WO2024091060A1 - Procédé de production d'un complexe organoïde cérebral de type cancer cérébral par co-culture d'un organoïde cérébral issu de cellules souches pluripotentes induites d'un patient atteint d'un cancer cérébral et d'un organoïde cérébral de patient atteint d'un cancer cérébral - Google Patents

Procédé de production d'un complexe organoïde cérebral de type cancer cérébral par co-culture d'un organoïde cérébral issu de cellules souches pluripotentes induites d'un patient atteint d'un cancer cérébral et d'un organoïde cérébral de patient atteint d'un cancer cérébral Download PDF

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WO2024091060A1
WO2024091060A1 PCT/KR2023/016862 KR2023016862W WO2024091060A1 WO 2024091060 A1 WO2024091060 A1 WO 2024091060A1 KR 2023016862 W KR2023016862 W KR 2023016862W WO 2024091060 A1 WO2024091060 A1 WO 2024091060A1
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brain
brain cancer
organoid
producing
composition
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양승호
이정은
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가톨릭대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

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  • the present invention relates to a method of producing brain cancer organoids from induced pluripotent stem cells derived from brain cancer patients.
  • cells derived from organs or tissues are artificially immortalized or two-dimensionally cultured to verify the efficacy and toxicity of candidate substances.
  • cells that are artificially cultured in two dimensions are separated from tissues and undergo changes in original cell shape and loss of cell function during the culture process, or the connection with the extracellular environment (ECM, microenvironment) is severed, causing phenomena that occur in actual living organisms. There is a fatal flaw that cannot be reflected.
  • ECM extracellular environment
  • the action and mechanism of the drug are often not the same as in humans due to differences between species, which hinders the development of efficient drug development and requires a lot of time and cost, so drug screening is difficult. and there is great difficulty in predicting its reactivity.
  • stem cell organoid production technology has recently attracted attention as a method of producing patient-derived similar organs and using them for disease modeling, pathology research, drug screening, toxicity evaluation, and genetic manipulation.
  • 3D organoid organ-like organ
  • various organoids such as brain organoid, heart organoid, liver organoid, lung organoid, and small intestine organoid, have been produced. Results are being reported showing that it can be done.
  • Organoids contain several specific cell populations that make up an organ or tissue, have a form and structural organization similar to an actual tissue or organ, and can reproduce the special functions of each organ. Organoids are formed through a series of common processes. Cells with the same function are grouped together and placed in an appropriate location. After the cell compartments are separated, more detailed differentiation occurs. This is called lineage specification, and is the differentiation of precursors into complete adult cells so that they can perform actual functions.
  • organoids represents the creation of a 3D environment close to the inside of a living body, allowing experiments to be conducted “outside the body” as if drugs were acting “inside the body,” making it possible to replicate the effects seen in actual human organs. It has the advantage of enabling new drug screening, drug toxicity testing, etc.
  • One object of the present invention is to provide a method for producing brain cancer organoids from induced pluripotent stem cells derived from brain cancer patients, comprising the following steps:
  • iPSC induced pluripotent stem cells
  • Another object of the present invention includes a first composition, a second composition, and a third composition, wherein the first composition is a medium composition for producing the induced pluripotent stem cells, and the second composition is a medium composition for producing the brain organoid. and the third composition is the medium composition for co-culture, and the first composition, the second composition, and the third composition are each used sequentially to provide a composition for producing brain cancer organoids.
  • Another object of the present invention is to provide a kit for preparing brain cancer organoids containing the composition and instructions describing the method.
  • the present invention provides a method for producing brain cancer organoids from induced pluripotent stem cells derived from brain cancer patients, comprising the following steps:
  • iPSC induced pluripotent stem cells
  • the brain organoid may be a cerebral organoid, but is not limited thereto.
  • the brain organoids may include, but are not limited to, the ventricular zone, external subventricular zone, intermediate zone, and cortical plate.
  • the brain organoid may express one or more of the ventricular zone, external subventricular zone, intermediate zone, and cortical plate markers corresponding to (a) to (d) below, respectively. , but is not limited to:
  • the brain organoid is composed of Nestin (neuroepithelial stem cell protein), GFAP (Glial fibrillary acidic protein), Tuj1 (Neuron-specific class III beta-tubulin), and NeuN (neuronal nuclear protein). It may express any one or more selected from the group consisting of, but is not limited to this.
  • the present invention provides a brain cancer therapeutic screening method comprising the following steps: producing brain cancer organoids by the above method; and confirming the treatment effect after treating the brain cancer organoid with a brain cancer treatment candidate material.
  • the method may further include the step of selecting a brain cancer treatment candidate when a treatment effect appears after treatment with the brain cancer treatment candidate, but is not limited thereto.
  • the brain cancer organoid may express Ki67 or Iba1, but is not limited thereto.
  • the brain cancer is glioblastoma, astrocytoma, ependymoma, oligodendroglioma, mixed glioma, brain stem glioma, Optic nerve glioma, pituitary adenoma, craniopharyngioma, medulloblastoma, primitive neuroectodermal tumors, pineal tumors, meningioma, schwannoma It may be one or more selected from the group consisting of (schwannoma), metastatic brain tumors, CNS lymphoma, neurofibromatosis, pseudotumor cerebri, and tuberous sclerosis. , but is not limited to this.
  • the present invention includes a first composition, a second composition, and a third composition, wherein the first composition is a medium composition for producing the induced pluripotent stem cells, and the second composition is a medium composition for producing the brain organoid,
  • the third composition is the medium composition for co-cultivation, and the first composition, the second composition, and the third composition are each used sequentially.
  • a composition for producing brain cancer organoids is provided.
  • the present invention provides a kit for preparing brain cancer organoids including instructions describing the method and the composition.
  • the present invention provides the use of organoids prepared according to the above method for screening brain cancer therapeutic agents.
  • the present invention provides a use of the composition or the kit for producing brain cancer organoids.
  • brain organoids with excellent manufacturing efficiency can be produced using patient-derived induced pluripotent stem cells and cancer cells. Since the manufacturing method of the present invention can produce patient-specific brain organoids, it is possible to not only screen effective drugs for patients, but also confirm responsiveness to various drugs, making it useful as an organoid for screening brain-related cancer treatments. It can be utilized.
  • Figure 1 shows the process for producing induced pluripotent stem cells from peripheral blood mononuclear cells derived from brain cancer patients.
  • Figure 2 shows the process for producing cerebral organoids from induced pluripotent stem cells derived from brain cancer patients.
  • Figure 3 is a diagram showing a method of producing a brain cancer organoid model by co-culturing organoids and cancer cells derived from a brain cancer patient.
  • Figure 4 is a photograph showing the results in which cancer cells proliferate and cerebral organoids are lost after a co-culture period when using 1 ⁇ 10 3 or more cancer cells.
  • Figure 5 is a photograph showing a brain cancer organoid and an orthotopic mouse model produced by the method for producing a brain cancer model derived from a brain cancer patient according to the present invention.
  • Figure 6 is a photograph showing a glioblastoma brain cancer organoid produced by the method for producing a brain cancer model derived from a brain cancer patient according to the present invention.
  • Figure 7a is a photograph showing the results of ki67 and Iba1 immunofluorescence staining when glioblastoma organoids and brain organoids produced by the method according to the present invention were co-cultured
  • Figure 7b is an enlarged photograph.
  • the present invention provides a method for producing brain cancer organoids from induced pluripotent stem cells derived from brain cancer patients, comprising the following steps:
  • iPSC induced pluripotent stem cells
  • induced pluripotent stem cells are also referred to as pluripotent stem cells, and are cells that return somatic cells or differentiated cells to the cell stage before differentiation through a specific method, and are pluripotent like embryonic stem cells. These are derived cells.
  • methods for inducing iPCS include, but are not limited to, methods such as genetic transformation by injecting cell differentiation-related genes or culturing under specific conditions.
  • Induced pluripotent stem cells can be produced from any somatic cell or any cell that has completed differentiation, and in the present invention, they were produced from peripheral blood mononuclear cells, but are not limited thereto.
  • organoids are organ-specific cell aggregates made by three-dimensionally cultivating, aggregating, or recombining stem cells, and are capable of self-renewal and are also called “mini-organs” or “pseudo-organs.” . Organoids are similar to actual organs and can carry out studies in vitro that are difficult to implement in animal models, such as regulating molecular signals, so they are very useful in basic research, as well as in human developmental processes, establishment of disease models, screening for drug effectiveness evaluation, etc. It can be very useful in various fields such as development of drug toxicity evaluation platform and cell therapy development, but is not limited to this.
  • brain cancer organoid is manufactured by co-culturing the brain organoid prepared from induced pluripotent stem cells derived from PBMC of a brain cancer patient according to the method of the present invention and the cancer cells collected and isolated from the brain cancer patient. It may mean an organoid that has been developed.
  • brain cancer organoid of the present invention may also be referred to as an “avatar model,” but is not limited thereto.
  • the brain organoid may be a cerebral organoid, but is not limited thereto.
  • the brain organoids may include, but are not limited to, the ventricular zone, external subventricular zone, intermediate zone, and cortical plate.
  • the brain organoid may express one or more of the ventricular zone, external subventricular zone, intermediate zone, and cortical plate markers corresponding to (a) to (d) below, respectively. , but is not limited to:
  • the brain organoid is composed of Nestin (neuroepithelial stem cell protein), GFAP (Glial fibrillary acidic protein), Tuj1 (Neuron-specific class III beta-tubulin), and NeuN (neuronal nuclear protein). It may express any one or more selected from the group consisting of, but is not limited to this.
  • nestin, GFAP, Tuj1, and NeuN may be neural lineage markers, but are not limited thereto.
  • Neural lineage markers are endogenous tags expressed in various nerve-related cells along differentiated cells such as neurogenesis or neurons. Cells expressing markers belonging to these tags can be detected and identified as specific cells according to the characteristics of each marker. . In other words, it may refer to an indicator used to detect and identify which cells exhibit which characteristics through various technologies, but is not limited thereto.
  • neural lineage markers are various types of neural lineage markers, such as neural stem cells, neural progenitor cells, neurons, glial cells, Neuroglia, Glia “Glue”, astrocytes, oligodendrocytes, microglia, and ependymal cells. It may include all markers for identifying cells, but is not limited thereto.
  • Neural lineage markers can be DNA, mRNA or RNA expressed in the cell of interest, as well as protein tags as partial proteins, proteins or epitopes that distinguish between different cell types or different states of a common cell. , but is not limited to this.
  • the ideal marker will depend on a given cell type during steady state and/or injury; cellular markers can identify disease and are very useful tools for examining cell function even under normal conditions.
  • the discovery of various proteins specific to specific cells can lead to the production of cell type-specific antibodies used to identify cells, which can be utilized in various fields, but is not limited to this.
  • Neural progenitor cells are distinguished from neural stem cells by their inability to continuously self-renew and their ability to generally generate only one type of differentiated progeny.
  • neural progenitor cells are tripotent cells that can generate neurons, astrocytes, and oligodendrocytes.
  • oligodendrocyte progenitor cells can generate oligodendrocytes until their mitotic ability is exhausted. It is not limited to this.
  • neural progenitor markers can track cells as they undergo expansion and differentiation into neurons in rosettes, where neural rosettes are radial arrays of columnar cells that express many of the proteins expressed in the neuroepithelial cells of the neural tube, the differentiating embryo. It may signal the development of neural progenitor cells in stem cell culture. Cells within the rosette may express several cellular markers, including but not limited to Nestin, NCAM, and Musashi-1, RNA binding proteins expressed in proliferating neural stem cells.
  • Nestin is a protein encoded by the NES gene, which may refer to neuroepithelial stem cell protein, and is expressed in large quantities in neural progenitor cells or neural stem cells. It may be used as a marker, but is not limited thereto.
  • nestin is a type VI intermediate filament (IF) protein, and these intermediate filament proteins can be expressed primarily in neurons involved in the radial growth of axons.
  • IF intermediate filament
  • brain organoids prepared by the method of the present invention abundantly express nestin, which is achieved by producing a large amount of intact neural progenitor cells using stem cells derived from brain cancer patients. This may mean that it is possible to produce fully functional brain organoids.
  • GFAP Global fibrillary acidic protein
  • CNS central nervous system
  • IF intermediate filament
  • GFAP is an intermediate microfilament protein of astrocytes, and the expression of GFAP is used as a marker for the regulation of astrogliosis.
  • the expression of GFAP is known to be regulated by various stages of post-traumatic signals and neural activity, but is not limited to this.
  • GFAP is expressed in brain organoids produced by the method according to an embodiment of the present invention, and based on this, neural progenitor cells are fully produced in large quantities using stem cells derived from brain cancer patients, and thus, functional It can be judged that the production of this complete brain organoid is possible.
  • Neuronal markers can detect neurons at various stages of development in the nucleus, cytoplasm, membrane, or perisynaptic products present in neurons, and are also possible to specifically label cholinergic, dopaminergic, serotonergic, GABAergic, or glutamatergic neurons.
  • Pan neuron markers have multiple targets (somatic, nuclear, dendritic, spine and axonal proteins) and consequently all parts of the neuron can be labeled, with specific markers not only marking specific regions of the neuron but also morphing the neuron. It can also be used for research.
  • Tuj1 Neuron-specific Class III ⁇ -tubulin
  • Tuj1 Neuron-specific Class III ⁇ -tubulin
  • neuron nuclear antigen or Fox-3 is a nuclear protein present in cells after mitosis, and may be expressed at the point of differentiation into mature cells, but is not limited thereto. Since it is expressed in almost all neuronal cell types except Purkinje cells of the substantia nigra, olfactory mitral cells, retinal photoreceptors, and dopaminergic neurons, it can be used as a marker to detect it, but is not limited to this.
  • brain organoids were derived from induced pluripotent stem cells derived from brain cancer patients. Brain organoids containing various types of nerve-related cells, particularly cerebral organoids, may be produced, but are not limited thereto.
  • the number of cancer cells may be from 1 to 1 ⁇ 10 3 per brain organoid, but is not limited thereto.
  • brain cancer cells or cancer organoids obtained through surgical treatment in a patient derived from the brain organoid are co-cultured. You can.
  • co-culture may be performed using 1 ⁇ 10 3 brain cancer cells or cancer organoids, and when culturing more than 1 ⁇ 10 3 cancer cells, brain cancer organoids may tend to disappear.
  • the brain cancer organoid may express Ki67 or Iba1, but is not limited thereto.
  • the present invention provides a brain cancer therapeutic screening method comprising the following steps: producing brain cancer organoids by the above method; and confirming the treatment effect after treating the brain cancer organoid with a brain cancer treatment candidate material.
  • the method may further include the step of selecting a brain cancer treatment candidate when a treatment effect appears after treatment with the brain cancer treatment candidate, but is not limited thereto.
  • screening may mean selecting a substance with a specific target property from a candidate group consisting of several substances using a specific manipulation or evaluation method.
  • the screening method of the present invention is to process candidate substances in the brain cancer organoid of the present invention in order to identify a brain cancer treatment agent that produces the best treatment effect for brain cancer patients, and the treatment response and effect of the brain cancer organoid therefor. It may refer to a series of processes including the step of determining the brain cancer treatment substance that produced the best treatment effect as a brain cancer treatment after confirming, but is not limited to this.
  • the step of confirming the treatment response and effect may be repeated several times depending on the therapeutic candidate, and additional substances or steps may be added to confirm the treatment response and effect, a step used in the art as a general screening method. may additionally include, but is not limited thereto.
  • the “confirmation” may correspond to the meaning of “analysis,” but is not limited thereto.
  • analysis may preferably mean “measurement”, the qualitative analysis may mean measuring and confirming the presence of the desired substance to confirm the treatment response, and the quantitative analysis may mean measuring and confirming the presence or absence of the desired substance to confirm the treatment response. may mean measuring and confirming changes in the presence level (expression level) or amount of the target substance.
  • analysis or measurement can be performed without limitation, including both qualitative and quantitative methods, and quantitative measurement may be performed.
  • the brain cancer treatment candidate includes all formulations and methods that can be used for the treatment of various brain cancers.
  • it may be an anticancer agent applied to the treatment of brain cancer, but is not limited thereto.
  • the anticancer agent is a general term for chemotherapy agents used to treat malignant tumors.
  • Most anticancer drugs are drugs that mainly inhibit nucleic acid synthesis or exhibit anticancer activity by interfering with various metabolic pathways of cancer cells.
  • Anticancer drugs currently used for cancer treatment are classified into six categories according to their biochemical mechanisms of action.
  • Alkylating agents are highly reactive substances that have the ability to introduce an alkyl group R-CH 2 into certain compounds. When applied to cells, most of them react with the N7 of guanine in DNA to modify the DNA structure. It causes chain cleavage and exhibits anti-cancer and cytotoxic effects. Drugs belonging to this group include: 1 Nitrogen mustard: Nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, etc.
  • Ethyleneimine Thiotepa 3 Alkyl sulfonate: Busulfan 4 Triazine, Hydrazine series: DTIC (dacarbazine), procarbazine 5 Nitrozourea series: BCNU, CCNU, methyl-CCNU, etc.
  • Antimetabolites These have the effect of inhibiting the metabolic process necessary for the proliferation of cancer cells.
  • 1 Folic acid derivative methotrexate (MTX) 2
  • Purine derivative 6-mercaptopurine (6-MP), 6 -Thioguanine 3
  • Pyrimidine derivatives 5-fluorouracil, cytarabine, etc.
  • Antibiotics include adriamycin, daunorubicin, bleomycin, mitomycin-C, and actinomycin-D.
  • Mitotic inhibitor (vinca alkaloid): It is a division-specific drug that stops cell division at metaphase during mitosis. These include vincristine, vinblastine, VP-16-213, and VM-26.
  • Hormones Some types of cancer can be treated by administering hormones. Male hormones are effective in breast cancer, female hormones are effective in prostate cancer, progesterone is effective in endometrial cancer, and adrenocortical hormones are effective in treating cancer. is used to treat acute lymphoblastic leukemia and lymphoma, and tamoxifen, an anti-female hormone, is used for breast cancer.
  • Anticancer agents as therapeutic substances of the present invention may include hormonal anticancer agents that can be applied to brain cancer.
  • Cisplatin L-asparaginase, o,p-DDD, etc.
  • Chemotherapeutic approaches can primarily be used to treat metastatic or particularly aggressive cancers.
  • Treatment candidates subject to the screening method according to the present invention may include not only specific drug agents such as the above-mentioned anticancer drugs, but also anticancer treatments such as radiation therapy, therapy using electricity, electromagnetic waves, etc., or chemotherapy.
  • the present invention provides a brain cancer treatment method in the screening method, further comprising administering the therapeutic agent to an individual in need thereof.
  • the brain cancer is glioblastoma, astrocytoma, ependymoma, oligodendroglioma, mixed glioma, brain stem glioma, Optic nerve glioma, pituitary adenoma, craniopharyngioma, medulloblastoma, primitive neuroectodermal tumors, pineal tumors, meningioma, schwannoma It may be one or more selected from the group consisting of (schwannoma), metastatic brain tumors, CNS lymphoma, neurofibromatosis, pseudotumor cerebri, and tuberous sclerosis. , but is not limited to this.
  • cancer refers to cells characterized by uncontrolled growth. Due to this abnormal cell growth, a cell mass called a tumor is formed, which infiltrates surrounding tissues and, in severe cases, may metastasize to other organs of the body. It says what to do.
  • brain cancer refers to primary brain cancer that occurs in brain tissue and the meninges surrounding the brain and secondary brain cancer that metastasizes from cancer that originates in the skull or other parts of the body, and occurs regardless of age, gender, or race. It means everything that happens.
  • the present invention includes a first composition, a second composition, and a third composition, wherein the first composition is a medium composition for producing the induced pluripotent stem cells, and the second composition is a medium composition for producing the brain organoid,
  • the third composition is the medium composition for co-cultivation, and the first composition, the second composition, and the third composition are each used sequentially.
  • a composition for producing brain cancer organoids is provided.
  • the medium for producing induced pluripotent stem cells is a medium for producing induced pluripotent stem cells from PBMC, and may include a composition for producing induced pluripotent stem cells from PBMC. Since the PBMCs are isolated from brain cancer patients, in addition to the components generally used to produce induced pluripotent stem cells from PBMCs, additional components for producing induced pluripotent stem cells may be included depending on the characteristics of the PBMCs of brain cancer patients. It is not limited to this.
  • the medium for producing brain organoids is a medium for producing (or producing) brain organoids from induced pluripotent stem cells, and may include a composition for producing brain organoids from induced pluripotent stem cells.
  • the composition may further contain any ingredients taking into account the type and characteristics of brain cancer, characteristics of brain cancer patients, etc. in order to improve the production efficiency of brain organoids from induced pluripotent stem cells derived from brain cancer patients, but is not limited thereto.
  • brain organoids were prepared using the TEMdiffTM Cerebral Organoid Kit according to a method described in a literature that discloses an organoid differentiation protocol using iPSC, but is not limited thereto.
  • the medium for the co-cultivation step may be a medium used in the step of producing a brain cancer organoid by co-culturing the brain organoid prepared according to the method of the present invention and cancer cells isolated from a brain cancer patient.
  • the medium for co-culture may contain any composition for co-culturing brain organoids prepared according to the method of the present invention and cancer cells isolated from brain cancer patients.
  • the composition may additionally contain suitable ingredients in comprehensive consideration of the type of brain cancer, the time of cancer progression, the type of treatment or treatment administered, the application period, and the brain cancer patient's response thereto, but is not limited thereto.
  • a medium in which the medium for brain organoid production and DMEM medium are mixed at a weight ratio of 1:1 was used as a co-culture medium for producing glioblastoma organoids, but is not limited thereto.
  • co-culture may mean that different cells are cultured together at a certain time
  • “co-culture” in the “one period” means when different cells are cultured simultaneously
  • “co-cultured together for some time or period of time” including cases where cells are sequentially added and cultured according to the generally applied time difference depending on the characteristics of the cells, and when specific cells are cultured first and then culture (liquid) of the cells
  • all cases affected by previously introduced cell culture may be included, but are not limited to this.
  • the present invention provides a kit for preparing brain cancer organoids including instructions describing the method and the composition.
  • the “kit” includes a composition for a medium for producing induced pluripotent stem cells from PBMC of a brain cancer patient, a composition for a medium for producing a brain organoid of a brain cancer patient from the induced pluripotent stem cells, and a brain organoid. It may mean, but is limited to, an agent that allows the production of brain cancer organoids derived from a brain cancer patient, including a composition for a medium used for co-culturing cancer cells derived from a brain cancer patient, and a tool including a manual describing the manufacturing method thereof. It doesn't work.
  • kit of the present invention may include other components, compositions, solutions, devices, etc. commonly required for the method of producing brain cancer organoids, and there is no limitation on the order and subsequent application of the above materials. Application of each material may occur simultaneously or at a microscopic level.
  • the kit may further include a container, etc., but is not limited thereto.
  • the container may serve to package the material, and may also serve to store and secure the material.
  • the material of the container may be, for example, plastic, glass bottle, etc., but is not limited thereto.
  • Example 1 Production of induced pluripotent stem cells derived from brain cancer patients
  • PBMC Peripheral blood mononuclear cells
  • PBMC medium Peripheral blood mononuclear cells
  • 5 ⁇ 10 5 PBMC cells were incubated with 8 MOI of CytoTune-iPS Sendai Reprogramming (Thermo Fisher scientific cat no. A16517) was used to induce pluripotent stem cells (iPS cell/iPSC, hereinafter referred to as iPSC).
  • iPSC pluripotent stem cells
  • the induced iPSCs were transferred to vitronectin-coated medium, and the medium was replaced with iPSC medium E8 media or mTesR every day from the next day ( Figure 1).
  • Example 2 Production of cerebral organoids derived from brain cancer patients using induced pluripotent stem cells derived from brain cancer patients
  • a brain cancer patient's cerebral organoid was produced from the brain cancer patient-derived iPSC produced in Example 1 (FIG. 2).
  • the STEMdiffTM Cerebral Organoid Kit (cat no. #08570) was used based on the literature (Lancaster MA et al. Nature, 2013 and Lancaster MA et al. Science, 2014) in which the organoid differentiation protocol using iPSC was disclosed (Lancaster MA et al. Nature, 2013 and Lancaster MA et al. Science, 2014). Cerebral organoids from brain cancer patients were cultured and produced.
  • induced pluripotent stem cell colonies were separated into small cell clusters using ReLeSRTM (Stem Cell Technology) and then separated into single cells using Accutase (ThermoFisher). To form embryoid bodies, 9,000 to 10,000 single cells were dispensed into each well of a U-bottom low-attachment 96-well plate (Corning).
  • STEMdiff Cerebral Organoid Kit replace with neural induction media, differentiate for 2 to 3 days, and place the embryoid bodies one by one in matrigel on parafilm. They were planted and fixed in an incubator at 37°C and 5% CO 2 for 20 minutes. The hardened Matrigel mass was placed in cerebral differentiation media and neuroepithelium was continuously induced for about 3 days. Then, on the fourth day, the organoids were transferred to a low-attachment 6-well plate (Corning) containing 3 ml of mature medium and incubated on an orbital shaker (88 rpm; ThermoFisher) until the day of analysis. Organoids were cultured. The medium was changed every 3 days. Brain organoids were prepared by growing for a total of 40 days.
  • organoids generated from iPSCs derived from brain cancer patient blood began with the embryoid body (EB) formation stage, with the neuroepithelium expanding, and Nestin (Nestin) representing neural progenitor cells in mature cerebral organoids cultured for a period of more than 40 days.
  • EB embryoid body
  • Nestin Nesin
  • GFAP Merck Millipore; AB5804
  • Tuj1 Biolegend; 801201
  • NeuN ab134014, Abcam
  • ventricular zone PAX6 + /SOX2 + /Ki-67 +
  • external subventricular zone Ki-67 + /p-Vimentin +
  • intermediate zone TBR2 +
  • cortical plate CTIP2 + /MAP2 + /TBR1 +
  • Example 2 it was confirmed that cerebral organoids showing the characteristics of brain cancer patients could be cultured from induced pluripotent stem cells derived from brain cancer patients, and that mature cerebral organoids could be produced when cultured for a period of 40 days or more. .
  • Example 4 Production of brain cancer organoids using cerebral organoids and brain cancer cells derived from brain cancer patients
  • Cancer cells were obtained from around the patient's tissue by chopping the patient's brain tissue isolated during the surgery of a brain cancer patient into small pieces and culturing them in DMEM medium for about 7 days. Patient-derived cancer cells grown after subculture were stored at -80°C.
  • the cerebral organoid prepared in Example 2 and about 1 ⁇ 10 3 brain cancer cells were prepared.
  • Organoid maturation medium and DMEM medium were mixed 1:1 in a 1.5 ml EP tube, then prepared patient-derived organoids and 1 ⁇ 10 3 brain cancer cells were added and spun down for 5 minutes.
  • the cerebral organoid itself tended to disappear as the cancer cells proliferated after the co-culture period (FIG. 4).
  • brain cancer organoids (avatar models) were produced using organoids derived from brain cancer patients by culturing them in an incubator for about 3 days.
  • the brain cancer organoid according to the present invention is not only almost similar to the structural characteristics of brain cells of patients with brain cancer compared to the xenograft mouse tumor model, It was confirmed that the tumor microenvironment within the tumor was well maintained (Figure 5).
  • Example 5 Production of glioblastoma organoids using glioblastoma patient-derived cerebral organoids and glioblastoma cells
  • Example 5-1 Construction of glioblastoma patient-derived glioblastoma organoids
  • glioblastoma organoids were produced using induced pluripotent stem cells and cancer cells derived from patients with glioblastoma, a type of brain cancer.
  • peripheral blood mononuclear cells were isolated from glioblastoma patients using the methods of Examples 1 and 2, and cultured with induced pluripotent stem cells to produce cerebral organoids.
  • cancer tissue was isolated from the surgery of the same glioblastoma patient using the method of Example 4, and it was co-cultured with the cerebral organoid to establish a glioblastoma model (FIG. 6).
  • Example 5-2 Confirmation of excellent reflection of in vivo characteristics of glioblastoma patient-derived glioblastoma organoids
  • Ki67 Green
  • Iba1 Red
  • Iba1 is used as a microglial marker because it is specifically expressed in microglial cells of the central nervous system, which are immune cells residing in the brain.
  • Iba1 is expressed in resting microglial cells but is expressed at a high level in activated microglial cells, allowing the state of the central nervous system to be confirmed with high accuracy.
  • the experiment was confirmed by co-culturing glioblastoma organoids and brain organoids, fixing them with PFA, and then immunofluorescently staining them.
  • the glioblastoma organoid prepared according to the method of the present invention was confirmed to exhibit all in vivo characteristics of the patient's glioblastoma from which the organoid was derived, so when using it, the patient's individual characteristics can be obtained. It is expected that it will be useful as a brain cancer organoid that reflects brain cancer.
  • brain organoids with excellent manufacturing efficiency can be produced using patient-derived induced pluripotent stem cells and cancer cells. Since the manufacturing method of the present invention can produce patient-specific brain organoids, it is possible to not only screen effective drugs for patients, but also confirm responsiveness to various drugs, making it useful as an organoid for screening brain-related cancer treatments. Since it can be utilized, its industrial applicability is recognized.

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Abstract

Selon un procédé de production d'un organoïde de type cancer cérébral à partir de cellules souches pluripotentes induites issues d'un patient atteint d'un cancer cérébral, un organoïde cérébral présentant une efficacité de production élevée peut être produit à l'aide de cellules souches pluripotentes induites et de cellules cancéreuses issues d'un patient. Le procédé de la présente invention permet la production d'un organoïde cérébral spécifique du patient, et il est donc possible non seulement de cribler des médicaments efficaces pour un patient, mais aussi de déterminer la réactivité à divers médicaments, ce qui le rend utile en tant qu'organoïde pour le criblage de traitements anticancéreux liés au cerveau.
PCT/KR2023/016862 2022-10-27 2023-10-27 Procédé de production d'un complexe organoïde cérebral de type cancer cérébral par co-culture d'un organoïde cérébral issu de cellules souches pluripotentes induites d'un patient atteint d'un cancer cérébral et d'un organoïde cérébral de patient atteint d'un cancer cérébral WO2024091060A1 (fr)

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KR10-2022-0140531 2022-10-27
KR20220140531 2022-10-27
KR10-2023-0144118 2023-10-25
KR1020230144118A KR20240059569A (ko) 2022-10-27 2023-10-25 뇌암 환자의 유도만능줄기세포 유래 뇌오가노이드와 환자 뇌암 오가노이드 공배양을 통한 뇌암-뇌오가노이드 복합체를 제작하는 방법

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200055673A (ko) * 2018-11-13 2020-05-21 한국화학연구원 세포 배양용 캐리어를 이용하여 제조된 오가노이드 및 이를 이용한 약물 독성 평가방법
KR20200061235A (ko) * 2018-11-23 2020-06-02 오가노이드사이언스 주식회사 뇌 오가노이드를 기반으로 하는 허혈성 뇌질환 모델의 제조방법 및 이의 용도
KR20210051508A (ko) * 2019-10-30 2021-05-10 가톨릭대학교 산학협력단 3차원 뇌 오가노이드를 이용한 뇌신경계질환 치료용 줄기세포 스크리닝 방법
WO2021138209A1 (fr) * 2020-01-02 2021-07-08 The Regents Of The University Of California Procédés de culture de cellules cancéreuses et d'inhibition de l'invasion du cancer
KR102296446B1 (ko) * 2018-10-17 2021-09-02 코아스템(주) 인간 만능 줄기세포로부터 제작된 3d 오가노이드를 해체하여 세포를 다량 확보하는 분화방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102296446B1 (ko) * 2018-10-17 2021-09-02 코아스템(주) 인간 만능 줄기세포로부터 제작된 3d 오가노이드를 해체하여 세포를 다량 확보하는 분화방법
KR20200055673A (ko) * 2018-11-13 2020-05-21 한국화학연구원 세포 배양용 캐리어를 이용하여 제조된 오가노이드 및 이를 이용한 약물 독성 평가방법
KR20200061235A (ko) * 2018-11-23 2020-06-02 오가노이드사이언스 주식회사 뇌 오가노이드를 기반으로 하는 허혈성 뇌질환 모델의 제조방법 및 이의 용도
KR20210051508A (ko) * 2019-10-30 2021-05-10 가톨릭대학교 산학협력단 3차원 뇌 오가노이드를 이용한 뇌신경계질환 치료용 줄기세포 스크리닝 방법
WO2021138209A1 (fr) * 2020-01-02 2021-07-08 The Regents Of The University Of California Procédés de culture de cellules cancéreuses et d'inhibition de l'invasion du cancer

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