WO2022059777A1 - Three-dimensional artificial tissue manufacturing apparatus and three-dimensional artificial tissue manufacturing method - Google Patents

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

Artificial 3D tissue manufacturing equipment and artificial 3D structure manufacturing method

The present invention relates to an artificial three-dimensional structure manufacturing apparatus and an artificial three-dimensional structure manufacturing method.
This application claims priority based on US Patent Provision 63 / 080,686 filed on September 19, 2020, the contents of which are incorporated herein by reference.

As a method of constructing tissue outside the body, research on a bottom-up method in which multiple types of cells are three-dimensionally cultured is underway. Patent Document 1 discloses that rectangular first cell modules and second cell modules containing cells are alternately laminated on a hydrogel. Non-Patent Document 1 discloses a technique of patterning and laminating a plurality of types of tissues by an inkjet method for a liquid mixture containing cells using a 3D printer.

Japanese Unexamined Patent Publication No. 2020-141573

In the technique of Patent Document 1, since the thickness of the laminated body in which the cell modules are laminated becomes large, there is a possibility that oxygen deficiency and nutritional deficiency of the cells located in the central portion in the thickness direction may occur. In the technique of Non-Patent Document 1, patterning in the same step is difficult due to the difference in culture conditions and maturation period between a plurality of types of cells, and tissue construction is inefficient.

The present invention has been made in view of the above problems, and it is an object of the present invention to provide an artificial three-dimensional tissue manufacturing apparatus and an artificial three-dimensional structure manufacturing method capable of dealing with various problems and constructing an artificial three-dimensional structure with high versatility. The purpose.

In order to solve the above problems, in the artificial three-dimensional tissue manufacturing apparatus according to one aspect of the present invention, the ends of the linear tissue modules that face each other apart from each other in the first direction and extend in the first direction are respectively the first. A pair of anchor members that support the tissue module from the outside in the direction and a holder member that holds the pair of the anchor members that support the tissue module in a positioned state in the first direction are provided, and the tissue module includes cells. Each of the anchor members having a gel body has an engaging portion that engages with the end portion of the tissue module from the inside in the first direction in a region facing each other, and the holder member has the structure. A plurality of the pair of anchor members supporting the module can be held side by side in a direction intersecting the first direction.

The method for producing an artificial three-dimensional tissue according to one aspect of the present invention is a region in which a linear tissue module having a gel body containing cells and extending in the first direction is opposed to each other in the first direction and faces each other. The engaging portion provided in the above is engaged from the inside of the first direction, and a pair of anchor members supporting the outside of the first direction are prepared, and the pair of anchor members supporting the tissue module are provided. A plurality of holder members that can be held side by side in a direction intersecting the first direction are held in a state of being positioned in the first direction, and the tissue module held by the holder member via the pair of anchor members. Includes culturing by immersing in a medium.

According to the artificial three-dimensional tissue manufacturing apparatus and the artificial three-dimensional tissue manufacturing method of the present invention, it is possible to deal with various problems such as lack of oxygen in cells, lack of nutrition, and inefficiency of tissue construction, and the artificial three-dimensional tissue is highly versatile. Can be built.

It is an external perspective view of the artificial three-dimensional structure manufacturing apparatus 1 which concerns on 1st Embodiment of this invention. FIG. 3 is an external perspective view of a mold member 30 constituting the artificial three-dimensional structure manufacturing apparatus 1. It is an external perspective view of the anchor member 10 constituting the artificial three-dimensional structure manufacturing apparatus 1. It is sectional drawing of the base material 21 in the plane orthogonal to the Z direction. It is a schematic cross-sectional view orthogonal to the X direction of a tissue module M. It is a schematic cross-sectional view orthogonal to the X direction of a tissue module M. It is a photograph figure of the tissue module M which has skeletal muscle tissue and aggregates. It is a photograph showing the skeletal muscle tissue at the beginning of the culture and after culturing for 11 days. It is a figure which shows the relationship between the culture days and the change amount of the width of a skeletal muscle tissue. It is a figure which shows the relationship between the time and displacement when the electric stimulus is applied to the skeletal muscle tissue. It is a figure which shows the fluorescence image in the long axis direction of the skeletal muscle tissue. It is a figure which shows the fluorescence image in the short axis direction of the skeletal muscle tissue. It is a photograph figure of the skeletal muscle tissue manufactured by arranging two tissue modules M side by side. 13 is a cross-sectional view taken along the line AB in FIG. It is a photograph figure which arranged the skeletal muscle module M1 and the hydrogel module M2. It is a photograph figure of the skeletal muscle module M1 and the hydrogel module M2 after culturing for two days. It is a photograph figure which arranged the skeletal muscle module M1 and the fat module M3. It is an enlarged photograph and fluorescence image of the skeletal muscle module M1 and the fat module M3. It is sectional drawing which shows the pair of anchor members 10A and the mold member 30A for forming a tissue module M having a perfusion flow path. It is sectional drawing along the X direction of a pair of anchor members 10A. It is a figure for demonstrating the procedure for forming a tissue module M having a perfusion flow path. It is a figure for demonstrating the procedure for forming a tissue module M having a perfusion flow path. It is a figure for demonstrating the procedure for forming a tissue module M having a perfusion flow path. It is a figure for demonstrating the procedure for forming a tissue module M having a perfusion flow path. It is a schematic cross-sectional view in which the vascular system module M4 is arranged in the center, and eight skeletal muscle modules M1 are arranged around it. It is a photograph figure which looked at the tissue module M in the Z direction. It is a photograph of the cross section orthogonal to the X direction of a tissue module M. It is a photographic view of the cross section along the X direction of the tissue module M. It is a photographic view of the cross section along the X direction of the artificial three-dimensional tissue in which a liquid containing a fluorescent substance flows in the lumen layer 36. It is a fluorescence image 15 minutes after flowing a liquid containing a fluorescent substance. It is a photograph figure of the skeletal muscle tissue cultured without perfusion. It is a photograph figure of the skeletal muscle tissue cultured with perfusion. It is a cross-sectional photograph of the skeletal muscle tissue cultured with and without perfusion. It is a figure which shows the relationship between the time and the amount of contraction when electrical stimulation is periodically applied to the skeletal muscle tissue cultured with perfusion and the skeletal muscle tissue cultured without perfusion, respectively. It is a photograph figure which cultured the vascular system module M4. It is a schematic cross-sectional view which shows the modification of the arrangement of the tissue module M. It is a schematic cross-sectional view which shows the modification of the arrangement of the tissue module M.

Hereinafter, embodiments of the artificial three-dimensional tissue manufacturing apparatus and the artificial three-dimensional structure manufacturing method of the present invention will be described with reference to FIGS. 1 to 37.
It should be noted that the following embodiments show one aspect of the present invention, do not limit the present invention, and can be arbitrarily changed within the scope of the technical idea of the present invention. Further, in the following drawings, in order to make each configuration easy to understand, the scale and number of each structure are different from the actual structure.

[First Embodiment of Artificial Three-Dimensional Tissue Manufacturing Equipment]
FIG. 1 is an external perspective view of the artificial three-dimensional tissue manufacturing apparatus 1 according to the first embodiment.
The artificial three-dimensional structure 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.

[Organization module M]
The tissue module M has a gel body 2 containing cells. The tissue module M contains a tissue T formed by culturing cells. The tissue module M is formed in a linear shape along the horizontal direction. The tissue module M of the present embodiment is formed in a square columnar shape extending linearly.

In the following, the direction in which the tissue module M extends along the horizontal direction is the X direction (first direction), the direction orthogonal to the X direction in the horizontal direction is the Y direction (second direction), and the X direction and the Y direction. The vertically orthogonal direction may be appropriately described as the Z direction (third direction). However, this defines the vertical direction and the horizontal direction only for convenience of explanation, and does not limit the relative positions of the artificial three-dimensional structure manufacturing apparatus and the artificial three-dimensional structure manufacturing method according to the present invention. do not have.

The tissue T is not particularly limited, and for example, skeletal muscle tissue in which myoblasts are cultured as cells, muscle tissue in which myocardial cells are used as cells, adipose tissue in which fat cells are cultured as cells, and vasculature cells as cells are used. The cultured vascular tissue can be exemplified.

Gel body 2 is, for example, an extracellular matrix component. The extracellular matrix component is not particularly limited, and for example, collagen (type I, type II, type III, type V, type XI, etc.), mouse EHS tumor extract (type IV collagen, laminin, heparan sulfate proteoglycan, etc.) can be used. Examples thereof include basement membrane components (trade name: Matrigel) reconstituted from (including), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan and the like.

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

As shown in FIG. 3, the anchor member 10 has a base 11 and an anchor portion 12. The substrate 11 is a square columnar extending in the X direction. The cross section of the substrate 11 orthogonal to the X direction is a substantially square. The substrate 11 has a fitting groove 11a located at the center in the X direction. The fitting groove 11a is formed at a position on the inner side of the both end faces located on the outer side in the X direction of the substrate 11. The fitting groove 11a is formed by being recessed in the Y direction from both side surfaces in the Y direction of the substrate 11. The fitting groove 11a penetrates the substrate 11 in the Z direction.

The anchor portion 12 is provided in a region of the substrate 11 facing another pair of anchor members 10. The anchor portion 12 projects in the X direction from the end surface 11b facing the other anchor member 10. The anchor portion 12 includes a pair of axial 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. Have.

The pair of axial portions 12a project in the X direction from the end faces 11b at intervals in the Y direction. The pair of axial portions 12a are located at the center of the end face 11b in the Z direction. The pair of axial portions 12a are parallel to each other. The cross-sectional shape of the shaft-shaped portion 12a is circular. The first engaging portion 12b extends in the Y direction and connects the tips of the shaft-shaped portions 12a to each other. The second engaging portion 12c is arranged with a gap between the end surface 11b and the first engaging portion 12b, respectively. The second engaging portion 12c extends in the Y direction and connects the axial portions 12a to each other. The cross-sectional shapes of the first engaging portion 12b and the second engaging portion 12c are circular.

The first through hole 13a is formed in a rectangular shape in a region surrounded by a pair of axial portions 12a, a first engaging portion 12b, and a second engaging portion 12c. The first through hole 13a penetrates the anchor portion 12 in the Z direction. The first engaging portion 12b engages with the tissue module M located in the first through hole 13a from the inside in the X direction.

The second through hole 13b is formed in a rectangular shape in a region surrounded by a pair of axial portions 12a, a second engaging portion 12c, and an end surface 11b. The second through hole 13b penetrates the anchor portion 12 in the Z direction. The second engaging portion 12c engages with the tissue module M located in the second through hole 13b from the inside in the X direction.

In the anchor portion 12 of the pair of anchor members 10, the first engaging 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 engaging portion 12c is the second. By engaging the tissue module M located in the through hole 13b from the inside in the X direction, the tissue module M located between the pair of anchor members 10 is suppressed from shrinking in the X direction.

[Holder member 20]
The holder member 20 holds a pair of anchor members 10 that support the tissue module M in a state of being positioned in the X direction. The holder member 20 has a pair of base materials 21 arranged apart from each other in the X direction. One of the pair of base materials 21 holds one of the pair of anchor members 10, and the other of the pair of base materials 21 holds the other of the pair of anchor members 10. Since the pair of base materials 21 are line-symmetrical with respect to the center line extending in the Y direction, the configuration of one base material 21 will be described below.

Returning to FIG. 1, the base material 21 has a base portion 22, a step portion 23, a fitting wall 24, and a facing wall 25. The base portion 22 has a rectangular plate shape extending in the Y direction. The step portion 23 projects upward on the + Z side at a position away from the end portion of the base material 21 in the Y direction. The upper surface of the step portion 23 supports the substrate 11 in the anchor member 10 from below. In the base portion 22 and the step portion 23, the position of the end portion on the tissue module M side in the X direction is substantially the same as the position of the end portion on the tissue module M side in the substrate 11. As a result, a space can be formed under the tissue module M and filled with the medium.

The fitting wall 24 projects from the step portion 23 to the + Z side and extends. The fitting wall 24 has a rectangular shape extending in the X direction when viewed from + Z. FIG. 4 is a cross-sectional view of the base material 21 on a plane orthogonal to the Z direction. As shown in FIG. 4, a plurality of fitting walls 24 are arranged facing each other apart from each other in the Y direction. Four fitting walls 24 of this embodiment are arranged. The fitting walls 24 adjacent to each other in the Y direction are fitted into the fitting grooves 11a provided on both sides of the anchor member 10 in the Y direction.

Each fitting wall 24 adjacent to each other in the Y direction is fitted into the fitting grooves 11a provided on both sides in the Y direction, so that the anchor member 10 sandwiched between the adjacent fitting walls 24 is a holder member 20. It is positioned in the Y direction with respect to. Further, one fitting wall 24 is fitted so as to straddle both of the facing fitting grooves 11a in the anchor members 10 adjacent to each other in the Y direction. Therefore, in the present embodiment, it is not necessary to provide two fitting walls 24 for each anchor member 10, which can contribute to the miniaturization of the device. In the present embodiment, the three anchor members 10 can be arranged, positioned and held in the Y direction using the four fitting walls 24. The number of fitting walls 24 may be appropriately selected according to the number of anchor members 10 and tissue modules M arranged in the Y direction.

Further, the fitting wall 24 is fitted into the fitting groove 11a, and the ends of the fitting wall 24 on both sides in the X direction are in contact with the substrate 11, so that the anchor member 10 is in the X direction with respect to the holder member 20. Positioned. The dimension of the fitting wall 24 in the Z direction is a dimension of fitting across a plurality of anchor members 10 arranged in the Z direction. The lowermost anchor member 10 is supported by the upper surface of the step portion 23 and is positioned in the Z direction. In this embodiment, the anchor members 10 are sized so that they can be arranged in three stages in the Z direction. Therefore, in the holder member 20 of the present embodiment, when the fitting wall 24 is fitted into the fitting groove 11a, three anchor members 10 are arranged in the Y direction and three anchor members are arranged in three stages in the Z direction, for a total of nine anchor members. The 10 and the tissue module M can be held in a state of being positioned in the X direction, the Y direction, and the Z direction. The dimensions of the fitting wall 24 in the Z direction may be appropriately selected according to the number of anchor members 10 and the tissue modules M arranged in the Z direction.

The facing wall 25 faces the end portion of the base 11 of the anchor member 10 opposite to the anchor portion 12. The facing wall 25 has a recess 25a in the center in the Y direction. The recess 25a penetrates the facing wall 25 in the X direction. As shown in FIG. 1, the recess 25a opens upward. The lower position of the recess 25a is a position facing the anchor member 10 in the second stage from the bottom in the X direction.

[Mold member 30]
The mold member 30 forms the tissue module M. The mold member 30 is made of a soft elastic material such as PDMS (polydimethylsiloxane). As shown in FIG. 2, the mold member 30 has a recess 30a that opens upward. The recess 30a has a holding portion 31 located on both sides in the X direction and a module forming portion 32 located between the holding portions 31. The holding portion 31 positions and holds the anchor member 10 inserted from above in the X direction.

The module molding unit 32 forms the outer contour of the tissue module M. The module molding section 32 is filled with a solution of extracellular matrix components containing cells. The dimension of the module molding portion 32 in the Y direction gradually increases from both ends in the X direction toward the center. The outer shape on both sides of the module molding portion 32 in the Y direction is a curve in which the dimension in the Y direction gradually becomes longer from the end portion of the holding portion 31 where the substrate 11 of the anchor member 10 is located toward the center in the X direction.

The dimension in the Y direction at the central end of the holding portion 31 where the substrate 11 of the anchor member 10 is located is L (mm), and the dimension in the longest Y direction in the module molding portion 32 at the center in the X direction is W (mm). And. The dimension L is preferably 0.8 mm or more and 3.5 mm, 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.

When the extracellular matrix component containing cells is cultured in the module molding unit 32, the matrix component contracts significantly from both ends in the X direction toward the center. By gradually increasing the dimensions of the module molding portion 32 in the Y direction from both ends in the X direction toward the center, the outer shapes on both sides in the Y direction are parallel to the X direction when the gel body 2 contracted as described above is formed. It can be molded into a square columnar shape.

[Artificial three-dimensional structure manufacturing method]
Next, a method for manufacturing an artificial three-dimensional structure using the above-mentioned artificial three-dimensional structure manufacturing apparatus 1 will be described with reference to FIGS. 5 to 6.
The artificial three-dimensional tissue manufacturing method according to the present invention is provided in a region facing each other and facing each other in the X direction with respect to a linear tissue module M having a gel body 2 containing cells and extending in the X direction. A pair of anchor members 10 for engaging the first engaging portion 12b and the second engaging portion 12c from the inside in the X direction and supporting them from the outside in the X direction are prepared, and a pair supporting the tissue module M. A plurality of anchor members 10 that can be held side by side in a direction intersecting the X direction are held in a state of being positioned in the X direction, and a tissue module held by the holder member 20 via a pair of anchor members 10. The present invention comprises immersing M in a medium and culturing it.

(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 member 10 is inserted and held in the holding portion 31 of the mold member 30 shown in FIG. Subsequently, a solution of the extracellular matrix component containing cells is injected into the module molding portion 32 of the mold member 30.

In this embodiment, myoblasts are used as cells, and a skeletal muscle module is produced as a tissue module M. The cell density of myoblasts is preferably 1.0 × 10 ⁶ to 1.0 × 10 ⁸ cells / mL, preferably 1.0 × 10 ⁷ to 5.0 × 10 ⁷ cells / mL. Is more preferable.

As the extracellular matrix component, a mixture of Matrigel (registered trademark) and Fibrinogen is used. The mixing ratio of Matrigel (registered trademark) and fibrinogen is preferably 20 to 80% for Matrigel (registered trademark) and 80 to 20% for fibrinogen, 40 to 60% for Matrigel (registered trademark), and 60 for fibrinogen. More preferably, it is -40%.

When a solution of the extracellular matrix component containing cells is injected into the module molding unit 32, the extracellular matrix component is cultured under predetermined conditions to gel (solidify) the extracellular matrix component to form a gel body 2. As an example of the culture conditions, the cells are cultured at a temperature of 37 ° C. for 30 minutes. By gelling the extracellular matrix component, the gel bodies 2 located at the ends on both sides in the X direction are engaged with and supported by the anchor portion 12. As a result, a tissue module M having a gel body 2 containing myoblasts is formed in which the outer side in the X direction is supported by a pair of anchor members 10.

In this state, since the culture time is short and the skeletal muscle tissue is immature, the tissue module M and the pair of anchor members 10 are taken out from the mold member 30 using tweezers or the like and cultured in a culture tank or the like to mature the skeletal muscle tissue. .. In the tissue module M in which the skeletal muscle tissue has matured, the contraction of the gel body 2 progresses. Since the outer shape of the module molding portion 32 on both sides in the Y direction is a curve in which the dimension in the Y direction gradually increases from the end of the holding portion 31 where the substrate 11 of the anchor member 10 is located toward the center in the X direction, the contraction shrinks. The advanced gel body 2 is a square columnar whose outer shape on both sides in the Y direction is parallel to the X direction.

[Retaining the tissue module M and the anchor member 10 to the holder member 20]
Before holding the tissue module M in which the skeletal muscle tissue has matured and the pair of anchor members 10 in the holder member 20, the pair of base materials 21 are placed in a culture tank (not shown) in which the medium is stored. The liquid level of the medium is the height at which the uppermost tissue module M held by the holder member 20 is immersed.

Next, as shown in FIG. 4, the tissue module M in which the skeletal muscle tissue has matured and the pair of anchor members 10 are fitted with the fitting groove 11a in the anchor member 10 into the fitting wall 24 in the base material 21 from above. Let me. As a result, the pair of anchor members 10 are positioned in the X direction, the Y direction, and the Z direction with respect to the base material 21.

[Culture of tissue module M held in holder member 20]
In the present embodiment, a total of nine tissue modules M and a pair of anchor members 10 in three rows in the Y direction and three stages in the Z direction are arranged side by side in each direction. 5 and 6 are schematic cross-sectional views orthogonal to the X direction of the tissue module M. As shown in FIG. 5, the tissue module M held on the base material 21 in three rows × three stages is easily fluctuated because it is immersed in the medium C, and it is difficult for the adjacent tissue modules M to fuse with each other. Therefore, the medium C is temporarily removed from the culture tank in which the pair of base materials 21 are installed.

By removing the medium C, as shown in FIG. 6, the tissue module M in three rows × three stages is aggregated by the action of surface tension. FIG. 7 is a photographic view of a tissue module M having skeletal muscle tissue and agglomerated. The aggregated tissue module M is supplied with a solution of the cell adhesive g (Hydrogel solue) to maintain the aggregated state. Examples of the cell adhesive g include hydrogels in a medium containing fibrinogen (4 mg / ml) and thrombin (50 UN / ml).

By culturing the tissue module M supplied with the cell adhesive g for 10 minutes, a layer of the cell adhesive g is formed around the tissue module M having three rows × three stages, as shown in FIG. After that, the medium C is stored in the culture tank again, and the tissue module M is immersed in the medium. Since the tissue module M having three rows and three stages is maintained in an aggregated state by the layer of the cell adhesive g, it fuses without shaking even when immersed in the medium C. As a result, the culture can be carried out in a state where the tissue modules M of three rows × three stages are fused.

[Shrinkage of tissue module M]
The contraction of the tissue module M having the skeletal muscle tissue manufactured by the above artificial three-dimensional tissue manufacturing method was confirmed. A three-row x one-stage tissue module M was used to confirm shrinkage. FIG. 8 is a photograph showing the initial stage of culturing (Day 0) cultivated using the artificial three-dimensional tissue manufacturing apparatus 1 and the skeletal muscle tissue after culturing for 11 days. As shown in FIG. 8, as the culture progressed and matured, one skeletal muscle tissue in which three tissue modules M contracted was observed.

In FIG. 9, the relationship between the number of culture days and the amount of change in the width of the skeletal muscle tissue was measured for each of the 1-row × 1-stage tissue module M, the 2-row × 1-stage tissue module M, and the 3-row × 1-stage tissue module M. .. In FIG. 9, the width of the skeletal muscle tissue at the initial 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 the increase in the number of culture days.

[Contraction movement of skeletal muscle tissue by electrical stimulation]
The function of the skeletal muscle tissue manufactured by the above artificial three-dimensional tissue manufacturing method was confirmed. To confirm the function, the contractile movement when electrical stimulation was periodically applied to the skeletal muscle tissue via electrodes was measured. FIG. 10 is a diagram showing the relationship between the time (sec) and the displacement (μm) when an electric stimulus is applied to the skeletal muscle tissue at an electric field strength of 1.8 V / mm, 2 ms, and 1.0 Hz. ..
As shown in FIG. 10, it was confirmed that the skeletal muscle tissue was displaced with the application of electrical stimulation. Therefore, the skeletal muscle tissue produced by the artificial three-dimensional tissue manufacturing method of the present embodiment can exhibit the function as skeletal muscle. In addition, it was visually confirmed that the skeletal muscle tissue was displaced with the application of electrical stimulation.

FIG. 11 is a diagram showing a fluorescence image in the long axis direction of skeletal muscle tissue produced by culturing for 7 days by an artificial three-dimensional tissue production method. FIG. 12 is a diagram showing a fluorescence image in the short axis direction of the skeletal muscle tissue produced by the artificial three-dimensional tissue production method.
As shown in FIG. 11, it was possible to observe that muscle fibers 4a and nuclei 4b were formed in the skeletal muscle tissue. Further, as shown in FIG. 12, it was observed that enucleation due to malnutrition or oxygen deficiency did not occur in the skeletal muscle tissue.

[Fusion of Organization Module M]
FIG. 13 is a photographic view of skeletal muscle tissue manufactured by arranging two tissue modules M side by side. FIG. 14 is a cross-sectional view taken along the line AB in FIG.
As shown in FIG. 14, it was possible to observe that the skeletal muscle tissues produced side by side were fused and formed without any gaps.

FIG. 15 is a photographic diagram of two tissue modules M manufactured side by side. As shown in FIG. 15, in the early stage of culture (Day 0), as one of the tissue modules M, the skeletal muscle module M1 for producing the skeletal muscle tissue and as the other one of the tissue modules M, only the extracellular matrix component was used. The hydrogel module M2 cultured in the above was arranged side by side.

FIG. 16 is a photographic diagram of the skeletal muscle module M1 and the hydrogel module M2 after culturing for two days, and an enlarged photographic diagram of the boundary between the skeletal muscle module M1 and the hydrogel module M2.
As shown in FIG. 16, it was possible to observe that the hydrogel of the hydrogel module M2 fuses with the skeletal muscle tissue due to the adhesive substance from the cells in the skeletal muscle module M1.

[Fusion of skeletal muscle module and fat module]
FIG. 17 is a photographic diagram of two tissue modules M manufactured side by side.
As shown in FIG. 17, as one of the tissue modules M, a skeletal muscle module M1 for producing skeletal muscle tissue, and as another one of the tissue modules M, a fat module M3 having adipose tissue in which adipocytes are cultured. Lined up. Adipose tissue takes longer to mature than skeletal muscle tissue. Therefore, when holding the holder member 20 for confirmation of fusion, the skeletal muscle module M1 in the initial stage of culture (Day 0) and the fat module M3 that had been cultured for 30 days and matured (Day 30) were arranged side by side (upper photo). ). The lower photograph shows the skeletal muscle module M1 and the fat module M3 that have been cultured for another 13 days while being held by the holder member 20.

FIG. 18 is an enlarged photograph (left photograph) of the skeletal muscle module M1 and the fat module M3 after culturing for 13 days, and a fluorescent image (on the right) of a further enlarged portion of the fusion of the skeletal muscle module M1 and the fat module M3. Photo) is shown.
As shown in FIG. 18, it was possible to observe that granular lipids were fused and formed in the skeletal muscle tissue having myotubes and nuclei.

As described above, in the artificial three-dimensional tissue manufacturing apparatus and the artificial three-dimensional tissue manufacturing method of the present embodiment, the tissue modules M having individually matured tissues can be arranged on the holder member 20 and cultured in the same process, so that the efficiency is high. It is possible to construct an artificial three-dimensional organization. Further, in the artificial three-dimensional tissue manufacturing apparatus and the artificial three-dimensional tissue manufacturing method of the present embodiment, at least two kinds of tissue modules M having different cells in the Y direction and the Z direction can be arranged and cultured in an arbitrary arrangement, so that various types can be obtained. It is possible to construct an artificial three-dimensional organization with high versatility corresponding to the problem.

[Second Embodiment of Artificial Three-Dimensional Tissue Manufacturing Equipment]
Subsequently, the second embodiment of the artificial three-dimensional structure manufacturing apparatus 1 will be described with reference to FIGS. 19 to 35.
In these figures, the same components as the components of the first embodiment shown in FIGS. 1 to 18 are designated by the same reference numerals, and the description thereof will be omitted.

In the second embodiment, a configuration for forming a tissue module M having a perfusion channel for constructing a tissue while perfusing the 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 flow path. FIG. 20 is a cross-sectional view of the pair of anchor members 10A along the X direction.

As shown in FIG. 20, the anchor member 10A has a through hole 14 penetrating in the X direction. In the pair of anchor members 10A, the through holes 14 are formed coaxially with the same diameter. The anchor portion 12 in the anchor member 10A has a shaft-shaped portion 15a and a third engaging portion (engaging portion) 15b. The shaft-shaped portion 15a protrudes from the end surface 11b of the substrate 11 and extends in the X direction. The third engaging portion 15b is provided at the tip of the shaft-shaped portion 15a. The dimension of the third engaging portion 15b in the direction orthogonal to the X direction is larger than that of the axial portion 15a. At least one of the shaft-shaped portion 15a and the third engaging portion 15b may be cylindrical or square tubular.

The gel body 2 of the tissue module M arranged in the space between the end surface 11b and the third engaging portion 15b on the outer peripheral side of the shaft-shaped portion 15a has a third engaging portion when contracted in the X direction. Engage with 15b from the outside in the X direction. That is, the third engaging portion 15b engages with the gel body 2 of the tissue module M from the inside in the X direction to suppress the contraction of the tissue module M. Further, the gel body 2 is crimped to the outer peripheral surfaces of the axial portion 15a and the third engaging portion 15b due to the contraction of the axial portion 15a in the central axial direction. Therefore, the frictional force between the gel body 2 and the anchor member 10A becomes large, and the shrinkage of the tissue module M in the direction orthogonal to the X direction is suppressed.

As shown in FIG. 19, a shaft-shaped member 33 is freely inserted and removed in the through holes of the pair of anchor members 10A held by the holding portion 31 of the 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.

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

First, as shown in FIG. 19, a pair of anchor members 10A in a state where the shaft-shaped member 33 is inserted into the through hole 14 is inserted into the holding portion 31 of the mold member 30A from above. As a result, the pair of anchor members 10A are positioned in the X direction, the Y direction, and the Z direction with respect to the mold member 30A.

Next, as shown in FIG. 21, a solution (suspension) of extracellular matrix components containing skeletal muscle cells as tissue S is injected into the module molding portion 32 of the mold member 30A. Then, for example, the cells are cultured at a temperature of 37 ° C. for 2 days (48 hours) to form a tissue module M composed of a gel body 2 having a skeletal muscle tissue as a gel layer.

Subsequently, as shown in FIG. 22, the pair of anchor members 10A and the tissue module M are removed from the mold member 30A. Further, the shaft-shaped member 33 penetrating the pair of anchor members 10A and the tissue module M is extracted. By extracting the shaft-shaped member 33, a perfusion flow path 34 that communicates coaxially with the through hole 14 of the pair of anchor members 10A and extends in the X direction is formed inside the tissue module M. The tissue module M at this point is a skeletal muscle module having a perfusion channel 34.

Subsequently, as shown in FIG. 23, the luminal layer 36 is cultured for a certain period of time by injecting (seeding) the vasculature cells 35 into the wall surface facing the perfusion flow path 34 through the through hole 14 of the anchor member 10A. To form. As a result, the perfusion channel 34 is surrounded by the luminal layer 36. That is, the tissue module M is formed in the shape of a square tube in which the gel body 2 having the skeletal muscle tissue on the outer peripheral side of the lumen layer 36 is provided as the gel layer. As the vasculature cell 35, vascular endothelial cells (HUVEC) are used as an example.

When the tissue module M having the perfusion flow path 34 is formed inside the skeletal muscle tissue, as shown in FIG. 24, the supply pipe 41 connected to the medium supply unit 40 is connected to one of the pair of anchor members 10A. , The discharge pipe 42 is connected to the other of the pair of anchor members 10A. Then, by supplying the medium C1 from the medium supply unit 40 to the perfusion flow path 34 through the supply pipe 41 and the through hole 14, the skeletal muscle tissue can be cultured while perfusing the medium C1.

In the above artificial three-dimensional tissue manufacturing method, a procedure for forming a tissue module M in which a perfusion flow path 34 is formed inside a skeletal muscle tissue is exemplified, but the procedure is not limited to this configuration, and for example, cells are included. A solution of extracellular matrix components may be injected into the module molding section 32 and then cultured to form a gel layer. In this case, as a subsequent procedure, the luminal layer 36 (perfusion flow path 34) is formed in the hydrogel by extracting the axial member 33, injecting and culturing the vascular cells 35 in the same manner as described above. The vascular system module M4 (see FIG. 25) can be manufactured.
Further, a solution (suspension) of extracellular matrix components containing vascular cells such as vascular endothelial cells (HUVEC) as tissue S is injected into the module molding portion 32 of the mold member 30A shown in FIG. 21 and cultured. May be good. In this case, vascular cells are connected to each other to form a vessel. Then, as a subsequent procedure, the vascular system module M4 (see FIG. 25) in which the luminal layer 36 (perfusion flow path 34) is formed can be manufactured by extracting the axial member 33 in the same manner as described above. ..

In FIG. 25, the above-mentioned vascular system module M4 is arranged in the center of a total of nine tissue modules M having three rows in the Y direction and three stages in the Z direction on the holder member 20, and eight around the vascular system module M4. It is a schematic cross-sectional view orthogonal to the X direction of the artificial three-dimensional tissue in which the skeletal muscle module M1 is arranged.

The skeletal muscle module M1 was cultured while perfusing the medium C1 from the medium supply unit 40 shown in FIG. 24 into the luminal layer 36 in the central vascular system module M4. FIG. 26 is a photographic view of an artificial three-dimensional structure viewed in the Z direction. FIG. 27 is a photographic view of a cross section orthogonal to the X direction of the artificial three-dimensional structure. FIG. 28 is a photographic view of a cross section of the artificial three-dimensional structure along the X direction.

As shown in FIG. 27, a vascular system module M4 in which the lumen layer 36 remains is formed in the center, and an artificial three-dimensional structure in which the skeletal muscle tissue formed by the eight skeletal muscle modules M1 covers the circumference of the vascular system module M4. I was able to observe that the tissue was being constructed. Further, as shown in FIG. 28, it was possible to observe that the luminal layer 36 was formed through the artificial three-dimensional tissue.

FIG. 29 is a photographic view of a cross section of an artificial three-dimensional tissue in which a liquid containing a fluorescent substance flows in the lumen layer 36 along the X direction. FIG. 30 is a fluorescence image 15 minutes after flowing a liquid containing a fluorescent substance. Diffusion of the fluorescent substance could not be confirmed immediately after flowing the liquid containing the fluorescent substance, but as shown in FIG. 30, 15 minutes after flowing the liquid containing the fluorescent substance, the skeletal muscle from the lumen layer 36 It was possible to observe that the fluorescent substance was diffused in the tissue.

[Comparison with and without perfusion]
FIG. 31 is a photograph of skeletal muscle tissue cultured without perfusion. FIG. 32 is a photograph of skeletal muscle tissue cultured with perfusion. As shown in FIGS. 31 and 32, changes were observed in that the skeletal muscle tissue cultured with perfusion became thinner than the skeletal muscle tissue cultured without perfusion. In this case, it is considered that the degree of orientation of the muscle tissue may be increased by the perfusion flow stimulation.

FIG. 33 is a cross-sectional photograph of the skeletal muscle tissue cultured with and without perfusion. As shown in FIG. 33, it was confirmed that the luminal layer remained in the case of perfusion. In the case of perfusion, it was confirmed that the nuclei were scattered as a whole. From these facts, it is considered that the survival rate is improved by culturing while perfusing.

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

As shown in FIG. 34, the skeletal muscle tissue cultured with perfusion had a larger amount of contraction when electrical stimulation was applied than the skeletal muscle tissue cultured without perfusion. Therefore, it is considered that the skeletal muscle tissue cultured with perfusion progresses faster than the skeletal muscle tissue cultured without perfusion.

[Blood vessel differentiation]
FIG. 35 is a photograph of the above-mentioned vasculature module M4 cultured.
As shown in FIG. 35, it was confirmed that new blood vessels were differentiated from the thick blood vessels forming the luminal layer 36. Thereby, it can be suitably used as a blood vessel module when forming a blood vessel in another tissue in combination with a tissue module M containing other cells.

As described above, in the artificial three-dimensional tissue manufacturing apparatus and the artificial three-dimensional tissue manufacturing method of the present embodiment, the holder member 20 has a perfusion flow path 34 near the tissue module M for forming an arbitrary tissue. By arranging the tissue module M, it becomes possible to suppress the occurrence of oxygen deficiency and nutritional deficiency of cells and to construct a good artificial three-dimensional tissue.

Although the preferred embodiments according to 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 the above examples. The various shapes and combinations of the constituent members shown in the above-mentioned example are examples, and can be variously changed based on design requirements and the like within a range not deviating from the gist of the present invention.

For example, in the above embodiment, as the tissue module M, 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 are combined with the holder member 20, respectively. However, the configuration is not limited to this combination. For example, as shown in FIG. 36, the vascular system module M4 may be arranged in the center, and the skeletal muscle module M1 and the fat module M3 may be arranged around the vascular system module M4. By adopting this configuration, it is possible to construct an artificial three-dimensional tissue in which adipose tissue is formed at an arbitrary position in the skeletal muscle tissue.

Further, in the above embodiment, as the tissue module M, a module having skeletal muscle tissue, a module having adipose tissue, and a vascular system module are exemplified, but the configuration is not limited to this. For example, a dermis tissue module having dermis cells as cells S and an epidermal tissue module having epidermal cells as cells S may be used. In this case, for example, the dermis tissue module can be placed around the vascular system module and the dermis tissue can be cultured while perfusing. It is also possible to place the dermis tissue module around the vascular system module and the epidermis tissue module around the dermis tissue module to cultivate the epidermis tissue while performing perfusion to form an artificial three-dimensional skin tissue. Is.

Further, in the above embodiment, a holder member capable of holding a three-row × three-stage tissue module M is illustrated, but the configuration is not limited to this. For example, as shown in FIG. 37, there is a configuration using a holder member capable of holding a seven-row × three-stage tissue module M, or a configuration using a holder member capable of holding another number of rows and stages of tissue module M. You may. When using a tissue module M having more than three rows × three stages, it is preferable to arrange a part of all the tissue modules M other than the vascular system module M4 in contact with the vascular system module M4 from the viewpoint of suppressing the death of the tissue. ..

The artificial three-dimensional tissue manufactured by the above-mentioned artificial three-dimensional tissue manufacturing apparatus and artificial three-dimensional tissue manufacturing method shall be used for drug screening (drug evaluation) such as the reaction of the tissue to the addition of the drug and the view of the interaction between the tissues. Can be done. Drugs include drugs such as pharmaceuticals, cosmetics and quasi-drugs. According to this drug screening, for example, the drug can be evaluated in an environment closer to the actual tissue as compared with the conventional method. Further, for example, it is extremely useful in the dynamic evaluation of drugs having various molecular weights in the creation of new drugs and the evaluation in the development of cosmetics and quasi-drugs.

Further, for the artificial three-dimensional structure manufactured by the above-mentioned artificial three-dimensional structure manufacturing apparatus and the artificial three-dimensional structure manufacturing method, an electrode may be provided on the anchor member 10 to apply electrical stimulation. By adopting this configuration, it is possible to observe and evaluate changes in blood vessels and adipose tissue due to contraction of skeletal muscle tissue.

The present invention can be applied to an artificial three-dimensional tissue manufacturing apparatus and an artificial three-dimensional tissue manufacturing method.

1 ... Artificial three-dimensional structure manufacturing device, 2 ... Gel body, 10, 10A ... Anchor member, 11a ... Fitting groove, 12b ... First engaging part (engaging part), 12c ... Second engaging part (engaging) Part), 15b ... Third engaging part (engaging part), 20 ... Holder member, 30, 30A ... Mold member, 33 ... Axial member, 34 ... Perfusion channel, 35 ... Vascular cells, 36 ... Cavity Layer, 40 ... medium supply part, g ... cell adhesive, M ... tissue module, T ... tissue

Claims (12)

1. A pair of anchor members that face each other apart from each other in the first direction and support the ends of the linear tissue modules extending in the first direction from the outside in the first direction, respectively.
   A holder member that holds the pair of anchor members that support the tissue module in a state of being positioned in the first direction, and a holder member.
   Equipped with
   The tissue module has a gel containing cells and
   The pair of anchor members each have an engaging portion that engages from the inside in the first direction with an end portion of the tissue module in a region facing each other.
   The holder member can hold a plurality of the pair of anchor members supporting the tissue module side by side in a direction intersecting the first direction.
   Artificial three-dimensional tissue manufacturing equipment.
2. The holder member is
   The pair of base materials arranged apart from each other in the first direction and the upper surface of the base material are arranged facing each other in the second direction orthogonal to the first direction, and the first direction and the second direction Has a fitting wall extending in a third direction orthogonal to
   Each of the pair of anchor members has a fitting groove penetrating in the third direction on both side surfaces in the second direction.
   The fitting wall fits into the fitting groove to position the anchor member in the first direction.
   The artificial three-dimensional tissue manufacturing apparatus according to claim 1.
3. The dimension of the fitting wall in the third direction is a dimension of fitting across the plurality of anchor members arranged in the third direction.
   The artificial three-dimensional tissue manufacturing apparatus according to claim 2.
4. Three or more of the fitting walls are arranged so as to face each other 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 fitting wall is fitted across both of the opposing fitting grooves in the anchor member adjacent to the second direction.
   The artificial three-dimensional tissue manufacturing apparatus according to claim 2 or 3.
5. The pair of anchor members each have a through hole coaxial with each other and penetrating in the first direction.
   It has a shaft-shaped member provided in the through hole of the pair of anchor members so as to be freely inserted and removed.
   The artificial three-dimensional tissue manufacturing apparatus according to any one of claims 1 to 4.
6. It has a culture medium supply unit that supplies a medium to the tissue module from the outside in the first direction through the through hole.
   The artificial three-dimensional tissue manufacturing apparatus according to claim 5.
7. With respect to the linear tissue module having a gel body containing cells and extending in the first direction, an engaging portion provided in a region facing each other and facing each other apart from each other in the first direction engages from the inside of the first direction. In addition to preparing a pair of anchor members that support from the outside in the first direction,
   A holder member capable of arranging and holding a plurality of the pair of anchor members supporting the tissue module in a direction intersecting the first direction is held in a state of being positioned in the first direction.
   The tissue module held by the holder member via the pair of anchor members is immersed in a medium and cultured.
   Artificial three-dimensional tissue manufacturing method including.
8. The holder member has a base material and has a base material.
   A plurality of the pair of anchor members supporting the tissue module can be arranged side by side in a second direction orthogonal to the first direction along the upper surface of the base material, and the first direction and the second direction Multiple pieces can be arranged side by side in the third direction orthogonal to
   The artificial three-dimensional structure manufacturing method according to claim 7.
9. A plurality of the tissue modules are arranged on the holder member via the anchor member along at least one of the second direction and the third direction while being immersed in the medium.
   To remove the medium in which the plurality of tissue modules are immersed to aggregate the plurality of tissue modules, and to aggregate the plurality of tissue modules.
   By supplying a cell adhesive to the aggregated tissue module to maintain the aggregated state,
   By immersing the tissue module in the medium again and culturing the aggregated state,
   including,
   The artificial three-dimensional structure manufacturing method according to claim 8.
10. At least two types of tissue modules with different cells are arranged on the holder member via the anchor member.
    The artificial three-dimensional structure manufacturing method according to claim 9.
11. Preparing the pair of anchor members to support the tissue module from the outside in the first direction can be done.
    To prepare a pair of anchor members having through holes coaxial with each other and penetrating in the first direction, and having a shaft-shaped member extending in the first direction straddling the through holes.
    The pair of anchor members into which the axial members are inserted is held by the mold member forming the tissue module in a state of being positioned in the first direction.
    By supplying at least an extracellular matrix to the mold member and culturing it to form a gel layer,
    The axial member is extracted from the pair of anchor members and the gel layer to form a perfusion flow path penetrating 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 artificial three-dimensional structure manufacturing method according to any one of claims 7 to 10.
12. An extracellular matrix containing the cells different from the vasculature cells is supplied to the mold member and cultured.
    The artificial three-dimensional structure manufacturing method according to claim 11.

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