Culture system

Abstract

A culture system that comprises: a support for stacking cell aggregates, said support being provided with needle-shaped parts that are arranged on a substrate like a kenzan (flower frog needles), in which the outermost needle-shaped parts on the substrate are arranged so that the distance from the needle-shaped parts to the outer edge of the substrate is shorter than the distance between adjacent needle-shaped parts; a culture tank for housing a culture unit in which cell aggregates are stacked on the support; a liquid culture medium container; and a plurality of liquid culture medium supply pipes that supply a liquid culture medium from the liquid culture medium container to the culture unit, wherein each of the plurality of liquid culture medium supply pipes is positioned between the needle-shaped part.

Description

Technical Field

[0001] The present invention relates to a culture system using a support for laminating cell aggregates in which acicular bodies are arranged in a pincushion shape on a substrate, and a method for manufacturing a large three-dimensional cell structure.

Background Art

[0002] A method is known for producing a cell construct consisting only of cells by arranging cell aggregates in an arbitrary three-dimensional space using a support for arranging the cell aggregates in an arbitrary space and fusing the cell aggregates together (Japanese Patent No. 4517125), and an automated apparatus for that method has also been developed (Non-Patent Document 1: D. Murata, K. Arai, and K. Nakayama, “Scaffold-Free Bio-3D Printing Using Spheroids as ‘Bio-Inks’ for Tissue (Re-)Construction and Drug Response Tests,” Adv. Healthcare Mater., vol. 9, no. 15, p. 1901831, Aug. 2020.; Non-Patent Document 2: K. Nakayama, Kenzan Method for Scaffold-Free Biofabrication. Cham: Springer International Publishing, 2021.).

[0003] The above apparatus can construct cell structures of various shapes as a bio-3D printer, and in the above apparatus, a support in which acicular bodies are arranged within a 1 cm square is used (Fig. 1). For this reason, the maximum size (planar area) for which lamination (printing) is possible is 1 cm 2 square. Therefore, in order to print a larger cell structure, it is necessary to increase the area for arranging the acicular bodies and also increase the number.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Literature

[0005]

Non-Patent Literature 1

Non-Patent Literature 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] In order to print a large cell structure, operations such as enlarging the support are required, which requires modification of the bioprinter itself and is time-consuming and costly. Therefore, a means for easily enlarging the cell structure has been demanded.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventor modularized (parts-ized) the kenzan-shaped support, divided the 3D data of the target cell structure, and stacked cell aggregates on the modularized support for each divided data, and succeeded in constructing a large cell structure by culturing these supports, thus completing the present invention. That is, the present invention is as follows. (1) A support for laminating cell aggregates, in which acicular bodies are arranged in a pincushion shape on a substrate, and the outermost acicular bodies on the substrate are arranged such that the distance from the acicular bodies to the outer edge of the substrate is shorter than the interval between adjacent acicular bodies. (2) The support according to (1), wherein the distance from the outermost acicular body to the outer edge is one-half of the interval between adjacent acicular bodies. (3) The support according to (1) or (2), wherein the substrate has a rectangular plane. (4) The support according to (3), wherein one side of the rectangular plane has a length of 10 mm. (5) A culture system for culturing cell aggregates, (1) A culture tank for accommodating a culture unit in which cell aggregates are laminated on a plurality of supports according to any one of (1) to (4), A culture solution container, and a plurality of culture solution supply pipes for supplying a culture solution from the culture solution container to the culture unit, wherein each of the plurality of culture solution supply pipes is disposed between the acicular bodies. (6) The culture system according to (5), wherein the culture unit is arranged such that the supports are adjacent to each other. (7) The culture system according to (5) or (6), wherein each of the plurality of culture solution supply pipes is disposed along the upper surface of the substrate. (8) The culture system according to any one of (5) to (7), wherein each of the plurality of culture solution supply pipes is disposed below the cell aggregate. (9) The culture system according to any one of (5) to (8), wherein each of the plurality of culture solution supply pipes has an opening through which the culture solution flows out. (10) The culture system according to (9), wherein the opening is formed so that the culture solution flows out toward the cell aggregate. (11) The culture system according to any one of (5) to (10), wherein each of the plurality of culture solution supply pipes extends on the substrate parallel to at least one side of the substrate. (12) Each of the plurality of culture solution supply pipes is arranged in parallel with each other, and the culture system according to any one of (5) to (11). (13) The culture tank includes a frame or a recess for accommodating the culture unit, and the culture system according to any one of claims 5 to 12. (14) A method for manufacturing a large-sized three-dimensional cell structure having a length of at least 10 mm in any one direction, characterized in that a culture unit in which cell masses are stacked on a plurality of supports according to any one of (1) to (4) is placed and cultured in the culture system according to any one of (5) to (13). (15) The stacking of the cell masses is designed by dividing the shape data of the tissue to be manufactured into a plurality of parts, and is stacked on each support based on the divided and designed data. The method according to (14). (16) The culture unit is arranged such that the stacked cell masses have the original shape of the three-dimensional cell structure to be manufactured. The method according to (14) or (15). (17) A large-sized three-dimensional cell structure manufactured by the method according to any one of (14) to (16) and having a length of at least 10 mm in any one direction.

Advantages of the Invention

[0008] According to the present invention, it has become possible to manufacture a three-dimensional structure having a size with a length of 10 mm or more in any direction.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Mode for Carrying Out the Invention

[0010] The present invention is a support for laminating cell aggregates (cell masses, also referred to as spheroids) in which acicular bodies are arranged in a pincushion shape on a substrate, and the outermost acicular body on the substrate is arranged such that the distance from the acicular body to the outer edge of the substrate is shorter than the interval between adjacent acicular bodies. The present invention relates to the support. Conventionally, a support in which acicular bodies are arranged within a 1 cm square has been used. However, since there was a margin on the substrate on which the support was arranged (FIG. 1), acicular bodies could not be arranged adjacent to the margin portion. The present invention provides a support that eliminates the above margin portion and loses an extra edge (becomes edgeless) and is modularized so that they can be arranged adjacent to each other. The present invention also relates to a culture system for culturing cell masses using the above support.

[0011] 1. Support for Laminating Cell Masses A side cross-sectional view of the support for laminating cell aggregates (cell masses) of the present invention is shown in FIG. 2. As shown in Fig. 2, the support 22 for cell mass lamination (also simply referred to as the "support" or "support 22") is composed of a plate-shaped substrate 24 having a plurality of through holes 24a and a plurality of needle-like bodies 26 attached to the substrate 24 through the plurality of through holes 24a. The needle-like body 26 has a sharp tip and a lower end portion having a diameter slightly larger than the diameter of the through hole 24a. As shown in Fig. 2, when the tip of the needle-like body 26 is inserted into the through hole 24a from below the substrate 24 and moved upward, the lower end portion of the needle-like body 26 fits into the through hole 24a. Thereby, the needle-like body 26 is fixed to the substrate 24. The plane of the substrate 24 can preferably be rectangular, more preferably square or rectangular. The length of one side of the rectangle (square or rectangle) can preferably be 0.5 cm to 3.0 cm, more preferably 0.8 cm to 1.2 cm, and even more preferably about 1 cm (10 mm).

[0012] In addition, in the support 22, among the needle-like bodies 26 attached to the substrate 24, the distance d from the outermost needle-like body 26 to the outer edge of the substrate 24 is designed to be shorter than the interval D between adjacent needle-like bodies (D > d). Preferably, the distance d is half of the interval D between adjacent needle-like bodies. In this way, by deleting the blank portion of the substrate (edgeless), for example, when two supports are arranged adjacent to each other, the distance between the outermost needle-like body of one support and the outermost needle-like body of the other support on the adjacent surface is almost the same as the interval D (Fig. 6b). When the distance d is half of the interval D between the needle-like bodies, the distance from the outermost needle-like body of one support to the outermost needle-like body of the other support on the adjacent surface is 2d = D. Thereby, the adjacent cell agglomerates between the supports are likely to adhere to each other.

[0013] As the material of the needle-like body 26, those made of stainless steel, polypropylene, nylon, etc. can be used, but it is not limited thereto. In addition, the substrate 24 and the sheet 28 (details of the sheet 28 will be described later (Figs. 7 and 8)) are preferably coated with a cell non-adhesive such as fluorine (for example, made of polydimethylsiloxane). However, in addition to those processed with fluorine, those processed with polyhydroxyethyl methacrylate polymer can also be used. Furthermore, those made of acrylic resin, ABS resin, polyester resin, polycarbonate resin, polypropylene, polyethylene, polyacetal, polyetheretherketone, nylon, etc. can also be used.

[0014] 2. Culture System The culture system 100 according to this embodiment is shown in Fig. 3. The culture system 100 includes a culture tank 20 that houses a plurality of supports 22, at least one culture solution container 30 that stores the culture solution, and a culture solution supply pipe 32 that supplies the culture solution from the culture solution container 30 to the culture tank 20.

[0015] On the bottom surface of the culture tank 20, in order to arrange the plurality of supports 22 adjacent to each other, a frame or a recess 28 that houses the plurality of supports 22 is provided. The frame or the recess 28 may be provided corresponding to each of the supports 22. The recess 28 is rectangular in accordance with the plane of the support 22, but it can also be a recess in which a protrusion (not shown) is provided on the bottom surface of the support 22 and the protrusion fits in. The side surfaces of the substrates 24 of the adjacent supports 22 can preferably be arranged to abut against each other.

[0016] In Fig. 3, five supports 22 are arranged adjacent to each other. In the region 22' indicated by the dashed line, no support 22 is arranged, but a support 22 can be additionally arranged to make a total of six. The number of supports 22 to be arranged is appropriately selected according to the size of the target three-dimensional structure.

[0017] As shown in FIG. 4, the culture solution supply pipe 32 has openings 32a arranged at regular intervals. The culture solution pumped from the culture solution container 30 flows out from the openings 32a. The shape of the openings 32a can preferably be circular or elliptical. However, the number of the openings 32a may be one or plural, and is not limited. Also, an injection needle may be used as the culture solution supply pipe 32 such that the tip of the needle forms the openings 32a.

[0018] As shown in FIG. 3, each of the plurality of culture solution supply pipes 32 extends on the substrate 24 parallel to at least one side of the rectangular plane of the substrate 24. Each of the plurality of culture solution supply pipes 32 is arranged in parallel with each other.

[0019] FIG. 5 shows a state in which a plurality of cell masses C are arranged through the respective acicular bodies 26 of the support 22. In this state, the culture solution supply pipe 32 is arranged between the lowermost cell mass C and the upper surface of the substrate 24, and the culture solution and other nutrient solutions are supplied upward from the openings 32a of the culture solution supply pipe 32. Examples of the nutrient solution include serum and growth factors (such as platelet-derived growth factor (PDGF), transforming growth factor (TGF), and bone morphogenetic protein (BMP)), and they can also be contained in the culture solution. In this specification, the "culture solution" includes a culture solution containing the above nutrient solution. Each of the plurality of culture solution supply pipes 32 is arranged along (preferably in parallel with) the upper surface of the substrate 24. Each of the plurality of culture solution supply pipes 32 is arranged below the cell mass C.

[0020] The type of cells used in the present invention is not particularly limited, and any cells that can form cell masses can be used. Examples of cells that can form cell masses include undifferentiated cells such as stem cells (ES cells, iPS cells, cord blood-derived cells, undifferentiated mesenchymal stem cells, adult mesenchymal stem cells, etc.) or their differentiated cells. Tissues from which these cells are derived include, for example, articular cartilage, bone, adipose tissue, ligament, tendon, tooth, auricle, nose, liver, kidney, blood vessel, nerve, heart, etc., and among them, cartilage and the like are preferable. Also, the cell mass does not necessarily have to be formed as an aggregate of a single type of cell, and as long as a cell mass is formed, it may be formed from a plurality of cell types.

[0021] In addition, the culture period until the cell mass is formed is generally about 3 days to 21 days under general culture conditions (for example, at 37°C in a 5% CO2 atmosphere).

[0022] 3. Manufacture of the three-dimensional structure FIG. 6 shows a design in which the shape data of the three-dimensional structure (tissue T) to be manufactured is divided into a plurality of parts, and cell masses are laminated on respective supports based on the divided and designed data. The shape of the tissue T is designed to be the shape of a human ear. In FIG. 6, by decomposing the shape of the ear into six parts, cell masses can be laminated for each part. If the support on which the cell masses are laminated is referred to as a "culture unit", FIG. 6a shows a state in which six culture units are arranged adjacent to each other so as to form the original shape of the ear. By culturing in the state of FIG. 6, the cell masses adjacent to each other between the respective culture units are integrated to form the shape of one tissue as a whole. FIG. 6b is a side cross-sectional view of FIG. 6a. The cell mass laminated on the left support and the cell mass laminated on the right support are fused to form one tissue T as a whole.

[0023] In the state of FIG. 6, the plurality of culture solution supply pipes 32 can supply the culture solution to the tissue T (a plurality of cell masses) from the broken line portion 32' located below the tissue T composed of cell masses. The larger the size of the tissue T becomes, the more difficult it is for the culture solution and other nutrients to reach the inside of the tissue T. To avoid this, the culture system of the present embodiment can supply sufficient culture solution from the culture solution supply pipe 32 to the regions where the culture units 22 are adjacent to each other while changing the arrangement of the culture units 22 according to the shape of the tissue T.

[0024] After the culture is completed, the formed tissue T is taken out from the culture unit and isolated. In FIG. 7, by laying the sheet 28 on the substrate 24 in advance (FIG. 7a), after the culture is completed, the sheet is lifted and the tissue T is pulled out from the needle-like body 26 (FIGS. 7b, c).

[0025] Alternatively, as shown in FIG. 8a, if the sheet 28 is laid on the bottom of the substrate 24 (the substrate 24 is arranged on the sheet 28) and the needle-like body 26 is passed through to form the support 22, after the culture is completed, by pulling out the sheet 28 from the substrate 24, the needle-like body 26 is also pulled out together and the tissue T is separated from the culture unit (FIGS. 8b, c). The tissue T separated in this way is a large cell structure in which the length in any one direction is at least 10 mm.

[0026] Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples. [Example 1]

[0027] FIG. 9 is a photograph showing an embodiment of the culture system shown in FIG. 3. Two supports were arranged in the center of the system, and the culture solution supply pipes were passed through from both sides. [Example 2]

[0028] A total of 676 needles with a diameter of 0.1 mm were arranged on a square substrate with a side length of 10 mm at intervals of 0.4 mm in a 26 x 26 pattern. This was taken as one support unit (module). The distance from the outermost needles to the edge of the substrate was 0.2 mm. After downloading the 3D data of the ear, the ear shape data was divided into six parts and data processing was performed to stack cell masses on each part. The left side of Figure 10 shows six supports with a substrate in the shape of a 1 cm square rectangle arranged (a total culture unit of 2 cm x 3 cm), and the ear data was displayed on these six supports. The right side of Figure 10 is a diagram showing the data of the six ear parts.

[0029] Fibroblasts were aggregated to form cell masses, and based on the above data, cell masses were stacked on each support. Figure 11a shows a diagram of six supports arranged before stacking. Figure 11b shows a diagram of the stacked supports arranged such that each part is in its original position. As shown in Figure 11b, a three-dimensional structure formed only of cell masses was formed in a region 2 cm wide and 3 cm long while maintaining the shape of the ear. [Example 3]

[0030] Porcine cartilage was 3D scanned, and three-dimensional data was created in the same manner as in Example 2 (upper part of Figure 12). This was divided into two parts, and data for stacking cell masses was created (lower part of Figure 12). Based on this data, cell masses were stacked on each support.

[0031] The cell masses were prepared by culturing mesenchymal stem cells MSC (iNCMSC) cells induced from human iPS cells via neural crest in a chondrocyte induction medium in a PrimeSurface (registered trademark) 96-well plate. The culture unit with the stacked cell masses was placed in the system of the present invention and cultured at 37 °C using the chondrocyte induction medium. The composition of the chondrocyte induction medium is as follows. Basal medium (Chondrogenic Differentiation Basal Medium (PT-392, LONZA)) Serum (Chondrogenic SingleQuots(PT-4121,LONZA)) Growth factors (Platelet-Derived Growth Factor-BB(PDGF-BB), Transforming Growth Factor-β3(TGF-β3), Bone Morphogenetic Protein 4(BMP-4)

[0032] The left of Fig. 13 shows the cell mass on the first day of lamination (Day0), and the center of Fig. 13 shows the cell mass on the 8th day of culture (Day8). It can be seen that on Day8, the cell masses have fused to form a cartilage tissue with a vertical length of about 2 cm. The right of Fig. 13 is a view on the 14th day of culture (Day14), and a cartilage tissue was formed as a whole. [Description of reference signs] 100 Culture system 20 Culture tank 22 Support 24 Substrate 24a Through-hole 26 Needle-like body 28 Frame or recess 30 Culture solution container 32 Culture solution supply pipe 32a Opening C Cell mass T Tissue

Claims

1. A culture system for culturing cell clusters, comprising: a culture tank for accommodating a culture unit in which cell clusters are stacked on a plurality of supports for stacking cell clusters, wherein a plurality of acicular bodies are arranged in a hedgehog shape on a plurality of substrates; a culture solution container; a plurality of culture solution supply pipes for supplying a culture solution from the culture solution container to the culture unit, wherein the outermost acicular bodies of the supports are arranged such that the distance from the acicular bodies to the outer edge of the substrate is shorter than the interval between adjacent acicular bodies; each of the plurality of culture solution supply pipes is disposed between the acicular bodies; the culture system.

2. The culture system according to claim 1, wherein the distance from the outermost acicular body of the support to the outer edge is half of the interval between adjacent acicular bodies.

3. The culture system according to claim 1, wherein the substrate has a rectangular plane.

4. The culture system according to claim 3, wherein one side of the rectangular plane has a length of 10 mm.

5. The culture unit according to claim 1, wherein the supports are arranged adjacent to each other.

6. The culture system according to claim 1, wherein each of the plurality of culture solution supply pipes is disposed along the upper surface of the substrate.

7. The culture system according to claim 1, wherein each of the plurality of culture solution supply pipes is disposed below the cell clusters.

8. The culture system according to claim 1, wherein each of the plurality of culture solution supply pipes has an opening for flowing out the culture solution.

9. The culture system according to claim 8, wherein the opening is formed so as to flow out the culture solution toward the cell clusters.

10. The culture system according to claim 1, wherein each of the plurality of culture solution supply pipes extends on the substrate in parallel with at least one side of the substrate.

11. The culture system according to claim 1, wherein the plurality of culture solution supply pipes are arranged in parallel with each other.

12. The culture system according to claim 1, wherein the culture tank includes a frame or a recess for accommodating the culture unit.

13. A plurality of supports for laminating cell aggregates, in which acicular bodies are arranged in a pincushion shape on a plurality of substrates, wherein the outermost acicular body of the support is arranged such that the distance from the acicular body to the outer edge of the substrate is shorter than the interval between adjacent acicular bodies. A method for manufacturing a large three-dimensional cell structure having an arbitrary one-way length of at least 10 mm, characterized in that a culture unit having cell aggregates laminated thereon is placed in the culture system according to any one of Claims 1 to 12 and cultured.

14. The method according to Claim 13, wherein the lamination of the cell aggregates is designed by dividing the shape data of the tissue to be manufactured into a plurality of parts, and the lamination is performed on each support based on the divided design data.

15. The method according to Claim 13, wherein the culture unit is arranged such that the laminated cell aggregates form the original shape of the three-dimensional cell structure to be manufactured.

Images