WO2019012622A1

WO2019012622A1 - Device for producing three-dimensional cell structure having arbitrary shape, and method for producing same

Info

Publication number: WO2019012622A1
Application number: PCT/JP2017/025400
Authority: WO
Inventors: 次郎大野
Original assignee: 次郎大野
Priority date: 2017-07-12
Filing date: 2017-07-12
Publication date: 2019-01-17

Abstract

[Problem] To provide a production device for producing a three-dimensional cell structure having an arbitrary shape. [Solution] A production device for producing a cell structure according to the present invention is provided with: multiple linear members 110; multiple columnar supports 120 for supporting the multiple linear members 110; multiple linear members 130; multiple columnar supports 140 for supporting the multiple linear members 130; a lower frame body 160A connected to the columnar supports 120; an upper frame body 170A connected to the columnar supports 140; a gripping member 400 for detaching the multiple columnar supports 120 from the multiple linear members 110; and a gripping member 420 for detaching the multiple columnar supports 140 from the multiple linear members 130.

Description

Device for producing three-dimensional cell structure of arbitrary shape and method for producing the same

The present invention relates to a method for producing a three-dimensional cell structure or a three-dimensional cell construct using a cell aggregate, and in particular, a three-dimensional cell using a strip-like, linear or planar member formed into an arbitrary shape. Apparatus and method for manufacturing a structure

Although the demand, demand and demand for medical practice are only increasing with the increase of population and long life span worldwide, in recent years, regenerative medicine using cells as a new method has attracted attention. Medical techniques for injecting individual cells directly into the body have already been put to practical use in other fields. Although this method is easy to perform, there is a problem that the injected cells are difficult to be established at the desired site.

On the other hand, methods for fusing a large number of cells to produce a three-dimensional structure have been developed. Cells are spatially retained in any shape with a petri dish, gel-like support, needle-like support or the like to form a three-dimensional structure. There are two major applications of this method.

The first is the preparation of artificial tissues and organs intended for human transplantation. Artificial tissue is produced in such a way that a portion of the artificial tissue expresses a function, and human transplantation is the final purpose. However, before transplantation into the human body, appropriate evaluation accreditation is required, and long-term efforts are required. Under the present circumstances, it is difficult to directly produce an organ having a complicated shape, and only an organ (such as a blood vessel) having a simple shape is created.

The second is a method of utilizing toxicity test, drug effect determination, pathology determination, developmental science, etc. using these three-dimensional cell structures as a test strip. By creating a three-dimensional cell structure using human cells only and performing the above-described test under or near the environment of the body, experiments simulating the body can be performed outside the body. This enables efficient drug discovery research, personalized medication diagnosis, observational research of each organ development, etc. Especially in the case of cancer medication, it is difficult to predict the timing and effect, but for example, the drug effect can be evaluated by first conducting a medication test assessment and determination outside the test strip created in the cancer tissue of the patient using this technology. It is expected to be an indicator of identification.

There are two types of cells: suspension cells and anchorage-dependent adherent cells. In the former, cells of blood system and immune system belong, and in the latter, cells of organs, skin, bone and the like belong. Adherent cells can not survive for a long time in the floating state in a solution, and need to survive and proliferate by adhering to a scaffold such as a glass petri dish or hydrogel. When the adherent cells are placed in a non-adherent environment, the cells adhere to each other in search of a scaffold, cell aggregates are formed, and cell aggregates are placed in an environment in which they are mutually contacted in some way. They adhere and fuse to form larger three-dimensional cell structures. This phenomenon is widely known, and Non-patent documents 1 to 6 show these specific examples. As described in Non-Patent Document 1, the phenomenon that cell aggregates (also referred to as Cluster in this document) are fused has long been known from the 1960s. In particular, Non-Patent Document 6 shows an idea of treating a three-dimensional cell structure as a "building block", and suggests that various cells can be used.

A cell mass (Spheroid) is a roughly circular aggregate composed of cells alone, and a cell aggregate (Cell Aggregate) is a aggregate composed of cell mass and cells and other substances. .

Patent Document 1 discloses a method for producing a tissue plug which can produce tissue of any shape with cells alone without using a carrier. Specifically, the cell aggregate is placed in a chamber having micropores through which the culture solution can pass only on the bottom surface, and the culture solution is contained in the chamber in an amount such that a part of the cell aggregate contacts the gas phase. The cell aggregates are cultured in a culture solution in an excess amount of the culture solution in the chamber.

In addition, as a method for producing a three-dimensional cell structure, a dispensing method in which cell aggregates are dispensed on a plane from a nozzle of a bioprinter shown in Patent Document 2 or a needle-like support shown in Patent Document 3 The Kenzan method of penetrating cell mass is known. Further, Patent Document 4 discloses a method of producing a three-dimensional cell by laminating a cultured cell cultured in a flat plane on a permeable sheet on a cultured cell in another planar culture together with the sheet.

Patent No. 4122280 U.S. Patent No. 8852932 Patent No. 4517125 (International Application No. PCT / JP2008 / 056268) International Publication WO2005 / 047496

In many of the dispensing methods shown in Patent Document 2, a method in which a mixture of a cell mass called bio ink and a connecting material such as hydrogel or collagen is discharged on a flat surface, or easily solidified such as hydrogel or collagen Although a cell mass is injected into the inside of a scaffold (scaffold) made of a shape-retainable material in advance to form a three-dimensional cell structure method, there is a drawback that contact between cells is prevented. In addition, since the shape of the three-dimensional cell structure produced by this method depends on the shape holding power of the connecting material, restrictions are imposed on the size and shape (especially in the height direction) of the three-dimensional cell structure. Further, since the binder remains in the three-dimensional cell structure, there remains a problem that the adverse effect of the binder on the cells can not be excluded, and additional evaluation and confirmation are required at the time of human transplantation or effect determination. Moreover, in the Kenzan method shown in Patent Document 3, the shape of the needle-like support is restricted.

An object of the present invention is to solve such conventional problems, and to provide a manufacturing apparatus capable of manufacturing a three-dimensional cell structure having an arbitrary shape, and a method for manufacturing the same.

In the method for producing a cell structure according to the present invention, a member capable of permeating a liquid such as a culture medium and forming a three-dimensional space for holding cell aggregates is prepared, and a plurality of cell aggregates are collected in the space Providing a collection and fusing the plurality of cell aggregates to form a cell structure.

A manufacturing apparatus for manufacturing a cell structure according to the present invention includes a first member defining at least a first space, and a device disposed opposite to the first member and defining at least a second space A plurality of cell aggregates can be accommodated in a third space defined by the first space and the second space, having at least two members, and at least both the first member and the second member Is permeable to liquids such as culture media.

According to the present invention, a three-dimensional space is formed by a member permeable to a liquid such as a culture medium, and a cell aggregate is supplied in the three-dimensional space to manufacture a cell structure. Three-dimensional structures can be manufactured. Furthermore, it becomes possible to freely set the medium supply channel.

FIG. 1 (A) is a perspective view showing a schematic configuration of a cell structure manufacturing apparatus according to an embodiment of the present invention, and FIG. 1 (B) is a view showing an example of connection between linear members and columns. . It is a flow which shows the manufacturing process of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other structural example of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the structural example of the detachment | leave mechanism of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows the other manufacture example of the cell structure which concerns on the Example of this invention. It is a block diagram which shows the electric constitution of the manufacturing apparatus of the cell structure which concerns on the Example of this invention. It is a figure which shows an example of the operation | movement flow of the manufacturing apparatus of the cell structure which concerns on the Example of this invention.

In the method of producing a three-dimensional cell construct according to the embodiment of the present invention, an arbitrary three-dimensional space for holding cell aggregates is formed by a linear member or a porous member or the like, and the three-dimensional space A plurality of cell aggregates are supplied to the inside to produce a three-dimensional cell structure according to a three-dimensional space. In a preferred embodiment, the member forming the three-dimensional space is permeable to a liquid such as a culture medium, so that the cell structure in the three-dimensional space can be supplied with the culture medium or the like from all directions. In a further preferred embodiment, the member forming the three-dimensional space can be formed using a three-dimensional printer. The three-dimensional printer generates a member that defines an arbitrary three-dimensional space based on the three-dimensional data. It should be noted that the scale of the drawings is exaggerated for the purpose of understanding the present invention, and may not necessarily be different from the size of an actual product or the like.

Next, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a view showing a schematic configuration of a cell structure manufacturing apparatus according to a first embodiment of the present invention. The manufacturing apparatus 100 of the cell structure of the present embodiment includes a plurality of linear members 110, a plurality of columns 120 supporting each of the plurality of linear members 110, a plurality of linear members 130, and a plurality of linear members 130. And a plurality of pillars 140 for supporting each of the linear members 130.

The plurality of linear members 110 may be of any shape, for example, the plurality of linear members 110 may have a circular or rectangular cross section, and may be two-dimensionally spaced at a predetermined interval substantially parallel to one another. Extends in a straight line. Alternatively, the plurality of linear members 110 may be curved or bent three-dimensionally at regular intervals substantially parallel to one another. Preferably, the plurality of linear members 110 are processed into an arbitrary shape and made of plastic or other soft material so as to facilitate removal from the cell structure. A plurality of linear members 110 arranged two-dimensionally or three-dimensionally, or processed two-dimensionally or three-dimensionally, form a two-dimensional space or three-dimensional space by the envelope surface of the plurality of lines. As defined above, this two-dimensional space or three-dimensional space provides a space for holding cell aggregates.

The plurality of linear members 130 are also configured in the same manner as the plurality of linear members 110, and are arranged two-dimensionally or three-dimensionally, or processed two-dimensionally or three-dimensionally, The envelopes of the lines of each of the linear members 130 define a two-dimensional space or a three-dimensional space, and these spaces provide a space for holding cell aggregates. In the example shown in the figure, the plurality of linear members 110 define a two-dimensional flat surface by their lines, and the plurality of linear members 130 have convex steps or bends in part thereof. By forming the part, a substantially convex three-dimensional space S is defined.

Further, each of the plurality of linear members 110 is not limited to the same shape, and may have different shapes. The same applies to the plurality of linear members 130. Furthermore, the spacing of each of the plurality of linear members 110 is not limited to being uniform, and each spacing may be different. The same applies to the distance between each of the plurality of linear members 130. Thus, the spacing between each of the plurality of linear members 110 and the spacing between each of the plurality of linear members 130 may be different.

The plurality of columns 120 are connected to the back side of each of the plurality of linear members 110. That is, on the back surface of one linear member, it is supported by the plurality of columns 120 along the direction in which the line extends, and as a result, on the back surface side of the plurality of linear members 110, the plurality of columns 120 are two-dimensionally arranged. Similarly, the plurality of columns 140 are connected to the back side of each of the plurality of linear members 130, and the plurality of columns 140 are two-dimensionally arranged on the back side of the plurality of linear members 130. .

Preferably, the plurality of linear members 110 and the plurality of columns 120 are integrally formed by the three-dimensional printer, and the plurality of linear members 130 and the plurality of columns 140 are also integrally formed by the three-dimensional printer.

FIGS. 1 (B), (C), and (D) are diagrams showing an example of connection between a support and a linear member. Although the connection method between the column and the linear member is arbitrary, in the example shown in FIG. 1B, the column 120/140 has a relatively large head 150 and the head 150 is a linear member. It is engaged in the groove on the back side of 110/130. In the example shown in FIG. 1C, the tip end portion 152 of the support 120/140 has a trapezoidal shape in which it gradually becomes thinner, and the material forming the support 120/140 and the material forming the linear member 110/130 are Differently, both materials are in an easy-to-peel relationship. In the example shown in FIG. 1D, the tip end portion 154 of the support column 120/140 has a shape that is easily broken from the linear member 110/130 or is made of a material that is easily broken. As will be described later, when the cell structure of a desired shape is produced by the production apparatus 100, the step of separating the columns 120/140 from the linear members 110/130 is included.

FIG. 2 is a schematic cross-sectional view for explaining the manufacturing process of the cell structure of the present example. As shown in FIG. 2A, the ends of the plurality of columns 120 are connected to the lower frame 160, and the ends of the plurality of columns 140 are connected to the upper frame 170. The lower frame 160 includes a recess space and accommodates the linear member 110 and the support 130 in the recess space, and similarly, the upper frame 170 includes the linear member 120 and the linear member 120 in the recess space. The post 140 is accommodated. The lower frame 160 and the upper frame 170 have the same size. In a preferred example, the lower frame 160 and the plurality of columns 120 are integrally formed by a three-dimensional printer, and the upper frame 170 and the plurality of columns 140 are integrally formed by a three-dimensional printer.

The lower frame 160 and the upper frame 170 are positioned such that the plurality of linear members 110 face the plurality of linear members 130. At this time, the plurality of linear members 110 and the plurality of linear members 130 are in close contact with each other, and a rectangular space S is formed therebetween. The space S is defined by the plurality of

linear members

110 and 130, but since there is a fixed gap between the plurality of

linear members

110 and 130, the space S is a linear member Exposed to the outside through the gap of Here, a rectangular space S is formed in order to manufacture a rectangular cell structure, and in the case of manufacturing a cell structure having another shape, a space corresponding to that is formed.

Cell aggregates are injected into the rectangular space S. Cell aggregates are a collection of multiple cells. In one preferable example, the distance D1 between the plurality of linear members 110 located on the lower side is smaller than the distance D2 between the plurality of linear members 130 located on the upper side (D1 <D2). The average size (particle size) R is in the relationship of D1 <R <D2. An opening (not shown) for injecting a cell aggregate is formed in a part of the upper frame 170, and the discharge part of the dispenser is directed into the space S into the cell aggregate solution through the opening. Discharge. The cell aggregates contained in the solution pass through the linear members 130 having a distance D2 larger than the particle diameter R, and are filled in the space S.

When the filling of the cell aggregates into the space S is completed, then, these are permeated into a medium suitable for cells to adhere and fuse the cell aggregates. For example, the assembly including the lower frame 160 and the upper frame 170 is immersed in a vessel containing the culture fluid. In this case, in either of the lower frame 160 and the upper frame 170, an opening through which the culture solution can enter is formed. The assembly may be subjected to constant vibration or oscillation to promote adhesion and fusion. The culture solution is sufficiently supplied from the three-dimensional direction through the gaps between the plurality of

linear members

110 and 130 between the plurality of cell aggregates in the space S, as a result, as shown in FIG. 2 (B). As such, adjacent cell aggregates adhere and fuse to form a cell structure T.

When the cell structure T is formed, next, the lower frame 160 and the upper frame 170 are pulled apart, and the plurality of

columns

120, 140 are separated from the plurality of

linear members

110, 130. As shown in FIGS. 1B to 1D, the

columns

120 and 140 are structured to be easily separated from the

linear members

110 and 130, and the lower frame 160 and the upper frame 170 are separated. By applying the force F in the direction, as shown in FIG. 2C, the

support columns

120 and 140 are separated from the back side of the

linear members

110 and 130. Next, as shown in FIG. 2D, the plurality of

linear members

110 and 130 can be removed from the cell structure T in the axial direction X to obtain a cell structure as a final product.

Next, another structural example of the manufacturing apparatus of this embodiment is shown in FIG. As shown in the figure, through

holes

200, 210, 220, and 230 are formed on the main surfaces of the lower frame 160 and the upper frame 170. The shape, number, and size of the through holes are arbitrary. Thereby, a passage which can access the space S through the through

holes

200 and 210 of the lower frame 160 and a passage which can access the space S through the through

holes

220 and 230 of the upper frame 170 And are formed. For example, a nutrient solution or a culture solution can be supplied to the cell aggregate in the space S through the through holes 200 to 230.

FIG. 3C is a further modification. By providing separation members 180 that separate the respective spaces of lower frame 160 and upper frame 170, the solution supplied from through

holes

200 and 210 and the solution supplied from through

holes

220 and 230 May be different. The separating member 180 does not have to completely shut off the upper and lower spaces, and it is sufficient if the solutions supplied from the upper and lower sides are prevented from being freely mixed without having a barrier. The separating member 180 can be configured, for example, as a rectangular frame, and is fixed, for example, between the end of the linear member and the inner wall of the frame by an adhesive or the like.

FIG. 4 shows a further modification of the manufacturing apparatus of this embodiment. In the example shown in FIGS. 4A and 4B, the through

holes

240 and 250 are formed only in the upper frame 170, and the culture fluid and the nutrient solution are supplied to the inside through the through holes 240, and the through holes 250 are formed. The culture fluid and nutrient solution are discharged to the outside through the By forming such a circulation route, it is possible to supply a fresh culture fluid or the like to the cell aggregate in the space S, or to supply a selected culture fluid or nutrient solution. Further, in the example shown in FIG. 4C, the culture fluid and the nutrient solution are supplied to the inside through the through holes 260 of the upper frame 170, and the culture fluid and the nutrition through the through holes 270 of the lower frame 160. The liquid may be discharged to the outside.

FIG. 5 shows a further modification of the manufacturing apparatus of this embodiment. In the example shown in the figure, a planar member 110A is used instead of the plurality of linear members 110. The planar member 110A may be a flat surface as shown in the figure, or may be a surface (a spherical recess or a rectangular recess) defining a three-dimensional space other than this. . A plurality of support columns 120 are connected to the back surface side of the planar member 110A in the same manner as described above. As shown in FIG. 5B, the space S when the planar member 110A is used is exposed to the outside through the gap of the upper linear member 130.

In FIG. 6, the further modification of the manufacturing apparatus of a present Example is shown. In the example shown in FIG. 1, the directions in which the plurality of linear members 110 and the plurality of linear members 130 extend are the same, but in the example shown in FIG. 6, the plurality of linear members 110 and The direction in which the plurality of linear members 130 extend is different, for example, the directions of both are orthogonal to each other. With such an array of linear members, when the linear members are removed from the cell structure, the linear members 110 and the linear members 130 are drawn out in orthogonal directions, so that the cell structure can be removed at the time of removal. The stress generated in the body can be relieved more than in one direction.

FIG. 7 shows a further modification of the manufacturing apparatus of this embodiment. In the example shown in the figure, the manufacturing apparatus further includes another plurality of linear members 300. That is, the plurality of linear members have a three-layer structure. Each of the plurality of intermediate linear members 300 may have any shape, any number, any spacing, etc., and extend in the same direction as the plurality of

linear members

110 and 130 It may extend in different directions. Furthermore, a plurality of columns 310 are connected to both ends of the intermediate linear member 300.

In one example, as shown in FIG. 7 (B), when the lower frame 160 and the upper frame 170 are assembled, the support 310 is formed on either the lower frame 160 or the upper frame 170. It is fixed. When the lower frame 160 and the upper frame 170 are pulled apart, the support 310 is separated from the linear member 300. Then, the

linear members

110, 130, and 300 are removed from the cell structure, respectively. An intermediate linear member 300 can be inserted to promote adhesion, fusion of cell aggregates, or inserted to promote the formation of cell structures.

FIG. 8 shows a further modification of the manufacturing apparatus of this embodiment. In the example shown in the figure, the manufacturing apparatus further includes a core 330. The plurality of linear members 110 define a semi-cylindrical concave space S1 in a portion thereof, and the plurality of linear members 130 define a semi-cylindrical convex space S2 in a portion thereof The two spaces S1 and S2 define a cylindrical space. The core 330 is a cylindrical member having a diameter smaller than the diameter of the cylindrical space of the spaces S1 and S2, and a part of the core 330 is supported in a floating state by the

linear members

110 or 130. And as shown to FIG. 8 (B), the cell aggregate was filled with space S1 and S2, and the cell structure was formed. Thereafter, the plurality of

linear members

110, 130 are withdrawn, and the cylindrical core 330 is withdrawn in the axial direction. As a result, as shown in FIG. 8C, for example, a hollow cylindrical cell structure such as a blood vessel can be obtained.

In FIG. 9, the further modification of the manufacturing apparatus of a present Example is shown. The example shown in the figure is a modification of the planar member 110A shown in FIG. 5, that is, a plurality of through holes 340 are formed in the member 110B on the surface. The shape, size, and number of the through holes 340 are arbitrary. FIG. 9B shows an example in which a semicircular recess 350 is formed in the member 110C on the surface, and a plurality of through holes 352 are formed in the recess 350.

FIG. 10 shows a further modification of the manufacturing apparatus of this embodiment. In the example shown in the figure, the window 360 which can see the inside is formed in a part of the upper side frame 170. The window 360 may be a through hole, or the window 360 may be attached with transparent glass or transparent plastic to seal the internal space. The adhesion state of the cell aggregate inside and the progress of fusion can be visually confirmed through the window 360. If the culture is confirmed to be insufficient, additional nutrient solution can be supplied.

FIG. 11 shows a further modification of the manufacturing apparatus of this embodiment. In the example shown in the figure, the porous membrane 380 is embossed toward the back surface of the plurality of linear members 120 by the embossing member 370. Thereby, a membrane 380 having the same shape as the three-dimensional space of the plurality of linear members 120 can be obtained. Membrane 380 can be, for example, decellularized or a biodegradable material such as collagen.

As shown in FIG. 11 (B), a plurality of linear members 120 can be separated from the embossed membrane 380, and the cell aggregate can be filled in the space of the membrane 380 to form a cell structure. . In this case, the membrane 380 can be a part of the cell structure, so there is no need to remove it. As described above, when the plurality of linear members or planar members are made of collagen or a decellularized porous membrane, the step of removing the plurality of linear members 120 can be omitted. , Production of cell structures can be facilitated.

Next, the separating mechanism of the lower side frame and the upper side frame in the manufacturing apparatus of the present embodiment will be described with reference to FIG. The detachment mechanism of the present embodiment includes a grip 400 and a plurality of connecting members 410. One end of the connecting member 410 is connected to the grip 400, and the other end is connected to the back surface side of the plurality of linear members 110, and the connecting members 410A on both sides are connected to the end of the upper frame 170A Be done. Moreover, the through-hole for making the some connection member 410 penetrate is formed in lower side frame 160A. The lower frame 160A is pulled away from the upper frame 170A by applying a force to the grip 400 so that the gap between the grip 400 and the lower frame 160A is narrowed, and at the same time, the plurality of columns 120 are a plurality of lines Are separated from the back surface of the second member 110.

The detachment mechanism further includes a grip 420 and a plurality of connection members 430. One end of the connection member 430 is connected to the grip 420 and the other end is connected to the back side of the plurality of linear members 130. Moreover, the through-hole for making the some connection member 410 penetrate is formed in the upper side frame 170A. The plurality of columns 140 are separated from the back surface of the plurality of linear members 130 by applying a force to the grip 420 so that the distance between the grip 420 and the upper frame 170A is narrowed.

FIG. 13 is a view for explaining an example of extraction of a plurality of linear members from the cell structure T. As shown in FIG. 13A, if the plurality of linear members 500 have a linear shape, removal thereof is easy. However, as shown in FIG. 13 (B), if there are steps, bending or the like in the plurality of linear members 500, removal from the cell structure T becomes difficult. Preferably, the linear member 500 is made of a soft material so that it can be easily removed even if it has an arbitrary shape, and at the same time, the strength that the linear member 500 does not break is required.

Therefore, the linear member 500 is coated with the strong cylindrical core 510 and the core 510 with the adhesive soft material 520. FIG. 13 (C) is a single-core linear member, and FIG. 13 (D) is an example of a multi-core linear member. When removing such a linear member, the linear member can be removed while rotating so as to facilitate removal from the cell structure.

FIG. 14 is a block diagram showing the electrical configuration of the cell aggregate production apparatus of this example. As shown in the figure, the manufacturing apparatus of this embodiment includes a supply source 610 for supplying a culture solution, a nutrient solution, and the like to the assembly 600 of the lower frame 160 and the upper frame 170, and an assembly from the supply source 610. A valve 620 for adjusting the flow rate of fluid supplied to 600, a valve control unit 630 for controlling the valve 620, a discharge source 640, and a valve 650 for adjusting the flow rate of fluid discharged from the assembly 600 to the discharge source 640 Valve controller 660 for controlling valve 650, temperature sensor 670 for detecting the temperature inside assembly 600, pressure sensor 680 for detecting the pressure inside assembly 600, and controller 690 for controlling the operation of each part. It comprises.

The controller 690 includes, for example, a RAM / ROM, a microprocessor or the like, and preferably controls each unit by executing a program that controls the manufacturing process of the cell aggregate. FIG. 15 shows an example of a control sequence of the manufacturing process by the controller 690.

As described above, the assembly 600 is a stack of the lower frame 160 and the upper frame 170, and the inner spaces of both frames are filled with cell aggregates. In one example, the supply source 610 is connected to the through hole 260 (see FIG. 4C) of the upper frame 170 by a pipe for conveying a fluid, and the discharge source 640 is penetrated by the lower frame 160 by a pipe. It is connected to the hole 270. From such a state, production of a cell structure shall be started.

First, the internal temperature of the assembly 600 is detected by the temperature sensor 670 (S100), and the internal pressure of the assembly 600 is detected by the pressure sensor 680 (S102). The controller 690 controls the flow rate of fluid supplied to the assembly 600 based on the detected temperature and pressure via the valve control unit 630 and controls the flow rate of fluid discharged from the assembly 600 via the valve control unit 660 (S104). For example, when the pressure of the assembly 600 is above a certain value, the flow rate of the supplied fluid may be decreased or the flow rate of the discharged fluid may be increased. Also, when the temperature in the assembly 600 is above a certain value, the flow rate of the supplied fluid is increased, or the flow rate of the discharged fluid is decreased. The controller 690 checks whether or not a predetermined time has elapsed (S106), and if it has not, the steps S100 to S104 are repeated. The fixed time is, for example, a time until cell aggregates adhere and fuse to form a cell structure.

Although FIG. 14 shows an example in which the fluid is supplied from one source 610 to the assembly 600, the present invention is not limited thereto. A plurality of sources are connected to the assembly 600 through a plurality of pipes, and a plurality of sources are connected. The valve may be controlled to supply the assembly 600 with a fluid selected from the following sources. For example, at the first temperature, the first type of fluid is supplied to the assembly 600, and at the second temperature, the supply of the first type of fluid is stopped, and the second type of fluid is assembled to the assembly. It may be supplied. Alternatively, during the first time period, the first type of fluid is supplied to the assembly 600, and during the second time period, the supply of the first type of fluid is stopped, and the second type of Fluid may be supplied to the assembly.

Although the preferred embodiments of the present invention have been described above in detail, the present invention is not limited to the specific embodiments, and various modifications may be made within the scope of the present invention as set forth in the claims. Modifications and changes are possible.

100: manufacturing apparatus 110:

linear members

110A, 110B: planar members 120: support members 130: linear members 140:

support members

150, 152, 154: connection means 160: lower frame 170: upper frame 200 210, 220, 230: through hole 300: intermediate linear member 310: post 330: core 340: through hole 350: recess 360: window 370: embossing member 380: membrane 400, 420: gripping member 410, 430: Connecting member T: Cell structure

Claims

A method for producing a cell structure, comprising
Preparing a member which is permeable to a liquid such as a culture medium and which forms a three-dimensional space for holding cell aggregates;
Supplying a plurality of cell aggregates in the space;
A method of producing a cell structure, comprising the step of fusing the plurality of cell aggregates to form a cell structure.
The method according to claim 1, further comprising the step of immersing the member supplied with the cell aggregate in a container containing a culture solution.
The method according to claim 1, wherein the method further comprises the step of separating the member from the cell structure.
The method according to claim 1, wherein the member includes at least an upper member and a lower member, and the upper member and the lower member form a three-dimensional space.
The method according to claim 4, wherein the member further includes a core between the upper and lower members, and the core forms a three-dimensional space.
The method according to claim 1, wherein the member comprises a plurality of linear members, and the step of drawing the plurality of linear members from the cell structure.
The method according to claim 5, wherein the upper member includes a plurality of linear members, and the lower member includes a plurality of linear members.
The method according to claim 6, wherein any one of the upper and lower members includes a flat surface.
The method according to claim 1, wherein the member is held by a lower frame member and an upper frame member.
The manufacturing method according to claim 9, wherein at least one through hole connected to the three-dimensional space is formed in at least one of the lower frame member and the upper frame member.
The manufacturing method according to any one of claims 1 to 10, wherein the member is formed by a three-dimensional printer.
12. A method according to any one of the preceding claims, wherein at least a portion of the member is comprised of a biodegradable material.
A manufacturing apparatus for manufacturing a cell structure, comprising:
A first member defining at least a first space;
A second member disposed opposite to the first member and defining at least a second space;
A plurality of cell aggregates can be accommodated in a third space defined by the first space and the second space,
The manufacturing apparatus, wherein at least the first member and the second member are both permeable to a liquid such as a culture medium.
In addition, the manufacturing equipment
A first support member including a plurality of columns connected to the first member;
A second support member comprising a plurality of posts connected to the second member;
An upper frame connected to the first support member;
And a lower frame connected to the second support;
The manufacturing apparatus according to claim 13, wherein the upper frame and the lower frame are positioned relative to each other.
15. The manufacturing apparatus according to claim 14, wherein the first member and the second member are composed of a plurality of linear members.
The manufacturing apparatus according to claim 15, wherein the plurality of linear members and the plurality of columns are separable.
The manufacturing apparatus according to claim 13, further comprising connection means for detachably connecting the upper frame and the lower frame.
The manufacturing apparatus according to claim 17, wherein the connection means includes a release mechanism for separating the upper frame and the lower frame.
The manufacturing apparatus according to claim 13, wherein at least one through hole connected to the three-dimensional space is formed in at least one of the upper frame and the lower frame.
20. The manufacturing apparatus according to claim 19, further comprising supply means for supplying a culture solution, such as a culture medium, a nutrient, or a growth factor, to the three-dimensional space through the through hole.
20. The manufacturing apparatus according to claim 19, further comprising: a discharge unit configured to discharge the liquid supplied to the three-dimensional space through the through hole.
The manufacturing apparatus according to claim 19, wherein the through hole is a window for observing cell aggregates in the three-dimensional space.