (v/v) ethanol. After that, the cross-linked alginate film was washed with deionized water. The alginate film was sandwiched between two glass plates, further clipped, and dried at 35°C. The thickness of the resulting alginic acid film was about 40 μm. Finally, a rectangle of 4.5 mm×5 mm was cut out to form the partition wall 5 in order to attach it to the second substrate.

A first substrate 100 was fabricated by soft lithography according to the procedure shown in FIG. A silicon wafer having a thickness of 100 μm was used as the support member 111 . SU-8 3050 (MicroChem, USA) was patterned into a pattern 112 on the support member 111 by photolithography. To facilitate later removal of the support member 111, it was treated with trichloro-(1H,1H,2H,2H-perfluorooctyl)silane (Sigma). Furthermore, on the patterned SU-8 3050 pattern 112 and the support member 111, polydimethylsiloxane (PDMS) prepolymer (PDMS main agent: curing agent = 10: 1 (weight ratio)) (Dow Corning Toray Co., Ltd.) company, Japan) was spin-coated at 500 rpm for 15 seconds, followed by degassing in a vacuum chamber for 30 minutes. Then, it was allowed to stand and the PDMS prepolymer was cured at 70° C. for 2 hours to obtain a cured product 113 (FIG. 8(a)).

The cured product 113 was peeled off from the pattern 112 made of SU-8 3050 and the support member 111 to obtain a first substrate 100 of 24 mm×24 mm×0.5 mm. The first substrate 100 was turned over and placed on a 24 mm×24 mm slide glass (Matsunami Glass Industry, Japan) as a support 114 (FIG. 8(b)).

The first substrate 100 supported by the fabricated support 114 has, as shown in FIG. It had two grooves 108 . The first wall portion 3 and the second wall portion 7 each have a length of 4.1 mm, and are provided with twenty first slits 21 or second slits 22 at regular intervals. The first and second grooves 108 in the first groove 104 had a length of 6.1 mm and a diameter of 1 mm. The depth of the concave portion 102, the first groove portion 104 and the second groove portion 108 was 100 μm.

A second substrate 200 was produced by the procedure shown in FIG. As the mold 302 of the partition wall 5, two pieces of cellophane tape (total thickness: 100 μm) of 4.5 mm×5 to 6 mm were used. After attaching two pieces of cellophane tape to the center of the plastic container 301, the PDMS prepolymer as the prepolymer 303 was poured into the container 301, degassed in a vacuum chamber for 1 hour, and cured at 80°C for 2 hours. ((a) of FIG. 9). The cured product is peeled from the container 301 and the through holes 206, two pairs of first fluid feed through holes 209, two pairs of second fluid feed through holes 210, and a pair of vascular endothelial cell feed through holes shown in FIG. Each hole 211 was punched with a biopsy punch (Sterile Dermal Biopsy Punch, Kaijirushi Co., Ltd., Japan) to obtain a polymer substrate 304, which is an example of the second substrate 200 (FIG. 9(b)). Through-holes 206, first fluid feed-through hole 209, second fluid feed-through hole 210, and vascular endothelial cell feed-through hole 211 in polymer substrate 304 have diameters of 3.5 mm, 6 mm, 2 mm, and 2 mm, respectively. have.

Subsequently, a PDMS prepolymer as a glue 305 was applied to the gaps for the barrier ribs 5 in the polymer substrate 304, and the membrane-shaped barrier ribs 306 prepared by the above method were placed on the glue 305 and heated at 70°C. By curing for 30 minutes, the polymer substrate 304 and the partition wall 5 were adhered.

Furthermore, the surfaces of the first substrate 100 and the polymer substrate 304 that are in contact with each other are plasma-processed by plasma treatment for 40 seconds with atmospheric plasma (50 sccm, 270 pa, 40 W, Femto Science Cute), and the two are tightly bonded, A microfluidic device 1 was fabricated.

<Example 2 Construction of vascular bed and culture of three-dimensional tissue>
(cell culture)
Human Umbilical Vein Endothelial Cells Expressing Green Fluorescent Protein (GFP-HUVEC, Angio Proteomie, USA) and Human Umbilical Vein Endothelial Cells Expressing Red Fluorescent Protein (RFP-HUVEC, Angio Proteomie) were cultured in endothelial cell growth medium-2 (EGM-2, Lonza, Switzerland) and cells at passages 4-6 were used for experiments. The EGM-2 used contained 1% penicillin and streptomycin (P/S, Thermo Fisher Scientific, USA) instead of gentamicin, one of the EGM-2 supplements. Human lung fibroblasts (hLF (Human Lung Fibroblasts), Lonza) were cultured in fibroblast growth medium-2 (FGM-2, Lonza) and passage 4-5 cells were used for experiments. To prepare co-culture spheroids, hLF and HUVEC were mixed in 200 μL of EGM-2 at a ratio of 4:1 (2.0×10 ⁴ cells:5.0×10 ³ cells). Spheroids were cultured in U-bottomed 96-well plates (Sumitomo Bakelite, Japan) for 1 day before introduction into the microfluidic device.

(Introduction of cells)
Using the microfluidic device 1 produced in Example 1, a three-dimensional tissue was cultured. First, the device was UV sterilized. Fibrinogen powder (Sigma) was dissolved in PBS to 2.8 mg/mL. Collagen I (Corning) was neutralized to pH 7 and dissolved in PBS to 3.0 mg/mL. Furthermore, 107.2 μL of the fibrinogen solution, 8.0 μL of the collagen I solution, and 3.6 μL of aprotinin (Sigma) (5 U/mL) were mixed to prepare a gel solution.

The cultured GFP-HUVEC and hLF were detached with trypsin, neutralized with DMEM medium containing 10% FBS to stop the action of trypsin, collected in a centrifugation tube, and centrifuged at 220×g for 3 minutes. GFP-HUVEC and hLF were suspended in the gel solution to 8×10 ⁶ cells/mL and 5×10 ⁶ cells/mL, respectively. 99 μL of the obtained cell suspension was dispensed, and 1 μL of 50 U/mL thrombin was added. The concentrations of fibrinogen, collagen I, aprotinin and thrombin contained in the obtained cell suspension were 2.5 mg/mL, 0.2 mg/mL, 0.15 U/mL and 0.5 U/mL, respectively.

As shown in FIG. 10, 30 μL of the GFP-HUVEC suspension was introduced into the vascular bed retention chamber 2 via the vascular endothelial cell supply 11 . 20 μL of hLF suspension was introduced into each of the pair of second channels 8 via the second fluid supply 10 . Incubated for 15 minutes in a _CO2 incubator. Subsequently, EGM-2 medium was introduced into the vascular bed holding chamber 2 and the pair of second channels 8 . To prevent drying, the microfluidic device 1 was placed in a 100 mm dish with wet Kimwipes® and incubated in a CO ₂ incubator.

On the second day of culture, a GFP-HUVEC suspension suspended in EGM-2 medium at a concentration of 5×10 ⁶ cells/mL was prepared, and via the first fluid supply unit 9, one of the first 20 μL was introduced into the channel 4 . The plate was incubated at a 90° tilt for 30 minutes, thereby allowing GFP-HUVEC to adhere to the gel surface. Similarly, 20 μL of the GFP-HUVEC suspension was introduced into the other first channel 4 to adhere HUVECs to the gel surface. Subsequently, the medium was changed every two days and cultured. From the 4th day of culture, it was observed that the vascular network (vascular bed) which was being formed in the vascular bed holding chamber 2 and the vascular endothelial cells adhered to the gel surface were joined together.

(Introduction of spheroids)
On day 7 of culture, 20 μL of 50 mM EDTA (Thermo Fisher Scientific)/DPBS (Dulbecco's phosphate-buffered saline) was put into the opening 6 and incubated at room temperature for 3 minutes to dissolve the alginate membrane of the septum 5 . After three washes with DPBS to remove excess EDTA, aspirate medium from all supplies (first fluid supply 9, second fluid supply 10, vascular endothelial cell supply 11);2. Spheroids constructed with 5×10 ⁴ RFP-HUVEC and 1×10 ⁵ hLF were introduced into vascular bed retention chamber 2 . Incubation at 37° C. for 30 minutes in a CO ₂ incubator allowed the spheroids to adhere to the gel. Fresh EGM-2 medium was supplied from all supplies to fill the vascular bed holding chamber 2, the first channel 4, and the second channel 8. The medium was replaced every two days, and spheroids were cultured on the constructed vascular bed.

<Example 3 Visualization of vascular bed perfusion>
To examine the perfusion properties of the vascular beds formed in Example 2, EGM-2 medium was removed from all supplies of the device on day 7 of culture, when the vascular beds were fully formed, before removing septum 5. After that, a DPBS solution of 10 μM rhodamine-dextran (70 kDa, Sigma), which is a fluorescent dye, was introduced into only one of the pair of first channels 4 . The fluorochrome solution was then perfused into the vascular lumen of the vascular bed by the liquid level difference between the pair of first channels 4 . We observed how the fluorescent dye solution flowed through the lumen of the blood vessel. Fluorescence images were taken using an inverted microscope (CKX53, Olympus) equipped with an imaging charge-coupled device camera (DP74) and a 4x objective (Olympus).

The results are shown in FIG. (a) of FIG. 12 is a fluorescence image obtained by observing the fluorescence (GFP) of the vascular endothelial cells themselves, and (b) is a fluorescence image obtained by observing the perfused fluorescent dye (rhodamine-dextran) in the lumen of the blood vessel. . From FIG. 12, it was confirmed that a vascular network having lumens was formed and no solution leaked from the vascular wall of the vascular network.

<Example 4 Analysis of three-dimensional structure>
Four days after spheroid introduction, time-lapse bright-field and fluorescence images were taken using an inverted microscope (CKX53, Olympus) equipped with an imaging charge-coupled device camera (DP74) and a 4x objective (Olympus). Cross-sectional images were acquired via a confocal microscope (FV3000, Olympus) equipped with 20x and 40x lenses and 488 and 561 wavelength spectral lasers. The resulting images were analyzed with ImageJ software (National Institutes of Health, Md.).

The results are shown in FIG. From FIG. 13, it was confirmed that the vascular network extends to the inside of the spheroid, and that the initially prepared vascular bed communicates with the vascular network inside the spheroid.

<Example 5 Examination of the action of the septum in the construction of the vascular bed>
Using the device prepared in Example 1 (device with partition walls) and a device without partition walls, which is the same as the device with partition walls except that the alginate membrane does not have partition walls, the effect of the presence or absence of partition walls was investigated.

Prior to the vascular bed construction experiment, the vascular bed retention chamber 2 of the septumless device was plugged with a chip. Specifically, as shown in FIG. 14, an appropriate amount of PDMS was put into a 1 mL pipette tip and hardened, and the tip of the tip was cut and put into the opening 6 of the septumless device to form the vascular bed holding chamber 2. blocked. After that, using a device with a septum and a device without a septum, construction of a vascular bed was attempted in the same manner as in Example 2. A GFP-HUVEC suspension and an hLF suspension were prepared as in Example 2, 50 μL of the GFP-HUVEC suspension was introduced into the vascular bed holding chamber 2, and 20 μL of the hLF suspension was introduced into a pair of second streams. After each introduction into channel 8, the chip was removed from the septumless device. It was then incubated in a _CO2 incubator for 15 minutes. In addition, EGM-2 medium was introduced into a pair of second channels. On the second day of culture, GFP-HUVEC was attached to the sidewalls of the pair of first flow channels 4 by the same procedure as in Example 2, and the medium was changed every two days and cultured. The vascular network (vascular bed) formed in the vascular bed holding chamber 2 was observed from day 4 of culture.

The results for the device with partitions are shown in FIG. 15(a), and the results for the device without partitions are shown in FIG. 15(b). Compared to the case where the septum was present, the construction of a sufficient vascular network could not be observed in the vascular bed holding chamber 2 when the septum was not present. Without the septum, much of the cell suspension flowed into the center of the vascular bed holding chamber 2, which had no ceiling. As a result, the number of cells in the central portion of the vascular bed holding chamber 2 increased, the supply of culture medium was insufficient, and the vascular network could not be successfully constructed.

DESCRIPTION OF SYMBOLS 1... Device main body 2... Vascular bed holding chamber 3... First wall part 4... First flow path 5... Partition wall 6... Opening part 7... Second wall part 8... Second wall part Flow path 9 First fluid supply unit 10 Second fluid supply unit 11 Vascular endothelial cell supply unit 21 First slit 22 Second slit 100 First substrate DESCRIPTION OF SYMBOLS 102... Concave part 104... First groove part 108... Second groove part 111... Support member 112... Pattern 113... Cured material 114... Support body 200... Second substrate 206... Through hole 209 ... first fluid supply through-hole, 210 ... second fluid supply through-hole, 211 ... vascular endothelial cell supply through-hole, 301 ... container, 302 ... mold, 303 ... prepolymer, 304 ... polymer substrate, 305 ... glue, 306... Membrane partition wall.

Claims (17)

A microfluidic device,
the device itself,
a first channel provided in the device body;
a vascular bed holding chamber adjacent to the first channel in the device body and provided via a first wall;
an opening provided in the device body and communicating with the vascular bed retention chamber;
A partition provided to block the opening,
A plurality of first slits are formed in the first wall,
A microfluidic device, wherein the septum is removable. A pair of the first flow paths are provided,
2. The microfluidic device of claim 1, wherein each of the pair of first channels is adjacent to the vascular bed retention chamber via the first wall. 3. The microfluidic device according to claim 1 or 2, wherein the septum is provided between the vascular bed holding chamber and the opening. The microfluidic device according to any one of claims 1 to 3, wherein the partition is removable by a chemical method or a physical method. The microfluidic device according to any one of claims 1 to 4, wherein the partition is a membrane partition made of a water-soluble material. 6. The microfluidic device according to any one of claims 1 to 5, wherein the first slits in the first wall are formed at regular intervals so that the slit width is 50 µm to 200 µm. The microfluidic device according to any one of claims 1 to 6, further comprising a first fluid supply unit communicating with said first channel. further comprising a second flow path provided in the device body,
The second channel is adjacent to the first channel via a second wall,
The microfluidic device according to any one of claims 1 to 7, wherein the second wall is formed with a plurality of second slits. a pair of the first flow paths are provided, each of the pair of the first flow paths being adjacent to the vascular bed holding chamber via the first wall;
A pair of the second flow paths are provided, and each of the pair of second flow paths is adjacent to each of the pair of first flow paths via the second wall portion. Item 9. The microfluidic device according to Item 8. 10. The microfluidic device according to claim 8, wherein the second slits in the second wall are formed at equal intervals so that the slit width is 50 μm to 200 μm. 11. The microfluidic device according to any one of claims 8 to 10, further comprising a second fluid supply section communicating with said second channel. A method for manufacturing the microfluidic device according to any one of claims 1 to 11,
A first substrate having a first groove, a recess adjacent to the first groove and provided through a first wall, a second substrate having a through hole, and a removable partition and
A step of forming the through hole as the opening by installing the partition so as to close the through hole on the surface of the second substrate that is in contact with the first substrate;
The first substrate is placed on the surface of the second substrate that is in contact with the first substrate through the partition so that the opening is located at a position corresponding to at least a part of the recess. placing to form said recess as said vascular bed retention chamber. In the step of placing the first substrate, each surface where the first substrate and the second substrate are in contact is plasma-processed so that the first substrate is bonded to the second substrate. 13. The manufacturing method according to claim 12, wherein the method is installed in a A method for culturing a three-dimensional tissue using the microfluidic device according to any one of claims 1 to 11,
supplying a fluid containing an angiogenic factor to the first channel, supplying a medium containing vascular endothelial cells to the vascular bed retention chamber, and forming a vascular bed in the vascular bed retention chamber;
removing the septum, placing a three-dimensional tissue on the formed vascular bed, and culturing. The microfluidic device is the microfluidic device according to any one of claims 8 to 11, and supplying an angiogenic factor to the first channel causes fibroblasts to flow into the second channel. 15. The method of culturing according to claim 14, wherein the method is carried out by supplying a medium containing. 16. The culture method according to claim 14 or 15, wherein the step of forming the vascular bed further comprises introducing vascular endothelial cells into the first channel. The culture method according to any one of claims 14 to 16, further comprising a step of isolating a three-dimensional tissue obtained by culturing.

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