WO2025182782A1 - Microfluidic device - Google Patents

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

This disclosure relates to microfluidic devices.

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.

In recent years, a method for evaluating angiogenic/vasculogenic potential has emerged in which the extension of vascular endothelial cells is assessed using three-dimensional tissue placed in a microfluidic device. This method is particularly useful for screening and evaluating drugs using three-dimensional tissue, such as tumor models, as well as for elucidating the mechanism by which endothelial cells form tubular structures.

As a device for forming such three-dimensional tissue, for example, Non-Patent Document 1 discloses a device (All-in-One-IMPACT) used to create three-dimensional tissue in which a tubular structure is formed through angiogenesis. Devices capable of creating three-dimensional tissue in which a tubular structure is formed through 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.

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.

Patent Document 3 also discloses a microfluidic device comprising a device main body, a first flow path provided in the device main body, a vascular bed holding chamber adjacent to the first flow path in the device main body and provided via a first wall portion, an opening provided in the device main body and communicating with the vascular bed holding chamber, and a partition wall provided to close the opening, wherein a plurality of first slits are formed in the first wall portion, and the partition wall is removable. With this device, angiogenesis can be evaluated by placing and culturing a three-dimensional tissue on the formed vascular bed after removing the partition wall.

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

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.

The device described in Non-Patent Document 1 is used by 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 endothelial cells are then introduced and cultured around the hydrogel, allowing the endothelial cells to form a luminal structure in the hydrogel and the spheroids within it. Therefore, if 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 point where luminal structure formation begins and the spheroid. This suggests that the properties and quality of the three-dimensional tissue produced and the luminal structure formed therein may vary. The devices disclosed in Non-Patent Documents 2-4 and Patent Documents 1 and 2 may also exhibit similar variations in quality and properties due to the inability to control the positional relationship.

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

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 other methods in already formed cell aggregates.

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 tissue created using the device described in Patent Document 5, changes in the morphology of the tubular structure occur due to passage through the porous membrane, and it may not be possible to accurately reproduce biological tissue.

The present disclosure aims to provide a microfluidic device with an internal tubular structure that can be used to create three-dimensional tissue of stable quality.

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 extends along the first direction and is spaced from the first flow path in a second direction perpendicular to 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.

In the microfluidic device of this embodiment, the first flow path includes a first cell adhesion surface, the second flow path includes a second cell adhesion 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 adhesion surface and/or the second cell adhesion 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 adhesion surface and/or the second cell adhesion surface. In the microfluidic device of this embodiment, a tubular structure extends from the endothelial cells adhered to the first cell adhesion surface and/or the second cell adhesion 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 of this embodiment, the positional relationship between the cell aggregates and the first cell adhesion surface and/or the second cell adhesion surface to which the endothelial cells adhere, which is the starting point for tubular structure formation, remains the same regardless of the trial and is maintained. As a result, the microfluidic device according to this embodiment can produce three-dimensional tissues with stable quality and high reproducibility.

In the microfluidic device of 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 of this embodiment, a luminal structure extends toward the cell aggregate stored in the cell aggregate storage section from the first cell adhesion surface of the first flow path and the second cell adhesion surface of the second flow path, which are positioned to sandwich the cell aggregate. Therefore, in the microfluidic device of this embodiment, 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 process of luminal structure formation.

In the microfluidic device of this embodiment, tubular structures extend from the endothelial cells adhered to the first cell adhesion surface and the second cell adhesion surface. Therefore, for example, if the endothelial cells are vascular endothelial cells, it is possible to create a three-dimensional tissue containing blood vessels formed by angiogenesis. Such three-dimensional tissue can be used for drug screening and evaluation and to elucidate the mechanism of tubular structure formation. For example, if the three-dimensional tissue is a tumor model, it can be suitably used for screening and evaluation of antitumor drugs and to elucidate the mechanism of angiogenesis or vasculogenesis in tumor tissue.

One embodiment of the present invention is [2] the microfluidic device described in [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. In the microfluidic device according to this embodiment, the first endothelial cells can pass through the first opening to extend a tubular structure toward the cell aggregate, and the second endothelial cells 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 tubular structures therein.

One embodiment of the present invention is [3] "a microfluidic device according to [2], wherein 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 the 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 from the cell aggregate introduction hole to the cell aggregate constraint section, and guides the cell aggregate introduced from 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 makes it easier to introduce cell aggregates into cell aggregate introduction holes than to manually place cell aggregates, allowing for more efficient (high-throughput) production of three-dimensional tissues. Furthermore, it makes it possible to produce three-dimensional tissues while eliminating the variability between experimenters that occurs when placing cell aggregates manually.

One embodiment of the present invention is [4] "a 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 results in a plurality of three-dimensional tissues as indicators.

One embodiment of the present invention is [5] "a 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 makes it possible to create three-dimensional tissue while restricting movement of the cell aggregate, particularly along the first direction.

One embodiment of the present invention is [6] "a 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 makes it possible to create three-dimensional tissue while restricting the movement of the cell aggregate, particularly along the second direction.

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 makes it possible to create three-dimensional tissue while restricting the movement of the cell aggregate, particularly along the third direction.

One embodiment of the present invention is [8] "A method for producing three-dimensional tissue having an internal tubular structure using the microfluidic device described in any one of [1] to [7], the method 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 connecting to the interior of the cell aggregate." The production method according to this embodiment makes it possible to produce three-dimensional tissue of stable quality having an internal tubular structure.

One embodiment of the present invention is [9] "A screening method for 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 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 connected 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." The screening method of this embodiment allows screening of test substances. In particular, the microfluidic device used in the screening method of this embodiment allows for the production of three-dimensional tissues of stable quality, as described above, enabling screening with high reproducibility.

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 as viewed from the first direction, wherein the first flow path includes a first cell adhesion surface to which the first endothelial cells are adhered, and 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. The microfluidic structure in the array of this embodiment corresponds to the configuration of the microfluidic device according to [1] above. The array of this embodiment makes it possible to create a large number of three-dimensional tissues. This allows for the highly efficient screening and evaluation of multiple test substances using a single array.

The present disclosure provides a microfluidic device that can be used to create three-dimensional tissue with stable quality and an internal tubular structure. The present disclosure also provides a method for creating three-dimensional tissue using such a device and a method for screening test substances.

FIG. 1 is a perspective view of a microfluidic device 1. FIG. 1 is a plan view of a microfluidic device 1. FIG. 2 is an enlarged cross-sectional view of the microfluidic device 1 taken along a cross section perpendicular to a third direction (direction A3). 1 is an enlarged cross-sectional view of a cross section of the microfluidic device 1 perpendicular to a first direction (direction A1). FIG. FIG. 2 is an enlarged cross-sectional view of the microfluidic device 1 taken along a cross section perpendicular to a second direction (direction A2). 1A is a schematic diagram showing an outline of a method for producing a three-dimensional tissue; FIG. 1A is a schematic diagram showing an overview of arranging a cell aggregate in a cell aggregate accommodation step; FIG. 1B is a schematic diagram showing a first endothelial cell adhesion step and a second endothelial cell adhesion step together in one diagram; and FIG. 1C is a schematic diagram showing a tubular structure formation step. FIG. 1 is a plan view of the array 100. A VI-axis cross-sectional view of the array 100. 1A and 1B are schematic diagrams illustrating an outline of a method for producing a microfluidic device 1 in Production Example 1. FIG. 1 shows a fluorescent image of a three-dimensional tissue prepared in Example 1 when the HUVEC concentration of the HUVEC suspension was 5.0×10 ⁶ cells/mL. 1 shows a fluorescent image of a three-dimensional tissue prepared in Example 1 when the HUVEC concentration of the HUVEC suspension was 1.0×10 ⁷ cells/mL. 1 shows a fluorescent image of a three-dimensional tissue prepared in Example 1 when the HUVEC concentration of the HUVEC suspension was 1.5×10 ⁷ cells/mL. 1 shows fluorescent images on days 1, 3, 5, 7 and 9 of a three-dimensional tissue prepared in Example 1 when the HUVEC concentration of the HUVEC suspension was 5.0×10 ⁶ cells/mL. FIG. 1 shows the transition of tumor area ratio in Example 1. FIG. 1 is a graph showing the change in the blood vessel area ratio in Example 1. FIG. 1 is a graph showing the change in the blood vessel area ratio within a tumor in Example 1. FIG. 1 is a diagram showing an outline of a method for preparing a frozen section containing a three-dimensional tissue in Example 2. FIG. 10 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 three-dimensional tissue in the cross section. 19 is an enlarged fluorescent image of the central area of the central visual field of FIG. 18 in Example 2. 1A is a diagram showing a fluorescent image of a cross section perpendicular to the third direction (A3 direction) alongside a schematic diagram showing an outline of the three-dimensional tissue in the cross section in Example 2. FIG. 1B is a diagram showing a fluorescent image of a cross section perpendicular to the second direction (A2 direction) alongside a schematic diagram showing an outline of the three-dimensional tissue in the cross section in Example 2. FIG. 10 is a diagram showing the blood vessel area ratio for each chamber in the device in Example 3. FIG. 10 is a diagram showing the blood vessel area ratio for each device in Example 3. 22 shows the standard deviation (Device) of the measurement results within each device in Example 3, and FIG. 23 shows the standard deviation (Chamber) of the measurement results between devices in FIG. 21. FIG. FIG. 10 shows fluorescent images of the three-dimensional tissues prepared in Examples 4-1, 4-2, 5, and 6 and Comparative Examples 1 and 2 in Example 7, taken 9 days after the start of culture. FIG. 10 is a diagram comparing the blood vessel area ratios in Examples 4-1, 5, and 6 in Example 7. FIG. 10 shows a fluorescent image of a three-dimensional tissue obtained 9 days after the start of culture in Example 8. FIG. 10 is a graph comparing the vascular area ratio for each concentration of vascular endothelial cells in Example 8. FIG. 10 shows a fluorescent image of a three-dimensional tissue obtained 9 days after the start of culture in Example 9. FIG. 10 is a diagram comparing the blood vessel area ratio under various conditions in Example 9.

A microfluidic device according to one 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 will be designated by the same reference numerals, and duplicate explanations will be omitted.

FIG. 1 is a perspective view of a microfluidic device 1 according to one 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.

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, a microfluidic device 1 formed from dimethylpolysiloxane can be suitably used for evaluation by slicing three-dimensional tissue.

The device flow path plate 2 has a rectangular shape in a plan view. The device flow path 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, the device flow path plate 2 has ten through-holes. The through-holes extend perpendicular to the flow path plate main surface 2a (first surface). The device substrate 3 has a rectangular shape in a plan view. The device substrate 3 has no grooves or through-holes. The device substrate 3 functions as the bottom plate of the microfluidic device 1.

The microfluidic device 1 has multiple cell aggregate storage sections 5A, 5B, 5C, a first flow path system 2R, and a second flow path system 2L. The multiple cell aggregate storage sections 5A, 5B, 5C are sandwiched between the first flow path system 2R and the second flow path system 2L. Each of the multiple cell aggregate storage sections 5A, 5B, 5C holds a cell aggregate 91 (see Figure 3) in a predetermined position. The first flow path system 2R and the second flow path system 2L hold endothelial cells 92 (first endothelial cells 92R, second endothelial cells 92L, see Figure 3) near the cell aggregate storage sections 5A, 5B, 5C. The endothelial cells 92 are the starting points of first tubular structures 93R and second tubular structures 93L that extend toward the cell aggregate 91. In the following description, when there is no need to distinguish between the first luminal structure 93R and the second luminal structure 93L, they may be referred to simply as "luminal structure 93" (see Figure 3). Similarly, when there is no need to separately describe the first endothelial cell 92R and the second endothelial cell 92L for convenience of explanation, they may be referred to simply as "endothelial cell 92."

<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 this first axis A1 as the axis of symmetry. Therefore, the following description will focus on the first flow path system 2R in detail. Regarding the second flow path system 2L, descriptions of the same aspects as those of the first flow path system 2R will be omitted as appropriate.

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

The shape of the first supply holes 27R, 28R is approximately 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. In other words, 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.

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 is defined that is perpendicular to the first axis A1. The first supply holes 27R, 28R are spaced apart from the cell aggregate storage section 5A, etc. 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.

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.

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

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 communicates with the space outside the microfluidic device 1 via the first supply holes 27R and 28R.
As a result, for example, first supply hole 27R can supply a cell suspension or culture medium to first flow path 20R. In this case, the cell suspension or culture medium supplied to first flow path 20R may be discharged from first supply hole 28R. That is, the cell suspension or culture medium can be perfused from first supply hole 27R through first flow path 20R toward first supply hole 28R. Note that the cell suspension or culture medium may also be introduced from first supply hole 28R. In this case, the cell suspension or culture medium can be perfused from first supply hole 28R toward 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.

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.

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

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

The first end of the first connection flow path portion 21R is connected to the first supply hole 27R. The first connection flow path portion 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. In other words, the first connection flow path portion 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 portion 21R may be located, for example, in the middle 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 portion 21R is connected to the first end of the first relay flow path portion 22R.

The first relay flow path section 22R connects the first connection flow path section 21R to the first supply flow path section 23R. The first relay flow path section 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 section 22R may be longer than the length of the first connection flow path section 21R. The second end of the first relay flow path section 22R is connected to the first end of the first supply flow path section 23R.

Endothelial cells are placed and adhered to the first supply flow path 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. In other words, the first supply flow path 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 flow path section 23R is connected to a second end of the first relay flow path section 22R. The first supply flow path section 23R extends along the first axis A1 from one outer edge 2e1 to the other outer edge 2e2 of the device flow path plate 2. For example, the length of the first supply flow path 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 flow path section 23R is connected to the first relay flow path section 24R.

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. The first end of the first relay flow path section 24R is connected to the 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. The second end of the first relay flow path section 24R is connected to the first connection flow path section 25R.

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 way, the first flow path 20R has, in this order from the first supply hole 27R, which communicates with the space outside the microfluidic device 1, toward the first supply hole 28R, a first connection flow path section 21R, a first relay flow path section 22R, a first supply flow path section 23R, a first relay flow path section 24R, and a first connection flow path section 25R.

As already mentioned, 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 sections 21L, 25L, a pair of second relay flow path sections 22L, 24L, and a second supply flow path section 23L (second flow path section). 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.

Here, attention is focused on the first supply flow path section 23R and the second supply flow path section 23L. Three cell aggregate storage sections 5A, 5B, and 5C are provided in the region between the first supply flow path section 23R and the second supply flow path 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, Figure 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 containers provided in the microfluidic device 1 may be either an odd number or an even number.

The three cell aggregate storage units 5A, 5B, and 5C shown in Figures 1 and 2 differ only in their placement positions; the individual structures are identical. Below, with reference to Figure 3, cell aggregate storage unit 5A will be described in detail, and a description of cell aggregate storage units 5B and 5C will be omitted.

<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.

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

Here, we will provide a detailed description of the first supply flow path 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. The first supply flow path section 23R is formed by a groove provided in the device flow path plate 2 being blocked by the device substrate 3. The first supply flow path section 23R is an area surrounded by the lower ceiling surface 211R, the first outer flow path wall surface 231R, the first inner flow path wall surface 232R, and the first flow path floor surface 31R. The lower ceiling surface 211R, the first outer flow path wall surface 231R, and the first inner flow path wall surface 232R are part of the device flow path plate 2. The first flow path floor surface 31R is part of the device substrate 3. More specifically, the first flow path floor surface 31R is part of the substrate main surface 3a.

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

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.

Furthermore, openings in the gap regions 55Ra, 55Rb, and 55Rc, described below, are formed in a portion of the first inner flow path wall surface 232R. The 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.

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

<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.

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.

<Ventilation hole 52S>
The deaeration 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 deaeration hole 52S, which is circular in plan view, overlaps with the first axis A1. In other words, the deaeration hole 52S and the cell aggregate introduction hole 51S are aligned apart from each other on the first axis A1.

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

<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.

More specifically, the cell aggregate holding area 53S is located 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 along the direction of the first axis A1 toward the degassing hole 52S. This portion extending along the direction of 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 when viewed in a plan view. The inner diameter of the cell aggregate restraint section 53S1 is the same as the width of the cell aggregate introduction section 53S2. The inner diameter of the cell aggregate restraint section 53S1 may be determined, for example, according to the size of the cell aggregate 91.

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

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 restriction wall surfaces), and an upper circumferential surface 291 (first restriction wall surface, see Figure 5).

The cell aggregate restraint portion 53S1 is defined by an upper circumferential surface 291 included in the device flow path 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 portion 53S1 is connected to the deaeration hole 52S by a region 56S sandwiched between the lower ceiling surface 211C of the device flow path plate 2 and the substrate main surface 3a. This region 56S can also be considered the region sandwiched between the upper wall surfaces 241R and 241L, which will be described later.

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 portion 53Su can be said to regulate the position of the cell aggregate 91 along the second axis A2. Furthermore, the other end of the upper holding area portion 53Su is the cell aggregate restraint portion 53S1 described above.

The upper ceiling surface 212C regulates the position of the cell aggregate 91 along 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.

In other words, the cell aggregate 91 is held in position along the first axis A1, second axis A2, and 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.

In contrast, the position of the lower holding area 53Sd in the negative direction of the third axis A3 is simply restricted 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 a gap is 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 tubular structure 93 can reach the cell aggregate 91 through this gap.

From the perspective of the cell aggregate 91, the cell aggregate 91 includes a restricting portion 91a surrounded by a pair of upper wall surfaces 241R, 241L and an upper circumferential surface 291, and an exposed portion 91b that is not surrounded by a pair of upper wall surfaces 241R, 241L and an upper circumferential surface 291. For example, the restricting portion 91a can be said to be the upper portion of the cell aggregate 91, and the exposed portion 91b can be said to be 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 tubular structure 93 can reach the exposed portion 91b from all directions about the third axis A3.

<First Access Area, Second Access Area>
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 allows the distance from the endothelial cells 92 to the cell aggregate 91 to be shortened.

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

The gap areas 55Ra, 55Rb, and 55Rc are provided in the first wall portion 26R, which separates the first supply flow path portion 23R and the cell aggregate storage portion 5A. Three gap areas 55Ra, 55Rb, and 55Rc are provided in the first wall portion 26R. The three gap areas 55Ra, 55Rb, and 55Rc are aligned along the direction of the first axis A1. For example, of the three gap areas 55Ra, 55Rb, and 55Rc, attention is focused on the centrally located gap area 55Rb. The gap area 55Rb of the first access area 54SR and the gap area 55Lb of the second access area 54SL sandwich the aforementioned cell aggregate restraint portion 53S1. In addition, attention is focused on the gap area 55Ra located on the side of the cell aggregate introduction hole 51S relative to the gap area 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 section 53S2.

The gap regions 55Ra, 55Rb, and 55Rc increase in width from the first supply flow path 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 said to be 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 flow path section 23R. For example, the gap region 55Ra is surrounded by a pair of inclined wall surfaces 551, the lower ceiling surfaces 211R and 211L included in the device flow path plate 2, and the substrate main surface 3a.

Note that Figure 5 shows 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.

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

As mentioned above, the first supply flow path section 23R is also sandwiched between the lower ceiling surfaces 211R, 211L and the substrate main surface 3a. In other words, the first supply flow path 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 flow path 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 flow path section 23R can easily reach the cell aggregate 91 by extending in the direction of the second axis A2.

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 greater than the width of the second outer opening 55Ls (second opening), which is the opening facing the second supply flow path section 23L. A detailed description of the second access region 54SL will be omitted.

<Action and effect>
The microfluidic device 1 includes a first supply channel portion 23R, which is 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, which is 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, which is the 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, which stores a cell aggregate 91 and is sandwiched between the first supply channel portion 23R, which is the first channel portion, and the second supply channel portion 23L, which is the second channel portion, as viewed in the direction of the first axis A1. The first supply channel portion 23R, which is the 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.

In 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, in 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. In 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 adjusted as desired, thereby stabilizing the quality of the three-dimensional tissue comprising the cell aggregate 91 and the tubular structure 93. Furthermore, the reproducibility of the three-dimensional tissue produced can be improved.

The first supply flow path section 23R, which is the first flow path section, includes a first cell adhesion surface 233R. The second supply flow path section 23L, which is the second flow path 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 an endothelial cell 92 adhered to at least one of the first cell adhesive surface 233R and the second cell adhesive surface 233L toward the cell aggregate 91. By being able to define the positional relationship between the cell aggregate 91 and the endothelial cell 92, the positional relationship between the endothelial cell 92, which is the starting point 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 cell 92, the reproduced positional relationship between the endothelial cell 92 and the cell aggregate 91 can be maintained throughout the period during which the tubular structure 93 extends toward the cell aggregate 91. This allows the microfluidic device 1 to produce three-dimensional tissues of consistent quality. Furthermore, the microfluidic device 1 allows for the production of three-dimensional tissues with high reproducibility.

When viewed from the direction of the first axis A1, the cell aggregate storage section 5A is 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. Therefore, the tubular structure 93 extends from the first cell adhesion surface 233R of the first supply flow path section 23R, which is the first flow path section, and the second cell adhesion surface 233L of the second supply flow path section 23L, which are positioned to sandwich the cell aggregate 91, toward the cell aggregate 91 stored in the cell aggregate storage section 5A. As a result, the tubular 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 tubular structure 93 can be formed that penetrates the cell aggregate 91.

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, if the endothelial cells 92 are vascular endothelial cells, it is possible to create a three-dimensional tissue containing blood vessels formed by angiogenesis. 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.

The first supply flow path section 23R, which is the first flow path 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 flow path section 23L, which is the second flow path 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 structure 93 toward the cell aggregate 91 by passing through the first outer opening 55Rs. Furthermore, the second endothelial cells 92L can extend their luminal structure 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 a luminal structure 93 therein.

The cell aggregate restraint section 53S1 overlaps the region sandwiched between the first outer opening 55Rs and the second outer opening 55Ls. The cell aggregate storage section 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 section 53S2 that includes a portion that does not overlap the region sandwiched between the first outer opening 55Rs and the second outer opening 55Ls, is connected from the cell aggregate introduction hole 51S to the cell aggregate restraint section 53S1, and guides the cell aggregate 91 introduced from the cell aggregate introduction hole 51S to the cell aggregate restraint section 53S1.
In this microfluidic device 1, by introducing a cell aggregate 91 through the cell aggregate introduction hole 51S, the cell aggregate 91 can be guided 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 positioning 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 positioning the cell aggregate 91. As a result, three-dimensional tissues can be produced with higher efficiency (high throughput). This also eliminates the variability that can occur between experimenters when manually positioning the cell aggregate 91.

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. When viewed from the direction of the first axis A1, 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. This microfluidic device 1 is capable of producing a plurality of three-dimensional tissues. As a result, a single microfluidic device 1 can be used to screen and evaluate drugs using the results in multiple three-dimensional tissues as indicators. Furthermore, a single microfluidic device 1 can also be used to elucidate the mechanism of formation of tubular structures 93 using the results in multiple three-dimensional tissues as indicators.

The cell aggregate restraint portion 53S1 includes an upper circumferential surface 291 that restricts the movement of the cell aggregate 91 along the direction of the first axis A1. This microfluidic device 1 makes it possible to create three-dimensional tissue while restricting the movement of the cell aggregate 91 along the direction of the first axis A1.

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. This microfluidic device 1 allows the creation of three-dimensional tissue while regulating the movement of the cell aggregate 91 along the second axis A2.

The cell aggregate restraint portion 53S1 includes an upper ceiling surface 212C that regulates 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 three-dimensional tissue while regulating the movement of the cell aggregate 91 along the third axis A3.

<Method for producing three-dimensional tissue with an internal tubular structure>
Another aspect of the present invention is a method for producing a three-dimensional tissue having an internal luminal structure using a microfluidic device according to one aspect of the present invention, the 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); 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). Hereinafter, a 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.

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

<Cell aggregate accommodation step>
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.

In this disclosure, a cell aggregate refers to a three-dimensional tissue (3D tissue) whose main component is 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. The artificially produced three-dimensional tissue may be, for example, a spheroid or an organoid. The cell aggregate may be an aggregate of cells, or may be an aggregate containing components that are sometimes contained in living tissue in addition to cells. Examples of such components include extracellular matrix constituent molecules (collagen, fibronectin, proteoglycan, etc.) and gel-forming polymer compounds. The cells contained in the cell aggregate may be labeled with a fluorescent protein, fluorescent dye, etc.

The size of the cell aggregate 91 used in the cell aggregate storage process is not particularly limited as long as it can remain in the cell aggregate holding area 53S of the microfluidic device 1. For example, the maximum diameter of the cell aggregate 91 may be 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 in the third direction of the cell aggregate restraint section 53S1, 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 the cell aggregate 91 may be 0.6 times or more, 0.7 times or more, or 0.8 times or more the thickness of the cell aggregate introduction section 53S2 in the third direction (direction A3), or may be 1.2 times or less, 1.1 times or less, or 1.0 times or less. If the maximum diameter of the cell aggregate 91 is 1.2 times or less the thickness of the cell aggregate introduction section 53S2 in the third direction, the cell aggregate 91 is less likely to clog the cell aggregate introduction section 53S2 when being guided into the cell aggregate constraint section 53S1, allowing for highly efficient placement of the cell aggregate.

When the cell aggregate is an organoid or ex vivo three-dimensional tissue, the tissue from which it is derived can be, for example, tumor tissue or normal tissue. For example, when the tissue from which it is derived is tumor tissue, by creating three-dimensional tissue using vascular endothelial cells, it is possible to obtain tumor tissue (tumor model) with internal blood vessels through angiogenesis. Furthermore, when the tissue from which it is derived is normal tissue, by creating three-dimensional tissue using lymphatic endothelial cells, it is possible to obtain a tissue model with internal lymphatic vessels.

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, these tumor cells or normal cells are the cells that primarily constitute the tissue that will serve 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.

If 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 producing spheroids in a medium, adding the cell suspension to a microwell plate that can be used for producing spheroids, and incubating the plate. Microwell plates commonly used by those skilled in the art can be used as appropriate for producing spheroids. Examples of such microwell plates include plates that are not cell-adhesive and have wells with concave bottoms. The culture period for producing spheroids may be, for example, one day or more and seven days or less, and may be, for example, two days.

The number of cells to be suspended in the cell culture medium when preparing spheroids is not particularly limited and can be appropriately selected by those skilled in the art depending on the type of cells used. Examples of such cell numbers include 1.00 x 10 ³ cells/mL to 1.00 x 10 ⁷ cells/mL, 1.00 x 10 ⁴ cells/mL to 1.00 x 10 ⁶ cells/mL, or 5.00 x 10 ⁴ cells/mL to 1.20 x 10 ⁵ cells/mL. 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. Examples of such cell ratios include tumor cells or normal cells:pericytes, which may be 1:10 to 50:1, 1:5 to 10:1, or 1:3 to 5:1, with specific examples including 1:1 and 3:1.

Gel-forming polymer compounds are polymer compounds that can exist as a solution (e.g., an aqueous solution) under certain conditions and that have the property of gelling when stimulated. Gel-forming polymer compounds may be natural or artificial. Natural polymer compounds that are gel-forming polymer compounds include those classified as proteins or polysaccharides, such as collagen (collagen I, collagen II, collagen III, collagen V, etc.), fibrinogen, chitosan, gelatin, hyaluronic acid, and alginic acid. Artificial polymer compounds that are gel-forming polymer compounds include synthetic polymers, such as agarose, polyacrylamide, and polyacrylic acid. Gel-forming polymer compounds may be composed of one or more of the above-described compounds. The stimuli that gel these gel-forming polymer compounds vary 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). For example, polyacrylic acid gels when the pH of an acidic solution containing polyacrylic acid is raised to a value at which the carboxyl groups are deprotonated (pH-sensitive).

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 is capable of retaining the cell aggregate 91 in the cell aggregate holding area 53S.

The cell aggregate-gel solution may further contain pericytes (e.g., pericytes) in addition to the cell aggregates and 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 with a structure 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 to 1.0 x 10 ⁸ cells/mL, 3.0 x 10 ⁵ cells/mL to 3.0 x 10 ⁷ cells/mL, or 1.0 x 10 ⁶ cells/mL to 1.0 x 10 ⁷ cells/mL, with 2.5 x 10 ⁶ cells/mL being an example.

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

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 the 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.

(a) of Figure 6 is a schematic diagram showing an overview of placing cell aggregates in the cell aggregate accommodation step. As shown in (a) of Figure 6, by introducing a cell aggregate-gel solution through the cell aggregate introduction hole 51S, the cell aggregates are guided to the cell aggregate constraint section 53S1. At this time, the inside 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 aggregates to the cell aggregate constraint section 53S1 and fill the inside 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. At this time, if a temperature-sensitive gel-forming polymer compound is used, the cell aggregates may be placed under temperature conditions (e.g., room temperature or on ice) at which the gel-forming polymer compound does not gel.

In the cell aggregate accommodation step, the cell aggregate-gel solution is placed in a 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 leave the microfluidic device 1 in an environment with temperature conditions at which the gel-forming polymer compound gels (e.g., in a 37°C incubator). 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 a cell aggregate accommodation section 5A or the like, and then the cell aggregate-gel solution can be placed in a 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, if 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 level 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, 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).

<First endothelial cell adhesion step>
FIG. 6B is a schematic diagram illustrating the first endothelial cell adhesion process and the second endothelial cell adhesion process. In the first endothelial cell adhesion process, 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.

A medium commonly used by those skilled in the art for culturing endothelial cells can be used. An example of such a medium is Endothelial Cell Growth Medium (EGM-2, Lonza, CC-3162).

The endothelial cells and culture medium can be placed in the first supply flow path section 23R 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 may be, for example, 1.0 x 10 ⁴ cells/mL to 1.0 x 10 ⁹ cells/mL, 1.0 x 10 ⁵ cells/mL to 1.0 x 10 ⁸ cells/mL, or 1.0 x 10 ⁶ cells/mL to 5.0 x 10 ⁷ cells/mL, examples of which include 5.0 x 10 ⁶ cells/mL, 1.0 x 10 ⁷ cells/mL, and 1.5 x 10 ⁷ cells/mL.

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, having an endothelial cell suspension disposed in the first supply flow channel section 23R, is placed so that the first inner flow channel wall surface 232R faces vertically downward (e.g., 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, e.g., 15 minutes. Furthermore, placing the microfluidic device 1 so that the first inner flow channel wall surface 232R faces vertically downward may be performed, for example, in an incubator at 37°C and 5% _CO2 .

<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.

The first endothelial cells and the second endothelial cells may be the same type of cell, or different types of cell. In the microfluidic device 1, luminal structures are formed inside the cell aggregate 91, connecting them from both the first inner flow path wall surface 232R and the second inner flow path wall surface 232L. Therefore, when the first endothelial cells and the second endothelial cells are the same type, it is possible to clarify the mechanism of interaction between the luminal structures in those endothelial cells. Furthermore, when the first endothelial cells and the second endothelial cells are different types, it can be used to clarify the interaction between the luminal structures formed by endothelial cells of different types.

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, in which an endothelial cell suspension is placed 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 standing time and conditions can be the same as those described above for the first endothelial cell adhesion process.

<Lumen structure formation process>
In the luminal structure formation 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 connecting to the interior of the cell aggregate. (c) of Fig. 6 is a schematic diagram showing the luminal structure formation step. As shown in (c) of Fig. 6, in the luminal structure formation step, the microfluidic device 1 after the second endothelial cell adhesion step is incubated to form a three-dimensional tissue having a luminal structure therein. Incubation may be performed by gently soaking or leaving the microfluidic device 1 stationary under an environment typically used for cell culture (e.g., in an incubator at 37°C and 5% _CO2 ).

Incubation in the tubular structure formation process 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 can 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. Furthermore, incubation can also 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 directions.

The incubation time in the tubular structure formation process can be appropriately selected by those skilled in the art depending on the three-dimensional tissue to be produced. The incubation time in the tubular structure formation process may be, for example, from half a day to 30 days or from one day to 15 days.

<Three-dimensional organization>
The three-dimensional tissue prepared according to the method of the present invention serves as a model that reproduces a three-dimensional biological tissue having an internal luminal structure. Such three-dimensional tissue can be used for drug screening and evaluation and for elucidating the mechanism of luminal 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.

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

Furthermore, in the production method according to one embodiment of the present invention, the luminal structures formed from 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 that penetrates the cell aggregate. For example, such a three-dimensional tissue can be suitably used to elucidate the interaction between luminal structures during the process of luminal structure formation.

<Evaluation of three-dimensional tissue>
Three-dimensional tissues prepared according to the method of one embodiment of the present invention may be evaluated by imaging the cell aggregates while they are maintained in the gel in the cell aggregate holding region 53S. When the device flow path 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 made of a material suitable for use as the bottom plate of an imaging dish, such as glass.

The three-dimensional tissue produced according to the production method of one embodiment of the present invention may be evaluated by retrieving it from the device. For example, the three-dimensional tissue can be retrieved from the device by applying a stimulus that causes the gel in the cell aggregate storage section 5A or the like to return 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 retrieved in this manner can be evaluated by methods commonly used by those skilled in the art, and may, for example, be evaluated by labeling with hematoxylin-eosin staining or immunoantibody staining and then imaging.

For example, when the microfluidic device 1 is formed from a material (e.g., dimethylpolysiloxane) that can be cut at room temperature, the three-dimensional tissue produced according to the production 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, first wall portion 26R, and second wall portion 26L. Such cryosectioning can be achieved 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, first wall portion 26R, and second wall portion 26L, and then embedding the recovered composite of the three-dimensional tissue and a portion of the device directly in OCT compound. Using cryosections produced in this manner, it is possible to evaluate fluorescent images of cross sections perpendicular to the first and second directions in addition to cross sections perpendicular to the third direction.

<Method for evaluating tubular structure formation inside 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 under "Evaluation of Three-Dimensional Tissue." This evaluation method allows for evaluation of changes in the morphology of the luminal structure formed depending on the type of cell aggregate or endothelial cell, or the conditions for preparing the three-dimensional tissue. Therefore, this evaluation method allows for elucidation of the mechanism by which endothelial cells form a luminal structure.

<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.

The test substance may be a substance known to have some effect on three-dimensional tissue, or it may be a substance with no known effect. Furthermore, 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. Furthermore, the test substance may 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, peptide library, mRNA library, or siRNA library.

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 the 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 flow path section 23R and the second supply flow path 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 of the test substance to be placed and the placement time (i.e., the time the three-dimensional tissue is exposed to the test substance) can be appropriately set by one skilled in the art to suit the test substance.

In another embodiment of the screening method, the 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 changes in the mode of luminal structure formation in the exposed three-dimensional tissue as an indicator. Therefore, this embodiment makes it possible to select 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 culture medium introduced from the first supply hole (27R and/or 28R) and/or the second supply hole (27L and/or 28L) in the luminal structure formation step. The concentration of the test substance to be placed can be appropriately set by one skilled in the art depending on the test substance. Furthermore, the test substance may be placed for the entire incubation time in the luminal structure formation step, or for only a portion of the time. In other words, the test substance may be exposed to the three-dimensional tissue throughout the entire luminal structure formation step, or for only a portion of the time during the step.

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 that has not been 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, and an increase or decrease in the frequency of luminal structure formation in the tested cell aggregate. 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 blood vessels and/or the frequency of blood vessel formation can be selected as a candidate antitumor drug that can inhibit angiogenesis or the expansion of intratumoral blood vessels.

<Array>
An array according to an 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.

The shape of the array 100 is a rectangular parallelepiped measuring 127.6 mm x 85.3 mm x 20.5 mm. The shape of the array 100 may be a plate standard that is compatible with a microwell plate reader commonly used for analyzing 96-well or 384-well microplates.

7 is a plan view of the array 100. As shown in FIG. 7, the array 100 has eight microfluidic structures 1S. Specifically, in a plan view, the array 100 has four microfluidic structures 1S arranged in a direction parallel to the 127.6 mm long side and two microfluidic structures 1S arranged in a direction parallel to the 85.3 mm long side. The structure of each of the eight microfluidic structures 1S is the same as that of the microfluidic device 1 (see FIG. 1) described above. The array 100 having eight microfluidic structures 1S can perform the same amount of assay as eight microfluidic devices 1. The number of microfluidic structures 1S arranged in the array 100 is not limited to eight. The number of microfluidic structures 1S arranged 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. Examples of the number of microfluidic structures 1S provided in the array 100 include 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, and 96.

Four microfluidic structures 1S are arranged in a direction parallel to the 127.6 mm long side, with multiple cell aggregate holding sections 5A, 5B, and 5C 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 sections 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 created in the multiple cell aggregate holding sections 5A, 5B, and 5C in the same manner as evaluation using a 96-well microplate.

Figure 8 is a cross-sectional view of the array 100. As shown in Figure 8, the array 100 is composed of an array flow channel plate 2S (first array plate portion) and an array substrate 3S (second array plate portion). The materials and manufacturing methods for the array flow channel plate 2S and array substrate 3S may be the same as those for the device flow channel plate 2 and device substrate 3 that make up the microfluidic device 1.

Array 100 is constructed as a single array plate by bonding two plate-like members together. For example, array 100 may be constructed as a single plate-like member. For example, array 100 can be constructed as a single plate-like member by using so-called three-dimensional modeling technology.

The array flow channel plate 2S is provided with multiple microfluidic structures 1S. The array flow channel plate 2S is rectangular, measuring 127.6 mm x 85.3 mm in plan view. The array flow channel plate 2S includes grooves formed on its back side, which is 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 multiple microfluidic structures 1S included in the array 100 are formed by the array substrate 3S covering the grooves and through-holes formed in the array flow channel plate 2S.

As described above, the array 100 comprises an array flow channel plate 2S, which is a first array plate portion, and an array substrate 3S, which is a 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 Figure 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 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. With this configuration, the array 100 can be used to perform screening and evaluation of multiple test substances with high efficiency.

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

<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 create a mold made of HTL resin (BMF) for fabricating the device channel plate 2 of microfluidic device 1, as shown in FIG. 9(a). Next, 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 PDMS prepolymer was then left to cure overnight at 70°C, yielding 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.

The microfluidic device 1 obtained in this manner had an approximately 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 area 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 area 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 area 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 deaeration hole 52S had an opening diameter of 1.0 mm.

In the microfluidic device 1 obtained in this manner, the width in the direction of the first axis A1 of the first outer opening 55Rs of the gap regions 55Ra, 55Rb, and 55Rc and the second outer opening 55Ls of the gap regions 55La, 55Lb, and 55Lc was 0.15 mm. Furthermore, the width in the direction of the first axis A1 of the first inner opening 55Rt of the gap regions 55Ra, 55Rb, and 55Rc and the second inner opening 55Lt of the gap regions 55La, 55Lb, and 55Lc was 0.275 mm. The length 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 area inner wall surface 22Rd, or the distance between the second inner flow path wall surface 232L and the second retaining area 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°.

Example 1: Preparation of three-dimensional tissue using tumor spheroids and vascular endothelial cells
[Preparation of spheroids]
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 standard methods. The fluorescently stained ASPS cells were suspended in IMDM medium (Wako, 098-06465) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at a concentration of 1.25 x ¹⁰ cells/mL. The resulting cell suspension was seeded into a 96-well plate (Sumitomo Bakelite, MS-9096U) at 200 μL/well (2.50 x ¹⁰ cells/well). The seeded cells were cultured in an incubator at 37°C and 5% CO ₂ for 2 days to prepare tumor spheroids.

[Introduction of spheroids into microfluidic devices]
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 thrombin-fibrin gel solution was obtained by mixing 1% by volume of 50 U/mL thrombin solution with the 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.

4 μL of the thrombin-fibrin gel solution was collected so that tumor spheroids could be collected. The tumor spheroids were introduced one by one through the cell aggregate introduction holes 51S of the cell aggregate containers 5A, 5B, and 5C of the microfluidic device 1 (hereinafter simply referred to as "device 1") prepared in Production Example 1. This placed the tumor spheroids in the cell aggregate holding area 53S, and the interiors of the cell aggregate containers 5A, 5B, and 5C were filled with the thrombin-fibrin gel solution. The device 1 was then placed in an incubator at 37°C and 5% _CO2 for 15 minutes, allowing 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 volume of 600 μL. This filled the first flow path 20R and the second flow path 20L of the device 1 with EGM-2. Thereafter, the device 1 was left standing overnight in an incubator at 37°C and 5% CO ₂ .

[Introduction of vascular endothelial cells into a microfluidic device]
Human umbilical vein endothelial cells (HUVECs) were fluorescently stained by expressing green fluorescent protein (GFP) using standard methods. The fluorescently stained HUVECs were suspended in EGM-2 to a concentration of 5.0 x ¹⁰ cells/mL, 1.0 x ¹⁰ cells/mL, or 1.5 x ¹⁰ cells/mL to obtain a HUVEC suspension. 10 μL of the obtained HUVEC suspension was added to the first supply hole 27R and introduced into the first supply channel section 23R. The device 1 was placed 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 placed 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. This filled the first channel 20R and the second channel 20L of the device 1 with EGM-2. The microfluidic device 1 was then placed on an Infinity Rocker Mini (Next Advance, hereinafter also referred to as "Rocker") installed in the incubator and incubated under conditions of a rocking angle of 5-7° and a rocking rate of 0.5 cycles/min. During incubation, the medium was replaced with fresh medium once a day.

[Evaluation of three-dimensional tissue]
The prepared 3D tissues were evaluated 1, 3, 5, 7, or 9 days after the start of incubation on the Rocker (Days 1, 3, 5, 7, and 9). Evaluation was performed by acquiring fluorescent images of the 3D tissues. Fluorescent images were acquired using a confocal microscope (FV3000, Evident) to capture the fluorescence of GFP and DsRed excited by 488 nm and 561 nm lasers, respectively. Simultaneously, differential interference contrast images using transmitted light were acquired. The results for Days 1, 5, and 9 when the HUVEC concentration of the HUVEC suspension was 5.0 × ¹⁰ cells/mL are shown in Figure 10. 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 11. The results for Days 1, 5, and 9 when the HUVEC concentration of the HUVEC suspension was 1.5 x ¹⁰ 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.

The results in Figures 10 to 12 show 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 extended into the tumor spheroids. This demonstrates that the microfluidic device of the present invention can be used to create three-dimensional tissues with internal tubular structures. In particular, the microfluidic device of the present invention demonstrated that angiogenesis occurred from vascular endothelial cells toward the tumor spheroids, making it possible to create tumor tissue models containing internal blood vessels.

Figure 13 shows the three-dimensional tissue results for Days 1, 3, 5, 7, and 9 in Chamber 2 when a 5.0 x ¹⁰ cells/mL HUVEC suspension was seeded. In Figure 13, the length of the scale bar is 200 μm. For each three-dimensional tissue, the area occupied by tumor or blood vessels 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 the results are shown in Figure 14. The ratio of the area occupied by blood vessels (HUVECs; green fluorescence) to the area of cell aggregate arresting section 53S1 (vascular area ratio, Ratio of vascular area) was calculated and is shown in Figure 15. The ratio of the area occupied by blood vessels (HUVECs; green fluorescence) to the area of the spheroid region (the area indicated by the white circle in the fluorescent image in Figure 13) (intratumor vascular area ratio, Ratio of invasive vascular area) was calculated and is shown in Figure 16. The results in Figures 14 to 16 are shown as the mean ± standard deviation for n = 8.

The results in Figures 13 to 16 show that the area occupied by the tumor increases over time, and that blood vessels extend into the tumor. This demonstrates that the microfluidic device of the present invention makes it possible to evaluate the process of creating three-dimensional tissue with an internal tubular structure from the perspectives of both tumor growth and blood vessel extension. It is believed that a microfluidic device capable of simultaneously evaluating tumor growth and blood vessel extension can be used to evaluate three-dimensional tissue and the interaction between tumors and blood vessels during its formation process.

Example 2: Preparation and evaluation of three-dimensional tissue slices from a microfluidic device
A microfluidic device 1 with a device substrate 3 made of polydimethylsiloxane (PDMS) was used to prepare a three-dimensional tissue using 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.

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 an image in which the fluorescence image and transmitted light image for a cross section perpendicular to the second direction (A2 direction) are superimposed, alongside a schematic diagram showing an overview of the three-dimensional tissue in the cross section. Figure 19 also shows an enlarged fluorescence image 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, by fluorescently observing the prepared frozen sections, it was possible to visualize the vascular lumen formed within the three-dimensional tissue.

Figure 20(a) shows a fluorescent image of a cross section perpendicular to the third direction (A3 direction). Figure 20(b) shows the fluorescent image of a cross section perpendicular to the second direction alongside a schematic diagram showing an overview of the three-dimensional tissue in the cross section. In Figure 20(a), the length of the scale bar is 200 μm. In Figure 20(b), the length of the scale bar is 50 μm. As shown in Figure 20, with the frozen section prepared using microfluidic device 1, 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.

Furthermore, when the fabricated three-dimensional tissue was observed, the infiltration of blood vessels into the tumor was clearly visualized in a cross section perpendicular to the third direction, and the formation of vascular cavities within the tumor was clearly visualized in a cross section perpendicular to the second direction. Three-dimensional tissue with blood vessels formed within the tumor can be suitably used to evaluate drug administration through blood vessels and the ability of tumors to form blood vessels. Therefore, it was demonstrated that the microfluidic device 1 can be used to fabricate three-dimensional tissue suitable for evaluating drug administration and the ability of tumors to form blood vessels.

Example 3: Comparison of blood vessel area error between cell aggregate storage sections (chambers) and between devices
In order to evaluate the stability of the quality of three-dimensional tissues fabricated using the microfluidic device 1, we investigated whether there was variation in the blood vessel area among the cell aggregate storage sections 5A, 5B, and 5C. For the same purpose, we also investigated whether there was variation in the blood vessel area among the devices.

Seven microfluidic devices 1 were used to create three-dimensional tissues in the same manner as in Example 1. However, cell aggregate storage section 5A (Chamber 1) was created in seven devices, 5B (Chamber 2) in seven devices, and 5C (Chamber 3) in five devices. In this case, three-dimensional tissues were created 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 taken, and the area percentage occupied by blood vessels was measured and calculated in the same manner as in Example 1.

Figure 21 shows 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 Figure 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 greater. Figure 22 also shows 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). Additionally, Figure 23 shows the standard deviation of the measurement results within each device (Device) in Figure 22 and the standard deviation of the measurement results within each chamber (Chamber, standard deviation of measurement results between devices, S.D.) in Figure 21.

The results in Figures 21 to 23 show that, first, in Figure 21, no significant difference was observed in the area occupied by blood vessels between the chambers within the device. This demonstrates that, in microfluidic device 1, there is no difference in the efficiency of blood vessel growth between cell aggregate storage sections 5A, 5B, and 5C, and that three-dimensional tissue of consistent quality can be produced regardless of which chamber is used. Furthermore, the results in Figures 22 and 23 show that the standard deviation was small, averaging less than 0.10, both within the device and between devices. This demonstrates that three-dimensional tissue produced using microfluidic device 1 can be used to perform assays such as drug screening and evaluation, and to elucidate the mechanism of tubular structure formation, under conditions with minimal device-related error.

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

<Example 4-2: Preparation of three-dimensional tissue from spheroids containing tumor cells and pericytes, and vascular endothelial cells 2>
Spheroids containing tumor cells and pericytes and ^three -dimensional tissues using vascular endothelial cells were prepared in the same manner as in Example 4-1, except that the cell concentrations in cell suspensions A, B, and C were half that of Example 4-1 (5.0 × 10 4 cells/mL).

Example 5: Preparation of three-dimensional tissue using spheroids containing tumor cells, 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 standard methods. The stained tRCC cells were suspended in cell culture medium (IMDM medium (Wako, 098-06465) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) at 1.0 x ¹⁰ cells/mL to obtain a cell suspension. The cell suspension was seeded into a 96-well plate (Sumitomo Bakelite, MS-9096U) at 200 μL per well (2.00 x ¹⁰ cells per well). The seeded cells were cultured in an incubator at 37°C and 5% _CO2 for 2 days to prepare spheroids containing tumor cells.

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

Example 6: Preparation of three-dimensional tissue using spheroids containing tumor cells and pericytes, pericytes in gel, and vascular endothelial cells
Spheroids containing tumor cells and pericytes were prepared according to the same method 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 x 10 ⁴ cells per well, pericytes: 5.0 x 10 ³ cells per well). A PC-fibrin gel solution was prepared according to the same method as in Example 5, and 1 volume% of 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. Three-dimensional tissues were then 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.

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

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

Example 7: Construction of an angiogenesis evaluation system using microfluidic device 1
We investigated whether microfluidic device 1 could be used to evaluate angiogenesis in tRCC cell spheroids. For the three-dimensional tissues prepared in Examples 4-1, 4-2, 5, and 6 and Comparative Examples 1 and 2, fluorescent images were acquired 9 days after the start of culture, as in Example 1, and the presence or absence of angiogenesis was evaluated. The results are shown in Figure 24. In Figure 24, K represents ¹⁰³ and M represents ^106. The length of the scale bar in Figure 24 is 200 μm. Figure 25 also shows a comparison of the ratio of vascular area to the entire image in Examples 4-1, 5, and 6. The results in Figure 25 represent the results of preparing and evaluating two or three three-dimensional tissues per device using three devices. The results in Figure 25 are shown as mean ± standard deviation.

As shown in Figure 24, no angiogenesis was observed under the conditions of Comparative Examples 1 and 2, where 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, where 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. These results also demonstrate that angiogenesis can be evaluated according to conditions by evaluation using microfluidic device 1. Furthermore, Figure 25 unexpectedly showed that, of the conditions of Examples 4-1, 5, and 6, the condition of Example 5, where pericytes were present in the gel but not within the spheroids, may be the most likely to result in vigorous angiogenesis. This demonstrates that evaluation using microfluidic device 1 can evaluate not only the presence or absence of angiogenesis but also the extent of angiogenesis that has occurred. Furthermore, because the microfluidic device 1 is capable of evaluating the degree of angiogenesis in this way, it has been 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.

Example 8: Preparation of three-dimensional tissue using spheroids containing tumor cells, pericytes in gel, 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 standard methods. The stained tRCC cells were suspended in cell culture medium (IMDM medium (Wako, 098-06465) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) at 1.0 x ¹⁰ cells/mL to obtain a cell suspension. The cell suspension was seeded into a 96-well plate (Sumitomo Bakelite, MS-9096U) at 200 μL per well (2.00 x ¹⁰ cells per well). The seeded cells were cultured in an incubator at 37°C and 5% _CO2 for 2 days to prepare spheroids containing tumor cells.

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

Fluorescent images of the three-dimensional tissues thus prepared were obtained 9 days after the start of culture, as in Example 1, and angiogenesis was evaluated. The results are shown in Figure 26. In Figure 26, K represents ¹⁰³ and M represents ^106. The scale bar in Figure 26 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 preparing and evaluating two or three three-dimensional tissues per device using three devices (N = 6-8). The results in Figure 27 are shown as mean ± standard deviation, and * indicates a p-value of less than 0.05 in the Tukey test after ANOVA (analysis of variance).

As shown in Figures 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 an assay using microfluidic device 1 can evaluate differences in blood vessel growth within tumors due to differences in cell density.

<Example 9: Creation of three-dimensional tissues using tumor cell-containing spheroids, various concentrations of pericytes in a gel, and vascular endothelial cells in the presence of various concentrations of fibrinogen>

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

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. Three-dimensional tissues were then prepared in the same manner as in Example 1, producing three-dimensional tissues consisting of tumor spheroids, pericytes in the gel, and vascular endothelial cells. A 1.5 x 10 ⁷ cell/mL suspension of fluorescently stained HUVECs was used as the vascular endothelial cells. In the three-dimensional tissues produced in this example, pericytes were present in the gel surrounding the spheroids.

For the three-dimensional tissues thus prepared, fluorescent images were taken 9 days after the start of culturing in the same manner as in Example 1, and angiogenesis was evaluated. The results are shown in Figure 28. In Figure 28, K represents ¹⁰³ and M represents ^106. The length of the scale bar in Figure 28 is 200 μm. Figure 29 also 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 ± standard deviation.

As shown in Figures 28 and 29, it was possible to quantitatively evaluate angiogenesis under all conditions. These results demonstrate that assays using microfluidic device 1 can evaluate angiogenesis under a combination of multiple conditions. Therefore, while the concentration of each biomolecule in a biological environment can vary widely, assays using microfluidic device 1 can quantitatively evaluate angiogenesis under conditions that simulate a variety of biological environments.

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...restricting 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 channel plate, 3S...array substrate, 2p...cured product.

Claims (10)

a first flow path in which a first endothelial cell is disposed and which extends in a first direction;
a second flow path having second endothelial cells arranged therein, the second flow path being spaced apart from the first flow path in a second direction perpendicular to the first direction and extending along the first direction;
a cell aggregate storage section that stores cell aggregates and is sandwiched between the first flow path and the second flow path when viewed from the first direction,
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;
A microfluidic device, wherein 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. the first flow path is formed on the first cell adhesive surface and includes a first opening for passing the first endothelial cells to the cell aggregate receiving portion;
The microfluidic device according to claim 1 , wherein 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. the cell aggregate arresting portion overlaps with a region sandwiched between the first opening and the second opening,
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;
The microfluidic device of claim 2, further comprising a cell aggregate introduction section that does not overlap the area sandwiched between the first opening and the second opening, that is connected from the cell aggregate introduction hole to the cell aggregate constraint section, and that guides the cell aggregate introduced from the cell aggregate introduction hole to the cell aggregate constraint section. a plurality of the cell aggregate storage units are provided between the first flow path and the second flow path;
The microfluidic device according to claim 1 , wherein each of the plurality of cell aggregate holding sections is sandwiched between the first flow channel and the second flow channel when viewed from the first direction. The microfluidic device of claim 1, wherein the cell aggregate constraint portion includes a first regulating wall surface that regulates movement of the cell aggregate along the first direction. The microfluidic device of 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. The microfluidic device of 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. A method for producing a three-dimensional tissue having a luminal structure therein, using the microfluidic device according to any one of claims 1 to 7, comprising:
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;
a step of 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 a step of 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 that connects to the inside of the cell aggregate. A method for screening a test substance using the microfluidic device according to any one of claims 1 to 7, comprising:
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;
A screening method comprising: a step of culturing the cell aggregate, the first endothelial cells, and the second endothelial cells to form a three-dimensional tissue having a luminal structure therein so that the first endothelial cells and the second endothelial cells can form a luminal structure that connects to the interior of the cell aggregate; and a step of 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. a first array plate portion;
a second array plate portion bonded to the first array plate portion,
the first array plate portion and the second array plate portion bonded together form a plurality of microfluidic structures;
The microfluidic structure comprises:
a first flow path in which a first endothelial cell is disposed and which extends in a first direction;
a second flow path having second endothelial cells arranged therein, the second flow path being spaced apart from the first flow path in a second direction perpendicular to the first direction and extending along the first direction;
a cell aggregate storage section that stores cell aggregates and is sandwiched between the first flow path and the second flow path when viewed from the first direction,
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;
The cell aggregate storage portion includes a cell aggregate restraint portion that maintains the position of the cell aggregate relative to the first cell adhesive surface and/or the second cell adhesive surface.