JP6880384B2 - Cell mass sheet manufacturing equipment - Google Patents

Description

The present invention relates to a cell mass sheet manufacturing apparatus, and more specifically, a cell mass sheet manufacturing apparatus in which a plurality of cell masses are arranged in a flat manner on a flat culture tray to produce a cell mass sheet in which the cell masses are cultured and fused with each other. Regarding.

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 laminated is known (Patent Document 1).
On the other hand, cell clusters (spheroids) in which cells are cultured in a substantially spherical shape in advance are arranged in contact with each other and subjected to suspension culture, and sheet-like cell clusters (cells) having a thickness of 50 to 300 μm in which the cell clusters are fused with each other are performed. A method for producing a mass sheet) is known (Patent Document 2).
Further, as a method for arranging the cell mass in an arbitrary space, a method for producing a three-dimensional structure of cells in which the cell mass is penetrated through a support made of filamentous or needle-like bodies is known (Patent Document 3). ).

Japanese Patent No. 4921353 Japanese Patent No. 5523830 Japanese Patent No. 4517125

However, when the cell sheets are laminated as in Patent Document 1, since the cell sheets are extremely thin, they are difficult to handle and difficult to automate.
Further, when a cell mass is suspended and cultured as in Patent Document 2 to obtain a cell mass sheet, the cell masses are irregularly fused with each other, so that the shape of the obtained cell mass sheet is not determined and the thickness is also uncertain. There was a problem of becoming uniform.
Further, when the cell mass is penetrated through a support made of a filamentous body or a needle-like body as in Patent Document 3, there is a risk of damaging the cells.
In view of such a problem, the present invention provides a cell mass sheet manufacturing apparatus capable of producing a cell mass sheet having a constant shape and a uniform thickness.

That is, the invention of claim 1 is a cell mass sheet manufacturing apparatus in which a plurality of cell masses are arranged in a plane on a placement surface in a culture vessel, and the cell masses are cultured to produce a cell mass sheet fused with each other. There,
A guide means having a plurality of accommodating portions having a size capable of individually accommodating the cell mass, a supply means for supplying the cell mass into the accommodating portion of the guide means installed on the above-mentioned mounting surface, and the above-described placement. It is equipped with a camera that captures a surface and a floating prevention means that prevents the cell masses aligned on the above-mentioned mounting surface from floating.
The cell mass is accommodated in the accommodating portion of the guide means by the supply means, the cell mass is aligned on the mounting surface, and it is confirmed by the camera whether or not the cell mass is accommodated in the accommodating portion, and then the above-mentioned ascent is performed. It is characterized in that the cell mass is prevented from floating by the preventive means, and the guide means is retracted from the mounting surface.

According to the above invention, by accommodating the cell mass in the accommodating portion of the guide means installed on the mounting surface, the cell mass can be regularly arranged according to the accommodating portion.
In this state, the guide means retracts from the mounting surface to maintain the state in which the cell masses are regularly aligned. Therefore, by culturing the cell masses in that state, the shape as aligned by the guide means. A cell mass sheet can be prepared in the cell mass, and its thickness can be kept constant.

Configuration diagram of the cell mass sheet manufacturing apparatus according to the first embodiment Sectional view of the containment vessel and suction nozzle Perspective view of culture tray and culture container Top view of culture trays and culture vessels Side view of the culture vessel support Side view of inspection means Side view of the culture solution supply means and the cell mass sheet moving means Top view of the culture trays that can be used in the first embodiment Top view and side view of pins for the culture trays that can be used in the first embodiment Top view of the culture trays that can be used in the first embodiment Side view of the culture tray and culture container that can be used in the first embodiment. Side view of the culture solution supply means that can be used in the first embodiment. Configuration diagram of the cell mass sheet manufacturing apparatus according to the second embodiment Top view of the culture vessel Side view of the culture vessel Top view explaining the holder Side view of cell mass sheet moving means Top view of the culture vessel that can be used in the second embodiment Front view of the isolator and incubator provided with the cell mass sheet manufacturing device Configuration diagram of the cell mass sheet manufacturing apparatus according to the third embodiment

Hereinafter, the illustrated examples will be described. FIGS. 1 to 7 show the cell mass sheet manufacturing apparatus 1 according to the first embodiment, and a plurality of cell masses 2 (spheroids: see FIG. 2) are placed in a culture vessel. The cell mass 2 is cultured and fused with each other to prepare a cell mass sheet 4 (see FIG. 6), which is arranged in a plane on a flat mounting surface provided in 3.
The cell mass 2 can be produced, for example, by the 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 adhere to each other in search of a scaffold to form a small cell mass, which is further fused to have an outer diameter of about 500 μm. A substantially spherical cell mass 2 up to is formed.
More efficiently, the cell mass 2 can be easily obtained by culturing in a non-adhesive well (substantially hemispherical housing portion) of the well plate. The method for producing the cell mass 2 is not limited to this, and a swirling culture method in which the cell suspension is placed in a swirling culture solution, a method in which the cell suspension is placed in a test tube and precipitated by a centrifuge, Alternatively, it can be produced by various known methods such as a method of culturing using alginate beads.
As described above, the cell mass 2 refers to a substantially spherical cell aggregate having an outer diameter of about 100 to 500 μm in which cells are aggregated and aggregated, and the cell mass sheet 4 in the present invention is described as described above. It is formed into a sheet by fusing a plurality of various cell masses, and it is possible to prepare a sheet having a thickness of about 100 to 500 μm depending on the size of the cell mass 2 to be used, and the planar sizes are aligned. It can be adjusted by the number of cell clusters 2 to be allowed.
The cell mass sheet 4 thus produced is different from the cell sheet obtained by culturing a single cell in a sheet shape by culturing the cell suspension in a plane.

The cell mass sheet manufacturing apparatus 1 of this embodiment is provided inside an isolator 61 whose inside is kept sterile as shown in FIG. 19 and an incubator 62 provided so as to be connectable to the isolator 61. Further, the isolator 61 is provided with a pass box 63 for decontaminating the carried-in material.
FIG. 1 shows a configuration provided inside the isolator 61, and shows a culture that supports a storage container support portion 6 that supports a storage container 5 that stores the cell mass 2 and a culture container 3 that supports the culture container 3 in which the cell mass sheet 4 is cultured. The container support portion 7, the suction nozzle 8 as a plurality of holding means for holding the cell mass 2, and the nozzle as a moving means for moving the suction nozzle 8 relative to the storage container 5 and the culture container 3. The moving means 9 and the inspection means 10 (see FIG. 6) for confirming the culture state of the cell mass sheet 4 in the culture vessel 3 are provided.
On the other hand, FIG. 7 shows the configuration provided inside the incubator 62, and the culture solution supply means 11 for supplying the culture solution to the cell mass 2 or the cell mass sheet 4 on the culture vessel 3 and the cell mass sheet being cultured. 4 The cell mass sheet moving means 12 for moving is provided. Since the cell mass 2 or the cell mass sheet 4 is cultured inside the incubator 62, the internal structure of the incubator 62 constitutes a cell mass culturing device or a cell mass sheet culturing device.
In the following description, the left-right direction shown in FIG. 1 is the X direction, the front-back direction is the Y direction, the up-down direction is the Z direction, and in FIG. 4, the left-right direction shown is the X direction and the up-down direction is the Y direction.

The inside of the isolator 61 is maintained in a sterile environment, and a unidirectional flow of sterile air is formed from above to below by a sterile air supply means.
Further, a glove 61a that can be worn by an operator is provided on the front surface of the isolator 61, 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 61 so that these operations are automatically performed.
Next, the inside of the incubator 62 is maintained in a sterile environment and is maintained at a predetermined temperature and humidity suitable for culturing the cell mass sheet 4, and the cell mass 2 is cultured and fused to the cell mass sheet 4. The incubator 62 is separated from the isolator 61, and the cell mass 2 can be cultured at a place away from the isolator 61.
Therefore, the isolator 61 and the incubator 62 are connected by a connecting means 64, and for example, as described in Japanese Patent No. 4656485, a connecting means for connecting the incubator 62 and the isolator 61 while maintaining an aseptic state. Can be used.

FIG. 2 shows a cross-sectional view of the storage container 5, in which the storage container 5 is provided with a plurality of storage recesses 5a in the vertical and horizontal directions, and inside each storage recess 5a, one substantially spherical cell mass 2 is provided together with the culture solution. It is contained.
The storage recesses 5a of the storage container 5 are aligned in predetermined numbers in the X and Y directions in a plan view, and in this embodiment, the X of the cell mass 2 constituting the cell mass sheet 4 produced in the culture container 3 The same number as the number in the direction and the Y direction, that is, 7 in the X direction and 7 in the Y direction are provided.

The storage container support portion 6 that supports the storage container 5 is configured by a Y-direction table 6a that positions the storage container 5 on its upper surface by a positioning piece 6b and constitutes the nozzle moving means 9.
As will be described later, since seven suction nozzles 8 of this embodiment are provided in the X direction and can be moved only in the X direction and the Z direction, the Y direction table 6a moves the storage container 5 in the Y direction. The suction nozzle 8 and the storage container 5 can be relatively moved in each direction of XYZ.
Specifically, the Y-direction table 6a first positions the first-row storage recess 5a of the storage container 5 below the suction nozzle 8, and the suction nozzle 8 causes the cell mass from the first-row storage recess 5a. When 2 is sucked and held, the Y-direction table 6a moves the storage container 5 in the Y direction by one row, and the storage recess 5a in the second row is positioned below the suction nozzle 8.

As shown in FIG. 1, a culture tray 13 is housed in the culture container 3 so as to be able to move up and down, and the culture container 3 is supported by the culture container support portion 7.
As shown in FIG. 3, the culture tray 13 has a substantially square plate shape, and as shown in FIG. 4, the upper surface thereof has seven cell clusters 2 in each of the X and Y directions as the mounting surface 13a. The size is set so that they can be aligned in contact with each other.
The placement surface 13a of the culture tray 13 on which the cell mass 2 is placed has a non-adhesive property of cells, and it is difficult for the cell mass 2 and the cell mass sheet 4 to adhere to each other. Since it is difficult for the cell mass 2 to adhere to the mounting surface 13a, the function of the adjacent cell masses 2 to bond with each other becomes stronger, and the formation of the cell mass sheet 4 is promoted.
The cell non-adhesiveness can be obtained by making the surface properties water-repellent or super-hydrophilic, and can be realized by adopting a known material having these properties, or by applying a surface treatment or a coating treatment. In addition, as a surface property of the mounting surface 13a, it is also possible to form a DLC (diamond-like carbon) film.
Further, the culture tray 3 is made of a material that transmits light, and when a coating or the like is formed on the above-mentioned mounting surface 13a, the coating also transmits light.

The culture vessel 3 has a bottomed box shape, and a culture solution is stored in the inside at a predetermined depth in advance, and a plurality of pins 14 as guide means G are regularly provided on the bottom surface.
The bottom surface of the culture vessel 3 is formed to have substantially the same shape as the culture tray 13, so that among the cell clusters 2 aligned on the mounting surface 13a of the culture tray 13, the cell mass 2 located on the outer peripheral side is the culture vessel 3. It comes into contact with the inner wall of the cell cluster, thereby supporting the aligned cell mass 2 so as not to move from the outside.
Further, the culture container 3 is made of transparent resin or glass and can transmit light, and the culture container 3 can be inspected by the above-mentioned inspection means 10.

The culture tray 13 is positioned at a lowering position in contact with the bottom surface of the culture container 3 and an ascending position raised with respect to the lowering position.
Support columns 15 are provided at the four corners of the culture tray 13, and adjacent columns 15 are connected to each other by reinforcing members 16 provided above the columns. Each support column 15 is allowed to move only in the vertical direction (Z direction) along the inner surface of the culture vessel 3, so that the culture tray 13 can move up and down inside the culture vessel 3 while maintaining a horizontal state. There is.
On the other hand, the culture vessel 3 is provided with a lock mechanism 17 which is provided at a position where any of the columns 15 passes and which appears and disappears by a spring or the like (not shown).
Of the columns 15, the column 15 passing through the lock mechanism 17 is formed with a recess 15a in which the lock mechanism 17 engages, and when the culture tray 13 is located in the raised position, the lock mechanism 17 protrudes and the recess is recessed. It engages with 15a and holds the culture tray 13 in the ascending position.
On the other hand, when moving the culture tray 13 up and down from this ascending position, a predetermined force may be applied to the support column 15 in the Z direction, whereby the lock mechanism 17 resists the urging force of the spring. Then, it retracts and separates from the recess 15a.

The pin 14 is fixed to the bottom surface of the culture vessel 3 so as to face upward, and a plurality of through holes 13b are bored in the culture tray 13 in accordance with the position of the pin 14.
Therefore, when the culture tray 13 is located in the descending position, the pin 14 penetrates the through hole 13b and protrudes to the upper surface of the culture tray 13, and the guide means G is installed on the culture tray 13.
On the other hand, when the culture tray 13 is located in the ascending position, the culture tray 13 is located above the tip of the pin 14 and the pin 14 is separated from the through hole 13b, so that the mounting surface 13a of the culture tray 13 The guide means G is retracted from above.

Next, as shown in FIG. 4, in this embodiment, seven cell clusters 2 are arranged vertically and horizontally in the X direction and the Y direction on the culture tray 13, and the above pins 14 are respectively arranged. A space provided around the cell mass 2 and formed by the plurality of pins 14 and the pins 14 constitutes an accommodating portion Ga for accommodating each cell mass 2.
Specifically, the circumference of one cell mass 2 is surrounded by four pins 14, and the distance between the adjacent pin 14 and the pin 14 is made narrower than the diameter of the cell mass 2.
As a result, by accommodating the cell mass 2 in the accommodating portion Ga, the cell masses 2 accommodating in the adjacent accommodating portions Ga can be brought into mutual contact with each other. The distance between the cell clusters 2 and the degree of contact at this time can be adjusted by the distance between the pin 14 and the pin 14 and the diameter of the pin 14.
Then, as in this embodiment, when the cell mass 2 is aligned on the mounting surface 13a of the culture tray 13, when the cell mass 2 and the wall surface of the culture container 3 come into contact with each other, the wall surface is concerned. And the pin 14 also form the accommodating portion Ga.

As shown in FIG. 5, the culture container support portion 7 is composed of a guide moving means 18 for moving the guide means G and a Y direction table 7a constituting the nozzle moving means 9.
The guide moving means 18 supports and raises and lowers the holding means 18a that holds the reinforcing member 16 of the support column 15 provided on the culture tray 13 and holds the culture tray 13 at a predetermined height, and the culture container 3. It is composed of an elevating means 18b.
While the holding means 18a holds the culture tray 13, the elevating means 18b raises and lowers the culture container 3 so that the culture container 3 and the culture tray 13 are relatively raised and lowered, and the culture tray 3 is lowered. And can be positioned in the ascending position.
Similar to the Y-direction table 6a constituting the storage container support portion 6, the Y-direction table 7a is provided each time the cell mass 2 adsorbed and held by the suction nozzle 8 is stored in the storage portion Ga of the culture tray 13. The storage portion Ga of the culture tray 3 is moved in a row in the Y direction together with the culture container 3.

Further, at a position adjacent to the culture container support portion 7, a holding lid supply means (not shown) for supplying a holding lid 19 as a floating prevention means is provided above the cell mass 2 arranged on the culture tray 13. There is.
A robot or the like can be used as the holding lid supply means, and when the cell mass 2 is aligned on the culture tray 13, the holding lid 19 is supplied to the upper part of the aligned cell mass 2. There is.
The holding lid 19 is a member having a mesh shape or a large number of through holes formed therein, so that the holding lid 19 does not obstruct the supply of the culture solution to the cell mass 2 and the cell mass sheet 4 on the culture tray 13. It has become.
Further, the holding lid 19 is made of a material that transmits light like the culture tray 13, and the side of the holding lid 19 that comes into contact with the cell mass 2 or the cell mass sheet 4 is the same as the mounting surface 13a of the culture tray 13. , Cell non-adhesive.

Next, the suction nozzle 8 as the holding means will be described. As shown in FIG. 2, a main body 8a connected to a negative pressure generating means (not shown) and a tubular suction provided at the lower end of the main body 8a. It is provided with a part 8b.
The inner diameter of the adsorption portion 8b is smaller than that of the cell mass 2, and when the cell mass 2 is adsorbed and held at the tip of the adsorption portion 8b in the storage recess 5a of the storage container 5, the cell mass 2 is adsorbed and held. It is designed not to be sucked into the inside of 8b.
Further, on the mounting surface 13a of the culture tray 13, the center position of the suction portion 8b is positioned substantially at the center of the accommodating portion Ga formed by the pin 14 and the pin 14, and in this state, the negative pressure generated by the negative pressure generating means is used. By eliminating the above, the cell mass 2 can be arranged in the accommodating portion Ga.
As the holding means, in addition to the suction nozzle 8 that sucks and holds the cell mass 2, the cell mass 2 may be held by a gripper or the like.

Further, the nozzle moving means 9 as a moving means for moving the suction nozzle 8 is provided along the X-direction rail 21 provided in the X direction over the storage container support portion 6 and the culture container support portion 7 and the X-direction rail 21. The X-direction moving means 22 that moves the suction nozzle 8 in the X direction, the Z-direction moving means 23 that is provided in the X-direction moving means 22 and raises and lowers the suction nozzle 8 in the Z direction, and the storage container support portion 6 and It is composed of the Y-direction tables 6a and 7a in the culture container support portion 7. Then, the holding means such as the suction nozzle 8 and the moving means thereof constitute a supply means for the cell mass 2.
Further, seven suction nozzles 8 of this embodiment are provided so as to be aligned in the X direction, and the intervals of the suction nozzles 8 are changed in the X direction by the interval changing means 24.
Specifically, when the suction nozzle 8 is located above the storage container support portion 6, the interval changing means 24 uses the suction nozzle 8 at the same interval as the storage recesses 5a aligned in the X direction in the storage container 5. Change the interval of.
On the other hand, when the suction nozzle 8 is located above the culture vessel support portion 7, the spacing changing means 24 has the same spacing as the spacing in the X direction in the housing portion Ga formed by the pins 14 protruding on the mounting surface 13a. The interval of the suction nozzle 8 is changed to.
It is possible to provide a plurality of rows of suction nozzles 8 in the Y direction, and when the distance between the storage recesses 5a of the storage container support portion 6 and the distance between the storage portions Ga formed on the culture tray 3 are the same. The interval changing means 24 can be omitted.
Further, for example, when the distance between the accommodating portions Ga is narrow, it is possible to accommodate the cell mass 2 in every other accommodating portion Ga by the suction nozzle 8, and in that case, an odd number on the mounting surface 13a is first obtained. The cell mass 2 may be placed on the third accommodating portion Ga, and then the cell mass 2 may be placed on the even-numbered accommodating portion Ga.
It is sufficient that at least one suction nozzle 8 is provided, and as a means for supplying the cell mass 2, in addition to the configuration in which the cell mass 2 is transferred as in the present embodiment, a large number of cell masses 2 may be provided. It may be configured to be fed out one by one from the contained container.

Further, the X-direction rail 21 is provided with a camera 25 that moves in the X direction, and when the cell mass 2 is arranged on the culture tray 13 by the suction nozzle 8, the camera 25 of the culture container support portion 7. The mounting surface 13a is photographed by moving to the upper side.
From the captured image, it is confirmed whether or not the cell mass 2 is arranged in all the accommodating portions Ga on the above-mentioned mounting surface 13a, and if necessary, with respect to the accommodating portion Ga in which the cell mass 2 is not accommodated. It can be instructed to contain the cell mass 2.
Further, when the nozzle moving means 9 positions the suction nozzle 8 above the culture container support portion 7, the camera 25 retracts from above the culture container support portion 7 in order to avoid contact with the suction nozzle 8. It has become.

The inspection means 10 shown in FIG. 6 includes a lighting means 26 provided below and a camera 27 provided above, and the culture vessel 3 is provided with the lighting means 26 and the camera 27 by a robot or other means (not shown). It is designed to be held between.
As described above, since the culture tray 13, the bottom of the culture container 3, and the holding lid 19 are each made of a light-transmitting material, the light emitted by the lighting means 26 is the culture tray 3 and the culture container. It penetrates the bottom of 3 and passes through the cell mass 2 or the cell mass sheet 4 on the culture tray 13 so that the light is received by the camera 27.
The camera 27 can confirm these culture states from the photographed cell mass 2 or cell mass sheet 4, and at that time, can determine whether to continue or stop the culture according to the quality of the culture state.
In this embodiment, the lighting means 26 and the camera 27 are provided inside the isolator 61, but a part of the isolator 61 is projected to the outside, and the upper and lower portions of the protruding portion are light-transmitting. As a member, the culture vessel 3 may be held between these members.

FIG. 7 shows a culture solution supply means 11 provided inside the incubator 62 for supplying the culture solution to the cell mass sheet 4, and a cell mass sheet movement for moving the cell mass sheet 4 being cultured on the culture tray 13. Means 12 and.
When a plurality of culture containers 3 are housed inside the incubator 62, the culture solution supply means 11 and the cell mass sheet moving means 12 can be provided for each of the culture containers 3.
The culture vessel 3 is provided with a connection port 3a below the connection port 3a. Specifically, the connection port 3a is located below the culture tray 13 with the culture tray 13 in the ascending position. It is provided as follows.
The culture solution supply means 11 has a pipe 31 having one end connected to the connection port 3a and the other end inserted into the liquid surface of the culture solution from above the culture container 3, and the culture solution provided in the pipe 31. It is composed of a liquid feeding pump 32 for feeding liquid.
The liquid feed pump 32 sucks the culture solution from the upper side of the culture container 3 and allows the culture solution to flow in from below the culture tray container 3 through the connection port 3a, whereby in the culture container 3. The culture solution is circulated.

On the other hand, the culture tray 13 is carried into the incubator 62 in a state of being positioned in the ascending position in the culture container 3, and therefore the pin 14 is located below the culture tray 13.
The culture tray 13 is provided with through holes 13b that penetrate vertically in accordance with the positions of the pins 14, and specifically, the cell mass 2 that is aligned on the mounting surface 13a of the culture tray 13. It is bored at a position between the cell mass 2 and the cell mass 2, and communicates the mounting surface 13a side and the lower surface side of the culture tray 13.
With such a configuration, the culture solution supplied by the culture solution supply means 11 passes through the through hole 13b and passes from the lower surface side of the culture tray 13 to the mounting surface 13a side, that is, the mounting surface 13a and the cell mass sheet. It is supplied between the back surface side of No. 4 and the through hole 13b can be used as a supply hole for the culture solution.
As a result, the culture solution can be supplied and brought into contact with the back surface side of the cell mass sheet 4 in contact with the culture tray 13, and the cells tend to be insufficiently supplied by contacting the culture tray 13. The back surface side of the mass sheet 4 can be cultivated satisfactorily.
When the liquid feeding direction of the liquid feeding pump 32 is reversed, the culture solution is sucked from the back surface side of the cell mass sheet 4 through the through hole 13b, so that the culture solution is cultured from the periphery of the cell mass sheet 4 to the back surface side. The liquid flows in and the culture liquid is supplied. In this case, the through hole 13b functions as a discharge hole.

As the cell mass sheet moving means 12, a through hole 13b formed in the culture tray 13 and a pin 14 provided in the culture container 3 and penetrating the through hole 13b can be used.
Further, in the incubator, a holding member 33 for holding the reinforcing member 16 of the support column 15 provided on the culture tray 13 and an elevating means 34 for raising and lowering the holding member 33 are provided, and the holding member is provided by the elevating means 34. As the 33 moves up and down, the culture tray 13 moves up and down in the culture container 3.
As shown in FIG. 7B, when the elevating means 34 lowers the culture tray 13 and the pin 14 penetrates from below the culture tray 13, the pin 14 slightly protrudes from the culture tray 13 and the cell mass sheet 4 Is designed to be pushed up from below.
Here, since the mounting surface 13a of the culture tray 3 is formed in a non-adhesive manner, the cell mass sheet 4 can be easily peeled off from the mounting surface 13a by the pin 14.
The cell mass sheet moving means 12 operates at predetermined intervals, and by acting in this way, the cell mass sheet 4 can be easily recovered from the culture vessel 3 when the culture is completed, and the culture can be completed. Inside, it is expected to stimulate the cell mass 2 and activate the cell mass 2.
The pin 14 does not have to be completely removed from the through hole 13b.

Hereinafter, a method for producing the cell mass sheet 4 using the cell mass sheet manufacturing apparatus 1 having the above configuration will be described.
First, the storage container 5 containing the cell mass 2 and the culture container 3 containing the culture tray 13 are carried into the isolator 61 via the pass box 63, or a predetermined amount is placed in the culture container 3. Perform a preparatory step to supply the culture solution of.
At that time, if a plurality of the storage containers 5 and the culture containers 3 are carried into the isolator 61, a plurality of cell mass sheets 4 can be continuously produced by using the plurality of culture containers 3.

Next, a guide installation step of installing the pin 14 as the guide means G on the mounting surface 13a of the culture tray 13 is performed. Specifically, in the culture container support portion 7, the guide moving means 18 positions the culture tray 13 in a descending position, whereby the pin 14 protrudes from the upper surface of the culture tray 13, and the storage portion Ga is formed. The guide means G will be installed on the mounting surface 13a.
The guide installation step can be performed at the same time as the preparatory step by carrying the culture container 3 in which the culture tray 13 is positioned in the descending position in advance into the isolator.

After the guide means G is installed on the culture tray 13 in this way, the suction nozzle 8 is subsequently moved to perform a storage step of storing the cell mass 2 in the storage portion Ga of the guide means G, respectively.
The nozzle moving means 9 moves the suction nozzle 8, and the interval changing means 24 sucks and holds the cell mass 2 from the accommodating recess 5a of the accommodating container 5 while changing the interval of the suction nozzle 8, and the aligned cell mass. 2 is stored in the storage portion Ga of the guide means G installed in the culture tray 13 of the culture container 3.
Since the storage portion Ga is composed of four pins 14 surrounding the cell mass 2, the cell mass 2 housed in the storage portion Ga is prevented from popping out, and the cell mass 2 is aligned. It is supposed to be maintained.
When the cell mass 2 is accommodated in all the accommodating portions Ga in this way, the camera 25 moves above the culture vessel support portion 7 to photograph the cell mass 2 on the mounting surface 13a, and the cell mass 2 is photographed. Is confirmed whether or not is contained in all the accommodating parts Ga.

Subsequently, a levitation prevention step of arranging the holding lid 19 on the upper part of the cell mass 2 arranged on the culture tray 13 is performed.
Specifically, an operator wearing a holding lid supply means (not shown) or a glove 61a puts the holding lid 19 into the inside of the culture container 3, and the holding lid 19 lifts the cell mass 2 on the mounting surface 13a. To prevent.

Next, a guide evacuation step is performed in which the pin 14 as the guide means G is retracted from the mounting surface 13a of the culture tray 13 from the state in which the cell mass 2 is accommodated in the accommodating portion Ga of the guide means G.
Specifically, the culture tray 13 is positioned at an elevated position with respect to the culture vessel 3 by the guide moving means 18 of the culture vessel support portion 7, whereby the pin 14 protruding from the upper surface of the above-mentioned mounting surface 13a is mounted. It is in a state of being retracted from the placing surface 13a.
Further, since the adjacent cell clusters 2 are brought into contact with each other when they are accommodated in the storage portion Ga, the vertically and horizontally aligned cell clusters 2 remain in contact with each other on the mounting surface 13a. It will remain.

When the cell mass 2 was arranged on the culture tray 13 in this way, the culture tray 13 was transferred to the incubator 62 together with the culture container 3 by the manual work of the robot or the operator, and was arranged on the culture tray 13. A culturing step of culturing the cell mass 2 is performed.
First, when the culture tray 13 is transferred to the incubator 62, the incubator 62 is separated from the isolator 61 and placed at a position separated from the isolator 61.
Then, the inside of the incubator 62 is maintained at a predetermined temperature and humidity, and a predetermined concentration of carbon dioxide gas and oxygen is maintained, and the cell mass 2 on the culture tray 13 is cultured, so that the adjacent cell masses 2 are fused with each other. Finally, one cell mass sheet 4 is formed.
At this time, since the cell mass 2 is aligned on the mounting surface 13a of the culture tray 13 and maintains a contact state, the formed cell mass sheet 4 has the shape of the mounting surface 13a of the culture tray 13. It will have substantially the same shape as, and its thickness can be made substantially uniform.

During the above culture step, in the incubator 62, the culture solution supply step of supplying the culture solution to the cell mass 2 or the cell mass sheet 4 on the culture tray 13 and the cell mass sheet 4 on the culture tray 13 are moved at predetermined intervals. A cell mass sheet transfer step is performed.
As the culture solution supply step, the culture solution supply means 11 operates at predetermined intervals to generate a flow of the culture solution from the bottom to the top in the culture vessel 3, whereby the cell mass 2 or the cells A new culture solution is supplied to the mass sheet 4.
Further, since the culture tray 13 is located in the ascending position in the culture vessel 3 and the through hole 13b as the supply hole is bored, the cell mass sheet 4 and the mounting surface 13a are formed through the through hole 13b. The culture solution can be supplied between the cells, and the back surface side of the cell mass sheet 4 can be efficiently cultured.
That is, when the cell mass 2 is aligned to prepare the cell mass sheet 4 as in this example, there is a problem that the culture solution is not sufficiently distributed on the back surface side of the cell mass 2 located at the center. By performing this culture solution supply step, it became possible to satisfactorily cultivate the cells located in the relevant portion.

Then, when the adjacent cell masses 2 are fused with each other to form the cell mass sheet 4, the culture tray 3 located at the ascending position is lowered by the elevating means 34 as the cell mass sheet moving step.
Then, the pin 14 protrudes from the upper surface of the culture tray 3, and the cell mass sheet 4 is pushed up from below by the pin 14 to peel the cell mass sheet 4 from the culture tray 3. At that time, since the surface of the culture tray 3 is formed non-adhesively, the cell mass sheet 4 can be peeled off and moved upward with a small pressing force.
By performing this cell mass sheet moving step at predetermined intervals, it is possible to maintain a state in which the cell mass sheet 4 does not adhere to the culture tray 3 or a state in which the cell mass sheet 4 can be peeled off from the culture tray 3 with a smaller force, and it is appropriate. Can give a stimulus.

When the cell mass sheet 4 is formed to some extent by the above culture step, the incubator 62 is connected to the isolator 61 at regular intervals to perform an inspection step of confirming the culture state of the cell mass sheet 4.
In the inspection step, the culture vessel 3 is transferred from the connected incubator 62 to the isolator 61, and further transferred to the inspection means 10 shown in FIG.
In the inspection means 10, the light irradiated by the lighting means 26 from below passes through the culture container 3, the culture tray 13, the cell mass sheet 4, and the holding lid 19, and the cell mass sheet 4 is cultured based on the image taken by the camera 27. The quality of the product is inspected.
When it is determined in the inspection step that the cell mass sheet 4 is sufficiently cultured, a recovery step of collecting the cell mass sheet 4 from the culture vessel 3 is subsequently performed.
By performing the cell mass sheet action step during the culture step, the cell mass sheet 4 can be peeled off from the mounting surface 13a and recovered without using a chemical such as trypsin in the recovery step, and the cells can be recovered. Damage to can be suppressed as much as possible.

Although the guide evacuation step in the above embodiment is performed before the culture tray 3 is transferred to the incubator 62, the guide evacuation step may be performed after the incubator 62 is transferred.
Specifically, the culture step is performed with the pin 14 as the guide means G protruding into the mounting surface 13a of the culture tray 13, and the adjacent cell masses 2 are fused to form the cell mass sheet 4. After that, the pin 14 can be pulled out from the formed cell mass sheet 4. Further, after the cell mass 2 has been cultured to some extent, a guide evacuation step may be performed to continue the culture.

Further, with respect to the culture solution supply step in the above embodiment, the culture tray 13 is moved up and down during the culture step, and the pin 14 is inserted into and removed from the through hole 13b. The culture solution may be pushed or pulled out into the cell mass sheet 4 to be distributed, and the culture solution may be supplied to the back surface side of the cell mass sheet 4.

Then, in the cell mass sheet moving step in the above embodiment, the culture solution is supplied at a predetermined pressure by the culture solution supply means 11, and the culture solution is ejected upward from the through hole 13b of the culture tray 13. , The cell mass sheet 4 may be pushed up from below.
In that case, the holding member 33 and the elevating means 34 as the cell mass sheet moving means 12 can be omitted, and the culture solution supplying means 11 can be used as the cell mass sheet moving means 12.

FIG. 8 illustrates variations of the culture tray 13 and the pin 14 as the guide means G that can be used in the cell mass sheet manufacturing apparatus 1 of the first embodiment.
The cell mass 2 is placed in a staggered pattern on the culture tray 13 shown in FIG. 8. Specifically, the cell mass 2 is linearly arranged in the X direction, and the cell mass 2 is arranged in the Y direction. It is arranged so that it is aligned by half.
In order to align the cell mass 2 in this way, the through hole 13b drilled in the culture tray 13 and the pin 14 as the guide means G accommodate six pins 14 located around each cell mass 2. It is arranged so as to form a portion Ga.
Further, also in this culture tray 13, the cell mass 2 comes into contact between the adjacent pin 14 and the pin 14, and as in the above embodiment, even if the pin 14 is removed, the cell mass 2 remains in contact with each other. The state of contact can be maintained.
The cells are not limited to the arrangement of the through holes 13b and the pins 14 in the culture tray 13 shown in FIGS. 4 and 8, for example, the cell mass 2 is arranged concentrically in the circular culture tray 13. The mass 2 can be aligned.

FIG. 9 also illustrates variations of the culture tray 13 and the pin 14 as the guide means G that can be used in the cell mass sheet manufacturing apparatus 1 of the first embodiment.
In the culture tray 13 of this example, as shown in FIG. 9A, four pins 14 are positioned around each cell mass 2 to vertically and horizontally the cell mass 2 as in the first embodiment. It is designed to be aligned with.
The pin 14 has a larger diameter than the pin 14 shown in FIG. 4, and the through hole 13b is formed to be larger than the diameter of the pin 14, and a gap is formed between the pin 14 and the opening of the through hole 13b. Is to be formed. Further, at each of the four corners of the through hole 13b, four arc-shaped relief portions are formed according to the shape of the cell mass 2.
By increasing the through hole 13b so that a gap is formed between the pin 14 and the opening of the through hole 3a in this way, more culture is performed from the through hole 3a as the supply hole in the culture solution supply step. The liquid can be supplied to the back surface side of the cell mass 2 or the cell mass sheet 4.
On the other hand, as shown in FIG. 9B, a tapered portion 14a is formed at the tip of the pin 14, and the tapered portion 14a hinders contact between the cell clusters 2 between the pin 14a and the pin 14a. It is not possible.

FIG. 10 also describes variations of the culture tray 13 that can be used in the cell mass sheet manufacturing apparatus 1 of the first embodiment, and here, the culture tray 13 used in FIG. 8 will be used for description.
The culture tray 13 of this example is provided with a supply hole 13c at a position corresponding to the storage portion Ga, that is, a position where the cell mass 2 is placed, in addition to the through hole 13b through which the pin 14 penetrates. It has become.
That is, in the culture solution supply step, the culture solution can be supplied to the back surface side of the cell mass sheet 4 from the through hole 3a and the supply hole 3b, and a large amount of the culture solution can be circulated. ing.
In any of the above examples, the pin 14 is not arranged on the outer peripheral side of the aligned cell mass 2, but the pin 14 is also arranged on the outer peripheral side so that the cell mass 2 does not touch the inner wall of the culture vessel 3. It can also be configured as.
In this case, the pins 14 on the outer peripheral side are configured to remain on the mounting surface 13a even if the other pins 14 are removed from the mounting surface 13a in the guide means evacuation step.
More specifically, when the pin 14 on the outer peripheral side is made longer than the other pins 14 and the culture tray 13 is positioned in the raised position with respect to the culture vessel 3 by the guide moving means 18, the pin 14 on the outer peripheral side is located on the outer peripheral side. The pin 14 is maintained in a state of protruding from the mounting surface 13a. When it is necessary to retract the pins 14 on the outer peripheral side as well, the ascending position may be set in two stages so that the culture tray 13 can be further ascended from the ascending position.

FIG. 11 illustrates variations of the culture tray 13 and the culture container 3 that can be used in the cell mass sheet manufacturing apparatus 1 of the first embodiment.
The culture tray 13 of this example has a bottomed box shape, and has a mounting surface 13a on which the cell mass 2 is placed and a side surface 13c provided adjacent thereto. Unlike the culture tray 13 and the culture vessel 3 in 3, the cell mass 2 aligned by the side surface 13c of the culture tray 13 is supported.
The bottom surface of the culture vessel 3 is formed to be one size larger than the culture tray 13, so that a gap is formed between the side surface of the culture tray 13 and the inner wall of the culture vessel 3.
The gap is provided with a slide member 41 provided on the side surface of the culture tray 13 and a guide rail 42 provided on the culture container 3 in the vertical direction, whereby the culture tray 13 is attached to the guide rail 42. The inside of the culture vessel 3 is moved up and down along the line.
The culture tray 13 is provided with a locking member 44 that is swingably provided via a hinge 43, and the hinge is in a state where the culture tray 13 is positioned in an ascending position as shown in FIG. 11 (b). The locking member 44 is tilted outward by the 43 and is locked to the upper end of the culture vessel 3.
As the guide means G, as in the case of the culture tray 13 and the culture container 3 shown in FIG. 3, the pin 14 provided on the bottom surface of the culture container 3 is passed through the through hole 13b provided in the culture tray 13. 14 has a structure in which 14 is projected onto the mounting surface 13a of the culture tray 13.
Then, as the guide moving means 18 for moving the culture tray 13 inside the culture container 3 and projecting the pin 14, a robot or the like can be used instead of the holding means 18a and the elevating means 18b in FIG. ..

FIG. 12 illustrates variations of the culture solution supply means 11 that can be used in the cell mass sheet manufacturing apparatus 1 of the first embodiment.
The culture solution supply means 11 shown in FIG. 7 has a configuration in which the culture solution in the culture vessel 3 is circulated by using the pipe 31 and the liquid feed pump 32 to supply the culture solution. The culture solution supply means 11 has a configuration capable of supplying a fresh culture solution into the culture vessel 3.
Specifically, the culture solution supply means 11 is a supply pipe connected between a culture solution tank 51 for storing fresh culture solution and a connection port 3a between the culture solution tank 51 and the culture container 3. 52, a liquid feed pump 53 provided in the supply pipe 52, a discharge pipe 54 having one end inserted into the culture solution of the culture container 3 and the other end connected to a drainage tank (not shown), and the drainage pipe. It is composed of a drainage pump 55 provided in 54.
According to the above configuration, the used culture solution in the culture container 3 is discharged by the drainage pump 55 while the fresh culture solution in the culture solution tank 51 is supplied to the culture container 3 by the liquid feed pump 53. Therefore, the fresh culture solution can be circulated in the culture container 3.
In this case, as shown in the drawing, the culture solution may be supplied below the culture tray 13 and discharged from above, but conversely, it may be supplied upward and discharged from below.

Next, FIGS. 13 to 16 explain the method for producing the cell mass sheet manufacturing apparatus 1 and the cell mass sheet 4 according to the second embodiment. The description of the configuration common to the configuration of the first embodiment will be omitted, and the common configuration will be described with the same reference numerals.
As shown in FIGS. 13 and 14, the culture vessel 103 of this example has a bottomed box shape and contains a culture solution in advance, and the cell mass 2 is on the mounting surface 103a which is the bottom surface of the culture vessel 103. It is designed to be placed in.
The guide means G of the present embodiment shown in FIG. 13A is placed on the mounting surface 103a of the culture vessel 103, and is held and retracted from the mounting surface 103a. There is.
Further, the guide means G is composed of a partition member 104 in which a plurality of accommodating portions Ga are partitioned, and the cell mass 2 is arranged vertically and horizontally in the X direction and the Y direction, respectively.
Here, the size of each storage portion Ga is set to be slightly larger than the outer diameter dimension of the cell mass 2.
After accommodating the cell mass 2 in the accommodating portion Ga, the guide means G can be retracted from the culture tray 103 so that the cell mass 2 is arranged in an aligned state on the mounting surface 103a of the culture container 103. it can.

The culture vessel 103 of this embodiment is provided with a pressing means 105 that presses the cell masses 2 arranged on the above-mentioned placing surface 103a from the lateral direction to bring the adjacent cell masses 2 into contact with each other.
The pressing means 105 approaches each other from the first facing direction (Y direction) facing the cell mass 2 arranged on the culture vessel 103, and presses the cell mass 2 from the Y direction. The pressing member 111, the second pressing member 112 that approaches each other from the second facing direction (X direction) orthogonal to the Y direction and presses the cell mass 2 in the X direction, and the culture vessel support portion 7 are provided. It is composed of a moving means 113 that moves the first and second pressing members 111 and 112 forward and backward.
Connecting rods 111a and 112a penetrating from the outside to the inside are slidably provided on the four side surfaces 103b of the culture container 103, respectively, and are provided at the inner end of the culture container 103 in the connecting rods 111a and 112a. The first and second pressing members 111 and 112 are provided, respectively.
The connecting rods 111a and 112a are provided with two stopper members 111b and 112b with the side surface 103b of the culture vessel 103 interposed therebetween, and the stroke amount of the connecting rods 111a and 112a is determined at intervals of the stopper members 111b and 112b. Has been done.
With such a configuration, the first and second pressing members 111 and 112 can reciprocate to the retracted position (FIG. 14 (a)) or the forward position (FIG. 14 (c) (d)), respectively. As shown in FIG. 15, the upper part of the first and second pressing members 111 and 112 is covered with the pressing lid 19 to prevent the cell mass 2 from floating during the pressing operation.

The distance between the first pressing member 111 and the second pressing member 112 located at the retracted position are set to be substantially the same as the lengths of the partition members 104 constituting the guide means G in the X direction and the Y direction, respectively. ..
When the first pressing member 111 is moved to the forward position as shown in FIG. 14 (c), the cell mass 2 is pressed in the Y direction by the first pressing member 111, and thus the gap in the Y direction in the aligned cell mass 2 Is resolved.
Similarly, when the second pressing member 112 is moved to the forward position as shown in FIG. 14D, the cell mass 2 is pressed in the X direction by the second pressing member 112, and the cell mass 2 aligned thereby. The gap in the X direction is eliminated.
Here, when moving the second pressing member 112 to the forward position, the first pressing member 111 is moved to the backward position in advance in order to avoid contact with the first pressing member 111.
Then, by pressing the cell mass 2 from the Y direction and the X direction by the first and second pressing members 111 and 112, respectively, the cell mass 2 on the culture vessel 103 is in contact with each other. It will be aligned in the state.

When the adjacent cell clusters 2 are aligned in a state of being in contact with each other by the pressing means 105 in this way, the holder 114 as shown in FIG. 16 is attached to the outer edge of the aligned cell mass 2. There is.
The holder 114 is a frame-shaped member set to be substantially the same as the width in the X direction and the Y direction of the cell mass 2 in contact with each other, and supports the cell mass 2 from the outside so as not to break the aligned state. It has become a thing.
Further, the holder 114 is provided with a mesh on the upper surface, so that the floating and movement of the cell mass 2 inside the holder 114 are restricted, and the flow of the culture solution is allowed so as not to hinder the culture of the cell mass 2 inside. It has become.

FIG. 17 shows the cell mass sheet moving means 121 provided inside the incubator 62, the first and second pressing members 111 and 112 provided on the culture tray 103, and the first and second pressing members 111 and 112 provided inside the incubator. It is composed of a moving means 115 that reciprocates the first and second pressing members 111 and 112.
The first and second pressing members 111 and 112 are reciprocated by the moving means 115 to press the cell mass 2 to which the holder 114 is attached from the lateral direction together with the holder 114.
Since the placement surface 103a of the culture vessel 103 is also non-adhesive to cells, the cell mass sheet 4 can be moved by pressing from the lateral direction.
Further, the culture container 103 may be provided with a connection port 103c so that the culture solution in the culture container 103 can be circulated in the same manner as in the culture solution supply means 11 described in the first embodiment.

A method for producing the cell mass sheet 4 using the cell mass sheet producing apparatus 1 of the second embodiment having the above configuration will be described.
First, a carry-in step of carrying the storage container 5 and the culture container 103 into the isolator and a preparatory step of storing the culture solution in the culture container 103 are performed. When the culture container 103 is placed on the culture container support portion 7, the moving means 113 of the culture container support portion 7 is connected to the connecting rods 111a and 112a of the first and second pressing members 111 and 112.
Next, as shown in FIG. 14A, a guide installation step of installing the guide means G in the culture container 103 is performed, and the accommodating portion Ga of the guide means G installed in the culture container 103 by the suction nozzle 8 is performed. A containment step of accommodating the cell mass 2 is performed.
After accommodating the cell mass 2 in the accommodating portion Ga of the guide means G by the accommodating step, a guide evacuation step of retracting the guide means G from the culture vessel 103 is performed as shown in FIG. 14 (b), and further on the culture vessel 3. A levitation prevention step is performed in which the holding lid 19 is placed on the upper part of the cell mass 2 arranged in the above.
The guide installation step, guide removal step, and levitation prevention step can be performed by an operator or a robot.

Following the guide evacuation step and the levitation prevention step, the culture vessel support portion 7 performs a pressing step of pressing the cell mass 2 on the culture vessel 103 from the lateral direction to bring them into contact with each other.
That is, in the state where the guide means G is evacuated in the guide evacuating step, although the cell clusters 2 are aligned, a gap may be formed between them, and even if the culture is performed in this state, the cell mass 2 may be formed. Fusion between cell clusters 2 may not occur.
Therefore, as shown in FIG. 14 (c), the cell mass 2 is first moved in the Y direction by the first pressing member 111, and then the cell mass 2 is moved by the second pressing member 112 as shown in FIG. 14 (d). The cells are moved in the X direction so that the cell masses 2 on the culture tray 103 are aligned in contact with each other.

When the cell clumps 2 come into contact with each other by the pressing step, the holding lid 19 provided on the upper part of the aligned cell clumps 2 is removed, and the second pressing member 112 is positioned at the retracted position, and then the aligned cell clumps are positioned. A holder mounting step of mounting the holder 114 on the outer periphery of 2 is performed.
By attaching the holder 114, the movement of the cell mass 2 inside the holder 114 is prevented, so that the cell mass 2 can maintain the aligned state in contact with each other.

Subsequently, the culture vessel 103 is transferred to the incubator 62, and the culture step is performed in the incubator 62.
When the culturing step is performed for a predetermined period of time and the cell masses 2 are fused inside the holder 114 to form the cell mass sheet 4, the cell mass sheet moving step of moving the cell mass sheet 4 is performed.
The cell mass sheet 4 to which the holder 114 is attached is located in the culture vessel 103, and the moving means 115 provided in the incubator 62 moves the first and second pressing members 111 and 112 to move the holder 114. The whole cell sheet 4 is pressed laterally.
By moving the cell mass sheet 4 in the cell mass sheet moving step, the culture solution enters between the cell mass sheet 4 and the culture vessel 103, so that the culture solution is used for the cells located on the back surface side of the cell mass sheet 4. It is possible to supply.
In this embodiment, the cell mass sheet 4 in the holder 114 can be moved in the X direction and the Y direction by the first and second pressing members 111 and 112, but the first pressing member 111 or the second pressing member 112. The moving means 115 may be connected to only one of them so that the moving means 115 can be moved in either the X direction or the Y direction.

FIG. 18 illustrates the culture vessel 103 that can be used in the cell mass sheet manufacturing apparatus 1 in the second embodiment, and illustrates variations of the culture vessel 103 in the second embodiment.
Both ends of the first pressing member 111 can be expanded and contracted by an urging force such as a spring, and in a state where the first pressing member 111 is extended, the guide means G is retracted by the guide retracting step. It has the width of cell clusters 2 in which gaps are formed with each other in the state.
Therefore, by moving the first pressing member 111 to the forward position, the cell mass 2 can be pressed in the Y direction, and the gap in the aligned cell mass 2 in the Y direction can be eliminated.
Since the first pressing member 111 can be expanded and contracted, the second pressing member 112 can be positioned in an approaching state while the first pressing member 111 is positioned in the forward position.
Then, while the second pressing member 112 presses the cell mass 2, it also presses the first pressing member 111, which makes it possible to shrink the first pressing member 111.
As a result, the cell mass 2 can be aligned with the first and second pressing members 111 and 112 both positioned in the forward position, and when the second pressing member 112 is positioned in the forward position, the cell mass 2 can be aligned. It is possible to prevent the cell from moving in the Y direction.

The contents of the first and second embodiments can be combined as appropriate.
For example, the culture vessel 103 in the second example was housed in the culture vessel 3 instead of the culture tray 3 in the first embodiment, and a through hole was formed in the bottom surface of the culture vessel 103 and provided on the bottom surface of the culture vessel 3. If the pin 14 is penetrated, the pin 14 can be used as the guide means G.
In this case, since the culture vessel 103 is provided with the pressing means 105, it is not necessary to bring the cell masses 2 housed in the housing portion Ga of the guide means G into contact with each other, so that the pins 14 and the pins 14 can be easily brought into contact with each other. The cell mass 2 can be accommodated in the accommodating portion Ga formed by the above, and then the cell mass 2 can be aligned in contact with each other by operating the pressing means pressing means 105 and performing the pressing step.
Further, with such a configuration, the culture solution is supplied to the back surface side of the cell mass sheet 4 by using the through hole provided in the culture vessel 103 as a supply hole, so that the cells located on the back surface side can be satisfactorily cultured. And the cell mass sheet can be pushed up from the culture vessel 103 by the above pins.
That is, in the cell mass sheet moving step, it is not necessary to move the cell mass sheet in the lateral direction as in the second embodiment, so that the first and second pressing members 111 and 112 are positioned at the forward positions in the pressing step. It can be accommodated in the incubator as it is, and in that case, the attachment of the holder 114 can be omitted.

FIG. 20 describes the culture vessel 3 used in the method for producing a cell mass sheet according to the third embodiment, and can be used, for example, in the cell mass sheet manufacturing apparatus 1 used in the first example.
The culture vessel 3 of this example has a bottomed box shape, and the bottom surface thereof constitutes the mounting surface 13a of the cell mass 2, and the mounting surface 13a has cell non-adhesiveness as in the above example. ..
The guide means G of the present embodiment is composed of a support frame 201 provided in a grid pattern and a pin 202 provided so as to hang down from an intersection portion in the grid, and the intersection portion of the grid and the position of the pin 202. Is provided in the same arrangement as the pins 14 shown in FIG. 4 in the first embodiment, and the four pins 202 form a storage portion Ga for accommodating the cell mass 2.
By arranging the pins 202 in this way, when the cell mass 2 is accommodated in the accommodating portion Ga, the cell mass 2 can be aligned in a state where the adjacent cell masses 2 are in contact with each other.
Here, the size of each lattice constituting the support frame 201 is set to be slightly smaller than the diameter of the cell mass 2 because the pins 202 are provided in the above arrangement, but the suction nozzle 8 It is possible to push the cell mass 2 from above.
Then, as in the above embodiment, when the cell mass 2 is accommodated in the accommodating portion Ga of the guide means G, the guide means G may be immediately taken out as a guide evacuation step, and the culture of the cell mass 2 has progressed to some extent. You may evacuate from.
Further, the cell container 3 of this embodiment is provided with a lid 203 on the upper portion thereof, and similarly to the above embodiment, a pipe 31 of a culture solution supply means 11 (not shown) is provided on the lid 203. , The culture solution is supplied.

Further, in each of the above examples, the cell mass 2 is arranged in a plane on the culture tray to culture the cell mass sheet 4 in one layer, but the cell mass 2 is placed on the culture tray 3 in the storage step. If a plurality of cell mass sheets 4 are laminated, a thicker cell mass sheet 4 can be cultured.
Further, in each of the above-described embodiments, one cell mass 2 is accommodated in each of the accommodating portions Ga of the guide means G, but a plurality, for example, about 2 to 4 cells are accommodated in one accommodating portion Ga. A preset number of cell masses 2 may be accommodated, and each accommodating portion Ga may individually accommodate a predetermined number of cell masses 2.

1 Cell mass sheet manufacturing device 2 Cell mass 3 Culture container 4 Cell mass sheet 5 Storage container 8 Suction nozzle 9 Nozzle moving means 10 Inspection means 11 Culture solution supply means 12 Cell mass sheet moving means 13 Culture tray 13a Mounting surface 13b Through hole 13c Supply hole 14 pin 19 Pressing lid G Guide means Ga Accommodating part

Claims (9)

A cell mass sheet manufacturing apparatus for arranging a plurality of cell masses in a plane on a placement surface in a culture vessel to produce a cell mass sheet in which the cell masses are cultured and fused with each other.
A guide means having a plurality of accommodating portions having a size capable of individually accommodating the cell mass, a supply means for supplying the cell mass into the accommodating portion of the guide means installed on the above-mentioned mounting surface, and the above-described placement. It is equipped with a camera that captures a surface and a floating prevention means that prevents the cell masses aligned on the above-mentioned mounting surface from floating.
The cell mass is accommodated in the accommodating portion of the guide means by the supply means, the cell mass is aligned on the mounting surface, and it is confirmed by the camera whether or not the cell mass is accommodating in the accommodating portion, and then the ascending A cell mass sheet manufacturing apparatus characterized in that the prevention means prevents the cell mass from floating and the guide means is retracted from the mounting surface. The supply means includes a holding means for holding the cell mass and a moving means for moving the holding means.
The first aspect of claim 1, wherein when the moving means moves the holding means and takes out the cell mass from the storage container for accommodating the cell mass, the cell mass is accommodated in the accommodating portion of the guide means on the above-mentioned mounting surface. Cell mass sheet manufacturing equipment. The cell according to claim 1 or 2, wherein the guide means is installed on the mounting surface and the guide moving means for retracting the guide means is provided on the mounting surface. Ingot sheet manufacturing equipment. Any of claims 1 to 3, wherein the guide means is provided with a pressing means for laterally pressing the cell clusters arranged on the mounting surface after the guide means is retracted from the mounting surface. The cell mass sheet manufacturing apparatus described in Crab. The guide means is composed of a plurality of pins provided so as to be located around each cell mass aligned on the above-mentioned mounting surface, and the accommodating portion is formed between the plurality of pins. The cell mass sheet manufacturing apparatus according to any one of claims 1 to 4. A culture tray on which the cell mass is placed is provided in the culture container, the above-mentioned placing surface is formed on the culture tray, and a plurality of through holes corresponding to the arrangement of the plurality of pins are formed in the culture tray. ,
The claim is characterized in that the guide means is installed on the mounting surface by projecting the plurality of pins from the through hole, and the guide means is retracted from the mounting surface by retracting into the through hole. 5. The cell mass sheet manufacturing apparatus according to 5. The plurality of pins are erected on the bottom surface of the culture vessel.
Guide movement to install a plurality of pins on the above-mentioned mounting surface by lowering the culture tray relative to the culture container, and retracting the plurality of pins from the mounting surface by raising the culture tray relatively. The cell mass sheet manufacturing apparatus according to claim 6, further comprising means. The cell mass sheet manufacturing apparatus according to any one of claims 1 to 7 , wherein the accommodating portion is formed so as to be able to accommodate a plurality of cultured lumps. The cell mass sheet manufacturing apparatus according to any one of claims 1 to 8 , wherein the above-mentioned placing surface has a cell non-adhesive property.

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