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

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]

[0001] 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. [Background technology]

[0002] Currently, regenerative medicine using myoblast sheets or cardiomyocyte sheets derived from induced pluripotent stem cells (iPS cells) has begun as a new treatment for heart failure (Non-Patent Documents 1 and 2). This treatment relies on the paracrine effect, whereby the numerous growth factors secreted by the cell sheets promote angiogenesis in the host heart and improve cardiac function. However, these treatments do not result in an increase in functional cardiomyocytes, and are therefore not applicable to severe heart failure. Therefore, the present inventors are aiming to develop a next-generation regenerative medicine treatment using three-dimensional myocardial tissue transplantation with mechanical pulsation-assistance function. This treatment involves transplanting three-dimensional myocardial tissue produced ex vivo by layering multiple cardiomyocyte sheets derived from human iPS cells. The realization of this treatment is expected to not only provide the conventional paracrine effect, but also mechanically assist the pump function of the cardiomyocytes themselves.

[0003] However, it has been reported that layered tissues obtained by fabricating three or more layers of cell sheets experience necrosis within the tissue due to lack of oxygen and nutrients and the accumulation of waste products (Non-Patent Document 3). Therefore, in order to construct three-dimensional myocardial tissue that exceeds the layering limit, it is important to establish a method for providing a vascular structure responsible for nutrient supply inside the three-dimensional tissue ex vivo.

[0004] In vitro vascular construction research can be broadly divided into research targeting hydrogels with low cell densities and research targeting three-dimensional tissues with high cell densities. In research targeting hydrogels, numerous methods have been reported for constructing blood vessels with perfusion-capable luminal structures from endothelial cells suspended in hydrogels using 3D printing or microfluidic devices (e.g., Non-Patent Documents 4 and 5). In contrast, in research targeting three-dimensional tissues such as organoids and cell sheets, most reports involve the creation of blood vessels with actual blood perfusion by transplanting tissues with endothelial cell networks constructed in vitro into living organisms (Non-Patent Documents 6 and 7). In other words, while methods for constructing luminal blood vessels in vitro have been established in hydrogels, they remain unestablished for tissues with high cell densities.

[0005] The present inventors have previously developed vascular beds using collagen gels and biological tissues, thereby enabling the introduction of a vascular network ex vivo into cell sheets, which are high-cell-density tissues (Patent Documents 1 and 2, and Non-Patent Documents 8 and 9). A vascular bed is a foundation for three-dimensional tissue culture, with a flow path structure that allows the perfusion of culture medium. By utilizing blood vessels constructed in collagen gel or blood vessels in three-dimensional biological tissue, and perfusing culture medium into the blood vessels in the vascular bed after engrafting a cell sheet onto the vascular bed, angiogenic factors in the culture medium are supplied to the cell sheet by diffusion through the blood vessels, and angiogenesis is induced within the cell sheet, thereby achieving the introduction of blood vessels into the sheet.

[0006] They also reported that they created a microfluidic device made of polydimethylsiloxane (PDMS) and introduced a perfusable vascular structure into spheroids suspended in the hydrogel by using a vascular structure constructed within the hydrogel (Non-Patent Document 10).These studies have reported that by utilizing the vascular network within hydrogels and three-dimensional biological tissues, it is possible to introduce a perfusable vascular network into high-cell-density tissues ex vivo. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2012 / 036225 [Patent Document 2] International Publication No. 2012 / 036224 [Non-patent literature]

[0008] [Non-Patent Document 1] Miyagawa S, et al.: Impaired Myocardium Regeneration With Skeletal Cell Sheets-A Preclinical Trial for Tissue-Engineered Regeneration Therapy. Transplantation, 2010 [Non-patent document 2] 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 [Non-patent document 3] Shimizu T, et al: Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. The FASEB Journal, 2006 [Non-patent document 4] Kolesky, DB, et al.: 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Adv. Mater. 26, 3124‐3130, 2014 [Non-Patent Document 5] Nguyen, D.-HT, et al.: Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc. Natl. Acad. Sci. USA 110, 6712-6717, 2013 [Non-patent document 6] Takebe T, et al.: Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature. 499, 481-485, 2013 [Non-Patent Document 7] 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 [Non-patent document 8] Sakaguchi K, et al.: In vitro engineering of vascularized tissue surrogates, Sci. Rep., 1316, 2013 [Non-Patent Document 9] Sekine H, et al.: In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels. Nat. Com, 2013 [Non-Patent Document 10] Nashimoto Y, et al.: Integrating perfusable vascular networks with a three-dimensional tissue in a microfluidic device. Integr. Biol., 2017 Summary of the Invention [Problem to be solved by the invention]

[0009] Conventional methods for introducing vascular networks into in vitro formed three-dimensional tissues (e.g., high-cell-density three-dimensional tissues such as layered cell sheets and organoids) that utilize vascular networks within the vascular beds of hydrogels or three-dimensional tissues have the drawback of requiring at least five days or more to introduce a vascular network, and even if a vascular network is introduced, the number of blood vessels formed is extremely small, resulting in poor efficiency of vascular network introduction. Furthermore, with conventional techniques, it is difficult to control the position of the neovascular network formed within the collagen gel used as the vascular bed and the position of the blood vessels within the collected tissue, resulting in differences in the physical distance from the vascular bed flow path to the three-dimensional tissue for each vascular bed sample, and therefore variations in the quality of the vascular bed itself. [Means for solving the problem]

[0010] The present inventors have conducted research and development in an effort to solve the above-mentioned problems, making various investigations. As a result, they have discovered that by controlling the physical distance and shape between the three-dimensional biological tissue and the flow channels constructed in the vascular bed, it is possible to improve the quality (e.g., increase the number of vascular networks) of the vascular network constructed in the three-dimensional biological tissue (e.g., a three-dimensional tissue with a high cell density, such as a layered cell sheet or an organoid), and that it is possible to not only shorten the time required to construct the vascular network, but also to construct the vascular network with good reproducibility, thereby completing the present invention. That is, the present invention may include the following aspects.

[0011] [1] An artificial vascular bed, a vascular bed formed from a composition including a biocompatible hydrogel, the vascular bed having a mounting surface for three-dimensional biological tissue; a first flow path and a second flow path provided within the vascular bed; a first hole and a second hole provided on the mounting surface, the first hole and the second hole communicating with the first flow path and the second flow path, respectively; a bridging channel that communicates between the first gap and the second gap and is formed on the mounting surface; a first fluid delivery unit in fluid communication with the first flow path for supplying a culture medium; a second fluid delivery unit in fluid communication with the second flow path for discharging the culture medium; An artificial vascular bed comprising: [2] The artificial vascular bed described in Item 1, wherein the biocompatible hydrogel is a crosslinked biocompatible hydrogel. [3] The artificial vascular bed described in Item 1, wherein the biocompatible hydrogel comprises a 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 delivery unit; a medium supply tank for supplying a medium to the medium supply line; a liquid supply pump for supplying a culture medium to the culture medium supply line; a culture medium discharge line connected to the second liquid supply unit; a culture medium discharge tank for storing the culture medium discharged from the culture medium discharge line; 5. 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 tank, and the medium is circulated.

[0012] [7] An artificial vascular bed according to any one of items 1 to 6, a three-dimensional biological tissue placed on the placement surface so as to cover the bridging channel; and an artificial three-dimensional biological tissue comprising: [8] The artificial three-dimensional biological tissue described in Item 7, wherein the three-dimensional biological tissue is a sheet-like tissue or an organoid containing cells. [9] The artificial three-dimensional biological tissue according to Item 7 or 8, wherein the three-dimensional biological tissue contains vascular endothelial cells.

[0013]

[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 described in any one of items 1 to 6; a step of perfusion culturing while supplying a culture medium to the first liquid supplying section and discharging the culture medium from the second liquid supplying section; A method comprising:

[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 comprises vascular endothelial cells. [Effects of the Invention]

[0014] The present invention can improve the quality of vascular networks constructed within three-dimensional biological tissues (e.g., by increasing the number of vascular networks and forming mature blood vessels with large diameters), and can also shorten the time required to construct a vascular network. Furthermore, because this technology directly utilizes pores and bridging channels in the vascular bed as channels in the vascular bed, it solves the problem of variations in channel structure among vascular bed specimens. Therefore, it is possible to construct a high-quality vascular network with good reproducibility. Furthermore, because it is possible to directly apply mechanical stimuli using fluids to three-dimensional biological tissues, it can be used as an experimental model to elucidate the effects of mechanical stimuli on angiogenesis within three-dimensional tissues with high cell density. [Brief explanation of the drawings]

[0015] [Figure 1-1] FIG. 1 is a diagram showing an outline of an artificial vascular bed of the present invention in one embodiment. [Figure 1-2] FIG. 1 shows an overview of the artificial vascular bed of the present invention in one embodiment, and an overview of the three-dimensional biological tissue during perfusion culture. [Figure 2]FIG. 1 is a schematic diagram showing the procedure for producing an artificial vascular bed of the present invention in one embodiment. [Figure 3] FIG. 1 shows an artificial vascular bed of the present invention in one embodiment. [Figure 4] 1 shows a fluorescent image and an optical coherence tomography (OCT) image of a three-dimensional biological tissue with a vascular endothelial network (a co-culture sheet of layered vascular endothelial cells and dermal fibroblasts) placed on an artificial vascular bed of the present invention in one embodiment. [Figure 5] A schematic diagram for selecting a hydrogel composition suitable for the fabrication of an artificial vascular bed is shown. [Figure 6-1] The results of evaluating the structural stability of artificial vascular beds made from different hydrogels after static culture of cell sheets for 5 days are shown. Top: Macroscopic observation images (BF), bottom: Cross-sectional observation images (OCT). [Figure 6-2] The results of evaluating the structural stability of artificial vascular beds when cell sheets were statically cultured for 5 days on artificial vascular beds made from different hydrogels are shown. The surface area and cross-sectional area were quantified from the images obtained in Figure 6-1, and the surface area ratio (left) and cross-sectional area ratio (right) are shown for each day, with the first day (Day 0) as the base. [Figure 7] FIG. 1 is a schematic diagram showing a method for static culture or perfusion culture using the artificial vascular bed of the present invention in one embodiment, and a method for evaluating the same. [Figure 8] 1 shows a fluorescence microscope image of a cell sheet containing GFP-positive human umbilical vein endothelial cells (GFP-HUVEC) when the cell sheet was subjected to static culture or perfusion culture using an artificial vascular bed of the present invention in one embodiment. [Figure 9] 1 shows the results of evaluating blood vessels formed in a cell sheet by perfusion with India ink after static culture or perfusion culture of the cell sheet using an artificial vascular bed of the present invention in one embodiment. [Figure 10] 1 shows the results of perfusion culture of a cell sheet using an artificial vascular bed of the present invention in one embodiment, followed by evaluation of blood vessels formed in the cell sheet by perfusion with rat blood. [Figure 11-1]1 shows an HE stained image of a cell sheet after static culture using an artificial vascular bed of the present invention in one embodiment. [Figure 11-2] 1 shows a fluorescence microscope image of a cell sheet after static culture using an artificial vascular bed of the present invention in one embodiment, where green indicates endothelial cells (CD31 positive), and blue indicates nuclei (DAPI). [Figure 12-1] 1 shows an HE staining image after perfusion culture of a cell sheet using the artificial vascular bed of the present invention in one embodiment. [Figure 12-2] 1 shows a fluorescence microscope image of a cell sheet after perfusion culture using an artificial vascular bed of the present invention in one embodiment, green: endothelial cells (CD31 positive), blue: nuclei (DAPI). [Figure 13] FIG. 1 is a schematic diagram showing the results of a comparison between the vascular beds of the prior art and the present invention. [Figure 14] 1 shows an example of application of an artificial vascular bed or an artificial three-dimensional biological tissue (three-dimensional tissue) of the present invention in one embodiment. [Figure 15] In one embodiment, a cell sheet was perfusion cultured using an artificial vascular bed of the present invention at different perfusion rates (25 μL / min (5 days) or 25 μL / min (3 days) → 100 μL / min (2 days)), and the blood vessels formed in the cell sheet were evaluated by perfusion with India ink. [Figure 16] 1 shows an example of an artificial vascular bed of the present invention in one embodiment. [Figure 17] 1 shows an outline of a method for constructing a three-dimensional tissue by stepwise lamination using an artificial vascular bed of the present invention in one embodiment. [Figure 18] 1 shows an OCT image of a three-dimensional tissue constructed using an artificial vascular bed of the present invention in one embodiment. [Figure 19] 1 shows enlarged images (left: bright field observation image, left: fluorescent observation image) of a three-dimensional tissue constructed using an artificial vascular bed of the present invention in one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, embodiments of the present invention will be described with reference to the drawings as needed. The configurations of the embodiments are examples, and the configuration of the present invention is not limited to the specific configurations of the embodiments.

[0017] In this specification, terms such as "first," "second," and "third" are used to distinguish one element from another; for example, a first element may be expressed as a second element, and similarly, a second element may be expressed as a first element, without departing from the scope of the present invention.

[0018] Unless otherwise defined, all terms (technical and scientific) used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0019] 1-1 and 1-2 are schematic diagrams showing an artificial vascular bed of the present invention in one embodiment. An artificial vascular bed, comprising: a vascular bed formed from a composition including a biocompatible hydrogel, the vascular bed having a mounting surface for three-dimensional biological tissue; a first flow path and a second flow path provided within the vascular bed; a first hole and a second hole provided on the mounting surface, the first hole and the second hole communicating with the first flow path and the second flow path, respectively; a bridging channel that communicates between the first gap and the second gap and is formed on the mounting surface; a first fluid delivery unit in fluid communication with the first flow path for supplying a culture medium; a second fluid delivery unit in fluid communication with the second flow path for discharging the culture medium; To provide an artificial vascular bed comprising:

[0020] As used herein, the term "artificial vascular bed" refers to a vascular bed that can be produced in vitro and is used to construct a vascular network in a three-dimensional biological tissue (also referred to as a "three-dimensional tissue" in this specification), and is distinct from conventional tissues rich in vascular networks (for example, biological tissues such as skin flaps described in WO 2012 / 036224).

[0021] In one embodiment, the artificial vascular bed of the present invention comprises a vascular bed portion having a support surface for supporting a three-dimensional biological tissue (also referred to herein as a "three-dimensional tissue") intended to construct a perfusable vascular network. The support surface may have any shape that allows the three-dimensional biological tissue to be supported thereon, but is preferably flat.

[0022] The vascular bed is formed from a composition containing a biocompatible hydrogel. Examples of "biocompatible hydrogels" applicable to the present invention include hydrogels obtained by chemically crosslinking water-soluble, water-philic, or water-absorbent synthetic polymers such as polyacrylamide, polyacrylic acid, polyhydroxyethyl methacrylate, polyvinyl alcohol, polylactic acid, and polyglycolic acid, as well as polysaccharides, proteins, and nucleic acids. Examples of polysaccharides include glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, starch, glycogen, agarose, pectin, and cellulose. Examples of proteins include collagen and its hydrolyzed products such as gelatin, proteoglycans, fibronectin, vitronectin, laminin, entactin, tenascin, thrombospondin, von Willebrand factor, osteopontin, and fibrinogen (e.g., fibrin gel prepared by reacting fibrinogen with thrombin). These biocompatible hydrogels may be crosslinked (e.g., polymerized) using known methods to produce crosslinked biocompatible hydrogels with increased strength. The biocompatible hydrogels that can be used in the present invention are preferably hydrogels that have mechanical properties and high cell adhesiveness that can withstand the contraction of a three-dimensional biological tissue (e.g., a sheet-like tissue containing cells) placed on the artificial vascular bed of the present invention. For example, they may be fibrin gels that further contain factor XIII. The inclusion of a fibrin gel stabilizing factor in the fibrin gel promotes crosslinking of the fibrin gel, preventing or delaying the degradation or contraction of the biocompatible hydrogel by the three-dimensional biological tissue placed on the mounting surface of the artificial vascular bed of the present invention, thereby maintaining the shape of the flow path within the vascular bed.

[0023] In one embodiment, the vascular bed portion of the artificial vascular bed of the present invention is provided with a first flow path inside that can deliver a fluid containing nutrients necessary for culture, such as a culture medium, from the outside of the vascular bed portion, and a second flow path inside that can discharge a fluid containing nutrients necessary for culture, such as a culture medium, that has been supplied to the three-dimensional biological tissue on the mounting surface.

[0024] In one embodiment, the artificial vascular bed of the present invention includes a bridging channel that connects the first and second pores and is formed on the mounting surface (see Figures 1-2). The bridging channel provides fluid communication between the first and second pores and has an open opening along the mounting surface, allowing culture medium from the first channel to be directly supplied to the three-dimensional biological tissue placed over the bridging channel. The supply of a large amount of biochemical factors and mechanical stimulation by perfusion from directly below the three-dimensional biological tissue allows the creation of a functional vascular network within the applied three-dimensional biological tissue, a level that has not been achieved with previous artificial or biological vascular beds.

[0025] The cross-sectional shapes of the first and second flow channels may be rectangular or approximately circular. In the case of a circular shape, the diameter is not particularly limited, as it can be adjusted depending on the shape and thickness of the three-dimensional biological tissue to be used in the artificial vascular bed. The width and depth of the bridging channel may be, for example, approximately 0.01 to 10 mm, preferably approximately 0.1 to 5 mm, as long as the culture medium delivered from the first flow channel can be exposed directly below the three-dimensional biological tissue. However, if the width is increased, the three-dimensional biological tissue on the mounting surface may sink in that area, increasing the resistance of the flow channel. Therefore, it is more preferable to adjust the width to a value that does not cause this. At the connection between the first or second flow channel and the bridging channel, a first vertical hole and a second vertical hole are provided, respectively, which connect the mounting surface with the first or second flow channel (Figures 1-1 and 1-2).

[0026] In one embodiment, the first and second flow paths may be arranged substantially parallel to each other as shown in FIG. 1-1, may be arranged linearly on both sides of the bridging flow path, or may be arranged in a V-shape on both sides of the bridging flow path, but are not limited thereto. In another embodiment, one or more bridging flow paths may be provided between the first and second flow paths (e.g., FIG. 17). In another embodiment, the combination of "first flow path-bridging flow path-second flow path" may be one or more. The bridging flow path may have a more complex structure, such as a mesh-like structure, as long as it connects the first and second flow paths, and is not limited to a linear structure. Furthermore, the outer peripheries of the first and second flow paths may be formed in contact with the mounting surface, or grooves may be formed on the mounting surface as the first and second flow paths. In these cases, the first and second holes and holes may not be provided.

[0027] In one embodiment, the artificial vascular bed of the present invention comprises a first fluid delivery section fluidly connected to the first flow path for supplying a culture medium, and a second fluid delivery section fluidly connected to the second flow path for discharging the culture medium. In one embodiment, the first flow path and the first fluid delivery section, and the second flow path and the second fluid delivery section, may be integrally formed, or may be a tube partially made of a material other than a biocompatible hydrogel (e.g., a silicone tube, a metal tube, a catheter, a Surflo needle, or an artificial blood vessel made of polyester (e.g., Dacron (registered trademark), Teflon (ePTEE) or a synthetic polymer, etc.).

[0028] For example, when using Surflow needles as the first and second fluid delivery units, a composition containing a biocompatible hydrogel is poured into a mold for forming a vascular bed, and the Surflow needles serving as the first and second fluid delivery units are positioned so that they protrude beyond the vascular bed (Figure 2). A channel-forming device having a bridging channel-forming unit and a pore-forming unit is then placed on top of the mold, and the pore-forming unit is positioned so that it contacts the Surflow needles serving as the first and second fluid delivery units, allowing the biocompatible hydrogel to harden. The channel-forming device and the Surflow needles can then be removed as needed, allowing the resulting artificial vascular bed to be used (Figure 2). The mold may be used together with the artificial vascular bed during culture, and can be removed as needed for transplantation or other applications. Furthermore, by moving the tip of the outer tube of the Surflow needle toward the first and second fluid delivery sections from the hole section and removing the inner tube of the Surflow needle, the outer tube of the Surflow needle can be used as the first flow path (or second flow path) and the first fluid delivery section (or second fluid delivery section). This makes it easy to connect a medium supply line (or medium discharge line) during perfusion culture. It also improves the pressure resistance of the flow path.

[0029] In one embodiment, the artificial vascular bed of the present invention comprises: a culture medium supply line connected to the first liquid transport unit; a medium supply tank for supplying a medium to the medium supply line; a liquid supply pump for supplying a culture medium to the culture medium supply line; a culture medium discharge line connected to the second liquid supply unit; The apparatus may further include a culture medium discharge tank for storing the culture medium discharged from the culture medium discharge line. These configurations enable the culture medium to be perfused continuously or intermittently. As the liquid delivery pump, any known pump can be used, including a tube pump (perista pump) or a piezoelectric pump, as long as it is capable of delivering a fluid.

[0030] In one embodiment, the medium supply tank and the medium discharge tank may be the same, allowing the medium to circulate. This allows liquid components, such as cell growth factors, produced by the three-dimensional biological tissue placed on the artificial vascular bed to circulate for a certain period of time before the medium is replaced.

[0031] By using the artificial vascular bed of the present invention, a three-dimensional biological tissue, an artificial vascular bed, and a perfusable vascular network between them can be constructed. As used herein, "three-dimensional biological tissue" refers to a three-dimensional structure containing cells, and includes, for example, biological tissue isolated from a living body (e.g., organs and tissues or parts thereof (e.g., skin tissue (e.g., skin tissue associated with hair roots), cardiac muscle tissue, skeletal muscle tissue, smooth muscle tissue, liver tissue, kidney tissue, digestive tract tissue, eye tissue (e.g., corneal tissue), brain tissue, thymus tissue, testis tissue, pancreatic tissue, thyroid tissue, mammary gland tissue, salivary gland tissue, lung tissue, etc.)), or a three-dimensional cell structure reconstructed using cells constituting biological tissue, such as an organoid, is applicable to the present invention. The biological tissue isolated from a living organism that can be applied to the present invention may be the biological tissue itself collected from a living organism, or may be a tissue piece obtained by processing biological tissue collected from a living organism into any shape. Furthermore, the three-dimensional cell structure that can be applied 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, a three-dimensional cell structure in which multiple sheet-like tissues containing cells (e.g., cell sheets) are stacked, or a sheet-like tissue containing cells on which organoids are placed.

[0032] As used herein, the term "organoid" refers to a three-dimensional tissue constructed in vitro from a collection of progenitor cells that contribute to the formation of an organ or organ system, and is synonymous with the term "organoid" as interpreted in the broadest sense by those skilled in the art. Organoids are generally smaller and simpler in structure than actual organs or organ systems, but exhibit anatomical and functional characteristics similar to those of organs or organ systems present in vivo. Examples of organoids include kidney organoids, liver organoids, gastrointestinal organoids (e.g., intestinal organoids, oral organoids, stomach organoids, etc.), optic cup organoids, brain organoids, thymus organoids, testicular organoids, pancreatic organoids, epithelial organoids, thyroid organoids, mammary gland organoids, salivary gland organoids, and lung organoids. These organoids also include "cancer organoids" containing cancer cells. Furthermore, as used herein, organoids may include organs and tissues collected from a living body or portions thereof (e.g., tissue fragments) (see, for example, Shamir ER., Nat. Rev. Mol. Cell Biol. 2014 Oct; 15(10): 647-64).

[0033] Methods for obtaining three-dimensional biological tissues applicable to the present invention, such as organoids, may be known methods, including but not limited to the following: for kidney organoids, see Takasato M., et al., Nature. 2015, Oct. 22, 526 (7574), pp. 564-568; for lung organoids, see Unbekandt M., Kidney Int., 2010, Mar., 77 (5), pp. 407-416; for thymus organoids, see Sheridan JM., et al., Genesis, 2009 May, 47 (5), pp. 346-351; for testicular organoids, see Sanjo H., et al., PLoS One. 2018, Feb 12, 13 (2), e0192884. Additionally, any organoid obtained by a method for forming organoids can also be used.

[0034] The three-dimensional biological tissue applicable to the present invention is, for example, a three-dimensional biological tissue of a mammal, a bird, an amphibian, a reptile, or a fish, and is preferably a three-dimensional biological tissue of a mammal, such as a mouse, a rat, a human, a monkey, a pig, a dog, a sheep, a cat, or a goat, containing cells derived from the mammal.

[0035] The cells contained in the three-dimensional biological tissue applicable to the present invention may be primary cells collected from the biological tissue, established cell lines, or cells induced to differentiate from pluripotent stem cells or tissue stem cells (e.g., mesenchymal stem cells).

[0036] As used herein, the term "pluripotent stem cells" 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, and the like. Any known pluripotent stem cells can be used, including, for example, the pluripotent stem cells described in International Publication No. WO 2009 / 123349.

[0037] Known methods can be used to differentiate tissue stem cells or pluripotent cells into cells that construct any three-dimensional biological tissue (for example, organoids). For example, but not limited to, cells for constructing kidney organoids are described in Takasato M., et al., Nature. 2015, Oct. 22, 526 (7574), pp. 564-568; cells for constructing liver organoids are described in Takebe T., et al., Nature, 2013, Jul. 25, 499 (7459), pp. 481-484; cells for constructing digestive tract organoids are described in Sato T., et al., Nature, 2009, May 14, 459 (7244), pp. 262-265; cells for constructing optic cup organoids are described in Eiraku M., et al., Nature, 2011, Apr. 7, 472 (7341), pp. 51-56; cells for constructing brain organoids are described in 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. For cells used to construct lung organoids, see Yamamoto Y.,et al.,Nat Methods.2017 Nov,14(11),pp.1097-1106. For cells used to construct pancreatic organoids, see Raikwar SP.,et al.,PLoS One.2015 Jan 28,10(1),e0116582. For cells used to construct thyroid organoids, see Ma R.,et al.,Front Endocrinol(Lausanne).2015 Apr 22,6,56. Additionally, any cells obtained by a method for differentiating them into cells that construct three-dimensional biological tissues can also be used.

[0038] As used herein, the term "sheet-like tissue containing cells" refers to biological tissue collected from a living organism or a thin-film tissue containing cells. The sheet-like tissue containing cells may be a tissue collected from a living organism itself, or a three-dimensional biological tissue obtained by processing tissue collected from a living organism into a sheet and laminating it into multiple layers (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, or more). Furthermore, the sheet-like tissue containing cells may be a sheet-like tissue formed by mixing a cell-containing suspension with a gel, or a cell sheet produced using a stimuli-responsive culture dish (e.g., a temperature-responsive culture dish). Furthermore, the sheet-like tissue containing cells may be formed by directly seeding cells on the upper surface of a membrane-like gel and culturing the cells.

[0039] As used herein, the term "cell sheet" refers to a single or multilayer sheet-like cell population obtained by culturing a cell population containing a plurality of cells on a cell culture substrate and then detaching the cell population from the cell culture substrate. Examples of methods for obtaining cell sheets include culturing cells on a stimuli-responsive culture substrate coated with a polymer whose molecular structure changes in response to stimuli such as temperature, pH, or light, and then altering the surface of the stimuli-responsive culture substrate by changing the stimuli such as temperature, pH, or light to detach the cells in a sheet form from the stimuli-responsive culture substrate while maintaining cell adhesion. Other methods include culturing cells on a culture substrate and then physically detaching them using tweezers or the like. Known stimuli-responsive culture substrates for obtaining cell sheets include temperature-responsive culture substrates coated with a polymer whose hydration strength changes over a temperature range of 0 to 80°C. Cells are cultured on the temperature-responsive culture substrate at a temperature range where the hydration strength of the polymer is weak, and then the culture medium is changed to a temperature where the hydration strength of the polymer is strong, allowing the cells to be detached as a sheet-like cell population.

[0040] The temperature-responsive culture substrate used to obtain a cell sheet is preferably a substrate that changes its surface hydration power within a temperature range in which cells can be cultured. This temperature range is preferably the temperature at which cells are generally cultured, for example, 33°C to 40°C. The temperature-responsive polymer coated on the culture substrate used to obtain a cell sheet may be either a homopolymer or a copolymer. Examples of such polymers include the polymers described in JP-A-2-211865.

[0041] The use of poly(N-isopropylacrylamide) as a stimuli-responsive polymer, particularly a temperature-responsive polymer, will be described using an example (temperature-responsive culture dish). Poly(N-isopropylacrylamide) is known to have a lower critical dissolution temperature of 31°C. In its free state, it dehydrates in water at temperatures above 31°C, causing the polymer chains to aggregate and become cloudy. Conversely, at temperatures below 31°C, the polymer chains become hydrated and dissolved in water. In the present invention, this polymer is coated and immobilized on the surface of a substrate such as a petri dish. Therefore, at temperatures above 31°C, the polymer on the surface of the culture substrate also dehydrates, but because the polymer chains are immobilized on the surface, the surface of the culture substrate becomes hydrophobic. Conversely, at temperatures below 31°C, the polymer on the surface of the culture substrate becomes hydrated, but because the polymer chains are coated on the surface, the surface of the culture substrate becomes hydrophilic. The hydrophobic surface is an appropriate surface for cells to attach and grow, while the hydrophilic surface prevents cells from attaching. Therefore, when the substrate is cooled to below 31°C, the cells detach from the substrate surface. If the cells are cultured to confluence over the entire culture surface, the cell sheet can be collected by cooling the substrate to below 31°C. The temperature-responsive culture substrate is not limited as long as it has the same effect, but examples include UpCell (registered trademark) available from CellSeed Inc. (Tokyo, Japan) and the smart temperature-responsive culture dish SSCW available from the Cell Sheet Regenerative Medicine Promotion Organization (Tokyo, Japan).

[0042] In one embodiment, the present invention provides an artificial three-dimensional biological tissue comprising the above-described artificial vascular bed and a three-dimensional biological tissue placed on the mounting surface so as to cover the bridging channel. The artificial three-dimensional biological tissue obtained by the present invention can be used, for example, as an angiogenesis research model to clarify the relationship between mechanical stimuli and angiogenesis in a high-cell-density three-dimensional biological tissue through the study of channel shape and perfusion conditions. It can also be used as a new drug discovery test model that allows for the administration of drugs via the blood vessels and allows evaluation using, for example, morphological changes and increases or decreases in the levels of their metabolites as indicators. Furthermore, the artificial three-dimensional biological tissue can be provided such that the first and second fluid delivery sections can be anastomosed as arteries or veins, respectively (see, for example, FIG. 14).

[0043] In one embodiment, the present invention provides placing a three-dimensional biological tissue on the placement surface of the artificial vascular bed so as to cover the bridging channel; a step of perfusion culturing while supplying a culture medium to the first liquid supplying section and discharging the culture medium from the second liquid supplying section; The present invention provides a method for producing an artificial three-dimensional biological tissue with a vascular network, comprising:

[0044] The rate at which the culture medium is supplied or discharged may be adjusted as appropriate as long as it is capable of supplying nutrients and oxygen to the three-dimensional biological tissue placed on the artificial vascular bed and promoting angiogenesis.

[0045] The three-dimensional biological tissue applied to the present invention preferably contains vascular endothelial cells, which may be vascular endothelial cells contained in the harvested three-dimensional biological tissue or vascular endothelial cells added exogenously. [Example]

[0046] The present invention will be described in more detail below with reference to examples, but these are not intended to limit the present invention.

[0047] Example 1 The artificial vascular bed of the present invention was fabricated by transferring the structure of a flow channel-forming device fabricated using a 3D printer onto the surface of a hydrogel. To construct the first and second flow channels, the hydrogel was injected into a silicone mold, which served as a flow channel-forming device with an outer tube and an inner needle inserted into a 20G Surflow tube. The 3D-printed device was then placed over the device and allowed to stand at room temperature for 30 minutes to allow gelation. After gelation was confirmed, the 3D-printed device and Surflow tube were removed, resulting in the fabrication of a hydrogel artificial vascular bed with the pore structure and bridging flow channel (Figure 2).

[0048] By transferring a 3D-printed device structure onto the hydrogel surface, we created a hydrogel artificial vascular bed with two 500 μm diameter pore structures (pores 1 and 2) that connect to the first and second flow paths, and a bridging flow path (300 μm wide, 800 μm deep, and 5 mm long) on ​​the surface that connects the two pore structures (Figure 3). Furthermore, by completely removing only the inner needle of the Surflow and leaving the outer sleeve in place, fluid can be easily delivered to the vascular bed flow path. Optical coherence tomography (OCT) (Santec Corporation, Japan) was used to observe the pore structure and bridging flow path, confirming that the above structure had been achieved on the hydrogel surface (Figure 3).

[0049] <Example 2> The cell sheet with vascular endothelial network to be layered on the artificial vascular bed of the present invention was prepared by co-culturing GFP-positive human umbilical vein endothelial cells (GFP-HUVEC) and human dermal fibroblasts (NHDF). GFP-HUVEC and NHDF were cultured at a ratio of 9:1, with 1.0 × 10 6A total of 2 mL of cell suspension prepared to a concentration of 1000 cells / mL was seeded onto a 35 mm diameter temperature-responsive culture dish (Up Cell®, CellSeed, Tokyo, Japan) or a smart temperature-responsive culture dish SSCW (Cell Sheet Regenerative Medicine Promotion Organization, Tokyo, Japan), and static culture was carried out for 3 days in a 37°C incubator to produce a cell sheet with a vascular endothelial network. The cell sheet was recovered from the temperature-responsive culture dish by static culture for 30 minutes to 1 hour in an incubator at 20°C.

[0050] The three-dimensional tissue cultured on the artificial vascular bed of the present invention was prepared by stacking three layers of cell sheets with vascular endothelial networks. First, the first cell sheet was adhered to a 100 mm diameter culture dish by static culture at 37°C for 1 hour. Next, the second cell sheet was layered on top of the first cell sheet, and the cell sheets were adhered to each other by culturing at 37°C for 30 minutes. The third cell sheet was similarly adhered by culturing at 37°C for 30 minutes, resulting in a tissue consisting of three cell sheets with vascular endothelial networks stacked together. The layered tissue was transferred onto the artificial vascular bed using an OHP film, placed so as to cover the pore structure and bridging channel, and then engrafted onto the artificial vascular bed by static culture at 37°C for 1 hour.

[0051] Observation of the cell sheet on the third day of culture using a fluorescence microscope confirmed the formation of a vascular endothelial cell network structure. Furthermore, after static culture for one hour with the laminated tissue placed over the pore structure and bridging channel of the artificial vascular bed of the present invention, and observation of the cross-sectional structure using OCT, it was confirmed that the cell sheet had taken root directly above the pore structure and bridging channel, realizing an artificial vascular bed in which the pore structure and bridging channel served as direct perfusion channels for the culture medium (Figure 4).

[0052] Example 3 We investigated the composition of hydrogels that can be used to fabricate the artificial vascular bed of the present invention. This artificial vascular bed utilizes the space formed between the bridging channel of the artificial vascular bed and the cell sheet as a direct flow path. Therefore, if the hydrogel shrinks or degrades due to the cell sheet layered on the artificial vascular bed, the porous structure of the vascular bed flow path and the bridging channel may be destroyed. If the vascular bed flow path is destroyed, not only will perfusion culture become difficult, but it will also be difficult to control the distance between the cell sheet and the flow path within the vascular bed. This will make it impossible to evaluate whether perfusion of culture medium directly below the cell sheet is effective as a method for vascularizing high-cell-density tissues in vitro. Therefore, we determined that a hydrogel whose structure is resistant to cell-induced gel shrinkage and degradation would be suitable for fabricating an artificial vascular bed using a 3D microfluidic system (Figure 5).

[0053] To identify suitable gels for the construction of artificial vascular beds of the present invention, we selected collagen gel or fibrin gel, which are hydrogels used in angiogenesis research. We constructed artificial vascular beds with porous structures using three types of gels: 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 fibrin gel containing factor XIII (0.5 U / mL thrombin, 5 mM CaCl2, 40 U / mL Factor XIII). A three-layer cell sheet was transplanted into the vascular beds constructed with each hydrogel, and the resulting tissues were cultured in VEC1 cells for 5 days. The top surface of the vascular beds was observed by optical microscopy, and the cross-sections of the bridging channels were observed by optical coherence tomography every day for 5 days. The structural stability of each hydrogel vascular bed was evaluated by measuring the surface area of ​​the upper surface of the vascular bed and the cross-sectional area of ​​the bridging channel, and calculating the ratio of the change over 5 days to the area immediately after implantation of the three-layer cell sheet.

[0054] Figure 6-1 shows bright-field images and cross-sectional images of the bridging channels when three-layered cell sheets were statically cultured on artificial vascular beds constructed with each hydrogel. The upper surface of the vascular bed fabricated with collagen gel contracted significantly on the first day of culture and continued to contract over the next four days. Furthermore, cross-sectional observation of the bridging channels confirmed that the bridging channels had almost completely disappeared by the first day of culture. For the vascular bed fabricated with fibrin gel, contraction of the upper surface and bridging channels was confirmed on the first day of culture. Subsequently, the upper surface remained unchanged from the first day to the fifth day of culture, but the bridging channels continued to contract over the next four days. On the other hand, observation of the vascular bed fabricated with fibrin gel containing factor XIII confirmed that both the upper surface and the bridging channels maintained their structures throughout the five-day culture period. Evaluation of the rate of change in each area over time revealed that fibrin gel containing factor XIII was the hydrogel that best maintained the structure of the upper surface of the artificial vascular bed and the bridging channels (Figure 6-2).

[0055] From the above results, we have clarified a method for producing a fibrin gel containing factor XIII whose structure is resistant to changes by cells, in order to produce the artificial vascular bed of the present invention with a pore structure and a bridging channel as a direct channel.

[0056] Example 4 We investigated the ex vivo effects of static and perfusion culture of cell sheets on an artificial vascular bed of the present invention, fabricated with fibrin gel containing factor XIII, to confer vascularity with a lumen within the layered cell sheet tissue. A three-layered layered tissue with vascular endothelial meshwork, constructed using the aforementioned protocol, was engrafted onto the porous structure of the artificial vascular bed, and then static and perfusion cultures were performed at 37°C and 5% CO2. For perfusion culture, endothelial cell medium VEC1 (Kohjin Bio, Japan) was perfused at a flow rate of 25 μL / min through the first channel using a microtube pump. The medium that passed through the bridging channel was then discharged into the chamber through the second channel, allowing perfusion directly below the cell sheet. The medium that had drained into the chamber was managed by daily draining until the cell sheet surface was no longer exposed to air. In static culture, perfusion was not performed directly below the cell sheet. Instead, the culture medium was perfused into the chamber using a microtube pump at a flow rate of 25 μL / min using VEC1. Similarly, the amount of medium was controlled by draining the liquid daily, using the same amount of medium as used in perfusion culture.

[0057] After 5 days of culture, the morphology of the vascular endothelial cells was evaluated by fluorescence observation, the vascular structure was visualized by perfusion of rat blood from the fluid supply section, and histological evaluation was performed by HE staining and immunostaining using CD31 to evaluate whether a vascular structure with a lumen had been created within the cell sheet (Figure 7).

[0058] The morphology of GFP-HUVECs was evaluated by observing them from directly above the cell sheet on the artificial vascular bed using a fluorescence microscope (Fig. 8).

[0059] Low-magnification images (left and center in Figure 8) of specimens cultured for 5 days after perfusion culture and static culture are shown, along with high-magnification images of the tissue after culture (right in Figure 8). Under both conditions, it was observed that the network of vascular endothelial cells was more promoted in the tissue after culture than before culture. Furthermore, in specimens cultured by perfusion culture, cells were observed to have accumulated in the pore structure compared to specimens cultured by static culture. Furthermore, high-magnification images confirmed that the network structure of endothelial cells in the tissue cultured by perfusion culture was thicker than that in static culture, and that lumens had been formed.

[0060] <Example 5> To verify the effect of providing blood vessels within the cell sheet, we visualized the vascular structure by perfusing India ink. India ink was diluted 2-fold with saline and perfused for 1 hour at a flow rate of 25 μL / min, the same as during perfusion culture, to evaluate whether a vascular structure with a perfusable lumen had been provided within the cell sheet. A syringe pump was used for perfusing the India ink (Figure 9).

[0061] Figure 9 shows images of tissues prepared by static culture and perfusion culture after one hour of India ink perfusion. In the tissue prepared by static culture, the India ink perfused from the first channel (bottom) was observed to pass through the pore structure, the bridging channel, and then flow out into the second channel. No vascular structures were observed within the cell sheet. Although a network of vascular endothelial cells was formed within the tissue, no India ink perfusion was observed, demonstrating that static culture does not allow the formation of a perfusable vascular network. In contrast, in the tissue prepared by perfusion culture, India ink was observed to flow out to the upper surface of the tissue, passing through the pore structure above the first channel and the vascular structure formed within the tissue originating from the bridging channel. Fluorescence observation of the vascular network into which India ink flowed confirmed that India ink was introduced into the vascular lumen composed of GFP-positive vascular endothelial cells, confirming the formation of a perfusable vascular network.

[0062] Tissues prepared by perfusion culture were perfused for 1 hour with conditioned blood, prepared by diluting rat blood two-fold with physiological saline, using a syringe pump. Figure 10 shows an image of the tissue after perfusion. It was confirmed that the conditioned blood flowing in through the first channel passed through the mesh-like structure within the cell sheet and exited the upper part. Furthermore, observation of the dotted area in the upper right with a blood flow microscope (TOKU Capillaro) confirmed that red blood cells in the conditioned blood flowed through the mesh-like structure within the cell sheet (Figure 10, lower left). These results demonstrate that perfusion culture on an artificial vascular bed utilizing the bridging channels (and pores, if possible) on the mounting surface formed a vascular network within the cell sheet that was visible by macroscopic observation, and further confirmed that the provided blood vessels were capable of perfusing red blood cells.

[0063] Example 6 Tissue sections were prepared from specimens subjected to static and perfusion culture, and histological evaluation was performed using HE staining and CD31 fluorescent immunostaining. After culture, the tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Tissue sections were then prepared from the pore structure and bridging channel regions and HE stained. Furthermore, immunostaining with CD31, an antibody against vascular endothelial cells, was performed to evaluate the distribution of vascular endothelial cells within the tissue. The tissue cross-sections were designated the inflow region, bridge region, and outflow region, taking into account the inflow and outflow regions during perfusion culture. Images of each region were shown at low and high magnification.

[0064] The HE staining image in static culture (Figure 11) and the results of immunostaining using CD31 (Figure 11-2) confirmed that static culture did not result in the formation of blood vessels with lumens within the cell sheet at any point.

[0065] On the other hand, HE staining images of the perfusion cultured specimen (Figure 12-1) confirmed significant cell proliferation in the outflow and bridge regions. Furthermore, high-magnification images confirmed the formation of numerous luminal structures containing red blood cells, as indicated by the red arrows, in both regions.

[0066] Furthermore, to evaluate the distribution of vascular endothelial cells, fluorescent immunostaining using CD31 was performed (Figure 12-2). As a result, it was confirmed that luminal structures composed of green-stained vascular endothelial cells were uniformly and densely present within the tissue. It was also confirmed that red blood cells were contained within the luminal structures composed of vascular endothelial cells. This demonstrated that the numerous luminal structures in the tissue containing red blood cells are blood vessels with lumens composed of endothelial cells.

[0067] The above results demonstrate that the perfusion culture method directly beneath the cell sheet using the artificial vascular bed of the present invention is an effective method for providing a large number of functional blood vessels to high-cell-density tissues ex vivo.

[0068] The artificial vascular bed of the present invention exhibited a greater vascularization effect than conventional vascular beds, likely due to the supply of large amounts of biochemical factors from the cell sheet proximal area and mechanical stimulation by perfusion (Figure 13). In conventional vascular beds, angiogenesis was induced within the tissue by the diffusion of biochemical factors such as cytokines from new blood vessels within the gel proximal and distal to the cell sheet engraftment surface or from blood vessels within muscle tissue. In contrast, in the present invention, the pore structure of the fibrin gel, whose structure is resistant to cell modification, and the bridging channels were utilized directly as vascular bed channels, enabling a stable supply of culture medium directly beneath the cell sheet. This enabled the stable supply of larger amounts of cytokines from proximal to the cell sheet engraftment surface. Furthermore, perfusion of culture medium enabled the application of mechanical stimulation, such as shear flow, directly to the cell sheet, which is thought to contribute to angiogenesis induction. It is believed that the above combined factors enabled unprecedented vascularization of high-cell-density tissues ex vivo. Furthermore, because the pore structure in the fibrin gel is directly used as a flow path, there is no variation in the shape of the vascular bed flow path, and it is possible to stably supply biochemical factors to the tissue and provide mechanical stimulation through perfusion, making this a highly reproducible method of vascularization (Figure 13).

[0069] The artificial vascular bed developed in this study is expected to be utilized as a new platform technology for ex vivo perfusion culture of three-dimensional tissues. Using a 3D printer, we were able to freely design pore structures and bridging channels on the fibrin gel surface. Furthermore, because the channels are cell-free, we were able to easily fabricate a vascular bed with a stable channel structure with minimal sample variation. By engrafting cell sheets, which have been used in clinical studies with various cell types, onto this vascular bed and performing perfusion culture, we were able to create three-dimensional tissues with a vascular network that can actually be perfused. Furthermore, by engrafting three-dimensional tissues created by tissue engineering techniques other than cell sheets, such as organoids, onto the cell sheet, we anticipate that ex vivo vascularization and perfusion culture of the engrafted three-dimensional tissues will be possible. This allows for direct application of fluid mechanical stimuli to three-dimensional biological tissues, making it useful as an experimental model for elucidating the effects of mechanical stimuli on angiogenesis within highly cell-dense three-dimensional tissues. Furthermore, it is anticipated that the three-dimensional tissue constructed ex vivo by perfusing drugs into the attached blood vessels may be used as a drug discovery test model, or as a research model for transplantation therapy using the three-dimensional tissue constructed ex vivo, and various applications for research on the construction of three-dimensional tissue ex vivo are expected.

[0070] Example 7 As an example of the application of the present invention, we investigated the potential use of this model as an angiogenesis research model by examining whether changing the perfusion rate during perfusion culture affected angiogenesis. (Condition 1) was a specimen perfused at 25 μL / min for 5 days, while (Condition 2) was a specimen perfused at 25 μL / min for 3 days, then increased fourfold to 100 μL / min for 2 days. By perfusing India ink into specimens cultured under each condition, we compared the perfusable vascular structures formed. (Condition 2, with its increased perfusion rate, exhibited a denser and more extensive vascular network than the previous model (Figure 15).) These results confirm that changing the perfusion rate affects angiogenesis. These results suggest that this model can be used as an angiogenesis research model to clarify the biochemical and mechanical conditions necessary for efficient angiogenesis in vitro into high-density, three-dimensional tissues.

[0071] Example 8 A key feature of this technique is that the vascular bed channels are constructed by transferring the device structure, which was designed using Three Dimensional Computer-Aided Design (3DCAD), onto the fibrin gel surface. This allows for the easy creation of various channels. Therefore, it is expected that the angiogenesis site can be designed for three-dimensional tissue, and multiple channels can be created to induce angiogenesis over a wider area. Furthermore, because the fibrin gel used is resistant to cell-induced structural changes, computational fluid dynamics analysis can be used to determine the speed of the culture medium flowing through each channel. By appropriately modifying the channel design, multiple channels with controlled flow rates can be constructed. Figure 16 shows an example of an artificial vascular bed with multiple channels.

[0072] Example 9 To demonstrate the usefulness of this invention as a three-dimensional tissue construction method, we investigated whether it is possible to construct three-dimensional tissue by stepwise stacking of cell sheets using the developed artificial vascular bed. "Stepwise stacking" is a method of introducing blood vessels into the cell sheets stepwise to construct three-dimensional tissue by repeatedly transplanting a set of three-layer cell sheets, which can be maintained by the diffusion of culture medium, onto the artificial vascular bed at regular intervals (Figure 17).

[0073] In this example, we used cell sheets prepared using the same method as in Example 2. After perfusion culture of a three-layered cell sheet on an artificial vascular bed for five days, the next set of three-layered cell sheets was transplanted and perfusion cultured for an additional three days. Optical coherence tomography (OCT) observation of the tissue cross-section after culture confirmed the formation of luminal structures within the additional three-layered cell sheets (Figure 18). Furthermore, perfusion of India ink into the cultured tissue revealed the formation of a large vascular network, with India ink perfusion evident, within the six-layered cell sheets (Figure 19). These results demonstrate that stepwise stacking using the artificial vascular bed of the present invention enables the construction of three-dimensional tissues with perfusable vascular networks. Therefore, increasing the number of layered layers to 3, 6, 9, or 12 may be applicable to the construction of millimeter-order three-dimensional tissues, which have previously been difficult to produce. These results suggest that this method is useful for constructing three-dimensional tissues in vitro.

Claims

1. An artificial vascular bed, comprising: a vascular bed formed of a composition including a biocompatible hydrogel, the vascular bed having a mounting surface for three-dimensional biological tissue; a first flow path and a second flow path provided within the vascular bed; a first hole and a second hole provided on the mounting surface, the first hole and the second hole communicating with the first flow path and the second flow path, respectively; a bridging channel that communicates between the first gap and the second gap and is formed on the mounting surface; a first fluid delivery unit in fluid communication with the first flow path for supplying a culture medium; a second fluid delivery unit in fluid communication with the second flow path for discharging the culture medium; An artificial vascular bed comprising:

2. The artificial vascular bed of claim 1 , wherein the biocompatible hydrogel is a crosslinked biocompatible hydrogel.

3. The artificial vascular bed of claim 1 , wherein the biocompatible hydrogel comprises a fibrin gel.

4. The artificial vascular bed of claim 3 , wherein the biocompatible hydrogel comprises a fibrin gel stabilizing factor.

5. a culture medium supply line connected to the first liquid transport unit; a medium supply tank for supplying a medium to the medium supply line; a liquid supply pump for supplying a culture medium to the culture medium supply line; a culture medium discharge line connected to the second liquid supply unit; a culture medium discharge tank for storing the culture medium discharged from the culture medium discharge line; The artificial vascular bed according to any one of claims 1 to 4, further comprising:

6. 6. The artificial vascular bed according to claim 5, wherein the medium supply tank and the medium discharge tank are the same tank, and the medium is circulated.

7. An artificial vascular bed according to any one of claims 1 to 4, a three-dimensional biological tissue placed on the placement surface so as to cover the bridging channel; and an artificial three-dimensional biological tissue comprising:

8. The artificial three-dimensional biological tissue of claim 7, wherein the three-dimensional biological tissue is a sheet-like tissue or an organoid containing cells.

9. The artificial three-dimensional biological tissue according to claim 7 , wherein the three-dimensional biological tissue comprises 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 on the placement surface of the artificial vascular bed according to any one of claims 1 to 4 so as to cover the bridging channel; a step of perfusion culturing while supplying a culture medium to the first liquid supplying part and discharging the culture medium from the second liquid supplying part; A method comprising:

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 , wherein the three-dimensional biological tissue comprises vascular endothelial cells.

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