JP2017195845A - Culture vessel, culture apparatus, and culture method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture vessel, a culture apparatus, and a culture method, which are capable of forming a tubular blood vessel-like structure angiogenesis using cultured cells to allow angiogenesis to be sufficiently evaluated.SOLUTION: A culture vessel according to one embodiment of the present invention comprises a first cell, a second cell, and a third cell. The first cell is able to contain a culture solution. The second cell is able to contain the culture solution. The third cell comprises a first partition adjacent to the first cell, a second partition wall adjacent to the second cell, and a first pore portion, the third cell being capable of connecting the first cell and the second cell and allowing a cell scaffold material to be filled in the flow passage. The first pore portion includes at least a pair of pores formed so as to respectively face the first partition wall and the second partition wall and communicate with the first cell and the second cell, the first partition wall being configured to define a flow path of the culture solution.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a culture vessel, a culture apparatus, and a culture method capable of producing a tubular blood vessel-like structure by culturing vascular endothelial cells and the like.

  Angiogenesis is deeply involved in the invasion and metastasis of cancer cells, and drugs that suppress this angiogenesis are being developed. In recent years, in order to evaluate angiogenesis in screening for drugs and the like, a technique for artificially producing tubular blood vessel-like cells in vitro has been attracting attention in place of animal experiments and the like.

For example, Non-Patent Document 1 discloses a method in which a tube structure is produced by piercing and pulling a needle into a gel-like cell scaffold, and then seeding vascular endothelial cells on the cell scaffold.
Patent Document 1 discloses a method of creating a blood vessel-like structure by creating a plurality of channels using a plurality of mandrels and seeding the cells, and a channel in which the mandrels are withdrawn after the cells are pre-seeded on the mandrels. A method of seeding cells on the inner wall is disclosed. In addition, in this document, after a tube structure is formed using a mandrel, a mechanical load is applied to the cell using a syringe pump to culture the cell seeded in the channel like a blood vessel, and the cell is cultured in the channel. A method for perfusing fluid is disclosed.
Furthermore, Non-Patent Document 2 discloses a method for manufacturing an artificial blood vessel structure device for monitoring invasion or metastasis of cancer cells, in which a blood vessel endothelial cell is injected after molding the tube structure on a collagen gel with a nickel titanium rod. ing. Further, the same document discloses a method of applying a mechanical load to vascular endothelial cells, injecting the cells, attaching a flow path to the device, and perfusing with a gravity flow.

Patent No. 5725859

Kenneth M. Chrobak and two others, “Formation of perfused, functional microvascular tubes in vitro”, Microvascular Research, February 2006, Vol. 71, No. 3, p.185-196 Andrew D. Wong et al., "Live-Cell Imaging of Invasion and Intravasation in an Artificial Microvessel Platform", Cancer Research, September 1, 2014, Vol. 74, No. 17, p.4937

However, in the method disclosed in Non-Patent Document 1, since a vascular endothelial cell is seeded after a tube structure is prepared with a cell scaffold, it is difficult to prepare a tubular structure in which the vascular endothelial cells themselves are dense.
Moreover, in the method disclosed in Patent Document 1, there is a possibility of damaging cells depending on the mandrel extraction method. In addition, when applying a mechanical load using a syringe pump, it is necessary to prepare and connect as many syringe pumps as the number of samples when considering a plurality of chemical liquid samples, which is complicated.
In the method disclosed in Non-Patent Document 2, since a vascular endothelial cell is injected after the tube structure is molded into a gel, it is difficult to form a dense blood vessel-like structure that can be used for drug screening. In addition, the device must be sterilized after the device is manufactured, and the method for applying a mechanical load is also complicated.
Thus, it is difficult to form a desired blood vessel-like structure that can be used for drug screening, regardless of the method disclosed in any document.

  In view of the circumstances as described above, an object of the present invention is to provide a culture vessel, a culture apparatus, and a culture method capable of forming a tubular blood vessel-like structure capable of sufficiently evaluating angiogenesis using cultured cells. There is to do.

In order to achieve the above object, a culture vessel according to one embodiment of the present invention includes a first cell, a second cell, and a third cell.
The first cell can contain a culture solution.
The second cell can contain the culture solution.
The third cell is
A first partition adjacent to the first cell;
A second partition adjacent to the second cell;
A first hole;
The first cell and the second cell are connected to each other, and the cell scaffolding material can be filled around the flow path.
The first hole portion includes at least a pair of holes formed to face the first partition wall and the second partition wall, respectively, and communicated with the first cell and the second cell, respectively. It is configured to be able to define a liquid flow path.

  According to the above configuration, since the flow path of the culture solution is defined between the first cell and the second cell by the first hole, the culture solution can be perfused into the flow channel. Therefore, it is easy to culture for constructing a blood vessel-like structure by seeding cells on the inner surface of the flow channel and perfusing the culture solution from one of the first cell and the second cell toward the other. It can be carried out.

The third cell may further include a second hole for injecting the cell scaffold.
Thereby, the cell scaffolding material can be filled around the flow path of the third cell.

The third cell may include a peripheral surface formed of a light transmissive material.
This facilitates observation of the blood vessel-like structure with a microscope.

Moreover, the culture apparatus which concerns on the other form of this invention comprises a culture container and a needle-shaped member.
The culture vessel connects a first cell capable of containing a culture solution, a second cell capable of containing the culture solution, the first cell, and the second cell. And a third cell.
The third cell is opposite to the first partition adjacent to the first cell, the second partition adjacent to the second cell, the first partition and the second partition, respectively. Including at least a pair of holes formed in communication with the first cell and the second cell, respectively, and capable of defining a flow path of the culture solution, The cell scaffold can be filled around the channel.
The acicular member is a acicular member configured to be movable between a first position penetrating the third cell and a second position extracted from the third cell. , And inserted into the first hole at the first position.

  According to the above configuration, the needle-like member is inserted into the first hole, the cell scaffolding material can be filled around the first hole, and the flow path can be formed in the void from which the needle-like member has been removed. This facilitates culturing to form a blood vessel-like structure by seeding cells on the inner surface of the flow channel and perfusing the culture solution from one of the first cell and the second cell toward the other. Can be done.

The culture container has a plurality of culture containers,
The acicular member has a plurality of acicular members,
Each of the plurality of needle-like members may be configured to be able to penetrate the third cell of one of the plurality of culture containers.
Thereby, a several blood vessel-like cell can be formed simultaneously and culture | cultivation efficiency can be improved.

In this case, the culture apparatus is
A first holding member for holding the plurality of culture vessels;
A second holding member for holding the plurality of needle-shaped members so that each of the plurality of needle-shaped members can penetrate the third cell of one of the plurality of culture containers;
May further be provided.
Thereby, alignment between a some culture container and a some acicular member can be performed easily.

In addition, the culture apparatus includes
A movement mechanism for moving the second holding member may be further provided so that each of the plurality of needle-like members can be moved between the first position and the second position.

Further, the first holding member holds the plurality of culture vessels such that the at least one pair of holes face each other in a uniaxial direction.
The second holding member holds the plurality of needle-like members so that the extending direction of each of the plurality of needle-like members matches the uniaxial direction,
The moving mechanism may move the second holding member along the uniaxial direction of the second holding member.
Thereby, the needle-like member can be removed while maintaining the extending direction and the moving direction of the needle-like member in parallel, and a tubular flow path along the uniaxial direction can be accurately formed. Therefore, a tubular blood vessel-like structure can be formed without disturbing the structure of the cells seeded on the inner surface of the flow path.

The culture method according to still another aspect of the present invention includes a first cell capable of containing a culture solution, a second cell capable of containing the culture solution, the first cell, and A culture method using a culture vessel having a third cell connecting the second cell, and penetrating the third cell through a needle-like member seeded on the surface with a cell serving as a base The step of inserting into the third cell.
A cell scaffold is filled around the needle-like member of the third cell.
The cells are transferred from the needle-like member to the cell scaffold.
A culture solution is added to at least one of the first cell and the second cell.
The needle member is removed from the third cell.
Based on the head difference of the culture solution between the first cell and the second cell, the culture solution is perfused into a flow path formed by removing the needle-like member.

  According to the above method, since the cells are seeded in advance on the needle-like member, by transferring the cells to the cell scaffold filled around, the tubular blood vessel-like structure in which the cells are densely seeded is formed. Easy to form. In addition, the culture solution can be perfused into the flow channel after the needle-like member is removed based on the liquid head difference between the two containers, and the culture for forming the blood vessel-like structure can be easily performed.

  In the step of adding the culture solution, a buffer solution is added to at least one of the first cell and the second cell, the needle-like member is removed from the third cell, and then the buffer solution is cultured. You may make it replace with a liquid.

The culture container has a plurality of culture containers,
The acicular member has a plurality of acicular members,
In the step of inserting the needle-like member into the third cell, each of the plurality of needle-like members may be inserted into the third cell of one of the plurality of culture vessels.
Thereby, culture | cultivation efficiency can be improved.

  In this case, in the step of removing the needle-like member from the third cell, the first cell and the second cell are respectively arranged in a plurality of culture vessels arranged so as to face each other in a uniaxial direction. The plurality of needle-like members whose extending directions coincide with the uniaxial direction may be moved along the uniaxial direction.

Further, in the step of perfusing the culture solution into the flow path, the plurality of culture vessels are respectively arranged so that the first cell and the second cell face each other in a uniaxial direction, and are orthogonal to the uniaxial direction. The culture solution may be perfused into the flow path by rotating the plurality of culture vessels around an axis to be rotated.
Thereby, even when a plurality of culture vessels are used, the culture medium can be perfused with an apparatus having a simple configuration.

The needle-like member is seeded with the cell via a compound capable of binding to the surface. In the step of transferring the cell, a voltage is applied to the needle-like member to The cell may be transferred from the acicular member to the cell scaffold by separating the surface from the surface.
As a result, the cells can be electrochemically transferred while suppressing the mechanical burden on the cells, and the disorder of the blood vessel-like structure can be prevented.

Further, in the step of inserting the needle-like member into the third container, the needle-like member is placed in the culture container in the first posture in which the first cell and the second cell are opposed in the vertical direction. Insert the needle-like member so that the extending direction of the
In the step of adding the culture solution, the culture solution or the buffer solution may be added to the culture vessel in a second posture in which the first cell and the second cell are horizontally opposed.
Thus, when the needle-like member is inserted, the needle-like member can be prevented from being deformed and the like can be easily aligned, and when the culture medium or the buffer solution is added, the needle-like member is changed to the second posture. It is possible to prevent the flow path from being exposed to the atmosphere by intrusion of the culture solution or the buffer into the flow path after the extraction of the member, and to easily form a liquid head difference.

  As described above, according to the present invention, it is possible to provide a culture vessel, a culture apparatus, and a culture method capable of forming a tubular blood vessel-like structure that can be sufficiently evaluated for angiogenesis using cultured cells. .

It is a figure which shows the culture apparatus which concerns on one Embodiment of this invention. It is a perspective view which shows the culture container of the said culture apparatus. It is a top view which shows the culture container of the said culture apparatus. It is sectional drawing which shows the culture container of the said culture apparatus. It is a perspective view which shows the some culture container and 1st holding member of the said culture apparatus. It is a perspective view which shows the several acicular member and 2nd holding member of the said culture apparatus. It is a side view which shows the relative positional relationship of the said acicular member and the said culture container. It is a side view which shows the relative positional relationship of the said acicular member and the said culture container. It is a flowchart explaining the culture | cultivation method which concerns on one Embodiment of this invention. It is a perspective view for demonstrating ST11 of the said culture method. It is a perspective view for demonstrating ST11 of the said culture method. It is a perspective view for demonstrating ST11 of the said culture method. It is a schematic diagram explaining cell seeding and detachment using a needle-like member. It is a perspective view for demonstrating ST12 of the said culture method. It is a perspective view for demonstrating ST13 of the said culture method. It is a perspective view for demonstrating ST14 of the said culture method. It is a perspective view for demonstrating ST15 of the said culture method. It is a perspective view for demonstrating ST16 of the said culture method. It is a perspective view for demonstrating ST19 of the said culture method. It is a side view for demonstrating ST19 of the said culture method. It is a perspective view of the culture container which concerns on the modification of the said embodiment. It is a typical figure at the time of inserting a needle-like member into the culture container concerning the modification of the above-mentioned embodiment. It is a typical figure at the time of cell culture using the culture container concerning the modification of the above-mentioned embodiment. It is a typical figure at the time of cell culture using the culture container concerning the modification of the above-mentioned embodiment. It is a typical figure at the time of cell culture using the culture container concerning the modification of the above-mentioned embodiment. It is a perspective view of the culture container which concerns on the other modification of the said embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In addition, the X-axis, Y-axis, and Z-axis directions in the figure indicate triaxial directions orthogonal to each other, the X-axis and Y-axis correspond to the horizontal direction, and the Z-axis corresponds to the height direction, respectively.

[Culture equipment]
FIG. 1 is a diagram showing a culture apparatus according to an embodiment of the present invention.
As shown in the figure, the culture apparatus 100 includes a plurality of culture containers 10, a container holding member (first holding member) 20, a plurality of needle-like members 30 (see FIG. 6), and a needle-like member 30. A holder (second holding member) 40 to be held and a moving mechanism 50 are provided.

The culture apparatus 100 of this embodiment is an apparatus capable of producing a blood vessel-like structure for evaluating angiogenesis in vitro. For example, in order to produce a blood vessel-like structure using vascular endothelial cells (hereinafter also simply referred to as “cells”), a tubular cell structure is first produced, and subsequently, the tubular cell structure is formed inside the tubular cell structure. Perfuse the culture. As a result, the cells constituting the tubular structure proliferate, and a blood vessel-like structure that simulates angiogenesis is formed. Hereinafter, the process of perfusing the culture solution into the tubular cell structure is also referred to as “perfusion culture”. Furthermore, the effect | action with respect to the angiogenesis of the said chemical | medical agent can be evaluated by adding the chemical | medical agent of a screening object to the said culture solution at the time of perfusion culture | cultivation.
According to the culture device 100, the needle-shaped member 30 seeded with cells in advance can be inserted into the culture container 10, and the cells can be transferred to the cell scaffold filled around the needle-shaped member 30. Then, the culture solution can be perfused into the flow path formed by removing the needle-like member 30 to form a blood vessel-like structure, and angiogenesis can be evaluated.

  The “cell scaffold” is typically a cell scaffold that can be gelled, and a material that easily gels by heating, chemical crosslinking, photocrosslinking, or the like can be used. Examples of such cell scaffolds include collagen, gelatin, elastin, proteoglycan, glycosaminoglycan, fibronectin, vitronectin, laminin, pectin, hyaluronic acid, chitin, chitosan, alginic acid, starch, polyethylene glycol, polydimethyl Polymer gels composed of siloxane, polylactic acid, polyvinyl pyrrolidone, polyvinyl alcohol, polyglutamic acid, polyglycolic acid, polycaprolactone, polyacrylic acid, and the like, and polymer gels obtained by appropriately modifying them can be used.

(Culture container)
2-4 is a figure which shows the culture container 10, FIG. 2 is a perspective view, FIG. 3 is a top view, FIG. 4 is sectional drawing. In the figure, the x-axis, y-axis, and z-axis directions indicate triaxial directions orthogonal to each other.
As shown in these drawings, the culture vessel 10 includes a first cell 11, a second cell 12, and a third cell 13. The first cell 11 and the second cell 12 may have, for example, the same volume, and the third cell 13 has a smaller volume than, for example, the first cell 11 and the second cell. It may be.
In the present embodiment, the culture vessel 10 is formed of a translucent material as a whole. The material of the culture container 10 is not specifically limited, For example, translucent resin materials, such as an acrylic resin, glass etc. can be used.

Each of the first cell 11 and the second cell 12 is configured to be able to contain a culture solution, and is disposed to face the third cell 13 in the y-axis direction, for example. The first cell 11 and the second cell 12 communicate with the third cell 13 through a first hole 131 described later, and can supply a culture solution to the third cell 13.
The first cell 11 and the second cell 12 include openings 11a and 12a orthogonal to the z-axis direction, for example, and are configured in a substantially rectangular parallelepiped shape. The openings 11a and 12a are used for adding, for example, a culture solution from above in FIGS. 2A and 2B.
As shown in FIGS. 2A and 2B, in the present embodiment, the first cell 11 can seal the hole 111 and the hole 111 on the surface facing the surface adjacent to the third cell 13. Plug 112. As will be described later, the hole 111 is configured such that the needle-like member 30 and the holder 40 that holds the needle-like member 30 can be inserted in the open state. Further, the hole 111 is sealed by the plug 112, so that leakage of the culture solution that can be accommodated in the first cell 11 during perfusion culture can be prevented.

  The “culture medium” referred to here may be a liquid medium used for cell culture, or may be an appropriate addition of a drug to be screened, a growth factor or the like to the medium. In addition, the first cell 11 and the second cell 12 may contain a buffer solution other than the culture solution, physiological saline, or the like, if necessary.

The third cell 13 connects the first cell 11 and the second cell 12 to each other. In the third cell 13, the flow path F is formed by the cell scaffold as shown virtually in FIG. 3 and FIG. 4, and the cells arranged around the flow path F are perfused to obtain a blood vessel-like structure. Is formed.
As shown in FIGS. 3 and 4, the third cell 13 includes a first partition wall 13a, a second partition wall 13b, a peripheral surface 13c, a top surface 13d, a first hole 131, 2 holes 132.

The first partition wall 13 a is a partition wall adjacent to the first cell 11 (an image wall that partitions the first cell 11 and the third cell 13).
The second partition wall 13 b is a partition wall adjacent to the second cell 12 (an image wall that partitions the second cell 12 and the third cell 13).
The first partition wall 13a and the second partition wall 13b oppose each other in the y-axis direction.
The peripheral surface 13c includes a surface (bottom surface) that forms the bottom of the third cell 13 during perfusion culture, and two side surfaces that face in the x-axis direction, and is connected to the first partition wall 13a and the second partition wall 13b. Arranged. In the present embodiment, the bottom surface of the third cell 13 is formed on substantially the same plane as the bottom surfaces of the first cell 11 and the second cell 12. In addition, since the peripheral surface 13c is formed of a translucent material, it is easy to observe the blood vessel-like structure with an optical microscope, a fluorescence microscope, or the like.
The top surface 13 d is connected to the peripheral surface 13 c and connects the first cell 11 and the second cell 12. The top surface 13d is disposed at a position lower than the height of the openings 11a and 12a in the z-axis direction (see FIG. 4).

The first hole 131 includes at least a pair of holes 131a formed in the first partition wall 13a and the second partition wall 13b so as to face each other in the y-axis direction. Each hole 131a communicates with the first cell 11 and the second cell 12, respectively, and is configured such that the needle-like member 30 can be inserted so as to penetrate the third cell 13.
In the present embodiment, the first hole 131 includes a plurality of pairs (for example, two pairs) of holes 131a. Thereby, a plurality of needle-like members 30 are inserted into one third cell 13 and a plurality of flow paths F are formed. Therefore, angiogenesis of a plurality of blood vessel-like structures can be evaluated for one type of culture solution, and efficient and accurate evaluation can be performed.
The first hole 131 is configured to be able to define a culture fluid flow path F. In the culture method using the culture apparatus 100, as will be described later, while the needle-like member 30 is inserted into the first hole 131, the gel-like cell scaffold is filled around the needle-like member 30. The That is, after the needle-shaped member 30 is removed, the region in which the needle-shaped member 30 has been inserted becomes a void that is not filled with the cell scaffold. Thereby, the tubular space | gap which connects between the pair of hole 131a formed after the needle-shaped member 30 extraction can become the flow path F at the time of perfusion of a culture solution.

The 2nd hole 132 is formed in the 1st partition 13a, and is comprised as a hole for inject | pouring a cell scaffold material. The second hole portion 132 may have one hole, or may have a plurality of holes 132a as in the present embodiment. In the case of having a plurality of holes 132a, each hole 132a may be capable of injecting a cell scaffold, and at least one hole 132a may be used for exhausting the third cell 13 or the like. It may be responsible for the function.
Moreover, the 2nd hole 132 is comprised in this embodiment so that engagement with the cell scaffold material injection port 412 of the holder 40 mentioned later is possible. Thereby, the cell scaffold can be injected into the third cell 13 through the second hole 132 without leaking to the first cell 11 side.

  With the culture container 10 having such a configuration, the flow path F of the culture solution is defined between the first cell 11 and the second cell 12 by the first hole 131, and the first cell 11 and the second cell The culture medium can be perfused from one of the cells 12 to the other. Therefore, the blood vessel-like structure can be cultured by a simple method without using a configuration such as a pump.

(Container holding member (first holding member))
FIG. 5 is a perspective view showing a plurality of culture containers 10 and a container holding member 20.
As shown in FIGS. 5 and 1, the container holding member 20 holds a plurality of culture containers 10.
In the present embodiment, the container holding member 20 holds the plurality of culture containers 10 so that at least a pair of holes 131a face each other in the Z-axis direction. Thereby, in each culture container 10, the extending direction of the flow path F can be prescribed | regulated.
Moreover, the container holding member 20 arranges the plurality of culture containers 10 along the X-axis direction, for example.
The container holding member 20 is not particularly limited as long as it can hold the plurality of culture containers 10 in the desired posture and arrangement as described above.

  In the present embodiment, the container holding member 20 is configured by a prismatic body that engages with the top surface 13 d of the third cell 13, the first cell 11, and the second cell 12, respectively, in each culture container 10. With such a configuration, the posture of each culture vessel 10 around the X axis can be defined.

(Needle member)
FIG. 6 is a perspective view showing the needle-like member 30 and the holder 40. 7 and 8 are side views showing the relative positional relationship between the holder 40 that holds the needle-like member 30 and the culture vessel 10.

The needle-shaped member 30 has conductivity and is configured to be able to penetrate the third cell 13 of one of the plurality of culture containers 10. Specifically, each needle-like member 30 is inserted into the pair of holes 131a.
The needle-like member 30 is made of a conductive material, and the surface 30a of the needle-like member 30 is made of, for example, gold (Au). Thereby, the detachment | desorption of the cell seed | inoculated on the surface 30a can be performed smoothly so that it may mention later.
The needle-like member 30 is configured, for example, in a bar shape extending in a uniaxial direction (for example, the Z-axis direction), and the cross-section of the needle-like member 30 (cross section orthogonal to the extending direction) is typically configured in a circular shape. The The diameter of the cross section can be appropriately set according to the diameter of the blood vessel-like structure to be produced, and can be configured to be, for example, about 0.1 mm to 1.0 mm.
As shown in FIGS. 7 and 8, the needle-like member 30 is movable between a first position that penetrates the third cell 13 and a second position that is removed from the third cell 13. Configured. 7 shows a state where the needle-like member 30 is in the first position, and FIG. 8 shows a state where the needle-like member 30 is in the second position.

(holder)
With reference to FIGS. 6 to 8, the holder 40 has a plurality of needle-like members 30 that can penetrate the third cell 13 of one culture container 10 out of the plurality of culture containers 10. The needle-like member 20 can be held.
The holder 40 has a plurality of main bodies 41 and a main body holder 42.

The main body 41 holds each needle-like member 30 so as to prevent the deformation of each needle-like member 30 and the displacement with respect to the main body 41. That is, the main body 41 is configured to be able to define the relative posture of each needle-like member 30 with respect to the main body 41.
In the present embodiment, each main body 41 is configured to correspond to one culture vessel 10, and is configured to hold, for example, two needle-like members 30. The main body 41 may have a hole into which each needle-like member 30 is inserted, for example. Or although not shown in figure, the main body 41 may have holding tools, such as a clip which can hold | grip each acicular member 30. FIG.
The main body 41 has a cell scaffold material introduction port 411 and a cell scaffold material injection port 412.

  The cell scaffold material introduction port 411 is configured to introduce a cell scaffold material that can be injected into the third cell 13 into the main body 41. The cell scaffold material introduction port 411 may have a tubular configuration as shown in FIG. 6, or may be an opening formed in the main body 41 although not shown.

As shown in FIGS. 7 and 8, the cell scaffold material injection port 412 has a configuration for injecting the cell scaffold material into the third cell 13. The cell scaffold material injection port 412 is configured as a discharge port for the cell scaffold material introduced into the main body 41 from the cell scaffold material introduction port 411 and communicates with the cell scaffold material introduction port 411 in the main body 41.
The cell scaffold material injection port 412 injects the cell scaffold material into the third cell 13 through the second hole 132, for example. The cell scaffold material injection port 412 may have, for example, a protrusion that can be engaged with the second hole 132 of the third cell 13, but is not limited thereto, and is an opening formed in the main body 41. It may be.

  As shown in FIG. 6, the main body holder 42 is configured to be able to hold a plurality of main bodies 41. The main body holder 42 defines the position of each main body 41 relative to the main body holder 42 so as to correspond to each culture vessel 10 and also defines the relative posture of each main body 41 with respect to the main body holder 42.

  The holder 40 configured as described above can define the extending direction of each of the plurality of needle-like members 30 by the main body 41 and the main body holder 42. Thereby, the holder 40 can hold | maintain the some acicular member 30 so that the extending direction of each of the some acicular member 30 may correspond to the Z-axis direction which is the opposing direction of the hole 131a.

(Movement mechanism)
With reference to FIG. 1, the moving mechanism 50 moves the holder 40 so that each of the plurality of needle-like members 30 can be moved between the first position and the second position. In this embodiment, the moving mechanism 50 moves the main body holder 42 of the holder 40 along the Z-axis direction while matching the extending direction of each needle-like member 30 with the Z-axis direction.
Here, the holder 40 is moved to the top position of the moving mechanism 50 when the plurality of needle-like members 30 are in the second position, and the movement mechanism is located when the plurality of needle-like members 30 are in the first position. It is assumed that it is moved to the 50 bottom position.
As shown in FIG. 1, the moving mechanism 50 may be composed of, for example, a ball screw. As a result, it is possible to perform a rectilinear movement with high accuracy along the Z-axis direction. Moreover, the structure of the moving mechanism 50 will not be limited if the holder 40 can be moved as mentioned above.
For example, the moving mechanism 50 includes a main body 51, a movable member 52, and a mounting base 53.
The movable member 52 is configured to hold the holder 40 and be able to move in the main body 51 along the Z-axis direction.
The mounting table 53 arranges the container holding member 20 and the like. The main body 51 may be formed with a configuration for fixing the position of the container holding member 20 or the like disposed on the mounting base 53. Examples of such a configuration include a protrusion, a hole, and a screw that can be engaged with the container holding member 20.

  The culture apparatus 100 configured as described above can be used in the following culture method.

[Culture method]
FIG. 9 is a flowchart for explaining the culture method according to the present embodiment, and FIGS. 10 to 20 are diagrams for explaining each step of the culture method. Each step of the culture method will be described with reference to these drawings.

  First, seed cells are seeded on the surface 30a of the needle-shaped member 30 (ST11).

As shown in FIG. 10, first, a container 60 having a plurality of wells 61 is prepared. Each well 61 is disposed so as to face each main body 41 of the holder 40 in the Z-axis direction, and each well 61 contains a cell-containing liquid L1 containing cells. The cell-containing liquid L1 here may be, for example, a liquid in which cells are contained in a liquid medium for culturing cells.
The holder 40 is prepared at the top position of the moving mechanism 50 while holding the plurality of needle-like members 30.
Subsequently, as shown in FIG. 11, the container 60 is fixed to the mounting base 53 of the moving mechanism 50 so that each well 61 corresponds to each main body 41. The fixing method is not particularly limited.
Then, as shown in FIG. 12, the holder 40 is moved to the bottom position of the moving mechanism 50, and each needle member 30 is immersed in the cell-containing liquid L <b> 1 in each well 61.

  Cell seeding may be performed without attaching the culture vessel 10 to the moving mechanism 50. The cell-containing liquid L1 may be placed in a separately prepared culture container, the needle-shaped member 30 may be immersed, and the cell may be sufficiently adsorbed, and then attached to the moving mechanism 50. The attachment to the moving mechanism 50 is preferably performed quickly in order to prevent the cells from drying, and it is clamped by a clamping mechanism charged in the moving mechanism 50 or attached to the holder 40 and the moving mechanism 50 by a magnetic force. May be.

In the present embodiment, the cells seeded on the surface 30a of the needle-like member 30 are electrochemically detached from the surface 30a. For this reason, cells are seeded and detached using a compound that can bind to the surface 30a and can be detached by applying a predetermined voltage to the surface 30a.
FIG. 13 is a schematic diagram illustrating cell seeding and detachment.
In the preparation stage of seeding of FIG. 10, FIG. 11, the compound P which can be couple | bonded with the surface 30a is couple | bonded with the surface 30a of each acicular member 30 (refer FIG. 13A). In this embodiment, this compound can be an oligopeptide (hereinafter simply referred to as “peptide”) P. When the surface of the needle-shaped member 30 is formed of Au, the peptide P may contain a sulfur-containing amino acid (for example, cysteine). Thereby, sulfur (S) of the peptide P and Au on the surface 30a can form a bond.
Then, by immersing each needle-like member 30 in the cell-containing liquid L1, the peptide P on the surface 30a of each needle-like member 30 and the cell C are adhered (see FIG. 13B), and the surface 30a of the needle-like member 30 is obtained. The cells C are seeded via the peptide P.

Subsequently, the culture vessel 10 is prepared (ST12). In this step, the culture vessel 10 is not filled with a culture solution, a cell scaffold, or the like, and the plug 112 for sealing the hole 111 is removed.
As shown in FIG. 14, for example, the plurality of culture containers 10 are held by the container holding member 20, and the container holding member 20 is fixed to the platform 53 of the moving mechanism 50.
On the other hand, in this step, the holder 40 is prepared at the top position of the moving mechanism 50 while holding the plurality of needle-like members 30. Each needle-like member 30 is disposed at the second position shown in FIG. 8 and is set in the holder 40 so as to extend along the Z-axis direction.
The culture container 10 in this step is arranged with respect to the needle-like member 30 (main body 41) as follows, for example. That is, each culture vessel 10 is disposed so as to face each main body 41 set in the moving mechanism 50 in the Z-axis direction. Specifically, in each culture vessel 10, the first hole 131 of the third cell 13 faces the needle-like member 30 in the Z-axis direction, and the second hole 132 of the third cell 13. Is arranged so as to face the cell scaffold inlet 412 in the Z-axis direction.
Each culture container 10 at this time is arrange | positioned so that the following attitude | positions may be taken, for example. That is, each culture vessel 10 is arranged so that the first cell 11 and the second cell 12 face each other in the Z-axis direction, and further, the paired holes 131a are arranged so as to face each other in the Z-axis direction. Is done. Moreover, the culture container 10 is arrange | positioned with the 1st attitude | position which the 1st cell 11 and the 2nd cell 12 oppose to a perpendicular direction. In the first posture, each container 10 has the x-axis direction shown in FIGS. 2A and 2B oriented in the Z-axis direction.

Subsequently, the needle-like member 30 is inserted into the third cell 13 so as to penetrate the third cell 13 (ST13). In the present embodiment, each of the plurality of needle-like members 30 is inserted into the third cell 13 of one of the plurality of culture containers 10. Each acicular member 30 moves from the second position to the first position penetrating the third cell 13 (see FIG. 7). Also in this step, the culture vessel 10 is maintained in the first posture.
As shown in FIGS. 15 and 7, in the present embodiment, the holder 40 is moved to the bottom position of the moving mechanism 50, and each needle member 30 is inserted into the pair of holes 131a. At this time, each needle-like member 30 is moved in the Z-axis direction while maintaining the extending direction in the Z-axis direction because the posture and the moving direction are regulated by the holder 40 and the moving mechanism 50.
Further, when the needle-like member 30 moves to the first position, the cell scaffold material inlet 412 is engaged with the second hole 132 of the third cell 13 as shown in FIG.

Subsequently, the cell scaffolding material S is filled around the needle-like member 30 of the third cell 13 (ST14).
As shown in FIG. 16, in this step, first, the liquid cell scaffold S is introduced into the cell scaffold inlet 411 of each main body 41. Thereby, the cell scaffold material S passes through the inside of each main body 41 and is discharged from the cell scaffold material injection port 412. The discharged cell scaffold S is filled into the third cell 13 through the second hole 132.
Then, the cell scaffold S is gelled. The method of gelling the cell scaffold S can be appropriately selected according to the properties of the cell scaffold S, such as heating, light irradiation, chemical reaction, and the like. Thereby, as shown in FIG. 13C, the cells C seeded on the surface 30a are covered with the gel-like cell scaffold S.
Also in this step, the first cell 11 is maintained in the first posture.

Subsequently, the cells are transferred from the needle-like member 30 to the cell scaffold S (ST15).
In this step, the cells C are transferred from the needle-like member 30 to the cell scaffold S by applying a voltage to the needle-like member 30 to separate the peptide P from the surface 30a. Specifically, as schematically shown in FIG. 17, an electric circuit is constructed so as to generate a potential difference between the potential of the needle-like member 30 and the potential of the cell scaffold S.
In FIG. 17, for the sake of explanation, a voltage is applied to one needle-like member 30, but all the needle-like members 30 are connected in parallel, and the same voltage is simultaneously applied to all the needle-like members 30. You may apply. The conditions such as the magnitude of voltage and time are not particularly limited, and for example, a voltage of -5V for 5 minutes can be applied.
As a result, as shown in FIG. 13D, the bond between the surface 30a of the needle-like member 30 and the peptide P is released, and the cells C can be transferred from the needle-like member 30 to the cell scaffold S.

Subsequently, as shown in FIG. 18, the posture of the culture vessel 10 is changed so that the first cell 11 and the second cell 12 are in the second posture in which they face each other in the horizontal direction (ST16). In the second posture, each container 10 has the z-axis direction shown in FIGS. 2A and 2B oriented in the Z-axis direction.
Furthermore, in this embodiment, not only the culture container 10 but the posture of the entire culture apparatus 100 is changed.

Subsequently, referring to FIG. 18, a culture solution is added to at least one of first cell 11 and second cell 12 (preferably second cell 12) (ST17). By adding the culture solution before the next step of removing the needle-like member 30, problems such as bubbles entering the flow path F at the time of removal can be prevented.
In the present embodiment, the culture solution may be added to one of the first cell 11 and the second cell 12. For example, the culture solution can be added to the second cell 12 that does not have the holes 111. The amount of the culture medium added may be an amount that can sufficiently cover the needle-like member 30 penetrating the third cell 13, for example.
In addition, since the culture vessel 10 is in the second posture, the culture solution can be added from at least one of the opening 11a and the opening 12a.

Referring to FIGS. 18 and 8, the needle-like member 30 is removed from the third cell 13 (ST18). By removing the needle-like member 30 in this step, a void that is not filled with the cell scaffolding material S is formed in the region in the third cell 13 defined by the pair of holes 131a. This gap can be a flow path F.
In this step, a plurality of needle-like members 30 having the extending direction aligned with the Y-axis direction are moved along the Y-axis direction with respect to the plurality of culture vessels 10. At this time, the plurality of culture vessels 10 are arranged such that the first cell 11 and the second cell 12 face each other in the Y-axis direction, and the pair of holes 131a also face each other in the Y-axis direction. For this reason, the tubular flow path F extending in the Y-axis direction can be formed.
In the present embodiment, the extending direction and moving direction of the needle-like member 30 are defined by the holder 40 and the moving mechanism 50, and the posture and position of the culture vessel 10 are defined by the container holding member 20. Therefore, the above operation can be performed only by moving the holder 40 from the bottom position of the moving mechanism 50 to the top position.

  Finally, based on the liquid head difference of the culture solution L2 between the first cell 11 and the second cell 12, the culture solution L2 is perfused into the flow path F formed by removing the needle-like member 30 (ST19). ). The “liquid head difference” here means a difference in height of the liquid surface along the vertical direction of the culture solution accommodated in the first cell 11 and the second cell 12. In this step, the culture vessel 10 is transferred from the moving mechanism 50 to the perfusion apparatus 200 and perfusion culture is performed using the perfusion apparatus 200.

19 and 20 are diagrams for explaining this process, FIG. 19 is a perspective view, and FIG. 20 is a schematic side view.
As shown in these drawings, the perfusion apparatus 200 includes a support member 210 and a drive mechanism 220.
The support member 210 arranges the culture vessel 10. In the present embodiment, the support member 210 is configured in a tray shape in which a plurality of culture vessels 10 are arranged. The first cell 11 and the second cell 12 of each culture vessel 10 are arranged to face each other in the horizontal direction (X-axis direction in the illustrated example).
As shown in FIG. 20, the drive mechanism 220 includes a mounting base 221 on which the support member 210 is disposed, a shaft portion 222 along the X-axis direction, and a drive portion (not shown). The shaft portion 222 is disposed, for example, at a central portion along the X-axis direction of the mounting table 221, and the mounting table 221 is configured to be able to rotate around the Y axis about the shaft portion 222 by a driving unit. The With such a drive mechanism 220, the plurality of culture vessels 10 can be rotated around the Y axis, and the culture medium L 2 can be perfused through the flow path F.

For example, perfusion culture using the perfusion apparatus 200 is performed as follows.
That is, first, the culture vessel 10 is rotated around the Y axis from a horizontal state until a predetermined rotation angle is obtained. In each culture vessel 10, a culture solution L2 is added to at least one of the first cell 11 and the second cell 12. Due to the above rotation, the head difference of the culture medium L2 between the first cell 11 and the second cell 12 occurs, and the flow from the cell to which the culture medium L2 has been added to the higher position to the other cell The culture solution L2 flows through the path F. By maintaining the state in which the culture vessel 10 is tilted, the culture solution L2 is continuously perfused into the flow path F. Then, the cell placed in the vertical direction can be switched to the other cell by rotating the culture vessel 10 again until the culture medium L2 in the cell arranged in the vertical direction disappears. Thus, the culture medium L2 can be perfused through the flow path F.
The flow velocity in the flow path F has a correlation with, for example, the cross-sectional area of the flow path F, the size of the rotation angle, the angular velocity of rotation, and the like. For this reason, the flow rate at the time of using the culture container 10 is controllable mainly by examining the conditions regarding rotation of the perfusion apparatus 200.

  Here, it is known that angiogenesis in vivo involves various factors such as mechanical stimulation from blood. According to this embodiment, the culture medium L2 is continuously perfused into the flow path F, so that appropriate mechanical stimulation can be applied to the cells C arranged in a tubular shape on the inner peripheral surface of the flow path F. The state of angiogenesis can be reproduced in a pseudo manner.

  After the perfusion culture, the blood vessel-like structure can be evaluated by observing the third cell 13 of each culture vessel 10 with a microscope or the like.

As described above, according to the present embodiment, by transferring the cells C previously seeded around the needle-like member 30 to the cell scaffold S, a tubular blood vessel-like structure in which the cells C are densely arranged is easily produced. can do. Further, it is possible to prevent the blood vessel-like structure from being damaged when the needle-like member 30 is removed. The flow rate in the perfusion culture can be easily controlled by perfusing the culture solution L2 into the flow path F based on the head difference of the culture solution L2 between the two cells. Conditions can be easily considered. Therefore, it becomes possible to culture a longer blood vessel-like structure and to evaluate angiogenesis with high accuracy in vitro.
In addition, according to the present embodiment, it is possible to culture a blood vessel-like structure in large quantities. Therefore, a large amount of specimens can be easily evaluated.
Furthermore, according to the culture method described above, a complicated operation for perfusion can be eliminated, and since there is no need to install an apparatus for perfusion inside the culture vessel 10, the possibility of contamination is also reduced. can do. In addition, the culture medium can be easily replaced during perfusion culture.

  The culture apparatus 100 and the culture method described above can be applied as follows.

[Application Example 1: Drug screening]
Drug screening can be performed using the culture apparatus 100 and the culture method described above.
The drug here is mainly a drug that suppresses or promotes angiogenesis, and specifically includes an anticancer agent that suppresses angiogenesis.
In this application example, first, in the perfusion culture step of ST19, a drug to be screened is added to the culture solution to perform perfusion culture. According to the present embodiment, since angiogenesis can be evaluated with high accuracy in vitro as described above, the effect of a drug on angiogenesis can be fully examined. In addition, since large-scale culture is possible, a large amount of screening can be efficiently performed using a plurality of conditions and a plurality of drugs.

[Application Example 2: Revascularization Treatment]
The culture apparatus 100 and the culture method may be used for research on revascularization therapy.
For blood vessel occlusion, etc., medication, treatment for dilating blood vessels using balloon catheters or stents, and surgical bypass surgery have been applied in the past. Research on treatments to be produced is ongoing.
Since the culture apparatus 100 and the culture method according to the present embodiment can cultivate long blood vessel-like structures, they can also be used for research of such treatment.

[Application Example 3]
The culture apparatus 100 and the culture method may be used as a technique for three-dimensional tissue regeneration in regenerative medicine. In order to create a tissue having a three-dimensional size and shape in regenerative medicine using cells, it is necessary to provide a blood vessel structure for supplying oxygen and nutrients simultaneously with tissue processing. By using this method, a three-dimensional tissue that can be easily perfused with a culture solution can be constructed. For example, by adding hepatocytes together with a cell scaffold, a three-dimensional tissue structure can be created while maintaining liver function.

[Modification]
The embodiment of the present invention has been described above, but the present invention is not limited to the above-described embodiment, and it is needless to say that various modifications can be made without departing from the gist of the present invention.

21A and 21B are perspective views of culture containers 10A and 10B according to a modification of the present embodiment.
In the above-described embodiment, the first cell 11 and the second cell 12 of the culture vessel 10 have been described as having a substantially rectangular parallelepiped shape having the same volume, but the present invention is not limited to this.
As shown in FIG. 21A, for example, both the first cell 11A and the second cell 12A may be configured in a tube shape. The material of the first cell 11A and the second cell 12A may be formed of a deformable material such as a silicone resin. The cross-sectional shapes of the first cell 11A and the second cell 12A are not particularly limited, such as a circular shape, an elliptical shape, and a rectangular shape. The third cell 13A may also be configured in a tube shape, and may be configured of a material such as glass or a light-transmitting resin (for example, acrylic resin). Although not shown, the third cell 13A includes a first partition, a second partition, a first hole, and the like.
Moreover, as shown to FIG. 21B, the volume of the 1st cell 11B and the 2nd cell 12B may differ. Although not shown, the third cell 13B includes a first partition, a second partition, a first hole, and the like.

22A and 22B are schematic views when the needle-like member 30 is inserted into the third cell 13B of the culture vessel 10B shown in FIG. 21B.
As shown in FIG. 22A, the first cell 11B may be configured to be detachable from the third cell 13B, and the first cell 11B may be removed and the needle-like member 30 may be inserted into the third cell 13B.
Or as shown to FIG. 22B, you may insert the acicular member 30 in the 3rd cell 13B of the state which connected the 1st cell 11B.

Even when the culture vessel 10B is used, perfusion culture can be performed based on the liquid head difference between the culture solutions L2 of the first cell 11B and the second cell 12B.
23 to 25 are schematic diagrams when the culture vessel 10B is perfused.
As shown in FIG. 23, similarly to the perfusion device 200, the culture vessel 10B is placed around an axis orthogonal to the Z-axis direction where the first cell 11B and the second cell 12B face each other with the third cell 13B interposed therebetween. A rotating perfusion device 200A can be used. The perfusion device 200A includes a mounting table 221A and a shaft portion 222A. In this case, as shown in FIG. 24, the axis of the third cell 13B of the culture vessel 10B may be orthogonal to, for example, the plane of the mounting table 221A of the perfusion apparatus 200A, or the perfusion apparatus 200A. It may be inclined at a predetermined angle around the X axis with respect to the plane of the mounting table 221A. By arranging the culture vessel 10B to be inclined around the X axis with respect to the mounting table 221A as described above, the flow rate of the culture fluid flowing through the cell 13B can be increased without causing an increase in the hydrostatic pressure in the third cell 13B. Enough can be secured.

  Alternatively, as shown in FIG. 25, a perfusion apparatus 200B having a first shaft portion 210B, a second shaft portion 220B, and a gripping member 230B that grips the culture vessel 20B may be used. The first shaft portion 210B is disposed along the Y-axis direction. The second shaft portion 220B is disposed on the first shaft portion 210B along the X-axis direction. The holding member 230B is attached to the second shaft portion 220B and holds the culture vessel 10B. Also with the perfusion apparatus 200B having such a configuration, the perfusion culture can be performed by rotating the culture vessel 10B around the first shaft portion 210B (Y axis).

  FIG. 26 is a view showing another modification of the culture vessel. As shown in the figure, the culture vessel 10C has a first cell 11C, a second cell 12C, and a third cell 13C. For example, the culture vessel 10C is entirely formed of a translucent material, and the containers 11C, 12C, and 13C having the same positional relationship as the culture vessel 10B are formed therein. Even with such a configuration, the same effect as that of the culture vessel 10 can be obtained.

  In the above-described culture method, it has been described that the culture medium is added in ST17 and then the needle-shaped member 30 is removed in ST18. However, the present invention is not limited to this. For example, the culture solution may be added after the needle-like member 30 is removed. Alternatively, before removing the needle-like member, a buffer solution or physiological saline may be added instead of the culture solution, the needle-like member may be removed, and then these solutions may be replaced with the culture solution.

  Moreover, in the above-mentioned culture method, although it demonstrated that a posture was changed by ST16, it is not limited to this. For example, the step of inserting the needle-like member 30 of ST13 into the third cell 13 is performed in such a posture that the Z-axis direction and the X-axis direction indicate the horizontal direction and the Y-axis direction indicates the vertical direction. Each step may be performed in the same posture. Alternatively, when the culture solution is not added before removing the needle-like member 30 as described above, the posture may be changed after the needle-like member 30 is removed.

  In addition, it has been described that the third cell 13 of the culture vessel 10 has the second hole 132 for injecting the cell scaffold material, but the cell scaffold material can be injected into the third cell 13. It is not limited to this. Moreover, it is not limited to the structure which inject | pours a cell scaffold through the main body 41 of the holder 40, You may inject | pour a cell scaffold using a separate apparatus or instrument.

DESCRIPTION OF SYMBOLS 10 ... Culture container 11 ... 1st cell 12 ... 2nd cell 13 ... 3rd cell 13a ... 1st partition 13b ... 2nd partition 131 ... 1st hole part 131a ... Hole 132 ... 2nd hole Part 20: Container holding member (first holding member)
30 ... acicular member 30a ... surface 40 ... holder (second holding member)
50 ... Movement mechanism 100 ... Culture apparatus

Claims (15)

A first cell capable of containing a culture solution;
A second cell capable of containing the culture solution;
A first partition adjacent to the first cell;
A second partition adjacent to the second cell;
Including at least a pair of holes formed so as to face the first partition and the second partition and communicating with the first cell and the second cell, respectively, and defining a flow path for the culture solution A first hole capable of:
And a third cell that connects the first cell and the second cell and can be filled with a cell scaffold around the flow path. The culture container according to claim 1,
The third cell further has a second hole for injecting the cell scaffold. The culture container according to claim 1 or 2,
The third cell includes a peripheral surface formed of a translucent material. A first cell capable of containing a culture solution, a second cell capable of containing the culture solution, and a third cell connecting the first cell and the second cell; A culture vessel having
A needle-like member configured to be movable between a first position penetrating the third cell and a second position removed from the third cell;
Comprising
The third cell is
A first partition adjacent to the first cell;
A second partition adjacent to the second cell;
Including at least a pair of holes formed so as to face the first partition and the second partition and communicating with the first cell and the second cell, respectively, and defining a flow path for the culture solution A first hole capable of:
The cell scaffolding material can be filled around the flow path,
The acicular member is inserted into the first hole at the first position. The culture apparatus according to claim 4, wherein
The culture container has a plurality of culture containers,
The acicular member has a plurality of acicular members,
Each of the plurality of needle-like members is configured to be able to penetrate the third cell of one of the plurality of culture containers. The culture apparatus according to claim 5, wherein
A first holding member for holding the plurality of culture vessels;
A second holding member that holds the plurality of needle-like members so that each of the plurality of needle-like members can be passed through the third cell of one of the plurality of culture vessels. A culture apparatus further provided. The culture apparatus according to claim 6,
A culture apparatus further comprising a moving mechanism for moving the second holding member such that each of the plurality of needle-like members can be moved between the first position and the second position. The culture apparatus according to claim 7,
The first holding member holds the plurality of culture vessels so that the at least one pair of holes face each other in a uniaxial direction,
The second holding member holds the plurality of needle-like members so that the extending direction of each of the plurality of needle-like members matches the uniaxial direction,
The moving mechanism moves the second holding member along the uniaxial direction of the second holding member. A first cell capable of containing a culture solution, a second cell capable of containing the culture solution, and a third cell connecting the first cell and the second cell; A culture method using a culture vessel having
Inserting a needle-like member seeded on the surface with cells serving as a base into the third cell so as to penetrate the third cell,
Filling a cell scaffolding material around the needle-like member of the third cell;
Transferring the cells from the needle-like member to the cell scaffold;
Adding a culture solution to at least one of the first cell and the second cell;
Removing the needle-like member from the third cell;
A culture method in which the culture solution is perfused into a flow path formed by removing the needle-like member based on a liquid head difference of the culture solution between the first cell and the second cell. The culture method according to claim 9, comprising:
The step of adding the culture solution includes:
Adding a buffer to at least one of the first cell and the second cell;
A culture method in which the buffer solution is replaced with a culture solution after the needle-like member is removed from the third cell. The culture method according to claim 9 or 10,
The culture container has a plurality of culture containers,
The acicular member has a plurality of acicular members,
In the step of inserting the needle-like member into the third cell, each of the plurality of needle-like members is inserted into the third cell of one of the plurality of culture vessels. The culture method according to claim 11, comprising:
In the step of removing the needle-like member from the third cell, the first cell and the second cell are extended with respect to the plurality of culture containers respectively arranged so as to face each other in a uniaxial direction. A culture method in which the plurality of needle-like members whose directions are aligned with the uniaxial direction are respectively moved along the uniaxial direction. The culture method according to claim 11 or 12,
In the step of perfusing the culture solution into the flow path, the plurality of culture vessels are respectively arranged so that the first cell and the second cell face each other in a uniaxial direction, and an axis orthogonal to the uniaxial direction A culture method in which the culture solution is perfused into the flow path by rotating the plurality of culture vessels around. A culture method according to any one of claims 9 to 13,
The needle-like member is seeded with the cell via a compound capable of binding to the surface,
In the step of transferring the cells, the cell is transferred from the needle-like member to the cell scaffold by applying a voltage to the needle-like member to separate the compound from the surface. The culture method according to any one of claims 9 to 14,
In the step of inserting the needle-like member into the third cell, the needle-like member is extended to the culture vessel in the first posture in which the first cell and the second cell face each other in the vertical direction. Insert the needle-like member so that the direction of orientation coincides with the vertical direction,
In the step of adding the culture solution, the culture solution is added to the culture vessel in a second posture in which the first cell and the second cell are horizontally opposed.

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