JP2023070669A - Artificial vascular bed, artificial three-dimensional biological tissue, and method for making artificial three-dimensional biological tissue including vascular network - Google Patents

Abstract

To provide an improved method for the introduction of a vascular network into three-dimensional biological tissue (for example, high-cell-density three-dimensional tissue such as a laminated cell sheet and an organoid).SOLUTION: An artificial vascular bed comprises: a vascular bed part having a mounting face for three-dimensional biological tissue, composed of a composition comprising biocompatible hydrogel; a first channel and a second channel provided inside the vascular bed part; a first gap and a second gap that are in communication with the first channel and the second channel, respectively, and provided at the mounting face; a bridging channel that brings the first gap and the second gap into communication with each other, formed at the mounting face; a first liquid feeding part that is in fluid communication with the first channel, feeding a medium; and a second liquid feeding part that is in fluid communication with the second channel, discharging the medium. There is also provided a method for making artificial three-dimensional biological tissue that comprises a vascular network comprising the artificial vascular bed.SELECTED DRAWING: Figure 1-2

Description

TECHNICAL FIELD The present invention relates to an artificial vascular bed, an artificial three-dimensional biological tissue, and a method for producing an artificial three-dimensional biological tissue having a vascular network.

Currently, regenerative medicine using a myoblast sheet or a cardiomyocyte sheet derived from induced pluripotent stem cells (iPS cells) has begun as a new treatment for heart failure (Non-Patent Documents 1 and 2). It is a treatment with a paracrine effect that promotes angiogenesis and the like in the host's heart by means of a large number of growth factors secreted by the cell sheet and improves cardiac function. However, these treatments do not lead to an increase in functional myocardial cells, and there is a problem that they cannot be applied to severe heart failure. Therefore, the present inventors are aiming for treatment by three-dimensional myocardial tissue transplantation having a mechanical pulsatile support function as next-generation regenerative medicine. This is a therapeutic method in which three-dimensional myocardial tissue prepared in vitro by laminating a plurality of human iPS cell-derived cardiomyocyte sheets is transplanted. By realizing this treatment method, in addition to the conventional paracrine effect, it is expected that the mechanical pump function of the myocardial cells themselves will be assisted.

However, it has been reported that a layered tissue obtained by preparing three or more layers of cell sheets undergoes necrosis inside the tissue due to lack of oxygen and nutrients and accumulation of waste products (Non-Patent Document 3). Therefore, in order to construct a three-dimensional myocardial tissue that exceeds the stacking limit, it is important to establish a method for providing a vascular structure that supplies nutrients to the three-dimensional tissue in vitro.

Ex vivo angiogenesis research is broadly divided into research targeting hydrogels with low cell density and research targeting three-dimensional tissues with high cell density. In research on hydrogels, many methods have been reported for constructing blood vessels with a luminal structure that can be perfused from endothelial cells suspended in hydrogels by using 3D printing and microfluidic devices ( For example, Non-Patent Documents 4 and 5). On the other hand, in research on three-dimensional tissues such as organoids and cell sheets, by transplanting tissue with a network structure of endothelial cells constructed in vitro into the living body, blood vessels that are actually perfused with blood are created. Most of the reports are (Non-Patent Documents 6 and 7). Thus, in vitro luminal angiogenesis methods have been established in hydrogels, but remain unestablished in high cell density tissues.

To date, the present inventors have developed a vascular bed using collagen gel and biological tissue, making it possible to introduce a vascular network in vitro into a cell sheet, which is a tissue with high cell density (Patent document 1 and 2, and Non-Patent Documents 8 and 9). A vascular bed is a base for three-dimensional tissue culture that has a channel structure that allows perfusion of a culture solution. Angiogenesis in the culture solution by perfusing the culture solution into the blood vessels in the vascular bed after engrafting the cell sheet on the vascular bed using the blood vessels constructed in the collagen gel or the blood vessels in the three-dimensional living tissue. Vascular introduction into the sheet is achieved by supplying factors to the cell sheet by diffusion through blood vessels and inducing angiogenesis within the cell sheet.

In addition, by fabricating a microfluidic device made of polydimethylsiloxane (PDMS) and using the vascular structure built in the hydrogel, we have introduced a vascular structure that can perfuse spheroids suspended in the hydrogel. (Non-Patent Document 10). Based on the above studies, it has been reported that it is possible to introduce a vascular network that can be perfused into high-cell-density tissues in vitro by using hydrogels or vascular networks in three-dimensional living tissues. .

WO2012/036225 WO2012/036224

Miyagawa S, et al.: Impaired Myocardium Regeneration With Skeletal Cell Sheets-A Preclinical Trial for Tissue-Engineered Regeneration Therapy. Transplantation, 2010 Kawamura M, et al.: Feasibility, Safety, and Therapeutic Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Sheets in a Porcine Ischemic Cardiomyopathy Model. Circulation, 2012 Shimizu T, et al: Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. The FASEB Journal, 2006 Kolesky, D.B., et al.: 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Adv. Mater. 26, 3124-3130, 2014 Nguyen, D.-H.T., et al.: Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc. Natl. Acad. Sci. USA 110, 6712-6717, 2013 Takebe T, et al.: Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature. 499, 481-485, 2013 Sasagawa T, et al.: Comparison of angiogenic potential between prevascular and non-prevascular layered adipose-derived stem cell-sheets in early post-transplanted period. J. Biomed. Mater. Res. A. 102, 358-65, 2014 Sakaguchi K, et al.: In vitro engineering of vascularized tissue surrogates, Sci. Rep., 1316, 2013 Sekine H, et al.: In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels. Nat. Com, 2013 Nashimoto Y, et al.: Integrating perfusable vascular networks with a three-dimensional tissue in a microfluidic device. Integr. Biol., 2017

To prior art in vitro formed 3D biomedical tissues (e.g., high cell density conformations such as layered cell sheets and organoids) that utilize vascular networks within vascular beds such as hydrogels and 3D biomedical tissues. In the vascular network introduction method, it takes at least 5 days or more to introduce the vascular network, and even if the vascular network is introduced, the number of blood vessels to be constructed is remarkably small, and the vascular network introduction efficiency There was a problem that it was bad. In addition, in the conventional technology, it is difficult to control the position of the neovascular network constructed in the collagen gel used as the vascular bed and the position of the blood vessels in the biological tissue to be harvested. The physical distance to the three-dimensional living tissue differs for each vascular bed sample, and the quality of the vascular bed itself is inconsistent.

In order to solve the above problems, the present inventors have conducted research and development by adding studies from various angles. As a result, by controlling the physical distance and shape between the three-dimensional biological tissue and the channel constructed in the vascular bed, three-dimensional biological tissue (e.g., high cell density such as layered cell sheets and organoids) can be achieved. It is possible to improve the quality of the vascular network built in a dense three-dimensional tissue) (for example, increase the number of vascular networks), and not only can shorten the period until the vascular network is built, but also can be reproduced. The inventors have found that a vascular network can be constructed with high efficiency, and have completed the present invention. That is, the present invention can include the following aspects.

[1] An artificial vascular bed,
a vascular bed formed of a composition containing a biocompatible hydrogel and having a three-dimensional biological tissue mounting surface;
a first channel and a second channel provided inside the vascular bed;
a first hole and a second hole provided in the mounting surface and communicating with the first channel and the second channel, respectively;
a bridging flow path communicating between the first gap and the second gap and formed in the mounting surface;
a first liquid delivery unit in fluid communication with the first channel for supplying culture medium;
and a second liquid delivery section in fluid communication with the second flow path for discharging culture medium.
[2] The artificial vascular bed according to item 1, wherein the biocompatible hydrogel is a crosslinked biocompatible hydrogel.
[3] The artificial vascular bed according to item 1, wherein the biocompatible hydrogel contains fibrin gel.
[4] The artificial vascular bed according to item 3, wherein the biocompatible hydrogel contains a fibrin gel-stabilizing factor.
[5] a culture medium supply line connected to the first liquid feeding section;
a medium supply tank for supplying a medium to the medium supply line;
a liquid feed pump that feeds the medium to the medium supply line;
a medium discharge line connected to the second liquid feeding section;
a medium discharge tank for storing the medium discharged from the medium discharge line;
The artificial vascular bed according to any one of items 1 to 4, further comprising:
[6] The artificial vascular bed according to item 5, wherein the medium supply tank and the medium discharge tank are the same, and the medium is circulated.

[7] The artificial vascular bed according to any one of items 1 to 6;
and a three-dimensional living tissue mounted on the mounting surface so as to cover the bridging channel.
[8] The artificial three-dimensional biological tissue according to item 7, wherein the three-dimensional biological tissue is a sheet-like tissue containing cells or an organoid.
[9] The artificial three-dimensional biological tissue according to item 7 or 8, wherein the three-dimensional biological tissue contains vascular endothelial cells.

[10] A method for producing an artificial three-dimensional biological tissue having a vascular network, comprising:
A step of placing a three-dimensional biological tissue so as to cover the bridging channel on the placement surface of the artificial vascular bed according to any one of items 1 to 6;
A step of supplying a medium to the first liquid feeding part and performing perfusion culture while discharging the medium from the second liquid feeding part;
A method, including
[11] The method according to item 10, wherein the three-dimensional biological tissue is a sheet-like tissue or organoid containing cells.
[12] The method according to item 10 or 11, wherein the three-dimensional biological tissue contains vascular endothelial cells.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to improve the quality of a vascular network constructed in a three-dimensional living tissue (for example, increase the number of vascular networks and form mature blood vessels with a large diameter, etc.). It is possible to shorten the period until the construction of In addition, since it is a technique that directly utilizes the holes provided in the vascular bed and the bridging channels as the channels of the vascular bed, it solves the problem of variations in channel structure for each vascular bed sample. Therefore, it is possible to construct a high-quality vascular network with good reproducibility. In addition, it can be used as an experimental model for elucidating the effect of mechanical stimulation on angiogenesis in three-dimensional tissue with high cell density, because it is possible to directly apply mechanical stimulation by fluid to three-dimensional living tissue. is.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram of an artificial vascular bed of the present invention in one embodiment; BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing an outline of an artificial vascular bed of the present invention in one embodiment, and an outline of perfusion culture of a three-dimensional living tissue. 1 is a schematic diagram showing the procedure for creating an artificial vascular bed of the present invention in one embodiment. FIG. FIG. 1 shows an artificial vascular bed of the invention in one embodiment. Fluorescence image and optical coherence tomography (OCT) of three-dimensional biological tissue with vascular endothelial network (layered co-culture sheet of vascular endothelial cells and skin fibroblasts) placed on the artificial vascular bed of the present invention in one embodiment Show the image. Schematic diagrams for selecting hydrogel compositions suitable for artificial vascular bed fabrication are shown. Fig. 3 shows the results of evaluating the structural stability of artificial vascular beds when cell sheets were statically cultured on artificial vascular beds composed of different hydrogels for 5 days. Upper row: macroscopic observation image (BF), lower row: cross-sectional observation image (OCT). Fig. 3 shows the results of evaluating the structural stability of artificial vascular beds when cell sheets were statically cultured on artificial vascular beds composed of different hydrogels for 5 days. The surface area and cross-sectional area were quantified from the images obtained in FIG. 6-1, and the surface area ratio (left) and cross-sectional area ratio (right) are shown for each day based on the first day (Day 0). 1 is a schematic diagram showing a static culture or perfusion culture method using the artificial vascular bed of the present invention in one embodiment, and a method for evaluating the same. FIG. Fig. 2 shows fluorescent microscope images of cell sheets containing GFP-positive human umbilical vein endothelial cells (GFP-HUVEC) that were statically cultured or perfusion cultured using the artificial vascular bed of the present invention in one embodiment. 1 shows the results of evaluating blood vessels formed in a cell sheet by static culture or perfusion culture using the artificial vascular bed of the present invention according to one embodiment, and then perfusion with Indian ink. 1 shows the results of evaluation of blood vessels formed in a cell sheet by perfusion with rat blood after perfusion culture of the cell sheet using the artificial vascular bed of the present invention in one embodiment. FIG. 2 shows HE-stained images after statically culturing a cell sheet using the artificial vascular bed of the present invention in one embodiment. FIG. 2 shows fluorescence microscopic images after statically culturing a cell sheet using the artificial vascular bed of the present invention in one embodiment. Green: endothelial cells (CD31 positive), blue: nuclei (DAPI). FIG. 2 shows HE-stained images after perfusion culture of a cell sheet using the artificial vascular bed of the present invention in one embodiment. 1 shows a fluorescence microscope image after perfusion culture of a cell sheet using the artificial vascular bed of the present invention in one embodiment. Green: endothelial cells (CD31 positive), blue: nuclei (DAPI). FIG. 2 is a schematic diagram showing the results of comparison between the prior art and the vascular bed of the present invention; An application example of the artificial vascular bed or artificial three-dimensional living tissue (three-dimensional tissue) of the present invention in one embodiment is shown. After perfusion culture of the cell sheet at different perfusion rates (25 μL/min (5 days) or 25 μL/min (3 days) → 100 μL/min (2 days)) using the artificial vascular bed of the present invention in one embodiment Figure 2 shows the results of evaluating the blood vessels formed in the cell sheet of B. india by ink perfusion. 1 shows an example of a synthetic vascular bed of the present invention in one embodiment. 1 shows an outline of a method for constructing a three-dimensional tissue by stepwise lamination using the artificial vascular bed of the present invention in one embodiment. 1 shows an OCT observation image of a three-dimensional tissue constructed using the artificial vascular bed of the present invention in one embodiment. Fig. 2 shows enlarged images (left: bright-field observation image, left: fluorescence observation image) of a three-dimensional tissue constructed using the artificial vascular bed of the present invention in one embodiment.

Hereinafter, embodiments of the present invention will be described with reference to the drawings as necessary. The configuration of the embodiment is an example, and the configuration of the present invention is not limited to the specific configuration of the embodiment.

As used herein, the terms “first,” “second,” “third,” etc. are used to distinguish one element from another, e.g. , and similarly the second element may be referred to as the first element, without departing from the scope of the invention.

Unless otherwise defined, all terms (technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art.

1-1 and 1-2 are schematic diagrams showing the artificial vascular bed of the present invention in one embodiment. In one embodiment, the invention provides
An artificial vascular bed,
a vascular bed formed of a composition containing a biocompatible hydrogel and having a three-dimensional biological tissue mounting surface;
a first channel and a second channel provided inside the vascular bed;
a first hole and a second hole provided in the mounting surface and communicating with the first channel and the second channel, respectively;
a bridging flow path communicating between the first gap and the second gap and formed in the mounting surface;
a first liquid delivery unit in fluid communication with the first channel for supplying culture medium;
a second fluid delivery section in fluid communication with the second flow path for discharging culture medium.

As used herein, the term "artificial vascular bed" is used to construct a vascular network in a three-dimensional biological tissue (also referred to herein as a "three-dimensional tissue"). It refers to a vascular bed that can be created in vitro, as distinct from tissue rich in blood (eg, living tissue such as the skin flap shown in WO2012/036224).

In one embodiment, the artificial vascular bed of the present invention is a mounting for mounting a three-dimensional biological tissue (herein also referred to as a "three-dimensional tissue") intended to construct a perfusable vascular network. It has a vascular bed with a surface. The placement surface may have any shape as long as the three-dimensional living tissue can be placed thereon, but is preferably planar.

The vascular bed is made of a composition containing a biocompatible hydrogel. Examples of "biocompatible hydrogels" applicable to the present invention include water-soluble, water-affinitive, or Examples include hydrogels obtained by chemically cross-linking water-absorbing synthetic polymers, polysaccharides, proteins, nucleic acids, and the like. Examples of polysaccharides include glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, starch, glycogen, agarose, pectin, and cellulose. As proteins, collagen and its hydrolysates gelatin, proteoglycan, fibronectin, vitronectin, laminin, entactin, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen (for example, fibrinogen and thrombin were reacted fibrin gel) and the like. These biocompatible hydrogels may be subjected to a cross-linking treatment (eg, polymerization treatment) using a known method to obtain cross-linked biocompatible hydrogels with increased strength. Biocompatible hydrogels that can be used in the present invention include mechanical properties that can withstand the contraction of a three-dimensional biological tissue (for example, a sheet-like tissue containing cells) placed on the artificial vascular bed of the present invention, and high cell density. It is preferably an adhesive hydrogel, for example it is a fibrin gel and may additionally contain factor XIII. By further containing a fibrin gel stabilizing factor in the fibrin gel, cross-linking of the fibrin gel is promoted, and a biocompatible hydrogel is formed by the three-dimensional biological tissue mounted on the mounting surface of the artificial vascular bed of the present invention. is prevented or retarded from breaking down or constricting, and the shape of the channel within the vascular bed is maintained.

In one embodiment, the interior of the vascular bed of the artificial vascular bed of the present invention is provided with a first channel capable of feeding a fluid containing nutrients necessary for culture, such as a culture medium, from the outside of the vascular bed. . Further, inside the vascular bed portion of the artificial vascular bed of the present invention, there is provided a second channel for discharging a fluid containing nutrients necessary for culture, such as a culture medium, supplied to the three-dimensional biological tissue on the mounting surface. It has

In one embodiment, the artificial vascular bed of the present invention includes a bridging flow channel formed in the mounting surface and communicating between the first gap and the second gap (Fig. 1-2). The bridging channel provides fluid communication between the first and second gaps and is open or has an opening along the mounting surface, thereby covering the bridging channel. The culture medium fed from the first channel can be directly supplied to the three-dimensional living tissue placed in the manner described above. At this time, by supplying a large amount of biochemical factors from directly under the three-dimensional living tissue and mechanical stimulation by perfusion, the inside of the applied three-dimensional living tissue is replaced with conventional artificial vascular beds and living blood vessels. It has become possible to provide a functional vascular network of an amount that could not be achieved with a bed.

The cross-sectional shapes of the first channel and the second channel may be rectangular or substantially circular. In the case of a circular shape, the diameter is not particularly limited because it can be adjusted according to the shape, thickness, etc. of the three-dimensional living tissue applied to the artificial vascular bed. In addition, the width and depth of the bridging channel may be any shape that allows the medium fed from the first channel to be exposed directly below the three-dimensional biological tissue, for example, about 0.01 to 10 mm, preferably may be provided with a width and/or depth of about 0.1 to 5 mm, but if the width is increased, the three-dimensional biological tissue on the mounting surface will sink at that portion, increasing the resistance of the flow path. Therefore, it is more preferable to adjust the width to such an extent that this does not occur. In the communicating portion between the first or second flow path and the bridging flow path, a vertical hole-shaped first gap and a second hole that communicate with the mounting surface and the first or second flow path, respectively. A gap is provided (Fig. 1-1, Fig. 1-2).

In one embodiment, the first flow channel and the second flow channel may be arranged substantially parallel as shown in FIG. It may be arranged in a V-shape on both sides of the path, but is not limited. In another aspect, one or more bridging channels may be provided between the first channel and the second channel (eg, FIG. 17). In another aspect, the number of combinations of "first flow path-bridge flow path-second flow path" may be one, or a plurality thereof may be provided. Moreover, the bridging channel may have a more complicated structure, such as a mesh-like structure, as long as it communicates with the first channel and the second channel, and is not limited to a linear one. Furthermore, the outer peripheries of the first and second flow paths may be formed at positions in contact with the mounting surface, or grooves may be formed on the mounting surface as the first and second flow paths. The gap and the second hole gap may not be provided.

In one embodiment, the artificial vascular bed of the present invention is in fluid communication with the first channel and includes a first liquid delivery part for supplying a culture medium, and is in fluid communication with the second channel and has a culture medium. is provided with a second liquid feeding unit for discharging the In one embodiment, the first channel and the first liquid feeding part, and the second channel and the second liquid feeding part may be formed integrally, and a part thereof may be Tubing made of materials other than compatible hydrogels (e.g., silicone tubing, metal tubing, catheters, Surfflow needles, and prosthetics made of polyester (e.g., Dacron®, Teflon® (ePTEE) or synthetic polymers) blood vessels, etc.).

For example, when surfflow needles are used as the first liquid-feeding part and the second liquid-feeding part, a composition containing a biocompatible hydrogel is poured into a mold that forms a vascular bed, and the first needle is protruded from the vascular bed. A surflow needle is arranged as a liquid feeder and a second liquid feeder (FIG. 2). From above, a channel forming device having a bridging channel forming portion and a hole forming portion is used so that the hole forming portion is in contact with the surf needle as the first liquid feeding portion and the second liquid feeding portion. Place and cure the biocompatible hydrogel. After that, if necessary, it can be used as an artificial vascular bed by removing the channel forming device and the surfflow needle (Fig. 2). The mold may be used together with the artificial vascular bed during culture, and may be removed as necessary when used for purposes such as transplantation. Further, by moving the tip of the outer cylinder of the surflow needle toward the first liquid feeding section and the second liquid feeding section from the hole and removing the inner cylinder of the surflow needle, The outer cylinder can also be used as the first flow path (or second flow path) and the first liquid feeding section (or second liquid feeding section). This makes it possible to easily connect a medium supply line (or medium discharge line) during perfusion culture. Moreover, the pressure resistance of the flow path is also improved.

In one embodiment, the synthetic vascular bed of the present invention comprises:
a medium supply line connected to the first liquid feeding unit;
a medium supply tank for supplying a medium to the medium supply line;
a liquid feed pump that feeds the medium to the medium supply line;
a medium discharge line connected to the second liquid feeding section;
A medium discharge tank for storing the medium discharged from the medium discharge line may be further provided. These configurations allow continuous or intermittent medium perfusion. A known pump can be used as the liquid-sending pump, and it may be a tube pump (peristaltic pump) or a piezo pump, and any pump that can send out a fluid can be used. can.

In one embodiment, the medium supply tank and the medium discharge tank may be the same, and the medium may be circulated. As a result, liquid components such as cell growth factors produced from the three-dimensional biological tissue placed on the artificial vascular bed can be circulated for a certain period of time until the medium is replaced.

By using the artificial vascular bed of the present invention, a three-dimensional living tissue to be placed, an artificial vascular bed, and a perfused vascular network between them can be constructed. As used herein, "three-dimensional biological tissue" refers to a three-dimensional structure containing cells, for example, biological tissue isolated from a living body (e.g., organs and tissues or parts thereof (e.g., skin tissue (e.g. hair root-associated skin tissue), cardiac muscle tissue, skeletal muscle tissue, smooth muscle tissue, liver tissue, kidney tissue, gastrointestinal tissue, eye tissue (e.g. corneal tissue), brain tissue, thymus tissue, testicular tissue, pancreatic tissue , thyroid tissue, mammary gland tissue, salivary gland tissue, lung tissue, etc.), or a three-dimensional cell structure reconstructed using cells that constitute biological tissue, such as an organoid, can be applied to the present invention. The applicable biological tissue isolated from a living body may be the biological tissue itself collected from the living body, or a tissue piece obtained by processing the biological tissue collected from the living body into an arbitrary shape. In addition, the three-dimensional cell structure applicable to the present invention may be a three-dimensional cell structure formed by mixing a suspension containing cells with a gel solution or a gelling agent. It may be a three-dimensional cell structure in which sheet-like tissues containing cells (for example, cell sheets) are laminated, or a sheet-like structure containing cells on which organoids are placed.

As used herein, the term "organoid" refers to a tissue body constructed three-dimensionally in vitro from aggregates such as progenitor cells that contribute to the formation of an organ or organ. Synonymous with "organoid" in the broadest sense of the trade. Organoids are generally smaller than actual organs or organs, have simple structures, but exhibit features that are anatomically and functionally similar to organs or organs that exist in vivo. Organoids include, for example, renal organoids, liver organoids, gastrointestinal organoids (e.g., intestinal organoids, oral organoids, gastric organoids, etc.), optic cup organoids, brain organoids, thymus organoids, testicular organoids, pancreatic organoids, epithelial organoids, and thyroid organoids. , mammary gland organoids, salivary gland organoids, lung organoids, and the like, and “cancer organoids” containing cancer cells in these organoids are also included. In addition, as used herein, organoids may include organs and tissues collected from living organisms or parts thereof (eg, tissue fragments) (eg, Shamir ER., Nat. Rev. Mol. Cell Biol. 2014 Oct;15(10):647-64).

As a method for obtaining a three-dimensional biological tissue applicable to the present invention, such as an organoid, a known method may be used. , et al. , Nature. 2015, Oct. 22, 526 (7574), pp. 564-568; lung organoids are from Unbekandt M.; , Kidney Int. , 2010, Mar. , 77(5), pp. 407-416; thymic organoids are described in Sheridan JM. , et al. , Genesis, 2009 May, 47(5), pp. 346-351; testicular organoids are described in Sanjo H.; , et al. , PLoS One. 2018, Feb 12, 13(2), e0192884, etc. may be referred to. In addition, any organoids obtained by a method of forming organoids can also be used.

Three-dimensional biological tissues applicable to the present invention are, for example, three-dimensional biological tissues of mammals, birds, amphibians, reptiles, and fish. Preferably, it is a three-dimensional living tissue of mammals, including cells derived from mammals such as mice, rats, humans, monkeys, pigs, dogs, sheep, cats, and goats.

The cells contained in the three-dimensional biological tissue applicable to the present invention may be primary cells collected from the biological tissue, may be established cells, and may be pluripotent stem cells or tissue stem cells (e.g. , mesenchymal stem cells) may be differentiation-induced cells.

As used herein, the term “pluripotent stem cell” is intended to collectively refer to stem cells that have the ability to differentiate into cells of any tissue (pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), trophoblast stem cells (TS cells), Epiblast stem cells (EpiS cells), embryonic germ cells (EG cells), multipotent germline stem cells (mGS cells), induced pluripotent stem cells : iPS cells), Muse cells, etc. Any known pluripotent stem cells can be used, and for example, the pluripotent stem cells described in International Publication No. 2009/123349 can be used.

As a method for differentiating tissue stem cells or pluripotent cells into cells that construct any three-dimensional biological tissue (eg, organoid), known methods can be used. For example, but not by way of limitation, cells that construct renal organoids are described in Takasato M.; , et al. , Nature. 2015, Oct. 22, 526 (7574), pp. 564-568; , et al. , Nature, 2013, Jul, 25, 499(7459), pp. 481-484; cells that construct gastrointestinal organoids are described by Sato T.; , et al. , Nature, 2009, May 14, 459(7244), pp. 262-265; , et al. , Nature, 2011, Apr 7, 472(7341) pp. 51-56; the cells that make up brain organoids are described by Eiraku M.; , et al. , Cell Stem Cell, 2008, Nov 6, 3(5), pp. 519-532 and Lancaster MA. , et al. , Nature, 2013, Sep 19, 501(7467), pp. 373-379, the cells that make up the lung organoids are described by Yamamoto Y.; , et al. , Nat Methods. 2017 Nov, 14(11), pp. 1097-1106; the cells that make up the pancreatic organoids are described in Raikwar SP. , et al. , PLoS One. 2015 Jan 28, 10(1), e0116582; , et al. , Front Endocrinol (Lausanne). 2015 Apr 22, 6, 56. etc. can be used as a reference. In addition, any cell obtained by a method of differentiating into a cell that constructs a three-dimensional biological tissue can also be used.

As used herein, the term "sheet-like tissue containing cells" refers to a biological tissue collected from a living body or a thin film-like tissue containing cells. The sheet-shaped tissue containing cells is the tissue itself collected from the living body, or the tissue collected from the living body is processed into a sheet and formed into multiple layers (for example, 3 layers, 4 layers, 5 layers, 6 layers, 7 layers, 8 layers). , 9 layers, 10 layers, 20 layers, 25 layers, 30 layers, or more). Further, the sheet-like tissue containing cells may be a sheet-like tissue formed by mixing a suspension containing cells and a gel, and a stimulus-responsive culture dish (for example, a temperature-responsive culture dish) is used. It may be a cell sheet prepared by Furthermore, the sheet-like tissue containing cells may be formed by directly seeding and culturing a group of cells on the upper surface of a membrane-like gel.

As used herein, the term “cell sheet” refers to a single-layer or multi-layer sheet obtained by culturing a cell group containing a plurality of arbitrary cells on a cell culture substrate and peeling off from the cell culture substrate. It refers to a group of cells with a shape. As a method for obtaining a cell sheet, for example, cells are cultured on a stimulus-responsive culture substrate coated with a polymer whose molecular structure changes with stimuli such as temperature, pH and light, and then subjected to stimuli such as temperature, pH and light. By changing the conditions to change the surface of the stimulus-responsive culture substrate, while maintaining the adhesion state between cells, a method of exfoliating cells from a stimulus-responsive culture substrate in a sheet form, or any culture substrate a method of culturing the cells on the surface and physically peeling them off with tweezers or the like. As stimulus-responsive culture substrates for obtaining cell sheets, temperature-responsive culture substrates coated with a polymer whose hydration force changes in the temperature range of 0 to 80° C. are known. Cells are cultured on a temperature-responsive culture substrate in a temperature range where the hydration force of the polymer is weak, and then the temperature of the culture solution is changed to a state where the hydration force of the polymer is strong. It can be exfoliated as a group of cells.

The temperature-responsive culture substrate used to obtain the cell sheet is preferably a substrate that changes the hydration force of its surface in the temperature range where cells can be cultured. The temperature range is preferably the temperature at which cells are generally cultured, eg, 33°C to 40°C. The temperature-responsive polymer coated on the culture substrate used to obtain the cell sheet may be either a homopolymer or a copolymer. Examples of such polymers include polymers described in JP-A-2-211865.

A case where poly(N-isopropylacrylamide) is used as a stimulus-responsive polymer, particularly a temperature-responsive polymer, will be described as an example (temperature-responsive culture dish). Poly(N-isopropylacrylamide) is known as a polymer having a lower critical solution temperature of 31° C. If it is in a free state, it undergoes dehydration in water at a temperature of 31° C. or higher, causing the polymer chains to aggregate and become cloudy. Conversely, at temperatures below 31°C, the polymer chains are hydrated and dissolved in water. In the present invention, the surface of a substrate such as a petri dish is coated with the polymer and fixed. Therefore, at a temperature of 31°C or higher, the polymer on the surface of the culture substrate is also dehydrated, but since the polymer chains are fixed on the surface of the culture substrate, the surface of the culture substrate appears to be hydrophobic. become. Conversely, at temperatures below 31° C., the polymer on the surface of the culture substrate hydrates, but the surface of the culture substrate exhibits hydrophilicity because polymer chains are coated on the surface of the culture substrate. The hydrophobic surface at this time is a suitable surface to which cells can adhere and proliferate, and the hydrophilic surface is a surface to which cells cannot adhere. Therefore, when the substrate is cooled below 31° C., the cells are detached from the substrate surface. If the cells are cultured until they become confluent on the entire culture surface, the cell sheet can be collected by cooling the substrate to less than 31°C. The temperature-responsive culture substrate is not limited as long as it has the same effect. Smart temperature-responsive culture dish SSCW provided by Medical Promotion Organization (Tokyo, Japan) and the like.

In one embodiment, the present invention provides an artificial three-dimensional living tissue comprising the artificial blood vessel bed described above and a three-dimensional living tissue mounted on the mounting surface so as to cover the bridging channel. obtain. The artificial three-dimensional biological tissue obtained by the present invention, for example, is angiogenesis, which clarifies the relationship between mechanical stimulation and angiogenesis in a three-dimensional biological tissue with high cell density through examination of channel shape and perfusion conditions. In addition to being used as a research model, it can also be used as a new drug discovery test model that enables transvascular drug administration and can be evaluated, for example, by using morphological changes and changes in metabolites as indicators. can do. Also, the first liquid-feeding part and the second liquid-feeding part can each be provided as an artificial three-dimensional living tissue that can be anastomosed as an artery or a vein (see FIG. 14, for example).

In one embodiment, the invention provides
placing the three-dimensional biological tissue so as to cover the bridging channel on the placement surface of the artificial vascular bed;
A step of supplying a medium to the first liquid feeding part and performing perfusion culture while discharging the medium from the second liquid feeding part;
To provide a method for fabricating an artificial three-dimensional living tissue with a vascular network, comprising:

The speed of supplying or discharging the medium may be any speed that can supply nutrients and oxygen to the three-dimensional biological tissue placed on the artificial vascular bed and promote angiogenesis, and can be adjusted as appropriate. be.

The three-dimensional biological tissue applied to the present invention preferably contains vascular endothelial cells, which may be vascular endothelial cells contained in the collected three-dimensional biological tissue, or externally added vascular endothelial cells. It may be a cell.

The present invention will be described in more detail below based on examples, but these are not intended to limit the invention.

<Example 1>
The artificial vascular bed of the present invention was produced by transferring the structure of a channel forming device produced by a 3D printer onto the hydrogel surface. In order to construct the first and second flow paths, after injecting hydrogel into a silicone mold, which is a flow path forming device with a 20 G surflow outer cylinder and an inner needle inserted, a device made by a 3D printer is covered. It was gelled by standing at room temperature for 30 minutes. After confirming the gelation, the hydrogel artificial vascular bed having the pore structure and the bridging channel was fabricated by removing the 3D printer device and SURFLOW (Fig. 2).

By transferring the device structure made by a 3D printer to the hydrogel surface, two hole structures (first hole and second hole) with a diameter of 500 μm leading to the first and second flow paths, and two A hydrogel artificial vascular bed having bridging channels with a width of 300 µm, a depth of 800 µm, and a length of 5 mm connecting the pore structures was fabricated (Fig. 3). In addition, in the hydrogel artificial vascular bed, only the inner needle of SURFLOW is completely removed, and the outer tube is left in place without being completely removed, so that the liquid can be easily fed to the vascular bed channel. The pore structure and the bridging channel part were observed by optical coherence tomography (OCT) (Santec Corporation, Japan), and it was confirmed that the above structure was realized on the hydrogel surface (Fig. 3).

<Example 2>
A cell sheet having a vascular endothelial network layered on the artificial vascular bed of the present invention was produced by co-culturing GFP-positive human umbilical vein endothelial cells (GFP-HUVEC) and human dermal fibroblasts (NHDF). A cell suspension prepared by mixing GFP-HUVEC and NHDF at a ratio of 9:1 to a concentration of 1.0 × 10 ⁶ cells/mL was placed in a temperature-responsive culture dish (Up Cell (registered trademark), CellSeed Inc., 35 mm in diameter). Tokyo, Japan)) or a smart temperature-responsive culture dish SSCW (General Incorporated Association Cell Sheet Regenerative Medicine Promotion Organization, Tokyo, Japan). A vascular endothelial reticulated cell sheet was prepared. The cell sheet was collected from the temperature-responsive culture dish by static culture in an incubator at 20° C. for 30 minutes to 1 hour.

The three-dimensional tissue for culturing on the artificial vascular bed of the present invention was prepared by laminating three layers of vascular endothelial meshed cell sheets. First, a first cell sheet was allowed to adhere to a culture dish having a diameter of 100 mm by static culture at 37° C. for 1 hour. Subsequently, a second cell sheet was layered on the first cell sheet, and cultured at 37° C. for 30 minutes to allow the cell sheets to adhere to each other. The third cell sheet was similarly adhered by culturing at 37° C. for 30 minutes to obtain a tissue in which three cell sheets with vascular endothelial networks were layered. The layered tissue was transferred onto the artificial vascular bed with an OHP film, placed so as to cover the pore structure and the bridging channels, and then left to stand on the artificial vascular bed at 37° C. for 1 hour. rice field.

As a result of observing the cell sheet on day 3 of culture with a fluorescence microscope, formation of a network structure of vascular endothelial cells was confirmed. In addition, the layered tissue was placed so as to cover the pore structure of the artificial vascular bed of the present invention and the bridging channels, and after static culture for 1 hour, the cross-sectional structure was observed using OCT. As a result, it was confirmed that the cell sheet was engrafted directly above the pore structure and the bridging channel, and an artificial vascular bed was realized in which the pore structure and the bridging channel were directly used as perfusion channels for the culture solution ( Figure 4).

<Example 3>
The compositions of hydrogels that can be used to fabricate the artificial vascular bed of the present invention were examined. This artificial vascular bed utilizes the space constructed between the bridging channel of the artificial vascular bed and the cell sheet as a direct channel. Therefore, if the hydrogel shrinks or decomposes due to the cell sheet laminated on the artificial vascular bed, there is a possibility that the pore structure and bridging channels that are vascular bed channels will collapse. If the vascular bed channel collapses, not only will it be difficult to perform perfusion culture, but it will also be difficult to control the distance between the cell sheet and the channel in the vascular bed. It is not possible to evaluate whether it is effective as a technique for adding blood vessels to tissues with high cell density. Therefore, we considered that a hydrogel that is resistant to structural changes due to gel contraction and degradation by cells would be suitable for fabricating an artificial vascular bed using a three-dimensional microfluidic system (Fig. 5).

As a suitable gel for the artificial vascular bed fabrication of the present invention, collagen gel or fibrin gel, which are hydrogels used in angiogenesis research, were selected as candidates. collagen gel (4 mg/mL collagen, 0.3 mmol/L _NaHCO3 , and 0.2 mmol/L HEPES), fibrin gel (5 mg/mL Fibrinogen, 0.5 U/mL thrombin, and 5 mM _CaCl2 ), and factor XIII An artificial vascular bed having a pore structure was prepared using three types of gels: fibrin gel (0.5 U/mL thrombin, 5 mM CaCl ₂ , 40 U/mL Factor XIII) containing Three layers of cell sheets were transplanted into each hydrogel vascular bed, and the resulting tissue was statically cultured in VEC1 for 5 days. Observation of the upper surface of the vascular bed with an optical microscope and observation of the bridging channel cross section with an optical coherence tomography were carried out every day for 5 days. The stability of the structure of each hydrogel vascular bed was measured by measuring the surface area of the upper surface of the vascular bed and the cross-sectional area of the bridging channel, respectively, and calculating the ratio of the change during 5 days to the area immediately after the three-layered cell sheet was implanted. evaluated by calculation.

FIG. 6-1 shows bright-field observation images and cross-sectional images of bridging channels, respectively, when a three-layer cell sheet was statically cultured on an artificial vascular bed composed of each hydrogel. The upper surface of the vascular bed made of collagen gel contracted significantly on the first day of culture, and continued to contract over the following 4 days. Furthermore, from observation of the cross section of the bridging channel, it was confirmed that the bridging channel had almost disappeared on the first day of culture. In addition, contraction of the upper surface and the bridging channel was confirmed on the first day of culture for the vascular bed made of fibrin gel. After that, it was confirmed that the upper surface did not change from the 1st day to the 5th day of culture, but the bridging channel continued to shrink over the next 4 days. On the other hand, observation results of the vascular bed made of fibrin gel containing factor XIII confirmed that the structure of both the upper surface and the bridging channel was maintained during the 5-day culture period. As a result of evaluating the rate of change in each area over time, it was found that the fibrin gel containing factor XIII is the hydrogel that can best retain the structure of the upper surface of the artificial vascular bed and the bridging channel (Fig. 6-2).

From the above results, a method for producing a fibrin gel containing factor XIII whose structure is difficult to be changed by cells for producing an artificial vascular bed of the present invention with a pore structure and bridging channels as direct channels was clarified. bottom.

<Example 4>
Verification of the in vitro effect of imparting blood vessels with lumens to the layered cell sheet tissue by static culture and perfusion culture of the cell sheet on the artificial vascular bed of the present invention made of fibrin gel containing factor XIII bottom. After engrafting the layered tissue of three layers of the vascular endothelial mesh-attached cell sheet constructed according to the protocol described above on the pore structure of the artificial vascular bed, static culture and perfusion were carried out at 37° C. in a 5% CO ₂ environment. Culture was performed. In the perfusion culture, the endothelial cell medium VEC1 (Kohjin Bio, Japan) is perfused from the first channel using a microtube pump at a flow rate of 25 μL / min, and the medium that has passed through the bridging channel is discharged from the second channel into the chamber. Perfusion directly below the cell sheet was performed by flowing out to the bottom of the cell sheet. The medium that flowed out and accumulated in the chamber was managed by draining the liquid every day until the surface of the cell sheet did not come into contact with the air. In static culture, perfusion directly below the cell sheet should not be performed, and VEC1 should be perfused into the chamber with a microtube pump at a flow rate of 25 μL/min. Therefore, the same amount of medium as that used for perfusion culture was used.

After culturing for 5 days, the morphology of vascular endothelial cells was evaluated by fluorescence observation, the vascular structure was visualized by perfusion of rat blood from the liquid feeding part, and histological evaluation was performed by HE staining and immunostaining using CD31. It was evaluated whether the impartation of a vascular structure with a lumen into the sheet had been achieved (Fig. 7).

The morphology of GFP-HUVEC was evaluated by observing with a fluorescence microscope directly above the cell sheet on the artificial vascular bed (Fig. 8).

Low-magnification images before and after culture (left and middle in FIG. 8) and high-magnification images of the tissue after culture (right in FIG. 8) are shown for each of the specimens that underwent perfusion culture and static culture for 5 days. Under any conditions, it was observed that the networking of vascular endothelial cells was promoted more in the tissue after culture than before culture. In addition, it was confirmed that in the perfusion-cultured specimen, cells were accumulated in the pore structure portion compared to the static culture specimen. Furthermore, from observation images at high magnification, it was confirmed that the network structure of endothelial cells was thicker and formed lumens in the perfusion-cultured tissue compared to the static culture structure.

<Example 5>
In order to verify the effect of adding blood vessels to the cell sheet, we visualized the blood vessel structure by perfusion of Indian ink. By perfusing India India ink, diluted twice with physiological saline, for 1 hour at a flow rate of 25 μL/min, which is the same as in the perfusion culture, it was evaluated whether a vascular structure with a perfusable lumen was provided in the cell sheet. bottom. In addition, a syringe pump was used for perfusion of Indian ink (Fig. 9).

FIG. 9 shows observation images after 1-hour ink perfusion of tissues prepared by static culture and perfusion culture. In the tissue prepared by static culture, it was observed that the ink perfused from the first flow path in the lower part of the figure passed through the bridging flow path from the pore structure and flowed out to the second flow path. No vascular structure was observed in the sheet. In addition, although a network structure of vascular endothelial cells was formed in the tissue, perfusion of Indian ink was not observed, and it was clarified that static culture could not introduce a perfusable vascular network. On the other hand, in the tissue prepared by perfusion culture, it was confirmed that India ink flowed out to the upper surface of the tissue through the pore structure on the first channel and the vascular structure in the tissue formed starting from the bridging channel. bottom. In addition, as a result of fluorescence observation of the blood vessel network into which India ink has flowed, it was confirmed that India ink was introduced into the vascular lumen composed of GFP-positive vascular endothelial cells. admitted.

The tissue prepared by perfusion culture was perfused for 1 hour with conditioned blood obtained by diluting rat blood twice with physiological saline using a syringe pump. An observation image of the tissue after perfusion is shown in FIG. It was confirmed that the prepared blood that flowed in from the first channel passed through the mesh-like structure in the cell sheet and flowed out to the top. Furthermore, as a result of observing the dotted line portion on the upper right with a blood flow scope (TOKU Capillaro), it was confirmed that erythrocytes in the prepared blood flowed through the mesh-like structure in the cell sheet (FIG. 10, lower left). Based on the above results, perfusion culture on the artificial vascular bed using the bridging channels (and, if possible, the holes) of the mounting surface creates a vascular network at a level that can be confirmed by macroscopic observation in the cell sheet. It was confirmed that the formed and added blood vessels were capable of perfusion of erythrocytes.

<Example 6>
Tissue sections were prepared from specimens subjected to static culture and perfusion culture, and histological evaluation was performed by HE staining and fluorescent immunostaining using CD31. The cultured tissue was fixed with 4% paraformaldehyde and then embedded in paraffin. After that, tissue sections were prepared in the pore structure part and the bridging channel part, and HE staining was performed. Furthermore, in order to evaluate the distribution of vascular endothelial cells in the tissue, immunostaining using CD31, which is an antibody for vascular endothelial cells, was performed. In addition, observation images of tissue cross sections are referred to as inflow, bridge, and outflow parts in consideration of the liquid supply part and outflow part in perfusion culture, and each part is shown at low magnification and high magnification. rice field.

From the HE-stained image in static culture (Fig. 11) and the results of immunostaining using CD31 (Fig. 11-2), in static culture at any point, blood vessels having a lumen in the cell sheet were not added. It was confirmed that it did not.

On the other hand, in the HE-stained image (FIG. 12-1) of the sample subjected to perfusion culture, it was confirmed that the cells proliferated significantly in the Outflow area and the Bridge area. Furthermore, from observation images at high magnification, it was confirmed that a large number of luminal structures containing erythrocytes, as indicated by red arrows, were formed in all sites.

In addition, in order to evaluate the distribution of vascular endothelial cells, fluorescence immunostaining using CD31 was performed (Fig. 12-2). As a result, it was confirmed that a luminal structure composed of vascular endothelial cells stained green was uniformly and densely present in the tissue. In addition, it was confirmed that erythrocytes were contained within the luminal structure composed of vascular endothelial cells. This revealed that many luminal structures in tissues containing erythrocytes are blood vessels with lumens composed of endothelial cells.

From the above results, it was clarified that the method of perfusion culture directly under the cell sheet using the artificial vascular bed of the present invention is effective as a method of providing a large amount of functional blood vessels to a tissue with a high cell density in vitro. .

The artificial vascular bed of the present invention exhibited a greater vascularity than the conventional vascular bed, and the supply of a large amount of biochemical factors from the proximal region of the cell sheet and the mechanical stimulation due to perfusion are thought to be factors (Fig. 13). . In conventional vascular beds, neovascularization within the gel proximal or distal to the cell sheet engraftment surface or diffusion of biochemical factors such as cytokines from blood vessels within muscle tissue induces angiogenesis within the tissue. bottom. On the other hand, in the present invention, the pore structure provided in the fibrin gel, whose structure is difficult to be changed by cells, and the bridging channel are used directly as the vascular bed channel, thereby stably supplying the culture solution directly below the cell sheet. realized. This made it possible to stably supply a larger amount of cytokine from the vicinity of the cell sheet engraftment surface. Furthermore, it was possible to apply mechanical stimuli such as shear flow directly to the cell sheet by perfusion of the culture medium, which are thought to contribute to the induction of angiogenesis. It is believed that the combination of factors described above made it possible to provide unprecedented blood vessels to tissues with high cell density in vitro. In addition, since the pore structure provided in the fibrin gel is used as a direct channel, there is no variation in the shape of the vascular bed channel, and it is possible to stably supply biochemical factors to the tissue and to give mechanical stimulation by perfusion is possible, it has high reproducibility as a technique for imparting blood vessels (Fig. 13).

The artificial vascular bed developed in this research is expected to be used as a new platform technology for three-dimensional tissue perfusion culture in vitro. By utilizing a 3D printer, it is possible to freely design the pore structure and bridging channel on the fibrin gel surface, and since the main channel has a channel structure that does not contain cells, there is almost no sample difference. A vascular bed having a stable flow path structure without a flow path can be easily produced. On this vascular bed, by engrafting a cell sheet that has a proven track record of clinical research with various cells and performing perfusion culture, a three-dimensional tissue with a vascular network that can actually be perfused is created. is possible. In addition, by engrafting a three-dimensional tissue such as an organoid on a cell sheet produced by a tissue engineering technique other than a cell sheet, the engrafted three-dimensional tissue can be provided with blood vessels in vitro and perfused. It is expected that culturing is possible. As a result, it is possible to directly apply mechanical stimulation by fluid to three-dimensional living tissue, so it can be used as an experimental model for clarifying the effect of mechanical stimulation on angiogenesis in three-dimensional tissue with high cell density. It is possible. In addition, it can be used as a drug discovery test model using a three-dimensional tissue constructed in vitro by perfusion of a drug into the applied blood vessel, or as a research model for transplantation treatment using a three-dimensional tissue constructed in vitro. It is expected to be applied to various uses of three-dimensional tissue construction research outside.

<Example 7>
As an example of the application of the present invention, the possibility of use as an angiogenesis research model was investigated by confirming the presence or absence of the influence of changes in the perfusion amount during perfusion culture on angiogenesis. The specimen was perfused cultured for 5 days at a perfusion rate of 25 μL / min under condition 1, the perfusion rate was increased to 25 μL / min for 3 days, and then the perfusion rate was increased four times to 100 μL / min for 2 days. The specimen cultured under each condition was treated as condition 2, and the perfused vascular structure was compared by perfusing India ink into the specimen cultured under each condition. An extensive vascular network was observed (Fig. 15). From the above results, it was confirmed that changing the perfusion rate has an effect on angiogenesis. In other words, it is suggested that this model can be used as an angiogenesis research model for clarifying the biochemical and mechanical conditions necessary for efficient angiogenesis into high-cell-density three-dimensional tissues in vitro. rice field.

<Example 8>
As a feature of this method, the structure of the device produced by the design using Three Dimensional Computer-Aided Design (3D CAD) is transferred to the surface of the fibrin gel to construct the flow path of the vascular bed, so it can be easily used in various ways. A channel can be constructed. Therefore, it is expected that angiogenesis can be induced in a wider range by designing the position of angiogenesis in a three-dimensional tissue or by providing a plurality of channels. In addition, since fibrin gel is used, the structure of which is difficult to change due to cells, by introducing computational fluid dynamics analysis, it is possible to grasp the speed of the culture solution flowing through each channel when multiple channels are used. By appropriately changing the design of the flow path accordingly, it is possible to construct a plurality of flow paths in which the flow rate is controlled. FIG. 16 shows an example of an artificial vascular bed with multiple channels.

<Example 9>
In order to demonstrate the usefulness of the present invention as a three-dimensional tissue construction method, it was verified whether three-dimensional tissue construction by stepwise lamination of cell sheets using the developed artificial vascular bed is possible. “Stepwise lamination” means that a set of three layers of cell sheets that can be maintained by the diffusion of the culture medium is set and repeatedly transplanted onto the artificial vascular bed at regular intervals, thereby gradually introducing blood vessels into the cell sheet. This is a technique for constructing a three-dimensional structure (Fig. 17).

In this example, a cell sheet prepared by the same method as in Example 2 was used. After the 3-layer cell sheet was perfusion cultured on the artificial vascular bed for 5 days, the next set of 3-layer cell sheets was transplanted, and additional 3-day perfusion culture was performed. As a result of observing the cross section of the tissue after culturing by OCT, formation of a tubular structure was also observed in the additionally laminated three-layered cell sheet (Fig. 18). Furthermore, as a result of perfusion of India ink into the cultured tissue, it was clarified that a large amount of vascular network, in which perfusion of India ink was observed, was introduced into the six-layered cell sheet (Fig. 19). From the above results, it was shown that by stepwise lamination using the artificial vascular bed of the present invention, a three-dimensional tissue having a vascular network capable of perfusion can be constructed. From this, it is expected that by increasing the number of stepwise lamination to 3, 6, 9, and 12 layers, it will be possible to apply to construction of millimeter-order three-dimensional structures, which has been difficult to fabricate conventionally. In other words, it was suggested that this method is useful as a three-dimensional tissue construction method in vitro.

Claims (12)

An artificial vascular bed,
a vascular bed formed of a composition containing a biocompatible hydrogel and having a three-dimensional biological tissue mounting surface;
a first channel and a second channel provided inside the vascular bed;
a first hole and a second hole provided in the mounting surface and communicating with the first channel and the second channel, respectively;
a bridging flow path communicating between the first gap and the second gap and formed in the mounting surface;
a first liquid delivery unit in fluid communication with the first channel for supplying culture medium;
and a second liquid delivery section in fluid communication with the second flow path for discharging culture medium. 2. The artificial vascular bed according to claim 1, wherein the biocompatible hydrogel is a crosslinked biocompatible hydrogel. 2. The artificial vascular bed of claim 1, wherein said biocompatible hydrogel comprises fibrin gel. 4. The artificial vascular bed of claim 3, wherein said biocompatible hydrogel comprises a fibrin gel stabilizing factor. a medium supply line connected to the first liquid feeding unit;
a medium supply tank for supplying a medium to the medium supply line;
a liquid feed pump that feeds the medium to the medium supply line;
a medium discharge line connected to the second liquid feeding section;
a medium discharge tank for storing the medium discharged from the medium discharge line;
The artificial vascular bed according to any one of claims 1 to 4, further comprising: 6. The artificial vascular bed according to claim 5, wherein the medium supply tank and the medium discharge tank are the same, and the medium is circulated. The artificial vascular bed according to any one of claims 1 to 6;
and a three-dimensional living tissue mounted on the mounting surface so as to cover the bridging channel. 8. The artificial three-dimensional biological tissue according to claim 7, wherein the three-dimensional biological tissue is a sheet-like tissue containing cells or an organoid. The artificial three-dimensional biological tissue according to claim 7 or 8, wherein the three-dimensional biological tissue contains vascular endothelial cells. A method for producing an artificial three-dimensional biological tissue having a vascular network, comprising:
A step of placing a three-dimensional biological tissue so as to cover the bridging channel of the placement surface of the artificial vascular bed according to any one of claims 1 to 6;
A step of supplying a medium to the first liquid feeding part and performing perfusion culture while discharging the medium from the second liquid feeding part;
A method, including 11. The method according to claim 10, wherein the three-dimensional biological tissue is a sheet-like tissue or organoid containing cells. 12. The method of claim 10 or 11, wherein the three-dimensional biological tissue comprises vascular endothelial cells.

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