Three-dimensional tumor tissue model, and production method thereof

Abstract

One purpose of the present disclosure is to provide a three-dimensional tumor tissue model having a main vessel-like structure and a tumor vessel-like structure, and a production method of the same. Another purpose of the present disclosure is to provide a method for evaluating the effect of an evaluation target on a tumor tissue using the three-dimensional tumor tissue model. The three-dimensional tumor tissue model of the present disclosure has a main vessel-like structure and a tumor vessel-like structure connected to the main vessel-like structure, and contains vascular cells, cancer cells, and extracellular matrix.

Description

Three-dimensional tumor tissue model and its manufacturing method

[0001] The present disclosure relates to a three-dimensional tumor tissue model and a method for producing the same.

[0002] In the development of new drugs for cancer treatment, it is important to accurately evaluate the efficacy of the drug, and non-clinical models, such as two-dimensionally cultured cancer cells and animal models, are used (e.g., Non-Patent Documents 1 and 2). However, when two-dimensionally cultured cells are used, the drug sensitivity of cancer cells may be overestimated. Furthermore, when animal models are used, there are problems such as differences in species, time and cost required for preparation.

[0003] In recent years, the development and use of artificial tissues (three-dimensional tissues) has progressed, and spheroids or organoids prepared by three-dimensional culture of cancer cells or the like are also used as non-clinical models for drug evaluation for cancer treatment (e.g., Non-Patent Document 3). However, because conventional spheroids or organoids using cancer cells lack blood vessels, it is not possible to evaluate the delivery and distribution of drugs through blood vessels or the behavior of blood cells such as T cells.

[0004] As an example of an artificial tissue having a blood vessel-like structure, a three-dimensional liver tissue model having a main blood vessel-like structure and a sinusoid-like structure is disclosed in Patent Document 1. However, a three-dimensional tumor tissue model having a blood vessel-like structure is not known.

[0005] International Publication No. 2021 / 066196

[0006] L. Austin Doyle, et al., “A multi drug resistance transporter from human MCF-7 breast cancer cells”, Proc. Natl. Acad. Sci. USA 95 (1998) 15665-15670.doi.org / 10.1073 / pnas.95.26.15665.Y. Il Kim, et al., “Enhanced efficacy of CKD-516 in combination with doxorubicin: Pre-clinical evaluation using a hepatocellular carcinoma xenograft model”, Anticancer Res.34 (2014) 1715-1722.N. Boucherit et al., “3D Tumor Models and Their Use for the Testing of Immunotherapies”, Front. Immunol. 2020,11:603640. doi: 10.3389 / fimmu.2020.603640.

[0007] The present disclosure aims to provide a three-dimensional tumor tissue model having a main blood vessel-like structure and a tumor blood vessel-like structure, a method for producing the same, and a method for evaluating the effect of the three-dimensional tumor tissue model on a target tumor tissue.

[0008] Tumor tissue is characterized by high proliferation, vascular attraction and mobility of cancer cells, the presence of interstitium that blocks oxygen and nutrient supply, and a special vascular network structure with blind ends.Tumor tissue differs significantly from non-tumor biological tissues (e.g., liver tissue) in terms of its requirements for nutrients and oxygen, the hardness of its extracellular matrix, and its responsiveness to mechanical conditions such as fluid stimuli.Therefore, it is generally believed that methods for constructing three-dimensional tumor tissue models of non-tumor biological tissues (e.g., liver tissue) cannot be used directly to construct tumor tissue models.

[0009] As a result of extensive research, the present inventors discovered that a three-dimensional tumor tissue model having a blood vessel-like structure can be constructed by the method shown in the Examples below, and have thus completed the present disclosure.

[0010] That is, the present disclosure provides, for example, the following: [1] A three-dimensional tumor tissue model having main vessel-like structures and tumor vessel-like structures connected to the main vessel-like structures, and comprising vascular cells, cancer cells, and extracellular matrix. [2] The three-dimensional tumor tissue model according to [1], comprising a vascular layer and a tumor layer in contact with the vascular layer, wherein the vascular layer has main vessel-like structures and tumor vessel-like structures connected to the main vessel-like structures, and comprises vascular cells and extracellular matrix, and the tumor layer comprises cancer cells and extracellular matrix. [3] The three-dimensional tumor tissue model according to [1] or [2], further comprising interstitial cells. [4] The three-dimensional tumor tissue model according to [3], wherein the interstitial cells comprise lymphocytes. [5] The three-dimensional tumor tissue model according to [4], wherein the lymphocytes are T cells. [6] A method for producing the three-dimensional tumor tissue model according to [1] and [3] to [5], comprising the steps of: forming an extracellular matrix gel having through-channels for forming the main blood vessel-like structures, the extracellular matrix gel containing first vascular cells and cancer cells; seeding second vascular cells on the inner walls of the through-channels to form the main blood vessel-like structures; and perfusing a culture medium through the main blood vessel-like structures to form the tumor blood vessel-like structures. [7] A method for producing the three-dimensional tumor tissue model according to [2] to [5], comprising the steps of: forming a vascular layer having through-channels for forming the main blood vessel-like structures, the vascular layer being made of an extracellular matrix gel containing first vascular cells; forming a tumor layer being made of an extracellular matrix gel containing cancer cells so as to contact at least a portion of the surface of the vascular layer opposite to the through-channel side; seeding second vascular cells on the inner walls of the through-channels to form the main blood vessel-like structures; and perfusing a culture medium through the main blood vessel-like structures to form the tumor blood vessel-like structures. [8] The method according to [6] or [7], wherein the medium contains 50% by mass or more of a medium for vascular cells relative to the total volume of the medium. [9] The method according to any one of [6] to [8], wherein the flow rate of the perfusion is 0.5 mL / hour or more and 20 mL / hour or less.

[10] A method for evaluating the effect of a target on tumor tissue, comprising the steps of perfusing a solution containing the target into the three-dimensional tumor tissue model according to any one of [1] to [5], and evaluating the effect of the target on the three-dimensional tumor tissue model.

[11] The evaluation method according to

[10] , wherein the target comprises lymphocytes.

[12] The evaluation method according to

[11] , wherein the lymphocytes are T cells.

[0011] According to the present disclosure, it is possible to provide a three-dimensional tumor tissue model having a main blood vessel-like structure and a tumor blood vessel-like structure, a method for manufacturing the same, and a method for evaluating the effect on a tumor tissue to be evaluated using the three-dimensional tumor tissue model.

[0012] 1 is a perspective view showing an example of a perfusion device.

[0023] FIG. 1B is a diagram showing the fabrication steps of a method for manufacturing a monolayer-type three-dimensional tumor tissue model according to one embodiment, with (i) to (vi) being cross-sectional views of the perfusion device of FIG. 1 along the A-A' axis.

[0024] FIG. 1C is a diagram showing the results of immunostaining of four types of three-dimensional tumor tissue constructed in Example 1 by varying the cell density, medium type, and cell type.

[0025] FIG. 1D is a diagram showing HE staining (A) and immunostaining (B) of three-dimensional tumor tissue constructed in Example 1 by maintaining a constant perfusion flow rate, and HE staining (C) and immunostaining (D) of three-dimensional tumor tissue constructed in Example 1 by varying the perfusion flow rate.

[0026] FIG. 1E is a diagram showing the results of immunostaining of three-dimensional tumor tissue constructed using various colon cancer cell lines in Example 2.

[0027] FIG. 1F is a diagram showing the results of fluorescence microscopic observation of the efficacy of doxorubicin hydrochloride using the three-dimensional tumor tissue constructed in Example 2, and FIG. 1B is a diagram showing the results of evaluating the number of colon cancer cells following administration of doxorubicin hydrochloride. 1 is a diagram showing the results of examining the effect on capillary vessel formation when evaluating the efficacy of doxorubicin hydrochloride using the three-dimensional tumor tissue constructed in Example 2. FIG. 2 is a diagram showing the results of fluorescence microscopy when measuring the distribution of doxorubicin hydrochloride and cetuximab using the three-dimensional tumor tissue constructed in Example 3. FIG. 3 is a diagram showing the results of examining the rejection reaction of T cells against HUVECs in Example 4. FIG. 4 is a graph showing the results of examining the rejection reaction of T cells against HUVECs in Example 4. FIG. 5 is a diagram showing the results of examining the medium conditions for culturing HUVECs in Example 4, using T cell medium (A), an equal volume mixed medium of T cell medium and EGM2 medium (B), and EGM2 medium (C). FIG. 6 is a graph showing the results of examining the medium conditions for culturing T cells in Example 4. FIG. 7 is a diagram showing the results when T cells are perfused into the three-dimensional tumor tissue in Example 5, showing the localization of HUVECs and T cells (A) and the localization of DiFi and T cells (B). 10 is a diagram showing the results of investigating the influence of the presence or absence of T cell perfusion on the appearance of three-dimensional tumor tissue in Example 5. FIG. 11 is a diagram showing the results of investigating the influence of the presence or absence of oscillation when T cells are perfused into three-dimensional tumor tissue in Example 5.20A to 20C are diagrams showing the production steps of a method for producing a bilayered three-dimensional tumor tissue model according to one embodiment, with (i) to (vii) being cross-sectional views of the perfusion device of FIG. 1 along the A-A' axis. This figure shows the results of HE staining and immunostaining of the bilayered three-dimensional tumor tissue constructed in Example 6. This figure shows immunostained images of nuclei and CK18 in the three-dimensional tumor tissue under conditions (A) where an EGFR-CD3 bispecific antibody (EGFR-CD3 antibody) is not added to the perfusion medium and under conditions (B) where an EGFR-CD3 bispecific antibody (EGFR-CD3 antibody) is added to the perfusion medium in Example 7. This figure shows a graph showing the cell density of CK18-positive cells in the three-dimensional tumor tissue under conditions (A) where an EGFR-CD3 bispecific antibody (EGFR-CD3 antibody) is not added to the perfusion medium and under conditions (B) where an EGFR-CD3 bispecific antibody (EGFR-CD3 antibody) is added to the perfusion medium in Example 7. This figure shows a perspective view of an example of a perfusion device. This figure shows an exploded perspective view of the perfusion device shown in FIG. 20C. This figure shows the results of an investigation into the effect on the appearance of a three-dimensional tumor tissue constructed using the perfusion device used in Example 8. 10 shows immunostained images of nuclei (A), CD31 (B), and CD3 (C) of the three-dimensional tumor tissue constructed in Example 8, and a merged image thereof (D). FIG. 11 shows the results of investigating the effect on the appearance of a three-dimensional tumor tissue constructed by the method in Example 9. FIG. 12 shows HE staining of the three-dimensional tumor tissue constructed in Example 9. FIG. 13 shows immunostained images of nuclei (A), CD31 (B), and CD3 (C) of the three-dimensional tumor tissue constructed in Example 9, and a merged image thereof (D). FIG. 14 shows immunostained images of nuclei (A), CK18 (B), and CD3 (C) of the three-dimensional tumor tissue constructed in Example 9, and a merged image thereof (D).

[0013] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0014] <Three-dimensional tumor tissue model> The three-dimensional tumor tissue model according to this embodiment has a main blood vessel-like structure and a tumor blood vessel-like structure connected to the main blood vessel-like structure, and includes vascular cells, cancer cells, and extracellular matrix.

[0015] As used herein, "three-dimensional tumor tissue" refers to a structure in which vascular cells and cancer cells are three-dimensionally arranged in an extracellular matrix, and refers to an artificially constructed three-dimensional tumor-like tissue that mimics tumor tissue. The shape of the three-dimensional tumor tissue model is not particularly limited and can vary depending on the shape of the container that contains the extracellular matrix, and examples include tubular, spherical, ellipsoidal, and rectangular parallelepiped shapes.

[0016] The term "vascular-like structure" refers to an artificially constructed tubular structure that mimics biological blood vessels and contains or is composed of vascular cells. The term "main vascular-like structure" refers to a tubular structure that penetrates a three-dimensional tumor tissue model, has an opening that allows perfusion from the outside, and has a larger diameter than the tumor vascular-like structure, capillary-like structure, etc., described below. An external perfusion device or the like can be connected to the opening of the main vascular-like structure to perfuse the three-dimensional tumor tissue model. The term "tumor vascular-like structure" refers to an artificially constructed tubular vascular-like structure that contains or is composed of vascular cells, mimicking capillaries that deliver oxygen, nutrients, etc. to cancer cells, and has an opening that communicates with the main vascular-like structure. Cancer cells exist in an extracellular matrix, and at least some of the cancer cells may be in contact with each other to form a tumor tissue-like structure. Furthermore, at least some of the cancer cells may be clustered around the main vascular-like structure and / or the tumor vascular-like structure. Therefore, when perfused by an external perfusion device, oxygen, nutrients, etc. are transported to cancer cells through the main vasculature-like structure and tumor vasculature-like structure.

[0017] The three-dimensional tumor tissue model according to this embodiment allows for accurate evaluation of the effectiveness of drugs for cancer treatment, for example. Furthermore, by perfusing a solution containing a drug through the main vascular-like structures in the three-dimensional tumor tissue model according to this embodiment, the distribution and delivery of a drug in tumor tissue can be evaluated. Furthermore, in a similar manner, it is also possible to evaluate the effect of a drug on blood vessels in tumor tissue. Additionally, the effect of cells infiltrating tumor tissue on tumor tissue can also be evaluated.

[0018] The thickness (diameter) of the main vessel-like structure is not particularly limited as long as it is thick enough to perfuse the three-dimensional tumor tissue model, but may be, for example, more than 50 μm, 100 μm or more, 200 μm or more, or 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, or 300 μm or less. The length of the main blood vessel-like structure in the three-dimensional tumor tissue model is not particularly limited, and may be, for example, 50,000 μm or less, 40,000 μm or less, 30,000 μm or less, 20,000 μm or less, 10,000 μm or less, 9,000 μm or less, 8,000 μm or less, 7,000 μm or less, 6,000 μm or less, 5,000 μm or less, 4,000 μm or less, 3,000 μm or less, 2,000 μm or less, 1,000 μm or less, 750 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more. The three-dimensional tumor tissue model may have one main blood vessel-like structure, or two or more main blood vessel-like structures.

[0019] Tumor vasculature-like structures are newly formed vasculature-like structures that branch off from main vasculature-like structures, and are also called capillary vasculature-like structures. Preferably, tumor vasculature-like structures have a blind-end structure. The size (diameter) of the tumor vasculature-like structures is not particularly limited as long as it is thick enough to transport oxygen, nutrients, and the like to cancer cells. For example, the size may be 1 μm or more, 2 μm or more, 3 μm or more, 4 μm or more, or 5 μm or more, or may be 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, or 10 μm or less. The length of the tumor blood vessel-like structure in the three-dimensional tumor tissue model is not particularly limited, and may be, for example, 5000 μm or less, 4000 μm or less, 3000 μm or less, 2000 μm or less, 1000 μm or less, 750 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, or 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more. The tumor blood vessel-like structure in the three-dimensional tumor tissue model may be one (one strand), but preferably there are multiple strands, and may be, for example, 3 or more, 5 or more, 7 or more, 10 or more, or 15 or more.

[0020] Vascular cells are not particularly limited as long as they are capable of forming blood vessel-like structures, and may be, for example, cells derived from mammals, such as humans, monkeys, dogs, cats, pigs, cows, horses, rabbits, mice, and rats. Vascular cells include, for example, vascular epithelial cells and vascular endothelial cells, with vascular endothelial cells being preferred and human-derived vascular endothelial cells being more preferred. Vascular cells may also be cell lines, primary cultured cells, or vascular cells obtained by inducing differentiation of pluripotent stem cells. Pluripotent stem cells include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like.

[0021] Examples of human-derived vascular endothelial cells include human umbilical vein endothelial cells (HUVEC), human umbilical artery endothelial cells (HUAEC), human coronary artery endothelial cells (HCAEC), human saphenous vein endothelial cells (HSaVEC), human pulmonary artery endothelial cells (HPAEC), human aortic endothelial cells (HAoEC), human dermal microvascular endothelial cells (HDMEC), human dermal blood endothelial cells (HDBEC), and human dermal lymphatic endothelial cells (Human Dermal Examples of human-derived vascular endothelial cells include human lymphatic endothelial cells (HDLEC), human pulmonary microvascular endothelial cells (HPMEC), human cardiac microvascular endothelial cells (HCMEC), human bladder microvascular endothelial cells (HBdMEC), human uterine microvascular endothelial cells (HUtMEC), and human brain microvascular endothelial cells. Human-derived vascular endothelial cells are preferably human umbilical vein endothelial cells. Vascular endothelial cells isolated from human tumors can also be used.

[0022] The vascular cells that form the main vessel-like structures and the vascular cells present in parts other than the main vessel-like structures, such as tumor vessel-like structures, may be of the same type or different types.

[0023] Cancer cells may be derived from, for example, mammals, including humans, mice, and rats. Examples of cancer cells include leukemia cells, malignant lymphoma cells, myeloma cells, lung cancer cells, breast cancer cells, gastric cancer cells, colon cancer cells (e.g., SW620, HT-29, and DiFi), uterine cancer cells, ovarian cancer cells, laryngeal cancer cells, pharyngeal cancer cells, tongue cancer cells, osteosarcoma cells, chondrosarcoma cells, rhabdomyosarcoma cells, leiomyosarcoma cells, fibrosarcoma cells, liposarcoma cells, angiosarcoma cells, liver cancer cells, pancreatic cancer cells, biliary tract cancer cells, brain tumor cells, glioma cells, prostate cancer cells, and skin cancer cells. Alternatively, cancer cell lines or cancer cells isolated from tumor tissue prepared by transplanting a tumor excised from a patient into a mouse may be used. In this case, only cancer cells may be isolated from the tumor tissue, or a mixture of cancer cells and stromal cells may be used. Alternatively, cancer cells isolated from cancer cell lines or tissues cultured as organoids from tumors excised from patients may be used. In this case, too, only cancer cells may be isolated from the organoids, or a mixture of cancer cells and stromal cells may be used.

[0024] Extracellular matrix (ECM) is an insoluble substance present outside cells in living organisms. The extracellular matrix in the three-dimensional tumor tissue model may be dispersed in an aqueous medium as described below, and is preferably solid (gel-like) at cell culture temperatures (e.g., 30-37°C). The extracellular matrix used in the three-dimensional tumor tissue model is preferably a solution of the extracellular matrix dissolved in an aqueous medium (hereinafter sometimes simply referred to as "extracellular matrix solution") that is liquid at temperatures lower than the cell culture temperature (e.g., 0-20°C) but gels at the cell culture temperature (e.g., 30-37°C) (hereinafter simply referred to as "extracellular matrix gel"). The concentration of the extracellular matrix in the extracellular matrix solution or extracellular matrix gel is not particularly limited, and may be any concentration that provides a hardness generally suitable for cell culture and cell migration upon gelation, and may be, for example, 0.1 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 1.5 mg / mL or more, 2 mg / mL or more, 2.5 mg / mL or more, or 3 mg / mL or more, or 50 mg / mL or less, 40 mg / mL or less, 30 mg / mL or less, 20 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, or 5 mg / mL or less.

[0025] Examples of extracellular matrices include collagen, fibrin, elastin, proteoglycan, fibronectin, hyaluronic acid, laminin, vitronectin, tenascin, entactin, fibrillin, glycosaminoglycan, and alginic acid. Collagen or fibrin is preferred, and collagen is more preferred. In the three-dimensional tumor tissue model, the extracellular matrix may contain one type alone or two or more types in combination. The collagen may be, for example, various types of collagen, such as type I, type II, type III, type IV, type V, or type XI. Basement membrane extracts, such as Matrigel and Geltrex, may also be used.

[0026] The extracellular matrix in the three-dimensional tumor tissue model according to this embodiment contains water unless otherwise specified. Here, water refers to the aqueous medium described below. The content of the extracellular matrix (including water) in the three-dimensional tumor tissue model according to this embodiment is not particularly limited, but may be, for example, 10% by mass or more, 15% by mass or more, 20% by mass or more, 25% by mass or more, or 30% by mass or more, or 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 20% by mass or less, or 15% by mass or less, based on the total mass of the three-dimensional tumor tissue model.

[0027] The thickness of the three-dimensional tumor tissue model according to this embodiment is not particularly limited, and may be, for example, 5,000 μm or less, 4,000 μm or less, 3,000 μm or less, 2,000 μm or less, 1,000 μm or less, 750 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, or 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more. Here, in this specification, the thickness of the three-dimensional tumor tissue model refers to the distance from the vascular wall surface (the outer wall surface of the blood vessel) of the main blood vessel-like structure to the surface of the three-dimensional tumor tissue model.

[0028] The three-dimensional tumor tissue model according to this embodiment may contain stromal cells. Stromal cells are cells that constitute the support tissue of epithelial cells. Examples of stromal cells include fibroblasts, immune cells, pericytes, nerve cells, mast cells, epithelial cells, cardiac myocytes, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells, and mesenchymal stem cells.

[0029] Examples of immune cells include lymphocytes, macrophages, granulocytes, and dendritic cells, with lymphocytes being preferred. Examples of lymphocytes include T cells, B cells, and natural killer cells, with T cells being preferred.

[0030] Cells present in the cancer stroma are called cancer stromal cells and are believed to have properties such as secreting cytokines or growth factors that promote cancer proliferation and promoting cancer progression. Furthermore, cancer stromal cells are believed to differentiate through activation of stromal cells within tumor tissue. Therefore, in order to construct three-dimensional tumor tissue more similar to tumor tissue, the three-dimensional tumor tissue model according to this embodiment may contain cancer stromal cells as stromal cells. Examples of cancer stromal cells include fibroblasts, immune cells, and vascular endothelial cells present in the cancer stroma. Examples of fibroblasts present in the cancer stroma include cancer-associated fibroblasts (CAFs). Examples of immune cells present in the cancer stroma include tumor-associated macrophages (TAMs). Examples of vascular endothelial cells present in the cancer stroma include tumor vascular endothelial cells.

[0031] The three-dimensional tumor tissue model according to this embodiment may be a single-layered three-dimensional tumor tissue model or a multi-layered three-dimensional tumor tissue model. A single-layered three-dimensional tumor tissue model is a three-dimensional tumor tissue model having one layer in which all cells and extracellular matrix are contained.

[0032] A multi-layered three-dimensional tumor tissue model is a three-dimensional tumor tissue model that includes two or more layers, i.e., two or more layers that are in contact with each other, i.e., stacked, and one of the two or more layers may not contain all types of cells. The multi-layered three-dimensional tumor tissue model is not particularly limited, but may be two-, three-, four-, or five-layered, and preferably a two-layered three-dimensional tumor tissue model. The multiple layers are concentric with the longitudinal axis of the main vascular-like structure. In the case of a two-layered three-dimensional tumor tissue model, the three-dimensional tumor tissue model according to one embodiment comprises a vascular layer and a tumor layer, the vascular layer surrounding the main vascular-like structure, and the tumor layer stacked on the main vascular-like structure (i.e., on the opposite side from the main vascular-like structure). By using a multi-layered three-dimensional tumor tissue, the three-dimensional tumor tissue model can be structured more similarly to tumor tissue in vivo.

[0033] The vascular layer is a layer containing main vessel-like structures and tumor vessel-like structures, and also containing vascular cells and extracellular matrix. The extracellular matrix contains main vessel-like structures containing or consisting of vascular cells, and tumor vessel-like structures connected to the main vessel-like structures. During the cultivation of the three-dimensional tumor tissue model, the proliferation of vascular cells increases the number of tumor vessel-like structures in the vascular layer, and the length of the tumor vessel-like structures also increases. As a result, the tumor vessel-like structures in the vascular layer may extend into the tumor layer. Furthermore, cancer cells proliferating in the tumor layer, as described below, may infiltrate into the vascular layer. The vascular layer may further contain the above-mentioned stromal cells.

[0034] The tumor layer is a layer containing cancer cells and extracellular matrix. During the cultivation of the 3D tumor tissue model, cancer cells may proliferate and infiltrate into the vascular layer or gather around tumor vasculature-like structures extending from the vascular layer. The tumor layer may further contain the above-mentioned stromal cells.

[0035] A multilayered 3D tumor tissue model according to one embodiment may have an outermost layer containing a biocompatible material (hereinafter also referred to as a "coating layer"). The coating layer is a gel layer formed by gelation of the biocompatible material. In this specification, "outermost layer" refers to the layer farthest from the longitudinal axis of the main vessel-like structure. Preferably, the coating layer does not contain cells. By providing the 3D tumor tissue model according to this embodiment with a coating layer, the structure of the 3D tumor tissue (e.g., the main vessel-like structure, the overall structure of the 3D tumor tissue, etc.) is more stably maintained even with continued perfusion. The perfusion period may be, for example, one day or more, two weeks, one month, two months, or three months, but is not limited to these periods.

[0036] As used herein, a "biocompatible material" refers to a material that does not adversely affect cell growth and does not interfere with the formation of a three-dimensional tumor tissue model. Examples of biocompatible materials include bio-derived materials such as extracellular matrix components (e.g., fibrin, Matrigel, collagen, and alginic acid), and synthetic polymers such as polyethylene glycol hydrogel. Bio-derived materials are preferred as biocompatible materials, with extracellular matrices being more preferred and fibrin being even more preferred.

[0037] In one embodiment, a three-dimensional tumor tissue model includes a tissue layer and a covering layer in contact with the tissue layer, the tissue layer having a main blood vessel-like structure and a tumor blood vessel-like structure connected to the main blood vessel-like structure, and including vascular cells, cancer cells, and an extracellular matrix, the covering layer including a biocompatible material and positioned as the outermost layer of the three-dimensional tumor tissue model. The tissue layer is similar to the blood vessel layer except that it includes cancer cells.

[0038] In one embodiment, a three-dimensional tumor tissue model is a three-dimensional tumor tissue model comprising a vascular layer and a tumor layer in contact with the vascular layer, and further comprising a coating layer, the coating layer comprising a biocompatible material and located as the outermost layer of the three-dimensional tumor tissue model.

[0039] <Method for manufacturing a three-dimensional tumor tissue model> The method for manufacturing a three-dimensional tumor tissue model (hereinafter, sometimes simply referred to as "three-dimensional tumor tissue") according to this embodiment is a method for manufacturing a three-dimensional tumor tissue model having a blood vessel-like structure and a tumor blood vessel-like structure, and has a through-flow channel for forming the main blood vessel-like structure, and includes the steps of: forming an extracellular matrix gel containing first vascular cells and cancer cells (gel formation step); seeding second vascular cells on the inner wall of the through-flow channel to form the main blood vessel-like structure (main blood vessel-like structure formation step); and perfusing a culture medium into the main blood vessel-like structure to form a tumor blood vessel-like structure (tumor blood vessel-like structure formation step).

[0040] A manufacturing method according to one embodiment will now be described with reference to the drawings. In the following description, the same or corresponding elements will be designated by the same reference numerals, and redundant description will be omitted.

[0041] (Perfusion Device) A manufacturing method according to one embodiment may be, for example, a manufacturing method using a perfusion device. A perfusion device according to one embodiment may be, for example, as shown in FIG. 1 . The perfusion device 1 shown in FIG. 1 includes a perfusion device main body 11 and a bottom cover 15. The perfusion device main body 11 is provided with at least a chamber 13 and tubular connectors 12 and 18. The shape, size, etc. of the perfusion device 1 are not particularly limited, and can be appropriately designed so that the 3D tumor tissue model has a desired shape and size. The perfusion device 1 can also be manufactured using, for example, 3D printer technology. The material of the perfusion device 1 is not particularly limited, but may be, for example, acrylic resin. Preferably, the perfusion device 1 is configured such that, after the 3D tumor tissue model is constructed, it can be connected to a perfusion apparatus or the like that delivers fluid from the outside of the perfusion device 1 to the inside of the 3D tumor tissue model via the tubular connectors 12 or 18, thereby perfusing the 3D tumor tissue model.

[0042] The chamber 13 is a chamber (space) provided in the perfusion device main body 11 for forming and culturing a three-dimensional tumor tissue model. The chamber 13 is surrounded by an inner wall 32 and has openings on both the upper and lower sides. The lower opening can be closed with a bottom cover 15, allowing the chamber 13 to contain a liquid. The perfusion device main body 11 may have a groove 16 corresponding to the lower opening of the chamber 13, and the bottom cover 15 can be fitted into the groove 16. The groove 16 in the perfusion device main body 11 makes it easier to close the lower opening of the chamber 13. The shape and size of the chamber 13 are not particularly limited as long as it allows the formation of a three-dimensional tumor tissue model.

[0043] At least one pair of tubular connectors 12 and 18 are provided on each of opposing side surfaces of the perfusion device main body 11, and each have through-holes 121 and 181 that communicate from the outside of the perfusion device main body 11 to the inside of the chamber 13. The through-holes 121 and 181 of the tubular connector 12 and the tubular connector 18 have the same diameter and are centered on the same axis. This allows a rod-shaped member 17 (described below) to be passed through the through-holes 121 and 181. Furthermore, because the main vessel-like structures in the three-dimensional tumor tissue model communicate with the through-holes 121 and 181, fluid can pass from the outside of the perfusion device main body 11 into the main vessel-like structures via the tubular connectors 12 and 18. The diameters of the through-holes 121 and 181 are not particularly limited and may be appropriately set to match the diameter of the main vessel-like structures.

[0044] The number of tubular connectors 12 and 18 is not particularly limited, but may be one pair (one tubular connector 12 and one tubular connector 18) or two or more pairs (two or more tubular connectors 12 and two or more tubular connectors 18). Preferably, each of the tubular connectors 12 and 18 has an anchor structure 14 on the side exposed to the chamber 13. By providing the anchor structure 14 on the tubular connectors 12 and 18, contraction of the three-dimensional tumor tissue can be minimized, facilitating the construction of a three-dimensional tumor tissue model of the desired shape, and preventing the three-dimensional tumor tissue model from detaching from the perfusion device 1. Furthermore, the surfaces of the tubular connectors 12 and 18 exposed to the chamber 13 may be subjected to atmospheric plasma treatment and / or fibronectin coating. This treatment further prevents the detachment of the cultured three-dimensional tumor tissue model.

[0045] A perfusion device according to another embodiment may be, for example, the perfusion device 1a shown in Fig. 20 . As shown in the exploded perspective view of Fig. 21 , the perfusion device 1a shown in Fig. 20 includes a top cover 81, a seal member 91, and a perfusion device main body 11a. The perfusion device main body 11a is an integrated unit of the perfusion device main body 11 and the bottom cover 15 of the perfusion device 1. In other words, the perfusion device main body 11a does not have the lower opening described in the description of the chamber 13 in the perfusion device 1. Other specific aspects of the perfusion device main body 11a are as described for the perfusion device main body 11.

[0046] The top cover 81 according to one embodiment includes a cover 811 and four legs 812 with claws 813 attached to the four corners of the cover 811. The top cover 81 is fixed to the upper side of the perfusion device main body 11a and serves to close the upper opening of the chamber 13 in the perfusion device main body 11a with the cover 811. Providing the perfusion device 1a with the top cover 81 enables perfusion while maintaining the structure of the three-dimensional tumor tissue (e.g., the main blood vessel-like structure, the entire structure of the three-dimensional tumor tissue, etc.) more stably.

[0047] The method for fastening the top cover 81 to the perfusion device main body 11a may be, for example, a snap-fit ​​mechanism, a clamp, or a screw. The top cover 81 shown in FIG. 21 includes legs 812, each of which has a claw 813, for fastening to the perfusion device 11a by a snap-fit ​​mechanism. The claw 813 engages with the protrusion 19 of the perfusion device main body 11a, thereby fastening the top cover 81 to the perfusion device 11a. The top cover 81 may have a main body recess 814 on either the tubular connector 12 side or the tubular connector 18 side of the perfusion device main body 11a, or may have a main body recess 814 on both the tubular connector 12 side and the tubular connector 18 side. The shape of the main body recess 814 may be, for example, rectangular, triangular, curved, or a combination thereof. Having the main body recess 814 on the top cover 81 improves operability with respect to the tubular connector.

[0048] The shape, size, etc. of the top lid 81 are not particularly limited as long as they can close the upper opening of the chamber 13, and can be designed as appropriate. The top lid 81 can also be manufactured using, for example, 3D printer technology. The material of the top lid 81 is not particularly limited, but may be, for example, acrylic resin.

[0049] The seal member 91 according to one embodiment is provided with a recess 911. The seal member 91 is a member interposed between the perfusion device main body 11a and the top cover 81 to improve the sealing performance of the top cover 81. The seal member 91 is, for example, a packing. The seal member 91 may have the recess 911 on either the tubular connector 12 side or the tubular connector 18 side of the perfusion device main body 11a, or may have the recess 911 on both the tubular connector 12 side and the tubular connector 18 side. The shape of the recess 911 may be, for example, a square shape, a triangular shape, a curved shape, or a combination thereof. When the top cover 81 has a main body recess 814, the recess 911 is preferably provided in the same direction. The recess 911 in the seal member 91 improves operability with respect to the tubular connector.

[0050] The shape, size, etc. of the sealing member 91 are not particularly limited as long as they can close the upper opening of the chamber 13, and can be designed as appropriate. The sealing member 91 can also be manufactured by methods such as 3D printer technology, cutting, or transfer using a mold. The material of the sealing member 91 is not particularly limited, and may be, for example, rubber, silicone (e.g., dimethylpolysiloxane (PDMS)), etc.

[0051] [Method for manufacturing a single-layered three-dimensional tumor tissue model] The steps of a method for manufacturing a single-layered three-dimensional tumor tissue model according to one embodiment are shown in Figure 2. Figure 2 is a diagram showing a three-dimensional tumor tissue model manufactured using perfusion device 1, and is a cross-sectional view of the perfusion device taken along the line AA' in Figure 1. Hereinafter, each step in Figure 2 will also be referred to as "step (2-i)" or the like.

[0052] (Gel formation step) In the gel formation step, an extracellular matrix gel having through-flow channels for forming main vascular-like structures and containing first vascular cells and cancer cells is formed. The main vascular-like structures are as described above. The first vascular cells are vascular cells that form the main vascular-like structures, and the types of the first vascular cells are as described above.

[0053] The gel-forming step may be, for example, a step including steps (2-i) to (2-iii) in FIG.

[0054] [Step (2-i)] Step (2-i) is a step of passing a rod-shaped member 17 through the through-hole 121 of one tubular connector 12 into the through-hole 181 of the other tubular connector 18 in the perfusion device main body 11 provided with the pair of tubular connectors 12 and 18, and is a step for forming a through-flow channel 31 in the gel (extracellular matrix gel 26) in the step (2-iii) described below.

[0055] The thickness of the rod-shaped member 17 corresponds to the outer diameter of the main vessel-like structure 52 in the three-dimensional tumor tissue 61 (described below), and therefore the thickness of the rod-shaped member 17 may be appropriately designed depending on the outer diameter of the desired main vessel-like structure. The material of the rod-shaped member 17 is not particularly limited, but may be made of metal, preferably stainless steel. The shape of the rod-shaped member 17 is not particularly limited, and may be, for example, needle-shaped (e.g., syringe needle) or wire-shaped, as long as it can construct a main vessel-like structure 52 with a predetermined inner diameter and length.

[0056] [Step (2-ii)] Step (2-ii) is a step of forming an extracellular matrix gel 26 containing first vascular cells 22 and cancer cells 23. More specifically, in step (2-ii), after step (2-i), a cell suspension in which first vascular cells 22 and cancer cells 23 are suspended in extracellular matrix solution 21 is filled into the chamber 13 with the lower opening blocked, and gelation is allowed to occur. This allows the construction of a base portion of the three-dimensional tumor tissue model according to this embodiment. The first vascular cells, cancer cells, and extracellular matrix are as described above.

[0057] The cell suspension in which vascular cells 22 and cancer cells 23 are suspended in the extracellular matrix solution 21 may further contain cells such as the above-mentioned interstitial cells, if necessary.

[0058] The extracellular matrix solution 21 can be prepared by dissolving, dispersing, etc. the extracellular matrix in an aqueous medium. The extracellular matrix to be dissolved, dispersed, etc. in an aqueous medium may be in the form of a solution, a lyophilized product, or a powder.

[0059] The concentration of the extracellular matrix in the extracellular matrix solution 21 may be 0.1 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 1.5 mg / mL or more, 2 mg / mL or more, 2.5 mg / mL or more, or 3 mg / mL or more, or may be 50 mg / mL or less, 40 mg / mL or less, 30 mg / mL or less, 20 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, or 5 mg / mL or less.

[0060] As used herein, aqueous medium refers to water or an aqueous solution. Examples of water include deionized water and distilled water, and examples of aqueous solutions include buffer solutions such as Tris-hydrochloric acid buffer or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), liquid media, etc. The liquid media are not particularly limited, and a suitable liquid medium can be selected depending on the type of cells to be cultured.

[0061] The cell density of the vascular cells in the cell suspension is not particularly limited as long as a three-dimensional tumor tissue model can be constructed. For example, ５ Cells / mL or more, 5 x 10 ５ Cells / mL or more, 1 x 10 ６ Cells / mL or more, 5 x 10 ６ cells / mL or greater, or 1 x 10 ７ cells / mL or more, and ８ Cells / mL or less, 1 x 10 ８ Cells / mL or less, 5 x 10 ７ Cells / mL or less, 3 x 10 ７ Cells / mL or less, 1 x 10 ７ Cells / mL or less, 5 x 10 ６ cells / mL or less, or 1 x 10 ６ It may be less than cells / mL.

[0062] The cell density of the cancer cells in the cell suspension is not particularly limited as long as a three-dimensional tumor tissue model can be constructed. For example, ５ Cells / mL or more, 5 x 10 ５ Cells / mL or more, 1 x 10 ６ Cells / mL or more, 5 x 10 ６ cells / mL or greater, or 1 x 10 ７ cells / mL or more, and ８ Cells / mL or less, 1 x 10 ８ Cells / mL or less, 5 x 10 ７ Cells / mL or less, 3 x 10 ７ Cells / mL or less, 1 x 10 ７ Cells / mL or less, 5 x 10 ６ cells / mL or less, or 1 x 10 ６ It may be less than cells / mL.

[0063] The cell density ratio of vascular cells to cancer cells in the cell suspension (vascular cells:cancer cells) may be 1:0.3 to 1:3, 1:0.5 to 1:2.5, 1:0.8 to 1:2, or 1:1 to 1:1.5.

[0064] The cell density of the stromal cells in the cell suspension is not particularly limited as long as a three-dimensional tumor tissue model can be constructed. For example, ４ Cells / mL or more, 5 x 10 ４ Cells / mL or more, 1 x 10 ５ Cells / mL or more, 5 x 10 ５ cells / mL or greater, or 1 x 10 ６ cells / mL or more, and ７ Cells / mL or less, 1 x 10 ７ Cells / mL or less, 5 x 10 ６ Cells / mL or less, 3 x 10 ６ Cells / mL or less, 1 x 10 ６ Cells / mL or less, 5 x 10 ５ cells / mL or less, or 1 x 10 ５ It may be less than cells / mL.

[0065] The gelation of the cell suspension can be achieved, for example, by heating the cell suspension. The temperature and heating time can be appropriately set depending on the type and concentration of the extracellular matrix component, but for example, the heating temperature may be 25 to 45°C, and the heating time may be 1 to 3 hours.

[0066] [Step (2-iii)] Step (2-iii) is a step of forming through-channels in the extracellular matrix gel 26 formed in step (2-ii) and containing the first vascular cells 22 and cancer cells 23. More specifically, step (2-iii) is a step of removing the rod-shaped member 17 passed through the perfusion device main body 11 in step (2-i) after step (2-ii), thereby forming through-channels 31 in the extracellular matrix gel 26 formed in step (2-ii) and containing the first vascular cells 22 and cancer cells 23. The through-channels 31 serve as the bases of the main vessel-like structures 52 in the three-dimensional tumor tissue model according to this embodiment.

[0067] The extracellular matrix gel 26 containing the first vascular cells 22 and cancer cells 23 in step (2-iii) may be either contracted or not contracted after the extracellular matrix in the cell suspension is gelled in step (2-ii). In step (2-iii), the extracellular matrix gel 26 containing the vascular cells 22 and cancer cells 23 is shown in a contracted state after step (2-ii). When the extracellular matrix gel 26 containing the vascular cells 22 and cancer cells 23 is in a contracted state, at least a portion of the inner wall 32 of the perfusion device main body 11 and the extracellular matrix gel 26 are not in contact with each other.

[0068] (Main vascular-like structure formation step) In the main vascular-like structure formation step, second vascular system cells are seeded on the inner walls of the through-flow channels in the extracellular matrix gel formed in the gel formation step to form the main vascular-like structure. The second vascular system cells are vascular system cells that form the main vascular-like structure, and the types of the second vascular system cells are as described above. The second vascular system cells may be the same type as the first vascular system cells, or may be a different type.

[0069] The main vessel-like structure forming step is not particularly limited, and may be, for example, a step including steps (2-iv) to (2-v).

[0070] [Step (2-iv)] In step (2-iv) in Figure 2, after the above step (2-ii) or the above step (2-iii), the extracellular matrix gel 26 containing the vascular cells 22 and the cancer cells 23 formed in step (2-ii) is immersed in a culture medium 41 containing a culture medium for vascular cells.

[0071] The gel 26 may be immersed in the medium by pouring the medium into the gel 26 so that the entire extracellular matrix gel 26 is immersed therein, or by immersing the entire extracellular matrix gel in the medium.

[0072] As used herein, "medium for vascular cells" refers to a medium in which vascular cells can grow. Examples of media for vascular cells include Promocell's Endothelial Growth Medium, Endothelial Growth Medium 2, Endothelial Growth Medium MV, Endothelial Growth Medium MV2, and Lonza's EGM Bullet Kit, EGM-2 Bullet Kit, EGM-2MV Bullet Kit, and EGM-Plus Bullet Kit. The medium 41 used in step (4) may be a serum-supplemented medium or a serum-free medium. Furthermore, the medium 41 used in step (4) may contain components used in cell culture, such as growth factors, differentiation-inducing factors, hormones, amino acids, sugars, salts, and antibiotics.

[0073] The medium 41 used in step (2-iv) preferably contains 50% by mass or more of a medium for vascular cells relative to the total amount of the medium, and more preferably 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 100% by mass.

[0074] [Step (2-v)] In step (2-v), after step (2-iv), second vascular cells 51 are seeded into the through-flow channels 31 formed in the extracellular matrix gel 26 containing the first vascular cells 22 and cancer cells 23 in step (2-iii), thereby forming main vessel-like structures 52.

[0075] The second vascular system cells 51 seeded in step (2-v) may be, for example, a cell suspension in which the vascular system cells 51 are suspended in an aqueous medium. The method for seeding the second vascular system cells 51 into the through-channel 31 is not particularly limited, but may include, for example, a method of injecting a suspension of the vascular system cells 51 into the through-channel 31 using a syringe and a syringe pump or the like.

[0076] In step (2-v), the second vascular cells 51 are seeded in the through-channels 31, and then incubated for a predetermined time to form main vessel-like structures 52, thereby adhering the vascular cells 51 to the inner walls of the through-channels 31. To allow the second vascular cells 51 to adhere uniformly to the inner walls of the through-channels 31, the orientation of the perfusion device main body 11 may be changed as necessary.

[0077] The incubation temperature is not particularly limited as long as the second vascular cells 51 adhere to the inner wall of the through-channel 31. For example, the incubation temperature may be 35 to 40°C, preferably 37°C. The incubation time may be, for example, about 15 minutes to 1 hour. ２ The condition may be, for example, 5%.

[0078] (Tumor vasculature-like structure formation step) In the tumor vasculature-like structure formation step, a culture medium is perfused into the main vasculature-like structures formed in the main vasculature-like structure formation step to form tumor vasculature-like structures. The second vasculature cells are vasculature cells that form the main vasculature-like structures, and the types of the second vasculature cells are as described above. The second vasculature cells may be the same type as the first vasculature cells, or may be a different type.

[0079] The tumor vascular-like structure formation step is not particularly limited, but may be, for example, a step including the following step (2-vi):

[0080] [Step (2-vi)] In step (2-vi), for example, after step (2-v), a vascular cell culture medium is perfused from the tubular connector 12 provided on the perfusion device main body 11 into the main vessel-like structure 52 in the perfusion direction 71, thereby perfusion-culturing the cells contained in the three-dimensional tumor tissue model 61. The medium perfused in step (2-vi) may be any medium containing a vascular cell culture medium, and may be the same type of medium as the vascular cell culture medium 41 used in step (2-iv) or a different type of medium. Examples of the vascular cell culture medium used in step (2-vi) include those described above.

[0081] By injecting culture medium into the tubular connector 12 or 18 while the three-dimensional tumor tissue model 61 to be cultured is not detached from the tubular connectors 12 and 18, the culture medium can be perfused into the main vascular-like structure 52. This provides nutrients from within the three-dimensional tumor tissue model 61 to be cultured, culturing the first vascular system cells 22, the second vascular system cells 51, the cancer cells 23, and the like in the three-dimensional tumor tissue model 61. As a result, the first vascular system cells 22 form tumor vascular-like structures 62 connected to the main vascular system 52, effectively constructing the entire three-dimensional tumor tissue model 61 according to this embodiment. It is also preferable to perfuse the culture medium into the three-dimensional tumor tissue model 61 from the tubular connector 12 or 18 using an external pump or the like.

[0082] Examples of the external pump include a peristaltic pump, a syringe pump, an air pressure driven pump, etc. From the viewpoint of more easily forming a culture medium circulation path described below, the external pump is preferably one that can suck and deliver liquid, and a peristaltic pump is more preferred.

[0083] When perfusing the medium into the three-dimensional tumor tissue model 61 using an external pump, a medium circulation path may be formed for perfusion. The medium circulation path can be formed, for example, by using a tube and an external pump. In one embodiment of the medium circulation path, for example, the perfusion device main body 11 holding the extracellular matrix gel 26 containing the first vascular cells 22 and cancer cells 23 is immersed in a culture dish containing the medium to be perfused, one end of a tube is placed in the medium in the culture dish, and the other end of the tube is connected to a tubular connector 12. Furthermore, a peristaltic pump is connected to the tube. In this way, the medium in the culture dish is sucked from the end of the tube by the peristaltic pump and delivered to the three-dimensional tumor tissue model 61 via the tubular connector 12 for perfusion. The medium delivered to the three-dimensional tumor tissue model 61 is then discharged into the culture dish via the tubular connector 18.

[0084] The material of the tube is not particularly limited, and examples thereof include silicone, Teflon (registered trademark), and vinyl chloride, with silicone being preferred.

[0085] The material of the culture dish is not particularly limited, and examples thereof include plastic and glass, with plastic being preferred, and polystyrene being more preferred. As the culture dish, it is preferable to use a polystyrene dish that is commonly used for cell culture.

[0086] The medium circulation path may be appropriately connected to a bubble trap or the like. The bubble trap may be, for example, a device in which bubbles mixed in the medium are separated from the flow of the medium by buoyancy and accumulated in the container. More specifically, it can be configured by using a tube for introducing the medium into the sealed container and a tube for discharging the medium from the sealed container, with the end of the outlet tube located in the sealed container being lower than the end of the inlet tube located in the sealed container. Alternatively, a bubble removal device such as a membrane separator using an air-permeable membrane such as polytetrafluoroethylene (PTFE) may be used.

[0087] In the medium circulation path, the culture dish in which the perfusion device main body 11 is immersed may be separated from the culture dish to which the medium delivered into the 3D tumor tissue model 61 is discharged. In this case, the peristaltic pump aspirates the medium from the culture dish to which the medium is discharged. Alternatively, instead of circulating the medium, a system may be used in which new medium is constantly aspirated, delivered to the tumor tissue, and then the waste liquid is discharged into a waste liquid bottle.

[0088] The conditions for the perfusion culture in step (2-vi) are not particularly limited as long as the main vessel-like structures 52 and the tumor vessel-like structures 62 are formed. For example, the temperature may be 35 to 40°C, preferably 37°C, and the culture period may be, for example, about 2 to 20 days. ２ The condition may be, for example, 5%.

[0089] The flow rate of the perfusion in step (2-vi) is not particularly limited, and may be, for example, 0.5 mL / hour or more, 1 mL / hour or more, 2 mL / hour or more, 3 mL / hour or more, 4 mL / hour or more, 5 mL / hour or more, 6 mL / hour or more, 7 mL / hour or more, 8 mL / hour or more, 9 mL / hour or more, or 10 mL / hour or more, or may be 20 mL / hour or less, 19 mL / hour or less, 18 mL / hour or less, 17 mL / hour or less, 16 mL / hour or less, 15 mL / hour or less, 14 mL / hour or less, 13 mL / hour or less, 12 mL / hour or less, 11 mL / hour or less, or 10 mL / hour or less.

[0090] The perfusion flow rate in step (2-vi) may be constant or may be varied. When the perfusion flow rate is varied in step (2-vi), the flow rate may be varied after perfusion at a constant flow rate for, for example, 1 to 7 days, 1 to 5 days, 1 to 3 days, 1 to 2 days, or 1 day. The number of times the flow rate is varied is not particularly limited, and may be, for example, 5 times or less, 3 times or less, 2 times or less, or once.

[0091] When the perfusion flow rate is changed in step (2-vi), for example, the flow rate may be increased or decreased, but is preferably increased. When the flow rate is increased, the flow rate may be increased by 2 to 15 times, 3 to 14 times, 4 to 13 times, 5 to 12 times, 6 to 11 times, 7 to 10 times, 8 to 10 times, or 9 to 10 times.

[0092] The method for producing three-dimensional tumor tissue according to one embodiment may include a step of forming the coating layer (coating layer formation step) at any timing after the gel formation step. The coating layer is as described above. By including the coating layer formation step in the method for producing three-dimensional tumor tissue according to this embodiment, the structure of the three-dimensional tumor tissue (e.g., the main blood vessel-like structure, the overall structure of the three-dimensional tumor tissue, etc.) can be perfused while maintaining it more stably.

[0093] The coating layer forming step is preferably carried out after the main vessel-like structure forming step. The coating layer forming step can be carried out, for example, by the same procedure as (2-ii) in the gel forming step. More specifically, the chamber 13 with the lower opening closed is filled with a solution containing a biocompatible material and gelled.

[0094] A solution containing a biocompatible material can be prepared by dissolving, dispersing, etc. the biocompatible material in an aqueous medium. The biocompatible material to be dissolved, dispersed, etc. in an aqueous medium may be in the form of a solution, a freeze-dried product, or a powder.

[0095] The concentration of the biocompatible material in the solution containing the biocompatible material may be 0.1 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 1.5 mg / mL or more, 2 mg / mL or more, 2.5 mg / mL or more, or 3 mg / mL or more, or 150 mg / mL or less, 140 mg / mL or less, 130 mg / mL or less, 120 mg / mL or less, 110 mg / mL or less, 100 mg / mL or less, 90 mg / mL or less, 80 mg / mL or less, 70 mg / mL or less, 60 mg / mL or less, 50 mg / mL or less, 40 mg / mL or less, 30 mg / mL or less, 20 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, or 5 mg / mL or less.

[0096] The solution can be gelled, for example, by heating the solution. The temperature and heating time can be appropriately set depending on the type, concentration, etc. of the biocompatible material, but for example, the heating temperature may be 25 to 45°C, and the heating time may be 1 second to 3 hours.

[0097] [Method for Producing a Bilayered Three-Dimensional Tumor Tissue Model] When producing a bilayered three-dimensional tumor tissue model, instead of the gel-forming step, the method includes a step of forming a vascular layer (vascular layer formation step) having through-holes for forming main blood vessel-like structures and made of an extracellular matrix gel containing first vascular cells, and a step of forming a tumor layer (tumor layer formation step) made of an extracellular matrix gel containing cancer cells so as to contact at least a portion of the surface of the vascular layer opposite the through-channel side. The method for producing a bilayered three-dimensional tumor tissue model further includes a main blood vessel-like structure formation step and a tumor blood vessel-like structure formation step similar to those in the method for producing a monolayered three-dimensional tumor tissue model. In one embodiment, the method for producing a bilayered three-dimensional tumor tissue may also include a coating layer formation step at any timing after the tumor layer formation step. The coating layer formation step is preferably performed after the main blood vessel-like structure formation step.

[0098] FIG. 16 shows the fabrication steps of a method for manufacturing a two-layered three-dimensional tumor tissue model according to one embodiment. FIG. 16 illustrates the steps for fabricating a two-layered three-dimensional tumor tissue model using a perfusion device 1, with (i) to (vii) each being a cross-sectional view of the perfusion device in FIG. 1 taken along the A-A' axis. Hereinafter, each step in FIG. 16 will also be referred to as "step (16-i)," etc. Step (2-i) and step (16-i) are similar steps. Step (16-iv) is similar to step (2-iii) except that step (16-iv) is performed on a vascular layer 24 made of an extracellular matrix gel 26 containing first vascular system cells 22, instead of an extracellular matrix gel 26 containing first vascular system cells 22 and cancer cells 23. Step (16-v) is similar to step (2-iv) except that step (16-v) is performed on an extracellular matrix gel made of a vascular layer 24 and a tumor layer 25, instead of an extracellular matrix gel 26 containing first vascular system cells 22 and cancer cells 23. Step (16-vi) is the same as step (2-v) except that it is performed on the vascular layer 24 instead of the extracellular matrix gel 26 containing the first vascular cells 22 and the cancer cells 23. Step (16-vii) is the same as step (2-vi) except that it is performed on the two-layer three-dimensional tumor tissue model 61 instead of the single-layer three-dimensional tumor tissue model 61.

[0099] (Vascular layer formation step) In the vascular layer formation step, a vascular layer is formed that has through-flow channels for forming main vessel-like structures and is made of an extracellular matrix gel containing first vascular cells. The main vessel-like structures, through-flow channels, and first vascular cells are as described above.

[0100] In the vascular layer formation step, for example, as in step (16-ii), the chamber 13 with the lower opening closed is filled with a first liquid and gelled to form the vascular layer 24. The first liquid is a cell suspension in which first vascular cells 22 are suspended in an extracellular matrix solution 21, and can be prepared in the same manner as the cell suspension in step (2-ii) above. Except for this, the vascular layer 24 can be formed in the same manner as in step (2-ii) above.

[0101] In the tumor layer formation step, a tumor layer is formed from an extracellular matrix gel containing cancer cells so as to contact at least a portion of the surface of the vascular layer opposite to the through-channel side. The cancer cells and tumor vascular-like structures are as described above.

[0102] The tumor layer formation step is not particularly limited, and may be, for example, the following step (16-iii). In step (16-iii), a second liquid is filled onto the blood vessel layer 24 and allowed to gel, thereby forming a tumor layer 25. The second liquid is a cell suspension in which cancer cells 23 are suspended in an extracellular matrix solution 21, and can be prepared in the same manner as the cell suspension in step (2-ii) above. Except for this, the tumor layer 25 can be formed in the same manner as step (2-ii) above.

[0103] The tumor layer 25 may be formed so as to contact at least a portion of the surface of the blood vessel layer 24 opposite the through-flow channel 31 side, or may be formed so as to contact the entire surface of the blood vessel layer 24 opposite the through-flow channel side. The degree of contact between the tumor layer 25 and the blood vessel layer 24 can be changed by appropriately adjusting the portion where the second liquid is filled on the blood vessel layer 24 and the amount of the second liquid to be filled.

[0104] The thickness of each of the vascular layer 24 and the tumor layer 25 is not particularly limited as long as it is a thickness that allows the formation of a blood vessel-like structure, and may be, for example, 5000 μm or less, 4000 μm or less, 3000 μm or less, 2000 μm or less, 1000 μm or less, 750 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, or 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more. Here, the thickness of the vascular layer refers to the distance from the vascular wall surface (outer wall surface of the blood vessel) of the main blood vessel-like structure to the surface of the vascular layer 24 that contacts the tumor layer 25, and the thickness of the tumor layer 25 refers to the distance from the surface that contacts the vascular layer 24 to the surface opposite the vascular layer.

[0105] In the three-dimensional tumor tissue model 61, the formation of the main blood vessel-like structure 52 and the tumor blood vessel-like structure 62 can be confirmed by, for example, immunostaining as shown in the examples described later.

[0106] <Method for evaluating the effect on tumor tissue of the evaluation target> The method for evaluating the effect on tumor tissue of the evaluation target according to this embodiment (hereinafter also referred to as the "evaluation method according to this embodiment") includes a step of perfusing a solution containing the evaluation target into the three-dimensional tumor tissue model according to this embodiment (perfusion step), and a step of evaluating the effect of the evaluation target on the three-dimensional tumor tissue model (evaluation step).

[0107] The perfusion step can be carried out under the same conditions as the tumor vascular-like structure formation step in the method for producing a three-dimensional tumor tissue model according to the present embodiment described above.

[0108] The subject of evaluation may be any substance, including, for example, a drug such as an anticancer drug, or a cell such as a lymphocyte. Examples of anticancer drugs include doxorubicin, cetuximab, cisplatin, rituximab, carboplatin, cyclophosphamide, docetaxel, tamoxifen, tegafur, erlotinib, axitinib, etoposide, dexamethasone, mitomycin C, bleomycin, paclitaxel, doxil, bevacizumab, afatinib, nivolumab, 5-FU, and T-cell engagers. T-cell engagers are pharmaceuticals or compounds that aim to treat disease by bringing immune cells, such as killer T cells, that have the role of eliminating foreign substances into contact with disease-causing cells or pathogens, thereby eliminating the cause of the disease. The lymphocyte may be, for example, a T cell. Furthermore, the T cell may be genetically modified, such as a CAR-T cell expressing a chimeric antigen receptor. The T cell engager may be, for example, an EGFR-CD3 bispecific antibody. The EGFR-CD3 bispecific antibody is a bispecific antibody that binds to both CD3, an antigen on T cells, and EGFR, an antigen on cancer cells (DiFi) (Asano, R., et al., Sci Rep 10, 4913 (2020)). The target of evaluation may be a single type, or two or more types may be used in combination.

[0109] The solution may contain a medium for culturing the three-dimensional tumor tissue model according to this embodiment. The medium is the same as that described in the tumor vasculature-like structure formation step.

[0110] In the evaluation step, for example, when the evaluation target is a drug, the efficacy of the drug on the three-dimensional tumor tissue according to this embodiment may be evaluated, the distribution of the drug in the three-dimensional tumor tissue may be evaluated, the delivery of the drug in the three-dimensional tumor tissue may be evaluated, or the effect of the drug on blood vessels in the three-dimensional tumor tissue may be evaluated.Furthermore, in the evaluation step, for example, when the evaluation target is a lymphocyte, the infiltration of lymphocytes in the three-dimensional tumor tissue according to this embodiment may be evaluated.

[0111] The evaluation step can be performed, for example, by comparing the three-dimensional tumor tissue immediately after or before perfusion of the evaluation target into the three-dimensional tumor tissue model according to this embodiment with the three-dimensional tumor tissue after perfusion of the evaluation target into the three-dimensional tumor tissue model according to this embodiment for a predetermined period of time. Specific methods for evaluation include, for example, the methods described in the Examples below (counting the number of cancer cells, immunostaining, etc.).

[0112] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0113] Example 1. Examination of Conditions for Monolayer-Type 3D Tumor Tissue Construction (1) (1-1. Cell Culture) In Example 1, each cell was cultured as follows. Colon cancer cells (SW620, HT-29, DiFi) were cultured in Dulbecco's Modified Eagle Medium (hereinafter referred to as FBS-DMEM) supplemented with 10% fetal bovine serum, 1% 100x non-essential amino acids, and 1% 100x penicillin streptomycin solution. HUVECs were cultured in endothelial cell medium (Endothelial Growth Medium 2; EGM2, Promocell) supplemented with the growth factors included in the kit and 1% 100x penicillin streptomycin solution. Immortalized MSCs were cultured in DMEM supplemented with 10% fetal bovine serum, 1% 100x non-essential amino acid solution, and 1% 100x penicillin streptomycin solution. CD8+ T cells were cultured in T cell medium (Stemcell Technologies, ImmunoCult-XF T Cell Expansion Medium) supplemented with 10 ng / mL IL-2 and 25 μL / mL ImmunoCult Human CD3 / CD28 / CD2 T Cell Activator. Lung cancer cells (SBC-3) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% 100x penicillin streptomycin solution. However, T Cell Activator was added only for 3 days after thawing, and only IL-2 was added during subsequent passages. All cells were cultured at 37°C and 5% CO ２ It was cultivated in the environment.

[0114] (1-2. Fabrication of Perfusion Device) The perfusion device 1 shown in Figure 1 was fabricated using a 3D printer (ProJet MJP 3600 MAX (3D Systems)) with an acrylic UV-curable resin (product name: VisiJet M3 Crystal, 3D Systems). The perfusion device was cleaned in an ultrasonic cleaner and then sterilized with 70% ethanol. After drying in a clean bench, the perfusion device was treated with vacuum plasma for 5 minutes to improve the adhesion of cells and collagen gel. It was then immersed in 10 μg / mL bovine fibronectin and coated at 37°C for 2 hours. After threading needles as rod-shaped members 17 through the through-holes 121 and 181 of the tubular connectors 12 and 18, the perfusion device was stored at 4°C for up to one week until use in constructing 3D tumor tissue.

[0115] (1-3. Construction and culture of monolayer-type three-dimensional tumor tissue) According to the procedure shown in Figure 2, monolayer-type three-dimensional tumor tissues of Samples 1 to 4 were constructed under the conditions shown in Table 1 below. Specifically, colon cancer cells (6 x 10 ７ or 3 x 10 ７ cells / mL), HUVEC (3×10 ７ cells / mL), MSCs (0 or 5 × 10 ６ cells / mL), the cells were collected, suspended in a 3 mg / mL neutralized type I collagen solution, and this was filled into the perfusion device. The neutralized type I collagen solution was prepared by mixing 5 mg / mL collagen acidic solution I-AC50 (Kokensha) with calcium- and magnesium-free 10x Dulbecco's phosphate buffer and each medium shown in Table 1 in a ratio of 9:1:5. After incubation at 37°C to allow the filled collagen to gel, the needles attached to the perfusion device were removed to form through-channels, and the perfusion device was immersed in tissue culture medium contained in a culture dish. A mixture of equal amounts of FBS-DMEM and EGM2, or EGM2, was used as the tissue culture medium. 100 μL of HUVEC suspension (4 × 10 ６ The cells / mL were injected using a syringe and syringe pump. ２After 20 minutes of incubation, the perfusion device was turned upside down and incubated for another 20 minutes to allow cells to adhere to the upper surface of the through-channels. The perfusion device was then returned to its original position. After 80 minutes, tubing, a peristaltic pump, and a bubble trap were connected to the perfusion device to form a medium circulation path, and the medium was perfused. The perfusion culture of the 3D tumor tissue was carried out for 5 days.

[0116]

[0117] (1-4. Histological Analysis) After 5 days of perfusion culture, the 3D tumor tissue model was fixed with 4% paraformaldehyde and embedded in paraffin. The 3D tumor tissue model was sliced ​​at 5 μm and subjected to hematoxylin-eosin (HE) staining and fluorescent immunostaining or enzyme antibody immunostaining of vascular endothelial cells. For HE staining, the thin sections were deparaffinized and rehydrated, and stained with Mayer's hematoxylin and eosin Y in ethanol. For fluorescent immunostaining, the rehydrated thin sections were immersed in citrate buffer solution at pH 6 and autoclaved at 121°C for 5 minutes to perform antigen retrieval. The sections were washed with ultrapure water and then blocked with 4% Block Ace for 20 minutes. Next, they were incubated overnight with a primary antibody diluted in phosphate buffer. Human anti-CD31 antibody (BBA7, R&D Systems, or ab28364, Abcam) was used as the primary antibody. After washing the sections with PBS, they were incubated for 1 hour with a secondary antibody of the animal species corresponding to the primary antibody (Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 555-conjugated anti-rabbit, Thermo Fisher Scientific). Counterstaining was then performed using Hoechst. For enzyme immunostaining, the rehydrated thin sections were immersed in citrate buffer (pH 6) and autoclaved at 121°C for 5 minutes to perform antigen retrieval. The sections were immersed in 3% hydrogen peroxide for 10 minutes to inactivate endogenous peroxidase, then washed with ultrapure water and blocked with horse serum for 20 minutes. They were then incubated overnight with a primary antibody diluted in phosphate buffer. Human anti-CD31 antibody (BBA7, R&D Systems, or ab28364, Abcam) was used as the primary antibody. The sections were washed with PBS and then incubated for 1 hour with a secondary antibody (ImmPRESS Reagent, VECTOR LABORATORIES) of the corresponding animal species. Color development was performed with ImmPACT DAB (VECTOR LABORATORIES), followed by counterstaining with hematoxylin. The results of the enzyme-linked immunostaining are shown in Figure 3.

[0118] As shown in Figure 3, the density of colon cancer cells was 6 × 10 ７ 3D tumor tissue constructed as 3 × 10 cells / mL ７ When comparing the 3D tumor tissues constructed as 3×10 cells / mL, the cell density was 3×10 ７ The formation of more capillaries (tumor vascular-like structures) was confirmed in the 3D tumor tissue constructed with 0 MSC cells / mL (no introduction) and 5 × 10 MSC cells / mL (Figures 3(B) and 3(D)). ６ When compared with 3D tumor tissue constructed at 100 cells / mL, there was no significant difference in capillary formation (Figures 3(B) and 3(C)). Although it is thought that the introduction of MSCs would enable evaluation of the effects of cancer-associated fibroblasts, in the following studies, a concentration of 3 × 10 cells / mL was used to evaluate the effect of cancer-associated fibroblasts. ７ cells / mL of colon cancer cells and 3 x 10 ７ A monolayer type three-dimensional tumor tissue was constructed using HUVECs at 1000 cells / mL.

[0119] When three-dimensional tumor tissue constructed using a tissue culture medium consisting of an equal mixture of FBS-DMEM and EGM2 was compared with three-dimensional tumor tissue constructed using EGM2, it was found that when constructed using an equal mixture of FBS-DMEM and EGM2, cancer cells were maintained but capillary blood vessels were not formed, whereas when constructed using only EGM2, both the maintenance of cancer cells and the formation of capillary blood vessels were achieved (Figures 3(A) and 3(B)). Therefore, in the following studies, EGM2 was used as the tissue culture medium.

[0120] We also compared the perfusion flow rate between 1 mL / h during the 3D tumor tissue culture period and 1 mL / h for the first day and 10 mL / h for the remaining period. Perfusion within the tissue was visualized by injecting a 2:1 mixture of India ink and PBS using a syringe pump. As shown in Figure 4, compared with the 1 mL / h perfusion rate during the 3D tumor tissue culture period (Figures 4(A) and (B)), the formation of more capillaries was observed in the 3D tumor tissue when perfusion was performed at 1 mL / h for the first day and 10 mL / h for the remaining period (Figures 4(C) and (D)). Because tumor tissue has high nutritional requirements, perfusion at 1 mL / h on the first day of culture followed by perfusion at a higher flow rate of 10 mL / h is considered preferable for constructing a 3D tumor tissue model. Therefore, in the following studies, perfusion was performed at 1 mL / h for the first day and 10 mL / h for the remaining period.

[0121] Furthermore, under the above culture conditions, 3D tumor tissues were constructed using colon cancer cells SW620 or DiFi in addition to HT-29. The 3D tumor tissues were then fixed and paraffin-embedded as described in 1-4 above, and immunostaining was performed using human anti-CD31 antibody as the primary antibody. As a result, as shown in Figure 5, capillaries were formed regardless of the cancer cell type used, demonstrating that 3D tumor tissues with main vessel-like structures and capillaries (tumor blood vessels) can be constructed.

[0122] Example 2: Evaluation of Drug Efficacy Using Three-Dimensional Tumor Tissue On the second day of perfusion culture of the three-dimensional tumor tissue constructed in 1-3 above, the circulating tissue culture medium was replaced with medium containing the small molecule anticancer drug doxorubicin hydrochloride (doxorubicin) at a final concentration of 0 μM (no addition), 1 μM, or 10 μM, and perfusion culture was continued. On the fifth day, the tissue was fixed in the same manner as in 1-4 above, paraffin-embedded sections were prepared, and fluorescent immunostaining of cancer cells was performed and photographed using a fluorescent microscope. Fluorescent immunostaining was also performed in the same manner as in 1-4 above, staining cancer cells using a human anti-CK18 antibody (MA5-12104, Thermo Fisher Scientific) as the primary antibody. To count cancer cells from the photographed images, the following image processing steps A to F were performed using ImageJ. The results are shown in FIG. 6. A: The CK18 and nuclei channels are processed using "Threshold" and binarized. B: A range of 100 μm from the main blood vessel wall is set as the analysis target (ROI). C: In the nuclear channel ROI, the "Analyze Particle" plugin is used to extract and list the nuclear region. D: "Dilate" is applied to the CK18 channel to enlarge the CK18 signal. Since the CK18 signal is localized to the cell membrane, it does not normally overlap with the nucleus. However, this process enlarges the signal area, making it possible to detect overlap with the nucleus in the next step. E: For each nuclear region listed in C, the sum of the brightness values ​​in the corresponding region of the CK18 channel is calculated. This calculates the extent to which the CK18 signal overlaps with each nucleus. F: Nuclei with a CK18 signal above a predetermined threshold are counted as cancer cells.

[0123] As shown in Figures 6(A) and (B), it was confirmed that the number of CK18-positive cells (cancer cells) decreased in a doxorubicin concentration-dependent manner, demonstrating that the three-dimensional tumor tissue model of the present invention can be used as a testing tool for anticancer drugs.

[0124] In addition, paraffin-embedded sections were prepared from 3D tumor tissues treated with 0 μM and 1 μM doxorubicin using the same method as in 1-4 above, and vascular endothelial cells were fluorescently immunostained using human anti-CD31 antibody as the primary antibody. This allowed us to evaluate the effect of anticancer drugs on capillary formation in 3D tumor tissues. The results are shown in Figure 7.

[0125] As shown in Figure 7, it was found that the administration of doxorubicin enhanced the formation of capillaries. The increase in capillary density due to the administration of doxorubicin has only been reported in mouse tumor models, indicating that the three-dimensional tumor tissue of the present invention is more similar to a living body than conventional in vitro models.

[0126] Example 3: Measurement of Drug Distribution Using Three-Dimensional Tumor Tissue On the second day of perfusion culture of the three-dimensional tumor tissue constructed in 1-3 above, the circulating tissue culture medium was replaced with medium supplemented with doxorubicin hydrochloride or cetuximab, and perfusion was continued. On the fifth day, the tissue was fixed, paraffin-embedded, and thin-sectioned using the same method as in 1-4 above. For the three-dimensional tumor tissue administered with doxorubicin, the nuclei were stained with Hoechst and then subjected to fluorescence microscopy. Because doxorubicin itself is fluorescent, fluorescence observation was possible without any staining other than nuclear staining. Furthermore, for the tissue administered with cetuximab, cetuximab was fluorescently immunostained using a human anti-IgG antibody (ab109489, Abcam) as in 1-4 above, and fluorescence microscopy was performed. The results are shown in Figure 8.

[0127] As shown in Figure 8, it was demonstrated that by performing such appropriate staining, it is possible to measure the distribution of anticancer drugs in three-dimensional tumor tissue.

[0128] Example 4. Examination of Conditions for Co-Culture of T Cells and 3D Tumor Tissue An experiment was conducted to examine various conditions for perfusing T cells into 3D tumor tissue. First, because the T cells and HUVECs used were derived from different donors, there was concern about rejection due to a mismatch in human leukocyte antigens. Therefore, HUVECs cultured in a culture dish were administered a medium containing suspended T cells, and cultured for 3 days under the conditions described in 1-1 above in an environment where HUVECs and T cells coexist. The cells were then washed with PBS to remove T cells, and the cell counts of HUVECs were compared with those of a group not administered T cells. The results are shown in Figures 9 and 10.

[0129] As shown in Figures 9 and 10, there was no significant difference in the number of HUVEC cells with or without administration of T cells, indicating that there was no rejection reaction of T cells against HUVECs, or that the degree of rejection was negligible.

[0130] We also investigated the medium conditions used when perfusing T cells into 3D tumor tissue. More specifically, HUVECs or T cells were cultured for 3 days under the conditions described in 1-1 above using T cell medium, EGM2, or an equal mixture of the two media. The results are shown in Figures 11 and 12.

[0131] As shown in Figure 11, holes formed on the culture surface of HUVECs in T cell medium (arrowhead in Figure 11(A)). On the other hand, the culture surface of HUVECs was maintained in EGM2 and the equal-volume mixed medium, and no holes like those observed in T cell medium were observed (Figures 11(B) and (C)). Furthermore, as shown in Figure 12, when T cells were cultured for 3 days in T cell medium, EGM2, and an equal-volume mixed medium of both media, the cell number was almost maintained in T cell medium, but decreased to about half in the equal-volume mixed medium and to about one-quarter in EGM2. Based on these results, we decided to use the equal-volume mixed medium as a medium for perfusing T cells while maintaining blood vessels in tissues formed by HUVECs.

[0132] <Example 5. Perfusion of T cells into three-dimensional tumor tissue> On the third day of perfusion culture of the three-dimensional tumor tissue constructed in 1-3 above, EGM2, the medium that had been circulating for the three-dimensional tumor tissue culture up to that point, was replaced with 1 x 10 T cells. ６The medium was replaced with an equal volume mixture of T cell medium and EGM2 suspended at a concentration of 1000 cells / mL. Perfusion was then continued. On day 5 of perfusion culture, the tissue was fixed using the same method as in 1-4 above, paraffin-embedded sections were prepared, and fluorescent immunostaining was performed. For fluorescent immunostaining, human anti-CD31, human CD3 antibody (SP7, Abcam), and human anti-CK18 antibody were used as primary antibodies to stain HUVECs, T cells, and cancer cells (DiFi), respectively. Nuclei were also stained with Hoechst. The results are shown in Figure 13. As shown in Figure 14, when observed before tissue fixation, there was no significant difference in the appearance of the 3D tumor tissue with or without T cell perfusion.

[0133] As shown in Figure 13, the presence of T cells near capillaries (Figure 13(A)) and among cancer cells (Figure 13(B)) was observed. This suggests that the perfusion and infiltration of T cells into tumors can be simulated, observed, and evaluated using the three-dimensional tumor tissue model of the present invention.

[0134] On the other hand, when T cells were simply suspended in medium and perfused in this manner, the T cells settled to the bottom of the culture dish or bubble trap, and only a small number of the introduced T cells perfused into the tissue. Therefore, to perfuse T cells more efficiently, a culture system consisting of a pump, culture dish, bubble trap, perfusion device, and silicone tubing was mounted on a shaker and perfusion culture was performed while shaking at a rotation speed of 30 rpm. Subsequently, HUVECs, T cells, and cancer cells (DiFi) were fluorescently immunostained using human anti-CD31, human CD3 antibody, and human anti-CK18 antibody as primary antibodies, respectively. The results are shown in Figure 15.

[0135] As shown in Figure 15, it was confirmed that more T cells were present in the 3D tumor tissue when perfusion culture was performed with rocking compared to when no rocking was performed, demonstrating that the proportion of T cells perfused into the 3D tumor tissue could be improved.

[0136] Example 6. Construction and culture of a two-layered three-dimensional tumor tissue In order to obtain a three-dimensional tumor tissue having a structure closer to that of a living tumor tissue, a two-layered three-dimensional tumor tissue was constructed according to the procedure shown in Figure 16. More specifically, a 3 x 10 ７ The perfusion device 1 was filled with a hydrogel solution containing a suspension of 3 × 10 cells / mL HUVECs, and the solution was incubated at 37°C to gelate the solution, forming a vascular layer. The hydrogel used was a mixture of 2 mg / mL type I collagen and 2.5 mg / mL fibrin (both concentrations after mixing). After the formation of the vascular layer, 3 × 10 ７ 2 mg / mL of type I collagen suspended with 100 cells / mL of lung cancer cells SBC-3 was filled on the vascular layer 24, and the collagen was incubated at 37°C to gel, forming a tumor layer 25. As SBC-3, a strain in which vascular endothelial growth factor (VEGF) was forcibly expressed and a strain in which no expression was forcibly performed (mock) were used. After this, the needle embedded in advance was removed to form a through-channel 31, and the device was immersed in a tissue culture medium. EGM2 was used as the tissue culture medium. 100 μL of HUVEC suspension (4 × 10 ６ A concentration of 1000 cells / mL was injected using a syringe and syringe pump. After 20 minutes of incubation, the device was turned over to allow the cells to adhere to the upper surface of the flow channel, and the device was incubated for another 20 minutes. The perfusion device 1 was then returned to its original position, and after 80 minutes, the medium was perfused using a peristaltic pump. Perfusion culture was carried out for 5 days. The tissue was fixed in the same manner as in 1-4 above, and paraffin-embedded sections were prepared and subjected to HE staining and fluorescent immunostaining. For fluorescent immunostaining, HUVECs were stained using a human anti-CD31 antibody as the primary antibody. The results are shown in Figure 17.

[0137] As shown in Figure 17, HE staining confirmed the formation of two layers: a vascular layer and a tumor layer. Furthermore, fluorescent immunostaining revealed that the tissue using the VEGF-forced expression strain showed more capillary formation between the tumor layer and the main blood vessels than the tissue using the mock strain. Furthermore, cancer cell proliferation and invasion into the capillaries were also observed. These results indicate that differences in the angiogenic ability of cancer cells, such as the amount of VEGF expression, are reflected in the morphology of the three-dimensional tumor tissue of the present invention, suggesting that the tissue can be used to evaluate anti-VEGF drugs used in cancer treatment.

[0138] Example 7: Evaluation of the efficacy of T cell engagers using three-dimensional tumor tissues On the third day of perfusion culture of the three-dimensional tumor tissue constructed in 1-3 above, EGM2, the medium that had been circulating for the three-dimensional tumor tissue culture, was replaced with 1 x 10 T cells. ６ The cells were suspended at a concentration of 100 ng / mL and then replaced with an equal volume mixture of T cell medium and EGM2 supplemented with an EGFR-CD3 bispecific antibody (hereinafter simply referred to as "EGFR-CD3 antibody") at a final concentration of 100 ng / mL. Perfusion was then continued. On day 5 of perfusion culture, the tissue was fixed in the same manner as in 1-4 above, paraffin-embedded sections were prepared, and fluorescent immunostaining was performed. For fluorescent immunostaining, human anti-CK18 antibody was used as the primary antibody to stain the cancer cells (DiFi). Nuclei were also stained with Hoechst. The results are shown in Figures 18 and 19.

[0139] As shown in Figures 18 and 19, the density of CK18-positive cells (DiFi) was reduced under the EGFR-CD3 antibody addition condition compared to the non-addition condition. This result is presumably due to the EGFR-CD3 antibody promoting the binding of DiFi to T cells that had infiltrated into the tissue from the perfusate, causing the T cells to kill DiFi. This suggests that the efficacy of bispecific antibodies can be evaluated using the three-dimensional tumor tissue of the present invention.

[0140] Example 8. Investigation of Conditions for Constructing Single-Layer 3D Tumor Tissue (2) (8-1. Fabrication of Perfusion Device) The main body 11a of the perfusion device shown in Figures 20-21 was fabricated using a 3D printer (ProJet MJP 3600 MAX (3D Systems)) with an acrylic photocurable resin (product name: VisiJet M3 Crystal, 3D Systems). The top cover 81 shown in Figures 20-21 was fabricated using a 3D printer (Form3, Formlabs) with an acrylic photocurable resin (product name: Clear Resin, Formlabs). A mold was fabricated, and silicone (PDMS) was poured into the mold and cured to fabricate a sealing member 91 (packing). 20-21 was produced, comprising the perfusion device main body 11a, the upper cover 81, and the sealing member 91. The device was then treated in the same manner as in 1-2 above and stored at 4°C for up to one week.

[0141] (8-2. Construction, Culture, and Histological Analysis of Monolayer-Type Three-Dimensional Tumor Tissue) Three-dimensional tumor tissue was constructed using the perfusion device main body 11a prepared in 8-1 above, and construction and culture of the three-dimensional tumor tissue was carried out in the same manner as in Example 5, except that the perfusion device main body 11a was fitted with a top cover 81 and a sealing member 91 to seal the tissue culture space (chamber). Furthermore, the tissue was fixed in the same manner as in Example 5, and paraffin-embedded sections were prepared and subjected to fluorescent immunostaining. The results are shown in FIG. 23 . Furthermore, as shown in FIG. 22 , when the top cover 81 and sealing member 91 were removed from the perfusion device main body 11a before tissue fixation and the three-dimensional tumor tissue was observed, the appearance of the three-dimensional tumor tissue did not differ significantly from that of the three-dimensional tumor tissue constructed using the perfusion device 1.

[0142] As shown in Figure 23, even when the perfusion device 1a prepared in 8-1 above was used, the formation of a main vessel-like structure (Figure 23(B)) and the presence of T cells near the capillaries (Figures 23(C) and (D)) were observed. Furthermore, it was shown that sealing the culture space for the three-dimensional tumor tissue makes it possible to evaluate the effect of perfusion and the target tissue on the tissue while maintaining, for example, the structure of the main vessel-like structure or the three-dimensional tumor tissue itself more stable. One factor behind this is thought to be that sealing the culture space can suppress tissue deformation due to internal pressure generated by perfusion into the main vessel-like structure.

[0143] Example 9. Investigation of conditions for constructing a monolayer-type three-dimensional tumor tissue (3) Three-dimensional tumor tissue was constructed and cultured in the same manner as in Example 5, except that a perfusion device main body 11a was fabricated and used as the perfusion device, and fibrin glue was injected into the culture space (chamber) for the three-dimensional tumor tissue in the perfusion device before perfusion culture to form a fibrin gel layer that covered the three-dimensional tumor tissue.

[0144] The fibrin gel layer was formed as follows. Bolheal® (KM Biologics) was used as the fibrin glue. First, 30 μL of solution A, which contains fibrinogen as its main component, was injected into the chamber of the perfusion device (surrounding the tissue) and stirred thoroughly to blend with the tissue. Next, 30 μL of solution B, which contains thrombin as its main component, was injected into the chamber of the perfusion device (surrounding the tissue), mixed thoroughly with the injected solution A, and then heated to form a fibrin gel layer covering the tissue. At this time, the concentration of fibrinogen, a precursor of fibrin, in the solution immediately after mixing of solutions B and A was 80 mg / mL.

[0145] The tissue was then fixed in the same manner as in Example 5, paraffin-embedded sections were prepared, and fluorescent immunostaining was performed. The tissue was fixed in the same manner as in 1-4 above, paraffin-embedded sections were prepared, and HE staining was performed. The results are shown in Figures 25 and 26. As shown in Figure 24, when the three-dimensional tumor tissue was observed before fixation, the appearance of the three-dimensional tumor tissue was not significantly different from the single-layer three-dimensional tumor tissue constructed in other Examples, except for the formation of a fibrin gel layer.

[0146] As shown in Figure 25, injection of fibrin glue around the 3D tumor tissue resulted in the formation of a fibrin gel coating layer around the 3D tumor tissue. Furthermore, as shown in Figure 26, even when a fibrin gel coating layer was formed around the 3D tumor tissue, the formation of a main blood vessel-like structure (Figure 26(B)) and the presence of T cells near the capillaries (Figures 26(C) and (D)) were observed. Furthermore, as in Example 8, it was demonstrated that the effects of perfusion and the tissue on the subject of evaluation could be evaluated while maintaining the main blood vessel-like structure or the structure of the 3D tumor tissue itself more stably. This is thought to be because the fibrin gel coating layer played a similar role to the top cover 81 and sealing member 91 used in Example 8.

[0147] Furthermore, the three-dimensional tumor tissue constructed in Example 9 was perfusion cultured by adding EGFR-CD3 antibody and T cells to the medium using the same procedure as in Example 7. As a result, T cell infiltration was observed as shown in Figure 27, and a cancer cell density was observed that was similar to that observed under the EGFR-CD3 antibody addition condition as shown in Figure 18. This demonstrates that even when tissue is covered with a fibrin gel layer, it is possible to use the tissue to perform an evaluation test of T cell engagers.

[0148] DESCRIPTION OF SYMBOLS 1, 1a...perfusion device, 11, 11a...perfusion device main body, 12, 18...tubular connector, 13...chamber, 14...anchor structure, 15...bottom lid, 16...groove, 17...rod-shaped member, 19...protrusion, 21...extracellular matrix solution, 22...first vascular system cells, 23...cancer cells, 24...vascular layer, 25...tumor layer, 26...extracellular matrix gel, 31...through-flow channel, 32...inner wall, 41...culture medium containing vascular system cell culture medium, 51...second vascular system cells, 52...main vessel-like structure, 61...three-dimensional tumor tissue model, 62...tumor vessel-like structure, 71...perfusion direction, 121, 181...through-hole, 81...upper lid, 91...sealing member, 811...lid, 812...legs, 813...claws, 814...main body recess, 911...recess.

Claims

1. A three-dimensional tumor tissue model having a main vessel-like structure and a tumor vessel-like structure connected to the main vessel-like structure, and including vascular cells, cancer cells, extracellular matrix, and T cells.

2. a vascular layer and a tumor layer in contact with the vascular layer, the vascular layer has a main vessel-like structure and a tumor vessel-like structure connected to the main vessel-like structure, and comprises vasculature cells, extracellular matrix, and T cells; The three-dimensional tumor tissue model of claim 1 , wherein the tumor layer comprises cancer cells, extracellular matrix, and T cells.

3. A three-dimensional tumor tissue model as described in claim 1, having a fibrin gel layer as the outermost layer.

4. A method for producing the three-dimensional tumor tissue model according to claim 1, forming an extracellular matrix gel containing first vascular cells and cancer cells, the extracellular matrix gel having a through-flow channel for forming the main vessel-like structure; forming the main vessel-like structure by seeding second vascular system cells on the inner wall of the through-channel; Perfusing a culture medium into the main vessel-like structure to form the tumor vessel-like structure; After the step of forming the tumor vasculature-like structures, perfusing T cells into the main vasculature-like structures. A manufacturing method comprising:

5. A method for producing a three-dimensional tumor tissue model according to claim 2, comprising: forming a vascular layer having a through-flow channel for forming the main vessel-like structure and made of an extracellular matrix gel containing first vascular system cells; forming a tumor layer made of an extracellular matrix gel containing cancer cells so as to contact at least a portion of the surface of the blood vessel layer opposite to the through-channel side; forming the main vessel-like structure by seeding second vascular system cells on the inner wall of the through-channel; Perfusing a culture medium into the main vessel-like structure to form the tumor vessel-like structure; After the step of forming the tumor vasculature-like structures, perfusing T cells into the main vasculature-like structures. A manufacturing method comprising:

6. The method according to claim 4 or 5, wherein the medium contains 50% by mass or more of a medium for vascular cells based on the total amount of the medium.

7. The method according to claim 4 or 5, wherein the perfusion flow rate is 0.5 mL / hour or more and 20 mL / hour or less.

8. The manufacturing method described in claim 4, further comprising a step of forming a fibrin gel layer as the outermost layer after the step of forming the extracellular matrix gel.

9. The manufacturing method described in claim 5, further comprising a step of forming a fibrin gel layer as the outermost layer after the step of forming the tumor layer.

10. A manufacturing method described in claim 4 or 5, wherein the step of perfusing the T cells is performed while shaking.

11. A method for evaluating the effect of an object to be evaluated on tumor tissue, comprising the steps of perfusing a solution containing the object to be evaluated through a three-dimensional tumor tissue model described in any one of claims 1 to 3, and evaluating the effect of the object to be evaluated on the three-dimensional tumor tissue model.

12. The evaluation method according to claim 11 , wherein the subject to be evaluated comprises a lymphocyte.

13. The evaluation method according to claim 12 , wherein the lymphocytes are T cells.

14. An evaluation method as described in claim 11, wherein the step of perfusing a solution containing the object to be evaluated is carried out while shaking.

15. The step of perfusing a solution containing an object to be evaluated is carried out using a perfusion device, The evaluation method according to claim 11 , wherein the perfusion device comprises a main body provided with a chamber and a connector communicating with the main vessel-like structure, and an upper cover.