Brain–vascular–brain tumor organoid assembly and method for preparing same
Patent Information
- Application Number
- PCT/KR2025/019439
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-25
- Filing Date
- 2025-11-21
- Publication Date
- 2026-10-01
Smart Images
Figure KR2025019439_01102026_PF_FP_ABST
Abstract
Description
Brain-vascular-brain tumor organoid assembly and method for manufacturing the same
[0001] [Cross-reference to related applications]
[0002] This application claims priority to Korean Patent Application No. 10-2025-0038119 filed on March 25, 2025, the entire contents of which are incorporated by reference into this application.
[0003] The present disclosure relates to a brain-vascular-brain tumor organoid assembly and a method for manufacturing the same.
[0004]
[0005] Gliomas (GBMs) are the most malignant and have the worst prognosis among brain tumors. Despite current standard treatments—maximum surgical resection, radiation therapy, and temozolomide (TMZ) therapy—GBMs are still classified as a fatal disease. One factor hindering drug development for GBM is the lack of appropriate models that represent the complexity of the patient tumor.
[0006] In addition, while recently reported glioblastoma organoids (GBOs) show similarities to patient tissue, the lack of blood vessels makes it difficult to evaluate drug efficacy, absorption, and distribution in a realistic biological environment.
[0007] Meanwhile, in order to manufacture organoid assemblies, optimal organoid culture conditions must be identified by considering the developmental stage and maturity of each organoid. Accordingly, the inventors arrived at the present invention while searching for optimal culture conditions for glioma organoids, cerebral organoids, and vascular organoids derived from patient brain tumor tissue, as well as culture conditions for organoid fusions.
[0008]
[0009] In one aspect, the present invention aims to provide a method for manufacturing a brain-vascular-brain tumor organoid assembly, comprising the steps of: preparing and culturing a brain organoid, a vascular organoid, and a brain tumor organoid from brain tumor tissue isolated from a patient, respectively; co-culturing the brain organoid and the brain tumor organoid in a first step; and co-culturing the vascular organoid in a brain-brain tumor organoid assembly obtained from the first step.
[0010] In another aspect, the present invention aims to provide a brain-vascular-brain tumor organoid assembly manufactured by the above-described method for manufacturing a brain-vascular-brain tumor organoid assembly.
[0011] In another aspect, the present invention aims to provide a composition for screening substances for preventing or treating brain tumors, comprising the organoid assembly.
[0012]
[0013] In one aspect, the present invention provides a method for manufacturing a brain-vascular-brain tumor organoid assembly, comprising the steps of: preparing and culturing a brain organoid, a vascular organoid, and a brain tumor organoid from brain tumor tissue isolated from a patient, respectively; co-culturing the brain organoid and the brain tumor organoid in a first step; and co-culturing the vascular organoid in a brain-brain tumor organoid assembly obtained from the first step.
[0014] In an exemplary embodiment, the brain organoid and blood vessel organoid may be differentiated from human induced pluripotent stem cells.
[0015] In an exemplary embodiment, the brain organoid may be a cerebral organoid.
[0016] In an exemplary embodiment, the brain tumor tissue may be glioma tissue.
[0017] In an exemplary embodiment, the step of manufacturing and culturing the brain organoid may be performed for 30 to 65 days.
[0018] In an exemplary embodiment, the differentiation medium in the step of preparing and culturing the brain organoid may include at least one selected from the group consisting of heparin, B-27, and N2.
[0019] In an exemplary embodiment, the step of preparing and culturing the vascular organoid may be performed for 25 to 60 days.
[0020] In an exemplary embodiment, the differentiation medium in the step of preparing and culturing the vascular organoid may include at least one selected from the group consisting of Y-27635, CHIR 99021, VEGF-A, FGF-2, SB43152, and FBS.
[0021] In an exemplary embodiment, the step of preparing and culturing the brain tumor organoid may be performed for 5 to 60 days.
[0022] In an exemplary embodiment, the differentiation medium for the step of preparing and culturing the brain tumor organoid may include at least one selected from the group consisting of B-27 and N2.
[0023] In an exemplary embodiment, the first co-culturing step may be performed for 20 to 40 days.
[0024] In an exemplary embodiment, the second co-culturing step may be performed for 20 to 40 days.
[0025] In another aspect, the present invention provides a brain-vascular-brain tumor organoid assembly manufactured by the above-described method for manufacturing a brain-vascular-brain tumor organoid assembly.
[0026] In another aspect, the present invention provides a composition for screening substances for preventing or treating brain tumors, comprising the organoid assembly.
[0027]
[0028] The present invention provides a method for manufacturing a brain-vascular brain tumor organoid assembly, in particular, a cerebral-vascular glioma organoid assembly.
[0029] The assembly of the present invention can provide a vascular microenvironment by including a vascular organoid. Furthermore, by providing a vascular microenvironment, oxygen and nutrients can be efficiently distributed to the assembly, and it can directly participate in the growth, development, and emergence of brain neurons. Additionally, since it can reproduce the environment in which actual drugs are distributed through blood vessels, it can more closely mimic the complex brain tumor environment of actual humans.
[0030] Since the assembly of the present invention includes patient-derived brain tumor tissue, the phenotype of the tissue specific to each patient is preserved, making it usable as a patient-specific brain tumor model.
[0031] The assembly of the present invention can be widely applied in the fields of research on brain tumor-related disease mechanisms, drug development, and screening.
[0032]
[0033] Figure 1 is data showing the fabrication process and differentiation status of a vascular organoid fabricated according to an embodiment of the present invention. Figure 1a is a schematic diagram of the vascular organoid fabrication process, and Figure 1b shows an optical image according to the differentiation date of the vascular organoid. Figure 1c is a fluorescence image of the fabricated vascular organoid using immunohistochemistry. Well-differentiated blood vessels were confirmed through the identification of CD31, a vascular differentiation marker.
[0034] Figure 2 shows data illustrating the fabrication process and differentiation status of a cerebral organoid fabricated according to an embodiment of the present invention. Figure 2a is a schematic diagram of the fabrication of the cerebral organoid. Figure 2b shows an optical image according to the differentiation date of the cerebral organoid. Figure 2c is a fluorescence image of the fabricated cerebral organoid using an immunohistochemistry method. A well-differentiated cerebrum was confirmed through the identification of cerebral differentiation markers Tuj1 and SOX2.
[0035] Figure 3 shows data regarding the fabrication and verification of the cerebral-vascular-GBO assembly. Figure 3a shows a schematic diagram of the fabrication process of the cerebral-vascular-GBO assembly, and Figure 3b shows optical images according to the differentiation date of the cerebral-vascular-GBO assembly. Figure 3c is a fluorescence image of the fabricated cerebral-vascular-GBO assembly using immunohistochemistry. It was confirmed that the organois are bound and interact with each other through the identification of the cerebral marker Tuj1 and the vascular marker CD31. Figure 3d is a fluorescence image of the fabricated cerebral-vascular-GBO assembly using immunohistochemistry. It was confirmed that the organois are bound and interact with each other through the identification of the cerebral marker Tuj1 and the brain tumor marker EGFR.
[0036] FIGS. 4a to 4f are schematic diagrams (top of each figure) of cases in which assemblies were prepared by varying the individual culture periods and the fusion timing conditions of cerebral organoids, vascular organoids, and glioma organoids according to an embodiment of the present invention, and photographs of the assemblies formed accordingly (bottom of each figure). Experimental results confirmed that the method of FIG. 4f is the most optimized method for preparing a cerebral-vascular-GBO assembly.
[0037] Figure 5 is a photograph showing an attempt to fuse a VO (Vessel organoid) and a GBM according to one embodiment of the present invention, confirming that when only the vascular organoid and patient-derived glioma tissue are fused and differentiated, the organoid assembly tends to unravel.
[0038] Figure 6 is a photograph showing the case where the fusion of the assembly was attempted during the early stages of cerebral organoid culture according to one embodiment of the present invention (an attempt to fuse the cerebral organoid, vascular organoid, and patient-derived glioma tissue on day 12 of culture), confirming that the brain-vascular-GBO assembly was not formed well and tended to unravel.
[0039] Figure 7 is a photograph showing the case in which an attempt was made to fuse an assembly in a long-term cultured cerebral organoid according to one embodiment of the present invention (an attempt to fuse a cerebral organoid, a vascular organoid, and a patient-derived glioma tissue at the 4th week of culture), in which the brain-vascular-GBO assembly appears to be fused within the Metrigel, but since each form is individually identified, it is confirmed that it is not completely fused.
[0040] FIG. 8 is a photograph (top) and a cross-sectional image (bottom) confirming that the area of the assembly decreases over time when Example 1 of a brain-vascular-GBO assembly according to one embodiment of the present invention is treated with a TMZ drug for 1 to 7 days.
[0041] FIGS. 9a to 9c are photographs (Fig. 9a), cross-sectional images (Fig. 9b), and volume analysis graphs (Fig. 9c) confirming that the area of an assembly decreases over time when two examples of a brain-vascular-GBO assembly according to one embodiment of the present invention are treated with a TMZ drug for 1 to 7 days.
[0042]
[0043]
[0044] The present invention will be described in detail below.
[0045]
[0046] In one aspect, the present invention may provide a method for manufacturing a brain-vascular-brain tumor organoid assembly, comprising the steps of: manufacturing and culturing a brain organoid, a vascular organoid, and a brain tumor organoid from brain tumor tissue isolated from a patient, respectively; co-culturing the brain organoid and the brain tumor organoid in a first step; and co-culturing the vascular organoid in a brain-brain tumor organoid assembly obtained from the first step.
[0047] The inventors have prepared a cerebral-vascular-GBO organoid (CVG-O) assembly, which is a fusion of a glioma organoid (GBO) from a patient's brain tumor tissue and cerebral and vascular organoids derived from human induced pluripotent stem cells (hiPSCs), and have confirmed that said assembly can very closely mimic the actual human brain tumor environment. Specifically, the method for producing said CVG-O is as follows: hiPSCs are injected into a 96-well plate, and the spheroids formed therefrom are induced to differentiate into cerebral and vascular organoids, respectively. A GBO obtained from the patient's tumor tissue is fused with the formed cerebral organoid using Metrigel, and the spheroids are differentiated for about 30 days to produce a cerebral-GBO assembly called CG-O. Next, the produced CG-O and the vascular organoid are fused with Metrigel and co-cultured for 30 days to produce a CVG-O. Consequently, CVG-O produced by the method of the present invention can be used to detect and treat brain tumors. More specifically, it enables personalized treatment for brain tumor diseases, allows for the presentation of a brain tumor model that is more similar to the in vivo environment compared to conventional in vitro brain tumor models, and can be utilized as a platform for studying brain tumor pathogenesis mechanisms and screening drug candidates.
[0048] Gliomas (GBMs) are brain tumors with the highest malignancy and worst prognosis. Despite current standard treatments—maximum surgical resection, radiation therapy, and temozolomide (TMZ) therapy—gliomas are still classified as fatal diseases. One aspect hindering drug development for GBM is the lack of appropriate models that represent the complexity of patient tumors. To construct human-like brain tumor models, three-dimensional structures containing cell types highly similar to in vivo organs or tissues are essential. Furthermore, the method of the present invention can provide an effective complementary model that reflects the biological characteristics and in vitro functions of human tumors more directly and accurately than traditional experimental models.
[0049] 2D culture of human brain tumor cell lines and cancer stem cells cannot reproduce the 3D tumor environment in vivo. Although tumor spherical models generated from brain tumor cell lines mimic 3D structures, they lack organ-like histology and interactions between tumors and normal tissues. Therefore, it was confirmed that the glioma-CO (CG-O) co-culture model can help preserve patient-specific tissue phenotypes through the three-dimensional (3D) cell culture of the present invention and the method of fusing cerebral organoids (CO) derived from human induced pluripotent stem cells (hiPSCs) with human brain tumor tissue.
[0050] Conventional glioblastoma organoids (GBOs) exhibit a very high degree of similarity to patient tissue, but they have the disadvantage of lacking blood vessels, making it difficult to evaluate the efficacy, absorption, and distribution of drugs in a biological environment similar to that of the actual body. Blood vessels not only supply nutrients to the brain but also play a crucial role in the development, regeneration, and migration of brain neurons by providing a vascular microenvironment. Brain tumor models with formed blood vessels are expected to provide more precise models of brain development and disease, and to aid in the development of therapeutic agents. The assembly manufactured by the method of the present invention combines a cerebral organoid and a vascular organoid to provide a model that includes the 3D structure and vascular network of the actual human brain, thereby accurately reproducing an environment where drugs are distributed through blood vessels and oxygen and nutrients are effectively supplied.
[0051] The term organoid refers to a three-dimensional (3D) multicellular, stem cell-derived microtissue designed to closely mimic the complex structure and functionality of human organs, such as the lungs, liver, or brain.
[0052] The above co-culture refers to a method of culturing two or more different types of organoids together in a single culture environment, meaning that the organoids are cultured to form an organoid assembly by inducing physical bonding and functional interaction between the tissues. The above co-culture method may adopt and use any known method for fusing two or more types of organoids without limitation from the perspective of a person skilled in the art. For example, co-culture may be performed by embedding the brain organoid and the brain tumor organoid, or the brain-brain tumor organoid assembly and the vascular organoid, in the same medium, but is not limited thereto. Additionally, for example, the medium used in the second co-culture step may be a mixture of cerebral differentiation medium and vascular differentiation medium in ratios of 1-20:1, 3-17:1, 5-15:1, 7-12:1, or 8-10:1, but is not limited thereto.
[0053] In an exemplary embodiment, the brain organoid and blood vessel organoid may be differentiated from human induced pluripotent stem cells.
[0054] In an exemplary embodiment, the brain organoid may be a cerebral organoid.
[0055] In an exemplary embodiment, the brain tumor tissue may be glioma tissue.
[0056] In an exemplary embodiment, the step of preparing and culturing the brain organoid may be performed for 30 to 65 days. For example, the step of preparing and culturing the brain organoid may be performed for 30 days or more, 35 days or more, 40 days or more, 45 days or more, 50 days or more, 52 days or more, 54 days or more, 56 days or more, 58 days or more, or 60 days or more, and may be performed for 65 days or less, 64.5 days or less, 64 days or less, 63.5 days or less, 63 days or less, 62.5 days or less, 62 days or less, 61.5 days or less, 61 days or less, 60.5 days or less, or 60 days or less, but is not limited thereto.
[0057] In an exemplary embodiment, the differentiation medium in the step of preparing and culturing the brain organoid may include at least one selected from the group consisting of heparin, B-27, and N2. Additionally, the differentiation medium may include, without limitation, any medium capable of differentiating a brain organoid including the cerebrum from the perspective of a person skilled in the art, such as, Stemfit04 medium, iMatrix-511 coated tissue culture plate, or DMEM / F-12:Nerobasal medium, but is not limited thereto.
[0058] In an exemplary embodiment, the step of preparing and culturing the vascular organoid may be performed for 25 to 60 days. For example, the step of preparing and culturing the vascular organoid may be performed for 25 days or more, 25.5 days or more, 26 days or more, 26.5 days or more, 27 days or more, 27.5 days or more, 28 days or more, 28.5 days or more, 29 days or more, 29.5 days or more, or 30 days or more, and may be performed for 60 days or less, 55 days or less, 50 days or less, 45 days or less, 40 days or less, 38 days or less, 36 days or less, 34 days or less, 32 days or less, or 30 days or less, but is not limited thereto.
[0059] In an exemplary embodiment, the differentiation medium in the step of preparing and culturing the vascular organoid may include at least one selected from the group consisting of Y-27635, CHIR 99021, VEGF-A, FGF-2, SB43152, and FBS. Additionally, the differentiation medium may include, without limitation, any medium capable of differentiating the vascular organoid from the perspective of a person skilled in the art, such as Stemfit04 medium or iMatrix-511 coated tissue culture plates, but is not limited thereto.
[0060] In an exemplary embodiment, the step of preparing and culturing the brain tumor organoid may be performed for 5 to 60 days. For example, the step of preparing and culturing the brain tumor organoid may be performed for 5 days or more, 5.25 days or more, 5.5 days or more, 5.75 days or more, 6 days or more, 6.25 days or more, 6.5 days or more, 6.75 days or more, or 7 days or more, and may be performed for 60 days or less, 50 days or less, 40 days or less, 45 days or less, 30 days or less, 35 days or less, 20 days or less, 15 days or less, 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, or 8 days or less, but is not limited thereto.
[0061] In an exemplary embodiment, the differentiation medium in the step of preparing and culturing the brain tumor organoid may include at least one selected from the group consisting of B-27 and N2. Additionally, the differentiation medium may include, without limitation, any medium capable of differentiating the brain tumor organoid from the perspective of a person skilled in the art, such as DMEM / F-12 medium, but is not limited thereto.
[0062] In an exemplary embodiment, the first co-culturing step may be performed for 20 to 40 days. For example, the first co-culturing step may be performed for 20 days or more, 21 days or more, 22 days or more, 23 days or more, 24 days or more, 25 days or more, 26 days or more, 27 days or more, 28 days or more, or 29 days or more, and may be performed for 40 days or less, 39 days or less, 38 days or less, 37 days or less, 36 days or less, 35 days or less, 34 days or less, 33 days or less, 32 days or less, or 31 days or less, but is not limited thereto.
[0063] In an exemplary embodiment, the second co-culturing step may be performed for 20 to 40 days. For example, the second co-culturing step may be performed for 20 days or more, 21 days or more, 22 days or more, 23 days or more, 24 days or more, 25 days or more, 26 days or more, 27 days or more, 28 days or more, or 29 days or more, and may be performed for 40 days or less, 39 days or less, 38 days or less, 37 days or less, 36 days or less, 35 days or less, 34 days or less, 33 days or less, 32 days or less, or 31 days or less, but is not limited thereto.
[0064] In another aspect, the present invention may provide a brain-vascular-brain tumor organoid assembly manufactured by the method for manufacturing the brain-vascular-brain tumor organoid assembly described above. The brain-vascular-brain tumor organoid assembly is as described above.
[0065] In another aspect, the present invention may provide a composition for screening substances for preventing or treating brain tumors, comprising the organoid assembly. The organoid assembly is as described above.
[0066]
[0067] The present invention will be described in more detail below through examples and the like. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.
[0068]
[0069] Experimental Example
[0070]
[0071] 1. Preparation and Characterization of Brain-Blood-GBO Assemblies
[0072] 1) Production of vascular organoids and verification of differentiated vascular organoids
[0073] Vascular organoids mimicking human-like blood vessels were constructed using human induced pluripotent stem cells (hiPSCs). First, hiPSCs (Stem Cell Bank, CMC3) were grown on iMatrix-511 coated tissue culture plates in Stemfit04 medium. Spheroids were formed from hiPSCs (1.0 × per well) 4Dog cells were mixed with Stemfit04 medium and seeded into a 96-well plate. 48 hours after cell seeding, the culture medium was replaced with angiogenesis medium containing 50 μM Y-27635. After 2 days of culture, the medium was replaced with angiogenesis medium containing 12 μM CHIR 99021, and subsequently, cultured for 6 days in angiogenesis medium containing 30 ng / mL VEGF-A and 30 ng / mL FGF-2. On day 11 of angiogenesis, the cells were cultured for 2 days in differentiation medium containing 30 ng / mL VEGF-A, 30 ng / mL FGF-2, and 10 μM SB43152. On day 13 of differentiation, spheroids were embedded using Matrigel, replaced with differentiation medium containing 100 ng / mL VEGF-A, 100 ng / mL FGF-2, and 15% FBS, and cultured for 15 days (Fig. 1b). To confirm vascular organoid differentiation, immunohistochemistry was performed using the differentiation marker CD31, and it was confirmed that the vascular gene CD31 was well expressed and the organoids were vascularized (Fig. 1c).
[0074]
[0075] 1) Fabrication of cerebral organoids and verification of differentiated cerebral organoids
[0076] Cerebral organoids mimicking a human cerebrum were constructed using human induced pluripotent stem cells (hiPSCs). First, hiPSCs (Stem Cell Bank, CMC3) were grown on iMatrix-511 coated tissue culture plates in Stemfit04 medium. Spheroids were formed from hiPSCs (1.0 × per well) 4Patient-derived brain tumor tissue was formed by mixing the cells with Stemfit04 medium and seeding the mixture into a 96-well plate. Twenty-four hours after cell seeding, the cells were cultured in Stemfit04 medium for five days. On day six of differentiation, the medium was replaced with differentiation medium containing 1 ng / ml heparin and cultured for five days. Ten days after differentiation, spheroids were embedded in Matrigel, and the medium was replaced with DMEM / F-12:Nerobasal (1:1) differentiation medium containing 1% B-27 (excluding vitamin A) and 0.5% N2. By culturing the tissue, an environment can be provided for brain tumor cells to grow. Cultured patient-derived brain tumor tissue was embedded in Matrigel to induce cell growth in a 3D environment. Four days after embedding, the cerebral organoids were cultured for 1 to 2 months in DMEM / F-12:Nerobasal (1:1) differentiation medium containing 1% B-27 (containing vitamin A) and 0.5% N2 for maturation (Fig. 2b). To confirm cerebral organoid differentiation, immunohistochemistry was performed using differentiation markers tuj1 and SOX2, and it was confirmed that gene expression occurred well (Fig. 2c).
[0077]
[0078] 2) Fabrication and verification of the cerebral-GBM-vascular assembly (assembleoid)
[0079] Brain tumor tissue collected from male patients (aged 60 and 61) diagnosed with glioma at Seoul National University Bundang Hospital was cut into very small pieces and cultured for 7 days. The medium used was a DMEM / F-12 (1:1) mixed medium containing 1% B-27 (excluding vitamin A) and 0.5% N2. The cultured patient-derived brain tumor tissue (GBM tumoroid) was embedded in Matrigel along with cerebral organoids to induce cell interaction and growth in a 3D environment. Subsequently, the tissue was cultured in a cerebral differentiation medium containing 1% B-27 (including vitamin A) and 0.5% N2. Cerebral-GBM organoids cultured for one month were embedded in Matrigel along with vascular organoids. This creates an environment where brain tumor cells and vascular cells can interact. Then, the two organoids were cultured together using cerebral differentiation medium and vascular differentiation medium (a 9:1 mixing ratio). This mixed medium provided nutrients that helped cerebral and vascular cells differentiate and grow appropriately, respectively. The organoids cultured in this way were cultured for an additional 30 days, during which time the two organoids were allowed to interact with each other and grow in a natural environment. Immunostaining was performed to confirm whether the cerebral, brain tumor tissue, and vascular organoids were well combined to form an assembly. The experimental results confirmed the expression of the cerebral marker (Tuj1), vascular marker (CD31), and brain tumor marker (EGFR), proving that they were well expressed. It was also visually confirmed that the cerebral-GBM-vascular assembly (Example 1) differentiated normally into neurons and that brain tumor cells were well expressed (Figs. 3c and 3d).
[0080]
[0081] 2. Design of Optimal Conditions for Manufacturing Brain-Blood-GBO Assemblies
[0082] In order to design optimal conditions for manufacturing a brain-vascular-GBO assembly, the inventors conducted the following experiments by varying the fusion timing of cerebral organoids, vascular organoids, and glioma organoids.
[0083] In addition, other than the fusion timing, other conditions (including the culture medium conditions during the preparation and differentiation of each organoid) were conducted under the same conditions as those in Item 1 of the above experimental examples.
[0084]
[0085] 2-1) Preparation of the assembly of Fig. 4a (Comparative Example 1) and analysis of its characteristics
[0086] At the 12th day of differentiation (d12) of each BO and VO, fusion was attempted by embedding them in Matrigel, and subsequently, at the 7th day (d19) of the BO and VO fusion attempt, a GBM tumoroid that had been differentiated for more than 7 days separately was additionally embedded in Matrigel and fused for more than 7 days.
[0087] The characteristics of the manufactured assembly are as follows.
[0088] It was confirmed that cysts formed within the organoids during the fusion process. The formation of cysts impairs the normal structure and function of the organoids and can reduce the reliability of experiments. In particular, reproducibility is critical in drug trials and oncology research; however, the presence of cysts increases the likelihood of distorted data interpretation, rendering the organoids unusable or limited for experimental purposes. Cyst formation can primarily occur due to cell necrosis, abnormal angiogenesis, influences from the tumor microenvironment, or issues with the culture environment.
[0089] Therefore, the above assembly was classified as Comparative Example 1.
[0090]
[0091] 2-1) Preparation of the assembly of Fig. 4b (Comparative Example 2) and analysis of its characteristics
[0092] On the 12th day of BO differentiation (BO_d12), fusion of BO and a GBM tumoroid differentiated for more than 7 days was attempted by embedding with Matrigel. Subsequently, on the 7th day of the BO and GBM tumoroid fusion attempt (BO_d19), the above-mentioned VO differentiated for more than one month was additionally fused and cultured for more than 7 days (BO_d26).
[0093] The characteristics of the manufactured assembly are as follows.
[0094] It was confirmed that cysts formed within the organoids during the fusion process. The formation of cysts impairs the normal structure and function of the organoids and can reduce the reliability of experiments. In particular, reproducibility is critical in drug trials and oncology research; however, the presence of cysts increases the likelihood of distorted data interpretation, rendering the organoids unusable or limited for experimental purposes. Cyst formation can primarily occur due to cell necrosis, abnormal angiogenesis, influences from the tumor microenvironment, or issues with the culture environment.
[0095] Therefore, the above assembly was classified as Comparative Example 2.
[0096]
[0097] 2-2) Preparation of the assembly of Fig. 4c (Comparative Example 3) and analysis of its characteristics
[0098] BO and VO were differentiated separately at the same time. On the 12th day (d12) of differentiation of the above BO and VO, BO, VO, and GBM tumoriods differentiated for more than 7 days were simultaneously embedded in Matrigel to attempt differentiation, and were additionally cultured for 14 days (d26).
[0099] The characteristics of the manufactured assembly are as follows.
[0100] It was confirmed that cysts were formed within the organoid during the fusion process. The formation of cysts impairs the normal structure and function of the organoid and can reduce the reliability of the experiment.
[0101] Therefore, the above assembly was classified as Comparative Example 3.
[0102]
[0103] 2-3) Preparation of the assembly of Fig. 4d (Comparative Example 4) and analysis of its characteristics
[0104] On day 30 of BO differentiation (BO_d30), fusion of BO and a GBM tumoroid differentiated for more than 7 days was attempted by embedding with Matrigel. Subsequently, on day 15 of the attempt to fuse BO and GBM tumoroid (BO_d45), the above-mentioned VO differentiated for more than one month was additionally fused and cultured for 15 days (BO_d60).
[0105] The characteristics of the manufactured assembly are as follows.
[0106] We confirmed the formation of cysts within the organoids during the fusion process and observed a tendency for them to unravel. The formation of cysts impairs the normal structure and function of the organoids and can reduce the reliability of the experiment. Furthermore, organoid unraveling may occur due to factors such as decreased intercellular cohesion, changes in the extracellular matrix (ECM), increased cellular stress, and imbalances in the culture environment.
[0107] Therefore, the above assembly was classified as Comparative Example 4.
[0108]
[0109] 2-4) Preparation of the assembly of Fig. 4e (Comparative Example 5) and analysis of its characteristics
[0110] On day 30 of VO differentiation (VO_d30), fusion of VO and a GBM tumoroid differentiated for more than 7 days was attempted by embedding with Matrigel. Subsequently, on day 15 of the attempt to fuse VO and GBM tumoroid (VO_d45), an additional BO differentiated separately for more than one month was fused and cultured for more than 15 days (VO_d60).
[0111] The characteristics of the manufactured assembly are as follows.
[0112] We confirmed the formation of cysts within the organoids during the fusion process and observed a tendency for them to unravel. The formation of cysts impairs the normal structure and function of the organoids and can reduce the reliability of the experiment. Furthermore, organoid unraveling may occur due to factors such as decreased intercellular cohesion, changes in the extracellular matrix (ECM), increased cellular stress, and imbalances in the culture environment.
[0113] Therefore, the above assembly was classified as Comparative Example 5.
[0114]
[0115] 2-5) Preparation of the assembly of FIG. 4f (Example 1) and analysis of its characteristics
[0116] On day 60 of BO differentiation (d60), fusion of BO and a GBM tumoroid differentiated for more than 7 days was attempted by embedding with Matrigel. Subsequently, on day 30 of the fusion attempt between BO and the GBM tumoroid (fusion attempt d30, BO_d90), the above-mentioned VO differentiated for more than one month was additionally fused and cultured for 30 days (fusion attempt d60, BO_d120).
[0117] The characteristics of the manufactured assembly are as follows.
[0118] Microscopic images confirmed that the organoid assemblies naturally combine and that the organoid structure maintains a smooth form without disintegrating.
[0119] Therefore, the above assembly was selected as an example.
[0120]
[0121] 2-1) Preparation of the assembly of FIG. 4f (Example 1) and analysis of its characteristics
[0122] On day 60 of BO differentiation (d60), fusion of BO and a GBM tumoroid differentiated for more than 7 days was attempted by embedding with Matrigel. Subsequently, on day 30 of the fusion attempt between BO and the GBM tumoroid (fusion attempt d30, BO_d90), the above-mentioned VO differentiated for more than one month was additionally fused and cultured for 30 days (fusion attempt d60, BO_d120).
[0123] The characteristics of the manufactured assembly are as follows.
[0124] Microscopic images confirmed that the organoid assemblies naturally combine and that the organoid structure maintains a smooth form without disintegrating.
[0125] Therefore, the above assembly was selected as an example.
[0126]
[0127] 3. Experiment to confirm fusion of brain-blood vessel-GBO assemblies according to organoid culture conditions
[0128] 3-1) Attempt to Fusion Vascular Organoid and Glioma Tissue
[0129] Fusion was attempted by embedding early differentiated (d13) vascular organoids and glioma tissues differentiated for more than 7 days in Matrigel. Fusion conditions were maintained in an incubator at 37°C and 5% CO₂, using cerebral differentiation medium and vascular differentiation medium (9:1 mixing ratio). Static suspension culture was performed on low-adhesion culture plates.
[0130] Other conditions are the same as those for vascular organoids and gliomas in Section 1 above.
[0131] Experimental results confirmed that when a vascular organoid and a glioma are directly fused during differentiation, the organoid tends to unravel (Fig. 5).
[0132]
[0133] 3-2) Attempt to Fusion Short-Term Cultured Cerebral Organoids
[0134] Fusion was attempted with cerebral organoids and vascular organoids differentiated for 12 days, and glioma tissue differentiated for 7 days or more, by embedding them in Matrigel. Fusion conditions were maintained in an incubator at 37°C and 5% CO₂, and cerebral differentiation medium and vascular differentiation medium (9:1 mixing ratio) were used. Static suspension culture was performed on low-adhesion culture plates.
[0135] Other conditions are the same as those for cerebral organoids, vascular organoids, and gliomas in Section 1 above.
[0136] Experimental results confirmed that the organoid assembly was not formed well and the organoid tended to unravel (Fig. 6).
[0137]
[0138] 3-3) Attempt to Fusion Long-Term Cultured Cerebral Organoids
[0139] Fusion was attempted by embedding cerebral organoids differentiated for more than 4 months with vascular organoids differentiated for more than 30 days and glioma tissue differentiated for more than 7 days using Matrigel. Fusion conditions were maintained in an incubator at 37°C and 5% CO₂, and cerebral differentiation medium and vascular differentiation medium (9:1 mixing ratio) were used. Static suspension culture was performed on low-adhesion culture plates.
[0140] Other conditions are the same as those for cerebral organoids, vascular organoids, and gliomas in Section 1 above.
[0141] Experimental results confirmed that the organoid assembly was not formed well and the organoid tended to unravel (Fig. 7).
[0142]
[0143] 4. Experiment to confirm the response of the brain-vascular-GBO assembly to TMZ drug treatment
[0144] The potential for utilizing the brain-vascular-GBO assembly prepared by the method of the present invention as an in vitro brain tumor model was evaluated by treating it with temozolomide (TMZ; Sigma-Aldrich), a glioma treatment drug. The experimental method is as follows.
[0145] First, the brain-vascular-GBO assembly obtained in Section 1 above was treated with TMZ drug at a dose of 100 μM every other day for 7 days. As a result of the experiment, it was confirmed that the area of the assembly decreased over time, and analysis with Image J confirmed that the area of the assembly decreased by a total of 10.4% over 7 days (Fig. 8).
[0146] In addition, TMZ drug was administered at a dose of 100 μM every other day for 7 days to four brain-vascular GBO assemblies prepared from brain tumor tissue derived from two male patients (aged 60 and 62) diagnosed with glioma among patients visiting Seoul National University Bundang Hospital (the preparation method was the same as described in Section 1 above; a total of four assemblies were prepared, with two from each patient's glioma tissue). As a result of the experiment, it was confirmed that the surface area of all assemblies decreased over time, and analysis with Image J confirmed that the volume of the brain-vascular GBO assemblies decreased by an average of approximately 9.022% (Figs. 9a to 9c).
[0147]
[0148] [National R&D projects that supported this invention]
[0149] [Project ID] 2460000153
[0150] [Project No.] RS-2022-KH129481
[0151] [Ministry Name] Ministry of Health and Welfare
[0152] [Name of Project Management (Specialized) Agency] Korea Health Industry Development Institute
[0153] [Research Project Name] Research-Oriented Hospital Development R&D Project / Bio-industry Technology Development
[0154] [Project Title] Establishment of a Multi-Omics and Organoid-Based Gene Therapy Efficacy Evaluation System
[0155] [Name of Project Performing Organization] Bundang Seoul National University Hospital
[0156] [Research Period] July 1, 2022 ~ December 31, 2030
[0157]
[0158] [National R&D projects that supported this invention]
[0159] [Project ID] 2410003544
[0160] [Assignment No.] 20018578
[0161] [Ministry Name] Ministry of Trade, Industry and Energy
[0162] [Project Management (Specialized) Agency Name] Korea Institute of Industrial Technology Planning and Evaluation
[0163] [Research Project Name] Bioindustry Technology Development - Customized Diagnostic and Therapeutic Products
[0164] [Research Project Title] Development of Analytical Tools for Quality Verification and Characterization of Organoid Regenerative Therapeutics and Production Standardization
[0165] [Name of Project Performing Organization] Bundang Seoul National University Hospital
[0166] [Research Period] July 1, 2022 ~ December 31, 2025
[0167]
[0168] [National R&D projects that supported this invention]
[0169] [Project Unique ID] Not Assigned
[0170] [Project No.] RS-2025-00519649
[0171] [Ministry Name] Ministry of Science and ICT
[0172] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea
[0173] [Research Project Name] Individual Basic Research - Mid-career Research Type 1
[0174] [Project Title] Novel Nanobiohybrid Organoids for Anticancer Treatment of Intractable Malignant Brain Tumors
[0175] [Name of Project Performing Organization] Bundang Seoul National University Hospital
[0176] [Research Period] 2025.03.01 ~ 2028.02.28
Claims
1. A step of preparing and culturing brain organoids, vascular organoids, and brain tumor organoids from brain tumor tissue isolated from a patient, respectively; A first co-culture step of the brain organoid and the brain tumor organoid; and A method for manufacturing a brain-vascular-brain tumor organoid assembly, comprising the step of co-culturing the blood vessel organoid in a second co-culture of the brain-brain tumor organoid assembly obtained from the first co-culture.
2. In Paragraph 1, The above brain organoid and blood vessel organoid are differentiated from human induced pluripotent stem cells.
3. In Paragraph 1, The above brain organoid is a cerebral organoid, method.
4. In Paragraph 1, The above brain tumor tissue is a method in which the tissue of a glioma is a method.
5. In Paragraph 1, A method in which the step of preparing and culturing the brain organoid is performed for 30 to 65 days.
6. In Paragraph 1, A method in which the differentiation medium for the step of preparing and culturing the brain organoid comprises at least one selected from the group consisting of heparin, B-27, and N2.
7. In Paragraph 1, A method in which the step of preparing and culturing the above-mentioned vascular organoid is performed for 25 to 60 days.
8. In Paragraph 1, A method in which the differentiation medium for the step of preparing and culturing the above-mentioned vascular organoid comprises at least one selected from the group consisting of Y-27635, CHIR 99021, VEGF-A, FGF-2, SB43152, and FBS.
9. In Paragraph 1, A method in which the step of preparing and culturing the brain tumor organoid is performed for 5 to 60 days.
10. In Paragraph 1, A method in which the differentiation medium for the step of preparing and culturing the brain tumor organoid comprises at least one selected from the group consisting of B-27 and N2.
11. In Paragraph 1, A method in which the first co-culture step is performed for 20 to 40 days.
12. In Paragraph 1, A method in which the second co-culturing step is performed for 20 to 40 days.
13. A brain-vascular-brain tumor organoid assembly manufactured by the method of any one of claims 1 to 12.
14. A composition for screening substances for the prevention or treatment of brain tumors comprising the organoid assembly of claim 13.