WO2023120710A1 - Method for producing synthetic three-dimensional tissue, device for producing synthetic three-dimensional tissue, and synthetic three-dimensional tissue body - Google Patents

Abstract

The present invention comprises: preparing hollow fibers having a hollow part; preparing a pair of holders that are disposed apart in a first direction with a culturing space therebetween, said pair of holders each comprising an engaging wall which faces the culturing space, and a second culturing space that is positioned oppositely from the culturing space in the first direction with respect to the engaging wall, the engaging wall having a plurality of holding sections which pass through the engaging wall in the first direction and which have intervals therebetween in an a plane orthogonal to the first direction, and a communication section which passes through the engaging wall in the first direction and which puts the culturing space and the second culturing space in communication, the holding sections holding an end part side of the hollow fibers in the first direction; holding the hollow fibers in each of the holding sections of the pair of holders; disposing the pair of holders, which hold the hollow fibers, apart from each other in the first direction with the culturing space therebetween; supplying to the culturing space and the second culturing spaces a non-cured scaffolding material that contains cells and curing the scaffolding material; and culturing the cells while irrigating the hollow parts with a culture medium.

Description

Artificial three-dimensional tissue manufacturing method, artificial three-dimensional tissue manufacturing device, and artificial three-dimensional tissue

TECHNICAL FIELD The present invention relates to an artificial three-dimensional tissue manufacturing method, an artificial three-dimensional tissue manufacturing apparatus, and an artificial three-dimensional tissue.
This application claims priority based on Japanese Patent Application No. 2021-209959 filed in Japan on December 23, 2021, the content of which is incorporated herein.

It is difficult for nutrients and oxygen to permeate tissue created by culture when it exceeds a thickness of several hundred μm. In order to prepare a larger tissue, a method of maturing a plurality of thin tissues of about several hundred μm and then assembling them, a method of forming perfusion-capable tubes in a large tissue and performing perfusion culture, and the like have been proposed. However, there is a problem that the method of assembling a plurality of thin structures is complicated and the strength of the structure after assembly is weak. On the other hand, there are problems such as that the tube made in a large tissue is easily crushed, and the piping to the perfusion pump is easily leaked.

In Patent Document 1, a culture medium and cell masses are introduced from a supply port of a channel space portion in which a plurality of fibers are arranged, and are accumulated on the outer surface of each fiber with the vicinity of the discharge port of the channel space portion serving as a growth starting point. Culturing is disclosed. Each fiber in Patent Document 1 is hollow or has a groove formed along its longitudinal direction, and has micropores communicating from the outer surface to the inside of the hollow or the inside of the groove. Each fiber of Patent Document 1 removes metabolic waste products from the cell mass through the micropores and supplies at least one of growth factors and nutrient factors to the cell mass through the micropores.

Japanese Patent No. 6715330

However, with the technique described in Patent Document 1, since cell clusters accumulate on the outer surface of each fiber, orientation is difficult to occur in the tissue after culturing the cell clusters. Therefore, it cannot be said that the cultured tissue is close to living tissue.

The present invention has been made in consideration of the above points, and aims to provide an artificial three-dimensional tissue manufacturing method and an artificial three-dimensional tissue manufacturing apparatus capable of easily manufacturing an oriented large-sized tissue.

Another object of the present invention is to provide an oriented large-sized artificial three-dimensional tissue.

According to the first aspect of the present invention, a hollow fiber having a hollow portion is prepared; and a second culture space located on the opposite side of the culture space in the first direction with respect to the engagement wall, wherein the engagement wall connects the engagement wall to the first culture space. a plurality of holding portions provided in a plane orthogonal to the first direction and penetrating in the direction perpendicular to the first direction; a communicating portion for communicating, wherein the holding portion prepares a pair of the holders for holding the end portions of the hollow fibers in the first direction; holding one end side of the hollow fiber in each of the holders, holding the other end side of the hollow fiber in each of the plurality of holding sections in the other of the pair of holders; Disposing a pair of holders in the first direction with the culture space interposed therebetween, and supplying a pre-cured scaffold material containing cells to the culture space and the second culture space and curing the scaffold material. and culturing the cells while perfusing the hollow portion with a medium.

According to the second aspect of the present invention, a hollow fiber having a hollow portion and a pair of holders spaced apart in the first direction across a culture space in which a cell-containing scaffold material is disposed are provided. The holder has an engagement wall facing the culture space, and a second culture space located on the opposite side of the engagement wall to the culture space in the first direction with respect to the engagement wall. and the engaging wall includes a plurality of holding portions provided at intervals in a plane perpendicular to the first direction and penetrating the engaging wall in the first direction, and the engaging wall. a communicating portion penetrating in a first direction and communicating between the culture space and the second culture space; and one of the pair of holders has a plurality of holding portions each holding one end side of the hollow fiber. and the other of the pair of holders is provided with an artificial three-dimensional tissue manufacturing apparatus in which each of the plurality of holding parts holds the other end side of the hollow fiber.

According to the third aspect of the present invention, a cell-containing scaffold material culture body, and hollow fibers having a hollow portion and provided through the culture body in a direction in which the culture body extends, are provided. An artificial three-dimensional tissue is provided, wherein the hollow portion is a perfusion channel, and the outer peripheral surface of the hollow fiber is a treated surface sterilized with plasma.

According to the present invention, an oriented large-sized tissue can be easily produced.

1 is a cross-sectional view of an artificial three-dimensional tissue manufacturing device according to a first embodiment of the present invention; FIG. FIG. 4 is an external perspective view of the anchor section viewed from the culture space side. FIG. 4 is a cross-sectional view showing a process related to formation of muscle tissue; FIG. 4 is a cross-sectional view showing steps related to culturing muscle tissue. FIG. 4 is a cross-sectional view of the main body of the culture body; FIG. 4 is a cross-sectional view along the channel of the main body of the culture body. FIG. 4 is a diagram showing a fluorescence image of a cell nucleus in a cross section perpendicular to the X direction of one hollow fiber in the main body. FIG. 8 is a diagram showing the relationship between the distance range from the center of the hollow fiber and the cell nucleus density in the fluorescence image of the cell nucleus in FIG. 7; FIG. 10 is a diagram showing a fluorescence image of muscle tissue in a cross section along the X direction in a hollow fiber; 10 is a diagram showing the relationship between the direction and amount of muscle tissue in the fluorescence image shown in FIG. 9. FIG. FIG. 10 is a diagram showing a fluorescence image of muscle tissue in a cross section along the X direction in a hollow fiber; 12 is a diagram showing the relationship between the direction and amount of muscle tissue in the fluorescence image shown in FIG. 11. FIG. FIG. 4 is a diagram showing a fluorescence image of muscle tissue T in a cross section perpendicular to the X direction of the hollow fiber. FIG. 4 is a photographic view of a muscle tissue T cultured using nine hollow fibers F in a cross section perpendicular to the X direction of the hollow fibers. FIG. 15 is a diagram showing a fluorescence image of the muscle tissue shown in FIG. 14; FIG. 16 is a diagram showing an enlarged fluorescence image of a portion of the muscle tissue shown in FIG. 15; 17 is a diagram showing a fluorescence image of cell nuclei around the hollow fibers shown in FIG. 16. FIG. FIG. 3 is an external perspective view of muscle tissue formed in an elongated shape. FIG. 3 is a perspective view of a muscle tissue block in which muscle tissue is stacked in 4 rows×2 layers. FIG. 10 is an exploded perspective view of the artificial three-dimensional tissue manufacturing device according to the second embodiment of the present invention, before passing the hollow fibers. It is a perspective view which shows one side of the anchor part which concerns on the artificial three-dimensional tissue manufacturing apparatus. It is a perspective view which shows the other of the anchor part which concerns on the artificial three-dimensional tissue manufacturing apparatus. It is a figure which shows the state by which the anchor part was connected. 7 is a graph showing the results of texture analysis of the artificial three-dimensional tissue according to the second embodiment; 4 is a table showing amino acid analysis results of the artificial three-dimensional tissue according to the second embodiment.

Embodiments of an artificial three-dimensional tissue manufacturing method, an artificial three-dimensional tissue manufacturing apparatus, and an artificial three-dimensional tissue structure according to a first embodiment of the present invention will be described below with reference to FIGS. 1 to 19. FIG.
It should be noted that each of the embodiments shown below is one aspect of the present invention, does not limit the present invention, and can be arbitrarily changed within the scope of the technical idea of the present invention.

FIG. 1 is a cross-sectional view of an artificial three-dimensional tissue manufacturing apparatus 1. FIG.
As shown in FIG. 1 , the artificial three-dimensional tissue manufacturing apparatus 1 has a support member 10 , a pair of holders 20 , hollow fibers F, and a culture medium supply section 40 .

The support member 10 supports the pair of holders 20 with a predetermined distance in the first direction (horizontal direction in FIG. 1). The support member 10 is a rectangular parallelepiped flat plate. The support member 10 has a groove portion 12 on its upper surface 11 . The grooves 12 are spaced apart in the first direction. The groove portion 12 extends in a second direction orthogonal to the first direction (a direction orthogonal to the plane of the paper in FIG. 1). The support member 10 is made of, for example, a soft elastic material such as PDMS (polydimethylsiloxane).

Hereinafter, the horizontal direction in which the pair of holders 20 are arranged apart from each other is defined as the X direction, the horizontal direction orthogonal to the X direction is defined as the Y direction, and the vertical direction is defined as the vertical direction. , the direction orthogonal to the X direction and the Y direction is defined as the Z direction.

Since the pair of holders 20 are symmetrical about the center line parallel to the Z direction, the same reference numerals are given to the pair of holders 20 below, and the holder 20 located on the +X side will be described, and the -X side will be described. The description of the holder 20 positioned at is omitted.

A culture space 2 is between the pair of holders 20 in the X direction. That is, the pair of holders 20 are spaced apart in the X direction with the culture space 2 interposed therebetween. The holder 20 has a holder body (facing portion) 21 , an anchor portion 22 and a fitting projection 23 . The fitting projection 23 projects downward from the holder body 21 . The fitting projection 23 fits into the groove 12 of the support member 10 from above. By fitting the fitting protrusion 23 into the groove 12 , the holder 20 is supported while being positioned by the support member 10 .

FIG. 2 is an external perspective view of the anchor part 22 viewed from the culture space 2 side.
As shown in FIG. 2, the anchor portion 22 has a cubic shape. The anchor part 22 has an engaging wall 30 , a side wall 35 and a second culture space 37 .

The engagement wall 30 is arranged facing the culture space 2 . The engagement wall 30 has a rectangular plate shape parallel to the YZ plane. The engagement wall 30 has a holding portion 31 and a communicating portion 32 . The holding portion 31 has a circular cross section and penetrates the engaging wall 30 in the X direction. A plurality of holding portions 31 are provided at intervals in the YZ plane. In the holder 20 of the present embodiment, the holding portions 31 are provided in total of nine pieces, three each in the Y direction and the Z direction, centering on the central position of the engaging wall 30 in the YZ plane, with regular intervals therebetween. . A hollow fiber F can be inserted through each of the plurality of holding portions 31 . In the present embodiment, the ends of the hollow fibers F on the +X side are inserted through each of the plurality of holding portions 31 . The plurality of holding portions 31 hold the outer peripheral surfaces Fb of the hollow fibers F that are inserted therethrough.

The communicating portion 32 penetrates the engaging wall 30 in the X direction, which is the thickness direction. The communication part 32 communicates the culture space 2 with a second culture space 37, which will be described later. A plurality of communicating portions 32 are provided at intervals in the YZ plane. In the holder 20 of the present embodiment, a plurality of communicating portions 32 are provided at positions that do not overlap with the holding portion 31 in the YZ plane at intervals. A total of 16 communication portions 32 are provided, four each in the Y direction and the Z direction at regular intervals.

The side wall 35 extends from each edge of the rectangular engagement wall 30 to the +X side opposite to the culture space 2 in the X direction. That is, the side wall 35 has a plate shape parallel to the XZ plane or parallel to the XY plane. Each side wall 35 has a plurality of second communication portions 36 . The second communicating portion 36 penetrates the side wall 35 in the thickness direction. The second communication part 36 allows the second culture space 37 and the outside of the holder 20 to communicate with each other. Four second communicating portions 36 are provided on each side wall 35 at regular intervals, for a total of 16 second communicating portions 36 .

The second culture space 37 is formed inside the anchor section 22 . The second culture space 37 is a cubic space surrounded by the engaging walls 30 , the side walls 35 and the holder body 21 . The second culture space 37 communicates with the culture space 2 via the communicating portion 32 . The second culture space 37 communicates with the outside of the holder 20 in the Y and Z directions via the second communicating portion 36 .

The holder main body 21 is positioned on the +X side, which is the outside of the anchor portion 22 in the X direction. The holder main body 21 faces the engagement wall 30 with the second culture space 37 interposed therebetween in the X direction. The holder main body 21 has an insertion hole 24 , an adhesive portion 25 , a culture medium storage portion 26 and a connection portion 27 .
The insertion hole 24 extends in the X direction. The insertion hole 24 opens to the second culture space 37 at the −X side end and opens to the medium reservoir 26 at the +X side end. The insertion hole 24 is arranged coaxially with the holding portion 31 . Accordingly, there are nine insertion holes 24 in total, three each in the Y direction and the Z direction at regular intervals. A hollow fiber F is inserted through the insertion hole 24 . By arranging the insertion hole 24 and the holding portion 31 coaxially, the hollow fiber F can be inserted across the holding portion 31 and the insertion hole 24 .

The bonding portion 25 extends in a direction perpendicular to the X direction (only the bonding portion 25 extending in the Z direction is shown in FIG. 1). The position of the adhesion portion 25 in the X direction is between the second culture space 37 and the medium reservoir portion 26 . The adhesive portion 25 has one end connected to the insertion hole 24 and the other end opened to the outside of the holder main body 21 . The bonding portion 25 is provided for each of the plurality of insertion holes 24 . The bonding portion 25 is filled with an adhesive 25A. By filling the bonding portion 25 with the adhesive 25A, the gap between the hollow fiber F inserted in the insertion hole 24 and the insertion hole 24 can be closed to prevent the culture medium from leaking out of the gap.

The medium reservoir 26 is an area in which the medium is reserved. The culture medium reservoir 26 is arranged on the +X side of the insertion hole 24 . The culture medium storage part 26 is formed in a range spanning the plurality of insertion holes 24 . Therefore, all of the plurality of insertion holes 24 open to the culture medium reservoir 26 . The culture medium reservoir 26 in the holder 20 located on the +X side stores the culture medium supplied from the culture medium supply section 40 and before perfusing the hollow portions Fa of the hollow fibers F described later. The medium reservoir 26 in the holder 20 located on the -X side stores the medium after the hollow portion Fa of the hollow fiber F has been perfused.

The connecting portion 27 protrudes from the holder main body 21 to the +X side. A pipe 28 is connected to the connecting portion 27 . A hole portion 29 is provided inside the connection portion 27 . The hole portion 29 extends in the X direction, one end of which is open to the culture medium storage portion 26 and the other end of which is open to the +X side end of the connection portion 27 .

The above holder 20 is manufactured using, for example, a 3D printer. The material of the holder 20 is not particularly limited as long as it does not adversely affect the three-dimensional structure, and an appropriate material can be appropriately used according to the selected manufacturing method.

The medium supply unit 40 supplies the medium. The medium supply unit 40 is, for example, a perfusion pump. The culture medium supplied from the culture medium supply unit 40 is introduced into the holder 20 located on the +X side through the pipe 28 . That is, the connecting portion 27 of the holder 20 located on the +X side constitutes an introducing portion 27A into which the culture medium for perfusing the hollow portion Fa of the hollow fiber F is introduced via the pipe 28 . On the other hand, the connection portion 27 of the holder 20 positioned on the −X side constitutes a discharge portion 27B through which the culture medium perfused in the hollow portion Fa of the hollow fiber F is discharged through the pipe 28 .

A hollow fiber F is a fiber having a hollow portion Fa. The hollow fiber F is not particularly limited as long as it has a semipermeable membrane structure. The inner diameter of the hollow portion Fa is preferably about 20-1000 μm, more preferably about 50-500 μm, even more preferably about 50-150 μm. The thickness of the hollow fiber F is preferably about 10 to 200 μm.

The interval between the hollow fibers F defined by the arrangement of the holding portion 31 and the insertion hole 24 is a minute interval of about several hundred μm, preferably 20 to 1000 μm, more preferably 50 to 500 μm, and even more preferably 50 to 150 μm. Regular arrangement is preferred.

The material of the hollow fiber F is not particularly limited as long as it does not have a detrimental effect on cells, scaffold materials (hydrogel, etc.), and the medium. can be mentioned. Also, the average pore size of the porous hollow fiber membrane is desirably large in consideration of the material exchange property, and is desirably about 0.1 to 5 μm. However, the pore size is not limited to this range, and can be appropriately set according to the purpose of use, such as a pore size of less than 0.1 μm. It is also desirable that the minimum average molecular weight (molecular weight cutoff) of standard molecules that do not effectively diffuse through the membrane is greater than 70 kDa.

Further, the outer peripheral surface Fb of the hollow fiber F and the anchor portion 22 are preferably subjected to hydrophilic treatment. By subjecting the outer peripheral surface Fb of the hollow fibers F and the anchor portions 22 to a hydrophilic treatment, when the hollow fibers F are arranged in an array, the inside of the array (between the plurality of hollow fibers F) is filled with a viscous scaffolding material. becomes possible.

As the hydrophilization treatment for the outer peripheral surface Fb of the hollow fiber F and the anchor portion 22, for example, O ₂ plasma treatment or Aqua Plasma (registered trademark) treatment can be employed.
Aquaplasma (registered trademark) treatment is plasma treatment using water vapor. When Aquaplasma (registered trademark) treatment is adopted, surface hydrophilization and sterilization of the artificial three-dimensional tissue manufacturing apparatus 1 can be performed.

In order to culture cells using the hollow fibers F and the anchors 22, it is necessary to sterilize the surfaces of the hollow fibers F and the anchors 22 that come into contact with the cells.
Conventionally, for example, a wet sterilization method using a liquid such as ethanol has been adopted. If the plurality of hollow fibers F arranged in an array are loosened, the distance between the hollow fibers F cannot be controlled, and it becomes difficult to culture the cells in the entire tissue with good viability while evenly supplying nutrients.
In this embodiment, the outer peripheral surface Fb of the hollow fiber F is subjected to a plasma treatment using water vapor. That is, the outer peripheral surface Fb of the hollow fiber F is a plasma-sterilized surface.
Therefore, the outer peripheral surface Fb can be sterilized without causing slack in the hollow fibers F arranged in an array.
As a result, in the present embodiment, cells contained in the scaffolding material can be cultured with high viability around the hollow fibers F whose outer peripheral surface Fb is sterilized, and a large oriented tissue can be easily produced. can.

Next, an artificial three-dimensional tissue manufacturing method using the artificial three-dimensional tissue manufacturing apparatus 1 will be described with reference to FIGS. 3 to 6. FIG.
The artificial three-dimensional tissue manufacturing method according to the present invention comprises preparing a hollow fiber F having a hollow portion Fa, and a pair of holders 20 spaced apart in a first direction with the culture space 2 interposed therebetween, Each has an engaging wall 30 facing the culture space 2 and a second culture space 37 positioned opposite to the culture space 2 in the first direction with respect to the engaging wall 30, and the engaging wall 30 is engaged a plurality of holding parts 31 that penetrate the wall 30 in the first direction and are provided in a plane perpendicular to the first direction at intervals; A communicating portion 32 that communicates with the space 37, and the holding portion 31 prepares a pair of holders 20 that hold the ends of the hollow fibers F in the first direction, and the pair of holders 20 are provided. holding one end side of the hollow fiber F in each of the plurality of holding portions 31 on one side and holding the other end side of the hollow fiber F in each of the plurality of holding portions 31 on the other side of the pair of holders 20; 2 and the second culture space 37 are supplied with a pre-cured scaffold material (e.g., hydrogel, etc.) containing cells and cured, and the cells are cultured while perfusing the medium in the hollow portion Fa; including.

[Preparation of hollow fiber F and holder 20]
As preparation for the hollow fibers F and the holder 20, the hollow fibers F having the outer peripheral surface Fb previously subjected to the above-described hydrophilic treatment are attached to the holding portions 31 and the insertion holes 24 of the pair of holders 20 as shown in FIG. Insert each. The hollow fibers F pass from the holding portion 31 through the second culture space 37 and communicate with the insertion holes 24 . The end of the hollow fiber F may be located at the end facing the culture medium reservoir 26 in the insertion hole 24 or may protrude into the culture medium reservoir 26 . The number of hollow fibers F to be inserted through the holding portion 31 and the insertion hole 24 can be arbitrarily selected from one to nine.

Regarding the holder 20 through which the hollow fiber F is inserted, the fitting protrusion 23 is fitted into the groove 12 of the support member 10 from above, and is supported while being positioned at a predetermined distance in the X direction. After that, the hollow fibers F inserted through the holding portion 31 and the insertion hole 24 are fixed to the insertion hole 24 by filling the bonding portion 25 with an adhesive 25A. As a result, the culture medium stored in the culture medium reservoir 26 can be prevented from flowing out through the gap between the insertion hole 24 and the hollow fibers F.

[Formation of muscle tissue]
After preparation of the hollow fibers F and the holder 20 is completed, muscle tissue is formed as an artificial three-dimensional tissue.
FIG. 3 is a cross-sectional view showing the steps involved in forming muscle tissue.
As shown in FIG. 3, a mixture of cells C suspended in a scaffolding material (for example, hydrogel) is poured into the culture space 2 . A mixture of cells C poured into the culture space 2 and suspended in a scaffolding material (for example, hydrogel) is supplied to the second culture space 37 via the communicating portion 32 and the second communicating portion 36 .

That is, in the formation of muscle tissue, first, a scaffold material (for example, hydrogel) G containing cells C before hardening is supplied to the culture space 2 and the second culture space 37 .
Myoblasts are used as cells C in this embodiment. In addition, an extracellular matrix component is used as a scaffold material (eg, hydrogel, etc.) G.

A mixture of Matrigel ((registered trademark)) and Collagen is used as the extracellular matrix component. The mixing ratio of Matrigel (registered trademark) and collagen is preferably 20 to 80% Matrigel (registered trademark) and 80 to 20% collagen, and 40 to 60% Matrigel (registered trademark) and 60% collagen. More preferably ~40%.

When the pre-hardening scaffolding material (eg, hydrogel, etc.) containing cells C is supplied to the culture space 2 and the second culture space 37, it is cultured under predetermined conditions to harden the extracellular matrix components. Regarding the scaffold material (for example, hydrogel) G supplied to the culture space 2, since the outer peripheral surface Fb of the hollow fibers F is subjected to a hydrophilic treatment, it is can also be filled and cured.

As for the culture conditions, as an example, it is cultured at a temperature of 37°C for 30 minutes. As a result, a culture B in which a scaffolding material (for example, hydrogel, etc.) G containing cells C is cultured is formed. The culture body B consists of a body part B1 cultured in the culture space 2, an outside culture body B2 located outside the body part B1 in the X direction and cultured in the second culture space 37, the body part B1 and the outside culture. It includes a bound culture B3 that is bound to the body B2 and cultured in the communicating section 32 . The outer culture B2 is held in the second culture space 37 as a holding space.

The main body B1, which is cultured in the culture space 2 and contacts the engagement wall 30 from the inside in the X direction, shrinks inward in the X direction without restraint as the scaffolding material (for example, hydrogel, etc.) G shrinks. On the outer culture body B2 held in the second culture space 37, a contractile force inward in the X direction by the body part B1 acts via the bound culture body B3. The engaging wall 30 engages with the outer culture body B2 from the inside in the X direction and acts as an anchor to suppress the contraction of the outer culture body B2 inward in the X direction.

[Culture of muscle tissue by perfusion]
FIG. 4 is a cross-sectional view showing the steps involved in culturing muscle tissue.
When the culture body B is formed by hardening the scaffolding material (for example, hydrogel) containing the cells C, as shown in FIG. be placed on.

After that, the medium is supplied from the medium supply unit 40 to the holder 20 located on the +X side. The medium supplied to the holder 20 perfuses the hollow portions Fa of the hollow fibers F after being temporarily stored in the medium reservoir 26 . FIG. 5 is a cross-sectional view of the body portion B1 in the culture body B. FIG. As shown in FIG. 5, muscle tissue T is formed around hollow fibers F by culturing a scaffolding material (for example, hydrogel, etc.) containing cells C. As shown in FIG. Nutrients, oxygen, and the like permeate the porous hollow fiber membrane from the culture medium that perfuses the hollow portion Fa and diffuse into the muscle tissue T, which is the culture body B. As shown in FIG. On the other hand, from the muscle tissue T, metabolic waste products permeate the porous hollow fiber membrane and diffuse into the culture medium. The medium in which the waste products have diffused by perfusing the hollow portion Fa is once stored in the medium storage section 26 in the holder 20 located on the -X side, and then discharged to the culture tank 100 via the discharge section 27B and the pipe 28. be done.

As described above, the scaffolding material (eg, hydrogel, etc.) G containing cells C before hardening is supplied to the outer periphery of the hollow fiber F, and then the scaffolding material (eg, hydrogel, etc.) G is hardened to form a culture body B. The muscle tissue T can be cultured by forming the muscle tissue T as , and perfusing the medium in the hollow portion Fa. As the culture of the muscle tissue T progresses, the contraction of the body portion B1 increases. However, the engagement wall 30 engages the outer culture body B2 from the inside in the X direction as an anchor, so that the outer culture body B2 contracts. suppressed.

FIG. 6 is a cross-sectional view of the main body B1 of the culture body B along the channel.
In the main body portion B1, since the contraction of the muscle tissue T in the X direction increases, the muscle tissue T is easily oriented along the X direction as shown in FIG.

[Cell nucleus around hollow fiber F]
FIG. 7 shows a fluorescence image of cell nuclei in a cross section perpendicular to the X direction of the hollow fiber F in the main body B1 of the muscle tissue T cultured for 4 days in a state where only one hollow fiber F is perfused with the above medium. FIG. 4 is a diagram showing;
As shown in FIG. 7, cell nuclei are present at high density in the region near the outer peripheral surface Fb of the hollow fiber F, but cell nuclei are present at low density in the region away from the outer peripheral surface Fb of the hollow fiber F. rice field. From this, it can be concluded that nutrients, oxygen, and the like diffuse from the medium that perfuses the hollow portion Fa into the muscle tissue T formed around the hollow fibers F, and the cells are cultured in a live state. I can judge.

[Cell nucleus density and perfusion rate]
FIG. 8 is a diagram showing the relationship between the distance range from the center of the hollow fiber F and the cell nucleus density in the fluorescence image of the cell nucleus in FIG. The distance range from the center of the hollow fiber F includes a ring-shaped range with a radius of 250-300 μm from the center of the hollow fiber F, a ring-shaped range with a radius of 300-350 μm from the center of the hollow fiber F, and a ring-shaped range with a radius of 300-350 μm from the center of the hollow fiber F. A ring-shaped range with a radius of 350-400 μm, a ring-shaped range with a radius of 400-450 μm from the center of the hollow fiber F, and a ring-shaped range with a radius of 450-500 μm from the center of the hollow fiber F are defined. In FIG. 8, the number of cell nuclei with respect to the area in each ring-shaped range is shown as the cell nucleus density.

In addition, in FIG. 8, the distance from the center of the hollow fiber F when the perfusion rate of the medium for perfusing the hollow portion Fa was 0 μL/min, 15 μL/min, 100 μL/min, and 500 μL/min. The relationship between range and cell nucleus density is shown.
As shown in FIG. 8, no significant correlation was observed between the distance range from the center of the hollow fiber F and the cell nucleus density. It was confirmed that the cell nucleus density became higher than the perfusion rate.

[Orientation of muscle tissue]
FIG. 9 shows the body portion B1 of muscle tissue T cultured for 10 days in a state where only one hollow fiber F was perfused with the medium at a perfusion rate of 15 μL/min. FIG. 4 is a diagram showing a fluorescent image of muscle tissue in a cross section; FIG. 10 is a diagram showing the relationship between the direction and amount of muscle tissue in the fluorescence image shown in FIG. The direction (Direction (°)) in FIG. 10 is 0° in the Y direction and 90° in the X direction.

FIG. 11 shows the body portion B1 of the muscle tissue T cultured for 10 days in a state where only one hollow fiber F was perfused with the above medium at a perfusion rate of 500 μL/min. FIG. 4 is a diagram showing a fluorescent image of muscle tissue in a cross section; 12 is a diagram showing the relationship between the direction and amount of muscle tissue in the fluorescence image shown in FIG. 11. FIG. The direction (Direction (°)) in FIG. 12 is 0° in the Y direction and 90° in the X direction.

As shown in FIGS. 9 and 11, muscle tissue T could be formed when the medium was perfused at a perfusion rate of 15 μL/min. When the medium was perfused at a perfusion rate of 500 μL/min, a muscle tissue T with a greater thickness could be formed than when the medium was perfused at a perfusion rate of 15 μL/min. Further, as shown in FIGS. 10 and 12, in the muscle tissue T formed using the scaffold material (eg, hydrogel, etc.) G containing the cells C, the medium was perfused at a perfusion rate of 15 μL/min or more and 500 μL/min. When the culture was perfused at a rate of min or less, the contraction of the culture allowed the formation of muscle tissue T that was largely oriented in the X direction.
From these results, muscle tissue T can be formed when the medium perfusion rate is 15 μL/min or higher. In this case, even in the range where the medium perfusion rate exceeds 500 μL/min, not only can the muscle tissue T be formed, but also the muscle tissue T can be formed with a larger film thickness, a higher cell nucleus density, and a greater degree of orientation in the X direction. is assumed.

[Muscle tissue around hollow fiber F]
FIG. 13 shows the fluorescence of the muscle tissue T in the cross section perpendicular to the X direction of the hollow fiber F in the main body B1 of the muscle tissue T cultured for 10 days in a state where only one hollow fiber F is perfused with the above medium. FIG. 4 is a diagram showing an image;
As shown in FIG. 13, since muscle tissue is differentiated and formed around the hollow fibers F, the muscle tissue T formed around the hollow fibers F is perfused with the hollow portion Fa. It can be determined that nutrients, oxygen, and the like diffuse from the cells, and the cells are cultured in a live state.

[Muscle tissue with 9 hollow fibers F]
FIG. 14 is a photograph of the muscle tissue T in the cross section perpendicular to the X direction of the hollow fibers F in the main body B1 of the muscle tissue T cultured for 4 days in the state of perfusion with the medium using the nine hollow fibers F. It is a diagram.
As shown in FIG. 14, the muscle tissue T was able to be formed in a size surrounding the nine hollow fibers F. The center-to-center distance of the hollow fibers F was 900 μm before culturing the muscle tissue T, but the center-to-center distance of the hollow fibers F in the muscle tissue T after culturing was approximately 600 μm due to contraction accompanying the culture. In addition, the muscle tissue T after culture had a minimum width of approximately 2300 μm (2.3 mm).

Therefore, the muscle tissue T cultured by the artificial three-dimensional tissue manufacturing method using the artificial three-dimensional tissue manufacturing apparatus 1 of the present embodiment has an orientation and can easily produce a larger tissue than the conventional artificial three-dimensional tissue. I was able to

[Fluorescent image of muscle tissue by nine hollow fibers F]
15 is a diagram showing a fluorescence image of the muscle tissue in FIG. 14. FIG.
As shown in FIG. 15, muscle tissue was formed around eight hollow fibers F out of the nine hollow fibers F, indicating that the eight hollow fibers F were cultured by perfusion. I can judge.

FIG. 16 is a diagram showing a fluorescence image in which a part of the muscle tissue shown in FIG. 15 is enlarged.
As shown in FIG. 16, cell nuclei are present at high density in the region near the outer peripheral surface Fb of the hollow fiber F, but cell nuclei are present at low density in the region away from the outer peripheral surface Fb of the hollow fiber F. . From this fact, nutrients, oxygen, etc. diffuse from the medium perfusing the hollow portion Fa into the muscle tissue T formed around the eight hollow fibers F out of the nine hollow fibers F, and the cells It can be judged that the culture is performed in a living state.

[Fluorescence image of cell nucleus by nine hollow fibers F]
FIG. 17 is a diagram showing a fluorescent image of cell nuclei around the hollow fibers F shown in FIG.
As shown in FIG. 17, similar to the case of using one hollow fiber F, in the region near the outer peripheral surface Fb of the hollow fiber F, cell nuclei exist at a high density, but the outer peripheral surface Fb of the hollow fiber F Cell nuclei were present at a low density in regions away from . From this, it can be concluded that nutrients, oxygen, and the like diffuse from the medium that perfuses the hollow portion Fa into the muscle tissue T formed around the hollow fibers F, and the cells are cultured in a live state. I can judge.

[Long muscle tissue]
FIG. 18 is an external perspective view of an elongated muscle tissue. The muscle tissue shown in FIG. 18 is formed by culturing using four hollow fibers F arranged in two rows in the Y and Z directions. Whereas the muscle tissue described above was approximately 5 mm in length of body portion B1, the muscle tissue shown in FIG. 18 is approximately 4 cm in length of body portion B1.

When electricity was applied to this muscle tissue at 1 Hz and 19 Vpp, contraction was observed. Further, when the muscle tissue was energized at 20 Hz and 19 Vpp, a contraction state was continuously observed. Further, when the above energization was performed after removing the hollow fiber F from this muscle tissue, contraction with a larger displacement was observed.

In addition, two muscle tissue Ts each having a length of 4 cm were prepared, and each block was cut into four blocks each having a length of 1 cm. As shown in FIG. bottom. As an example, the muscle tissue block TB has a cross-sectional side of 2-2.5 mm. Therefore, the muscle tissue block TB can produce a large tissue on the order of centimeters with a width of 8-10 mm and a height of 4-5 mm. Also, by removing the hollow fibers F from the muscle tissue, it becomes possible to construct a large-sized artificial three-dimensional tissue suitable for meat.

As described above, in the present embodiment, by inserting the hollow fibers F through the holding portion 31 and the insertion hole 24 provided in the holder 20, a hardened scaffold material (for example, hydrogel, etc.) containing the cells C can be obtained. Hollow fibers F are passed through G, and the medium is perfused into the hollow portions Fa of the hollow fibers F for culturing. It becomes possible to manufacture to

In addition, in the present embodiment, by introducing the adhesive 25A into the adhesive portion 25 of the holder 20, the gap between the hollow fiber F and the insertion hole 24 can be closed to prevent the culture medium from leaking out of the gap.

In addition, in this embodiment, by fitting the fitting protrusions 23 of the holders 20 into the grooves 12 of the support member 10, the distance between the pair of holders 20 can be accurately defined. Therefore, in the present embodiment, it is possible to define the length in the X direction of the body portion B1 of the culture body B with high accuracy.

Further, in the present embodiment, since the outer peripheral surface Fb of the hollow fibers F and the anchor portion 22 are subjected to a hydrophilic treatment, when the hollow fibers F are arranged in an array, a viscous scaffolding material (for example, hydrogel, etc.) ) can be filled in the array (between a plurality of hollow fibers F), and the muscle tissue T can be easily produced even in an array with narrow gaps between the hollow fibers F.

In addition, in this embodiment, the artificial three-dimensional tissue structure M can be constructed by removing the pair of holders 20 containing the culture B (muscle tissue T) and the hollow fibers F from the support member 10. Of the artificial three-dimensional tissue structure M, the fitting protrusion 23 on one side of the holder 20 is supported by, for example, a fixed portion of the biological model, and the fitting protrusion 23 on the other side of the holder 20 is supported by the movable portion of the biological model. It can be used as an actuator in a biological model by supporting and contracting the culture body B (muscle tissue T) by energizing it.

When the artificial three-dimensional tissue structure M is used as an actuator, it is preferable to culture animal-derived cells C having a large contractile force in the muscle tissue T.
As animal-derived cells C, for example, C2C12 cells can be used. The use of C2C12 cells makes it possible to construct actuators without sacrificing animals.

Furthermore, in the present embodiment, by separating the pair of holders 20 from the artificial three-dimensional tissue structure M, the artificial three-dimensional tissue structure N in which the hollow fibers F penetrate the culture body B (muscle tissue T) in the longitudinal direction is formed. can get. In this artificial three-dimensional tissue N, since the outer peripheral surface Fb of the hollow fiber F is a treated surface sterilized with plasma, the outer peripheral surface Fb is sterilized without causing slack, and the cells contained in the scaffold material survive. It becomes possible to culture efficiently, and it becomes a large-sized tissue with orientation.

A second embodiment of the present invention will be described with reference to FIGS. 20 to 25. FIG. In the following description, the same reference numerals are given to the same configurations as those already described, and redundant descriptions will be omitted.

FIG. 20 shows an exploded view of the artificial three-dimensional tissue manufacturing device 101 according to this embodiment. The artificial three-dimensional tissue manufacturing device 101 includes a holder 120 in place of the holder 20 . Each holder 120 has a structure in which the guide 102 is arranged between the anchor portion and the holder body 121 .

In this embodiment, one holder is provided with an anchor portion 122A, and the other holder is provided with an anchor portion 122B. The anchor portion 122A and the anchor portion 122B are slightly different in shape.

FIG. 21 shows the anchor part 122A viewed from the front side facing the culture space. The anchor part 122A has 50 cylindrical holding parts 131A, and is configured so that more hollow fibers can be inserted than in the first embodiment. In the present embodiment, the holding portions 131A are arranged in a two-dimensional matrix of 5×10, but this is not essential, and by arranging them in another manner such as a honeycomb shape, the per unit cross-sectional area It is also possible to increase the arrangement density of the hollow fibers.
The holding portion 131A has a portion with a small inner diameter inside, thereby having a step 202 inside.

FIG. 22 shows the anchor part 122B viewed from the front side. The anchor portion 122B also has 50 cylindrical holding portions 131B like the anchor portion 122A. The outer diameter of the holding portion 131B is smaller than the inner diameter of the end opening of the holding portion 131A, and can be inserted into the holding portion 131A. Since the outer diameter of the holding portion 131B is larger than the inner diameter of the step 202 inside the holding portion 131A, it cannot enter deeper than the step 202 .

In this embodiment, an example of the procedure for passing the hollow fibers through the holder will be described.
The anchor part 122A and the anchor part 122B are brought close to each other with their front sides facing each other, and the ends of the respective holding parts 131B are inserted into the opposing holding parts 131A. Thereby, the anchor portion 122A and the anchor portion 122B are connected as shown in FIG. During the connecting operation, when the holding portion 131B enters the holding portion 131A by a certain amount, the end portion of the holding portion 131B abuts against the step 202, so excessive entry is suppressed.

With the anchor parts 122A and 122B connected, a hollow fiber is passed from the rear side of one anchor part through the holes that communicate with the holding parts, and protrudes from the rear side of the other anchor part. The hollow fiber may be inserted from either the anchor portion 122A or the anchor portion 122B.

After passing the hollow fibers through all the holding portions, the guides 102 are attached to the anchor portions 122A and 122B while passing the plurality of hollow fibers protruding from the rear side of the anchor portions 122A and 122B through different guides 102 respectively. . The attachment method is not particularly limited, and examples include mechanical fitting and adhesion.
As shown in FIG. 20, the guide 102 has an inclined surface 102a, and the width of the space through which the hollow fibers pass gradually narrows as the distance from the anchor portion increases. For this reason, the hollow fibers passing through the guide 102 are bundled into one while gradually narrowing the distance between them and guided to the holder main body 121 .

When the distance between the anchor portion 122A and the anchor portion 122B is adjusted to the distance when the anchor portion 122B is installed on the support member 10, and the holder body 121 is fixed to the guide 102, the anchor portion, the guide, and the holder body are integrated to form a pair. becomes the holder 120 of The operation for adjusting the distance between the anchor portions can be performed at any timing after the hollow fibers are passed through the two anchor portions in the connected state.
After that, the anchor portions 122A and 122B are inserted into the grooves 10a provided in the support member 10. As shown in FIG. As a result, the holder 120 holding the hollow fibers is attached to the support member 10 to complete the artificial three-dimensional tissue manufacturing apparatus 101, and perfusion and culture can be performed in the same manner as in the first embodiment.

In the artificial three-dimensional tissue manufacturing apparatus according to the present invention, it is possible to produce a larger artificial three-dimensional tissue by increasing the number of hollow fibers. becomes complicated. In particular, when a hollow fiber is passed through two holders that are spaced apart, the hollow fiber passed through one holder may be bent, making it difficult to pass through the other holder, or may be held in a non-corresponding (non-face-to-face) manner. The possibility of having to pass it through a part and need to redo it will increase, and the difficulty of the work will also increase.

In the artificial three-dimensional tissue manufacturing device 101 according to this embodiment, as described above, the anchor portions 122A and 122B are configured to be connectable. Therefore, by separating the anchor portion 122A and the anchor portion 122B after passing the hollow fiber in a connected state, it is possible to significantly suppress the occurrence of the above-described error when passing the hollow fiber.
As a result, even if the number of hollow fibers increases, it is possible to prevent the assembly of the artificial three-dimensional structure manufacturing apparatus and the execution of the artificial three-dimensional structure manufacturing method from becoming complicated. With such a configuration, the artificial three-dimensional tissue manufacturing apparatus can be expected to be automatically manufactured by a robot.

In this embodiment, the number of hollow fibers is not limited to 50 as described above, and it is of course possible to increase the number. Even if the number of anchors increases, the difficulty of assembling the artificial three-dimensional tissue manufacturing device and executing the artificial three-dimensional tissue manufacturing method does not change significantly by passing the hollow fibers while the anchor portions are connected.

In this embodiment, it is not essential to provide the step 202 inside the holding portion. As another method, an engaging wall around the holding portion can be used as a stopper to prevent excessive intrusion during connection.

In the above example, a plurality of hollow fibers are bundled together in the guide 102, but by changing the number and arrangement of slopes provided on the guide, the hollow fibers can be bundled into two or more bundles. The guide may be configured as follows. Such a mode is suitable when the number of hollow fibers increases, for example, 100 or more.

The evaluation results of the artificial three-dimensional tissue manufactured using the artificial three-dimensional tissue manufacturing apparatus 101 according to the present embodiment are shown.
An artificial three-dimensional tissue was produced using chicken myoblasts using an artificial three-dimensional tissue manufacturing apparatus configured using 50 hollow fibers. Two types of artificial three-dimensional tissues were prepared: a perfused sample cultured for 9 days with perfusion and a non-perfused sample cultured for 9 days without perfusion.

After culturing, the hollow fiber was pulled out and measured, and the weight of the perfused sample was 470 mg.
In the perfused sample, it was confirmed that cell nuclei were present at high density around the hollow fibers F, as in the first embodiment. In addition, although the tissue was larger than that of the first embodiment, the orientation of the muscle tissue was well aligned in the longitudinal direction of the hollow fiber, and good contraction was observed by energization.

Further analyzes were performed using perfused and non-perfused samples.
(Evaluation 1: Tissue texture analysis)
Using a texture analyzer (TA.XTPlus), a stress-time curve was acquired by measuring the reaction force of the tissue over time from pressing the tissue to half the thickness with a probe that moves up and down at a constant speed until it returns. . Each sample was divided into three, and each sample was measured three times.

The results are shown in FIG. In the graph, the horizontal axis is the elapsed time (seconds), and the vertical axis is the magnitude of the reaction force (g/mm ² ) that the probe received from the tissue.
All perfused samples had a longer time to peak reaction force than non-perfused samples. This indicates that the perfused samples are thicker than the non-perfused samples. In addition, the peak values of the reaction force of the perfused samples were all higher than the non-perfused samples. This indicates that the perfused samples have higher elasticity than the non-perfused samples, suggesting a denser tissue.
Evaluation 1 indicates that, for example, when an artificial three-dimensional tissue is used for meat, the elasticity of the tissue can be moderately improved by culturing the tissue while perfusion is performed, and the texture can be improved.

(Evaluation 2: Tissue amino acid analysis)
A post-column derivatization method was used to determine the amount of amino acids contained in each sample. Furthermore, amino acids mainly involved in sweetness (threonine, serine, glycine, alanine, proline), amino acids mainly involved in umami (glutamic acid, aspartic acid, glutamine, asparagine), amino acids mainly involved in bitterness ( tyrosine, valine, leucine, phenylalanine, methionine, isoleucine, arginine, cysteine, histidine, lysine, and tryptophan), and the ratio was calculated.

The results are shown in FIG. The total amount of amino acids per 100 grams of tissue in perfused samples was approximately double that in non-perfused samples. Also, the proportions of umami amino acids did not differ between samples, but there was a higher proportion of sweet amino acids and a lower proportion of bitter amino acids in the perfused samples.
Evaluation 2 indicates that, for example, when the artificial three-dimensional tissue is used for meat, the nutritional value and flavor can be improved by culturing while performing perfusion.

Although the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, it goes without saying that the present invention is not limited to such examples. The various shapes, combinations, etc., of the constituent members shown in the above examples are merely examples, and various modifications can be made based on design requirements and the like without departing from the gist of the present invention.

For example, hollow fibers that branch midway may be used. In this case, it is conceivable to have one holder on one side of the hollow fiber and two or more holders on the other side. can be identified as a pair of holders, and the direction in which the pair of holders are separated can be identified as the first direction.

The present invention can be suitably applied to an artificial three-dimensional tissue and its manufacture.

1, 101... artificial three-dimensional tissue manufacturing apparatus, 2... culture space, 10... support member, 20, 120... holder, 21, 121... holder main body (facing part), 22, 122A, 122B... anchor part, 24... insertion Hole 25 Adhering portion 25A Adhesive 27A Introduction portion (connecting portion) 27B Discharging portion (connecting portion) 30 Engaging wall 31, 131A, 131B Holding portion 32 Communicating portion 35... side wall, 36... second communication part, 37... second culture space (holding space), B... culture body, B1... body part, B2... outer culture body, B3... bound culture body, C... cell, F... Hollow fiber, Fa... Hollow portion, Fb... Peripheral surface, G... Scaffolding material, M... Artificial three-dimensional tissue structure

Claims (13)

1. preparing a hollow fiber having a hollow portion;
   A pair of holders spaced apart in a first direction with a culture space interposed therebetween, comprising an anchor portion having an engaging wall facing the culture space, and the culture space in the first direction with respect to the engaging wall. a space and a second culture space located on the opposite side, and the engaging walls penetrate the engaging walls in the first direction and are spaced apart from each other in a plane orthogonal to the first direction a plurality of holding portions; and a communicating portion that penetrates the engaging wall in the first direction and communicates the culture space and the second culture space, wherein the holding portion is the hollow fiber. preparing a pair of said holders for holding end sides in the first direction;
   One end side of the hollow fiber is held by each of the plurality of holding portions of one of the pair of holders, and the other end side of the hollow fiber is held by each of the plurality of holding portions of the other of the pair of holders. and
   arranging the pair of holders holding the hollow fibers so as to be spaced apart in the first direction with the culture space interposed therebetween;
   supplying an uncured scaffold material containing cells to the culture space and the second culture space and curing the scaffold material;
   culturing the cells while perfusing the hollow portion with a medium;
   including,
   An artificial three-dimensional tissue manufacturing method.
2. the holder has a facing portion facing the engagement wall across the second culture space in the first direction;
   The facing portion includes an insertion hole extending in the first direction and through which the hollow fiber is inserted;
   a bonding portion extending in a direction orthogonal to the first direction, having one end connected to the insertion hole and the other end opening outward;
   has
   holding the hollow fibers in the pair of holders includes introducing an adhesive through the bonding portion to fix the hollow fibers in the insertion holes;
   The method for producing an artificial three-dimensional tissue according to claim 1.
3. Preparing the pair of holders includes supporting the pair of holders on a support member while being separated from each other by a predetermined distance in the first direction,
   The method for producing an artificial three-dimensional tissue according to claim 1 or 2.
4. Preparing the hollow fiber includes:
   including subjecting the outer peripheral surface of the hollow fiber and the anchor portion to a hydrophilic treatment,
   The method for producing an artificial three-dimensional tissue according to any one of claims 1 to 3.
5. culturing the cells while perfusing the medium into the hollow portion at a rate of 15 μL/min or more;
   The method for producing an artificial three-dimensional tissue according to any one of claims 1 to 4.
6. a hollow fiber having a hollow portion;
   a pair of holders spaced apart in a first direction across a culture space in which a scaffold material containing cells is arranged;
   has
   The holder is
   an anchor portion having an engagement wall facing the culture space;
   each having a second culture space positioned opposite to the culture space of the engagement wall in the first direction with respect to the engagement wall;
   The engagement wall is
   a plurality of holding portions that penetrate the engagement wall in the first direction and are provided in a plane orthogonal to the first direction at intervals;
   a communicating portion that penetrates the engaging wall in the first direction and communicates the culture space and the second culture space;
   In one of the pair of holders, each of the plurality of holding portions holds one end side of the hollow fiber,
   In the other of the pair of holders, each of the plurality of holding portions holds the other end side of the hollow fiber.
   Artificial three-dimensional tissue manufacturing device.
7. one of the pair of holders has an introduction part into which a medium for perfusing the hollow part is introduced;
   The other of the pair of holders has a discharge part through which the medium perfused in the hollow part is discharged,
   The artificial three-dimensional tissue manufacturing apparatus according to claim 6.
8. the holder has a facing portion facing the engagement wall across the second culture space in the first direction;
   The facing portion includes an insertion hole extending in the first direction and through which the hollow fiber is inserted;
   a bonding portion extending in a direction orthogonal to the first direction, having one end connected to the insertion hole and the other end opening outward;
   has
   The hollow fiber is fixed to the insertion hole by an adhesive applied to the bonding portion,
   The artificial three-dimensional tissue manufacturing device according to claim 6 or 7.
9. Having a support member that supports the pair of holders in a state separated by a predetermined distance in the first direction,
   The artificial three-dimensional tissue manufacturing device according to any one of claims 6 to 8.
10. The hollow fiber is subjected to hydrophilic treatment on the outer peripheral surface,
    The artificial three-dimensional tissue manufacturing device according to any one of claims 6 to 9.
11. The engagement wall has a rectangular shape when viewed in the first direction,
    the holder has a side wall extending from each edge of the engagement wall in the first direction in a direction opposite to the culture space and surrounding the second culture space;
    The side wall has a second communication part that penetrates the side wall and communicates the second culture space with the outside of the holder.
    The artificial three-dimensional tissue manufacturing device according to any one of claims 6 to 10.
12. a culture of a scaffold material containing cells;
    a hollow fiber having a hollow portion and penetrating the culture body in a direction in which the culture body extends;
    has
    the hollow portion is a perfusion channel,
    The outer peripheral surface of the hollow fiber is a plasma-sterilized surface,
    An artificial three-dimensional tissue.
13. The culture has muscle tissue,
    The artificial three-dimensional tissue according to claim 12.
    

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