Artificial three-dimensional tissue manufacturing device and artificial three-dimensional tissue manufacturing method

Abstract

The purpose of the present invention is to provide a three-dimensional artificial tissue manufacturing apparatus and a three-dimensional artificial tissue manufacturing method which can cope with various problems and can construct a three-dimensional artificial tissue with high versatility. The present invention comprises: a pair of anchor members that are spaced apart from each other to face each other in a first direction, and support, from the outside in the first direction, an end of a linear tissue module extending in the first direction; and a holder member that holds the pair of anchor members supporting the tissue module while the pair of anchor members are positioned in the first direction. The tissue module has a gel body containing cells. The pair of anchor members each have an engagement part engaged with the end of the tissue module from the inside in the first direction in a region in which the anchor members face each other. The holder member can hold the pair of anchor members supporting the tissue module while arranging multiple pairs of anchor members side by side in a direction intersecting the first direction.

Description

[Technical Field]

[0001] The present invention relates to an apparatus and a method for producing an artificial three-dimensional tissue. This application claims priority to U.S. Provisional Patent Application No. 63 / 080,686, filed September 19, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Research is being conducted into bottom-up methods for culturing multiple types of cells in three dimensions as a method for constructing tissue outside the body. Patent Document 1 discloses alternating lamination of rectangular first and second cell modules containing cells in a hydrogel. Non-Patent Document 1 discloses a technology for using a 3D printer to inkjet-type a liquid mixture containing cells to pattern and laminate multiple types of tissue. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2020-141573 [Non-patent literature]

[0004] [Non-Patent Document 1] 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration, SCIENTIFIC REPORTS, (2018) 8:12307, DOI:10.1038 / s41598-018-29968-5 Summary of the Invention [Problem to be solved by the invention]

[0005] With the technology of Patent Document 1, the thickness of the laminate of stacked cell modules increases, which can lead to oxygen and nutrient deficiencies in cells located at the center of the thickness.With the technology of Non-Patent Document 1, differences in culture conditions and maturation periods between multiple types of cells make it difficult to pattern them in the same process, resulting in inefficient tissue construction.

[0006] The present invention has been made in consideration of the above problems, and aims to provide an artificial three-dimensional tissue manufacturing device and an artificial three-dimensional tissue manufacturing method that can address various problems and construct artificial three-dimensional tissue with high versatility. [Means for solving the problem]

[0007] In order to solve the above problem, one embodiment of the artificial three-dimensional tissue manufacturing apparatus of the present invention comprises a pair of anchor members that face each other at a distance in a first direction and support the ends of a linear tissue module extending in the first direction from the outside of the first direction, and a holder member that holds the pair of anchor members supporting the tissue module in a positioned state in the first direction, wherein the tissue module has a gel body containing cells, and the pair of anchor members each have engaging portions in opposing regions that engage with the ends of the tissue module from the inside of the first direction, and the holder member is capable of holding multiple pairs of anchor members supporting the tissue modules lined up in a direction intersecting the first direction.

[0008] A method for manufacturing an artificial three-dimensional tissue according to one embodiment of the present invention includes preparing a pair of anchor members that are spaced apart and face each other in the first direction, and that support a linear tissue module having a gel body containing cells and extending in a first direction, with engaging portions provided in opposing regions that engage with the tissue module from the inside in the first direction and support the tissue module from the outside in the first direction; holding the pair of anchor members supporting the tissue module in a holder member that can hold multiple tissue modules arranged in a direction intersecting the first direction, while positioning the pair of anchor members in the first direction; and immersing the tissue module held in the holder member via the pair of anchor members in a culture medium and culturing it. [Effects of the Invention]

[0009] The artificial three-dimensional tissue manufacturing device and method of the present invention can address various problems such as oxygen deficiency, nutrient deficiency, and inefficiency of tissue construction in cells, and can construct highly versatile artificial three-dimensional tissues. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is an external perspective view of an artificial three-dimensional tissue manufacturing apparatus 1 according to a first embodiment of the present invention. [Figure 2] FIG. 2 is an external perspective view of a mold member 30 that constitutes the artificial three-dimensional tissue manufacturing apparatus 1. [Figure 3] FIG. 2 is a perspective view of the appearance of an anchor member 10 constituting the artificial three-dimensional tissue manufacturing device 1. [Figure 4] 2 is a cross-sectional view of the base material 21 taken along a plane perpendicular to the Z direction. [Figure 5] 10 is a schematic cross-sectional view of the tissue module M, perpendicular to the X direction. FIG. [Figure 6] 10 is a schematic cross-sectional view of the tissue module M, perpendicular to the X direction. FIG. [Figure 7] FIG. 1 is a photograph of an aggregated tissue module M containing skeletal muscle tissue. [Figure 8] FIG. 1 is a photograph showing skeletal muscle tissue at an early stage of culture and after 11 days of culture. [Figure 9] FIG. 10 is a graph showing the relationship between the number of days of culture and the amount of change in width of skeletal muscle tissue. [Figure 10] FIG. 10 is a diagram showing the relationship between time and displacement when an electrical stimulus is applied to skeletal muscle tissue. [Figure 11] FIG. 1 shows a fluorescent image of skeletal muscle tissue in the longitudinal direction. [Figure 12] FIG. 1 shows a fluorescent image of skeletal muscle tissue in the short axis direction. [Figure 13] FIG. 1 is a photograph of skeletal muscle tissue fabricated by arranging two tissue modules M side by side. [Figure 14] 14 is a cross-sectional view taken along line AB in FIG. 13. [Figure 15]FIG. 1 is a photograph showing a skeletal muscle module M1 and a hydrogel module M2 arranged side by side. [Figure 16] FIG. 1 is a photograph of a skeletal muscle module M1 and a hydrogel module M2 after two days of culture. [Figure 17] FIG. 1 is a photograph showing a skeletal muscle module M1 and an adipose module M3 arranged side by side. [Figure 18] These are enlarged photographs and fluorescent images of the skeletal muscle module M1 and the adipose module M3. [Figure 19] FIG. 10 is a cross-sectional view showing a pair of anchor members 10A and a mold member 30A for forming a tissue module M having a perfusion channel. [Figure 20] 3 is a cross-sectional view of a pair of anchor members 10A taken along the X direction. FIG. [Figure 21] 10A and 10B are diagrams for explaining the procedure for forming a tissue module M having a perfusion channel. [Figure 22] 10A and 10B are diagrams for explaining the procedure for forming a tissue module M having a perfusion channel. [Figure 23] 10A and 10B are diagrams for explaining the procedure for forming a tissue module M having a perfusion channel. [Figure 24] 10A and 10B are diagrams for explaining the procedure for forming a tissue module M having a perfusion channel. [Figure 25] This is a schematic cross-sectional view of a vascular system module M4 placed in the center, with eight skeletal muscle modules M1 placed around it. [Figure 26] FIG. 1 is a photograph of the tissue module M viewed in the Z direction. [Figure 27] FIG. 10 is a photograph of a cross section of the tissue module M perpendicular to the X direction. [Figure 28] FIG. 10 is a photograph of a cross section of the tissue module M along the X direction. [Figure 29] FIG. 10 is a photograph of a cross section along the X direction of an artificial three-dimensional tissue in which a liquid containing a fluorescent substance flows in a luminal layer 36. [Figure 30] This is a fluorescent image taken 15 minutes after a liquid containing a fluorescent substance was poured. [Figure 31] FIG. 1 is a photograph of skeletal muscle tissue cultured without perfusion. [Figure 32] FIG. 1 is a photograph of skeletal muscle tissue cultured with perfusion. [Figure 33] Photographs of cross sections of skeletal muscle tissue cultured with and without perfusion. [Figure 34] FIG. 1 shows the relationship between time and the amount of contraction when electrical stimulation is applied periodically to skeletal muscle tissue cultured with perfusion and skeletal muscle tissue cultured without perfusion. [Figure 35] FIG. 10 is a photograph of the cultured vascular module M4. [Figure 36] 10 is a schematic cross-sectional view showing a modified example of the arrangement of the tissue module M. FIG. [Figure 37] 10 is a schematic cross-sectional view showing a modified example of the arrangement of the tissue module M. FIG. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the artificial three-dimensional tissue manufacturing apparatus and the artificial three-dimensional tissue manufacturing method of the present invention will be described with reference to FIGS. The following embodiment shows one aspect of the present invention, does not limit the present invention, and can be modified as desired within the scope of the technical concept of the present invention. In addition, in the following drawings, the scale and number of each structure are different from the actual structure to make each configuration easier to understand.

[0012] [First embodiment of the artificial three-dimensional tissue manufacturing device] FIG. 1 is a perspective view showing the appearance of an artificial three-dimensional tissue manufacturing apparatus 1 according to the first embodiment. The artificial three-dimensional tissue manufacturing apparatus 1 includes a pair of anchor members 10 for supporting the tissue module M, a holder member 20, and a mold member 30 shown in FIG.

[0013] [Organization Module M] The tissue module M has a gel body 2 containing cells. The tissue module M includes a tissue T formed by culturing cells. The tissue module M is formed linearly along the horizontal direction. The tissue module M of this embodiment is formed in the shape of a linear rectangular prism.

[0014] In the following description, the direction in which the tissue module M extends horizontally may be referred to as the X direction (first direction), the horizontal direction perpendicular to the X direction as the Y direction (second direction), and the vertical direction perpendicular to the X and Y directions as the Z direction (third direction). However, this definition of the vertical and horizontal directions is given merely for the sake of convenience of explanation, and does not limit the relative positions of the artificial three-dimensional tissue manufacturing apparatus and artificial three-dimensional tissue manufacturing method according to the present invention.

[0015] Examples of tissue T include, but are not limited to, skeletal muscle tissue in which myoblasts are cultured as cells, muscle tissue in which cardiac muscle cells are used as cells, adipose tissue in which adipocytes are cultured as cells, and vascular tissue in which vascular cells are cultured as cells.

[0016] The gel body 2 is, for example, an extracellular matrix component. Examples of the extracellular matrix component include, but are not limited to, collagen (type I, type II, type III, type V, type XI, etc.), a basement membrane component (trade name: Matrigel) reconstituted from mouse EHS tumor extract (containing type IV collagen, laminin, heparan sulfate proteoglycan, etc.), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, and proteoglycan.

[0017] [Anchor member 10] The pair of anchor members 10 face each other and are spaced apart in the X direction. The pair of anchor members 10 each support an end of the tissue module M from the outside in the X direction. The pair of anchor members 10 are symmetrical with respect to a center line extending in the Y direction, so the configuration of one of the anchor members 10 will be described below.

[0018] As shown in FIG. 3, the anchor member 10 has a base 11 and an anchor portion 12. The base 11 is a quadrangular prism extending in the X direction. A cross section of the base 11 perpendicular to the X direction is approximately square. The base 11 has a fitting groove 11a located in the center in the X direction. The fitting groove 11a is formed at a position further inward than both end faces of the base 11 located on the outer sides in the X direction. The fitting groove 11a is formed by being recessed in the Y direction from both side faces of the base 11 in the Y direction. The fitting groove 11a penetrates the base 11 in the Z direction.

[0019] The anchor portion 12 is provided in a region of the base 11 facing the other anchor member 10 that forms a pair. The anchor portion 12 protrudes in the X direction from the end face 11b that faces the other anchor member 10. The anchor portion 12 has a pair of shaft-shaped portions 12a, a first engaging portion (engaging portion) 12b, a second engaging portion (engaging portion) 12c, a first through hole 13a, and a second through hole 13b.

[0020] The pair of shaft-shaped portions 12a protrude in the X direction from the end face 11b with a gap in the Y direction. The pair of shaft-shaped portions 12a are located at the center of the end face 11b in the Z direction. The pair of shaft-shaped portions 12a are parallel to each other. The cross-sectional shape of the shaft-shaped portions 12a is circular. The first engagement portion 12b extends in the Y direction and connects the tips of the shaft-shaped portions 12a to each other. The second engagement portions 12c are arranged with a gap between the end face 11b and the first engagement portions 12b. The second engagement portions 12c extend in the Y direction and connect the shaft-shaped portions 12a to each other. The cross-sectional shapes of the first engagement portion 12b and the second engagement portion 12c are circular.

[0021] The first through-hole 13a is formed in a rectangular shape in an area surrounded by the pair of shaft-shaped portions 12a, the first engagement portion 12b, and the second engagement portion 12c. The first through-hole 13a penetrates the anchor portion 12 in the Z direction. The first engagement portion 12b engages with the tissue module M located in the first through-hole 13a from the inside in the X direction.

[0022] The second through-hole 13b is formed in a rectangular shape in an area surrounded by the pair of shaft-shaped portions 12a, the second engagement portion 12c, and the end surface 11b. The second through-hole 13b penetrates the anchor portion 12 in the Z direction. The second engagement portion 12c engages with the tissue module M located in the second through-hole 13b from the inside in the X direction.

[0023] In the anchor portion 12 of the pair of anchor members 10, the first engagement portion 12b engages with the tissue module M located in the first through hole 13a from the inside in the X direction, and the second engagement portion 12c engages with the tissue module M located in the second through hole 13b from the inside in the X direction, thereby preventing the tissue module M located between the pair of anchor members 10 from contracting in the X direction.

[0024] [Holder member 20] The holder member 20 holds a pair of anchor members 10 supporting tissue modules M, positioned in the X direction. The holder member 20 has a pair of base members 21 spaced apart from each other in the X direction. One of the pair of base members 21 holds one of the pair of anchor members 10, and the other of the pair of base members 21 holds the other of the pair of anchor members 10. The pair of base members 21 are symmetrical with respect to a center line extending in the Y direction, so the configuration of one of the base members 21 will be described below.

[0025] Returning to FIG. 1 , substrate 21 has a base portion 22, a step portion 23, a fitting wall 24, and an opposing wall 25. Base portion 22 is a rectangular plate extending in the Y direction. Step portion 23 protrudes upward, on the +Z side, at a position away from the end of substrate 21 in the Y direction. The upper surface of step portion 23 supports substrate 11 of anchor member 10 from below. The position of the end of base portion 22 and step portion 23 on the tissue module M side in the X direction is approximately the same as the position of the end of substrate 11 on the tissue module M side. This forms a space below tissue module M that can be filled with culture medium.

[0026] The fitting wall 24 extends and protrudes from the step portion 23 toward the +Z side. The fitting wall 24 has a rectangular shape extending in the X direction when viewed from the +Z direction. FIG. 4 is a cross-sectional view of the base material 21 taken along a plane perpendicular to the Z direction. As shown in FIG. 4, a plurality of fitting walls 24 are arranged facing each other and spaced apart in the Y direction. In this embodiment, four fitting walls 24 are arranged. Adjacent fitting walls 24 in the Y direction fit into fitting grooves 11a provided on both sides of the anchor member 10 in the Y direction.

[0027] Each of the engaging walls 24 adjacent to each other in the Y direction engages with the engaging grooves 11a provided on both sides in the Y direction, thereby positioning the anchor member 10 held between the adjacent engaging walls 24 in the Y direction relative to the holder member 20. Furthermore, one engaging wall 24 is engaged across both of the opposing engaging grooves 11a of the anchor members 10 adjacent to each other in the Y direction. Therefore, in this embodiment, there is no need to provide two engaging walls 24 for each anchor member 10, which contributes to the miniaturization of the device. In this embodiment, four engaging walls 24 are used to position and hold three anchor members 10 lined up in the Y direction. The number of engaging walls 24 may be selected appropriately depending on the number of anchor members 10 and tissue modules M to be lined up in the Y direction.

[0028] Furthermore, the engagement wall 24 engages with the engagement groove 11a, and both ends of the engagement wall 24 in the X direction contact the base 11, thereby positioning the anchor member 10 in the X direction relative to the holder member 20. The Z direction dimension of the engagement wall 24 is a dimension that allows it to fit across multiple anchor members 10 lined up in the Z direction. The lowest anchor member 10 is supported on the upper surface of the step portion 23 and positioned in the Z direction. In this embodiment, the dimension allows the anchor members 10 to be lined up in three tiers in the Z direction. Therefore, by engaging the engagement wall 24 with the engagement groove 11a, the holder member 20 of this embodiment can hold a total of nine anchor members 10 and tissue modules M, three of which are lined up in the Y direction and three tiers in the Z direction, positioned in the X, Y, and Z directions. The Z direction dimension of the engagement wall 24 can also be selected appropriately depending on the number of anchor members 10 and tissue modules M to be lined up in the Z direction.

[0029] The opposing wall 25 faces the end of the base 11 of the anchor member 10 opposite the anchor portion 12. The opposing wall 25 has a recess 25a in the center in the Y direction. The recess 25a penetrates the opposing wall 25 in the X direction. As shown in FIG. 1, the recess 25a opens upward. The lower side of the recess 25a is positioned opposite the second-lowest anchor member 10 in the X direction.

[0030] [Molding member 30] The mold member 30 forms the tissue module M. The mold member 30 is formed of a soft elastic material such as PDMS (polydimethylsiloxane). As shown in FIG. 2, the mold member 30 has a recess 30a that opens to the upper side. The recess 30a has holding portions 31 located on both sides in the X direction and a module forming portion 32 located between the holding portions 31. The holding portions 31 position and hold the anchor members 10 inserted from above in the X direction.

[0031] The module forming section 32 forms the outer contour of the tissue module M. The module forming section 32 is filled with a solution of extracellular matrix components containing cells. The dimension of the module forming section 32 in the Y direction gradually increases from both ends in the X direction toward the center. The outer contours of both sides of the module forming section 32 in the Y direction are curves whose dimension in the Y direction gradually increases from the ends of the holding section 31 where the base 11 of the anchor member 10 is located toward the center in the X direction.

[0032] The dimension in the Y direction of the center end of the holding portion 31 where the base 11 of the anchor member 10 is located is defined as L (mm), and the longest dimension in the Y direction of the module forming portion 32 at the center in the X direction is defined as W (mm). The dimension L is preferably 0.8 mm or more and 3.5 mm or less, and more preferably 1.0 mm or more and 1.6 mm or less. The dimension W is preferably 1.0 mm or more and 4.0 mm or less, and more preferably 1.6 mm or more and 2.0 mm or less.

[0033] When extracellular matrix components containing cells are cultured in the module molding part 32, the matrix components shrink significantly from both ends in the X direction toward the center. By gradually increasing the Y direction dimension of the module molding part 32 from both ends in the X direction toward the center, when the gel body 2 is formed in the above-described contracted state, the outer shape on both sides in the Y direction can be formed into a rectangular prism parallel to the X direction.

[0034] [Artificial three-dimensional tissue manufacturing method] Next, a method for producing an artificial three-dimensional tissue using the above-described artificial three-dimensional tissue producing apparatus 1 will be described with reference to FIGS. The method for producing an artificial three-dimensional tissue according to the present invention includes: preparing a pair of anchor members 10 that support a linear tissue module M having a gel body 2 containing cells and extending in the X direction, with first engaging portions 12b and second engaging portions 12c disposed in opposing regions spaced apart from each other in the X direction, engaging the linear tissue module M from the inside in the X direction; holding the pair of anchor members 10 supporting the tissue modules M in a holder member 20 capable of holding multiple tissue modules arranged in a direction intersecting the X direction, while positioning the pair of anchor members 10 in the X direction; and immersing the tissue module M held by the holder member 20 via the pair of anchor members 10 in a culture medium for culturing.

[0035] (Preparation of tissue module M and pair of anchor members 10) To prepare the tissue module M and the pair of anchor members 10, first, the anchor members 10 are inserted into and held in the holding portion 31 of the mold member 30 shown in Figure 2. Next, a solution of extracellular matrix components containing cells is injected into the module forming portion 32 of the mold member 30.

[0036] In this embodiment, myoblasts are used as cells, and skeletal muscle modules are produced as tissue modules M. The cell density of myoblasts is 1.0 × 10 6 ~1.0×10 8 cells / mL, preferably 1.0 x 10 7 ~5.0×10 7 More preferably, it is cells / mL.

[0037] The extracellular matrix component used is a mixture of Matrigel (registered trademark) and fibrinogen. The mixing ratio of Matrigel (registered trademark) and fibrinogen is preferably 20-80% Matrigel (registered trademark) and 80-20% fibrinogen, and more preferably 40-60% Matrigel (registered trademark) and 60-40% fibrinogen.

[0038] A solution of extracellular matrix components containing cells is injected into the module molding part 32, and the extracellular matrix components are cultured under predetermined conditions to gel (solidify) and form a gel body 2. One example of culture conditions is culturing at a temperature of 37°C for 30 minutes. As the extracellular matrix components gel, the gel bodies 2 located on both ends in the X direction engage with and are supported by the anchor parts 12. This forms a tissue module M having a gel body 2 containing myoblasts, with its outer sides in the X direction supported by a pair of anchor members 10.

[0039] In this state, the culture time is short and the skeletal muscle tissue is immature, so the tissue module M and the pair of anchor members 10 are removed from the mold member 30 using tweezers or the like, and the skeletal muscle tissue is cultured in a culture tank or the like to mature. In the tissue module M where the skeletal muscle tissue has matured, contraction of the gel body 2 progresses. Because the outer shape on both sides of the Y direction of the module molding section 32 is a curve in which the dimension in the Y direction gradually increases from the end of the holding section 31 where the base 11 of the anchor member 10 is located toward the center in the X direction, the gel body 2 that has progressed in contraction has an outer shape on both sides of the Y direction that is parallel to the X direction.

[0040] [Holding of tissue module M and anchor member 10 to holder member 20] Before the tissue module M with mature skeletal muscle tissue and the pair of anchor members 10 are held by the holder member 20, the pair of substrates 21 are placed in a culture tank (not shown) that stores culture medium. The liquid level of the culture medium is set to a height that allows the uppermost tissue module M held by the holder member 20 to be immersed.

[0041] Next, as shown in Fig. 4, the tissue module M with mature skeletal muscle tissue and the pair of anchor members 10 are fitted from above so that the fitting grooves 11a of the anchor members 10 are fitted into the fitting walls 24 of the base material 21. This positions the pair of anchor members 10 in the X, Y, and Z directions relative to the base material 21.

[0042] [Culturing of tissue module M held in holder member 20] In this embodiment, a total of nine tissue modules M and pairs of anchor members 10 are arranged in three rows in the Y direction and three rows in the Z direction. Figures 5 and 6 are schematic cross-sectional views of the tissue modules M, perpendicular to the X direction. As shown in Figure 5, the tissue modules M held on the substrate 21 in three rows and three rows are prone to shaking because they are immersed in the culture medium C, making it difficult for adjacent tissue modules M to fuse together. Therefore, the culture medium C is temporarily removed from the culture vessel in which the pair of substrates 21 is installed.

[0043] By removing the medium C, the three rows and three columns of tissue modules M are aggregated by the action of surface tension, as shown in Figure 6. Figure 7 is a photograph of the aggregated tissue modules M containing skeletal muscle tissue. A solution of cell adhesive g (Hydrogel glue) is supplied to the aggregated tissue modules M to maintain the aggregated state. One example of the cell adhesive g is a hydrogel medium containing fibrinogen (4 mg / ml) and thrombin (50 UN / ml).

[0044] By culturing the tissue modules M supplied with the cell adhesive g for 10 minutes, a layer of cell adhesive g is formed around the three rows x three layers of tissue modules M, as shown in Figure 6. After this, medium C is again stored in the culture tank, and the tissue modules M are immersed in the medium. Because the three rows x three layers of tissue modules M are maintained in an aggregated state by the layer of cell adhesive g, they fuse without shaking even when immersed in medium C. This allows the three rows x three layers of tissue modules M to be cultured in a fused state.

[0045] [Tissue module M contracts] Contraction of tissue modules M containing skeletal muscle tissue produced by the above-described artificial three-dimensional tissue production method was confirmed. Three rows and one layer of tissue modules M were used to confirm contraction. Figure 8 is a photograph showing skeletal muscle tissue cultured using the artificial three-dimensional tissue production device 1 at the initial stage of culture (Day 0) and after 11 days of culture. As shown in Figure 8, as the culture progressed and matured, one skeletal muscle tissue was observed in which the three tissue modules M contracted.

[0046] Figure 9 shows the relationship between the number of days in culture and the amount of change in skeletal muscle tissue width, measured for each of the one-row x one-tier tissue module M, the two-row x one-tier tissue module M, and the three-row x one-tier tissue module M. In Figure 9, the width of the skeletal muscle tissue at the early stage of culture (Day 0) is shown as 100%. As shown in FIG. 9, it was confirmed that the contraction of the skeletal muscle tissue in the tissue module M progressed with increasing days of culture.

[0047] [Contraction of skeletal muscle tissue by electrical stimulation] The functionality of the skeletal muscle tissue produced by the above-described artificial three-dimensional tissue production method was confirmed. To confirm functionality, electrical stimulation was applied periodically to the skeletal muscle tissue via electrodes, and the contractile movement was measured. Figure 10 shows the relationship between time (sec) and displacement (μm) when electrical stimulation was applied to the skeletal muscle tissue with an electric field strength of 1.8 V / mm, 2 ms, and 1.0 Hz. As shown in Figure 10, it was confirmed that the skeletal muscle tissue was displaced in response to the application of electrical stimulation. Therefore, the skeletal muscle tissue produced by the artificial three-dimensional tissue production method of this embodiment is capable of expressing the functions of skeletal muscle. Furthermore, it was also visually confirmed that the skeletal muscle tissue was displaced in response to the application of electrical stimulation.

[0048] Figure 11 shows a fluorescent image of the long axis direction of skeletal muscle tissue produced by the artificial three-dimensional tissue production method after 7 days of culture, and Figure 12 shows a fluorescent image of the short axis direction of skeletal muscle tissue produced by the artificial three-dimensional tissue production method. As shown in Figure 11, it was observed that muscle fibers 4a and nuclei 4b were formed in the skeletal muscle tissue. Furthermore, as shown in Figure 12, it was observed that no enucleation due to nutrient deficiency or oxygen deficiency had occurred in the skeletal muscle tissue.

[0049] [Merge of organizational module M] 13 is a photograph of a skeletal muscle tissue produced by arranging two tissue modules M. FIG. 14 is a cross-sectional view taken along the line AB in FIG. As shown in FIG. 14, it was observed that the two skeletal muscle tissues prepared side by side were fused together without any gaps.

[0050] Figure 15 is a photograph of two tissue modules M arranged side by side. As shown in Figure 15, at the early stage of culture (Day 0), one of the tissue modules M was a skeletal muscle module M1 produced from skeletal muscle tissue, and the other was a hydrogel module M2 cultured using only extracellular matrix components.

[0051] FIG. 16 shows photographs of the skeletal muscle module M1 and the hydrogel module M2 after two days of culture, and an enlarged photograph of the boundary between the skeletal muscle module M1 and the hydrogel module M2. As shown in FIG. 16, it was observed that the hydrogel of the hydrogel module M2 was fused with the skeletal muscle tissue due to adhesive substances from the cells in the skeletal muscle module M1.

[0052] [Fusion of skeletal muscle module and fat module] FIG. 17 is a photograph of two tissue modules M fabricated side by side. As shown in Figure 17, a skeletal muscle module M1, in which skeletal muscle tissue was produced, was lined up as one of the tissue modules M, and an adipose module M3, in which adipose tissue in which adipocytes were cultured, was lined up as another of the tissue modules M. Adipose tissue takes longer to mature than skeletal muscle tissue. Therefore, when holding them in the holder member 20 to confirm fusion, the skeletal muscle module M1 at the initial stage of culture (Day 0) was lined up with the adipose module M3 that had been cultured for 30 days and matured (Day 30) (top photo). The bottom photo shows the skeletal muscle module M1 and adipose module M3 that had been cultured for an additional 13 days while held in the holder member 20.

[0053] Figure 18 shows an enlarged photograph (left photo) of skeletal muscle module M1 and fat module M3 after 13 days of culture, and an even more enlarged fluorescent image (right photo) of the area where fat module M3 has fused to skeletal muscle module M1. As shown in FIG. 18, it was observed that granular lipids were fused to and formed in skeletal muscle tissue containing myotubes and nuclei.

[0054] As described above, in the artificial three-dimensional tissue manufacturing apparatus and artificial three-dimensional tissue manufacturing method of this embodiment, tissue modules M having individually matured tissues can be arranged on the holder member 20 and cultured in the same process, allowing for efficient construction of an artificial three-dimensional tissue. Furthermore, in the artificial three-dimensional tissue manufacturing apparatus and artificial three-dimensional tissue manufacturing method of this embodiment, at least two types of tissue modules M having different cells in the Y and Z directions can be arranged in any arrangement and cultured, allowing for highly versatile construction of an artificial three-dimensional tissue that can address a variety of problems.

[0055] [Second embodiment of the artificial three-dimensional tissue manufacturing device] Next, a second embodiment of the artificial three-dimensional tissue manufacturing apparatus 1 will be described with reference to FIGS. In these figures, the same elements as those in the first embodiment shown in FIGS. 1 to 18 are designated by the same reference numerals, and the description thereof will be omitted.

[0056] In the second embodiment, a configuration for forming a tissue module M having a perfusion channel for constructing tissue while perfusing a culture medium will be described. Fig. 19 is a cross-sectional view showing a pair of anchor members 10A and a mold member 30A for forming a tissue module M having a perfusion channel. Fig. 20 is a cross-sectional view of the pair of anchor members 10A along the X direction.

[0057] As shown in FIG. 20, anchor member 10A has through-hole 14 penetrating in the X direction. In a pair of anchor members 10A, through-holes 14 are formed coaxially and with the same diameter. An anchor portion 12 in anchor member 10A has a shaft-shaped portion 15a and a third engagement portion (engagement portion) 15b. Shaft-shaped portion 15a protrudes from end surface 11b of base body 11 and extends in the X direction. Third engagement portion 15b is provided at the tip of shaft-shaped portion 15a. The dimension of third engagement portion 15b in a direction perpendicular to the X direction is larger than that of shaft-shaped portion 15a. At least one of shaft-shaped portion 15a and third engagement portion 15b may be cylindrical or rectangular.

[0058] When the gel body 2 of the tissue module M, which is located on the outer periphery of the shaft-shaped portion 15a and in the space between the end surface 11b and the third engagement portion 15b, contracts in the X direction, it engages with the third engagement portion 15b from the outside in the X direction. That is, the third engagement portion 15b engages with the gel body 2 of the tissue module M from the inside in the X direction, suppressing contraction of the tissue module M. Furthermore, as the shaft-shaped portion 15a contracts in the direction of its central axis, the gel body 2 is pressed against the outer periphery of the shaft-shaped portion 15a and the third engagement portion 15b. Therefore, the frictional force between the gel body 2 and the anchor member 10A increases, suppressing contraction of the tissue module M in a direction perpendicular to the X direction.

[0059] 19, a shaft-shaped member 33 is provided so as to be insertable and removable in the through holes of a pair of anchor members 10A held by holding portions 31 of a mold member 30A. The shaft-shaped member 33 extends in the X direction and can be inserted across the through holes 14 of the pair of anchor members 10A.

[0060] A procedure for forming a tissue module M having a perfusion channel using a pair of anchor members 10A and mold members 30A having the above-described configuration will be described. In this embodiment, an example of forming a perfusion channel inside skeletal muscle tissue will be described.

[0061] 19, a pair of anchor members 10A with shaft members 33 inserted in through holes 14 are inserted into holding portions 31 of mold member 30A from above. As a result, the pair of anchor members 10A are positioned in the X, Y, and Z directions relative to mold member 30A.

[0062] 21, a solution (suspension) of extracellular matrix components containing skeletal muscle cells as tissue S is poured into module molding section 32 of mold member 30A. After this, the tissue is cultured for 2 days (48 hours) at a temperature of 37°C, for example, to form a tissue module M composed of a gel body 2 having skeletal muscle tissue as a gel layer.

[0063] 22, the pair of anchor members 10A and the tissue module M are removed from the mold member 30A. Also, the shaft-shaped member 33 that penetrates the pair of anchor members 10A and the tissue module M is removed. By removing the shaft-shaped member 33, a perfusion channel 34 that is coaxial with and communicates with the through-holes 14 of the pair of anchor members 10A and extends in the X direction is formed inside the tissue module M. At this point, the tissue module M is a skeletal muscle module having a perfusion channel 34.

[0064] 23, vascular cells 35 are injected (seeded) through the through-holes 14 of the anchor member 10A onto the wall surface facing the perfusion channel 34 and cultured for a certain period of time to form a luminal layer 36. As a result, the perfusion channel 34 becomes surrounded by the luminal layer 36. That is, the tissue module M is formed in the shape of a square tube with a gel body 2 containing skeletal muscle tissue provided as a gel layer on the outer periphery of the luminal layer 36. As an example, vascular endothelial cells (HUVECs) are used as the vascular cells 35.

[0065] Once a tissue module M having a perfusion channel 34 has been formed inside the skeletal muscle tissue, a supply pipe 41 connected to a culture medium supply unit 40 is connected to one of a pair of anchor members 10A, and a discharge pipe 42 is connected to the other of the pair of anchor members 10A, as shown in Figure 24. Then, by supplying culture medium C1 from the culture medium supply unit 40 through the supply pipe 41 and the through-hole 14 to the perfusion channel 34, the skeletal muscle tissue can be cultured while perfusing the culture medium C1.

[0066] In the above-described method for producing an artificial three-dimensional tissue, a procedure for forming a tissue module M in which a perfusion channel 34 is formed inside skeletal muscle tissue has been exemplified, but the present invention is not limited to this configuration. For example, a solution of extracellular matrix components that does not contain cells may be injected into the module molding section 32 and then cultured to form a gel layer. In this case, the subsequent procedures, similar to those described above, include removing the shaft-shaped member 33 and injecting and culturing vascular cells 35, thereby producing a vascular module M4 (see FIG. 25) in which a luminal layer 36 (perfusion channel 34) is formed in the hydrogel. Alternatively, a solution (suspension) of extracellular matrix components containing vascular cells such as vascular endothelial cells (HUVECs) may be injected into the module molding section 32 of the mold member 30A shown in Figure 21 and cultured as tissue S. In this case, the vascular cells connect with each other to form blood vessels. Then, as a subsequent step, the shaft-shaped member 33 is removed in the same manner as above, thereby producing a vascular module M4 (see Figure 25) in which a luminal layer 36 (perfusion channel 34) has been formed.

[0067] Figure 25 is a schematic cross-sectional view perpendicular to the X direction of an artificial three-dimensional tissue in which the above-mentioned vascular system module M4 is placed in the center of a total of nine tissue modules M arranged in three rows in the Y direction and three stages in the Z direction on a holder member 20, and eight skeletal muscle modules M1 are arranged around the vascular system module M4.

[0068] The skeletal muscle module M1 was cultured while perfusing the lumen layer 36 of the central vascular system module M4 with medium C1 from the medium supply unit 40 shown in Figure 24. Figure 26 is a photograph of the artificial three-dimensional tissue viewed in the Z direction. Figure 27 is a photograph of a cross section of the artificial three-dimensional tissue perpendicular to the X direction. Figure 28 is a photograph of a cross section of the artificial three-dimensional tissue along the X direction.

[0069] As shown in Figure 27, it was observed that an artificial three-dimensional tissue was constructed in which a vascular module M4 was formed in the center with a remaining luminal layer 36, and skeletal muscle tissue formed from eight skeletal muscle modules M1 was surrounding the vascular module M4. Furthermore, as shown in Figure 28, it was observed that the luminal layer 36 was formed penetrating the artificial three-dimensional tissue.

[0070] Figure 29 is a photograph of a cross section along the X direction of an artificial three-dimensional tissue in which a liquid containing a fluorescent substance is flowing through the luminal layer 36. Figure 30 is a fluorescent image 15 minutes after the liquid containing the fluorescent substance was flowed. Immediately after the liquid containing the fluorescent substance was flowed, no diffusion of the fluorescent substance was observed, but as shown in Figure 30, 15 minutes after the liquid containing the fluorescent substance was flowed, it was possible to observe that the fluorescent substance had diffused from the luminal layer 36 into the skeletal muscle tissue.

[0071] [Comparison with and without perfusion] Figure 31 is a photograph of skeletal muscle tissue cultured without perfusion. Figure 32 is a photograph of skeletal muscle tissue cultured with perfusion. As shown in Figures 31 and 32, skeletal muscle tissue cultured with perfusion became thinner than skeletal muscle tissue cultured without perfusion. It is also thought that the flow stimulation of perfusion may increase the degree of orientation of the muscle tissue.

[0072] Figure 33 shows cross-sectional photographs of skeletal muscle tissue cultured with and without perfusion. As shown in Figure 33, with perfusion, it was confirmed that the luminal layer remained. With perfusion, it was confirmed that nuclei were scattered throughout. From these findings, it is believed that culturing with perfusion improves survival rate.

[0073] [Comparison of electrical stimulation with and without perfusion] FIG. 34 shows the relationship between time and the amount of contraction when electrical stimulation is applied periodically to skeletal muscle tissue cultured with perfusion and skeletal muscle tissue cultured without perfusion.

[0074] As shown in Figure 34, skeletal muscle tissue cultured with perfusion contracted more when electrically stimulated than skeletal muscle tissue cultured without perfusion, suggesting that skeletal muscle tissue cultured with perfusion matures more rapidly than skeletal muscle tissue cultured without perfusion.

[0075] [Vascular differentiation] FIG. 35 is a photograph of the cultured vascular module M4 described above. As shown in Figure 35, it was possible to confirm that new blood vessels differentiated from the thick blood vessels that form the luminal layer 36. This allows the cells to be combined with tissue modules M containing other cells and to be suitably used as vascular modules when forming blood vessels in other tissues.

[0076] As described above, in the artificial three-dimensional tissue manufacturing apparatus and artificial three-dimensional tissue manufacturing method of this embodiment, by arranging a tissue module M having a perfusion flow path 34 near a tissue module M for forming any tissue in the holder member 20, it is possible to suppress oxygen and nutrient deficiency in cells and construct good artificial three-dimensional tissue.

[0077] While the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, it goes without saying that the present invention is not limited to these examples. The shapes and combinations of the components shown in the above examples are merely examples, and various modifications can be made based on design requirements, etc., without departing from the spirit of the present invention.

[0078] For example, in the above embodiment, a configuration was illustrated in which tissue modules M were combined and held by holder member 20, such as modules having skeletal muscle tissue, a module having skeletal muscle tissue and a hydrogel module, and a module having skeletal muscle tissue and a module having adipose tissue, but the configuration is not limited to this. For example, as shown in Figure 36, a configuration may be used in which a vascular system module M4 is placed in the center, and a skeletal muscle module M1 and adipose module M3 are placed around the vascular system module M4. By adopting this configuration, an artificial three-dimensional tissue can be constructed in which adipose tissue is placed at any position in the skeletal muscle tissue.

[0079] In the above embodiment, a module having skeletal muscle tissue, a module having adipose tissue, and a vascular system module are exemplified as tissue modules M, but the present invention is not limited to this configuration. For example, a configuration using a dermal tissue module having dermal cells as the cells S and an epidermal tissue module having epidermal cells as the cells S may be used. In this case, for example, the dermal tissue module may be arranged around the vascular system module, and dermal tissue may be cultured while perfusion is performed. Furthermore, it is also possible to arrange the dermal tissue module around the vascular system module and the epidermal tissue module around the dermal tissue module, and to culture epidermal tissue while perfusion is performed to form artificial three-dimensional skin tissue.

[0080] Furthermore, in the above embodiment, a holder member capable of holding three rows and three columns of tissue modules M was exemplified, but this configuration is not limited to this. For example, as shown in Figure 37, a configuration using a holder member capable of holding seven rows and three columns of tissue modules M, or a configuration using a holder member capable of holding tissue modules M with other numbers of rows and columns, is also possible. When using tissue modules M with more than three rows and three columns, it is preferable from the perspective of preventing tissue death to arrange all tissue modules M other than vascular system module M4 so that a portion of the tissue modules M contacts vascular system module M4.

[0081] The artificial three-dimensional tissues produced by the above-described artificial three-dimensional tissue production device and artificial three-dimensional tissue production method can be used for drug screening (drug evaluation) to examine tissue responses to drug addition, interactions between tissues, etc. Drugs include pharmaceutical drugs, cosmetics, quasi-drugs, etc. This drug screening allows, for example, drug evaluation in an environment closer to that of actual tissue compared to conventional methods. Furthermore, it is extremely useful, for example, for evaluating the kinetics of drugs of various molecular weights in the creation of new drugs, and for evaluation in the development of cosmetics, quasi-drugs, etc.

[0082] Furthermore, the artificial three-dimensional tissue produced by the above-mentioned artificial three-dimensional tissue production device and artificial three-dimensional tissue production method may be configured to provide electrical stimulation by providing electrodes on the anchor member 10. By adopting this configuration, it is possible to observe and evaluate changes in blood vessels and fat tissue due to contraction of skeletal muscle tissue. [Industrial Applicability]

[0083] The present invention can be applied to an apparatus for manufacturing an artificial three-dimensional tissue and a method for manufacturing an artificial three-dimensional tissue. [Explanation of symbols]

[0084] REFERENCE SIGNS LIST 1...artificial three-dimensional tissue manufacturing device, 2...gel body, 10, 10A...anchor member, 11a...engagement groove, 12b...first engagement portion (engagement portion), 12c...second engagement portion (engagement portion), 15b...third engagement portion (engagement portion), 20...holder member, 30, 30A...mold member, 33...shaft-shaped member, 34...perfusion channel, 35...vascular cells, 36...luminal layer, 40...culture medium supply unit, g...cell adhesive, M...tissue module, T...tissue

Claims

1. a pair of anchor members that face each other and space from each other in a first direction, and support ends of the linear tissue module extending in the first direction from the outside in the first direction; a holder member that holds the pair of anchor members supporting the tissue module in a state where the anchor members are positioned in the first direction; Equipped with the tissue module has a gel body containing cells; The pair of anchor members each have an engaging portion in an opposing region that engages with an end of the tissue module from the inside in the first direction, the holder member is capable of holding a plurality of pairs of the anchor members supporting the tissue modules arranged in a direction intersecting the first direction, The holder member is a pair of substrates spaced apart from each other in the first direction; a fitting wall disposed on an upper surface of the base material and spaced apart from each other in a second direction perpendicular to the first direction, and extending in a third direction perpendicular to the first and second directions; Each of the pair of anchor members has a fitting groove on both side surfaces in the second direction, the fitting groove extending through the both side surfaces in the third direction, The fitting wall fits into the fitting groove to position the anchor member in the first direction.

2. The dimension of the fitting wall in the third direction is a dimension that allows the fitting wall to fit across a plurality of the anchor members that are arranged in the third direction. The artificial three-dimensional tissue manufacturing apparatus according to claim 1 .

3. Three or more of the fitting walls are arranged opposite to each other and spaced apart from each other in the second direction, Two or more of the pair of anchor members supporting the tissue module are arranged in the second direction, One of the fitting walls is fitted across both of the opposing fitting grooves in the anchor members adjacent to each other in the second direction. The apparatus for producing an artificial three-dimensional tissue according to claim 1 or 2.

4. The pair of anchor members each have a through hole that is coaxial with each other and penetrates in the first direction, a shaft-shaped member provided so as to be insertable into and removable from the through holes of the pair of anchor members; The apparatus for producing an artificial three-dimensional tissue according to any one of claims 1 to 3.

5. a culture medium supply unit that supplies a culture medium to the tissue module through the through-hole from the outside in the first direction; The apparatus for producing an artificial three-dimensional tissue according to claim 4.

6. preparing a pair of anchor members that are spaced apart from each other in the first direction and opposed to each other in regions that face each other, and that engage with a linear tissue module having a gel body containing cells and extending in a first direction from the inside in the first direction and support the linear tissue module from the outside in the first direction; a holder member that can hold a pair of the anchor members supporting the tissue modules in a state where the pair of anchor members is positioned in the first direction and can hold the plurality of anchor members in a direction intersecting the first direction; immersing the tissue module held by the holder member via the pair of anchor members in a culture medium and culturing the tissue module; Including, the holder member has a base material; a pair of the anchor members supporting the tissue module can be arranged in a row in a second direction perpendicular to the first direction along the upper surface of the base material, and can be arranged in a row in a third direction perpendicular to the first direction and the second direction; arranging the plurality of tissue modules on the holder member via the anchor members along at least one of the second direction and the third direction while immersing the tissue modules in a culture medium; removing the medium in which the plurality of tissue modules are immersed to aggregate the plurality of tissue modules; Supplying a cell adhesive to the aggregated tissue module to maintain the aggregated state; immersing the tissue module in the aggregated state in a culture medium again and culturing it; A method for producing an artificial three-dimensional tissue, comprising:

7. The at least two types of tissue modules having different cells are arranged on the holder member via the anchor members. The method for producing an artificial three-dimensional tissue according to claim 6.

8. Providing a pair of anchor members that support the tissue module from outside in the first direction includes: preparing a pair of anchor members each having a through hole that is coaxial with each other and penetrates in the first direction, and a shaft-shaped member extending in the first direction being inserted across the through hole; holding the pair of anchor members into which the axial members are inserted in a state where the anchor members are positioned in the first direction on a mold member that forms the tissue module; supplying at least an extracellular matrix to the mold member and culturing the matrix to form a gel layer; removing the shaft-shaped member from the pair of anchor members and the gel layer to form a perfusion channel that penetrates the pair of anchor members and the gel layer; culturing vascular cells on the surface of the perfusion channel to form a luminal layer; Including, The method for producing an artificial three-dimensional tissue according to claim 6 or 7.

9. supplying an extracellular matrix containing the cells different from the vascular cells to the mold component and culturing the cells; The method for producing an artificial three-dimensional tissue according to claim 8.

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