JP2021023289A - Artificial three-dimensional tissue composite, production method of artificial three-dimensional tissue composite, and production device of artificial three-dimensional tissue composite - Google Patents

Abstract

To provide an artificial three-dimensional tissue composite (TC) comprising high functionality and equivalence to a living body and a production method thereof, and a production device thereof.SOLUTION: A production method of an artificial three-dimensional tissue composite comprises: a preparing step for preparing a substrate which comprises: a restriction region 71 which has a holding plane 11 having first regions 61 for holding a first gel body including a first cell respectively, and a second region 62 for holding a second gel body including a second cell, in which the first regions protrude from the holding plane and can restrict the first gel body; and a non-restriction region 72 not for restricting the first gel body; and a culturing step for culturing the first gel bodies including the first cell for forming first tissues 51, and culturing the second gel body including the second cell for forming a second tissue 52 on each of whose both ends, the first tissue is connected. In the culturing step, the second tissue which is oriented in a first direction by shrinkage on the second region is formed, and the first tissues which are oriented in the first direction by extension by shrinkage force of the second tissue and are formed on the first regions are formed on the non-restriction region.SELECTED DRAWING: Figure 1

Description

The present invention relates to an artificial three-dimensional tissue complex, an artificial three-dimensional tissue complex manufacturing method, and an artificial three-dimensional tissue complex manufacturing apparatus.
This application claims priority based on US Patent Provisional Application No. 62 / 880,808 filed on July 31, 2019, the contents of which are incorporated herein by reference.

Attempts have been made to construct skeletal muscle tissue outside the body and use it as a drug discovery model for evaluating drug response, industrially producible cultured meat, and an actuator element for a biorobot. However, it was not possible to reproduce the function and morphology of the muscle-tendon complex between the muscle tissue and the tendon tissue realized in the living body only by constructing the skeletal muscle tissue, and the equivalence with the living body was low.

Non-Patent Document 1 discloses that a scaffolding material, muscle cells, and tendon cells are used to form a junction. Non-Patent Document 2 discloses that it imparts extension stimulation to tendon cell differentiation of human bone marrow-derived mesenchymal stem cells. Non-Patent Document 3 discloses that a 3D printer is used to join the tendon tissues arranged on the left and right and the muscle tissue arranged in the center.

Co-electrospun dual scaffolding system with potential for muscle-tendon junction tissue engineering, Biomaterials 32 (2011) 1549-1559 Tendon Tissue Engineering: Effects of Mechanicaland Biochemical Stimulation on Stem Cell Alignment on Cell-Laden Hydrogel Yarns, Adv Healthcare Mater. 2019, 8, 1801218 A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues, SLAS Technology 2018, Vol.23 (6) 599-613

However, Non-Patent Document 1 discloses a technique using skin fibroblasts, and cannot be said to be a technique relating to a muscle-tendon complex. Further, considering the equivalence with a living body, the orientation of the muscle tissue and the tendon tissue is also necessary, but Non-Patent Document 1 does not clearly describe the orientation. In Non-Patent Document 2, although there is a description regarding orientation by imparting a stretch stimulus, it cannot be said that cells and tissues are functional because the expression of collagen is not high. In Non-Patent Document 3, the cut at the interface between the tendon tissue and the muscle tissue cannot be completely eliminated, and the orientation with respect to the tendon tissue is not specified.

The present invention has been made in consideration of the above points, and is an artificial three-dimensional tissue complex, an artificial three-dimensional tissue complex manufacturing method, and an artificial three-dimensional tissue complex having high functionality and equivalence with a living body. It is an object of the present invention to provide a manufacturing apparatus.

According to the first aspect of the present invention, it is arranged between the first region which is arranged apart from each other in the first direction and holds the first gel body containing the first cell, and the separated first region. It has a holding surface having a second region for holding the second gel body containing the second cell, and each of the first regions is located outside the first direction and has a gap from the position of the holding surface. A restraint region provided with a plurality of anchors capable of restraining the first gel body, and a non-constraint region located inside the restraint region in the first direction and unconstraining the first gel body. In the preparatory step of preparing a base material having a region, a liquid containing the first cell is supplied to each of the first regions and gelled to form the first gel body, and the second region is formed. A gelation step of supplying a liquid containing the second cell and gelling to form the second gel body, and culturing the first gel body containing the first cell to form a first tissue. At the same time, a culture step of culturing the second gel body containing the second cell to form a second tissue in which the first tissue is connected to both ends is included, and in the culture step, contraction occurs in the second region. The second structure oriented in the first direction is formed by the method, and among the first structures formed in the first region, the first structure is stretched by the contraction force of the second structure and oriented in the first direction. Provided is a method for producing an artificial three-dimensional tissue complex in which a first structure is formed in the unconstrained region.

According to the second aspect of the present invention, a base material having a holding surface is provided, and the holding surfaces are arranged apart from each other in a first direction and each hold a first gel body containing a first cell. It has one region and a second region that is located between the separated first regions and holds a second gel body containing a second cell, and each of the first regions is outside the first direction. A restraint region provided with a plurality of anchors located at the position of the holding surface and protruding from the position of the holding surface with a gap from each other and capable of restraining the first gel body, and a restraint region located inside the restraint region in the first direction. An artificial three-dimensional tissue complex manufacturing apparatus having an unconstrained region in which the first gel body is unconstrained is provided.

According to the third aspect of the present invention, the first tissue arranged apart from each other in the first direction and the second structure oriented in the first direction and extending and connecting the separated first structure are provided. Each of the first tissues is located outside the first direction and is located between the non-oriented portion that is not oriented in the first direction and the second structure and the non-oriented portion. An artificial three-dimensional tissue complex having an oriented portion oriented in one direction is provided.

In the present invention, an artificial three-dimensional tissue complex having high functionality and high equivalence with a living body can be obtained.

It is a figure which shows the embodiment of this invention, is the external perspective view which the artificial three-dimensional tissue complex TC is manufactured and held by the artificial three-dimensional tissue complex manufacturing apparatus 1. It is an external perspective view of the manufacturing apparatus 1 which concerns on this invention. It is an external perspective view which shows the process which concerns on the manufacturing of the artificial three-dimensional structure complex TC. It is an external perspective view which shows the process which concerns on the manufacturing of the artificial three-dimensional structure complex TC. It is an external perspective view which shows the process which concerns on the manufacturing of the artificial three-dimensional structure complex TC. It is an external perspective view which shows the process which concerns on the manufacturing of the artificial three-dimensional structure complex TC. It is a photograph figure which imaged the culture state of the tendon tissue 51 and the muscle tissue 52. It is a figure which shows the density which contains the tendon fibroblast, and the HE stained image and the Hoechst stained image of the tendon tissue 51 produced at the density. It is a figure which shows the density which contains the tendon fibroblast, and the HE stained image and the Hoechst stained image of the tendon tissue 51 produced at the density. It is a figure which shows the density which contains the tendon fibroblast, and the HE stained image and the Hoechst stained image of the tendon tissue 51 produced at the density. It is a figure which shows the density which contains the tendon fibroblast, and the HE stained image and the Hoechst stained image of the tendon tissue 51 produced at the density. It is a figure which shows the HE stained image and the Hoechst stained image of the tendon tissue 51 of the restraint area 71. It is a figure which shows the breaking strength of the tendon tissue 51 and the muscle tissue 52. It is a figure which shows the relationship between the culture days in the tendon tissue 51 and the change rate of the diameter. It is a figure which shows the HE stained image and the Hoechst stained image of the tendon tissue 51 of the restraint area 71. It is a figure which shows the relationship between the time when the electric stimulus is applied to the tissue at a certain cycle, and the amount of movement of the tissue by the electric stimulus. It is a figure which shows the relationship between the time when the electric stimulus is applied to the tissue at a certain cycle, and the amount of movement of the tissue by the electric stimulus.

Hereinafter, embodiments of the artificial three-dimensional tissue complex, the artificial three-dimensional tissue complex manufacturing method, and the artificial three-dimensional tissue complex manufacturing apparatus of the present invention will be described with reference to FIGS. 1 to 17.
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.

FIG. 1 is a diagram showing an embodiment of the present invention, in which an artificial three-dimensional tissue complex TC is manufactured (cultured) by an artificial three-dimensional tissue complex manufacturing apparatus 1 (hereinafter referred to as a manufacturing apparatus 1). It is an external perspective view which is held.

(Artificial three-dimensional tissue complex)
First, the artificial three-dimensional tissue complex TC according to the present invention will be described.
As shown in FIG. 1, the artificial three-dimensional tissue complex TC has a first tissue 51 and a second tissue 52. At least a part of the artificial three-dimensional tissue complex TC is held from below on the holding surface 11 (details will be described later) in the manufacturing apparatus 1. The first tissues 51 are provided in pairs in a separated state. The second tissue 52 extends in a direction in which the first tissue 51 is separated from each other. The second organization 52 connects the separated first organizations 51 to each other.

Each of the first structures 51 is located outside in a direction away from each other and is not oriented in that direction, and is located between the second structure 52 and the non-oriented portion 51A and is oriented in the direction of separation. It has an oriented portion 51B (details will be described later).

In the following, the pair of first structures 51 are separated from each other, the direction in which the second structure 52 extends is the Y direction, the direction parallel to the holding surface 11 and orthogonal to the Y direction is the X direction, and the Y direction and The direction orthogonal to the X direction will be appropriately described as the Z direction. Further, the Z direction will be appropriately described with the side on which the artificial three-dimensional tissue complex TC is held as the upper side and the opposite side as the lower side with respect to the manufacturing apparatus 1. However, this defines the vertical direction only for convenience of explanation, and does not limit the relative positions of the artificial three-dimensional tissue complex TC and the manufacturing apparatus 1 according to the present invention.

(Manufacturing equipment 1)
Next, the manufacturing apparatus 1 according to the present invention will be described with reference to FIG.
FIG. 2 is an external perspective view of the manufacturing apparatus 1.
As shown in FIG. 2, the manufacturing apparatus 1 includes a base material 10 and a pair of holders 20.

In the following, the direction in which the pair of holders 20 are separated is the Y direction (first direction), and the direction parallel to the holding surface 11 described later and orthogonal to the Y direction is the X direction (second direction). And the direction orthogonal to the X direction will be described as appropriate as the Z direction. Further, the Z direction will be appropriately described with the side on which the artificial three-dimensional tissue complex TC is held as the upper side and the opposite side as the lower side with respect to the manufacturing apparatus 1. However, this defines the vertical direction only for convenience of explanation, and does not limit the relative positions of the artificial three-dimensional tissue complex TC and the manufacturing apparatus 1 according to the present invention.

As an example, the base material 10 is an elastomer, and has an artificial three-dimensional structure described later, such as a soft elastic material that is elastically deformable such as silicone rubber and PDMS (polydimethylsiloxane), and a hard elastic material such as a resin material and a metal material. It is made of a material that can be elastically deformed by the tension of the composite TC.
The base material 10 is formed in a rectangular parallelepiped shape extending in the Y direction. In the following description, the side of the base material 10 facing the center in the Y direction is referred to as the inside, and the side of the base material 10 facing in the direction opposite to the inside is appropriately referred to as the outside.

The base material 10 has a holding surface 11 on the upper side. The holding surface 11 holds a part of the first structure 51 and the second structure 52 from below. The holding surface 11 has a first region 61 located outside in the Y direction for culturing and forming the first tissue 51, and a first region 61 located inside the Y direction for culturing and forming the second tissue 52. It has two regions 62.

The base material 10 has a recess 12 that opens into the holding surface 11. The recess 12 has a first recess 13 and a second recess 14. The first recesses 13 are provided on both ends in the Y direction at equidistant positions from the center of the base material 10 in the Y direction. The first recess 13 extends in the X direction and opens on both side surfaces of the base material 10 in the X direction. The first recess 13 has a rectangular cross section orthogonal to the X direction. A part of the holder 20 can be detachably fitted to the first recess 13.

The second recess 14 extends inward in the Y direction from the center of the first recess 13 in the X direction. That is, the first recess 13 and the second recess 14 are formed in a T shape when viewed from above. The position of the inner end of the second recess 14 is outside the boundary between the first region 61 and the second region 62. The second recess 14 has a rectangular cross section orthogonal to the Y direction. A part of the holder 20 can be detachably fitted to the second recess 14.

The holder 20 is formed in a T shape when viewed from above. The holder 20 has a first portion 20A extending in the X direction and a second portion 20B extending inward in the Y direction from the center of the first portion 20A in the X direction. The first portion 20A can be detachably fitted into the first recess 13, and is positioned in the Y direction when fitted into the first recess 13. The second portion 20B can be detachably fitted into the second recess 14, and is positioned in the X direction when fitted into the second recess 14. As an example, the holder 20 is made of a resin material such as epoxy or acrylic.

Each holder 20 has a second holding surface 22 and a plurality of anchors 21 provided on the second holding surface 22 with a gap from each other and projecting to the + Z side. The second holding surface 22 is flush with the holding surface 11 when the holder 20 is fitted and mounted in the recess 12.

The anchor 21 is a columnar shape having a circular cross-sectional shape, and is formed as an example having a diameter of several tens to several hundreds μm and a length of about 1 mm. The anchors 21 are arranged in a matrix as an example. The anchors 21 are arranged in a grid pattern or a staggered pattern. From the viewpoint of restraining the first tissue 51 in the Y direction, the anchors 21 are more staggered in that the adjacent rows are arranged in a half pitch offset in the Y direction with respect to the rows arranged at intervals in the X direction. preferable. The holder 20 is coated with at least the surface of the second holding surface 22 and the anchor 21 with a material that does not adversely affect cells, for example, polyparaxylylene (so-called parylene).

The first region 61 for culturing and forming the first tissue 51 is located in the region where the anchor 21 is arranged outside in the Y direction, and the restraint region 71 in which the first tissue 51 is restrained by the anchor 21 and It has an unconstrained region 72 that is located inside the region where the anchor 21 is arranged in the Y direction and makes the first structure 51 unconstrained with respect to the anchor 21. The recess 12 is formed in the restraint region 71.

Subsequently, a method of manufacturing the artificial three-dimensional tissue complex TC by the above-mentioned manufacturing apparatus 1 will be described.
In the method for producing an artificial three-dimensional tissue complex according to the present invention, a first region 61, which is arranged apart from each other in the first direction and holds a first gel body containing a first cell, and a separated first region 61, respectively. The holding surface 11 has a second region 62 which is arranged between the two and holds the second gel body containing the second cell, and each of the first regions 61 is located outside in the first direction and holds the holding surface 11. A restraint region 71 provided with a plurality of anchors 21 that protrude from each other with a gap from each other and can restrain the first gel body, and a restraint region 71 that is located inside the restraint region 71 in the first direction and does not restrain the first gel body. In addition to the preparatory step of preparing the base material 10 having the non-restraint region 72, and supplying a liquid containing the first cells to each of the first regions 61 to gel them to form the first gel body. The gelation step of supplying a liquid containing the second cells to the second region 62 and gelling the gel to form the second gel body, and culturing the first gel body containing the first cells to form the first tissue. Including a culture step of culturing a second gel body containing the second cells to form a second tissue 52 in which the first tissue 51 is connected to both ends, the culture step contracts in the second region 62. A second tissue 52 oriented in the first direction is formed by the above method, and of the first tissue 51 formed in the first region 61, the first structure 51 is extended by the contraction force of the second tissue 52 and oriented in the first direction. The tissue 51 is formed in the unconstrained region 72.

Hereinafter, a method for producing the artificial three-dimensional tissue complex TC will be described in detail with reference to FIGS. 3 to 6. 3 to 6 are external perspective views showing a process related to the production of the artificial three-dimensional tissue complex TC.
In the preparation step, as described above, the manufacturing apparatus 1 in which the holder 20 is fitted in the recess 12 of the base material 10 is prepared.

In the subsequent gelation step, as shown in FIG. 3, first, a liquid containing the first cell 31 is supplied to each of the holding surface 11 in the first region 61 and the second holding surface 22 of the holder 20, and then gelation is performed. Let (harden).

As the first cell 31, tendon fibroblasts are used as an example. As the liquid containing tendon fibroblasts, an extracellular matrix component for cell culture is preferably used. 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.

In this embodiment, a mixed solution of fibrin and Matrigel (registered trademark) is used as the liquid before gelation. The liquid containing the first cells 31 placed on the second holding surface 22 of the holder 20 is filled in the gap between the anchors 21 so as to surround the periphery of the plurality of anchors 21.

The density of tendon fibroblasts ^{is preferably 1 × 10 5} cells / ml or more and 1 × 10 ⁶ cells / ml or less.
If density tendon fibroblasts are contained is less than 1 × 10 ⁵ cells / ml, resulting in a sparse structure, sufficient collagen is produced to volume.
If the density of tendon fibroblasts are contained exceeds 1 × 10 ⁶ cells / ml, there is a possibility that the orientation of the collagen produced from tendon fibroblasts becomes insufficient. Therefore, by setting the density of tendon fibroblasts to 1 × 10 ⁵ cells / ml or more and 1 × 10 ⁶ cells / ml or less, it is a mature and dense tissue, and the produced collagen bundles are oriented. Can form aligned tendon tissue.

The supplied liquid containing the first cells 31 is held on the holding surface 11 of the base material 10 and the second holding surface 22 of the holder 20. After that, the manufacturing apparatus 1 is heated to a predetermined temperature (for example, 37 ° C.). As a result, the extracellular matrix component containing the tendon fibroblasts is gelled in the first region 61 to form the first gel body 41. At this time, of the first gel body 41, the first gel body 41 located in the restraint region 71 is in a state of being restrained by the anchor 21.

The gelled first gel body 41 before culturing satisfies the relationship of 0.4 ≦ W / L ≦ 0.55, where L is the length in the Y direction and W is the length in the X direction. By satisfying this relationship, the tendon tissue after culturing can be oriented (details will be described later). As an example, the length L in the Y direction is 5 to 7 mm, and the length W in the X direction is about 3 mm (2.5 mm or more and less than 4 mm).

In the gelation step, after the first gel body 41 is formed, the second cell 32 is contained in the second region 62 between the first gel bodies 41 separated on the holding surface 11 as shown in FIG. A liquid is supplied and gelled. As the second cell 32, myoblasts are used as an example. As the liquid containing myoblasts, the extracellular matrix component is preferably used.

After that, the manufacturing apparatus 1 is heated to a predetermined temperature (for example, 37 ° C.). As a result, similarly to the first gel body 41, the extracellular matrix component containing myoblasts is gelled in the second region 62 to form the second gel body 42. The second gel body 42 is in a state where both ends in the Y direction are in contact with the first gel body 41.
This completes the gelling step.

When the gelation step is completed, in the present embodiment, the process proceeds to a detachment step in which the first gel body 41 and the second gel body 42 are separated from the holding surface 11.
Specifically, first, the holder 20 is moved upward to be separated from the recess 12 of the base material 10 as shown in FIG. As a result, the first gel body 41 that was adhered to the holding surface 11 of the base material 10 at the time of gelation by heating and the second gel body 42 suspended between the separated first gel bodies 41 are separated from the holding surface 11. Leave.

Next, the detached holder 20 is lowered and refitted into the recess 12. At this time, the first gel body 41 and the second gel body 42 separated from the holding surface 11 come into contact with the holding surface 11 again, but once separated from the holding surface 11, they do not adhere to each other. Therefore, the first gel body 41 and the second gel body 42 can be contracted by the subsequent culture without causing any trouble as in the case where the first gel body 41 and the second gel body 42 are adhered to the holding surface 11.

Further, when the holder 20 is not provided with the second portion 20B and is composed of the first portion 20A, the holder 20 is positioned in the Y direction by fitting into the recess 12 of the base material 10, but X. Not positioned in the direction. In this case, when the detached holder 20 is lowered and refitted into the recess 12, the relative positions of the pair of retainers 20 in the X direction fluctuate with respect to before the retainer 20 is detached, and the gel body. May adversely affect the culture (manufacturing accuracy of artificial three-dimensional tissue complex). In the present embodiment, the holder 20 is provided with the second portion 20B, and the holder 20 is positioned in the X direction by fitting the second portion 20B into the second recess 14 of the base material 10. , The relative positions of the pair of holders 20 before being detached and after being refitted can be maintained. Therefore, in the present embodiment, it is possible to avoid adversely affecting the culture of the gel body (the accuracy of producing the artificial three-dimensional tissue complex).

In order to prevent the first gel body 41 and the second gel body 42 from adhering to the holding surface 11, for example, the holding surface 11 may be coated with an MPC (2-methacryloyloxyethyl phosphorylcholine) polymer or the like.

The withdrawal step may be carried out immediately after the completion of the gelation step or the culture step after the completion of the gelation step. When the withdrawal step is carried out in the middle of the culturing step, it is preferable that the culturing of the first gel body 41 and the second gel body 42 proceeds before the contraction starts.

In the culturing step, the first gel body 41 containing the first cell 31 and the second gel body 42 containing the second cell 32 are cultured at a temperature of, for example, 37 ° C. for a predetermined period (for example, several days). As the cells of the first cell 31 and the second cell 32 adhere to each other by culturing, the first gel body 41 and the second gel body 42 contract as shown in FIG.

Since both ends of the second gel body 42 are connected to the first gel body 41, the second gel body 42 becomes thinner as it shrinks. On the other hand, the first gel body 41 of the non-restraint region 72 is not restrained by the anchor 21 of the holder 20, and thus becomes thinner with contraction, but the first gel body 41 of the restraint region 71 is restrained by the anchor 21. It doesn't get thinner.

As the culture further progresses and the first cells 31 adhere to each other in the first gel body 41, as shown in FIG. 1, the tendon tissue (hereinafter referred to as tendon tissue 51) is used as the first tissue 51 in the first region 61. Is formed. Further, as the culture progresses and the second cells 32 adhere to each other in the second gel body 42, muscle tissue (hereinafter referred to as muscle tissue 52) is formed as the second tissue 52 in the second region 62.

The muscle tissue 52 is in a state of being oriented in the Y direction because tension is applied in the Y direction by contraction in a state where both ends are connected to the first gel body 41 during culturing. On the other hand, of the tendon tissues 51, the tendon tissue 51 in the restraint region 71 is restrained by the anchor 21 and does not extend due to the contraction of the muscle tissue 52, so that it is not oriented in the Y direction. Of the tendon tissues, the tendon tissue 51 of the non-restraint region 72 is connected to the tendon tissue 51 restrained by the anchor 21 on the outside and is connected to the contracted muscle tissue 52 on the inside, so that the contractile force of the muscle tissue 52 Extends inward by acting as tension. As a result, the tendon tissue 51 of the unrestrained region 72 is in a state of being oriented in the Y direction. As a result, the artificial three-dimensional tissue complex TC shown in FIG. 1 is manufactured.

FIG. 7 is a photograph of the cultured state of the tendon tissue 51 and the muscle tissue 52. In FIG. 7, the culture states after 1-day culture (Day 1), 3-day culture (Day 3), 5-day culture (Day 5), and 7-day culture (Day 7) are shown. Even in a state where the culture time is short and the gel body is not organized, it will be referred to as tendon tissue 51 and muscle tissue 52 for convenience.

As shown in FIG. 7, the tendon tissue 51 and the muscle tissue 52 contracted after about 3 days of culturing, and discoloration of the muscle tissue 52 was observed. Further, from the 5-day culture to the 7-day culture, the contraction further progressed, and the muscle tissue 52 and the tendon tissue 51 of the unrestrained region 72, which is lighter in color than the muscle tissue 52 in FIG. 7, were constructed without peeling. It was observed that the artificial three-dimensional tissue complex TC was produced.

8 to 11 are diagrams showing the density of tendon fibroblasts contained in the extracellular matrix component and the HE-stained image and Hoechst-stained image of the tendon tissue 51 of the non-restraint region 72 produced at the density. is there. As shown in FIGS. 8 and 9, when the density of tendon fibroblasts contained is 1 × 10 ⁵ cells / ml or more and 1 × 10 ⁶ cells / ml or less, the tendon has a good collagen orientation. Tissue 51 was observed. On the other hand, as shown in FIGS. 10 and 11, when the density of tendon fibroblasts contained is 1 × 10 ⁷ cells / ml or more and 1 × 10 ⁸ cells / ml or less, the number of voids increases, which is good. A tendon tissue 51 having a collagen orientation degree of not so much was observed.

FIG. 12 is a diagram showing an HE-stained image and a Hoechst-stained image of the tendon tissue 51 of the restraint region 71. In Figure 12, the density of tendon fibroblasts are contained in, 1 × 10 ⁵ cells / ml in culture tendon tissue 51 is shown. As shown in FIG. 12, the orientation of collagen could not be confirmed in the tendon tissue 51 of the restraint region 71 restrained during culturing.
From the above observation results, the tendon tissue 51 has a non-oriented portion 51A that is non-oriented in the Y direction in the restraint region 71 located outside in the Y direction, and as shown in FIG. 7, the non-oriented portion 51A and the muscle It was confirmed that the unconstrained region 72 between the tissue 52 and the tissue 52 had an oriented portion 51B located in the Y direction and oriented in the Y direction.

FIG. 13 is a diagram showing the breaking strength of the tendon tissue 51 and the muscle tissue 52. For the tendon tissue 51, the density (cells / ml) containing the tendon fibroblasts was measured for each sample ^{having a density of 1 × 10 5} , 5 × 10 ⁵ , and 1 × 10 ^6. For muscle tissue 52, density myoblasts is contained (cells / ml) were measured for samples of 1 × 10 ^8.
As shown in FIG. 13, it was confirmed that the tendon tissue 51 having a higher breaking strength than the muscle tissue 52 could be produced.

FIG. 14 is a diagram showing the relationship between the number of days of culture and the rate of change in diameter in the tendon tissue 51.
In FIG. 14, the density (cells / ml) containing the tendon fibroblasts is sample A: 5 × 10 ⁶ cells / ml, sample B: 1 × 10 ⁶ cells / ml, sample C: 5 × 10 ⁵ cells. / ml, sample C: was measured for each of the 1 × 10 ⁵ cells / ml. As shown in FIG. 14, it was confirmed that the cells shrank to about 20% after 14 days of culture regardless of the cell density.

FIG. 15 is a diagram showing a Hoechst-stained image of the tendon tissue 51 of the restraint region 71. In FIG. 15, an image of a sample in which the first gel body 41 gelled and before culturing has a length W = 4 mm in the X direction and a length W = 3 mm in the X direction is shown. Each sample had a length L = 6 mm in the Y direction. As shown in FIG. 15, a tendon tissue 51 having a good degree of collagen orientation was observed in a sample having a length W = 3 mm satisfying the relationship of 0.4 ≦ W / L ≦ 0.55. On the other hand, in the sample having a length W = 4 mm that does not satisfy the relationship of 0.4 ≦ W / L ≦ 0.55, a tendon tissue 51 having a collagen orientation degree which is not good due to many voids was observed.

16 and 17 show the time when electrical stimulation is applied at regular intervals and the movement of tissue by electrical stimulation using an artificial three-dimensional tissue complex TC having tendon tissue 51 and muscle tissue 52 and muscle tissue. It is a figure which shows the relationship with the quantity. FIG. 16 shows the amount of movement in the Y direction (contraction direction, orientation direction). FIG. 17 shows the amount of movement in the X direction (diameter direction). The electrical stimulation was applied at 1 Hz and an electric field of 1 V / mm.

As shown in FIG. 16, in the Y direction (contraction direction, orientation direction), the results of movement during contraction were obtained in both the artificial three-dimensional tissue complex TC shown in Graph E and the muscle tissue shown in Graph F. .. Regarding the amount of movement, the result was obtained that the artificial three-dimensional tissue complex TC had a larger amount than that of the muscle tissue alone. On the other hand, as shown in FIG. 17, in the X direction (diameter direction), the artificial three-dimensional tissue complex TC was found to move (swell) during contraction, but in the case of only muscle tissue, it almost moved during contraction. No results were obtained. From this, it was confirmed that the artificial three-dimensional tissue complex TC can move with a change in length equivalent to that of a living body even when both ends are fixed.

As described above, in the present embodiment, it is possible to obtain an artificial three-dimensional tissue complex TC having tendon tissue 51 and muscle tissue 52, both of which have orientation and are excellent in functionality and equivalence with a living body. ..

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, a procedure for starting the culture / production of tendon tissue and the culture / production of muscle tissue at almost the same time has been exemplified, but the procedure is not limited to this procedure. For example, tendon tissue has a longer time to mature and optimal than muscle tissue. Therefore, when the tendon tissue becomes mature, the muscle tissue becomes mature and then left unattended, which may deteriorate. Therefore, when culturing and producing a plurality of tissues having a large time difference until they reach a mature state, the tissue (for example, tendon tissue) that takes a longer time to reach a mature state is first started to be cultured. However, it is preferable to start culturing the tissue (for example, tendon tissue) that takes less time to reach a mature state after the above time difference elapses. As a result, it is possible to easily obtain an artificial three-dimensional tissue complex TC in which both tissues are matured and in an optimum state.

For example, when culturing muscle tissue after starting culturing tendon tissue, as described above, after starting culturing tendon tissue in each of the first regions 61 separated in the Y direction, in the second region 62. It may be a procedure for starting the culture of the muscle tissue, or after starting the culture of the tendon tissue in all of the first region 61 and the second region 62, the muscle tissue is excised in the space where the tendon tissue of the second region 62 is excised. It may be a procedure for starting the culture of.

Further, in the above embodiment, a configuration for producing an artificial three-dimensional tissue complex having a tendon tissue and a muscle tissue has been exemplified, but the configuration is not limited to this configuration, and a configuration may be formed including other tissues. For example, it may be configured to produce an artificial three-dimensional tissue complex having tendon tissue and muscular tissue in addition to bone (cartilage or the like) tissue connected to the outside of the tendon tissue.

Further, in the above embodiment, a configuration in which tendon tissue is produced using tendon fibroblasts as the first cell 31 and myoblasts are produced as the second cell 32 has been exemplified. Not limited. For example, it can be applied to a configuration in which chondrocytes are used to produce bone-cutting tissue and ligament cells are used to produce ligament tissue between cartilage tissues.

1 ... Artificial three-dimensional tissue complex manufacturing equipment (manufacturing equipment), 10 ... Base material, 11 ... Holding surface, 12 ... Recessed, 20 ... Holder, 20A ... 1st part, 20B ... 2nd part, 21 ... Anchor, 22 ... 2nd holding surface, 31 ... 1st cell, 32 ... 2nd cell, 41 ... 1st gel body, 42 ... 2nd gel body, 51 ... 1st tissue (tendon tissue), 51A ... non-oriented part, 51B … Orientation part, 52… 2nd tissue (muscle tissue), 61… 1st region, 62… 2nd region, 71… restraint region, 72… non-restraint region, TC… artificial three-dimensional tissue complex

Claims (12)

A second gel body arranged apart from each other in the first direction and holding a first gel body containing a first cell, and a second gel body arranged between the separated first regions and containing a second cell body. Each of the first regions is located outside the first direction and protrudes from the position of the holding surface with a gap from each other to restrain the first gel body. A preparatory step for preparing a substrate having a restraint region provided with a plurality of possible anchors and a non-constraint region located inside the restraint region in the first direction and unconstraining the first gel body. When,
A liquid containing the first cells is supplied to each of the first regions and gelled to form the first gel body, and a liquid containing the second cells is supplied to the second region to gel. In the gelation step of forming the second gel body,
The first gel body containing the first cell is cultured to form a first tissue, and the second gel body containing the second cell is cultured and the first tissue is connected to both ends. Including a culture step to form two tissues
In the culture step, a second tissue oriented in the first direction is formed by contraction in the second region, and the contraction force of the second tissue among the first tissues formed in the first region A method for producing an artificial three-dimensional tissue complex in which the first structure that is stretched and oriented in the first direction is formed in the unconstrained region. The gelling step and the culturing step are started first for the tissue of the first tissue and the second tissue, whichever takes longer to culture.
After the time difference between the time required for culturing the first tissue and the time required for culturing the second tissue has elapsed, the gelation step and the culture step are started for the tissue having the shorter time required for culturing.
The method for producing an artificial three-dimensional tissue complex according to claim 1. In the culture step, tendon fibroblasts in the extracellular matrix component are cultured in the first region to form tendon tissue, and myoblasts in the extracellular matrix component are cultured in the second region to form muscle tissue. Form,
The method for producing an artificial three-dimensional tissue complex according to claim 1 or 2. The density of the tendon fibroblasts is 1 × 10 ⁵ cells / ml or more and 1 × 10 ⁶ cells / ml or less.
The method for producing an artificial three-dimensional tissue complex according to claim 3. Let L be the length of the gelled extracellular matrix component containing the tendon fibroblasts in the first direction, and let W be the length of the second direction parallel to the holding surface and orthogonal to the first direction. ,
0.4 ≤ W / L ≤ 0.55
Satisfy the relationship,
The method for producing an artificial three-dimensional tissue complex according to claim 3 or 4. The base material is formed of an elastomer having a recess provided in the restraint region and a holder detachably fitted in the recess.
The holder is provided with a second holding surface that is flush with the holding surface when fitted into the recess, and an anchor that projects from the second holding surface with a gap from each other.
After the gelation step, the first gel body in which the holder is separated from the recess and is restrained by the anchor, and the second gel body suspended between the separated first gel bodies are formed. A step of separating the holder from the holding surface and a step of refitting the detached holder into the recess are included.
The method for producing an artificial three-dimensional tissue complex according to any one of claims 1 to 5. The holder includes a first portion parallel to the holding surface and extending in a second direction orthogonal to the first direction, and the second region side of the first portion from the middle of the second direction to the first direction. Has a second part that extends to
The holder fitted in the recess is positioned in the first direction in the first portion and in the second direction in the second portion with respect to the base material.
The method for producing an artificial three-dimensional tissue complex according to claim 6. It has a base material with a holding surface
The holding surface is arranged between the first region which is arranged apart from each other in the first direction and holds the first gel body containing the first cell, and the first region which is separated and contains the second cell. Has a second region that holds the second gel body
Each of the first regions is a restraint region located outside the first direction and protruding from the position of the holding surface with a gap from each other, and a restraint region provided with a plurality of anchors capable of restraining the first gel body. An artificial three-dimensional tissue complex manufacturing apparatus having an unconstrained region located inside the first direction with respect to the constrained region and having the first gel body unconstrained. The base material is formed of an elastomer having a recess provided in the restraint region and a holder detachably fitted in the recess.
The holder is provided with a second holding surface that is flush with the holding surface when fitted into the recess, and an anchor that projects from the second holding surface with a gap from each other.
The artificial three-dimensional tissue complex manufacturing apparatus according to claim 8. The holder includes a first portion parallel to the holding surface and extending in a second direction orthogonal to the first direction, and the second region side of the first portion from the middle of the second direction to the first direction. Has a second part that extends to
The holder fitted in the recess is positioned in the first direction in the first portion and in the second direction in the second portion with respect to the base material.
The artificial three-dimensional tissue complex manufacturing apparatus according to claim 9. With the first tissue arranged apart in the first direction,
It has a second tissue oriented and extending in the first direction and connecting the first tissues separated from each other.
Each of the first structures is located outside the first direction and is located between the non-oriented portion that is not oriented in the first direction and the second structure and the non-oriented portion and is located in the first direction. An artificial three-dimensional tissue complex having an oriented oriented portion. The first tissue is a tendon tissue
The second tissue is muscle tissue,
The artificial three-dimensional tissue complex according to claim 11.