Microfluidic device

Abstract

Provided is a microfluidic device. The microfluidic device includes: a first supply channel portion with a first endothelial cell arranged therein, the first supply channel portion extending in a first axial direction; a second supply channel portion with a second endothelial cell arranged therein, the second supply channel portion extending along the first axial direction, away from the first supply channel portion in a second axial direction orthogonal to the first axial direction; and a cell aggregate housing portion that houses a cell aggregate and is sandwiched between the first supply channel portion and the second supply channel portion, which are first channel portions when viewed along the first axial direction. Also, the cell aggregate housing portion includes a cell aggregate restraining portion that maintains the position of the cell aggregate on at least one of a first cell adhesion surface on the first supply channel portion and a second cell adhesion surface on the second supply channel portion.

Description

Microfluidic Devices

[0001] The present disclosure relates to microfluidic devices.

[0002] In living organisms, luminal structures such as blood vessels and lymphatic vessels are formed by endothelial cells. For example, blood vessel formation by vascular endothelial cells includes angiogenesis, in which new blood vessels develop or grow from existing blood vessels, and vasculogenesis, in which new blood vessels are formed in areas where there are no blood vessels. Angiogenesis and vasculogenesis are known to contribute to biological processes such as morphogenesis and tumor growth.

[0003] Recently, a method for evaluating angiogenesis / vasculogenesis activity has been proposed, which involves assessing the extension of vascular endothelial cells using three-dimensional tissues arranged in a microfluidic device. This method is particularly useful for screening and evaluating drugs using three-dimensional tissues, such as tumor models, and for elucidating the mechanism of endothelial cell formation.

[0004] As a device for forming such three-dimensional tissue, for example, Non-Patent Document 1 discloses a device (All-in-One-IMPACT) used to prepare three-dimensional tissue in which a tubular structure is formed by angiogenesis. Devices capable of preparing three-dimensional tissue in which a tubular structure is formed by angiogenesis are also disclosed in Non-Patent Documents 2 to 4. Furthermore, Non-Patent Document 5 discloses a device (OrganoPlate Graft) intended for evaluating angiogenesis.

[0005] Devices for forming such three-dimensional tissues are also disclosed in Patent Documents 1 to 5. For example, Patent Document 2 discloses a cell culture device including a microfluidic network, the microfluidic network including a microfluidic layer including a substrate, a microfluidic channel, and a cover, an organoid compartment extending into the microfluidic layer through a hole provided in the cover and fluidically communicating with the microfluidic channel, and a capillary pressure barrier substantially aligned with the hole and dividing the microfluidic network into a first subvolume including the organoid compartment and a second subvolume including at least a portion of the microfluidic channel.

[0006] Patent Literature 3 discloses a microfluidic device comprising: a device body, a first flow path provided in the device body, a vascular bed holding chamber adjacent to the first flow path in the device body and provided via a first wall portion, an opening provided in the device body and communicating with the vascular bed holding chamber, and a partition wall provided to close the opening, the first wall portion having a plurality of first slits, and the partition wall being removable. With this device, angiogenesis can be evaluated by removing the partition wall, placing a three-dimensional tissue on the formed vascular bed, and culturing it.

[0007] International Publication No. 2016 / 081751 Special Publication No. 2019-517808 International Publication No. 2020 / 262656 International Publication No. 2019 / 191111 Japanese Patent Application Laid-Open No. 2020-188723

[0008] Youngtaek Kim et al., "All-in-one microfluidic design to integrate vascularized tumor spheroid into high-throughput platform", Biotechnol Bioeng., 2022, 119, 3678-3693.Stephanie J. Hachey et al., "An in vitro vascularized micro-tumor model of human colorectal cancer recapitulates in vivo responses to standard-of-care therapy", Lab Chip, 2021, 21, 1333.Kristina Haase et al., " Endothelial Regulation of Drug Transport in a 3D Vascularized Tumor Model", Adv Funct Mater. 2020, 30, 48.Joonha Park et al., "Enabling perfusion through multicellular tumor spheroids promoting lumenization in a vascularized cancer model", Lab on a Chip 2022, 22, 4335-4348.Flavio Bonanini et al., "In vitro grafting of hepatic spheroids and organoids on a microfluidic vascular bed", Angiogenesis 2022, 25, 455-470.

[0009] The device described in Non-Patent Document 1 involves introducing a hydrogel solution containing suspended endothelial cells and interstitial cells into spheroids and gelling them. The spheroids are then embedded in the hydrogel containing endothelial cells and interstitial cells, and then endothelial cells are introduced and cultured around the hydrogel, allowing the endothelial cells to form a luminal structure in the hydrogel and the spheroids within it. Therefore, when the endothelial cells are vascular endothelial cells, the luminal structure formed by the device described in Non-Patent Document 1 is a vascular structure formed by angiogenesis. Furthermore, because the endothelial cells and interstitial cells in the hydrogel are concentrated around the spheroids, it is difficult to control the positional relationship between the site where luminal structure formation begins and the spheroids. Therefore, it is believed that the properties and quality of the three-dimensional tissues produced and the luminal structures formed therein may vary. The devices disclosed in Non-Patent Documents 2-4 and Patent Documents 1 and 2 may also exhibit variability in quality and properties due to the inability to control the positional relationship.

[0010] In the devices described in Non-Patent Document 5 and Patent Document 3, the extension of the luminal structure relative to the cell aggregate occurs only in one direction below the cell tissue.

[0011] The device described in Patent Document 4 can only simultaneously form cell aggregates and vascular tissue, and is not suitable for forming tubular structures through angiogenesis or the like in already formed cell aggregates.

[0012] In the device described in Patent Document 5, the formation of blood vessels from vascular endothelial cells toward tissue involves passage through a porous membrane. Therefore, in tissues prepared using the device described in Patent Document 5, the morphology of the tubular structure changes due to passage through the porous membrane, and there is a possibility that biological tissues cannot be accurately reproduced.

[0013] An object of the present disclosure is to provide a microfluidic device that has an internal tubular structure and can be used to create three-dimensional tissues with stable quality.

[0014] One embodiment of the present invention is [1] "a microfluidic device comprising: a first flow path in which first endothelial cells are arranged and which extends in a first direction; a second flow path in which second endothelial cells are arranged and which is spaced from the first flow path in a second direction perpendicular to the first direction and extends along the first direction; and a cell aggregate storage section in which cell aggregates are stored and which is sandwiched between the first flow path and the second flow path when viewed from the first direction, wherein the first flow path includes a first cell adhesion surface to which the first endothelial cells are adhered, the second flow path includes a second cell adhesion surface to which the second endothelial cells are adhered, and the cell aggregate storage section includes a cell aggregate constraint section that maintains the position of the cell aggregate relative to the first cell adhesion surface and / or the second cell adhesion surface."

[0015] In the microfluidic device according to this embodiment, the first flow path includes a first cell adhesive surface, the second flow path includes a second cell adhesive surface, and the cell aggregate storage section includes a cell aggregate constraint section that maintains the position of the cell aggregate relative to the first cell adhesive surface and / or the second cell adhesive surface. This defines and maintains the positional relationship between the cell aggregates constrained by the cell aggregate constraint section and the endothelial cells adhered to the first cell adhesive surface and / or the second cell adhesive surface. In the microfluidic device according to this embodiment, a tubular structure extends from the endothelial cells adhered to the first cell adhesive surface and / or the second cell adhesive surface toward the cell aggregate. Therefore, defining and maintaining the positional relationship between the cell aggregates and the endothelial cells means that in a three-dimensional tissue prepared using the microfluidic device according to this embodiment, the positional relationship between the cell aggregates and the first cell adhesive surface and / or the second cell adhesive surface to which the endothelial cells adhere (which serves as the starting point for tubular structure formation) remains the same regardless of the trial. As a result, the microfluidic device according to this embodiment can produce three-dimensional tissues of stable quality and high reproducibility.

[0016] In the microfluidic device according to this embodiment, the cell aggregate storage section is sandwiched between the first flow path and the second flow path when viewed from the first direction. Therefore, in the microfluidic device according to this embodiment, a luminal structure extends toward the cell aggregate stored in the cell aggregate storage section from the first cell adhesive surface of the first flow path and the second cell adhesive surface of the second flow path, which are positioned to sandwich the cell aggregate. Therefore, in the microfluidic device according to this embodiment, the luminal structures can be connected to each other from both sides inside the cell aggregate, forming a luminal structure that penetrates the cell aggregate. This makes the microfluidic device suitable for use in elucidating the interactions between luminal structures during the luminal structure formation process.

[0017] In the microfluidic device according to this embodiment, endothelial cells adhered to the first and second cell adhesion surfaces form tubular structures. Therefore, for example, when the endothelial cells are vascular endothelial cells, a three-dimensional tissue containing blood vessels formed by angiogenesis can be produced. Such a three-dimensional tissue can be used for drug screening and evaluation and for elucidating the mechanism of tubular structure formation. For example, when the three-dimensional tissue is a tumor model, it can be suitably used for screening and evaluation of antitumor drugs and for elucidating the mechanism of angiogenesis or vasculogenesis in tumor tissue.

[0018] One embodiment of the present invention is [2] "the microfluidic device according to [1], wherein the first flow path is formed on the first cell adhesion surface and includes a first opening for passing the first endothelial cell to the cell aggregate storage section, and the second flow path is formed on the second cell adhesion surface and includes a second opening for passing the second endothelial cell to the cell aggregate storage section." In the microfluidic device according to this embodiment, the first endothelial cell can pass through the first opening to extend a tubular structure toward the cell aggregate, and the second endothelial cell can pass through the second opening to extend a tubular structure toward the cell aggregate. Therefore, the microfluidic device according to this embodiment can be suitably used to create three-dimensional tissues having a tubular structure therein.

[0019] One embodiment of the present invention is the microfluidic device described in [2], [3] in which "the cell aggregate constraint section overlaps the region between the first opening and the second opening, and the cell aggregate storage section includes a cell aggregate introduction hole connected to an introduction opening formed on a first surface of the microfluidic device, and a cell aggregate introduction section that does not overlap the region between the first opening and the second opening, connects the cell aggregate introduction hole to the cell aggregate constraint section, and guides the cell aggregate introduced through the cell aggregate introduction hole to the cell aggregate constraint section." According to the microfluidic device of this embodiment, by introducing cell aggregates through the cell aggregate introduction hole, the cell aggregates can be guided to the cell aggregate constraint section via the cell aggregate introduction section. Therefore, the cell aggregates can be guided from the cell aggregate introduction hole to the cell aggregate constraint section without manually positioning the cell aggregates held by, for example, tweezers or a pipette. This allows for easier introduction of cell aggregates into the cell aggregate introduction holes than manual placement of cell aggregates, enabling the fabrication of three-dimensional tissues with higher efficiency (high throughput).Furthermore, it allows the fabrication of three-dimensional tissues while eliminating the variability between experimenters that occurs when cell aggregates are placed manually.

[0020] One embodiment of the present invention is [4] "the microfluidic device according to any one of [1] to [3], wherein a plurality of cell aggregate storage sections are provided between the first flow path and the second flow path, and each of the plurality of cell aggregate storage sections is sandwiched between the first flow path and the second flow path when viewed from the first direction." According to the microfluidic device of this embodiment, a single microfluidic device can be used to create a plurality of three-dimensional tissues. This makes it possible to use a single microfluidic device to screen and evaluate drugs and to elucidate the mechanism of tubular structure formation using the results in the plurality of three-dimensional tissues as indicators.

[0021] One embodiment of the present invention is [5] "the microfluidic device according to any one of [1] to [4], wherein the cell aggregate constraint portion includes a first restriction wall surface that restricts movement of the cell aggregate along the first direction." The microfluidic device according to this embodiment allows for the creation of a three-dimensional tissue while restricting movement of the cell aggregate, particularly along the first direction.

[0022] One embodiment of the present invention is [6] "the microfluidic device according to any one of [1] to [5], wherein the cell aggregate constraint portion includes a pair of second restriction wall surfaces that restrict the position of the cell aggregate along the second direction." The microfluidic device according to this embodiment allows the creation of a three-dimensional tissue while restricting the movement of the cell aggregate, particularly along the second direction.

[0023] One embodiment of the present invention is [7] "the microfluidic device according to any one of [1] to [6], wherein the cell aggregate constraint portion includes a third restriction wall surface that restricts the position of the cell aggregate along a third direction perpendicular to the first direction and the second direction." The microfluidic device according to this embodiment allows for the creation of a three-dimensional tissue while restricting the movement of the cell aggregate, particularly along the third direction.

[0024] One embodiment of the present invention is [8] "a method for producing a three-dimensional tissue having an internal tubular structure using the microfluidic device according to any one of [1] to [7], the method comprising the steps of: placing a liquid containing a cell aggregate and a gel-forming polymer compound in the cell aggregate storage section and gelling the liquid so that the cell aggregate remains in the cell aggregate constraint section; placing the first endothelial cells and a first culture medium in the first flow path and adhering the first endothelial cells to the first cell adhesion surface; placing the second endothelial cells and a second culture medium in the second flow path and adhering the second endothelial cells to the second cell adhesion surface; and culturing the cell aggregate, the first endothelial cells, and the second endothelial cells so that the first endothelial cells and the second endothelial cells can form a tubular structure connecting to the interior of the cell aggregate." The production method according to this embodiment makes it possible to produce a three-dimensional tissue having an internal tubular structure with stable quality.

[0025] One embodiment of the present invention is [9] "a method for screening a test substance using the microfluidic device described in any one of [1] to [7], comprising the steps of: placing a liquid containing cell aggregates and a gel-forming polymer compound in the cell aggregate storage section, and gelling the liquid so that the cell aggregates remain in the cell aggregate constraint section; placing the first endothelial cells and a first culture medium in the first flow path and adhering the first endothelial cells to the first cell adhesion surface; placing the second endothelial cells and a second culture medium in the second flow path and adhering the second endothelial cells to the second cell adhesion surface; culturing the cell aggregates, the first endothelial cells, and the second endothelial cells so that the first endothelial cells and the second endothelial cells can form a tubular structure connecting to the interior of the cell aggregate, thereby forming a three-dimensional tissue having a tubular structure therein; and placing the test substance in the first flow path and / or the second flow path, and evaluating the effect of the test substance on the three-dimensional tissue." According to the screening method of this embodiment, it is possible to screen a test substance. In particular, the microfluidic device used in the screening method of this embodiment allows the preparation of three-dimensional tissues of stable quality, as described above, and therefore allows screening to be performed with high reproducibility.

[0026] One embodiment of the present invention is

[10] "an array comprising a first array plate portion and a second array plate portion bonded to the first array plate portion, wherein the first array plate portion and the second array plate portion bonded to each other form a plurality of microfluidic structures, wherein the microfluidic structure has a first flow path in which first endothelial cells are arranged and extending in a first direction, a second flow path in which second endothelial cells are arranged and which is spaced from the first flow path in a second direction perpendicular to the first direction and extends along the first direction, and a cell aggregate storage portion that stores cell aggregates and is sandwiched between the first flow path and the second flow path when viewed from the first direction, wherein the first flow path includes a first cell adhesion surface to which the first endothelial cells are adhered, the second flow path includes a second cell adhesion surface to which the second endothelial cells are adhered, and the cell aggregate storage portion includes a cell aggregate restraint portion that maintains the position of the cell aggregate with respect to the first cell adhesion surface and / or the second cell adhesion surface." The microfluidic structure of the array of this embodiment corresponds to the configuration of the microfluidic device according to [1] above. The array of this embodiment allows for the fabrication of multiple three-dimensional tissues. This allows for the efficient screening and evaluation of multiple test substances using a single array.

[0027] The present disclosure provides a microfluidic device that can be used to produce three-dimensional tissues with stable quality and internal tubular structures, as well as a method for producing three-dimensional tissues and a method for screening test substances using such a device.

[0028] 1 is a perspective view of a microfluidic device 1; FIG. 2 is a plan view of the microfluidic device 1; FIG. 3 is an enlarged cross-sectional view of a cross section of the microfluidic device 1 perpendicular to the third direction (A3 direction); FIG. 4 is an enlarged cross-sectional view of a cross section of the microfluidic device 1 perpendicular to the first direction (A1 direction); FIG. 5 is an enlarged cross-sectional view of a cross section of the microfluidic device 1 perpendicular to the second direction (A2 direction); FIG. 6 is a schematic diagram showing an outline of a method for producing a three-dimensional tissue; (a) is a schematic diagram showing an overview of arranging cell aggregates in a cell aggregate accommodation step; (b) is a schematic diagram showing a first endothelial cell adhesion step and a second endothelial cell adhesion step together in one diagram; (c) is a schematic diagram showing a tubular structure formation step; (a) is a plan view of an array 100; (b) is a VI-axis cross-sectional view of the array 100; FIG. 7 is a schematic diagram showing an outline of a method for producing a microfluidic device 1 in Production Example 1; ６ In Example 1, the HUVEC concentration of the HUVEC suspension was 1.0 × 10 cells / mL. ７ In Example 1, the HUVEC concentration of the HUVEC suspension was 1.5 × 10 cells / mL. ７ 1 shows a fluorescent image of a three-dimensional tissue prepared when the HUVEC concentration of the HUVEC suspension was 5.0×10 cells / mL in Example 1. ６18A and 18B are fluorescent images of three-dimensional tissues prepared at Days 1, 3, 5, 7, and 9 in the case of 1000 cells / mL. This is a diagram showing the change in tumor area ratio in Example 1. This is a diagram showing the change in blood vessel area ratio in Example 1. This is a diagram showing the change in blood vessel area ratio in a tumor in Example 1. This is a diagram showing an outline of a method for preparing a frozen section containing three-dimensional tissue in Example 2. This is a diagram showing an image in which a fluorescent image and a transmitted light image are superimposed on a cross section perpendicular to the second direction (A2 direction) in Example 2, alongside a schematic diagram showing an overview of the three-dimensional tissue in the cross section. This is a fluorescent image of an enlarged area near the center of the central field of view in Example 2 of FIG. 18. (a) is a diagram showing a fluorescent image of a cross section perpendicular to the third direction (A3 direction) alongside a schematic diagram showing an overview of the three-dimensional tissue in the cross section in Example 2. (b) is a diagram showing a fluorescent image of a cross section perpendicular to the second direction (A2 direction) alongside a schematic diagram showing an overview of the three-dimensional tissue in the cross section in Example 2. 21 。 Example 3: A diagram showing the vascular area ratio for each chamber in the device. Example 3: A diagram showing the vascular area ratio for each device. Example 3: A diagram showing the standard deviation of the measurement results within each device (Device) in FIG. 22 and the standard deviation of the measurement results between devices (Chamber) in FIG. 21. Example 7: A diagram showing fluorescent images of the three-dimensional tissues prepared in Examples 4-1, 4-2, 5, and 6 and Comparative Examples 1 and 2, acquired 9 days after the start of culture. Example 7: A diagram comparing the vascular area ratios in Examples 4-1, 4-5, and 4-6. Example 8: A diagram showing a fluorescent image of the three-dimensional tissue acquired 9 days after the start of culture. Example 8: A diagram comparing the vascular area ratio for each concentration of vascular endothelial cells. Example 9: A diagram showing a fluorescent image of the three-dimensional tissue acquired 9 days after the start of culture. Example 9: A diagram comparing the vascular area ratios under various conditions.

[0029] A microfluidic device according to an embodiment of the present disclosure will be described in detail below with reference to the accompanying drawings. In the description of the drawings, identical elements are designated by the same reference numerals, and duplicated explanations will be omitted.

[0030] 1 is a perspective view of a microfluidic device 1 according to an embodiment of the present disclosure. The microfluidic device 1 has a rectangular shape in plan view. The microfluidic device 1 is formed by bonding a device flow channel plate 2 and a device substrate 3 together.

[0031] The size and shape of the microfluidic device 1 are not particularly limited. The shape of the microfluidic device 1 is a rectangular parallelepiped. The size of the microfluidic device 1 is, for example, approximately 23 mm in width, approximately 27 mm in length, and approximately 5.5 mm in thickness. The materials of the device flow path plate 2 and the device substrate 3 are not particularly limited. The materials of the device flow path plate 2 and the device substrate 3 may be, for example, dimethylpolysiloxane or glass. For example, a microfluidic device 1 formed from dimethylpolysiloxane can be cut with a relatively weak force using a cutting tool. With such a microfluidic device 1, as described below, a portion of the microfluidic device 1 can be cut out using a cutting tool such as a biopsy trephine so as to include the prepared three-dimensional tissue. In other words, the microfluidic device 1 formed from dimethylpolysiloxane can be suitably used for evaluation by slicing three-dimensional tissue.

[0032] The device flow channel plate 2 has a rectangular shape in a plan view. The device flow channel plate 2 includes grooves formed on the back side to be bonded to the device substrate 3, and through-holes connected to the grooves. For example, ten through-holes are provided in the device flow channel plate 2. The through-holes extend perpendicular to the flow channel plate main surface 2a (first surface). The device substrate 3 has a rectangular shape in a plan view. The device substrate 3 does not have grooves or through-holes. The device substrate 3 functions as the bottom plate of the microfluidic device 1.

[0033] The microfluidic device 1 has a plurality of cell aggregate storage units 5A, 5B, and 5C, a first flow path system 2R, and a second flow path system 2L. The plurality of cell aggregate storage units 5A, 5B, and 5C are sandwiched between the first flow path system 2R and the second flow path system 2L. Each of the plurality of cell aggregate storage units 5A, 5B, and 5C holds a cell aggregate 91 (see FIG. 3 ) at a predetermined position. The first flow path system 2R and the second flow path system 2L hold endothelial cells 92 (first endothelial cells 92R and second endothelial cells 92L; see FIG. 3 ) near the cell aggregate storage units 5A, 5B, and 5C. The endothelial cells 92 are the starting points of a first luminal structure 93R and a second luminal structure 93L that extend toward the cell aggregate 91. In the following description, when it is not necessary to distinguish between the first luminal structure 93R and the second luminal structure 93L, they may be simply referred to as "luminal structure 93" (see FIG. 3). Similarly, when it is not necessary to separately describe the first endothelial cell 92R and the second endothelial cell 92L for convenience of description, they may be simply referred to as "endothelial cell 92."

[0034] <First Flow Path System, Second Flow Path System> The first flow path system 2R includes a pair of first supply holes 27R, 28R and a first flow path 20R that interconnects the first supply holes 27R, 28R. Similarly, the second flow path system 2L includes a pair of second supply holes 27L, 28L and a second flow path 20L that interconnects the second supply holes 27L, 28L. A first axis A1 is defined that passes through the centers of the cell aggregate storage sections 5A, 5B, and 5C. The first flow path system 2R and the second flow path system 2L are symmetrical with respect to each other with respect to this first axis A1. Therefore, the following description will focus on the first flow path system 2R in detail. Regarding the second flow path system 2L, descriptions of features common to the first flow path system 2R will be omitted as appropriate.

[0035] The first supply holes 27R and 28R are for supplying a cell suspension or a culture medium. The cell suspension or the culture medium may be supplied from one of the first supply holes 27R or the other first supply hole 28R.

[0036] The shape of the first supply holes 27R, 28R is generally circular in plan view. The inner diameter of the first supply hole 27R may be the same as the inner diameter of the first supply hole 28R. That is, the shape of the first supply hole 27R may be the same as the shape of the first supply hole 28R. The inner diameter of the first supply holes 27R, 28R is not particularly limited. The inner diameter of the first supply holes 27R, 28R may be, for example, approximately 2 mm.

[0037] The first supply hole 27R is spaced apart from the first supply hole 28R along the direction of the first axis A1. Here, a second axis A2 perpendicular to the first axis A1 is defined. The first supply holes 27R, 28R are spaced apart from the cell aggregate storage section 5A and the like along the second axis A2. For example, the distance from the first axis A1 to the center of the first supply hole 27R is the same as the distance from the first axis A1 to the center of the first supply hole 28R. The first supply holes 27R, 28R are spaced apart from the outer edges 2e1, 2e2 of the device flow path plate 2 in a plan view.

[0038] The size of the second supply holes 27L, 28L can be explained in the same way as the first supply holes 27R, 28R. Furthermore, when the first axis A1 is used as the reference, the arrangement of the second supply holes 27L, 28L can be explained in the same way as the first supply holes 27R, 28R. Therefore, a detailed explanation of the second supply holes 27L, 28L will be omitted.

[0039] The first supply holes 27R, 28R and the second supply holes 27L, 28L may be arranged so that all four holes can overlap with the wells of a 96-well microplate at the same time. In this case, a supply device commonly used for 96-well microplates (e.g., a multi-channel pipette or an automatic dispenser) can be used to supply the cell suspension or medium from the first supply holes 27R, 28R and / or the second supply holes 27L, 28L.

[0040] A first supply hole 27R is connected to a first end of the first flow path 20R. A first supply hole 28R is connected to a second end of the first flow path 20R. With this connection configuration, the first flow path 20R is connected to a space outside the microfluidic device 1 via the first supply holes 27R and 28R. As a result, for example, the first supply hole 27R can supply a cell suspension or culture medium to the first flow path 20R. In this case, the cell suspension or culture medium supplied to the first flow path 20R may be discharged from the first supply hole 28R. That is, the cell suspension or culture medium can be perfused from the first supply hole 27R through the first flow path 20R toward the first supply hole 28R. Note that the cell suspension or culture medium may be introduced from the first supply hole 28R. In this case, the cell suspension or culture medium can be perfused from the first supply hole 28R toward the first supply hole 27R. The second flow path system 2L formed by the second flow path 20L and the second supply holes 27L and 28L has the same structure and function. The cell suspension or culture medium may be supplied from the second supply hole 27L and discharged from the second supply hole 28L via the second flow path 20L. Alternatively, the cell suspension or culture medium may be supplied from the second supply hole 28L and discharged from the second supply hole 27L via the second flow path 20L.

[0041] The cross-sectional shape of the first flow path 20R is rectangular. The size of the cross-sectional shape of the first flow path 20R may be constant. The size of the cross-sectional shape of the first flow path 20R is not particularly limited. The width of the first flow path 20R may be 0.5 mm. The height of the first flow path 20R may be 0.25 mm.

[0042] As shown in FIG. 2, the first flow path 20R includes a pair of first connection flow path portions 21R and 25R, a pair of first relay flow path portions 22R and 24R, and one first supply flow path portion 23R (first flow path portion).

[0043] Of these, the first connecting flow path portions 21R, 25R exist on a virtual reference line A1K that is parallel to the first axis A1. The first relay flow path portions 22R, 24R extend in the direction of the second axis A2. For example, the length of the first relay flow path portion 22R along the second axis A2 is the same as the length of the first relay flow path portion 24R along the second axis A2.

[0044] A first end of the first connection flow path section 21R is connected to the first supply hole 27R. The first connection flow path section 21R extends from the first supply hole 27R toward the outer edge 2e1 of the device flow path plate 2 along the first axis A1. That is, the first connection flow path section 21R is located between the first supply hole 27R and the outer edge 2e1 of the device flow path plate 2. The second end of the first connection flow path section 21R may be located, for example, in the center between the first supply hole 27R and the outer edge 2e1 of the device flow path plate 2. The second end of the first connection flow path section 21R is connected to a first end of the first relay flow path section 22R.

[0045] The first relay flow path portion 22R connects the first connection flow path portion 21R to the first supply flow path portion 23R. The first relay flow path portion 22R extends along the second axis A2 in a direction approaching the first axis A1. For example, the length of the first relay flow path portion 22R may be longer than the length of the first connection flow path portion 21R. A second end of the first relay flow path portion 22R is connected to a first end of the first supply flow path portion 23R.

[0046] Endothelial cells are arranged and adhered to the first supply channel section 23R. The adhered endothelial cells form a tubular structure toward the cell aggregates 91 in the multiple cell aggregate storage sections 5A, 5B, and 5C. That is, the first supply channel section 23R is the location where tubular structure formation begins when creating a three-dimensional tissue having an internal tubular structure. A first end of the first supply channel section 23R is connected to a second end of the first relay channel section 22R. The first supply channel section 23R extends along the first axis A1 from one outer edge 2e1 to the other outer edge 2e2 of the device channel plate 2. For example, the length of the first supply channel section 23R along the first axis A1 is longer than the distance from the center of the first supply hole 27R to the center of the first supply hole 28R. A second end of the first supply channel section 23R is connected to the first relay channel section 24R.

[0047] The first relay flow path section 24R transfers the cell suspension or culture medium received from the first supply flow path section 23R to the first connection flow path section 25R. A first end of the first relay flow path section 24R is connected to a second end of the first supply flow path section 23R. The first relay flow path section 24R extends along the second axis A2, away from the first axis A1. The length of the first relay flow path section 24R is the same as that of the first relay flow path section 22R. A second end of the first relay flow path section 24R is connected to the first connection flow path section 25R.

[0048] The first connection flow path section 25R transfers the cell suspension or culture medium received from the first relay flow path section 24R to the first supply hole 28R. A first end of the first connection flow path section 25R is connected to a second end of the first relay flow path section 24R. The first connection flow path section 25R extends along the first axis A1 from the outer edge 2e2 of the device flow path plate 2 toward the first supply hole 28R. For example, the length of the first connection flow path section 25R may be the same as that of the first connection flow path section 21R. A second end of the first connection flow path section 25R is connected to the first supply hole 28R. In this manner, the first flow path 20R includes, in this order, the first connection flow path section 21R, the first relay flow path section 22R, the first supply flow path section 23R, the first relay flow path section 24R, and the first connection flow path section 25R from the first supply hole 27R, which communicates with the space outside the microfluidic device 1, toward the first supply hole 28R.

[0049] As already described, the second flow path system 2L formed by the second flow path 20L and the second supply holes 27L, 28L also has a similar structure. For example, the second flow path 20L has a pair of second connection flow path portions 21L, 25L, a pair of second relay flow path portions 22L, 24L, and a second supply flow path portion 23L (second flow path portion). A detailed description of the second flow path system 2L formed by the second flow path 20L and the second supply holes 27L, 28L will be omitted. Note that the shapes of the first flow path 20R and the second flow path 20L described above are merely examples. The shapes of the first flow path 20R and the second flow path 20L are not limited to those described above.

[0050] Here, attention is focused on the first supply channel section 23R and the second supply channel section 23L. Three cell aggregate storage sections 5A, 5B, and 5C are provided in the region between the first supply channel section 23R and the second supply channel section 23L. Of the three cell aggregate storage sections 5A, 5B, and 5C, the central cell aggregate storage section 5B may be provided approximately in the center from the outer edge 2e1 to the outer edge 2e2 along the first axis A1. For example, the distance along the first axis A1 from the cell aggregate storage section 5A to the cell aggregate storage section 5B may be approximately the same as the length of the cell aggregate storage section 5A along the first axis A1. Furthermore, FIG. 2 illustrates three cell aggregate storage sections 5A, 5B, and 5C. However, the number of cell aggregate storage sections provided in one microfluidic device 1 is not limited to three. For example, the number of cell aggregate storage sections provided in the microfluidic device 1 may be two or four. Furthermore, the number of cell aggregate storage sections included in the microfluidic device 1 may be either an odd number or an even number.

[0051] The three cell aggregate storage units 5A, 5B, and 5C shown in Figures 1 and 2 have the same structure as each other, except for their positions in the cell aggregate storage units. Hereinafter, with reference to Figure 3, the cell aggregate storage unit 5A will be described in detail, and a description of the cell aggregate storage units 5B and 5C will be omitted.

[0052] <Cell Aggregate Storage Section> The cell aggregate storage section 5A is a space formed between the flow path plate rear surface 2b of the device flow path plate 2 and the substrate main surface 3a of the device substrate 3. The cell aggregate storage section 5A includes a cell aggregate introduction hole 51S (cell aggregate introduction hole), a degassing hole 52S, a cell aggregate holding area 53S, a first access area 54SR, and a second access area 54SL. The cell aggregate storage section 5A also includes additional areas not included in these. The additional areas connect the cell aggregate introduction hole 51S, the degassing hole 52S, the cell aggregate holding area 53S, the first access area 54SR, and the second access area 54SL to each other. These areas can be filled with hydrogel.

[0053] Cell aggregates 91 are introduced through cell aggregate introduction hole 51S. Cell aggregates 91 introduced into cell aggregate introduction hole 51S move to cell aggregate holding area 53S. Cell aggregates 91 that have moved to cell aggregate holding area 53S remain in place. First access area 54SR connects first supply flow path section 23R to cell aggregate holding area 53S. A tubular structure 93 extends from endothelial cells 92 held in first supply flow path section 23R. The tubular structure 93 reaches cell aggregates 91 held in cell aggregate holding area 53S via first access area 54SR. The same is true for second access area 54SL.

[0054] Here, the first supply channel section 23R, which is not a component of the cell aggregate storage section 5A but is closely related to the cell aggregate storage section 5A, will be described in detail. The first supply channel section 23R is formed by a groove provided in the device channel plate 2 being blocked by the device substrate 3. The first supply channel section 23R is an area surrounded by a lower ceiling surface 211R, a first outer channel wall surface 231R, a first inner channel wall surface 232R, and a first channel floor surface 31R. The lower ceiling surface 211R, the first outer channel wall surface 231R, and the first inner channel wall surface 232R are part of the device channel plate 2. The first channel floor surface 31R is part of the device substrate 3. More specifically, the first channel floor surface 31R is part of the substrate main surface 3a.

[0055] A part of the first inner flow channel wall surface 232R is a first cell adhesion surface 233R configured to allow adhesion of endothelial cells 92. The surface being configured to allow adhesion may mean, for example, that the device flow channel plate 2 is formed of a material, such as glass or dimethylpolysiloxane, to which endothelial cells 92 can adhere. Furthermore, the surface being configured to allow adhesion may mean, for example, that the surface is coated with a coating agent, such as collagen, that enhances cell adhesion.

[0056] The method for adhering cells to the first inner channel wall surface 232R is as follows: First, a cell suspension containing first endothelial cells 92R is introduced into the first channel 20R. Next, the microfluidic device 1 is placed so that the first inner channel wall surface 232R is vertically downward. By performing this procedure, the first endothelial cells 92R can be adhered to the first inner channel wall surface 232R.

[0057] Furthermore, openings of gap regions 55Ra, 55Rb, and 55Rc (described later) are formed in parts of the first inner flow path wall surface 232R. A tubular structure 93 extending from the cells adhered to the first inner flow path wall surface 232R passes through these gap regions 55Ra, 55Rb, and 55Rc and extends toward the cell aggregate 91 held in the cell aggregate storage section 5A.

[0058] The details of the second supply channel section 23L are the same as those described above. The second supply channel section 23L is an area surrounded by a lower ceiling surface 211L, a second outer channel wall surface 231L, a second inner channel wall surface 232L, and a second channel floor surface 31L. Similarly to the first inner channel wall surface 232R, the second inner channel wall surface 232L is configured to allow endothelial cells 92 to adhere thereto. In other words, a portion of the second inner channel wall surface 232L is a second cell adhesion surface 233L configured to allow cells to adhere thereto.

[0059] <Cell aggregate introduction hole 51S> The cell aggregate introduction hole 51S is for introducing the cell aggregate 91. The center of the cell aggregate introduction hole 51S, which is circular in plan view, overlaps with the first axis line A1.

[0060] The cell aggregate introduction hole 51S is an area surrounded by the supply peripheral wall surface 241 provided on the device flow path plate 2 and the substrate main surface 3a. One end of the cell aggregate introduction hole 51S is an opening provided on the flow path plate main surface 2a, and the other end of the cell aggregate introduction hole 51S is the substrate main surface 3a. The inner diameter of the cell aggregate introduction hole 51S is, for example, 1 mm. The inner diameter of the cell aggregate introduction hole 51S is smaller than the inner diameter of the first supply hole 27R.

[0061] <Ventilation hole 52S> The vent hole 52S is for releasing air from the cell aggregate storage section 5A when a solution containing cell aggregates is introduced into the cell aggregate storage section 5A. The center of the vent hole 52S, which is circular in plan view, overlaps with the first axis A1. In other words, the vent hole 52S and the cell aggregate introduction hole 51S are aligned apart from each other on the first axis A1.

[0062] The deaeration hole 52S has a structure similar to that of the cell aggregate introduction hole 51S. The deaeration hole 52S is an area surrounded by the deaeration peripheral wall surface 251 provided on the device flow path plate 2 and the substrate main surface 3a. One end of the deaeration hole 52S is an opening provided on the flow path plate main surface 2a, and the other end of the deaeration hole 52S is on the substrate main surface 3a.

[0063] <Cell aggregate holding area 53S> The cell aggregate holding area 53S is for holding the position of the cell aggregate 91. The cell aggregate holding area 53S is a groove provided in the device flow path plate 2 extending in the direction of the first axis A1. The cell aggregate holding area 53S includes a cell aggregate constraint portion 53S1 that determines the final position of the cells, and a cell aggregate introduction portion 53S2 that guides the cell aggregate 91 from the cell aggregate introduction hole 51S to the cell aggregate constraint portion 53S1. In other words, one end of the cell aggregate holding area 53S is connected to the cell aggregate introduction hole 51S. The other end of the cell aggregate holding area 53S is the cell aggregate constraint portion 53S1.

[0064] More specifically, the cell aggregate holding area 53S is provided between the cell aggregate introduction hole 51S and the degassing hole 52S. One end of the cell aggregate holding area 53S is connected to the cell aggregate introduction hole 51S. The cell aggregate holding area 53S extends toward the degassing hole 52S along the first axis A1. This portion extending along the first axis A1 may be defined as the cell aggregate introduction section 53S2. The width of the cell aggregate introduction section 53S2 is smaller than the inner diameter of the cell aggregate introduction hole 51S. The cell aggregate constraint section 53S1, which is the other end of the cell aggregate holding area 53S, is located in the center between the cell aggregate introduction hole 51S and the degassing hole 52S. In other words, the cell aggregate constraint section 53S1 is not connected to the degassing hole 52S. The cell aggregate constraint section 53S1 has a semicircular shape in a plan view. The inner diameter of cell aggregate restraint section 53S1 is the same as the width of cell aggregate introduction section 53S2. The inner diameter of cell aggregate restraint section 53S1 may be determined according to the size of cell aggregate 91, for example.

[0065] Furthermore, as shown in FIG. 4, the cell aggregate holding region 53S includes an upper holding region portion 53Su and a lower holding region portion 53Sd.

[0066] The upper holding area 53Su is surrounded by an upper ceiling surface 212C included in the device flow path plate 2, a pair of upper wall surfaces 241R, 241L (second regulating wall surfaces), and an upper circumferential surface 291 (first regulating wall surface, see Figure 5).

[0067] The cell aggregate restraint section 53S1 is defined by an upper circumferential surface 291 included in the device flow channel plate 2. This upper circumferential surface 291 regulates the position of the cell aggregate 91 along the first axis A1. As shown in Fig. 5, the cell aggregate restraint section 53S1 and the deaeration hole 52S are connected by a region 56S sandwiched between the lower ceiling surface 211C of the device flow channel plate 2 and the substrate main surface 3a. This region 56S can also be considered to be the region sandwiched between upper wall surfaces 241R and 241L, which will be described later.

[0068] The width of the cell aggregate introduction portion 53S2 described above corresponds to the distance between the pair of upper wall surfaces 241R, 241L. The pair of upper wall surfaces 241R, 241L regulate the position of the cell aggregate 91 along the second axis A2. In other words, the upper holding area 53Su regulates the position of the cell aggregate 91 along the second axis A2. Furthermore, the other end of the upper holding area 53Su is the cell aggregate restraint portion 53S1 described above.

[0069] The upper ceiling surface 212C regulates the position of the cell aggregate 91 along the direction of the third axis A3. The direction from the device substrate 3 toward the device flow path plate 2 along the third axis A3 is defined as the positive direction. The upper ceiling surface 212C (third regulation wall surface) regulates the position in the positive direction of the third axis A3. The direction from the device flow path plate 2 toward the device substrate 3 along the third axis A3 is defined as the negative direction. The position in the negative direction of the third axis A3 is regulated by the substrate main surface 3a. In short, the position of the cell aggregate 91 along the third axis A3 is regulated by the upper ceiling surface 212C of the device flow path plate 2 and the substrate main surface 3a of the device substrate 3.

[0070] In other words, the cell aggregate 91 is maintained in position along the first axis A1, the second axis A2 and the third axis A3 by the upper circumferential surface 291, the pair of upper wall surfaces 241R, 241L, the upper ceiling surface 212C and the substrate main surface 3a.

[0071] In contrast, the position of the lower holding region 53Sd in the negative direction of the third axis A3 is restricted only by the substrate main surface 3a. In other words, the pair of upper wall surfaces 241R, 241L and the upper circumferential surface 291 do not reach the substrate main surface 3a, and gaps are formed between the ends of the pair of upper wall surfaces 241R, 241L and the upper circumferential surface 291 and the substrate main surface 3a. The luminal structure 93 can reach the cell aggregate 91 through these gaps.

[0072] From the perspective of the cell aggregate 91, the cell aggregate 91 includes a restricting portion 91a surrounded by the pair of upper wall surfaces 241R, 241L and the upper circumferential surface 291, and an exposed portion 91b that is not surrounded by the pair of upper wall surfaces 241R, 241L and the upper circumferential surface 291. For example, it can be said that the restricting portion 91a is the upper portion of the cell aggregate 91, and the exposed portion 91b is the lower portion of the cell aggregate 91. The exposed portion 91b is not surrounded by walls in all directions about the third axis A3. Therefore, the luminal structure 93 can reach the exposed portion 91b from all directions about the third axis A3.

[0073] <First Access Region, Second Access Region> The first access region 54SR is for allowing the tubular structure 93 extending from the endothelial cells 92 held in the first supply flow path section 23R to reach the cell aggregate 91. The first access region 54SR is provided between the cell aggregate introduction hole 51S and the degassing hole 52S in the direction along the first axis A1. From another perspective, the first access region 54SR and the second access region 54SL sandwich the cell aggregate restraint section 53S1 in the direction of the second axis A2. This arrangement can shorten the distance from the endothelial cells 92 to the cell aggregate 91.

[0074] The first access region 54SR includes gap regions 55Ra, 55Rb, and 55Rc and a connecting region 55Rd.

[0075] The gap regions 55Ra, 55Rb, and 55Rc are provided on the first wall 26R, which separates the first supply flow path 23R and the cell aggregate storage section 5A. The first wall 26R is provided with three gap regions 55Ra, 55Rb, and 55Rc. The three gap regions 55Ra, 55Rb, and 55Rc are aligned along the first axis A1. For example, of the three gap regions 55Ra, 55Rb, and 55Rc, focus on the central gap region 55Rb. The gap region 55Rb of the first access region 54SR and the gap region 55Lb of the second access region 54SL sandwich the aforementioned cell aggregate restraint section 53S1. Also, focus on the gap region 55Ra located on the side of the cell aggregate introduction hole 51S relative to the gap region 55Rb. The gap region 55Ra of the first access region 54SR and the gap region 55La of the second access region 54SL sandwich the cell aggregate introduction portion 53S2.

[0076] The gap regions 55Ra, 55Rb, and 55Rc increase in width from the first supply channel section 23R toward the cell aggregate holding region 53S. In other words, the planar shape of the gap regions 55Ra, 55Rb, and 55Rc can also be considered tapered. With this planar shape, the width of the first inner opening 55Rt, which is the opening on the cell aggregate storage section 5A side, is greater than the width of the first outer opening 55Rs (first opening), which is the opening facing the first supply channel section 23R. For example, the gap region 55Ra is surrounded by the pair of inclined wall surfaces 551, the lower ceiling surfaces 211R and 211L included in the device channel plate 2, and the substrate main surface 3a.

[0077] 5 illustrates three gap regions 55Ra, 55Rb, and 55Rc. However, the number of gap regions included in the first access region 54SR is not limited to three. The number of gap regions included in the first access region 54SR may be two or four. Furthermore, the number of gap regions included in the first access region 54SR may be either an odd number or an even number.

[0078] Connection region 55Rd connects gap regions 55Ra, 55Rb, and 55Rc to cell aggregate holding region 53S. Connection region 55Rd is a region surrounded by lower ceiling surfaces 211R and 211L of device flow path plate 2, substrate main surface 3a, and first holding region inner wall surface 22Rd. The width from first holding region inner wall surface 22Rd on the first supply flow path section 23R side to second holding region inner wall surface 22Ld on the second supply flow path section 23L side may be larger than the inner diameter of cell aggregate introduction hole 51S.

[0079] As described above, the first supply channel section 23R is also sandwiched between the lower ceiling surfaces 211R, 211L and the substrate main surface 3a. In other words, the first supply channel section 23R, the gap regions 55Ra, 55Rb, 55Rc, and the connecting region 55Rd are a continuous, continuous region along the second axis A2. In other words, there are no steps in the direction of the third axis A3 between the first supply channel section 23R, the gap regions 55Ra, 55Rb, 55Rc, and the connecting region 55Rd. The tubular structure 93 extending from the endothelial cells 92 adhered to the first supply channel section 23R can easily reach the cell aggregate 91 by extending in the direction of the second axis A2.

[0080] The second access region 54SL has a structure similar to that of the first access region 54SR. For example, the second access region 54SL includes gap regions 55La, 55Lb, and 55Lc and a connecting region 55Ld. Furthermore, the width of the second inner opening 55Lt, which is the opening on the cell aggregate storage section 5A side, is larger than the width of the second outer opening 55Ls (second opening), which is the opening facing the second supply channel section 23L. A detailed description of the second access region 54SL will be omitted.

[0081] <Effects> The microfluidic device 1 includes a first supply channel portion 23R as a first channel portion in which a first endothelial cell 92R is arranged and which extends in the direction of a first axis A1, a second supply channel portion 23L as a second channel portion in which a second endothelial cell 92L is arranged and which extends along the first axis A1 and is spaced apart from the first supply channel portion 23R as a first channel portion in the direction of a second axis A2 perpendicular to the direction of the first axis A1, and a cell aggregate storage portion 5A that stores a cell aggregate 91 and is sandwiched between the first supply channel portion 23R as a first channel portion and the second supply channel portion 23L as a second channel portion as viewed from the direction of the first axis A1. The first supply channel portion 23R as a first channel portion includes a first cell adhesion surface 233R to which the first endothelial cell 92R is adhered. The second supply flow path section 23L, which is the second flow path section, includes a second cell adhesion surface 233L to which second endothelial cells 92L are adhered. The cell aggregate storage section 5A includes a cell aggregate restraint section 53S1 that maintains the position of the cell aggregate 91 relative to at least one of the first cell adhesion surface 233R and the second cell adhesion surface 233L.

[0082] According to the microfluidic device 1, a tubular structure 93 extends from a first flow path system 2R provided on one side of the cell aggregate storage units 5A, 5B, and 5C toward the cell aggregate 91. Furthermore, according to the microfluidic device 1, a tubular structure 93 extends from a second flow path system 2L provided on the other side of the cell aggregate storage units 5A, 5B, and 5C toward the cell aggregate 91. In other words, a three-dimensional tissue is formed by the tubular structure 93 extending from both sides of the cell aggregate 91. According to the microfluidic device 1, the position of the cell aggregate 91 is maintained by the cell aggregate storage units 5A, 5B, and 5C. Furthermore, the position of the endothelial cells 92 is maintained by the first flow path system 2R and the second flow path system 2L. Therefore, the relative positions of the cell aggregate 91 and the endothelial cells 92 can be set as desired, thereby stabilizing the quality of the three-dimensional tissue including the cell aggregate 91 and the tubular structure 93. Furthermore, the reproducibility of the three-dimensional tissue produced can be improved.

[0083] The first supply flow channel section 23R, which is the first flow channel section, includes a first cell adhesion surface 233R. The second supply flow channel section 23L, which is the second flow channel section, includes a second cell adhesion surface 233L. The cell aggregate storage section 5A includes a cell aggregate constraint section 53S1 that maintains the position of the cell aggregate 91 relative to at least one of the first cell adhesion surface 233R and the second cell adhesion surface 233L. This defines and maintains the positional relationship between the cell aggregate 91 constrained by the cell aggregate constraint section 53S1 and the endothelial cells 92 adhered to at least one of the first cell adhesion surface 233R and the second cell adhesion surface 233L. In the microfluidic device 1, a tubular structure 93 extends from the endothelial cells 92 adhered to at least one of the first cell adhesion surface 233R and the second cell adhesion surface 233L toward the cell aggregate 91. By being able to define the positional relationship between the cell aggregate 91 and the endothelial cells 92, the positional relationship between the endothelial cells 92, which are the starting points of the tubular structure 93, and the cell aggregate 91 can be accurately reproduced with each repeated trial. Furthermore, by being able to maintain the positional relationship between the cell aggregate 91 and the endothelial cells 92, the reproduced positional relationship between the endothelial cells 92 and the cell aggregate 91 can be maintained throughout the period in which the tubular structure 93 extends toward the cell aggregate 91. As a result, the microfluidic device 1 can produce three-dimensional tissues of stable quality. Furthermore, the microfluidic device 1 can produce three-dimensional tissues with high reproducibility.

[0084] The cell aggregate storage section 5A is sandwiched between a first supply flow path section 23R, which is a first flow path section, and a second supply flow path section 23L, which is a second flow path section, when viewed from the direction of the first axis A1. Therefore, the luminal structure 93 extends from the first cell adhesion surface 233R of the first supply flow path section 23R, which is a first flow path section, and the second cell adhesion surface 233L of the second supply flow path section 23L, which is a second flow path section, positioned to sandwich the cell aggregate 91, toward the cell aggregate 91 stored in the cell aggregate storage section 5A. As a result, the luminal structures 93 extending from both sides of the cell aggregate 91 toward the cell aggregate 91 are connected inside the cell aggregate 91. As a result, a luminal structure 93 penetrating the cell aggregate 91 can be formed.

[0085] The extension of the luminal structure 93 occurs from the endothelial cells 92 adhered to the first cell adhesion surface 233R and the second cell adhesion surface 233L. Therefore, for example, when the endothelial cells 92 are vascular endothelial cells, a three-dimensional tissue including blood vessels formed by angiogenesis can be produced. Such a three-dimensional tissue can be used for drug screening and evaluation. Furthermore, such a three-dimensional tissue can also be used to elucidate the mechanism of formation of the luminal structure 93. For example, a three-dimensional tissue that is a tumor model can be suitably used for screening and evaluating antitumor drugs. Furthermore, a three-dimensional tissue that is a tumor model can be suitably used to elucidate the mechanism of angiogenesis in tumor tissue. Furthermore, a three-dimensional tissue that is a tumor model can also be suitably used to elucidate the mechanism of vasculogenesis.

[0086] The first supply channel section 23R, which is the first channel section, is formed on the first cell adhesion surface 233R and includes a first outer opening 55Rs (first opening) for passing the first endothelial cells 92R to the cell aggregate storage section 5A. The second supply channel section 23L, which is the second channel section, is formed on the second cell adhesion surface 233L and includes a second outer opening 55Ls (second opening) for passing the second endothelial cells 92L to the cell aggregate storage section 5A. The first endothelial cells 92R can extend their luminal structures 93 toward the cell aggregate 91 by passing through the first outer opening 55Rs. The second endothelial cells 92L can extend their luminal structures 93 toward the cell aggregate 91 by passing through the second outer opening 55Ls. Therefore, this microfluidic device 1 can be suitably used to create three-dimensional tissues having luminal structures 93 therein.

[0087] The cell aggregate constraint portion 53S1 overlaps the area between the first outer opening 55Rs and the second outer opening 55Ls. The cell aggregate storage portion 5A includes a cell aggregate introduction hole 51S (cell aggregate introduction hole) connected to an introduction opening formed on the main surface 2a (first surface) of the flow path plate of the microfluidic device 1, and a cell aggregate introduction portion 53S2 that includes a portion that does not overlap the area between the first outer opening 55Rs and the second outer opening 55Ls and is connected from the cell aggregate introduction hole 51S to the cell aggregate constraint portion 53S1 and guides the cell aggregate 91 introduced from the cell aggregate introduction hole 51S to the cell aggregate constraint portion 53S1. By introducing the cell aggregate 91 through the cell aggregate introduction hole 51S, the microfluidic device 1 can guide the cell aggregate 91 to the cell aggregate constraint portion 53S1 via the cell aggregate introduction portion 53S2. Therefore, the cell aggregate 91 can be guided from the cell aggregate introduction hole 51S to the cell aggregate constraint portion 53S1 without manually placing the cell aggregate 91 held with, for example, tweezers or a pipette. This makes it easier to introduce the cell aggregate 91 into the cell aggregate introduction hole 51S than manually placing the cell aggregate 91. As a result, three-dimensional tissue can be produced with higher efficiency (high throughput). It also eliminates the variability between experimenters that can occur when placing the cell aggregate manually.

[0088] A plurality of cell aggregate storage sections 5A are provided between the first supply flow path section 23R, which is the first flow path section, and the second supply flow path section 23L, which is the second flow path section. The plurality of cell aggregate storage sections 5A are sandwiched between the first supply flow path section 23R, which is the first flow path section, and the second supply flow path section 23L, which is the second flow path section, as viewed from the direction of the first axis A1. This microfluidic device 1 is capable of producing a plurality of three-dimensional tissues. This allows for drug screening and evaluation using the results in the plurality of three-dimensional tissues as an indicator. Furthermore, a single microfluidic device 1 can also be used to elucidate the mechanism of formation of a luminal structure 93 using the results in the plurality of three-dimensional tissues as an indicator.

[0089] The cell aggregate restraint portion 53S1 includes an upper circumferential surface 291 that restricts the movement of the cell aggregate 91 along the first axis A1. According to this microfluidic device 1, it is possible to create a three-dimensional tissue while restricting the movement of the cell aggregate 91 along the first axis A1.

[0090] The cell aggregate constraint portion 53S1 includes a pair of upper wall surfaces 241R, 241L that regulate the position of the cell aggregate 91 along the second axis A2. According to this microfluidic device 1, it is possible to produce a three-dimensional tissue while regulating the movement of the cell aggregate 91 along the second axis A2.

[0091] The cell aggregate restraint portion 53S1 includes an upper ceiling surface 212C that restricts the position of the cell aggregate 91 along a third axis A3 that is perpendicular to the first axis A1 and the second axis A2. This microfluidic device 1 allows the creation of a three-dimensional tissue while restricting the movement of the cell aggregate 91 along the third axis A3.

[0092] <Method for Producing Three-Dimensional Tissue Having a Luminal Structure Therein> Another aspect of the present invention is a method for producing a three-dimensional tissue having a luminal structure therein using a microfluidic device according to one aspect of the present invention, the method comprising the steps of: disposing a liquid containing cell aggregates and a gel-forming polymer compound in a cell aggregate storage section and gelling the liquid so that the cell aggregates remain in the cell aggregate restraint section (cell aggregate storage step); disposing first endothelial cells and a first culture medium in a first flow path and adhering the first endothelial cells to the first cell adhesion surface (first endothelial cell adhesion step); disposing second endothelial cells and a second culture medium in a second flow path and adhering the second endothelial cells to the second cell adhesion surface (second endothelial cell adhesion step); and culturing the cell aggregates, the first endothelial cells, and the second endothelial cells so that the first endothelial cells and the second endothelial cells form a luminal structure connecting to the interior of the cell aggregate (luminal structure formation step). Below, the production method according to one embodiment of the present invention will be described using the microfluidic device 1 described above as an example. Also, in the following, for the sake of convenience, there are some places where the cell aggregates, endothelial cells, and luminal structures are not given symbols, but these are the same elements as the cell aggregates 91, endothelial cells 92, and luminal structures 93 described above.

[0093] Fig. 6 is a schematic diagram showing an outline of a fabrication method according to one embodiment of the present invention. In Fig. 6, the left diagram shows an outline of the fabrication method for a cross section perpendicular to the second direction (A2 direction), i.e., the cross section shown in Fig. 5. In Fig. 6, the right diagram shows an outline of the fabrication method for a cross section perpendicular to the third direction (A3 direction), i.e., the cross section shown in Fig. 3.

[0094] <Cell aggregate storage process> In the cell aggregate storage process, a liquid (cell aggregate-gel solution) containing cell aggregates 91 and a gel-forming polymer compound is placed in a cell aggregate storage section 5A, etc., and the liquid is gelled so that the cell aggregates 91 remain in the cell aggregate holding area 53S.

[0095] In the present disclosure, a cell aggregate refers to a three-dimensional tissue (3D tissue) primarily composed of cells. The cell aggregate may be an artificially produced three-dimensional tissue or a living tissue (ex vivo three-dimensional tissue) collected from a human or non-human animal. Examples of artificially produced three-dimensional tissues include spheroids and organoids. The cell aggregate may be an aggregate of cells, or may be an aggregate containing components that may be contained in living tissue in addition to cells. Examples of such components include extracellular matrix components (collagen, fibronectin, proteoglycan, etc.) and gel-forming polymer compounds. The cells contained in the cell aggregate may be labeled with a fluorescent protein, a fluorescent dye, or the like.

[0096] The size of the cell aggregate 91 used in the cell aggregate accommodation step is not particularly limited as long as it can remain in the cell aggregate holding region 53S of the microfluidic device 1, and may be, for example, 100 μm or more, 200 μm or more, 300 μm or more, or 400 μm or more, or 600 μm or less, 550 μm or less, or 500 μm or less. Furthermore, for example, the maximum diameter of the cell aggregate 91 may be 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, or 1.6 times or more the thickness of the cell aggregate restraint portion 53S1 in the third direction, or may be 2.4 times or less, 2.3 times or less, or 2.0 times or less. Furthermore, for example, the maximum diameter of cell aggregate 91 may be 0.6 times or more, 0.7 times or more, or 0.8 times or more, or 1.2 times or less, 1.1 times or less, or 1.0 times or less, the thickness of cell aggregate introduction section 53S2 in the third direction (direction A3). If the maximum diameter of cell aggregate 91 is 1.2 times or less the thickness of cell aggregate introduction section 53S2 in the third direction, cell aggregate 91 is less likely to clog cell aggregate introduction section 53S2 when guiding cell aggregate 91 to cell aggregate constraint section 53S1, and cell aggregate placement can be performed with high efficiency.

[0097] When the cell aggregate is an organoid or ex vivo three-dimensional tissue, the tissue from which it originates can be, for example, tumor tissue or normal tissue.For example, when the tissue from which it originates is tumor tissue, by using vascular endothelial cells to prepare three-dimensional tissue, tumor tissue (tumor model) with blood vessels inside can be obtained by angiogenesis.Also, for example, when the tissue from which it originates is normal tissue, by using lymphatic endothelial cells to prepare three-dimensional tissue, a tissue model with lymphatic vessels inside can be obtained.

[0098] When the cell aggregate is a spheroid, the spheroid may be a spheroid made from tumor cells or normal cells. The spheroid may contain one type of cell or multiple types of cells. Preferably, the tumor cells or normal cells are the cells that primarily constitute the tissue that serves as a model for the three-dimensional tissue to be created. Furthermore, when the cell aggregate is a spheroid, the spheroid may be a spheroid made from tumor cells or normal cells and pericytes. Pericytes are cells that exist in tissues containing luminal structures, adhering to the walls of the luminal structures so as to surround the luminal structures. In tissues, pericytes contribute to the permeability, structural stability, and contractility of the luminal structures. Therefore, when the spheroid contains pericytes, the three-dimensional tissue to be created becomes a model that more closely resembles biological tissue. Pericytes for vascular structures are called pericytes (vascular pericytes, PCs). In a preferred embodiment, the spheroid may be a spheroid made from tumor cells and pericytes.

[0099] When the cell aggregates are spheroids or organoids, the production method may further include a step of producing spheroids or organoids before the cell aggregate containing step. Spheroids or organoids can be produced by methods commonly used by those skilled in the art, and the method is not particularly limited. For example, spheroids can be produced by suspending the cells to be used for spheroid production in a culture medium, adding the cell suspension to a microwell plate that can be used for spheroid production, and incubating the suspension. Microwell plates commonly used by those skilled in the art can be used as appropriate for spheroid production. Examples of such microwell plates include plates that are not cell-adhesive and have wells with concave bottoms. The culture period for spheroid production may be, for example, one to seven days, for example, two days.

[0100] The number of cells to be suspended in the cell culture medium for producing spheroids is not particularly limited and can be appropriately selected by those skilled in the art depending on the type of cells to be used. ３ cells / mL or more 1.00×10 ７ cells / mL or less, 1.00×10 ４ cells / mL or more 1.00×10 ６ cells / mL or less, or 5.00 x 10 ４ cells / mL or more 1.20×10 ５ The concentration may be 1000 cells / mL or less. Furthermore, when preparing spheroids from tumor cells or normal cells and pericytes, the ratio of the number of cells suspended in the cell culture medium can be freely selected by those skilled in the art. The ratio of the number of such cells may be, for example, 1:10 to 50:1, 1:5 to 10:1, or 1:3 to 5:1 (tumor cells or normal cells:pericytes), and specific examples include 1:1 and 3:1.

[0101] Gel-forming polymer compounds are polymer compounds that can exist as a solution (e.g., an aqueous solution) under certain conditions and that gel when stimulated. Gel-forming polymer compounds may be natural or artificial. Known natural polymer compounds that are gel-forming polymer compounds include those classified as proteins or polysaccharides, such as collagen (e.g., collagen I, collagen II, collagen III, collagen V), fibrinogen, chitosan, gelatin, hyaluronic acid, and alginic acid. Known artificial polymer compounds that are gel-forming polymer compounds include synthetic polymers, such as agarose, polyacrylamide, and polyacrylic acid. As gel-forming polymer compounds, one of the compounds described above may be used alone, or two or more may be used. The stimulus that gels these gel-forming polymer compounds varies depending on the type of gel-forming polymer compound. For example, fibrinogen gels when exposed to a certain concentration of thrombin. For example, collagen I and agarose do not gel at low temperatures, but gel at physiological temperatures such as 37°C (temperature sensitivity). For example, alginic acid gels when calcium ions are added to a solution containing alginic acid (ion-sensitive), while polyacrylic acid gels when the pH of an acidic solution containing polyacrylic acid is increased to a value at which the carboxyl groups are deprotonated (pH-sensitive).

[0102] The cell aggregate-gel solution contains one cell aggregate per solution introduced into one cell aggregate storage section 5A, etc. The concentration of the gel-forming polymer compound in the cell aggregate-gel solution is not particularly limited as long as it can retain the cell aggregate 91 in the cell aggregate holding area 53S.

[0103] The cell aggregate-gel solution may further contain pericytes (e.g., pericytes) in addition to the cell aggregates and the gel-forming polymer compound. When the cell aggregate-gel solution contains pericytes, the pericytes are present in the gel surrounding the cell aggregates. This makes it easier for endothelial cells to form a tubular structure in the production of three-dimensional tissue, and can induce the formation of a tubular structure that is closer to that of living tissue. The concentration of pericytes in the cell aggregate-gel solution is not particularly limited, but may be, for example, 1.0 x 10 ５ cells / mL or more 1.0×10 ８ cells / mL or less, 3.0×10 ５ cells / mL or more 3.0×10 ７ cells / mL or less, or 1.0 x 10 ６ cells / mL or more 1.0×10 ７ cells / mL or less, and ６ An example is cells / mL.

[0104] In addition to the above components, the cell aggregate-gel solution may further contain components that may be used in the creation of three-dimensional tissues. Examples of such components include protein degradation inhibitors, extracellular matrix components, culture medium components, growth factors, and cytokines. Examples of protein degradation inhibitors include aprotinin and collagenase inhibitors. Examples of extracellular matrix components include fibronectin and laminin.

[0105] The solvent in the cell aggregate-gel solution is an aqueous solvent, and may be, for example, water or an aqueous solution (e.g., a buffer solution), or a mixed solvent of water and a hydrophilic solvent (e.g., dimethyl sulfoxide or ethanol). The cell aggregate-gel solution can be prepared by adding its components to a solvent and mixing them. For example, the cell aggregate-gel solution can be prepared by adding cell aggregates to a solution containing components other than cell aggregates.

[0106] FIG. 6A is a schematic diagram illustrating an overview of the placement of cell aggregates in the cell aggregate accommodation step. As shown in FIG. 6A, the cell aggregate-gel solution is introduced through the cell aggregate introduction hole 51S, leading the cell aggregate to the cell aggregate constraint section 53S1. At this time, the interior of the cell aggregate accommodation section 5A, etc., is filled with a liquid containing a gel-forming polymer compound. The amount of cell aggregate-gel solution introduced through the cell aggregate introduction hole 51S is not particularly limited, as long as it is an amount that can guide the cell aggregate to the cell aggregate constraint section 53S1 and fill the interior of the cell aggregate accommodation section 5A, etc., with a liquid containing a gel-forming polymer compound. An example of such an amount is 4 μL. In this case, for example, when a temperature-sensitive gel-forming polymer compound is used, the placement of the cell aggregate may be performed under temperature conditions (e.g., room temperature or on ice) at which the gel-forming polymer compound does not gel.

[0107] In the cell aggregate accommodation step, the cell aggregate-gel solution is placed in the cell aggregate accommodation section 5A or the like, and then the solution is gelled. The gelling conditions can be appropriately selected by those skilled in the art depending on the type of gel-forming polymer compound. For example, if the gel-forming polymer compound is temperature-sensitive, the gelling conditions may be to place the microfluidic device 1 in an environment (e.g., in a 37°C incubator) at a temperature that causes the gel-forming polymer compound to gel. For example, if the gel-forming polymer compound is ion-sensitive, ions that cause gelation (e.g., calcium ions) can be added to the cell aggregate-gel solution just before placing the cell aggregate-gel solution in the cell aggregate accommodation section 5A or the like, and then the cell aggregate-gel solution can be placed in the cell aggregate accommodation section 5A or the like and incubated, thereby gelling the solution within the cell aggregate accommodation section 5A or the like. For example, when the gel-forming polymer compound is pH-sensitive, an acid or base can be added to the cell aggregate-gel solution immediately before placing the cell aggregate-gel solution in the cell aggregate storage unit 5A or the like to lower or raise the pH of the cell aggregate-gel solution to a range where gelation occurs, and the cell aggregate-gel solution can then be placed in the cell aggregate storage unit 5A or the like and incubated, thereby gelling the solution within the cell aggregate storage unit 5A or the like. In the cell aggregate storage step, after the solution has gelled, a culture medium can be introduced into the first flow path 20R and the second flow path 20L and further incubated (for example, for one day).

[0108] <First Endothelial Cell Adhesion Step> Figure 6(b) is a schematic diagram illustrating the first endothelial cell adhesion step and the second endothelial cell adhesion step together. In the first endothelial cell adhesion step, endothelial cells (first endothelial cells) and a culture medium (first culture medium) are placed in the first supply channel section 23R, and the endothelial cells (first endothelial cells) are adhered to the first inner channel wall surface 232R. The type of endothelial cells can be selected according to the three-dimensional tissue to be prepared, and may be, for example, vascular endothelial cells or lymphatic endothelial cells. For example, if the three-dimensional tissue to be prepared is a tissue having a vascular structure therein, the endothelial cells may be vascular endothelial cells. The endothelial cells may be labeled with a fluorescent protein, a fluorescent dye, or the like.

[0109] The medium may be any medium commonly used by those skilled in the art for culturing endothelial cells, such as Endothelial Cell Growth Medium (EGM-2, Lonza, CC-3162).

[0110] The placement of endothelial cells and culture medium in the first supply flow path section 23R can be achieved by introducing a culture medium in which endothelial cells are suspended (endothelial cell suspension) into the first flow path 20R through the first supply holes 27R and / or 28R. The concentration of endothelial cells in the endothelial cell suspension is not particularly limited and can be appropriately selected by those skilled in the art depending on the type of endothelial cells and the three-dimensional tissue to be prepared. The concentration of endothelial cells in the endothelial cell suspension is, for example, 1.0 x 10 ４ cells / mL or more 1.0×10 ９ cells / mL or less, 1.0×10 ５ cells / mL or more 1.0×10 ８ cells / mL or less, or 1.0 x 10 ６ cells / mL or more 5.0×10 ７ cells / mL or less, and ６ cells / mL, 1.0×10 ７ cells / mL and 1.5 x 10 ７ An example is cells / mL.

[0111] The first inner flow channel wall surface 232R of the microfluidic device 1 is formed to allow cells to adhere thereto. Thus, when the microfluidic device 1, in which an endothelial cell suspension is disposed in the first supply flow channel section 23R, is placed so that the first inner flow channel wall surface 232R faces vertically downward (for example, by placing the microfluidic device 1 against a wall), the endothelial cells in the endothelial cell suspension adhere to the first inner flow channel wall surface 232R. The time for which the microfluidic device 1 is placed so that the first inner flow channel wall surface 232R faces vertically downward may be, for example, 1 minute to 48 hours, 3 minutes to 24 hours, 5 minutes to 6 hours, or 10 minutes to 2 hours, with 15 minutes being an example. Furthermore, placing the microfluidic device 1 so that the first inner flow channel wall surface 232R faces vertically downward can be performed, for example, at 37°C and 5% CO ２The incubation may be carried out in an incubator.

[0112] <Second Endothelial Cell Adhesion Step> In the second endothelial cell adhesion step, endothelial cells (second endothelial cells) and a culture medium (second culture medium) are placed in the second supply flow channel section 23L, and the endothelial cells (second endothelial cells) are adhered to the second inner flow channel wall surface 232L. The endothelial cells, the culture medium, and the endothelial cell suspension may be the same as those described above in the first endothelial cell adhesion step.

[0113] The first endothelial cells and the second endothelial cells may be the same type of cell or different types of cells. In the microfluidic device 1, luminal structures are formed inside the cell aggregate 91 so as to connect each other from both the first inner channel wall surface 232R and the second inner channel wall surface 232L. Therefore, when the first endothelial cells and the second endothelial cells are the same type, the mechanism of interaction between the luminal structures in the endothelial cells can be elucidated. Furthermore, when the first endothelial cells and the second endothelial cells are different types, the method can be used to elucidate the interaction between the luminal structures formed by the different types of endothelial cells.

[0114] The second inner flow channel wall surface 232L of the microfluidic device 1 is formed to allow cells to adhere thereto. As a result, when the microfluidic device 1 having an endothelial cell suspension disposed in the second supply flow channel section 23L is left standing with the second inner flow channel wall surface 232L facing vertically downward, the endothelial cells in the endothelial cell suspension adhere to the second inner flow channel wall surface 232L. The time and conditions for leaving the device standing can be the same as those described above in the first endothelial cell adhesion step.

[0115] <Luminous structure forming step> In the luminal structure forming step, the cell aggregate, the first endothelial cells, and the second endothelial cells are cultured so that the first endothelial cells and the second endothelial cells can form a luminal structure that connects to the inside of the cell aggregate. Fig. 6(c) is a schematic diagram showing the luminal structure forming step. As shown in Fig. 6(c), in the luminal structure forming step, the microfluidic device 1 after the second endothelial cell adhesion step is incubated to form a three-dimensional tissue having a luminal structure inside. The incubation is carried out under an environment in which cell culture is usually carried out (for example, 37°C, 5% CO ２This may be done by gently shaking or standing in a 200°C incubator.

[0116] Incubation in the luminal structure formation step is performed with the first flow path 20R and the second flow path 20L filled with culture medium. Incubation with the first flow path 20R and the second flow path 20L filled with culture medium may be performed, for example, by adding culture medium through the first supply hole (27R and / or 28R) and the second supply hole (27L and / or 28L) and changing the culture medium every 1 to 3 days. Alternatively, the incubation may be performed by perfusing the culture medium from the first supply hole 27R to the first supply hole 28R and from the second supply hole 27L to the second supply hole 28L, or in the opposite direction.

[0117] The incubation time in the tubular structure forming step can be appropriately selected by those skilled in the art depending on the three-dimensional tissue to be prepared. The incubation time in the tubular structure forming step may be, for example, from half a day to 30 days or from 1 day to 15 days.

[0118] <Three-dimensional tissue> A three-dimensional tissue prepared according to a method of one embodiment of the present invention serves as a model that reproduces three-dimensional biological tissue having an internal tubular structure. Such three-dimensional tissue can be used for drug screening and evaluation and for elucidating the mechanism of tubular structure formation. For example, when the three-dimensional tissue is a tumor model, it can be suitably used for screening and evaluation of antitumor drugs and for elucidating the mechanism of angiogenesis or vasculogenesis in tumor tissue.

[0119] Furthermore, the three-dimensional tissues fabricated according to the method of the present invention have luminal structures formed from starting points whose positional relationship with the cell aggregates is defined and maintained. As a result, the three-dimensional tissues fabricated according to the method of the present invention have stable quality. For example, such three-dimensional tissues enable highly reproducible drug screening and evaluation.

[0120] Furthermore, in a production method according to one embodiment of the present invention, the luminal structures formed on both sides are connected inside the cell aggregate. As a result, a three-dimensional tissue produced according to the production method according to one embodiment of the present invention has a luminal structure penetrating the cell aggregate. For example, such a three-dimensional tissue can be suitably used to elucidate the interaction between luminal structures during the formation process of the luminal structure.

[0121] <Evaluation of Three-Dimensional Tissue> Three-dimensional tissues prepared according to the preparation method of one embodiment of the present invention may be evaluated by applying imaging while the cell aggregate holding region 53S is maintained in the gel. When the device flow channel plate 2 and / or the device substrate 3 are configured to be light-transmitting, the microfluidic device 1 can be directly applied to a fluorescence microscope such as an epi-illumination microscope or a confocal microscope. Therefore, for example, by fluorescently labeling endothelial cells, the shape of the luminal structure within the formed three-dimensional tissue can be evaluated as a fluorescent image. When evaluating such three-dimensional tissues, the device substrate 3 may be formed of a material suitable for use as the bottom plate of an imaging dish, such as glass.

[0122] Three-dimensional tissues prepared according to the method of one embodiment of the present invention may be recovered from the device and evaluated. For example, the three-dimensional tissue can be recovered from the device by applying a stimulus to return the gel in the cell aggregate storage section 5A or the like to a solution state, then supplying a liquid such as culture medium through the degassing hole 52S, and pushing the three-dimensional tissue confined in the cell aggregate holding area 53S out of the cell aggregate introduction hole 51S via the cell aggregate introduction section 53S2. The three-dimensional tissue thus recovered can be evaluated by methods commonly used by those skilled in the art, such as by labeling with hematoxylin-eosin staining or immunological antibody staining and then imaging.

[0123] For example, when the microfluidic device 1 is formed of a material (e.g., dimethylpolysiloxane) that can be cut at room temperature, the three-dimensional tissue prepared according to the method of one embodiment of the present invention may be cryosectioned by directly cutting the three-dimensional tissue held in the cell aggregate holding region 53S along the third direction (A3 direction) to include the three-dimensional tissue, the first wall portion 26R, and the second wall portion 26L. Such cryosectioning can be performed by cutting and recovering the microfluidic device 1 after the tubular structure formation step along the third direction (A3 direction) using a biopsy trephine or the like, including the three-dimensional tissue, the first wall portion 26R, and the second wall portion 26L, and then embedding the recovered composite of the three-dimensional tissue and a portion of the device in OCT compound. Using cryosections prepared in this manner, it is possible to evaluate fluorescent images of cross sections perpendicular to the first and second directions in addition to the cross section perpendicular to the third direction.

[0124] <Method for Evaluating the Formation of a Luminal Structure Within a Three-Dimensional Tissue> One aspect of the present invention may be a method for evaluating the formation of a luminal structure within a three-dimensional tissue, comprising the steps of preparing a three-dimensional tissue containing a luminal structure therein according to a preparation method of one embodiment of the present invention, and evaluating the three-dimensional tissue (three-dimensional tissue evaluation step). The evaluation in the three-dimensional tissue evaluation step can be performed by the method described above in the section <Evaluation of Three-Dimensional Tissue>. Such an evaluation method can evaluate changes in the morphology of the formed luminal structure depending on the type of cell aggregate or endothelial cell, or the conditions for preparing the three-dimensional tissue. Therefore, such an evaluation method can elucidate the mechanism by which endothelial cells form a luminal structure.

[0125] <Test substance screening method> One aspect of the present invention is a method for screening a test substance using a microfluidic device according to one aspect of the present invention, the screening method comprising the steps of: placing a liquid containing cell aggregates and a gel-forming polymer compound in a cell aggregate storage section and gelling the liquid so that the cell aggregates remain in the cell aggregate restraint section (cell aggregate storage step); placing first endothelial cells and a first culture medium in a first flow path and adhering the first endothelial cells to the first cell adhesion surface (first endothelial cell adhesion step); placing second endothelial cells and a second culture medium in a second flow path and adhering the second endothelial cells to the second cell adhesion surface (second endothelial cell adhesion step); culturing the cell aggregates, the first endothelial cells, and the second endothelial cells so that the first endothelial cells and the second endothelial cells can form a luminal structure connecting to the interior of the cell aggregate, thereby forming a three-dimensional tissue with a luminal structure therein (luminal structure formation step); and placing a test substance in the first flow path and / or the second flow path and evaluating the effect of the test substance on the three-dimensional tissue (test substance evaluation step). The cell aggregate accommodation step, the first endothelial cell adhesion step, and the second endothelial cell adhesion step can be carried out in the same manner as in the method for producing a three-dimensional tissue according to one embodiment of the present invention.

[0126] The test substance may be a substance known to have some effect on three-dimensional tissue, or may be a substance with no known effect. The category of the test substance is not particularly limited and may be, for example, an organic molecule or its salt, a protein, a peptide, a nucleic acid, or a complex thereof. The test substance may also be, for example, a substance contained in a library composed of a large number of substances that have or may have a pharmacological effect, such as a compound library, a peptide library, an mRNA library, or an siRNA library.

[0127] A screening method according to one embodiment may include a test substance evaluation step following the tubular structure formation step. In this embodiment, the prepared three-dimensional tissue is exposed to a test substance to evaluate the effect of the test substance. In this case, the tubular structure formation step can be performed in the same manner as in the three-dimensional tissue preparation method according to one aspect of the present invention. In the test substance evaluation step, the test substance can be placed in the first supply channel section 23R and the second supply channel section 23L by being introduced, dissolved in culture medium, through the first supply hole (27R and / or 28R) and the second supply hole (27L and / or 28L). The concentration and placement time of the test substance to be placed (i.e., the time the three-dimensional tissue is exposed to the test substance) can be appropriately determined by one skilled in the art depending on the test substance.

[0128] In another embodiment of the screening method, a test substance evaluation step may be performed simultaneously with the luminal structure formation step. In this embodiment, the effect of the test substance is evaluated using a change in the mode of luminal structure formation in the exposed three-dimensional tissue as an indicator. Therefore, this embodiment allows for the selection of substances that have an effect on the formation of luminal structures in three-dimensional tissue. In this case, the test substance can be placed in the first supply channel section 23R and / or the second supply channel section 23L by dissolving the test substance in the medium introduced through the first supply hole (27R and / or 28R) and / or the second supply hole (27L and / or 28L) during the luminal structure formation step. The concentration of the test substance to be placed can be appropriately determined by those skilled in the art depending on the test substance. Furthermore, the test substance may be placed during the entire incubation time during the luminal structure formation step, or only during part of the incubation time. In other words, the test substance may be exposed to the three-dimensional tissue throughout the entire luminal structure formation step, or only during a portion of the step.

[0129] In the screening method, the effect of a test substance on three-dimensional tissue can be evaluated by evaluating the three-dimensional tissue after exposure to the test substance in a manner similar to the three-dimensional tissue evaluation step of the evaluation method of one embodiment of the present invention. Through such evaluation, a test substance that causes a change in the luminal structure compared to three-dimensional tissue not exposed to the test substance can be selected as a substance that may have an effect on three-dimensional tissue. For example, such changes in the luminal structure can include an increase or decrease in the area occupied by the luminal structure, a change in the shape of the luminal structure, an increase or decrease in the frequency of luminal structure formation in the tested cell aggregate, etc. As a specific example of evaluation, if the three-dimensional tissue is tumor tissue containing blood vessels, a test substance that causes a decrease in the area occupied by the blood vessels and / or the frequency of angiogenesis can be selected as a candidate antitumor drug that can inhibit angiogenesis or the expansion of intratumoral blood vessels.

[0130] <Array> An array according to one embodiment of the present invention will be described in detail below with reference to the accompanying drawings. In the description of the drawings, the same elements are given the same reference numerals and duplicated explanations will be omitted.

[0131] The shape of the array 100 is a rectangular parallelepiped of 127.6 mm × 85.3 mm × 20.5 mm. The shape of the array 100 may be a plate standard that is applicable to a microwell plate reader that is widely used for analyzing 96-well or 384-well microplates.

[0132] FIG. 7 is a plan view of the array 100. As shown in FIG. 7, the array 100 is provided with eight microfluidic structures 1S. Specifically, the array 100 is provided with four microfluidic structures 1S in a direction parallel to a side with a length of 127.6 mm and two microfluidic structures 1S in a direction parallel to a side with a length of 85.3 mm in a plan view. The structure of each of the eight microfluidic structures 1S is the same as that of the microfluidic device 1 (see FIG. 1 ). The array 100 provided with eight microfluidic structures 1S can perform the same amount of assays as eight microfluidic devices 1. The number of microfluidic structures 1S provided in the array 100 is not limited to eight. The number of microfluidic structures 1S provided in the array 100 may be, for example, 2 or more, 3 or more, 4 or more, 6 or more, 8 or more, or 12 or more, or may be 96 or less, or 24 or less. The number of the microfluidic structures 1S provided in the array 100 may be, for example, 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, or 96.

[0133] Four microfluidic structures 1S are arranged in a direction parallel to the 127.6 mm long side, and multiple cell aggregate holding portions 5A, 5B, and 5C are arranged on an axis parallel to the 127.6 mm long side in a planar view. The microfluidic structure 1S is arranged so that the multiple cell aggregate holding portions 5A, 5B, and 5C overlap the positions of wells W10 of a 96-well microplate in a planar view. This allows the array 100 to be used with a microwell plate reader to evaluate (e.g., acquire fluorescent images of) three-dimensional tissues prepared in the multiple cell aggregate holding portions 5A, 5B, and 5C in the same manner as evaluation using a 96-well microplate.

[0134] 8 is a cross-sectional view of the array 100. As shown in Fig. 8, the array 100 is composed of an array flow path plate 2S (first array plate portion) and an array substrate 3S (second array plate portion). The materials and manufacturing methods of the array flow path plate 2S and the array substrate 3S may be the same as those of the device flow path plate 2 and the device substrate 3 that constitute the microfluidic device 1.

[0135] The array 100 is configured as a single array plate by bonding two plate-like members together. For example, the array 100 may be configured as a single plate-like member. For example, the array 100 can be configured as a single plate-like member by using so-called three-dimensional modeling technology.

[0136] A plurality of microfluidic structures 1S are provided on the array flow channel plate 2S. The array flow channel plate 2S has a rectangular shape of 127.6 mm x 85.3 mm in plan view. The array flow channel plate 2S includes grooves formed on the back surface thereof to be bonded to the array substrate 3S, and through-holes connected to the grooves. The array substrate 3S does not have any grooves or through-holes. The array substrate 3S functions as the bottom plate of the array 100. The plurality of microfluidic structures 1S included in the array 100 are formed by the array substrate 3S covering the grooves and through-holes provided in the array flow channel plate 2S.

[0137] As described above, the array 100 includes the array flow channel plate 2S, which is the first array plate portion, and the array substrate 3S, which is the second array plate portion. The array flow channel plate 2S and the array substrate 3S are bonded together to form eight microfluidic structures 1S. The microfluidic structures 1S have a structure similar to that of a microfluidic device (see FIG. 1). That is, the microfluidic structure 1S has a first supply channel portion 23R as a first channel portion in which a first endothelial cell 92R is arranged and which extends in the direction of a first axis A1, a second supply channel portion 23L as a second channel portion in which a second endothelial cell 92L is arranged and which is spaced apart from the first supply channel portion 23R as a first channel portion in the direction of a second axis A2 perpendicular to the direction of the first axis A1 and extends along the first axis A1, and a cell aggregate storage portion 5A that stores a cell aggregate 91 and is sandwiched between the first supply channel portion 23R as a first channel portion and the second supply channel portion 23L as a second channel portion as viewed from the direction of the first axis A1. The first supply channel portion 23R as a first channel portion includes a first cell adhesion surface 233R to which the first endothelial cell 92R is adhered. The second supply flow path section 23L, which is the second flow path section, includes a second cell adhesion surface 233L to which second endothelial cells 92L are adhered. The cell aggregate storage section 5A includes a cell aggregate restraint section 53S1 that maintains the position of the cell aggregate 91 relative to at least one of the first cell adhesion surface 233R and the second cell adhesion surface 233L. By using this configuration, the array 100 can be used to perform screening and evaluation of multiple test substances with high efficiency.

[0138] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0139] <Production Example 1: Fabrication of Microfluidic Device 1> Microfluidic device 1 was fabricated according to the method outlined in FIG. 9 . A 3D printer (microArch® S140, BMF) was used to fabricate a mold made of HTL resin (BMF) for fabricating the device channel plate 2 of microfluidic device 1, as shown in FIG. 9(a) . Subsequently, as shown in FIG. 9(b) , polydimethylsiloxane (PDMS) prepolymer (PDMS base:curing agent = 10:1 (weight ratio)) (Dow Corning Toray Co., Ltd., Japan) was added to the mold, followed by degassing for at least 1 hour in a vacuum chamber. The mixture was then left overnight at 70°C to cure the PDMS prepolymer, yielding a cured product 2p. After the obtained cured product 2p was peeled from the mold, a biopsy trephine was used to form through-holes corresponding to the pair of first supply holes 27R, 28R, the pair of second supply holes 27L, 28L, and the cell aggregate introduction hole 51S and degassing hole 52S of the three cell aggregate storage sections 5A, 5B, and 5C, to obtain a device flow path plate 2. Then, as shown in (c) of FIG. 9 , a glass or polydimethylsiloxane device substrate 3 was attached to the surface of the device flow path plate 2 that had been in contact with the mold, to obtain a microfluidic device 1. Finally, as shown in (d) of FIG. 9 , a reservoir with an outer diameter of 8 mm and an inner diameter of 6 mm for introducing culture medium was attached to the flow path plate main surface 2a of the microfluidic device 1, surrounding the pair of first supply holes 27R, 28R and the pair of second supply holes 27L, 28L.

[0140] The microfluidic device 1 thus obtained had a roughly rectangular parallelepiped shape measuring 23 mm x 27 mm x 5.5 mm. The width of the first flow path 20R and the second flow path 20L was 0.5 mm and the height was 0.25 mm. In the cell aggregate storage sections 5A, 5B, and 5C, the length of the lower holding region 53Sd in the direction of the first axis A1 was 1.3 mm, the width in the direction of the second axis A2 was 1.268 mm, and the height was 0.25 mm. The length of the upper holding region 53Su in the direction of the first axis A1 was 1.3 mm, the width in the direction of the second axis A2 was 0.6 mm, and the height was 0.25 mm. The length of the region 56S in the direction of the first axis A1 was 0.7 mm, the width in the direction of the second axis A2 was 1.268 mm, and the height was 0.25 mm. The cell aggregate introduction hole 51S and the deaeration hole 52S were openings with a diameter of 1.0 mm.

[0141] In the microfluidic device 1 thus obtained, the first outer openings 55Rs of the gap regions 55Ra, 55Rb, and 55Rc and the second outer openings 55Ls of the gap regions 55La, 55Lb, and 55Lc had widths of 0.15 mm in the direction of the first axis A1, and the first inner openings 55Rt of the gap regions 55Ra, 55Rb, and 55Rc and the second inner openings 55Lt of the gap regions 55La, 55Lb, and 55Lc had widths of 0.275 mm in the direction of the first axis A1. The length of each of the gap regions 55Ra, 55Rb, 55Rc, 55La, 55Lb, and 55Lc in the direction of the second axis A2 (the distance between the first inner flow path wall surface 232R and the first retaining region inner wall surface 22Rd, or the distance between the second inner flow path wall surface 232L and the second retaining region inner wall surface 22Ld) was 0.216 mm. The angle formed by the pair of inclined wall surfaces 551 with the first flow path floor surface 31R and the second flow path floor surface 31L was 85°.

[0142] Example 1: Preparation of three-dimensional tissues from tumor spheroids and vascular endothelial cells [Spheroid preparation] ASPS cells (an established cell line of alveolar soft part sarcoma, described in Miwa Tanaka et al., "Modeling alveolar soft part sarcoma unveils novel mechanisms of metastasis," Cancer research, 77(4), 897-907 (2017)) were fluorescently stained by expressing the red fluorescent protein DsRed using a standard method. The fluorescently stained ASPS cells were cultured at 1.25 x 10 in IMDM medium (Wako, 098-06465) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. ５ The resulting cell suspension was placed in a 96-well plate (Sumitomo Bakelite, MS-9096U) at 200 μL / well (2.50 × 10 ４ The seeded cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 2 days to prepare tumor spheroids.

[0143] [Introduction of spheroids into a microfluidic device] Fibrinogen dissolved in Dulbecco's phosphate-buffered saline (Nacalai Tesque, 14249-24) was mixed at 2.5 mg / mL, neutralized collagen I at 0.2 mg / mL, and aprotinin at 0.15 U / mL to obtain a fibrin gel solution. A 50 U / mL thrombin solution was added to the fibrin gel solution at a volumetric concentration of 1% to obtain a thrombin-fibrin gel solution. One tumor spheroid was added to the thrombin-fibrin gel solution prepared above. The same process was repeated to prepare multiple thrombin-fibrin gel solutions containing similar tumor spheroids.

[0144] 4 μL of the above thrombin-fibrin gel solution was collected so that tumor spheroids could be collected, and the tumor spheroids were introduced one by one through the cell aggregate introduction holes 51S of the cell aggregate storage sections 5A, 5B, and 5C of the microfluidic device 1 (hereinafter simply referred to as "device 1") prepared in Production Example 1. As a result, the tumor spheroids were placed in the cell aggregate holding region 53S, and the insides of the cell aggregate storage sections 5A, 5B, and 5C were filled with the thrombin-fibrin gel solution. Thereafter, device 1 was incubated at 37°C and 5% CO ２ The device 1 was then placed in an incubator at 37°C for 15 minutes to allow the thrombin-fibrin gel solution to gel. EGM-2 (Lonza, CC-3162), an endothelial cell culture medium, was introduced from each medium reservoir to a total of 600 μL. As a result, the first flow path 20R and the second flow path 20L of the device 1 were filled with EGM-2. Thereafter, the device 1 was placed in an incubator at 37°C and 5% CO ２ The plate was left standing overnight in an incubator.

[0145] [Introduction of vascular endothelial cells into the microfluidic device] Human umbilical vein endothelial cells (HUVECs) were stained with green fluorescent protein (GFP) by a standard method. The stained HUVECs were then introduced into the microfluidic device at a concentration of 5.0 × 10 ６ cells / mL, 1.0×10 ７ cells / mL or 1.5 x 10 ７The HUVECs were suspended in EGM-2 to a concentration of 100 cells / mL, yielding a HUVEC suspension. 10 μL of the resulting HUVEC suspension was added to the first supply hole 27R and introduced into the first supply channel section 23R. The device 1 was left in an incubator for 15 minutes with the first inner channel wall surface 232R facing vertically downward, allowing the HUVECs to adhere to the first inner channel wall surface 232R. Next, 10 μL of the HUVEC suspension was added to the second supply hole 27L and introduced into the second supply channel section 23L. The device 1 was left in an incubator for 15 minutes with the second inner channel wall surface 232L facing vertically downward, allowing the HUVECs to adhere to the second inner channel wall surface 232L. Then, a total of 600 μL of EGM-2 was introduced from each medium reservoir. As a result, the first flow path 20R and the second flow path 20L of the device 1 were filled with EGM-2. Thereafter, the microfluidic device 1 was placed on an Infinity Rocker mini (Next Advance, hereinafter also referred to as "Rocker") installed in an incubator, and incubated under conditions of a rocking angle of 5 to 7° and a rocking rate of 0.5 cycles / min. During incubation, the medium was replaced with a fresh medium once a day.

[0146] [Evaluation of Three-Dimensional Tissue] The three-dimensional tissues prepared were evaluated 1, 3, 5, 7, or 9 days (Day 1, 3, 5, 7, and 9) after the start of incubation on the Rocker. The evaluation was performed by acquiring fluorescent images of the three-dimensional tissues. Fluorescent images were acquired using a confocal microscope (FV3000, Evident) to capture the fluorescence of GFP and DsRed excited by lasers of 488 nm and 561 nm, respectively. At the same time, differential interference contrast images using transmitted light were acquired. When the HUVEC concentration of the HUVEC suspension was 5.0 × 10 ６ The results for Days 1, 5, and 9 when the HUVEC concentration of the HUVEC suspension was 1.0 × 10 cells / mL are shown in Figure 10. ７ The results for Days 1, 5, and 9 when the HUVEC concentration of the HUVEC suspension was 1.5 × 10 cells / mL are shown in Figure 11. ７The results for Days 1, 5, and 9 in the case of 0.15 cells / mL are shown in Figure 12. In Figures 10 to 12, Chamber 1, Chamber 2, and Chamber 3 correspond to cell aggregate storage sections 5A, 5B, and 5C, respectively. In Figures 10 to 12, the length of the scale bar is 200 μm.

[0147] 10 to 12, it was observed that, at all HUVEC seeding densities, blood vessels gradually extended from the HUVECs on the first inner channel wall surface 232R and the second inner channel wall surface 232L toward the tumor spheroids and even into the tumor spheroids. This demonstrates that the microfluidic device of the present invention can produce three-dimensional tissues with internal tubular structures. In particular, it was demonstrated that the microfluidic device of the present invention can produce tumor tissue models containing internal blood vessels by vascularizing vascular endothelial cells toward the tumor spheroids.

[0148] Also, 5.0 × 10 ６ Figure 13 shows the results of three-dimensional tissue analysis on Days 1, 3, 5, 7, and 9 in Chamber 2 when a 1000 cells / mL HUVEC suspension was seeded. The scale bar in Figure 13 is 200 μm. The area occupied by tumor or blood vessels for each three-dimensional tissue was analyzed using ImageJ software (National Institutes of Health, Maryland). The ratio of the area occupied by tumor (ASPS cells; red fluorescence) to the area of ​​cell aggregate restriction region 53S1 (tumor area ratio, Ratio of tumor area) was calculated and shown in Figure 14. The ratio of the area occupied by blood vessels (HUVECs; green fluorescence) to the area of ​​cell aggregate restriction region 53S1 (vascular area ratio, Ratio of vascular area) was calculated and shown in Figure 15. The ratio of the area occupied by blood vessels (HUVECs; green fluorescence) to the spheroid area (area indicated by a white circle in the fluorescent image in Figure 13) (ratio of invasive vascular area within the tumor) was calculated and the results are shown in Figure 16. The results in Figures 14 to 16 are shown as the mean ± standard deviation for n = 8.

[0149] 13 to 16, it was observed that the area occupied by the tumor increased over time, and that blood vessels extended toward the interior of the tumor. This demonstrates that the microfluidic device of the present invention makes it possible to evaluate the process of creating a three-dimensional tissue containing a luminal structure from the perspectives of both tumor growth and vascular extension. It is believed that such a microfluidic device, which can simultaneously evaluate tumor growth and vascular extension, can be used to evaluate the interaction between tumors and blood vessels during the three-dimensional tissue formation process.

[0150] Example 2: Preparation and Evaluation of Three-Dimensional Tissue Slices from a Microfluidic Device A three-dimensional tissue was prepared using a microfluidic device 1 with a device substrate 3 made of polydimethylsiloxane (PDMS) by the same method as in Example 1. Using the three-dimensional tissue on day 9 of culture, frozen sections were prepared according to the method outlined in FIG. 17 . First, the cell aggregate storage section 5A of the microfluidic device 1 and the three-dimensional tissue prepared therein were cut and collected along the third direction (A3 direction) using a 2 mm diameter biopsy trephine (BPP-20F, Kai Industries) so as to include the three-dimensional tissue, the first wall section 26R, and the second wall section 26L (Cutting, Collecting). The collected spheroids were transferred to a cryomold (Sakura, 4565) together with the PDMS covering the outside. The cryomold was then filled with O.C.T. A compound (Optimal Cutting Temperature compound, Sakura, 4583) was added. The cryomold was frozen in a cryostat (Thermo Fisher Scientific, HM525NX) for more than 2 hours (Freezing). The frozen block was cut by the cryostat across the three-dimensional tissue and in a cross section perpendicular to the second direction or a cross section perpendicular to the third direction to prepare 10 μm thick sections (Sectioning). When preparing a cross section perpendicular to the second direction, the PDMS film was first placed in the cryomold so that it was in contact with the bottom surface of the cryomold, and the O.C.T. compound was frozen. The orientation of the frozen block was then changed so that the cross section perpendicular to the second direction was parallel to the bottom surface of the cryomold, and the O.C.T. was then again applied. The microfluidic device 1 was formed from dimethylpolysiloxane, a mildly cuttable material, and thus it was possible to easily prepare frozen sections containing three-dimensional tissue by cutting the device.

[0151] Fluorescence images were obtained for the obtained sections in the same manner as in Example 1. Transmitted light images were also obtained. Figure 18 shows a cross-section perpendicular to the second direction (A2 direction), with the fluorescent image and transmitted light image superimposed on it, alongside a schematic diagram showing an overview of the three-dimensional tissue in the cross-section. Figure 19 also shows a fluorescent image enlarged near the center of the central field of view in Figure 18. In Figure 18, the length of the scale bar is 100 μm. In Figure 19, the length of the scale bar is 50 μm. In Figures 18 and 19, the left side is the device substrate 3 side, and the right side is the device channel plate 2 side. As shown in Figure 19, the vascular lumen formed in the three-dimensional tissue could be visualized by fluorescent observation of the prepared frozen sections.

[0152] FIG. 20(a) shows a fluorescent image of a cross section perpendicular to the third direction (A3 direction). FIG. 20(b) shows the fluorescent image of a cross section perpendicular to the second direction, alongside a schematic diagram outlining the three-dimensional tissue in the cross section. In FIG. 20(a), the length of the scale bar is 200 μm. In FIG. 20(b), the length of the scale bar is 50 μm. As shown in FIG. 20, it was possible to obtain fluorescent images of both the cross section perpendicular to the third direction and the cross section perpendicular to the second direction in the frozen section prepared using the microfluidic device 1.

[0153] Furthermore, when the fabricated three-dimensional tissue was observed, the cross section perpendicular to the third direction clearly showed blood vessels penetrating into the tumor, and the cross section perpendicular to the second direction clearly showed the formation of vascular cavities within the tumor. This three-dimensional tissue with blood vessels formed within the tumor can be suitably used to evaluate drug administration through blood vessels and the angiogenic potential of tumors. Therefore, it was demonstrated that the microfluidic device 1 can be used to fabricate three-dimensional tissue suitable for such evaluations of drug administration and angiogenic potential.

[0154] Example 3 Comparison of Blood Vessel Area Error Between Cell Aggregate Containers (Chambers) and Between Devices In order to evaluate the stability of the quality of three-dimensional tissues fabricated using the microfluidic device 1, it was investigated whether variations in blood vessel area occur between the cell aggregate containers 5A, 5B, and 5C. For the same purpose, it was also investigated whether variations in blood vessel area occur between devices.

[0155] Three-dimensional tissues were prepared using seven microfluidic devices 1 in the same manner as in Example 1. However, cell aggregate storage section 5A (Chamber 1) was prepared using seven devices, 5B (Chamber 2) using seven devices, and 5C (Chamber 3) using five devices. In this case, three-dimensional tissues were prepared in all of the cell aggregate storage sections 5A, 5B, and 5C in five of the seven devices. Using three-dimensional tissues on day 9 of culture, fluorescent images were obtained, and the area percentage of the region occupied by blood vessels was measured and calculated in the same manner as in Example 1.

[0156] FIG. 21 shows the results of the ratio of the area occupied by the measured blood vessels (vascular area ratio) for each chamber, i.e., cell aggregate storage section 5A (Chamber 1), 5B (Chamber 2), and 5C (Chamber 3). The results in FIG. 21 are shown as mean ± standard deviation, and ns (not significant) in the figure indicates that the p-value in the Tukey test after ANOVA (analysis of variance) was 0.05 or higher. Furthermore, FIG. 22 shows the results of the ratio of the area occupied by the measured blood vessels for each device (i.e., for example, Device 1 shows the results for cell aggregate storage sections 5A, 5B, and 5C of Device 1). FIG. 23 shows the standard deviation of the measurement results within each device (Device) in FIG. 22 and the standard deviation of the measurement results within each chamber (Chamber, standard deviation of the measurement results between devices, S.D.) in FIG. 21.

[0157] 21 to 23, first, the result of FIG. 21 showed no significant difference in the area occupied by blood vessels between the chambers within the device. This revealed that in microfluidic device 1, there was no difference in the efficiency of blood vessel growth between cell aggregate storage sections 5A, 5B, and 5C, and that three-dimensional tissues of consistent quality could be produced regardless of which chamber was used. Furthermore, the results of FIGS. 22 and 23 showed that the standard deviations were small, averaging less than 0.10, both within and between devices. This demonstrated that three-dimensional tissues produced using microfluidic device 1 could be used to perform assays such as drug screening and evaluation and to elucidate the mechanism of luminal structure formation, with minimal device-related error.

[0158] Example 4-1: Preparation of three-dimensional tissue from spheroids containing tumor cells and pericytes, and vascular endothelial cells (1) tRCC cells (an established cell line of translocation renal cell carcinoma) were fluorescently stained by expressing the red fluorescent protein DsRed using a standard method. The fluorescently stained tRCC cells were cultured at 1.0 × 10 in IMDM medium (Wako, 098-06465) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. ５ Pericytes (PC, human placental microvascular pericytes, Angio-Proteomie, cAP-0029) were cultured in the above cell culture medium at a concentration of 1.0 × 10 cells / mL to obtain cell suspension A. ５ The cells were suspended in a volumetric ratio of 1:1 to obtain cell suspension B. Cell suspension A and cell suspension B were mixed at a volume ratio of 1:1 to obtain cell suspension C. Cell suspension C was added to a 96-well plate (Sumitomo Bakelite, MS-9096U) at 200 μL / well (1.0 × 10 ４ The seeded cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator at 150°C for 2 days to prepare spheroids containing tumor cells and pericytes. Thereafter, a three-dimensional tissue consisting of spheroids containing tumor cells and pericytes and vascular endothelial cells was prepared according to the same method as in Example 1.

[0159] Example 4-2: Preparation of three-dimensional tissue from spheroids containing tumor cells and pericytes, and vascular endothelial cells 2> The cell concentrations in cell suspensions A, B, and C were reduced to half of that in Example 4-1 (5.0 × 10 ４ A three-dimensional tissue was prepared from spheroids containing tumor cells and pericytes and vascular endothelial cells in the same manner as in Example 4-1, except that the concentration of the culture medium was changed to 1000 μg / mL.

[0160] Example 5: Preparation of three-dimensional tissue from tumor cell-containing spheroids, pericytes in gel, and vascular endothelial cells. tRCC cells (a translocation renal cell carcinoma cell line) were stained by expressing the red fluorescent protein DsRed using a standard method. The stained tRCC cells were cultured in a cell culture medium (IMDM medium (Wako, 098-06465) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) at a concentration of 1.0 × 10 ５ The cell suspension was seeded into a 96-well plate (Sumitomo Bakelite, MS-9096U) at 200 μL per well (2.00 × 10 cells per well). ４ The seeded cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 2 days to prepare spheroids containing tumor cells.

[0161] Fibrinogen was mixed with Dulbecco's phosphate buffered saline (Nacalai Tesque, 14249-24) at a concentration of 5.0 mg / mL, neutralized collagen I at a concentration of 0.4 mg / mL, and aprotinin at a concentration of 0.3 U / mL to obtain a fibrin gel solution. ６The pericytes were suspended in EGM-2 to a concentration of 1000 cells / mL. The resulting pericyte suspension was mixed with fibrin gel solution at a volume ratio of 1:1 (PC-fibrin gel solution). 1% by volume of 50 U / mL thrombin solution was mixed with the PC-fibrin gel solution to obtain a PC-fibrin-thrombin gel solution. The tumor spheroids prepared above were added to the PC-fibrin-thrombin gel solution. The tumor spheroids prepared above were added. Subsequently, three-dimensional tissue was prepared according to the same method as in Example 1, and a three-dimensional tissue consisting of tumor spheroids, pericytes in the gel, and vascular endothelial cells was produced. In the three-dimensional tissue prepared in this example, pericytes were present in the gel surrounding the spheroids.

[0162] Example 6: Preparation of three-dimensional tissues from spheroids containing tumor cells and pericytes, pericytes in gel, and vascular endothelial cells. Spheroids containing tumor cells and pericytes were prepared in the same manner as in Example 4-1, except that cell suspension A and cell suspension B were mixed at a volume ratio of 3:1 (tRCC: 1.5 × 10 per well). ４ Pericytes per well: 5.0 x 10 ３ A PC-fibrin gel solution was prepared according to the same method as in Example 5, and 1 volume % of a 50 U / mL thrombin solution was mixed with the resulting PC-fibrin gel solution to obtain a PC-fibrin-thrombin gel solution. The tumor spheroids prepared above were added to the PC-fibrin-thrombin gel solution. Spheroids containing tumor cells and pericytes were then added. Thereafter, three-dimensional tissues were prepared according to the same method as in Example 1, and three-dimensional tissues containing spheroids containing tumor cells and pericytes, pericytes in the gel, and vascular endothelial cells were prepared.

[0163] Comparative Example 1: Preparation of three-dimensional tissue from tumor cell-containing spheroids and vascular endothelial cells 1. A cell suspension containing 1.25 × 10 of stained ASPS cells was used. ５ Instead of a cell suspension of 5.0 × 10 cells / mL, 5.0 × 10 stained tRCC cells were ４Spheroids containing tumor cells and three-dimensional tissues containing vascular endothelial cells were prepared in the same manner as in Example 1, except that a cell suspension of 1000 cells / mL (a solution obtained by diluting cell suspension A in Example 4-1 by two times) was used.

[0164] Comparative Example 2: Preparation of three-dimensional tissue from spheroids containing tumor cells and vascular endothelial cells 2. As a cell suspension, 1.25 × 10 of stained ASPS cells were used. ５ Instead of a cell suspension of 1.0 × 10 cells / mL, 1.0 × 10 stained tRCC cells were ５ Spheroids containing tumor cells and three-dimensional tissues using vascular endothelial cells were prepared in the same manner as in Example 1, except that a cell suspension of 1000 cells / mL (cell suspension A in Example 4-1) was used.

[0165] Example 7: Construction of an angiogenesis evaluation system using microfluidic device 1 An investigation was conducted to determine whether angiogenesis in tRCC cell spheroids can be evaluated using microfluidic device 1. For the three-dimensional tissues prepared in Examples 4-1, 4-2, 5, and 6 and Comparative Examples 1 and 2, fluorescent images were obtained 9 days after the start of culture in the same manner as in Example 1, and the presence or absence of angiogenesis was evaluated. The results are shown in Figure 24. In Figure 24, K is 10 ３ , M is 10 ６ and , respectively. In Figure 24, the length of the scale bar is 200 µm. Figure 25 shows the results of comparing the ratio of vascular area to the entire image in Examples 4-1, 5, and 6. The results in Figure 25 are the results of using three devices to create and evaluate two or three three-dimensional tissues per device. The results in Figure 25 are shown as mean values ​​± standard deviation.

[0166] As shown in Figure 24, no angiogenesis was observed under the conditions of Comparative Examples 1 and 2, in which pericytes were absent both within the spheroids and the gel, whereas angiogenesis was observed under the conditions of Examples 4-1, 4-2, 5, and 6, in which pericytes were present within the spheroids and / or the gel. This demonstrates that angiogenesis is unlikely to occur in tRCC cell spheroids in the absence of pericytes. Furthermore, these results demonstrate that angiogenesis can be evaluated according to conditions by evaluation using the microfluidic device 1. Furthermore, as shown in Figure 25, among the conditions of Examples 4-1, 5, and 6, the condition of Example 5, in which pericytes were present in the gel but not within the spheroids, unexpectedly showed that angiogenesis may be most likely to occur actively. This demonstrates that evaluation using the microfluidic device 1 can evaluate angiogenesis according to conditions, not only by determining whether angiogenesis occurs but also by assessing the extent of angiogenesis that occurs. Furthermore, because the microfluidic device 1 can evaluate the degree of angiogenesis in this way, it was strongly suggested that it can be suitably used to screen and evaluate drugs and to elucidate the mechanism of tubular structure formation, using the degree of angiogenesis as an indicator.

[0167] Example 8: Preparation of three-dimensional tissues using tumor cell-containing spheroids, pericytes in gels, and vascular endothelial cells at various concentrations. tRCC cells (a translocation renal cell carcinoma cell line) were stained by expressing the red fluorescent protein DsRed using a standard method. The stained tRCC cells were cultured in a cell culture medium (IMDM medium (Wako, 098-06465) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) at a concentration of 1.0 × 10 ５ The cell suspension was seeded into a 96-well plate (Sumitomo Bakelite, MS-9096U) at 200 μL per well (2.00 × 10 cells per well). ４ The seeded cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 2 days to prepare spheroids containing tumor cells.

[0168] Fibrinogen was mixed with Dulbecco's phosphate buffered saline (Nacalai Tesque, 14249-24) at a concentration of 5.0 mg / mL, neutralized collagen I at a concentration of 0.4 mg / mL, and aprotinin at a concentration of 0.3 U / mL to obtain a fibrin gel solution. ６ The pericytes were suspended in EGM-2 to a concentration of 1.0 x 10 cells / mL. The resulting pericyte suspension was mixed with fibrin gel solution at a volume ratio of 1:1 (PC-fibrin gel solution). 1% by volume of 50 U / mL thrombin solution was mixed with the PC-fibrin gel solution to obtain a PC-fibrin-thrombin gel solution. The tumor spheroids prepared above were added to the PC-fibrin-thrombin gel solution. Thereafter, three-dimensional tissue was prepared in the same manner as in Example 1, and a three-dimensional tissue consisting of tumor spheroids, pericytes in the gel, and vascular endothelial cells was prepared. As in Example 1, 5.0 x 10 fluorescently stained HUVECs were used as vascular endothelial cells. ６ cells / mL, 1.0×10 ７ cells / mL or 1.5 x 10 ７ In the three-dimensional tissue prepared in this example, pericytes were present in the gel surrounding the spheroids.

[0169] For the three-dimensional tissue thus prepared, fluorescent images were taken 9 days after the start of culture in the same manner as in Example 1, and angiogenesis was evaluated. The results are shown in Figure 26. In Figure 26, K is 10 ３ , M is 10 ６ and , respectively. In Figure 26, the length of the scale bar is 200 μm. Figure 27 shows the results of comparing the ratio of vascular area to the entire image at each HUVEC concentration. The results in Figure 27 were obtained by using three devices and preparing and evaluating two or three three-dimensional tissues per device (N = 6-8). The results in Figure 27 are shown as mean ± standard deviation, and * in the figure indicates that the p-value in the Tukey test after ANOVA (analysis of variance) was less than 0.05.

[0170] 26 and 27, the higher the concentration of vascular endothelial cells, the larger the area of ​​the blood vessels formed. Therefore, it was demonstrated that the assay using the microfluidic device 1 can evaluate the difference in blood vessel growth within a tumor due to differences in cell density.

[0171] Example 9: Preparation of three-dimensional tissues from tumor cell-containing spheroids, various concentrations of pericytes in gels, and vascular endothelial cells in the presence of various concentrations of fibrinogen.

[0172] Fibrinogen was mixed with Dulbecco's phosphate buffered saline (Nacalai Tesque, 14249-24) at a concentration of 5.0 mg / mL or 10.0 mg / mL, neutralized collagen I at a concentration of 0.4 mg / mL, and aprotinin at a concentration of 0.3 U / mL to obtain a fibrin gel solution. ６ cells / mL or 5.00 x 10 ６ The pericytes were suspended in EGM-2 to a final concentration of 1.25 × 10 cells / mL. The resulting pericyte suspension was mixed with fibrin gel solution at a volume ratio of 1:1 (PC-fibrin gel solution). Thus, fibrinogen was contained at a final concentration of 2.5 mg / mL (Low Fib) or 5.0 mg / mL (High Fib), and pericytes (PC) were contained at a final concentration of 1.25 × 10 cells / mL. ６ cells / mL (Low PC) or 2.50 x 10 ６ Four types of PC-fibrin gel solutions containing 1000 cells / mL (High PC) were obtained.

[0173] Each PC-fibrin gel solution was mixed with 1% by volume of 50 U / mL thrombin solution to obtain a PC-fibrin-thrombin gel solution. The tumor spheroids prepared above were added to the PC-fibrin-thrombin gel solution. Tumor spheroids prepared in the same manner as in Example 8 were added. Thereafter, three-dimensional tissue was prepared in the same manner as in Example 1, and a three-dimensional tissue consisting of tumor spheroids, pericytes in the gel, and vascular endothelial cells was prepared. Note that 1.5 x 10 fluorescently stained HUVECs were used as vascular endothelial cells. ７In the three-dimensional tissue prepared in this example, pericytes were present in the gel surrounding the spheroids.

[0174] For the three-dimensional tissue thus prepared, 9 days after the start of culture, fluorescent images were taken in the same manner as in Example 1, and angiogenesis was evaluated. The results are shown in Figure 28. In Figure 28, K is 10 ３ , M is 10 ６ and , respectively. In Figure 28, the length of the scale bar is 200 μm. Figure 29 shows the results of comparing the ratio of vascular area to the entire image under each condition. The results in Figure 29 are the results of evaluating 13 to 22 three-dimensional tissues per condition. The results in Figure 29 are shown as mean values ​​± standard deviation.

[0175] 28 and 29, it was possible to quantitatively evaluate angiogenesis under all conditions. These results demonstrate that assays using the microfluidic device 1 can evaluate angiogenesis under a combination of multiple conditions. Therefore, although the concentrations of each biomolecule in a biological environment can vary widely, assays using the microfluidic device 1 can quantitatively evaluate angiogenesis under conditions that simulate various biological environments.

[0176] DESCRIPTION OF SYMBOLS 1... Microfluidic device, 2... Device channel plate, 3... Device substrate, 2a... Main surface of channel plate, 5A... Cell aggregate housing section, 5B... Cell aggregate housing section, 5C... Cell aggregate housing section, 2R... First channel system, 2L... Second channel system, 91... Cell aggregate, 92... Endothelial cell, 9 2R...first endothelial cell, 92L...second endothelial cell, 93...lumen structure, 93R...first lumen structure, 93L...second lumen structure, 27R, 28R...first supply hole, 20R...first channel, 27L, 28L...second supply hole, 20L...second channel, A1...first axis, A2...second axis, 2 e1...outer edge, 2e2...outer edge, 21R, 25R...first connection flow path section, 22R, 24R...first relay flow path section, 23R...first supply flow path section, A1K...virtual reference line, 21L, 25L...second connection flow path section, 22L, 24L...second relay flow path section, 23L...second supply flow path section, 2b...rear surface of flow path plate, 3a...main surface of substrate, 51S...cell aggregate introduction hole, 52S...vent hole, 53S...cell aggregate holding area, 54SR...first access area, 54SL...second access area, 211R...lower ceiling surface, 231R...first outer flow path wall surface, 232R...first inner flow path wall surface , 31R...first flow path floor surface, 233R...first cell adhesion surface, 55Ra...gap area portion, 55Rb...gap area portion, 55Rc...gap area portion, 211L...lower ceiling surface, 231L...second outer flow path wall surface, 232L...second inner flow path wall surface, 31L...second flow path floor surface, 233L...second cell adhesion surface, 241...supply peripheral wall surface, 251...deaeration peripheral wall surface, 53S1...cell aggregate restraint portion, 53S2...cell aggregate introduction portion, 53Su...upper holding area portion, 53Sd...lower holding area portion, 212C...upper ceiling surface, 241R...upper wall surface, 241L...upper wall surface, 291...upper circle Circumferential surface, 211C...lower ceiling surface, 56S...area, A3...third axis, 91a...regulating portion, 91b...exposed portion, 55Rd...connecting region, 26R...first wall portion, 55Lb...gap region, 55La...gap region, 55Rs...first outer opening, 55Rt...first inner opening, 55Lt...second inner opening, 551...inclined wall surface, 22Rd...inner wall surface of first holding region, 22Ld...inner wall surface of second holding region, 55Ls...second outer opening, 100...array, 1S...microfluidic structure, W10...well, 2S...array flow path plate, 3S...array substrate, 2p...cured product.

Claims

1. A microfluidic device comprising: a first flow path in which first endothelial cells are arranged and which extends in a first direction; a second flow path in which second endothelial cells are arranged and which is spaced from the first flow path in a second direction perpendicular to the first direction and extends along the first direction; and a cell aggregate storage section in which cell aggregates are stored and which is sandwiched between the first flow path and the second flow path when viewed from the first direction, wherein the first flow path includes a first cell adhesion surface to which the first endothelial cells are adhered; the second flow path includes a second cell adhesion surface to which the second endothelial cells are adhered; and the cell aggregate storage section includes a cell aggregate restraint section that maintains the position of the cell aggregate relative to the first cell adhesion surface and / or the second cell adhesion surface.

2. The microfluidic device of claim 1, wherein the first flow path is formed on the first cell adhesion surface and includes a first opening for passing the first endothelial cells to the cell aggregate storage section, and the second flow path is formed on the second cell adhesion surface and includes a second opening for passing the second endothelial cells to the cell aggregate storage section.

3. A microfluidic device as described in claim 2, wherein the cell aggregate constraint portion overlaps the area sandwiched between the first opening and the second opening, and the cell aggregate storage portion includes: a cell aggregate introduction hole connected to an introduction opening formed on the first surface of the microfluidic device; and a cell aggregate introduction portion that does not overlap the area sandwiched between the first opening and the second opening, is connected from the cell aggregate introduction hole to the cell aggregate constraint portion, and guides the cell aggregate introduced from the cell aggregate introduction hole to the cell aggregate constraint portion.

4. A microfluidic device as described in claim 1, wherein a plurality of cell aggregate holding sections are provided between the first flow path and the second flow path, and each of the plurality of cell aggregate holding sections is sandwiched between the first flow path and the second flow path when viewed from the first direction.

5. A microfluidic device as described in claim 1, wherein the cell aggregate constraint portion includes a first regulating wall surface that regulates the movement of the cell aggregate along the first direction.

6. A microfluidic device as described in claim 1, wherein the cell aggregate constraint portion includes a pair of second regulating wall surfaces that regulate the position of the cell aggregate along the second direction.

7. A microfluidic device as described in claim 1, wherein the cell aggregate constraint portion includes a third regulating wall surface that regulates the position of the cell aggregate along a third direction perpendicular to the first direction and the second direction.

8. A method for producing a three-dimensional tissue having an internal tubular structure using the microfluidic device described in any one of claims 1 to 7, comprising the steps of: placing a liquid containing cell aggregates and a gel-forming polymer compound in the cell aggregate storage section and gelling the liquid so that the cell aggregates remain in the cell aggregate restraint section; placing the first endothelial cells and a first culture medium in the first flow path and adhering the first endothelial cells to the first cell adhesion surface; placing the second endothelial cells and a second culture medium in the second flow path and adhering the second endothelial cells to the second cell adhesion surface; and culturing the cell aggregates, the first endothelial cells, and the second endothelial cells so that the first endothelial cells and the second endothelial cells can form a tubular structure that connects to the interior of the cell aggregate.

9. A screening method for a test substance using the microfluidic device of any one of claims 1 to 7, comprising the steps of: placing a liquid containing cell aggregates and a gel-forming polymer compound in the cell aggregate storage section, and gelling the liquid so that the cell aggregates remain in the cell aggregate restraint section; placing the first endothelial cells and a first culture medium in the first flow path and adhering the first endothelial cells to the first cell adhesion surface; placing the second endothelial cells and a second culture medium in the second flow path and adhering the second endothelial cells to the second cell adhesion surface; culturing the cell aggregates, the first endothelial cells, and the second endothelial cells so that the first endothelial cells and the second endothelial cells can form a tubular structure connecting to the interior of the cell aggregate, thereby forming a three-dimensional tissue with an internal tubular structure; and placing the test substance in the first flow path and / or the second flow path, and evaluating the effect of the test substance on the three-dimensional tissue.

10. An array comprising: a first array plate portion; and a second array plate portion bonded to the first array plate portion, wherein the first array plate portion and the second array plate portion bonded to each other form a plurality of microfluidic structures, wherein the microfluidic structure has: a first flow path in which first endothelial cells are arranged and extending in a first direction; a second flow path in which second endothelial cells are arranged and which is spaced from the first flow path in a second direction perpendicular to the first direction and extends along the first direction; and a cell aggregate storage portion that stores cell aggregates and is sandwiched between the first flow path and the second flow path when viewed from the first direction, wherein the first flow path includes a first cell adhesion surface to which the first endothelial cells are adhered; the second flow path includes a second cell adhesion surface to which the second endothelial cells are adhered; and the cell aggregate storage portion includes a cell aggregate restraint portion that maintains the position of the cell aggregate relative to the first cell adhesion surface and / or the second cell adhesion surface.

Images