TW201831676A - Method and apparatus both for producing cell aggregate structure - Google Patents

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 for producing aggregate structure of cells and manufacturing device  

The present invention relates to a method and apparatus for producing a collection structure of cells, and more particularly to a method of arranging a plurality of cell aggregates in a culture container in which a culture solution is accommodated, and culturing the plurality of cell aggregates. A method and apparatus for producing a collection structure of cells of a collection structure of cells in which a plurality of cell aggregates are fused to each other.

In a regenerative medicine that restores the function of a damaged organism using stem cells or the like, a cell sheet in which cells are cultured at a high density is used. Since the cell sheet is extremely thin, a method for producing a laminated cultured cell sheet in which a plurality of cell sheets are superposed is known (Patent Document 1).

Further, in the method for producing a laminated cultured cell sheet in which the cell sheets are superposed, a method is also known in which a hydrogel particle is placed between the cell sheet to be laminated and the cell sheet to spread oxygen or nutrients. The cells in the layered portion are more reliably produced into a cell sheet (Patent Document 2).

On the other hand, a method is also known in which a cell is cultured into a substantially spherical shape to form a cell agglutination block (spheroid), and the cell aggregates are arranged in contact with each other to perform phytoplankton culture. A sheet cell aggregate having a thickness of 50 to 300 μm which is fused by a cell aggregate is prepared (Patent Document 3).

[Previous Technical Literature]

[Patent Literature]

[Patent Document 1] Japanese Patent No. 4921353

[Patent Document 2] Japanese Patent No. 5629915

[Patent Document 3] Japanese Patent No. 5523830

However, when the cell sheet is laminated as in the case of the patent documents 1 and 2, since the cell sheet is extremely thin, it is difficult to handle and it is difficult to automate it. Further, as in Patent Document 3, it is desirable to float the cell agglutination block and hope When the agglomerate of the sheet-like cells is obtained, the cell agglomerates are irregularly fused with each other due to uneven density between the cells arranged in a neat arrangement, so that the shape of the obtained agglomerate of the sheet-like cells is not fixed, and the thickness is not fixed. Also uneven.

In view of the above problems, the present invention provides a method and apparatus for producing a collective structure of cells capable of obtaining a desired shape and size and capable of increasing functionality, and corresponding to automation.

In the method of producing the aggregate structure of the cell of the invention of claim 1, the plurality of cell aggregates are placed in a culture container in which the culture solution is accommodated, and the plurality of cell aggregates are cultured to produce a plurality of cells agglutinated. a collection structure of cells in which the blocks are fused together, and characterized in that: a supply step of holding a cell aggregate by a holding portion composed of a plurality of pins erected inside the culture container; and a culturing step And cultivating the aggregated cells held in the holding portion to form the aggregate structure; the detaching step of taking out the aggregate structure formed in the culturing step from the holding portion; and the injecting step of injecting the implant into the set The through hole formed by the pin is removed from the aggregate structure taken out in the step.

Further, in the apparatus for producing a collection structure of cells according to the invention of claim 3, a plurality of cell aggregates are placed in a culture container containing the culture solution, and the plurality of cell aggregates are cultured to produce a plurality of cells agglutinated. a collection structure of cells in which the blocks are fused together, and characterized in that: a culture holder provided inside the culture container and provided with a plurality of pins erected; and a supply means for the culture holder a plurality of holding portions formed by the pins hold the cell aggregates; and a separating means for taking out the aggregate structure formed by culturing the cell aggregates held in the culture holder from the holding portion of the culture holder; and injecting And means for injecting the injection into the through hole formed by the pin formed in the extracted aggregate structure.

According to the inventions of claim 1 and claim 3, the plurality of pins and the pins provided in the culture holder are used as the holding portions, and the cell agglomerates are held by the plurality of adjacent holding portions, so that the cells are aggregated. By merging the holding portions in a culture, it is possible to produce a collection structure of cells of a desired shape and size, and to automate them.

Further, in the aggregate structure of the cells thus obtained, the through-holes are formed by the pins constituting the holding portion, so that the injection structure can be injected into the through-holes, whereby the aggregate structure of the cells having increased functionality can be produced. .

1‧‧‧Financial collection device

2‧‧‧cell agglutination

3‧‧‧ culture container

4‧‧‧healing pad (collective structure of cells)

10‧‧‧ suction nozzle

12‧‧‧Injection

16‧‧ ‧ sales

17‧‧‧Support board

G‧‧‧ gap

H‧‧‧Holding Department

Fig. 1 is a front view of an isolator and an incubator of a manufacturing apparatus for setting a collection structure of cells.

Fig. 2 is a configuration diagram of a manufacturing apparatus for a collection structure of cells.

Figure 3 is a cross-sectional view of the storage container and the suction nozzle.

Figure 4 is a cross-sectional view of the culture vessel.

Fig. 5 is a plan view of a cell aggregate disposed in a culture container.

Fig. 6 is a cross-sectional view of the injection nozzle, and is a view illustrating a step of injecting the injection into the through hole of the aggregate structure of the cells.

Fig. 7 is a view for explaining the arrangement of pins constituting the first and second pin groups in the second embodiment.

Fig. 8 is a view showing the state of culture in the second embodiment.

Fig. 9 is a view showing the state of culture in the third embodiment.

Fig. 10 is a plan view showing a cell aggregate in a culture container in the third embodiment.

Fig. 11 is a view showing the state of culture in the fourth embodiment.

Fig. 12 is a view showing the state of culture in the fifth embodiment.

Fig. 13 is a view for explaining the thickness of the pin and the size of the cell aggregate.

Hereinafter, the illustrated embodiment will be described, and FIGS. 1 to 6 are views showing the apparatus 1 for producing a cell assembly according to the first embodiment, which shows a plurality of cell agglutination blocks 2 (spheroid cell blocks). With reference to Fig. 3), the cell agglutination block 2 is placed in the inside of the culture container 3, and a healing pad 4 (see Fig. 4) which is a collection structure of cells formed by merging the cell aggregates 2 is prepared.

The cell aggregate 2 can be produced, for example, by the method disclosed in Japanese Patent No. 4517125. That is, the cells are sown in a container in which the inner surface is non-adhesive, and the cells find a foothold and then follow each other, thereby agglutinating to form a cell aggregate 2, which is further fused to form an outer diameter of about 500 μm. A substantially spherical cell aggregate 2 .

More efficiently, the cells are cultured by non-adhesive pores (substantially hemispherical accommodating portions) of the well culture plate, whereby the cell aggregate 2 can be easily obtained. Further, the method for producing the cell aggregate 2 is not limited thereto, and can be produced by various known methods, for example, a spin culture method in which a cell suspension is added to a rotating culture solution, and a cell suspension is added to the test tube. A method in which a turbid liquid is precipitated by a centrifugal separator or a method of culturing using alginic acid granules.

In summary, the cell agglutination block 2 is a substantially spherical cell aggregate having an outer diameter of about 100 to 700 μm formed by aggregating and aggregating cells, and the cell agglomerates 2 are planar or three-dimensional. When it is placed in contact or close to each other, the cells of each cell aggregate 2 are proliferated and fused with the adjacent cell agglutination block 2, whereby a planar or stereoscopic cell aggregate structure can be obtained.

The healing pad 4 produced in the present embodiment is made of a tissue such as bone, muscle, viscera, blood vessel, skin, or somatic cells, organs or precursor cells thereof, stem cells, etc., which fills bones or cartilage. Use the defect or attach it to the surface of an organ or organ.

Further, the healing pad 4 is formed by arranging and condensing a plurality of cell aggregates 2 in a planar manner, and the thickness thereof can be made to be about 100 to 700 μm depending on the size of the cell aggregate 2 to be used, and the planar size can be determined according to The number of cell agglutination blocks 2 arranged neatly is adjusted.

The above-described healing pad 4 thus produced is different from a cell sheet obtained by culturing a cell suspension in a planar manner to culture a cell monomer into a sheet.

The apparatus 1 for producing a collective structure of cells of the present embodiment is provided inside the isolator 5 which is maintained in an aseptic state as shown in Fig. 1, and inside the incubator 6 which is connectable to the isolator 5 . Further, a pass box 5a for removing the contamination of the carry-in is provided in the isolator 5.

2 is a view showing a configuration provided inside the isolator 5, and includes a storage container support portion 8 that supports a storage container 7 that houses the cell aggregate 2; and a culture container support portion 9 that supports cultivation of the culture healing pad 4. a container 3; a plurality of suction nozzles 10, which hold the cell aggregate 2; and a suction nozzle moving means 11 for relatively moving the suction nozzle 10 with respect to the storage container 7 and the culture container 3; The injection nozzle 12 of the means injects the injection into the cultured healing pad 4; and the injection nozzle moving means 13 which relatively moves the injection nozzle 12 relative to the culture container 3.

In the following description, in FIG. 2, the left-right direction is shown as the X direction, the front-rear direction is the Y direction, and the vertical direction is the Z direction. In FIG. 5, the left-right direction is shown as the X direction. , set the up and down direction to the Y direction.

The isolator 5 is internally maintained in a sterile environment and is formed by a sterile air supply means to form a unidirectional sterile air stream from above.

Further, a glove 5b that can be worn by an operator is provided on the front surface of the isolator 5, so that various operations can be performed. Further, a robot or a transfer means having a desired configuration may be provided inside the isolator 5 to automatically perform the operations.

Next, the inside of the incubator 6 is maintained in a sterile environment and maintained at a specific temperature and humidity suitable for culturing the healing pad 4, and the cell can be made while the cell agglutination block 2 is fused to form the healing pad 4. 6 is separated from the isolator 5 and placed at a position away from the isolator 5.

Therefore, the isolator 5 and the incubator 6 are connected by the connecting means 5c. For example, the following connecting means as described in Japanese Patent No. 4654648 can be used to maintain the incubator 6 and the isolator 5 in a sterile state. Connect/separate.

Fig. 3 is a cross-sectional view showing the above-described storage container 7, in which the above-mentioned hole culture plate can be used. The storage container 7 is provided with a plurality of housing recesses 7a in a vertical direction and a horizontal direction in plan view, and each of the housing recesses 7a accommodates a culture solution and accommodates a substantially spherical cell aggregate 2 therein.

The storage recessed portion 7a of the storage container 7 is arranged in a predetermined number in the X direction and the Y direction in the plan view. In the present embodiment, the cell agglutination block 2 of the healing pad 4 produced in the culture container 3 is formed. The number of the X direction and the Y direction are the same, that is, five in the X direction and five in the Y direction.

The storage container support portion 8 that supports the storage container 7 is configured by positioning the storage container 7 on the upper surface thereof by the positioning piece 8b, and is constituted by the Y-direction platform 8a constituting the suction nozzle moving means 11.

As described below, the suction nozzle 10 of the present embodiment is provided in five in the X direction, and can be moved only in the X direction and the Z direction. Therefore, the storage container 7 can be moved in the Y direction by the Y direction platform 8a. The suction nozzle 10 and the storage container 7 are relatively moved in various directions of XYZ.

Specifically, first, the Y-direction stage 8a is such that the first row housing recess 7a of the storage container 7 is positioned below the suction nozzle 10, and if the suction nozzle 10 is sucked from the first row housing recess 7a, the cell aggregation is maintained. In the block 2, the Y-direction platform 8a moves the storage container 7 by only one row of the housing recess 7a in the Y direction, and the second row housing recess 7a is positioned below the suction nozzle 10.

As shown in FIG. 4, the culture container 3 has a bottomed box shape, and accommodates the culture liquid at a specific depth inside, and is provided with a bottom panel 14 which is disposed on the bottom surface, and a culture holder which is disposed on the bottom panel 14 The pedestal 15 and the plurality of pins 16 that are erected on the pedestal 15 are formed; and the support plate 17 as a support member moves up and down along the pin 16.

The bottom panel 14 has substantially the same shape as the bottom surface of the culture container 3, and is positioned such that the bottom panel 14 does not move inside the culture container 3. Further, the pedestal 15 has a rectangular parallelepiped shape and is fitted to the concave portion 14a formed on the upper surface of the bottom panel 14 to be positioned.

The pins 16 are regularly erected on the installation surface 15a formed on the upper surface of the pedestal 15 so as to face the Z direction.

5 is a plan view showing the pin 16 provided on the pedestal 15 and the cell aggregate 2 held by the pin 16. In the present embodiment, there are five cell aggregates 2 in the X direction and the Y direction to contact each other. The states are arranged neatly in a planar manner.

The pin 16 is provided so as to be located around each cell agglutination block 2. In the present embodiment, the four pins 16 constitute a holding portion H for holding the cell aggregate 2 . More specifically, the block in which the four pins 16 are located at substantially four squares in plan view is referred to as the above-described holding portion H.

In the present embodiment, the interval between the two pins 16 at the respective diagonal positions of the four pins 16 constituting the holding portion H is arranged to be shorter than the diameter of the cell aggregate 2 held by the same. In other words, when the cell aggregate 2 is produced, the diameter is made larger than the interval between the two pins 16 at the respective diagonal positions.

When the cell agglutination block 2 is held by the holding portion H thus constituted, the cell aggregate block 2 can be sandwiched between the plurality of pins 16 and the pin 16, and the cell aggregate 2 can be placed as described above in Fig. 4(a). The upper and lower intermediate portions between the top end of the pin 16 and the root.

In other words, the holding portion H can be set at a position apart from the installation surface 15a on which the plurality of pins 16 are erected, and can be formed between the cell aggregate 2 and the installation surface 15a held by the holding portion H. The gap g between the culture fluids.

Further, by making the interval between the two pins 16 at the respective diagonal positions shorter than the diameter of the cell aggregate 2, the cell aggregates 2 of the respective holding portions H can be cultured in contact with each other, but also depending on the pin. 16 diameter.

The bottom panel 14 is provided with two rod-shaped guide members 18 disposed at positions facing each other across the pedestal 15 and erected in the Z direction to vertically penetrate the support plate 17; The support plate 17 is provided with a through hole 17a through which the pin 16 passes.

According to the above configuration, the support plate 17 is provided so as to be movable up and down along the pin 16 and the guide member 18 in the Z direction, and is normally placed at a lowering position by the weight of the body, and the lowered position is placed on the pedestal 15 The position of the base end side of the pin 16 of the setting surface 15a.

When the support plate 17 shown in FIG. 4(a) is in the lowered position, a gap between the cell agglomerate 2 held in the lower middle portion of the pin 16 and the installation surface 15a of the pedestal 15 forms a gap for the culture liquid to flow. g.

On the other hand, as shown in FIG. 4(b), when the support plate 17 is raised, the support plate 17 can support the cell aggregate 2 held between the pin 16 and the pin 16 from below by the support plate. 17 is located at a rising position on the distal end side of the pin 16, and the healing pad 4 in which the cell aggregate 2 is cultured can be taken out from the holding portion H between the pins 16.

The rise of the above-described support plate 17 can be performed by a robot or the like in addition to the worker wearing the glove 5b. In this case, although the holding member that protrudes upward from the liquid surface of the culture liquid is provided on the upper surface of the support plate 17, the support plate 17 can be raised without contacting the culture liquid.

As shown in Fig. 2, the culture container support portion 9 is constituted by the Y-direction land 9a constituting the suction nozzle moving means 11, and is held and held in the same manner as the Y-direction platform 8a constituting the storage container support portion 8. The cell agglutination block 2 of the suction nozzle 10 is supplied to the holding portion H constituted by the above-described pin 16, and the holding portion H set between the pins 16 is moved in line with the culture container 3 in the Y direction.

Next, the suction nozzle 10 will be described. As shown in Fig. 3, the suction nozzle 10 includes a main body portion 10a connected to a negative pressure generating means (not shown), and a tubular adsorption portion 10b provided at a lower end portion of the main body portion 10a. .

The inner diameter of the adsorption unit 10b is smaller than the diameter of the cell aggregate 2, and when the storage recess 7a of the storage container 7 is adsorbed and held by the tip end of the adsorption unit 10b, the cell aggregate 2 is not The inside of the adsorption portion 10b is sucked.

Further, in the culture container 3, the center position of the adsorption portion 10b is located substantially at the center of the holding portion H formed between the pin 16 and the pin 16, and the cell aggregate 2 is placed in the lower middle position of the pin 16 above. Next, the negative pressure generated by the above negative pressure generating means is eliminated.

In addition, a cell agglutination block 2 may be held by using a suction nozzle 10 that has a cell aggregate 2 or the like held by a holder or the like instead of adsorbing and holding the cell aggregate 2 .

Further, the suction nozzle moving means 11 for moving the suction nozzle 10 is constituted by an X-direction rail 21 which is provided in the X direction across the storage container support portion 8 and the culture container support portion 9; a moving means 22 for moving the suction nozzle 10 in the X direction along the X-direction rail 21; a 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 Y-direction platforms 8a and 9a in the container support portion 8 and the culture container support portion 9 are housed. Further, the suction nozzle moving means 11 may have a configuration in which the X-direction rail 21 is moved in the Y direction instead of the Y-direction stages 8a, 9a. With such a configuration, the suction nozzle 10, the injection nozzle 12, or the camera 25 described below can be moved in the X-Y direction.

Further, the suction nozzles 10 of the present embodiment are provided in five rows in the X direction, and the 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 portion 8, the interval changing means 24 changes the interval between the suction nozzles 10 to the accommodation recessed portion 7a aligned with the X direction in the storage container 7. The same interval.

On the other hand, when the suction nozzle 10 is positioned above the culture container support portion 9, the interval changing means 24 changes the interval between the suction nozzles 10 to be formed between the pin 16 and the pin 16 with the inside of the culture container 3. The interval between the X directions in the holding portion H is the same.

Further, the suction nozzle 10 may be provided in a plurality of rows in the Y direction, and the interval between the housing recesses 7a of the storage container support portion 8 and the holding portion H formed between the pins 16 and the pins 16 are the same. In the case of the above, the interval changing means 24 can be omitted.

Further, for example, when the interval between the holding portions H is narrow, the holding portion H may hold the cell aggregate 2 every other time by the suction nozzle 10. In this case, the odd number is first kept. The portion H holds the cell aggregate 2, and then the even-numbered holding portion H is held in the cell aggregate 2.

In addition, as long as at least one of the suction nozzles 10 is provided, as a means for supplying the cell aggregate 2, in addition to the configuration in which the cell aggregate 2 is transferred as in the present embodiment, a plurality of cells may be aggregated. Block 2 is constructed one by one from the container in which it is housed.

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 an lower end portion of the main body portion 12a, and the injection material is as described below. The through hole 4a opened in the healing pad 4 to be cultured is injected.

In the above-described infusion product supply means, the following substances are supplied as an infusion in the form of a liquid, a gel, a gel, a particle, or a particle: a nutrient component that promotes the culture of the cell, and promotes the growth or growth of the healing pad 4 after being transplanted to the patient, and the periphery. a growth factor or a proliferative factor of the tissue, a cell suspension for promoting the regeneration of blood vessels, a drug for the living organism after transplantation, etc.; and the like, which is discharged from the tip end of the injection portion 12b and injected into the through hole 4a of the healing pad 4 . Further, the injection may be performed above the through hole 4a, or the injection nozzle 12 may be inserted into the through hole 4a.

By injecting the through hole 4a in this way, the implant can be buried inside the healing pad 4, and more infusion can be saturated to the healing than when the infusion is dropped only on the surface of the healing pad 4. The inside of the mat 4.

Further, the healing pad 4 to which the infusion material is injected may be cultured to block the through-hole 4a, or may be directly recovered for transplantation.

As shown in FIG. 2, the injection nozzles 12 are integrally arranged in the X direction, and specifically, at the same interval as the interval between the pins 16 arranged in the X direction in the inside of the culture container 3, that is, It is provided in the same interval as the through hole 4a of the healing pad 4.

The injection nozzle moving means 13 for moving the injection nozzle 12 is constituted by an X-direction rail 21 which can be used in common with the suction nozzle moving means 11 and a left-right movement means 25 along which the X-direction rail 21; moving; the vertical movement means 26 is provided in the left and right movement means 25 to raise and lower the injection nozzle 12 in the Z direction; and the Y-direction platform 9a in the culture container support portion 9.

Further, the injection nozzle 12 may be provided with at least one of the injection nozzles, and the injection nozzle moving means 13 may be configured to move the injection nozzle 12 in the X direction and the Y direction without providing the Y-direction platform 9a.

Further, it is also possible to provide a plurality of injection nozzles 12 and to move them individually, whereby different injections can be injected.

In the X-direction track 21, a camera 28 is provided in the lateral movement means 27 that moves in the X direction, and the camera 28 is moved above the culture container support portion 9 to take the inside of the culture container 3.

When the cell aggregate 2 is held in the holding portion H by the suction nozzle 10, the image captured by the camera 28 can be used to confirm whether or not all of the holding portions H have the cell aggregate 2 held, if necessary. An instruction is given to supply the cell aggregate 2 to the holding portion H in which the cell aggregate 2 is not held.

Further, when the injection material is injected into the healing pad 4 by the injection nozzle 12, the image taken by the camera 28 is used to recognize the position of the through hole 4a of the healing pad 4.

Further, when the suction nozzle moving means 11 is positioned above the culture container support portion 9 and the injection nozzle moving means 13 places the injection nozzle 12 above the culture container support portion 9, the camera 28 self-cultivates The upper side of the container support portion 9 is retracted to avoid contact with it.

Hereinafter, a method of producing the healing pad 4 using the manufacturing apparatus 1 having the aggregate structure of the cells having the above-described configuration will be described.

First, a preparation step is performed in which the storage container 7 or the culture container 3 in which the cell aggregate 2 is accommodated is carried into the isolator 5 via the transfer window 5a, or a specific amount of the culture solution is supplied to the culture container 3.

Further, the culture container 3 is provided with a culture holder composed of the pedestal 15 and the plurality of pins 16, and the support plate 17 is placed at a lowered position in contact with the installation surface 15a of the pedestal 15 by its own weight.

Further, in the preparation step, when a plurality of the storage containers 7 or the culture containers 3 are carried into the separator 5, a plurality of the healing pads 4 can be continuously produced using the plurality of culture containers 3.

After the preparation step is completed, a supply step is performed in which the cell aggregate 2 is moved from the storage container 7 to the culture container 3 by the suction nozzle 10, and is set to the pin 16 of the culture container 3. The holding portion H is supplied to the cell aggregate 2 .

The suction nozzle moving means 11 moves the suction nozzle 10, and the interval changing means 24 changes the interval between the suction nozzles 10. Then, the storage container support portion 8 sucks and holds the cell aggregate 2 from the housing recess 7a of the storage container 7. .

Then, the suction nozzle moving means 11 moves the suction nozzle 10 to the culture container support portion 9, and the interval changing means 24 changes the suction nozzle 10 in accordance with the interval between the holding portions H formed between the pin 16 and the pin 16. The interval is then lowered by the suction nozzle 10.

In this manner, the cell aggregates 2 held in the suction nozzle 10 are inserted into the holding portion H formed between the four pins 16, so that the cell agglomerates 2 are formed so as to form a gap g below the cell aggregate 2 It is held in the lower middle position above the above pin 16.

When all the holding portions H are held in the cell aggregate 2 as described above, the camera 28 moves to the upper side of the culture container support portion 9 to photograph the cell aggregate 2 in the culture container 3, and confirms whether or not the cell aggregate 2 has been placed. The required holding portion H.

After the cell aggregate 2 is placed in the culture container 3, the culture step is performed, that is, the culture container 3 is transferred to the incubator 6 by manual operation by a robot or a worker, and the culture container 3 is cultured. Cell agglutination block 2.

First, after the culture container 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 interior at a specific temperature and humidity, and maintain the specific The concentration of carbon dioxide or oxygen.

In the culture container 3, when the cell aggregate 2 is cultured, the cell aggregates 2 adjacent to the holding portion H are fused to each other, whereby the healing pad 4 as a collection structure of cells can be obtained as the aggregate structure of the cells. The healing pad 4 maintains the shape in which the cell aggregate 2 is planarly arranged.

That is, since the movement of each cell aggregate 2 is restricted by the pin 16, the size of the formed healing pad 4 can be made substantially the same as the size when the cell aggregate 2 is initially placed, and the same can be made when the cell agglutination block 2 is placed. Healing pad 4 of any shape.

The reason for this is that the cell aggregate 2 is proliferated or moved in close contact with the periphery of the pin 16 in contact therewith, and fills the gap formed between the adjacent cell aggregates 2; if the cells are not agglutinated by the pin 16 As the block 2 is arranged in a planar manner and cultivated, the overall size is hardly reduced.

Further, in this case, since the gap g is formed below the cell aggregate 2, the culture solution can be supplied between the healing pad 4 and the installation surface 15a of the pedestal 15, and the back surface side of the healing pad 4 can be efficiently used. Cultivate.

In other words, when the cell aggregate 2 is placed on the installation surface 15a to form the healing pad 4, there is a problem that the culture solution cannot sufficiently spread over the lower surface side of the central cell agglomerate 2, but the cells are agglomerated. The state in which the gap g is formed below the block 2 is maintained, and the cells located in the portion are also well cultured.

Further, if the support plate 17 is moved up and down during the culturing, the flow of the culture solution is promoted and the effect is further improved.

When the culture medium is changed while the culture medium is being exchanged in the above-described culture step, the cell aggregates 2 are fused to each other to obtain the healing pad 4 of a desired size, but the healing pad 4 is held in the holding portion H, in other words, A state in which the above-mentioned pin 16 penetrates the healing pad 4 is formed.

Therefore, the detachment step of taking out the healing pad 4 held by the holding portion H from the holding portion H is performed. Specifically, the incubator 6 is connected to the isolator 5, and the culture container 3 is placed on the culture vessel support portion 9 inside the isolator 5, and the support plate 17 located at the descending position is raised by a worker or a robot or the like. position.

Thereby, the healing pad 4 is raised along the pin 16, and as shown in FIG. 4(b), the support plate 17 is lifted upward, and in this state, the support plate 17 is fixed by a fixing member 17b such as a jig, or The support plate 17 is moved quietly toward the lowered position, and the healing pad 4 is supported from below by the distal end portion of the pin 16, whereby the healing pad 4 is positioned above the pin 16.

After the healing pad 4 is placed above the pin 16 by the above-described disengaging step, the following implantation step is performed, that is, the injection is injected into the healing pad 4 by the injection nozzle 12 to form the pin 16 Through hole 4a.

Specifically, first, the camera 28 is moved above the culture container support portion 9 and the healing pad 4 in the culture container 3 is photographed, and the position of the through hole 4a of the healing pad 4 is recognized.

Then, the injection nozzle moving means 13 moves the injection nozzle 12 above the culture container support portion 9 so as to be positioned above the through hole 4a of the healing pad 4, and then injects the injection nozzle 12 as shown in FIG. The portion 12b is adjacent to the healing pad 4.

When the injection material is released from the injection portion 12b of the injection nozzle 12 in this state, the injection material is injected into the inside of the through hole 4a.

After the injection material is injected into the through hole 4a of the healing pad 4 in this way, in order to directly use the healing pad 4, it can be taken out from the culture container 3 and moved to the container for carrying out, and can be carried out from the isolator 5, and the culture container can also be used. 3 is again transferred from the isolator 5 to the incubator 6 to continue the culture, and the through hole 4a is completely blocked.

Figs. 7 and 8 are views showing a method of producing a collection structure of cells according to a second embodiment. In the culture container 3 used in the present embodiment, the pin 16 fixed to the pedestal 15 is held by the healing pad 4. The first pin group (16a) in the center portion and the second pin group (16b) which is black in the outer peripheral portion of the healing pad 4 are formed.

Specifically, in FIG. 7, the pin 16b constituting the second pin group is arranged in a frame shape, and the frame shape is formed by arranging the pin 16b on the outermost circumference and the outermost circumference. Therefore, when the cell aggregate 2 is housed in the culture container 3 in the supply step, the cell aggregate 2 placed on the outermost periphery thereof is held by the holding portion H formed by the pin 16b of the second pin group.

Further, as shown in FIG. 8, the pin 16a constituting the first pin group is shorter than the pin 16b constituting the second pin group, and the height of the holding portion H holding the cell aggregate 2 is set to be the same as the first pin group. The height of the top end of the pin 16a is similar.

A method of producing a collection structure of cells using the culture container 3 will be described. As shown in FIG. 8(a), in the supply step, the first and second pin groups are used in the same manner as in the first embodiment. The holding portion H formed by the pins 16a, 16b holds the cell aggregate 2 .

Then, the culturing step is performed, and if the cell agglutination block 2 is fused to some extent to form the healing pad 4 being cultured, the culture container 3 containing the cultured healing pad 4 is transferred from the incubator 6 to the isolator 5. .

Then, as shown in FIG. 8(b), the detaching step is performed, that is, the support plate 17 at the lowered position is moved up and down, and the cultured healing pad 4 is moved to be close to the tip end portion of the pin 16b of the second pin group. The height is then taken out from the holding portion H of the pin 16a of the first pin group.

Thereby, the healing pad 4 is supported by the pin 16b of the second pin group, and the through hole 4a formed by the pin 16a of the first pin group is opened in the healing pad 4.

In this state, an injection step is performed in which the injection material is injected into the through hole 4a of the healing pad 4 by the pin 16a of the first pin group by the injection nozzle 12.

Then, the healing pad 4 is again moved from the isolator 5 to the incubator 6 to restart the culturing step, and the culturing is continued, whereby the through-holes 4a are filled with the cells obtained by the culture.

At this time, the healing pad 4 is contracted and bent toward the center portion during the process of blocking the through hole 4a, but it also depends on the size of the through hole 4a.

In this regard, in the present embodiment, the outer peripheral portion of the healing pad 4 is held by the pin 16b of the second pin group, so that the healing pad 4 in which the contraction of the central portion of the healing pad 4 is prevented and the thickness without bending is uniform can be obtained. .

When the above-described culturing step is completed, the through hole 4a formed in the central portion of the healing pad 4 is clogged, and the culture container 3 is moved from the incubator 6 to the isolator 5 to perform the detachment step.

Specifically, the support plate 17 is again raised from the lowered position to the raised position, and then the healing pad 4 is taken out from the holding portion H of the second pin group.

Since the through hole 4a formed by the pin 16b of the second pin group that has been penetrated before is formed in the peripheral portion of the healing pad 4 thus obtained, the outer peripheral portion of the healing pad 4 can be cut as necessary. In addition, only the central portion is recovered, and the injection step may be further performed to inject an injection different from the through hole 4a formed by the pin 16a of the first pin group.

Further, the injection material may be not inserted into the through hole 4a formed by the pin 16a of the first pin group, and the injection material may be injected into the through hole 4a formed by the pin 16b of the second pin group. The first pin group and the second pin group are selectively disposed, and the injection position of the implant is designed.

Fig. 9 is a view for explaining a method of fabricating the aggregate structure of the cells of the third embodiment, which corresponds to a case where the diameter of the cell aggregate 2 is smaller than the pin 16 of each diagonal position of the holder H. The spacing is a small diameter so that 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 healing pad 4 to be thinned, it is conceivable to use a very small cell aggregate 2, or the size of the produced cell agglomerate 2 varies, including The case of the cell aggregate 2 which was dropped between the pin 16 and the pin 16 was not maintained.

Therefore, in the supply step of the present embodiment, as shown in FIG. 9(a), the cell aggregate 2 is accommodated as the holding portion H between the pins 16, and is placed on the installation surface 15a of the pedestal 15. The upper surface of the support plate 17 is lowered in the upper position while the individual cell agglomerates 2 are held.

Then, in a state where the cell aggregate 2 is placed on the support plate 17, the culture container 3 is transferred to the incubator 6 to start the above-described culturing step, and culture is carried out as shown in Fig. 9 (b) until the cell aggregate 2 is fused. .

After the cell aggregate 2 is fused to a certain 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 is separated from the installation surface 15a on which the plurality of pins 16 are erected. The position as the holding portion H holds the cell aggregate 2 .

Moreover, in the present embodiment, after the healing pad 4 of a certain size is obtained, the transition from the culture step to the detachment step and the injection step is performed.

Fig. 11 is a view for explaining a method of producing the aggregate structure of cells according to the fourth embodiment. Unlike the above-described respective embodiments, the tip end of the pin 16 which is erected in the culture container 3 is formed sharply so that the pin 16 is fastened. The cell aggregate 2 is passed through and held.

The apparatus 1 for producing a collection structure of cells can be the same as the apparatus used in each of the above embodiments, but when the pin 16 is passed through the cell aggregate 2 by the suction nozzle 10, it is penetrated. The pin 16 is inserted into the suction portion 10b, and the suction nozzle 10 is stopped at a specific position to hold the cell aggregate 2 .

When the cell aggregate 2 is held in this manner, the cell aggregates 2 adjacent to the holding portion H in the above-described culturing step are fused to each other, and the same healing pad 4 as in the above embodiments can be obtained.

Further, in the detaching step, as shown in FIG. 11(b), the support plate 17 can be moved to the upper position so that the cell aggregate 2 is positioned above the pin 16; in the injection step, the injection nozzle 12 can be used. The injection is injected into each of the through holes 4a.

Further, in the present embodiment, the through hole 4a is formed at a position where the pin 16 penetrates, and the injection nozzle 12 is moved to the position of the pin 16 to be injected.

Fig. 12 is a view showing a method of producing a collection structure of cells according to a fifth embodiment, and a planar healing pad 4 as a collection structure of cells produced in the first to fourth embodiments, In the examples, a method of producing a collection structure of cells in which the cells of the cell agglutination block 2 are aligned in the Z direction will be described.

In the case of producing the aggregate structure of the three-dimensional cells, a device having the same configuration as the production device 1 of the aggregate structure of the cells used in the first embodiment may be used, and a device may be used. The steps are basically the same, and the detailed description will be omitted.

In the culture container 3 used in the present embodiment shown in Fig. 12, the length of the pin 16 is set according to the height of the desired three-dimensional aggregate structure of the cells, and can be formed by the four pins 16. The holding portion H holds a plurality of cell aggregates 2 in the Z direction.

Further, in the above-described supply step, the suction nozzle 10 supplies the cell aggregate 2 to the holding portion H formed by the pin 16 based on the three-dimensional arrangement of the cell agglomerates 2 set in advance.

Specifically, first, the culture container holder 9 sequentially moves the culture container 3 in the Y direction, supplies the cell aggregate 2 to be placed in the lowermost stage, and sequentially supplies the cell agglutination to the upper portion of the cell aggregate 2 held in the holding portion H. Block 2 is OK.

At this time, as in the above-described respective embodiments, a gap g is formed below the cell aggregate 2 located at the lowermost stage, whereby the culture liquid can be supplied to the lower surface of the aggregate structure of the steric cells. Further, a gap is formed between the side portion of the aggregate structure of the cells supporting the three-dimensionality of the pin 16 and the side portion of the culture container 3, so that the culture liquid can be supplied to the side portion of the aggregate structure of the cells. A stereoscopic aggregate structure can be formed in the culturing step.

Further, in the present embodiment, the injection hole may 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 the through hole of the collecting structure, and the injection object may be injected into a desired position above the through hole 4a.

Further, in each of the above embodiments, as the arrangement of the pins 16 for supporting the cell aggregate 2, the cell aggregate 2 can be supported by the plurality of pins 16, and for example, six pins 16 may be disposed around each other. Cell agglutination block 2.

In this case, the holding portion H is arranged in a zigzag shape, and a three-dimensional assembled structure of cells as in the fifth embodiment can be produced by using the arrangement.

Further, in each of the above embodiments, the thickness of the pin 16 provided in the culture container 3, the size of the holding portion H formed by the pin 16, or the diameter of the cell aggregate 2 to be used may be as shown in FIG. Consider a variety of aspects.

The figure shows a case where the diameter of the pin 16 shown in Fig. 13 is larger than the diameter of the pin 16 shown in each of the above embodiments, and the diameter of the cell aggregate 2 is larger than 2 of the diagonal position. At the interval of the pins 16, the cell aggregates 2 adjacent to each other held by the holding portion H are not in contact with each other.

In this case, the cell aggregate 2 is also cultured in the above-described culture step so that the adjacent cell aggregate 2 is fused, so that the healing pad 4 as described above can be obtained, and since the pin 16 is large in diameter, it is formed in Since the through hole 4a of the healing pad 4 also has a large diameter, it is possible to inject a large amount of the injection into the inside of the through hole 4a, and the injection amount can be specified by the thickness of the pin 16.

When a nutrient component, a growth factor, a proliferative factor, a drug, or the like is injected as an injection into the through hole 4a formed in the healing pad 4, it may be performed according to the action of the implant, for example, every other through hole 4a. The injection is performed to disperse the effect, and it is also conceivable to inject only the injection into the through hole 4a of the healing pad 4 concentrated at a specific position to make the specific position of the healing pad 4 effective.

Further, the cell suspension containing the cells constituting the blood vessel may be selectively injected as an infusion into the through hole 4a to form a blood vessel at a desired portion of the healing pad 4, or may be selected according to the use of the healing pad 4. The location of the holes 4a injects different types of implants.

Claims (6)

 A method for producing a collection structure of cells, wherein a plurality of cell aggregates are disposed in a culture container in which a culture solution is accommodated, and the plurality of cell aggregates are cultured, and the plurality of cell aggregates are fused together a collection structure of cells; and characterized by: a supply step of holding a cell aggregate by a holding portion composed of a plurality of pins erected inside the culture container; and a culture step of maintaining the culture in the above-mentioned retention a cell agglutinating block to form the aggregate structure; a detaching step of taking out the aggregate structure formed in the culturing step from the holding portion; and an injecting step of injecting the implant into the detaching step The through hole formed by the pin is formed in the aggregate structure.    The method for producing a collection structure of cells according to claim 1, wherein the collection structure is further cultured after the injection step.    An apparatus for producing a collection structure of cells, wherein a plurality of cell aggregates are placed inside a culture container in which a culture solution is accommodated, and the plurality of cell aggregates are cultured, and the plurality of cell aggregates are fused together a collection structure of cells; and characterized in that: a culture holder provided inside the culture container and provided with a plurality of pins erected; and a supply means for constituting the plurality of pins of the culture holder Each of the holding portions holds a cell aggregate; the detaching means takes out the aggregate structure formed by culturing the cell aggregates held in the culture holder from the holding portion of the culture holder; and the injection means, which is injected The object is injected into a through hole formed in the removed aggregate structure by the pin.    The apparatus for producing a collection structure of cells according to claim 3, wherein the holding portion of the culture holder is formed between the plurality of pins and the pin, and the supply means sandwiches or houses the cells between the pin and the pin. Agglutination block.    The apparatus for producing a collection structure of cells according to claim 3, wherein the holding portion of the culture holder is constituted by the pin whose tip end is sharply formed, and the supply means causes the pin to pass through the cell aggregate. And stopping the cell agglutination block at a specific position of the pin.    The apparatus for producing a collection structure of cells according to any one of claims 3 to 5, wherein the separation means includes a support member movable along a pin of the culture holder, and the holding means is provided by the supply means When the cell aggregate is held, the support member is located on the proximal end side of the pin, and when the aggregate structure formed by fusing the cell aggregate is taken out from the holding portion, the support member is located on the distal end side of the pin.