WO2018105255A1

WO2018105255A1 - Method and apparatus both for producing cell aggregate structure

Info

Publication number: WO2018105255A1
Application number: PCT/JP2017/038129
Authority: WO
Inventors: 健二米田; 一朗越田
Original assignee: 澁谷工業株式会社
Priority date: 2016-12-05
Filing date: 2017-10-23
Publication date: 2018-06-14
Also published as: TW201831676A; JP2018088879A; JP6889363B2

Abstract

A method for producing a cell aggregate structure comprises: a supply step of holding a cell agglutinate clump 2 on a holding section H composed of multiple pins 16 that are provided so as to stand in a culture vessel 3; a culturing step of culturing the cell agglutinate clump 2 held on the holding section H to form a cell aggregate structure (an adhesion pad 4); a detachment step of detaching the adhesion pad 4 formed in the culturing step from the holding section H; and an injection step of injecting an injectant into the adhesion pad 4 detached in the detachment step through through-holes 4a that are formed by the pins 16 and open into the adhesion pad 4. It becomes possible to produce a cell aggregate structure having a desired shape and a desired dimension.

Description

Method and apparatus for producing cell assembly structure

The present invention relates to a method and apparatus for producing a cell aggregate structure, and more specifically, a plurality of cell aggregates are arranged inside a culture vessel containing a culture solution, and the plurality of cell aggregates are cultured and fused together. The present invention relates to a method and apparatus for manufacturing a cell aggregate structure.

In regenerative medicine in which damaged biological functions are restored using stem cells or the like, cell sheets in which cells are cultured at high density are used. Since this cell sheet is extremely thin, a method for producing a laminated cultured cell sheet in which several cell sheets are superposed is known (Patent Document 1).
In addition, in the method for producing a laminated cultured cell sheet in which such cell sheets are superposed, hydrogel particles are arranged between the laminated cell sheets so that oxygen, nutrients, etc. are applied to the cells in the laminated part. A method for producing a cell sheet more reliably is also known (Patent Document 2).
On the other hand, cell aggregates (spheroids) in which cells have been cultured in a substantially spherical shape are arranged in contact with each other and subjected to suspension culture to produce a sheet-like cell aggregate having a thickness of 50 to 300 μm in which the cell aggregates are fused. A method is also known (Patent Document 3).

Japanese Patent No. 4931353 Japanese Patent No. 5862915 Japanese Patent No. 5523830

However, when cell sheets are laminated as in

Patent Documents

1 and 2, since the cell sheets are extremely thin, handling is difficult and it is difficult to automate, and cell aggregates are suspended and cultured as in Patent Document 3 When trying to obtain a sheet-like cell aggregate, the density of the aligned cell aggregate is generated and the cell aggregate aggregates irregularly, so the shape of the resulting sheet-like cell aggregate is not determined, There was a problem that the thickness became non-uniform.
In view of such a problem, the present invention can obtain a required shape and size, and can add functionality, and a method and apparatus for manufacturing a cell aggregate structure corresponding to automation Is to provide.

That is, in the method for producing a cell aggregate structure according to the invention of claim 1, a plurality of cell aggregates are arranged inside a culture vessel containing a culture solution, and the plurality of cell aggregates are cultured and fused to each other. In a method for producing a cell aggregate structure,
A supply step of holding a cell aggregate in a holding part constituted by a plurality of pins erected inside the culture vessel, and culturing the cell aggregate held in an adjacent holding part to form the aggregate structure A culturing step to be formed, a detaching step of taking out the aggregate structure formed in the culturing step from the holding portion, and injecting an injection into a through-hole by the pin opening in the aggregate structure taken out in the detaching step And an injection step.

According to a third aspect of the present invention, there is provided an apparatus for producing a cell aggregate structure, in which a plurality of cell aggregates are arranged inside a culture vessel containing a culture solution, and the plurality of cell aggregates are cultured and fused together. In an apparatus for producing a cell aggregate structure,
A culture holder provided inside the culture vessel and provided with a plurality of pins, a supply means for holding a cell aggregate in a plurality of holding parts constituted by the pins of the culture holder, and the culture holder The detachment means for taking out the aggregate structure formed by culturing the cell agglomerates held in the cell from the holding part of the culture holder, and the injection into the through-hole by the pin opening to the extracted aggregate structure And injection means for injecting.

According to the first and third aspects of the present invention, each cell agglomeration is held by a plurality of adjacent holding portions with a plurality of pins provided in the culture holder as a holding portion. The clumps are fused with each other by being cultured in the holding part, and a cell aggregate structure having a required shape and size can be produced, and this can be automated.
Furthermore, since the through-hole is formed in the aggregate structure of the cells cultured in this way by the pins constituting the holding portion, functionality is added by injecting an injection into the through-hole. An aggregate structure of cells can be produced.

Front view of an isolator and an incubator provided with an apparatus for manufacturing a cell aggregate structure Configuration diagram of cell assembly structure manufacturing equipment Cross-sectional view of the storage container and suction nozzle Cross section of culture vessel Plan view of cell clumps placed in the culture vessel It is sectional drawing about an injection nozzle, Comprising: The figure explaining the injection | pouring process which inject | pours an injection material into the through-hole of the assembly structure of a cell The figure explaining arrangement | positioning of the pin which comprises the 1st, 2nd pin group in 2nd Example. The figure which shows the culture state in 2nd Example The figure which shows the culture state in 3rd Example The top view about the cell agglomerate arrange | positioned in the culture container in 3rd Example The figure which shows the culture state in 4th Example The figure which shows the culture state in 5th Example Diagram explaining pin thickness and cell clump size

Hereinafter, the illustrated embodiment will be described. FIGS. 1 to 6 illustrate a cell aggregate structure manufacturing apparatus 1 according to the first embodiment, which includes a plurality of cell aggregates 2 (spheroids: see FIG. 3). ) Is placed inside the culture vessel 3, and the cell aggregate 2 is cultured to produce a fusion pad 4 (see FIG. 4) as an aggregate structure of cells fused together.
The cell aggregate 2 can be produced by, for example, a method disclosed in Japanese Patent No. 4517125. That is, when cells are seeded and cultured in a container whose inner surface is non-adhesive, the cells aggregate to form a cell aggregate 2 by adhering to each other in search of a scaffold, and these further fuse to form an outer diameter. A substantially spherical cell aggregate 2 having a dimension of about 500 μm is formed.
More efficiently, the cell aggregate 2 can be easily obtained by culturing in a non-adhesive well (substantially hemispherical accommodating portion) of the well plate. The method for producing the cell agglomerate 2 is not limited to this, and a swirl culture method in which the cell suspension is put in the swirling culture solution, or a method in which the cell suspension is put in a test tube and precipitated with a centrifuge Alternatively, it can be prepared by various known methods such as a culture method using alginate beads.
Thus, the cell aggregate 2 refers to a substantially spherical cell aggregate having an outer diameter of about 100 to 700 μm in which cells are aggregated and aggregated. These cell aggregates 2 are planar or three-dimensional. If the cells are placed in contact or close to each other, the cells of each cell aggregate 2 proliferate and fuse with the adjacent cell aggregate 2 to obtain a planar or three-dimensional aggregate structure of cells. .
The fusion pad 4 produced in the present embodiment is made from, for example, tissues such as bone, muscle, internal organs, blood vessels, skin, organs, somatic cells constituting the organs, precursor cells thereof, stem cells, etc., and bone or cartilage defects. It is intended to be used by filling the part or pasting it on the surface of an organ or organ.
Further, the above-mentioned fusion pad 4 is obtained by arranging a plurality of cell aggregates 2 in a plane and fusing them, and a thickness of about 100 to 700 μm is produced according to the size of the cell aggregates 2 to be used. The planar size can be adjusted by the number of cell aggregates 2 to be aligned.
The fusion pad 4 thus produced is different from a cell sheet obtained by culturing a cell suspension in a planar form by culturing a cell suspension in a planar manner.

The cell assembly structure manufacturing apparatus 1 of the present embodiment is provided inside an isolator 5 in which the interior shown in FIG. 1 is maintained in a sterile state and an incubator 6 provided so as to be connectable to the isolator 5. Yes. In addition, the isolator 5 is provided with a pass box 5a for decontaminating the incoming material.
FIG. 2 shows a configuration provided inside the isolator 5, and a container supporting unit 8 that supports the container 7 that stores the cell aggregate 2 and a culture that supports the culture container 3 in which the fusion pad 4 is cultured. A container support 9, a plurality of suction nozzles 10 for holding the cell aggregate 2, suction nozzle moving means 11 for moving the suction nozzle 10 relative to the storage container 7 and the culture container 3, and culture An injection nozzle 12 as injection means for injecting an injection into the fusion pad 4 and an injection nozzle moving means 13 for moving the injection nozzle 12 relative to the culture vessel 3 are provided.
In the following description, the horizontal direction shown in FIG. 2 is the X direction, the front-rear direction is the Y direction, the vertical direction is the Z direction, and in FIG. 5, the horizontal direction is the X direction and the vertical direction is the Y direction.

The interior of the isolator 5 is maintained in a sterile environment, and a one-way flow is generated by aseptic air from above to below by the sterile air supply means.
In addition, a glove 5b that can be worn by an operator is provided in front of the isolator 5, so that various operations can be performed. It is also possible to provide a robot or a transfer means having a required configuration inside the isolator 5 so that these operations are automatically performed.
Next, the inside of the incubator 6 is maintained in a sterile environment and is maintained at a predetermined temperature and humidity suitable for culturing the fusion pad 4, and the cell aggregate 2 is fused to form the fusion pad 4. During this time, the incubator 6 can be separated from the isolator 5 and placed at a location away from the isolator 5.
Therefore, the isolator 5 and the incubator 6 are connected by the connecting means 5c. For example, as described in Japanese Patent No. 4656485, the connecting means for contacting and separating the incubator 6 and the isolator 5 while maintaining the sterility. Can be used.

FIG. 3 shows a cross-sectional view of the container 7, and the well plate described above can be used. The storage container 7 includes a plurality of storage recesses 7a vertically and horizontally in a plan view, and each of the storage recesses 7a stores a substantially spherical cell aggregate 2 together with a culture solution one by one.
The storage recesses 7a of the storage container 7 are arranged in a predetermined number in the X direction and the Y direction in plan view. In this embodiment, the X of the cell aggregate 2 constituting the fusion pad 4 produced in the culture container 3 is used. The number is the same as the number in the direction and the Y direction, that is, five in the X direction and five in the Y direction.

The storage container support 8 that supports the storage container 7 is configured by a Y-direction table 8a that constitutes the suction nozzle moving means 11 while positioning the storage container 7 on the upper surface thereof by a positioning piece 8b.
As will be described later, five suction nozzles 10 of the present embodiment are provided in the X direction, and are movable only in the X direction and the Z direction. Therefore, the Y direction table 8a moves the container 7 in the Y direction. The suction nozzle 10 and the container 7 can be relatively moved in each direction of XYZ.
Specifically, the Y-direction table 8a first positions the first row of storage recesses 7a of the storage container 7 below the suction nozzle 10, and the suction nozzle 10 aggregates cells from the first row of storage recesses 7a. When the lump 2 is sucked and held, the Y-direction table 8a moves the storage container 7 in the Y direction by one row, and the second row of storage recesses 7a is positioned below the suction nozzle 10.

As shown in FIG. 4, the culture vessel 3 has a bottomed box shape, in which a culture solution is accommodated at a predetermined depth, and a bottom plate 14 installed on the bottom surface, and the bottom plate 14 and a culture holder comprising a plurality of pins 16 erected on the pedestal 15 and a support plate 17 as a support member that moves up and down along the pins 16 are provided. ing.
The bottom plate 14 has substantially the same shape as the bottom surface of the culture vessel 3 and is positioned so that the bottom plate 14 does not move inside the culture vessel 3. The pedestal 15 has a rectangular parallelepiped shape, and is positioned by being fitted into a recess 14 a formed on the upper surface of the bottom plate 14.

The pins 16 are regularly erected on an installation surface 15 a formed on the upper surface of the pedestal 15 so as to face the Z direction.
FIG. 5 shows a plan view of the pin 16 provided on the pedestal 15 and the cell aggregate 2 held by the pin 16, and in this embodiment, five cell aggregates 2 in the X direction and Y direction, respectively. Are arranged in a plane in contact with each other.
The pin 16 is provided so as to be located around each cell aggregate 2, and in this embodiment, the holding part H that holds the cell aggregate 2 is configured by four pins 16. More specifically, a substantially square section where the four pins 16 are located at the four corners in plan view is the holding portion H.
In the present embodiment, among the four pins 16 constituting the holding portion H, the interval between the two pins 16 at each diagonal position is arranged to be shorter than the diameter of the cell aggregate 2 to be held. ing. In other words, when the cell aggregate 2 is produced, the diameter is made larger than the interval between the two pins 16 at each diagonal position.
When the cell aggregate 2 is held in the holding portion H configured as described above, the cell aggregate 2 can be held between the plurality of pins 16 and the pins 16, and the cell aggregate 2 as shown in FIG. The lump 2 can be positioned at the upper and lower intermediate portion between the tip and the root of the pin 16.
In other words, the holding portion H can be set at a position separated from the installation surface 15a where the plurality of pins 16 are erected, and between the cell aggregate 2 held by the holding portion H and the installation surface 15a. In addition, a gap g through which the culture solution can be circulated can be formed.
Further, although depending on the diameter of the pins 16, the cell aggregates 2 of the holding portions H are brought into contact with each other by making the interval between the two pins 16 at the diagonal positions shorter than the diameter of the cell aggregate 2. It can be cultured in a state of being allowed to stand.

The bottom plate 14 is provided with two rod-shaped guide members 18 which are provided at positions opposed to each other with the pedestal 15 interposed therebetween and which are erected in the Z direction and penetrate the support plate 17 up and down. The support plate 17 has a through hole 17a through which the pin 16 passes.
With the above configuration, the support plate 17 is provided so as to be movable up and down in the Z direction along the pin 16 and the guide member 18, and usually the base end of the pin 16 placed on the installation surface 15 a of the pedestal 15 by its own weight. It is located at the lowered position.
When the support plate 17 shown in FIG. 4 (a) is positioned at the lowered position, the culture solution is interposed between the cell aggregate 2 held on the upper and lower intermediate portions of the pin 16 and the installation surface 15a of the pedestal 15. The gap | interval g which can be distribute | circulated is formed.
On the other hand, as shown in FIG. 4B, when the support plate 17 is raised, the support plate 17 can support the cell aggregate 2 held between the

pins

16 and 16 from below, Since the support plate 17 is positioned up to the ascending position on the tip side of the pin 16, the fusion pad 4 on which the cell aggregate 2 is cultured can be taken out from the holding portion H between the pins 16. Yes.
The support plate 17 can be raised by an operator wearing the globe 5b, or by a robot or the like. At this time, although not shown, by providing a grip member protruding above the surface of the culture solution on the upper surface of the support plate 17, the support plate 17 can be raised without touching the culture solution.

As shown in FIG. 2, the culture vessel support portion 9 is constituted by a Y-direction table 9 a that constitutes the suction nozzle moving means 11, and is similar to the Y-direction table 8 a that constitutes the storage vessel support portion 8. Each time the adsorbed and held cell aggregate 2 is supplied to the holding part H constituted by the pin 16, the holding part H set between the pins 16 together with the culture vessel 3 is moved in a row in the Y direction. It has become.

Next, the suction nozzle 10 will be described. As shown in FIG. 3, a main body portion 10a connected to a negative pressure generating means (not shown) and a tubular suction portion 10b provided at the lower end portion of the main body portion 10a. I have.
The inner diameter of the adsorbing portion 10b is smaller than that of the cell aggregate 2 and when the cell aggregate 2 is adsorbed and held at the tip of the adsorbing portion 10b in the accommodating recess 7a of the accommodating container 7, the cell aggregate 2 The suction portion 10b is not sucked into the interior.
In the culture vessel 3, the center position of the adsorbing portion 10 b is positioned at the approximate center of the holding portion H formed between the pin 16 and the pin 16, and the cell aggregate 2 is positioned at the upper and lower intermediate positions of the pin 16. The negative pressure by the negative pressure generating means is eliminated in the positioned state.
Instead of the suction nozzle 10 for adsorbing and holding the cell aggregate 2, the cell aggregate 2 may be held using a structure having a configuration such as holding the cell aggregate 2 with a gripper or the like.

The suction nozzle moving means 11 for moving the suction nozzle 10 includes an X-direction rail 21 provided in the X direction over the storage container support 8 and the culture container support 9, and a suction nozzle along the X-direction rail 21. X direction moving means 22 for moving 10 in the X direction, Z direction moving means 23 provided in the X direction moving means 22 for raising and lowering the suction nozzle 10 in the Z direction, the container holding unit 8 and the culture vessel support The unit 9 is composed of the Y-direction tables 8a and 9a. The suction nozzle moving means 11 may be configured to move the X-direction rail 21 in the Y direction instead of the Y-direction tables 8a and 9a. With such a configuration, the suction nozzle 10, the injection nozzle 12, and the camera 25 described later can be moved in the XY direction.

Further, five suction nozzles 10 according to the present embodiment are arranged in the X direction, and the intervals of these suction nozzles 10 are changed in the X direction by the interval changing means 24.
Specifically, when the suction nozzle 10 is positioned above the storage container support 8, the interval changing means 24 is arranged at the same interval as the storage recesses 7 a aligned in the X direction in the storage container 7. Change the interval.
On the other hand, when the suction nozzle 10 is positioned above the culture vessel support 9, the interval changing means 24 is set to X in the holding portion H formed between the pins 16 inside the culture vessel 3. The interval of the suction nozzle 10 is changed to the same interval as the direction interval.
The suction nozzles 10 can be provided in a plurality of rows in the Y direction, and the interval between the accommodation recesses 7a of the accommodation container support 8 and the holding portion H formed between the

pins

16 and 16 can be provided. When the interval is the same, the interval changing means 24 can be omitted.
Further, for example, when the interval between the holding portions H is narrow, it is possible to hold the cell aggregate 2 in every other holding portion H by the suction nozzle 10, and in this case, the odd-numbered holding portions are initially set. The cell aggregate 2 may be held by H, and then the cell aggregate 2 may be held by the even-numbered holding part H.
In addition, it is sufficient that at least one suction nozzle 10 is provided, and as a means for supplying the cell aggregate 2, in addition to the configuration for transferring the cell aggregate 2 as in this embodiment, a number of cell aggregates are provided. The configuration may be such that one by one is delivered from the container containing the lump 2.

As shown in FIG. 6, the injection nozzle 12 includes a main body portion 12a connected to an injection supply means (not shown) and a tubular injection portion 12b provided at the lower end of the main body portion 12a. Thus, the injection is injected into the through-hole 4a opened in the fusion pad 4 thus cultured.
From the above-mentioned injection supply means, nutritional components that promote cell culture as injections, engraftment and growth after transplantation of the fusion pad 4 to a patient, growth factors and growth factors that promote regeneration of surrounding tissues, blood vessel A cell suspension that promotes neoplasia, a drug that acts on the living body after transplantation, and the like are supplied in the form of a liquid, gel, gel, particle, granule, and the like. 4 through holes 4a. The injection may be performed above the through hole 4a, or may be performed by inserting the injection nozzle 12 into the through hole 4a.
By injecting into the through-hole 4a in this way, the injection can be embedded in the fusion pad 4, and a larger amount of the injection than in the case where the injection is simply dropped on the surface of the fusion pad 4 4 can penetrate.
And about the fusion pad 4 in which the said injection was inject | poured, it may culture | cultivate continuously and may block the said through-hole 4a, or it can collect | recover as it is and can also be used for a transplant.

As shown in FIG. 2, four injection nozzles 12 are arranged in the X direction, specifically, the same interval as the interval of the pins 16 aligned in the X direction inside the culture vessel 3, that is, fusion. The pads 4 are provided so as to have the same spacing as the through holes 4 a of the pads 4.
The injection nozzle moving means 13 for moving the injection nozzle 12 includes an X-direction rail 21 that is used in common with the suction nozzle movement means 11, a left-right movement means 25 that moves along the X-direction rail 21, The moving unit 25 includes a vertical moving unit 26 that moves the injection nozzle 12 up and down in the Z direction, and the Y direction table 9 a in the culture vessel support 9.
It is sufficient that at least one injection nozzle 12 is provided, and the injection nozzle moving means 13 may be configured not to include the Y-direction table 9a but to move the injection nozzle 12 in the X direction and the Y direction. it can.
It is also possible to inject different injections by providing a plurality of injection nozzles 12 and moving them individually.

The X-direction rail 21 is provided with a camera 28 in a lateral movement means 27 that moves in the X direction, and the camera 28 is moved to above the culture vessel support 9 to photograph the inside of the culture vessel 3. It has become.
When the cell aggregate 2 is held in the holding part H by the suction nozzle 10, it is confirmed from the images taken by the camera 28 whether or not the cell aggregate 2 is held in all the holding parts H. If necessary, it can be instructed to supply the cell aggregate 2 to the holding part H where the cell aggregate 2 is not held.
Furthermore, when the injection is injected into the fusion pad 4 by the injection nozzle 12, the position of the through hole 4a of the fusion pad 4 is recognized from the image taken by the camera 28.
The camera 28 is also used when the suction nozzle moving means 11 positions the suction nozzle 10 above the culture vessel support 9 and when the injection nozzle movement means 13 positions the injection nozzle 12 above the culture vessel support 9. In order to avoid contact with these, they are retracted from above the culture vessel support 9.

Hereinafter, a method for producing the fusion pad 4 using the cell assembly structure producing apparatus 1 having the above-described configuration will be described.
First, a preparation step of carrying the container 7 or the culture container 3 containing the cell aggregate 2 through the pass box 5a into the isolator 5 or supplying a predetermined amount of culture solution to the culture container 3 I do.
The culture vessel 3 is provided with a culture holder comprising the pedestal 15 and a plurality of pins 16, and the support plate 17 is located at a lowered position in contact with the installation surface 15 a of the pedestal 15 by its own weight.
Furthermore, in the preparation step, if a plurality of the storage containers 7 and culture containers 3 are carried into the isolator 5, a plurality of fusion pads 4 can be continuously produced using the plurality of culture containers 3.

When the preparation step is completed, the cell aggregate 2 is subsequently moved from the container 7 to the culture vessel 3 by the suction nozzle 10, and the cells are placed in the holding part H set by the pins 16 of the culture vessel 3. A supplying step for supplying the aggregate 2 is performed.
The suction nozzle moving means 11 moves the suction nozzle 10, and the interval changing means 24 changes the interval of the suction nozzle 10, so that the cell aggregate 2 is removed from the accommodating recess 7 a of the accommodating container 7 in the accommodating container support 8. Hold by adsorption.
Subsequently, the suction nozzle moving means 11 moves the suction nozzle 10 to the culture vessel support portion 9, and the interval changing means 24 is adjusted to the interval of the holding portion H formed between the

pins

16 and 16. The interval of the suction nozzle 10 is changed, and the suction nozzle 10 is lowered.
Then, each cell aggregate 2 held by the suction nozzle 10 is inserted into the holding portion H formed between the four pins 16, and the pin g is formed so that a gap g is formed below the cell aggregate 2. The cell aggregate 2 is held at 16 upper and lower intermediate positions.
When the cell aggregates 2 are held in all the holding parts H in this way, the camera 28 then moves above the culture container support part 9 to photograph the cell aggregates 2 in the culture container 3, and the cells It is confirmed whether or not the agglomerate 2 is arranged in the required holding part H.

When the cell aggregate 2 is arranged in the culture container 3 in this way, the culture container 3 is transferred to the incubator 6 by a robot or an operator's manual operation, and the cell aggregate 2 in the culture container 3 is removed. A culture step of culturing is performed.
First, when the culture vessel 3 is transferred to the incubator 6, the incubator 6 is separated from the isolator 5 and placed at a position separated from the isolator 5 to maintain the inside at a predetermined temperature and humidity, and a predetermined carbon dioxide gas. And maintain oxygen concentration.
In the culture vessel 3, when the cell aggregate 2 is cultured, the cell aggregates 2 of the adjacent holding portions H are fused with each other, so that the cell aggregate 2 is maintained in a planar arrangement. The fusion pad 4 as a collective structure can be obtained.
That is, since the movement of each cell aggregate 2 is regulated by the pin 16, the size of the formed fusion pad 4 can be made substantially the same as the size when the cell aggregate 2 is first arranged, It becomes possible to produce the fusion pad 4 having any shape as the cell aggregate 2 is arranged.
This is because the cell aggregate 2 grows or moves so as to be in close contact with the periphery of the pin 16 in contact with the cell aggregate 2 and fills the gap formed between the adjacent cell aggregates 2. As in the case where the cell agglomerates 2 are arranged in a plane and cultured without intervention, the overall dimensions hardly shrink.
At that time, since the gap g is formed below the cell aggregate 2, the culture solution is supplied between the fusion pad 4 and the installation surface 15 a of the pedestal 15, and the back side of the fusion pad 4. Can also be efficiently cultured.
That is, when the fusion pad 4 is produced by placing the cell aggregate 2 on the installation surface 15a, there is a problem that the culture solution does not sufficiently spread to the lower surface side of the cell aggregate 2 located in the center. By holding the cell aggregate 2 in a state in which the gap g is formed below, the cells located in the part can be well cultured.
If the support plate 17 is moved up and down during the culture, the flow of the culture solution is promoted, which is more effective.

Thus, when culture | cultivation is advanced while exchanging a culture solution in the said culture | cultivation process, the cell aggregate 2 fuse | melts and the fusion pad 4 of a required magnitude | size is obtained, but the fusion pad 4 is hold | maintained at the holding part H. In other words, the pin 16 remains penetrating the fusion pad 4.
Therefore, a detachment process for taking out the fusion pad 4 held by the holding part H from the holding part H is performed. Specifically, the incubator 6 is connected to the isolator 5, the culture vessel 3 is placed on the culture vessel support portion 9 inside the isolator 5, and the support plate 17 positioned at the lowered position is raised by an operator or a robot or the like. To position.
As a result, the fusion pad 4 is raised along the pins 16 and the support plate 17 is lifted upward as shown in FIG. 4B. In this state, the support plate 17 is fixed with a fixture 17b such as a clip. The fusion pad 4 is positioned above the pin 16 by fixing or by gently moving the support plate 17 to the lowered position and supporting the fusion pad 4 from below by the tip of the pin 16.

After the fusion pad 4 is positioned above the pin 16 by the above-described detachment process, an injection process for injecting an injection into the through-hole 4a by the pin 16 that is opened to the fusion pad 4 by the injection nozzle 12 is performed. .
Specifically, first, the camera 28 moves above the culture vessel support 9 to photograph the fusion pad 4 in the culture vessel 3 and recognize the position of the through hole 4 a of the fusion pad 4.
Subsequently, after the injection nozzle moving means 13 moves the injection nozzle 12 above the culture vessel support 9 and further positioned above the through-hole 4a of the fusion pad 4, as shown in FIG. The injection part 12 b of the injection nozzle 12 is brought close to the fusion pad 4.
When the injection is discharged from the injection portion 12b of the injection nozzle 12 in this state, the injection is injected into the through hole 4a.
In this way, when the injection is injected into the through-hole 4a of the fusion pad 4, in order to use the fusion pad 4 as it is, the fusion pad 4 is taken out from the culture vessel 3 and transferred to the carrying-out vessel and carried out from the isolator 5. Alternatively, the culture vessel 3 may be transferred again from the isolator 5 to the incubator 6 and the culture may be continued to completely close the through-hole 4a.

7 and 8 are diagrams for explaining a method for producing a cell aggregate structure as a second embodiment. In the culture vessel 3 used in this embodiment, the pin 16 fixed to the pedestal 15 is a fusion pad. 4 comprises a first pin group (16a) that holds the central part of the pin 4 and a second pin group (16b) shown in black that holds the outer peripheral edge part of the fusion pad 4.
Specifically, in FIG. 7, the pins 16 b constituting the second pin group are arranged in a frame shape arranged on the outermost periphery and on the inner side in one row. For this reason, when the cell aggregate 2 is accommodated in the culture container 3 in the supply step, the cell aggregate 2 arranged on the outermost periphery thereof is held by the holding portion H formed by the pin 16b of the second pin group. It has become.
As shown in FIG. 8, the pin 16 a constituting the first pin group is shorter than the pin 16 b constituting the second pin group, and the height of the holding portion H that holds the cell aggregate 2. Is set to a height near the tip of the pin 16a of the first pin group.

The method for producing a cell aggregate structure using the culture vessel 3 will be described. As shown in FIG. 8A, in the supplying step, the pins of the first and second pin groups are the same as in the first embodiment. The cell aggregate 2 is held in the holding portion H formed by 16a and 16b.
Thereafter, a culture process is performed, and when the cell aggregate 2 is fused to some extent to form the fusion pad 4 in the middle of the culture, the culture vessel 3 containing the cultured fusion pad 4 is transferred from the incubator 6 to the isolator 5. To do.
Then, as shown in FIG. 8B, the support plate 17 positioned at the lowered position is moved up and down to move the cultured fusion pad 4 to a height near the tip of the pin 16b of the second pin group, A detaching step for removing the pin 16a from the holding portion H of the first pin group is performed.
As a result, the fusion pad 4 is supported by the pin 16b of the second pin group, and the through hole 4a by the pin 16a of the first pin group is opened in the fusion pad 4.

In this state, the injection nozzle 12 performs an injection process of injecting an injection into the through hole 4a by the pin 16a of the first pin group of the fusion pad 4.
Thereafter, the fusion pad 4 is moved again from the isolator 5 to the incubator 6 to resume the culturing process, followed by culturing, whereby the through-hole 4a is filled with the cultured cells.
At that time, although depending on the size of the through hole 4a, the fusion pad 4 may contract toward the center and bend in the process of closing the through hole 4a.
On the other hand, in this embodiment, the outer peripheral edge of the fusion pad 4 is held by the pin 16b of the second pin group, so that the central portion of the fusion pad 4 is prevented from contracting and the thickness without bending is uniform. Can be obtained.

When the culture process is completed and the through-hole 4a formed in the central portion of the fusion pad 4 is closed, the culture container 3 is moved from the incubator 6 to the isolator 5 to perform the detachment process.
Specifically, the support plate 17 is raised again from the lowered position to the raised position, and the fusion pad 4 is taken out from the holding portion H of the second pin group.
Since the through-hole 4a by the pin 16b of the said 2nd pin group penetrated until then is formed in the outer periphery part of the fusion pad 4 obtained in this way, the fusion pad 4 of the fusion pad 4 is needed as needed. The outer peripheral edge portion may be cut and removed, and only the central portion may be collected, or an injection process may be performed to inject an injection different from the through hole 4a by the pin 16a of the first pin group. It may be.
In addition, the through hole 4a formed by the pin 16a of the first pin group can be closed without being injected, and can be injected into the through hole 4a formed by the pin 16b of the second pin group. The injection position of the injection can be designed by selectively arranging the first pin group and the second pin group.

FIG. 9 is a diagram for explaining a method for producing a cell aggregate structure as a third embodiment, in which the diameter of the cell aggregate 2 is smaller than the interval between the pins 16 at each diagonal position of the holding portion H. This corresponds to the case where the cell aggregate 2 cannot be held between the pin 16 and the pin 16.
Specifically, as shown in FIG. 10, in order to make the produced fusion pad 4 thin, there is a variation in the case of using the smallest cell aggregate 2 or the size of the produced cell aggregate 2. It is assumed that the pin 16 and the pin 16 include a cell aggregate 2 that cannot be held and falls.
Therefore, in the supply process of the present embodiment, as shown in FIG. 9A, the cell aggregate 2 is accommodated with the space between the pins 16 as the holding portion H, and is positioned at the lowered position on the installation surface 15a of the pedestal 15. Each cell aggregate 2 is held by being placed on the upper surface of the support plate 17.
Then, while the cell aggregate 2 is placed on the support plate 17, the culture vessel 3 is transferred to the incubator 6 to start the culture process, and as shown in FIG. Incubate until mass 2 is fused.
When the cell aggregate 2 is fused to some extent, the support plate 17 is raised to an intermediate position between the proximal end side and the distal end side of the pin 16, and separated from the installation surface 15a where the plurality of pins 16 are erected. The cell aggregate 2 is held with the position being the holding portion H.
Also in this embodiment, when the fusion pad 4 having a predetermined size is obtained, the culture process shifts to the withdrawal process and the injection process.

FIG. 11 is a diagram for explaining a method for producing a cell aggregate structure as a fourth embodiment. Unlike the above embodiments, the tip of a pin 16 erected in the culture vessel 3 is formed with a sharp point. The pin 16 is penetrated and held in the cell aggregate 2.
The cell assembly structure manufacturing apparatus 1 may be the same as that used in each of the above embodiments. However, when the cell aggregate 2 is penetrated by the pin 16 by the suction nozzle 10, The penetrating pin 16 is inserted into the suction portion 10b, and the lowering of the suction nozzle 10 is stopped at a predetermined position to hold the cell aggregate 2.
Even when the cell aggregate 2 is held in this manner, the cell aggregates 2 of the holding portions H adjacent to each other are fused in the culturing step, and the same fusion pad 4 as in the above embodiments can be obtained. .
Further, in the separation step, as shown in FIG. 11B, the support plate 17 can be moved to the upper position so that the cell aggregate 2 can be positioned above the pin 16. In the injection step, the injection nozzle 12, the injection can be injected into each through hole 4a.
In this embodiment, the through hole 4a is formed at the position where the pin 16 penetrates, and the injection nozzle 12 moves to the position of the pin 16 and injects.

FIG. 12 is a diagram for explaining a method for producing a cell aggregate structure according to the fifth embodiment. The planar union pad 4 as the cell aggregate structure produced in the first to fourth embodiments is shown in FIG. On the other hand, in this embodiment, a method for producing a three-dimensional aggregate structure of cells in which cell aggregates 2 are aligned in the Z direction will be described.
However, even in the case of producing the three-dimensional cell assembly structure, it is possible to use an apparatus having the same configuration as the cell assembly structure preparation apparatus 1 used in the first embodiment. The detailed description is omitted because it is possible and can be manufactured using substantially the same process.
In the culture vessel 3 used in this embodiment shown in FIG. 12, the length of the pin 16 is set according to the desired height of the three-dimensional cell assembly, and is formed by four pins 16. The holding portion H can hold a plurality of cell aggregates 2 in the Z direction.
In the supplying step, the suction nozzle 10 supplies the cell aggregate 2 to the holding portion H formed by the pins 16 based on a preset three-dimensional arrangement of the cell aggregate 2.
Specifically, first, the culture vessel support unit 9 sequentially moves the culture vessel 3 in the Y direction to supply the cell aggregate 2 to be arranged at the bottom, and then the cell aggregate held in the holding unit H What is necessary is just to supply the cell aggregate 2 to the upper part of 2 sequentially.
At this time, as in each of the above embodiments, a gap g is formed below the cell aggregate 2 located at the lowermost stage, whereby the culture solution on the lower surface of the three-dimensional cell assembly structure is formed. To be supplied. Further, since a gap is also formed between the side surface portion of the three-dimensional cell assembly structure supported by the pin 16 and the side surface portion of the culture vessel 3, the side surface portion of the cell assembly structure is also formed. The culture solution is supplied, and a three-dimensional aggregate structure can be formed in the culture process.
Also in the present embodiment, the injection can be injected from the injection nozzle 12 into the through hole of the aggregate structure taken out from the holding portion H by performing the above-described detachment step and the above-described injection step. In this case, the injection nozzle 12 may be inserted into a through-hole of the assembly structure to inject the injection into a required position in the vertical direction of the through-hole 4a.

In each of the above-described embodiments, the pin 16 that supports the cell aggregate 2 may be arranged with, for example, six pins 16 as long as the cell aggregate 2 can be supported by a plurality of pins 16. You may arrange | position so that the cell aggregate 2 may be surrounded.
In that case, the holding portions H are arranged in a staggered manner, and it is also possible to produce a three-dimensional aggregate structure of cells as in the fifth embodiment using this arrangement.

Further, in each of the above embodiments, as shown in FIG. 13, depending on the thickness of the pin 16 provided in the culture vessel 3, the size of the holding portion H formed by the pin 16, or the diameter of the cell aggregate 2 to be used. Various aspects are conceivable.
The diameter of the pin 16 shown in FIG. 13 is larger than the diameter of the pin 16 shown in the above embodiments, and the diameter of the cell aggregate 2 is larger than the interval between the two pins 16 at the diagonal positions. However, the case where the adjacent cell aggregate 2 hold | maintained at the said holding | maintenance part H does not contact is shown.
Even in such a case, since the cell aggregate 2 is cultured and the adjacent cell aggregate 2 is fused in the culturing step, the fusion pad 4 as described above can be obtained, and the pin 16 is large. Since the diameter of the through-hole 4a formed in the fusion pad 4 is large, it is possible to inject a large amount of an injection into the through-hole 4a. Can also be defined.

When injecting nutrients, growth factors, growth factors, drugs, or the like as injections into the through holes 4a formed in the fusion pad 4, depending on the action of these injections, for example, injection into every other through hole 4a Thus, the effect can be dispersed, and the injection is injected only into the through-holes 4a concentrated at a specific position in the fusion pad 4 so that the specific position of the fusion pad 4 is functional. It is also possible.
It is also possible to selectively inject the cell suspension containing the cells constituting the blood vessel as an injection into the through-hole 4a so that a blood vessel is formed in a required portion of the fusion pad 4. Depending on the application of 4, it is possible to inject different kinds of injections by selecting the positions of the through holes 4a.

DESCRIPTION OF SYMBOLS 1 Production apparatus of cell aggregate structure 2 Cell aggregate 3 Culture container 4 Healing pad (cell aggregate structure)
10 Suction nozzle 12 Injection nozzle 16 Pin 17 Support plate H Holding part g Gap

Claims

In a method for producing a cell aggregate structure, a plurality of cell aggregates are arranged inside a culture vessel containing a culture solution, and a plurality of cell aggregates are cultured to produce a cell aggregate structure fused together ,
A supply step of holding the cell aggregate in a holding part constituted by a plurality of pins standing inside the culture container, and culturing the cell aggregate held in the holding part to form the aggregate structure A culturing step, a detaching step of taking out the aggregate structure formed in the culturing step from the holding portion, and an injection for injecting an injection into the through-hole by the pin opening in the ensemble structure taken out in the detaching step A process for producing an aggregate structure of cells.
The method for producing an aggregate structure of cells according to claim 1, wherein the aggregate structure is continuously cultured after the injection step.
In a cell assembly structure manufacturing apparatus, a plurality of cell aggregates are arranged inside a culture vessel containing a culture solution, and the plurality of cell aggregates are cultured to produce a cell aggregate structure fused together ,
A culture holder provided inside the culture vessel and provided with a plurality of pins, a supply means for holding a cell aggregate in a plurality of holding parts constituted by the pins of the culture holder, and the culture holder The detachment means for taking out the aggregate structure formed by culturing the cell agglomerates held in the cell from the holding part of the culture holder, and the injection into the through-hole by the pin opening to the extracted aggregate structure And an injecting means for injecting the cell.
The cell assembly according to claim 3, wherein the holding part of the culture holder is formed between the plurality of pins and the supply means sandwiches or accommodates a cell aggregate between the pins. Structure manufacturing apparatus.
The holding part of the culture holder is constituted by the pin having a sharp tip, and the supply means penetrates the cell aggregate with the pin and stops at a predetermined position of the pin. The apparatus for producing a cell aggregate structure according to claim 3.
The detachment means includes a support member movable along the pin of the culture holder, and the support member is positioned on the proximal end side of the pin when the supply means holds the cell aggregate in the holding portion. The cell assembly according to any one of claims 3 to 5, wherein when the aggregate structure formed by fusing the cell agglomerates is taken out from the holding portion, the cell aggregate is located on the tip side of the pin. Structure manufacturing apparatus.