Method for manufacturing branched artificial blood vessel model

Abstract

In the present invention, smooth muscle production molds for forming a main blood vessel part and a branched blood vessel part are prepared. For the main blood vessel part, a smooth muscle layer is formed on a first core rod that has at least one hole, and for the branched blood vessel part, a smooth muscle layer is formed on a surface of a second core rod that has a diameter which enables fitting to the hole of the first core rod. Thereafter, the second core rod having the smooth muscle layer is fitted to the hole of the first core rod, thereby bringing the two smooth muscle layers into contact to form a branched structure, and thus a branched artificial blood vessel model is manufactured. The present invention makes it possible to provide a branched artificial blood vessel model which has a branched blood vessel shape and which constricts and relaxes according to physiological conditions.

Description

Manufacturing method of branched artificial blood vessel model

[0001] The present invention relates to an artificial blood vessel model having a bifurcation produced using cultured cells, and a method for producing the same.

[0002] There is a demand for tissue models that can reproduce not only the shape of tissues and organs but also the functions of biological tissues. If biological functions can be reproduced, it will be possible to reproduce and analyze pathological conditions at the tissue or organ level. In the search for therapeutic drugs, screening using tissue models consisting of multiple cell types may select different compounds than screening using a single cultured cell, which is expected to lead to the development of effective drugs.

[0003] Attempts have been made to construct models of various tissues, and these models are being used for analyzing pathologies and developing pharmaceuticals. Living tissues and organs are not composed of a single cell, and the usefulness of tissue models has been recognized. Three-dimensional tissue models constructed in vitro using multiple cells are thought to be closer to living tissues and contribute to elucidating biological functions and predicting drug responses. Cardiovascular diseases (CVD) in particular have a large number of patients, and there are no drugs to treat the dysfunction of the blood vessels themselves. Therefore, there are high expectations for the construction of vascular models and the use of these models to screen for pharmaceuticals.

[0004] CVD is said to account for approximately one-third of deaths worldwide. However, while progress has been made in developing drugs and treatment methods for diseases such as hypertension and diabetes that are secondary to cardiovascular disease, no treatments targeting the dysfunction of the blood vessels themselves have yet been developed. Because blood vessels have a layered structure composed of multiple cells, such as endothelial cells and smooth muscle cells, it is difficult to select compounds that are effective against the dysfunction of blood vessels, which are tissues composed of multiple types of cells, using screening using cultured cells.

[0005] Furthermore, the tissue deformation of blood vessels, specifically their contraction and dilation, is deeply linked to their physiological functions. Furthermore, blood vessels do not have a simple tubular structure; rather, they have a complex, branched structure. Stenosis is likely to occur near the branching points of blood vessels, and stenosis is known to cause diseases such as hypertension. For example, it is well known that renal vascular hypertension is closely related to the enhancement of the renin-angiotensin system due to renal artery stenosis (Non-Patent Document 1). However, no tools have been developed to date for analyzing the physiological functions of blood vessel structure. Therefore, a vascular model that has structural branches and contracts and dilates in response to physiological stimuli would be useful for analyzing vascular function and potentially contributing to drug screening.

[0006] Tissue engineered blood vessels (TEBV) using vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) have already been reported (Patent Documents 1 to 3, Non-Patent Document 2). Patent Document 1 discloses vascular tissue that is at least partially composed of cells and has all the necessary structures, including a branched structure. Patent Document 2 discloses a vascular model for analyzing drug-induced vascular damage that causes smooth muscle cell death and bleeding, which has an endothelial cell layer on one side of a porous membrane and a smooth muscle cell layer on the other side, and also discloses a branched structure. Patent Document 3 discloses an artificial blood vessel with a branched structure composed of a tube made of a woven or knitted thermoplastic resin fiber. Non-Patent Document 2 reports that a blood vessel with a branched structure was fabricated using a 3D printer and implanted in a pig. The inventors also disclosed an artificial blood vessel that contracts in response to physiological stimuli, but it does not have a branched structure (Non-Patent Document 3).

[0007] International Publication No. 2014 / 030418 US Patent Application Publication No. 2011 / 0053207 JP 2002-017758 A

[0008] Safian, RD, etal., N. Engl. J. Med., 2001, Vol.344, No.6, pp.431-442.Yeung, E. et al.,J. Thorac. Cardiovasc. Surg. 2020, Vol.159(5), pp.1971-1981,doi:10.1016 / j.jtcvs.2019.09.138.Itai, S. &Onoe H., 2022 IEEE 35th International Conference on MicroElectro Mechanical Systems Conference (MEMS), 10.1109 / MEMS51670.2022.9699803Tsujimoto H, et.al., Cell Rep., 2020, 31, 10746

[0009] However, the invention described in Patent Document 1 is a method for forming vascular tissue by introducing sodium alginate containing suspended cells into a flow channel structure and creating a hydrogel layer. Depending on the flow channel structure used, it is possible to create blood vessels with branched structures, but because the hydrogel layer is in close contact with the flow channel structure, the created blood vessels cannot contract or expand. The invention described in Patent Document 2 has a structure in which an endothelial cell layer is coated on one side of a porous membrane and a smooth muscle layer on the other side, so although it has a branched structure, it does not have a vascular structure that can contract or expand. The invention described in Patent Document 3 has a branched structure, but is composed of a tube made of a woven or knitted thermoplastic resin fiber, so it is not possible to analyze the biological functions of the blood vessel caused by the branched structure. The invention described in Non-Patent Document 2 has a branched structure, but is manufactured using a 3D printer, so the blood vessel itself does not have the ability to contract or expand. The artificial blood vessel described in Non-Patent Document 3 has physiological functions, but is manufactured by wrapping a smooth muscle layer around it, so it cannot have a branched structure. An object of the present invention is to provide an artificial blood vessel that has the physiological function of a blood vessel, that is, contracts or expands under physiological conditions, and further has a branched structure.

[0010] The present invention relates to the following TEBV and its manufacturing method: (1) A method for manufacturing a branched artificial blood vessel model, including a smooth muscle layer formation step of preparing a first mandrel having at least one hole and at least one second mandrel having a diameter that can fit into the hole, and forming smooth muscle layers on the surfaces of the first and second mandrels, and a branched structure formation step of fitting the second mandrel having a smooth muscle layer into the hole of the first mandrel having a smooth muscle layer, thereby bringing the smooth muscle layer formed on the surface of the first mandrel into contact with the smooth muscle layer formed on the surface of the second mandrel to form a branched structure. A smooth muscle layer that will become the main blood vessel portion is formed on the first mandrel, and a smooth muscle layer that will become the branched blood vessel portion is formed on the second mandrel. A branched artificial blood vessel model of a desired shape can be produced by adjusting the position of the hole formed in the first mandrel and the angle between the first and second mandrels.

[0011] (2) A branched artificial blood vessel model having at least one branched structure produced by the above-mentioned production method. The artificial blood vessel model produced by the above-mentioned production method not only has a branched structure, but also has a smooth muscle layer, so it can mimic the contraction and relaxation that occurs in vivo in response to physiological stimuli. As a result, the structure has a branched shape that exists in vivo and can faithfully reproduce physiological deformation reactions, resulting in an artificial blood vessel model that is closer to blood vessels in vivo.

[0012] (3) A method for screening drugs for treating diseases resulting from vascular dysfunction, comprising perfusing a candidate compound through the branched artificial blood vessel model and selecting the candidate compound by monitoring the physiological response of the branched artificial blood vessel model. Regarding cardiovascular disease, progress has been made in the development of drugs for treating secondarily induced hypertension and diabetes, but no drugs have been developed to treat vascular dysfunction itself. By using the branched artificial blood vessel model, it is possible to screen for compounds effective against vascular dysfunction.

[0013] 1 shows one embodiment of a branched artificial blood vessel model. 2 shows the appearance of a branched artificial blood vessel model. 3 shows a schematic representation of the cross-sectional structure of a branched artificial blood vessel model. 4 shows the cross-sectional structure of both the main blood vessel and the branched blood vessels. 5 shows a perspective view, a top view, and a side view of one embodiment of a mold for producing smooth muscle tissue. 6 shows a perspective view and a top view of another embodiment of a mold for producing smooth muscle tissue. 7 shows a top view and a side view of a first core rod 13a, and a top view of a second core rod 13b. 8 shows a schematic representation of the process for producing smooth muscle fibers. 9 shows a schematic representation of the winding process. 10 shows a diagram illustrating the process of producing a branched portion. 11 shows a diagram illustrating the process of covering with endothelial cells and extracellular matrix.

[0014] In this specification, an artificial blood vessel model is an artificially produced artificial blood vessel containing cellular tissue, which reproduces the biological structure of blood vessels that contract and relax. A branched artificial blood vessel model refers to an artificial blood vessel model that has a branched structure. It also includes structures that are more similar to biological tissues, such as those that are joined with connectors or the like to allow perfusion, and those that contain endothelial cells and extracellular matrix. The terms "tissue engineered blood vessel (TEBV)," "artificial blood vessel," and "artificial blood vessel model" can be used interchangeably.

[0015] The cells used to prepare the branched artificial blood vessel model may be cell lines of cells that constitute blood vessels, such as smooth muscle cell lines and endothelial cell lines, or stem cells with self-replication capacity, such as iPS cells (induced pluripotent stem cells), ES cells (embryonic stem cells), somatic stem cells, tissue stem cells, etc. In particular, iPS cells can be used as patient-derived cells, and therefore are preferably used for pharmaceutical screening, elucidation of pathological conditions, etc.

[0016] Figure 1A shows the appearance of a branched artificial blood vessel model according to one embodiment of the present invention, and Figure 1B shows a cross-section. The branched artificial blood vessel model 1 has a structure similar to that of blood vessels in vivo, in which a main blood vessel 2, which is long in the longitudinal direction, branches into a branched blood vessel 3, which is shorter than the main blood vessel 2. While a single branched blood vessel 3 is shown here, the number and location of branches can be adjusted to suit the actual blood vessel configuration. The branched blood vessel 3 shown in the embodiment has a smaller diameter than the main blood vessel 2, but it can also have the same diameter. The branching angle is approximately 45° here, but any angle possible in vivo can be used. The artificial blood vessel model has a helically oriented smooth muscle layer 4 in each of the main blood vessel 2 and the branched blood vessel 3, forming a branched shape that is tightly attached at approximately the center. An extracellular matrix layer 5 is provided on the outside of the smooth muscle layer. As shown in Figure 1B, the inside of the smooth muscle layer is covered by endothelial cells 6.

[0017] Although the smooth muscle layer 4 shown here is wound in a spiral shape, a ring-shaped one may also be used. The inner diameter of the tubular vascular structure is adjusted by the contraction or relaxation of the smooth muscle. By arranging smooth muscle cells in a spiral or ring shape without gaps around the circumference of the artificial blood vessel model, the artificial blood vessel model can mimic the contraction and relaxation caused by physiological stimuli, faithfully reproducing the deformation reaction in vivo.

[0018] As shown in Figure 1, it is preferable that the outside of the smooth muscle layer 4 is covered with an extracellular matrix layer 5, and the inside of the lumen is covered with endothelial cells 6. If the smooth muscle layer 4 is formed from smooth muscle cells derived from stem cells, it is possible to use stem cells derived from patients as well as stem cells derived from healthy individuals, which makes it possible to analyze the mechanisms of disease onset and screen pharmaceuticals.

[0019] The smooth muscle layer 4 contains not only smooth muscle cells but also extracellular matrix materials. The extracellular matrix materials are not particularly limited, but examples thereof include collagen, laminin, gelatin, cadherin, hyaluronic acid, fibronectin, fibrin, elastin, chitin, chitosan, vitronectin, and proteoglycan. Of these extracellular matrix materials, collagen is particularly preferred. One type of extracellular matrix material may be used alone, or two or more types may be used in combination.

[0020] It is also preferable to have an extracellular matrix layer 5 on the periphery of the smooth muscle layer. The extracellular matrix layer 5 is a layer laminated on the outer peripheral surface side of the smooth muscle layer, and may be composed of, for example, an extracellular matrix material and adventitial cells such as fibroblasts. As the extracellular matrix material, materials similar to the extracellular matrix material contained in the smooth muscle layer described above can be preferably used. Furthermore, the material used may be the same as the extracellular matrix contained in the smooth muscle layer 4, or a different material. The extracellular matrix layer 5 fills the space around the periphery of the smooth muscle layer 4, thereby serving as a physical support and a scaffold for cell adhesion.

[0021] Furthermore, it is preferable that the inner wall of the smooth muscle layer 4 contains endothelial cells 6. The endothelial cells 6 are present on the inner peripheral surface side of the smooth muscle layer 4, and in the artificial blood vessel model, a monolayer is formed by tightly bonding multiple endothelial cells. The endothelial cell layer can be formed by forming a tubular smooth muscle layer and then seeding the endothelial cells on the inner wall portion.

[0022] As will be explained in detail below, the manufacturing method of the branched artificial blood vessel model of the present invention can be summarized as follows: smooth muscle cells are induced from stem cells, smooth muscle fibers (smooth muscle tissue) are formed from the smooth muscle cells by culturing, and the smooth muscle tissue is wrapped around a core rod to create smooth muscle layers for the main blood vessel portion and the branched blood vessel portion. Two smooth muscle layers are then arranged to form a branched structure. Furthermore, connectors are attached for perfusion, and the smooth muscle layers are covered with an extracellular matrix. To form an endothelial cell layer on the inner wall, endothelial cells are induced from stem cells and seeded on the inner wall to form an endothelial cell layer. Regardless of whether an endothelial cell layer is present, the term "branched artificial blood vessel model" is used; however, a branched artificial blood vessel model with an endothelial cell layer is a model that more closely resembles a living body.

[0023] [Method for Producing a Branched Artificial Blood Vessel Model] (1) Method for Producing Smooth Muscle Fibers To form smooth muscle tissue, two molds for producing smooth muscle tissue, one for the main blood vessel and one for the branch blood vessel, were produced using a 3D printer. The two molds were essentially identical in shape, but the mold for the branch blood vessel was slightly smaller than the mold for the main blood vessel, allowing the production of a branch blood vessel with the appearance shown in Figure 1A. Figure 2A shows a perspective view, top view, and side view of one embodiment of the mold. The smooth muscle tissue production mold was produced here using polydimethylsiloxane (PDMS), but materials commonly used in this field, such as polylactic acid (PLA), can also be used. The smooth muscle tissue production mold 11 is a flat substrate with a certain thickness, and includes a first groove 12 for culturing smooth muscle cells to produce smooth muscle fibers, and a second groove 14 that intersects the first groove and accommodates a core rod 13 for winding up smooth muscle fibers located at the end of the first groove 12. Additionally, an anchor 15 is provided in the first groove 12 at the end opposite to the second groove 14. The anchor is used to wind the prepared smooth muscle fibers into a fibrous form. Although a circular anchor is shown here as an example, the anchor may have any shape, and the mold may be provided with a groove for setting the anchor.

[0024] 2B shows a perspective view and a top view of another embodiment of the mold for producing smooth muscle fibers. The second groove 14, in which the core rod 13 is placed in the mold 11 for producing smooth muscle tissue, intersects the first groove 12 at an angle. By providing a predetermined angle between the first groove 12 and the second groove 14, the produced smooth muscle fibers can be wrapped around the mold without any gaps.

[0025] The materials constituting the core rod and anchor are not particularly limited, but may include, for example, well-known metals used in medical devices such as tungsten, stainless steel, titanium, aluminum, and niobium, as well as resins such as fluororesins such as polytetrafluoroethylene (PTFE), silicon resins such as silicone, polylactic acid (PLA), glycol-modified polyethylene terephthalate (PETG), and thermoplastic polyurethane (TPU).As will be described later, it is preferable to use a resin such as PTFE because holes will be formed in the first core rod.

[0026] The molds for producing smooth muscle tissue are shaped similarly to those for the main blood vessel and the branch blood vessel, although their sizes differ. However, different core rods are prepared for the main blood vessel and the branch blood vessel. As shown in Figure 3, the first core rod 13a, which is the core rod for the main blood vessel, has at least one hole 16. The hole 16 has a diameter that allows it to fit into the second core rod 13b, which is the core rod for the branch blood vessel. The hole can be located anywhere to accommodate the branch blood vessel to be produced, and the number of holes does not need to be one. Except in cases where blood vessels branching from the branch blood vessel are to be produced, the second core rod 13b does not need to have a hole.

[0027] [Preparation of cell suspension] Below, an example of producing a branched artificial blood vessel model using iPS cells is shown, but ES cells, tissue stem cells, etc. can also be used to produce a branched artificial blood vessel model using known culture methods and differentiation induction methods.

[0028] In the following examples, iPS cells derived from healthy individuals or patients with Werner syndrome were used for the branched artificial blood vessel models. Undifferentiated iPS cells were cultured in Stemflex culture medium, and induction into smooth muscle cells and endothelial cells was performed according to the protocol described in Non-Patent Document 4. Specifically, for smooth muscle cells, undifferentiated iPS cells were first induced to mesoderm using N2B27 medium (a mixture of neurobasal medium, DMEM / F12 Glutamax medium, N2 supplement, B27 supplement, and 2-mercaptethanol), CHIR, and hBMP4, and then induced to smooth muscle using N2B27 medium, PDGF-BB, and Activin-A. Then, Type-I collagen coating, SmGM-2 (Smooth muscle growth medium), M-231 medium, and SMDS (Smooth Muscle Growth Medium) were added. The tissue was maintained using a muscle differentiation supplement.

[0029] Endothelial cells were first induced into mesoderm using DMEM / F12 Glutamax medium, B27 supplement, CHIR99021, hBMP4, RA (Retinoic Acid), and FGF-2, and then induced into vascular endothelium using Stempro medium, Laminin 411 coating, Y27632, DAPT, IWR-1, VEGF, hBMP4, and FGF-2, and then maintained using fibronectin coating and EGM-2 (Endothelial Growth Medium). For the co-culture of iPS-derived vascular smooth muscle cells and vascular endothelial cells, a mixture of M231 medium (containing SMDS) and EGM-2 at a volume ratio of 3:1 was used. Prior to cell seeding, the cells were incubated in a dish at 37°C and 5% CO. ２ The culture medium was changed every two days. iPS cell-derived differentiated cells were dispersed in a trypsin-EDTA solution, and smooth muscle cells were dispersed in a collagen pregel solution (Type-I acidic collagen solution, reconstitution buffer (NaOH, NaHCO ３, prepared from HEPES), a mixture of 10X HANKS balanced salt solution), and endothelial cells were suspended in EGM-2.

[0030] The method for producing a branched artificial blood vessel model is divided into the following steps: forming smooth muscle fibers for the main blood vessel and the branched blood vessel, winding the model to obtain an artificial blood vessel model with an embedded smooth muscle layer, forming a branched structure, and coating the model with endothelial cells and extracellular matrix. Details of the smooth muscle fiber formation step are as follows:

[0031] [Formation of Smooth Muscle Fibers] [1] A first or second mandrel and anchor are placed in each of two smooth muscle tissue preparation molds, one for the main blood vessel and one for the branch blood vessel. Here, the first mandrel is made of PTFE, and the second mandrel and anchor are made of tungsten wire. The mold for smooth muscle tissue preparation for the main blood vessel has a first mandrel with holes formed therein, while the mold for smooth muscle tissue preparation for the branch blood vessel has a second mandrel without holes. [2] A collagen pre-gel solution containing iPS cells suspended in it is poured into each mold (Figure 4A(i)). A collagen pre-gel solution (cell suspension) containing iPS cells induced to smooth muscle cells is poured into the mold for smooth muscle tissue preparation for the main blood vessel and the mold for smooth muscle tissue preparation for the branch blood vessel. The cell suspension is dripped onto the mold without touching the area around the mandrel (the area indicated by the arrow in the figure). If smooth muscle cells are present around the core rod, the strong contractile force of the smooth muscle cells will make it difficult to remove the core rod in a later step. After the cell suspension is dripped, the solution is incubated at 37°C to solidify. [3] A collagen pre-gel solution alone is added around the core rod to bond the core rod and collagen gel (Figure 4A(ii)). The collagen is then incubated at 37°C to solidify. This bonds the core rod with the cell-free collagen gel, preventing the application of large forces to the core rod when winding the smooth muscle fibers in a later step. [4] The resulting tissue is cultured in culture medium (M231 medium (+SMDS)) for 24 hours to induce axial alignment of the cells and form dense fibers (Figure 4A(iii)).

[0032] [Preparation of a smooth muscle layer-embedded artificial vascular model] The high-density smooth muscle fibers for the main and branch vessel sections obtained using two molds were wound around their respective mandrels and embedded in collagen gel to prepare a flexible and deformable branched artificial vascular model with circumferentially arranged smooth muscle cells (Figure 4B). [5] Rotate each mandrel in culture medium to wind the smooth muscle fibers around the mandrel. When winding the smooth muscle fibers around each mandrel, winding them at a predetermined inclination angle prevents the smooth muscle fibers from overlapping and allows them to spontaneously wind in a spiral around the mandrel. Because the smooth muscle tissue preparation mold has an inclined side, rolling the mandrel along the inclination allows the smooth muscle fibers to be wound in a spiral without overlapping (Figure 4B(i)-(iii)). Alternatively, smooth muscle fibers can be wound using a base that allows the mandrel to be positioned at an appropriate inclination.

[0033] [Fabrication of Branched Structures] [6] Once smooth muscle fibers for the main blood vessel and branched blood vessel are prepared and the smooth muscle layer 4 is wound around the mandrel, the branched structure is formed. As described above, the first mandrel 13a for the main blood vessel has a hole 16 into which the second mandrel 13b fits. The second mandrel 13b is then fitted into the hole 16 of the first mandrel 13a along with the wound smooth muscle layer (Figure 5). Because the branched section is fabricated by fitting the second mandrel into the hole of the first mandrel, it can be set at any angle relative to the main blood vessel. Actual blood vessels also branch at various angles, making it possible to fabricate models that mimic the angles in vivo.

[0034] [Endothelial Cell and Extracellular Matrix Coating] [7] Connectors are attached when performing endothelial cell seeding, perfusion, or addition of physiologically active substances. Connectors consisting of a silicone tube 21 and a glass capillary 22 are inserted into the ends of the first and second mandrels 13a and 13b (Figure 6, top). [8] An extracellular matrix coating mold 23 is prepared. Because branched artificial blood vessel models vary in shape, such as the angle of the branched blood vessels relative to the main blood vessel, an extracellular matrix coating mold is prepared to match the shape of the artificial blood vessel model. The extracellular matrix coating mold can be made using an easily fabricated material such as agarose (1-2 wt%) to suit the appropriate model shape. Since the mold is used to coat the collagen around the fabricated model, it should be several hundred micrometers larger than the smooth muscle layer, as long as it can completely coat the extracellular matrix. A collagen pregel solution (4 mg / mL) is poured around the smooth muscle layer and connector to coat the collagen. The collagen was then solidified by incubation at 37°C (Figure 6, top). [9] The extracellular matrix coating mold was removed to obtain a branched artificial blood vessel model in which smooth muscle fibers A were circumferentially aligned and coated with collagen gel B (Figure 6, bottom). Endothelial cells C were seeded inside the artificial blood vessel using a microsyringe to obtain a branched artificial blood vessel model equipped with endothelial cells.

[0035] Many diseases are thought to be related to vascular dysfunction and are therefore potential targets for drug screening using artificial blood vessels. Examples of such diseases include arteriosclerosis, including myocardial infarction, cerebral infarction, nephrosclerosis, and retinopathy; coronary spasm; diabetes (including conditions that cause vascular proliferation); vasculitis (hypertension), including various diseases such as ANCA-associated vasculitis, Takayasu's arteritis, and IgG4 vasculitis; embolism, which is intravascular thrombus formation; vascular malformation (moyamoya disease); and genetic disorders (mitochondrial disease). This branched artificial blood vessel model is particularly useful for screening therapeutic agents for diseases in which thrombus formation and infarction occur at vascular bifurcations. Specifically, candidate compounds can be perfused through the branched blood vessel model and physiological responses, such as contraction and relaxation, of the branched blood vessels can be monitored, and changes in gene expression in the smooth muscle cells and endothelial cells that make up the model can be analyzed to screen for compounds suitable for treating the target disease. Because the branched blood vessel model more closely resembles in vivo blood vessels, it is expected that compounds that directly affect the blood vessels themselves can be selected.

[0036] 1...branched artificial blood vessel model, 2...main blood vessel section, 3...branched blood vessel section, 4...smooth muscle layer, 5...extracellular matrix, 6...endothelial cells, 11...mold for producing smooth muscle tissue, 12...first groove section, 13...core rod, 13a...first core rod (core rod for main blood vessel section), 13b...second core rod (core rod for branched blood vessel section), 14...second groove section, 15...anchor, 16...hole, 21...silicone tube, 22...glass capillary, 23...mold for coating extracellular matrix, A...smooth muscle fiber, B...collagen gel, C...endothelial cells

Claims

1. A method for manufacturing a branched artificial blood vessel model, comprising: a smooth muscle layer formation step of preparing a first core rod having at least one hole and at least one second core rod having a diameter that can fit into the hole, and forming a smooth muscle layer on the surfaces of the first and second core rods; and a branched structure formation step of fitting the second core rod, also having a smooth muscle layer, into the hole of the first core rod, also having a smooth muscle layer, to bring the smooth muscle layer formed on the surface of the first core rod into contact with the smooth muscle layer formed on the surface of the second core rod, thereby forming a branched structure.

2. The manufacturing method according to claim 1, wherein the smooth muscle layer forming step is a step of winding a fibrous smooth muscle layer spirally around each mandrel while the mandrel is tilted.

3. The manufacturing method according to claim 1, further comprising a smooth muscle tissue preparation step of preparing the fibrous smooth muscle tissue before the smooth muscle layer formation step, the smooth muscle tissue preparation step comprising the steps of: preparing a smooth muscle tissue preparation mold for a main blood vessel portion and a branch blood vessel portion; placing the first core rod in a first groove of the smooth muscle tissue preparation mold for the main blood vessel portion and the second core rod in the smooth muscle tissue preparation mold for the branch blood vessel portion; injecting a liquid containing smooth muscle cells into a second groove that intersects with the first groove of each smooth muscle tissue preparation mold; and culturing the smooth muscle cells to obtain fibrous smooth muscle tissue.

4. The manufacturing method according to claim 1, further comprising, after the branched structure forming step, a step of placing the branched structure in a mold for extracellular matrix coating, and a step of forming an extracellular matrix by injecting a liquid containing an extracellular matrix into the mold for extracellular matrix coating and immersing the branched structure in the extracellular matrix material to cover the surface of the smooth muscle layer of the branched structure.

5. The manufacturing method according to claim 4, further comprising the steps of: after the extracellular matrix formation step, removing the first core rod and the second core rod; seeding and culturing endothelial cells on the inner wall of the smooth muscle layer; and covering at least a portion of the inner surface of the smooth muscle layer with endothelial cells.

6. A branched artificial blood vessel model having at least one branched structure produced by the manufacturing method described in claims 1 to 5.

7. A method for screening drugs for treating diseases resulting from vascular dysfunction, comprising using the branched artificial blood vessel model described in claim 6, perfusing a candidate compound, and selecting the candidate compound by monitoring the physiological response of the branched artificial blood vessel model or by analyzing changes in gene expression in the smooth muscle cells and endothelial cells that constitute the model.

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