WO2024091060A1 - Method for producing brain cancer-brain organoid complex by co-culturing brain organoid derived from induced pluripotent stem cells of brain cancer patient with patient brain cancer organoid - Google Patents

Method for producing brain cancer-brain organoid complex by co-culturing brain organoid derived from induced pluripotent stem cells of brain cancer patient with patient brain cancer organoid 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
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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

According to a method for producing a brain cancer organoid from induced pluripotent stem cells derived from a brain cancer patient, a brain organoid having high production efficiency can be produced using patient-derived induced pluripotent stem cells and cancer cells. The method of the present invention enables the production of a patient-specific brain organoid, and thus it is possible to not only screen effective drugs for a patient, but also determine responsiveness to various drugs, making it useful as an organoid for screening brain-related cancer treatments.

Description

뇌암 환자의 유도만능줄기세포 유래 뇌오가노이드와 환자 뇌암 오가노이드 공배양을 통한 뇌암-뇌오가노이드 복합체를 제작하는 방법Method for producing a brain cancer-brain organoid complex through co-culturing brain organoids derived from induced pluripotent stem cells of brain cancer patients and brain cancer organoids of patients.
본 발명은 뇌암 환자 유래 유도만능줄기세포로부터 뇌암 오가노이드를 제작하는 방법에 관한 것이다.The present invention relates to a method of producing brain cancer organoids from induced pluripotent stem cells derived from brain cancer patients.
본 출원은 2022년 10월 27일에 출원된 한국특허출원 제10-2022-0140531호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다. This application claims priority based on Korean Patent Application No. 10-2022-0140531 filed on October 27, 2022, and all contents disclosed in the specification and drawings of the application are incorporated in this application.
본 출원은 2023년 10월 25일에 출원된 한국특허출원 제10-2023-0144118호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다. This application claims priority based on Korean Patent Application No. 10-2023-0144118 filed on October 25, 2023, and all contents disclosed in the specification and drawings of the application are incorporated in this application.
현재 실험실에서 사용되는 일차 배양 세포 모델의 경우 장기 또는 조직에서 유래한 세포를 인공적으로 불멸화 (immortalized) 시키거나 2차원적으로 평면 배양하면서 후보물질의 효능 및 독성을 검증하게 된다. 하지만 2차원적에서 인공적으로 배양되는 세포들은 조직에서 분리된 후 배양 과정에서 본래 세포 형태 변화 및 세포 기능 상실이 발생하거나 세포외환경 (ECM, microenvironment)과의 연결이 단절되어 실제 생체에서 나타나는 현상을 반영하지 못하는 치명적인 단점이 있다. 뿐만 아니라 인간 세포가 아닌 경우 종간 차이로 인해 약물의 작용 및 기전이 인간과 동일하지 않은 경우가 많아 이로 인해 효율적인 약물개발의 발전이 저해되고 시간과 비용 또한 많이 소요된다는 등의 여러 문제점으로 인하여 약물 스크리닝 및 이에 대한 반응성 예측에 큰 어려움이 있다. In the case of primary culture cell models currently used in laboratories, cells derived from organs or tissues are artificially immortalized or two-dimensionally cultured to verify the efficacy and toxicity of candidate substances. However, 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. In addition, in the case of non-human cells, 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.
이를 해결하기 위한 수단으로 최근 환자 유래 유사 장기를 제작하여 질환 모델링, 병리 연구, 약물 스크리닝, 독성평가, 유전자조작에 활용하는 방법으로 줄기세포 오가노이드 제작 기술 개발이 관심을 받고 있다. 줄기세포의 다분화능 및 내재적 자기조직화를 이용하여 3D 상태로 분화를 유도하였을 때 신체 장기와 유사한 모양의 구조를 갖는 3D 상태의 오가노이드 (Organoid; 유사장기) 구조체가 형성되는 것이 확인되었고, 이를 바탕으로 현재까지 뇌 오가노이드 (brain organoid), 심장 오가노이드 (heart organoid), 간 오가노이드 (liver organoid), 폐 오가노이드 (lung organoid), 소장 오가노이드 (small intestine organoid) 등 다양한 오가노이드가 제작될 수 있다는 결과가 보고되고 있다. As a means of solving this problem, the development of 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. When differentiation into a 3D state was induced using the multipotency and intrinsic self-organization of stem cells, it was confirmed that a 3D organoid (organ-like organ) structure with a structure similar to that of a body organ was formed. Based on this, To date, 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.
이러한 오가노이드는 기관 또는 조직을 구성하는 여러 특이적 세포 집단들을 포함하고 있고, 실제 조직 또는 기관과 유사한 형태 및 구조적 조직화가 이루어져 있으며, 각 기관이 가지는 특수한 기능을 재현할 수 있다. 오가노이드는 공통적인 일련의 과정에 의해 형성되는데, 기능이 같은 세포들끼리 뭉쳐 적절한 위치로 배치되며, 세포들의 구획이 분리된 후에는 더욱 세부적인 분화가 일어난다. 이를 계통 특성화 (lineage specification)라고 하며, 실제 기능을 수행할 수 있도록 전구체가 완전한 성체 세포로 분화되는 것이다.These 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.
이러한 오가노이드의 개발은 생체 내와 가까운 3D 환경의 조성을 나타내 “몸 밖”의 실험 상황에서도 약물이 마치 “몸 안”에서 작용하듯이 실험할 수 있어 실제 사람의 장기에서 나타나는 효과를 그대로 재현할 수 있다는 장점이 있고, 신약 스크리닝, 약물 독성 테스트 등을 가능하게 한다.The development of these 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.
특히, 신체의 장기 중에서도 태아 단계 및 출생 초기에 발달이 완성되는 뇌의 경우 직접적인 접근이 거의 불가능하여 연구를 하기 위해 현실적으로 많은 제약이 따르는 문제가 있어 약물 스크리닝뿐만 아니라 약물에 대한 반응성을 예측하기 어렵다는 문제가 있다. In particular, among the organs of the body, the brain, whose development is completed during the fetal stage and early after birth, is almost impossible to access directly, so there are many practical limitations for research, making it difficult to predict drug responsiveness as well as drug screening. There is.
이러한 문제를 해결하기 위해 줄기세포를 신경세포가 포함된 뇌의 구성세포로 분화시키는 기술로 발달시키기 위한 연구가 진행 중이나, 환자 유래의 뇌 오가노이드의 제작은 성공률이 현저히 저조하여 효과적인 제작 방법 개발이 필요한 실정이다. To solve this problem, research is in progress to develop a technology to differentiate stem cells into brain constituent cells containing neurons, but the success rate in producing patient-derived brain organoids is significantly low, making it difficult to develop an effective production method. It is necessary.
본 발명의 하나의 목적은 하기의 단계를 포함하는 뇌암 환자 유래 유도만능줄기세포로부터 뇌암 오가노이드를 제작하는 방법을 제공하는 것이다: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:
뇌암 환자 유래 유도만능줄기세포 (iPSC)를 제조하는 단계;Preparing induced pluripotent stem cells (iPSC) derived from a brain cancer patient;
상기 유도만능줄기세포로부터 뇌 오가노이드를 제작하는 단계; 및Creating brain organoids from the induced pluripotent stem cells; and
상기 뇌 오가노이드 및 상기 뇌암 환자에서 분리된 암세포를 공배양하는 단계.Co-culturing the brain organoid and cancer cells isolated from the brain cancer patient.
본 발명의 다른 목적은 제1 조성물, 제2 조성물, 및 제3 조성물을 포함하고, 상기 제1 조성물은 상기 유도만능줄기세포 제조용 배지 조성물이고, 상기 제2 조성물은 상기 뇌 오가노이드 제작용 배지 조성물이고, 상기 제3 조성물은 상기 공배양용 배지 조성물이고, 상기 제1 조성물, 상기 제2 조성물, 및 상기 제3 조성물은 각각 순차적으로 사용되는 것인 뇌암 오가노이드 제작용 조성물을 제공하는 것이다.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.
본 발명의 또 다른 목적은 상기 방법이 기술된 설명서 및 상기 조성물을 포함하는 뇌암 오가노이드 제작용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for preparing brain cancer organoids containing the composition and instructions describing the method.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art to which the present invention belongs from the description below. There will be.
본 발명은 하기의 단계를 포함하는 뇌암 환자 유래 유도만능줄기세포로부터 뇌암 오가노이드를 제작하는 방법을 제공한다: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:
뇌암 환자 유래 유도만능줄기세포 (iPSC)를 제조하는 단계;Preparing induced pluripotent stem cells (iPSC) derived from a brain cancer patient;
상기 유도만능줄기세포로부터 뇌 오가노이드를 제작하는 단계; 및Creating brain organoids from the induced pluripotent stem cells; and
상기 뇌 오가노이드 및 상기 뇌암 환자에서 분리된 암세포를 공배양하는 단계.Co-culturing the brain organoid and cancer cells isolated from the brain cancer patient.
본 발명의 일 실시예에 있어서, 상기 뇌 오가노이드는 대뇌 오가노이드 (cerebral organoid)일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the brain organoid may be a cerebral organoid, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 뇌 오가오이드는 심실대, 외부 뇌실하대, 중간 영역, 및 피질판을 포함할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the brain organoids may include, but are not limited to, the ventricular zone, external subventricular zone, intermediate zone, and cortical plate.
본 발명의 일 실시예에 있어서, 상기 뇌 오가노이드는 하기의 (a) 내지 (d)에 해당하는 심실대, 외부 뇌실하대, 중간 영역, 및 피질판 마커 중 어느 하나 이상을 각각 발현할 수 있으나, 이에 제한되는 것은 아니다:In one embodiment of the present invention, 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:
(a) PAX6+, SOX2+, 및 Ki-67+; (a) PAX6 + , SOX2 + , and Ki-67 + ;
(b) Ki-67+ 또는 p-Vimentin+; (b) Ki-67 + or p-Vimentin + ;
(c) TBR2+; 및 (c) TBR2 + ; and
(d) CTIP2+, MAP2+, 및 TBR1+. (d) CTIP2 + , MAP2 + , and TBR1 + .
본 발명의 일 실시예에 있어서, 상기 뇌 오가노이드는 Nestin (neuroepithelial stem cell protein), GFAP (Glial fibrillary acidic protein), Tuj1 (Neuron-specific class III beta-tubulin), 및 NeuN (neuronal nuclear protein)으로 이루어진 군으로부터 선택되는 어느 하나 이상을 발현할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, 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.
본 발명은 하기의 단계를 포함하는 뇌암 치료제 스크리닝 방법을 제공한다: 상기 방법으로 뇌암 오가노이드를 제작하는 단계; 및 상기 뇌암 오가노이드에 뇌암 치료 후보 물질을 처리한 후 치료 효과를 확인하는 단계.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.
본 발명의 일 실시예에 있어서, 상기 방법은 상기 뇌암 치료 후보 물질 처리 후 치료 효과가 나타나는 경우 뇌암 치료제로 선정하는 단계를 더 포함할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, 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.
본 발명의 일 실시예에 있어서, 상기 뇌암 오가노이드는 Ki67 또는 Iba1를 발현할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the brain cancer organoid may express Ki67 or Iba1, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 뇌암은 교모세포종 (glioblastoma), 성상세포종 (astrocytoma), 상의세포종 (ependymoma), 핍지교종 (oligodendroglioma), 혼합교종 (mixed glioma), 뇌간 교종 (brain stem glioma), 시신경교종 (optic nerve glioma), 뇌하수체 종양 (pituitary adenoma), 두개인두종 (craniopharyngioma), 수모세포종 (medulloblastoma), 원시신경외배엽종양 (primitive neuroectodermal tumors), 송과체 종양 (pineal tumors), 수막종 (meningioma), 신경초종 (schwannoma), 전이성 뇌종양 (metastatic brain tumors), 중추신경 임파종 (CNS lymphoma), 신경섬유종증 (neurofibromatosis), 가성뇌종양 (pseudotumor cerebri), 및 결절성 경화증 (tuberoussclerosis)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, 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.
본 발명은 제1 조성물, 제2 조성물, 및 제3 조성물을 포함하고, 상기 제1 조성물은 상기 유도만능줄기세포 제조용 배지 조성물이고, 상기 제2 조성물은 상기 뇌 오가노이드 제작용 배지 조성물이고, 상기 제3 조성물은 상기 공배양용 배지 조성물이고, 상기 제1 조성물, 상기 제2 조성물, 및 상기 제3 조성물은 각각 순차적으로 사용되는 것인 뇌암 오가노이드 제작용 조성물을 제공한다.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.
본 발명은 상기 방법이 기술된 설명서 및 상기 조성물을 포함하는 뇌암 오가노이드 제작용 키트를 제공한다.The present invention provides a kit for preparing brain cancer organoids including instructions describing the method and the composition.
또한, 본 발명은 상기 방법에 따라 제조된 오가노이드의 뇌암 치료제를 스크리닝하기 위한 용도를 제공한다.Additionally, the present invention provides the use of organoids prepared according to the above method for screening brain cancer therapeutic agents.
또한, 본 발명은 상기 조성물 또는 상기 키트의 뇌암 오가노이드를 제조하기 위한 용도를 제공한다.Additionally, the present invention provides a use of the composition or the kit for producing brain cancer organoids.
뇌암 환자 유래 유도만능줄기세포로부터 뇌암 오가노이드를 제조하는 방법에 따르면, 환자 유래 유도만능줄기세포 및 암세포를 사용하여 제조 효율이 우수한 뇌 오가노이드를 제작할 수 있다. 본 발명의 제조 방법으로 환자 맞춤형 뇌 오가노이드를 제작할 수 있으므로 환자에게 효과적인 약물을 스크리닝할 수 있을 뿐만 아니라, 다양한 약물에 대한 반응성을 확인할 수 있다는 점에서 뇌와 관련된 암 치료제 스크리닝용 오가노이드로 유용하게 활용될 수 있다.According to the method for producing brain cancer organoids from induced pluripotent stem cells derived from brain cancer patients, 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.
도 1은 뇌암 환자 유래 말초혈액단핵세포로부터 유도만능줄기세포를 제조하는 방법 과정을 나타낸 것이다.Figure 1 shows the process for producing induced pluripotent stem cells from peripheral blood mononuclear cells derived from brain cancer patients.
도 2는 뇌암 환자 유래 유도만능줄기세포로부터 대뇌 오가노이드를 제작하는 방법 과정을 나타낸 것이다.Figure 2 shows the process for producing cerebral organoids from induced pluripotent stem cells derived from brain cancer patients.
도 3은 뇌암 환자 유래 오가노이드 및 암세포를 공배양하여 뇌암 오가노이드 모델을 제작하는 방법을 나타낸 그림이다.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.
도 4는 1×103개 이상의 암세포 사용 시 공배양 기간이 지나면 암세포가 증식하여 대뇌 오가노이드가 소실되는 결과를 나타낸 사진이다.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.
도 5는 본 발명에 따른 뇌암 환자 유래 뇌암 모델 제작 방법으로 제조된 뇌암 오가노이드 및 orthotopic 마우스 모델을 나타낸 사진이다.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.
도 6은 본 발명에 따른 뇌암 환자 유래 뇌암 모델 제작 방법으로 제조된 교모세포종 뇌암 오가노이드를 나타낸 사진이다.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.
도 7a는 본 발명에 따른 방법으로 제작된 교모세포종 오가노이드와 뇌 오가노이드를 공배양하였을 때의 ki67 및 Iba1 면역형광염색 결과를 나타낸 사진이고, 도 7b는 이를 확대한 사진이다.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, and Figure 7b is an enlarged photograph.
본 발명은 하기의 단계를 포함하는 뇌암 환자 유래 유도만능줄기세포로부터 뇌암 오가노이드를 제작하는 방법을 제공한다: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:
뇌암 환자 유래 유도만능줄기세포 (iPSC)를 제조하는 단계;Preparing induced pluripotent stem cells (iPSC) derived from a brain cancer patient;
상기 유도만능줄기세포로부터 뇌 오가노이드를 제작하는 단계; 및Creating brain organoids from the induced pluripotent stem cells; and
상기 뇌 오가노이드 및 상기 뇌암 환자에서 분리된 암세포를 공배양하는 단계.Co-culturing the brain organoid and cancer cells isolated from the brain cancer patient.
본 발명에서, “유도만능줄기세포 (iPCS)는 역분화줄기세포로도 지칭되며, 체세포 또는 분화가 끝난 세포를 특정 방법을 통해 분화 이전의 세포 단계로 되돌린 세포로서 배아줄기세포처럼 만능성을 유도해 낸 세포이다. 이 때, iPCS를 유도하기 위한 방법은 세포 분화 관련 유전자를 주입하는 유전적 변환 또는 특정 조건으로 배양하는 등의 방법을 포함하나 이에 제한되는 것은 아니다.In the present invention, “induced pluripotent stem cells (iPCS) 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. At this time, 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.
유도만능줄기세포는 체세포 또는 분화가 끝난 세포라면 어떤 임의의 세포로부터라도 제조될 수 있고, 본 발명에서는 말초혈액단핵세포로부터 제조되었으나, 이에 제한되는 것은 아니다.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.
본 발명에서, “오가노이드”는 줄기세포를 3차원적으로 배양하거나 응집 또는 재조합하여 만든 장기 특이적 세포 집합체로 자기재생 (self-renewal)이 가능하며 “미니 장기” 또는 “유사 장기”라고도 불린다. 오가노이드는 실제 장기와 유사한 형태로 분자 신호 조절 등과 같은 동물 모델에서는 구현하기 힘든 연구를 in vitro 상에서 구현할 수 있어 기초 연구에 매우 유용함은 물론, 인간의 발생과정, 질환 모델 확립, 의약품 유효성 평가 스크리닝, 의약품 독성 평가 플랫폼 개발, 세포치료제 개발 등 다양한 분야에 매우 유용하게 사용될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, “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.
본 발명에서, “뇌암 오가노이드”는 본 발명의 방법에 따라 뇌암 환자의 PBMC에서 유래된 유도만능줄기세포로부터 제조된 상기 뇌 오가노이드와, 상기 뇌암 환자로부터 채취하여 분리된 암세포를 공배양하여 제조된 오가노이드를 의미할 수 있다.In the present invention, “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.
또한, 본 발명의 뇌암 오가노이드는 “아바타 모델”로도 지칭될 수 있으나, 이에 제한되는 것은 아니다.Additionally, the brain cancer organoid of the present invention may also be referred to as an “avatar model,” but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 뇌 오가노이드는 대뇌 오가노이드 (cerebral organoid)일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the brain organoid may be a cerebral organoid, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 뇌 오가오이드는 심실대, 외부 뇌실하대, 중간 영역, 및 피질판을 포함할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the brain organoids may include, but are not limited to, the ventricular zone, external subventricular zone, intermediate zone, and cortical plate.
본 발명의 일 실시예에 있어서, 상기 뇌 오가노이드는 하기의 (a) 내지 (d)에 해당하는 심실대, 외부 뇌실하대, 중간 영역, 및 피질판 마커 중 어느 하나 이상을 각각 발현할 수 있으나, 이에 제한되는 것은 아니다:In one embodiment of the present invention, 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:
(a) PAX6+, SOX2+, 및 Ki-67+; (a) PAX6 + , SOX2 + , and Ki-67 + ;
(b) Ki-67+ 또는 p-Vimentin+; (b) Ki-67 + or p-Vimentin + ;
(c) TBR2+; 및 (c) TBR2 + ; and
(d) CTIP2+, MAP2+, 및 TBR1+. (d) CTIP2 + , MAP2 + , and TBR1 + .
본 발명의 일 실시예에 있어서, 상기 뇌 오가노이드는 Nestin (neuroepithelial stem cell protein), GFAP (Glial fibrillary acidic protein), Tuj1 (Neuron-specific class III beta-tubulin), 및 NeuN (neuronal nuclear protein)으로 이루어진 군으로부터 선택되는 어느 하나 이상을 발현할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, 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.
본 발명에서, 상기 nestin, GFAP, Tuj1, 및 NeuN은 신경 계통 마커 (Neuronal lineage marker)일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, nestin, GFAP, Tuj1, and NeuN may be neural lineage markers, but are not limited thereto.
신경 계통 마커란, 신경 발생 또는 뉴런과 같은 분화된 세포를 따라 다양한 신경 관련 세포에서 발현되는 내인성 태그로, 이에 속하는 마커가 발현되는 세포는 각 마커에 특징에 따라 특정한 세포로 감지 및 식별될 수 있다. 즉, 다양한 기술을 통해 어떤 세포가 어떤 특성을 나타내는 세포인지 감지하고 식별하기 위하여 사용되는 지표를 의미할 수 있으나, 이에 제한되는 것은 아니다.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.
예를 들어, 신경 계통 마커는 신경 줄기세포, 신경 전구세포, 뉴런, 신경교세포 (Glial cell, Neuroglia, Glia “Glue”), 성상교세포, 희돌기세포, 소교세포, 상의세포 등 다양한 종류의 신경 계통 세포를 식별하기 위한 마커를 모두 포함할 수 있으나, 이에 제한되는 것은 아니다.For example, 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.
신경 계통 마커는 관심 세포에서 발현되는 DNA, mRNA 또는 RNA일 수 있을 뿐만 아니라, 부분 단백질로서 단백질 태그일 수 있으며, 상이한 세포 유형 또는 공통 세포의 상이한 상태를 구별하는 단백질 또는 에피토프 (epitope)일 수 있으나, 이에 제한되는 것은 아니다. 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.
이상적인 마커는 정상 상태 및/또는 부상 중에 주어진 세포 유형에 따라 다르며, 세포 마커는 질병 여부를 확인할 수 있고, 정상 상태에서도 세포의 기능을 검사하는 데 매우 유용한 도구이다. 또한, 특정 세포에 특이적인 다양한 단백질의 발견은 세포를 식별하는 데 사용되는 세포 유형 특이적 항체의 생산으로 이어져 여러 방면으로 활용될 수 있으나, 이에 제한되는 것은 아니다.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. In addition, 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.
신경 전구세포 (neural progenitor cell)는 지속적인 자가 재생이 불가능하고 일반적으로 한 부류의 분화된 자손만 생성할 수 있는 능력이 있다는 점에서 신경 줄기 세포와 구별된다. 또한, 신경 전구세포는 뉴런, 성상교세포 및 희돌기교세포를 생성할 수 있는 삼능 세포로, 예를 들어 희돌기아교 전구세포는 유사분열 능력이 소진될 때까지 희돌기아교세포를 생성할 수 있으나, 이에 제한되는 것은 아니다. 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. In addition, neural progenitor cells are tripotent cells that can generate neurons, astrocytes, and oligodendrocytes. For example, oligodendrocyte progenitor cells can generate oligodendrocytes until their mitotic ability is exhausted. It is not limited to this.
일부 신경 전구 마커는 로제트에서 뉴런으로의 확장 및 분화를 겪을 때 세포를 추적할 수 있는데, 이때 신경 로제트는 신경관의 신경 상피 세포에서 발현되는 많은 단백질을 발현하는 원주 세포의 방사형 배열로, 분화하는 배아 줄기 세포의 배양에서 신경 전구 세포의 발달 신호를 의미할 수 있다. 로제트 내의 세포는 증식하는 신경 줄기 세포에서 발현되는 RNA 결합 단백질인 Nestin, NCAM, 및 Musashi-1을 비롯한 여러 세포 마커를 발현할 수 있으나, 이에 제한되는 것은 아니다.Some 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.
본 발명에서, Nestin (neuroepithelial stem cell protein)은 NES 유전자에 의해 암호화되는 단백질로, 신경 상피 줄기 세포 단백질 (neuroepithelial stem cell protein)을 의미할 수 있고, 신경 전구세포 또는 신경 줄기세포에서 다량으로 발현되어 마커로 사용될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, Nestin (neuroepithelial stem cell protein) 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.
또한, nestin은 VI형 중간 필라멘트 (IF) 단백질이며, 이러한 중간 필라멘트 단백질은 축삭의 방사상 성장과 관련된 신경 세포에서 주로 발현될 수 있다. 예를 들어 본 발명의 일 실시예에 따르면, 본 발명의 방법으로 제조된 뇌 오가노이드는 nestin이 풍부히 발현되는데, 이는 뇌암 환자 유래 줄기세포를 이용하여 신경 전구 세포가 다량으로 온전히 제조되고, 이에 따라 기능성이 완전한 뇌 오가노이드의 제작이 가능한 것을 의미할 수 있다.Additionally, 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. For example, according to one embodiment of the present invention, 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.
본 발명에서, GFAP (Glial fibrillary acidic protein)는 GFAP 유전자에 의해 암호화되는 단백질로, 발달 중 성상세포와 뇌실막 세포를 포함하여 중추신경계 (CNS)의 수많은 세포 유형, 특히 신경 세포에 의해 발현되는 III형 중간 필라멘트 (IF) 단백질이며, 성상세포의 특이적인 단백질을 의미할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, GFAP (Glial fibrillary acidic protein) is a protein encoded by the GFAP gene, a type III protein expressed by numerous cell types of the central nervous system (CNS), especially neurons, including astrocytes and ependymal cells during development. It is an intermediate filament (IF) protein and may refer to an astrocyte-specific protein, but is not limited thereto.
GFAP는 성상교세포의 중간미세섬유단백으로 GFAP의 발현이 astrogliosis 조절의 표식자로 이용되고 있고, GFAP의 발현은 여러 단계의 외상 후 신호와 신경활성도에 의해 조절된다고 알려져 있으나, 이에 제한되는 것은 아니며, 본 발명에서는 nestin과 마찬가지로 본 발명의 일 실시예에 따른 방법으로 제조된 뇌 오가노이드에서는 GFAP가 발현되므로, 이를 근거로 뇌암 환자 유래 줄기세포를 이용하여 신경 전구 세포가 다량으로 온전히 제조되고, 이에 따라 기능성이 완전한 뇌 오가노이드의 제작이 가능한 것으로 판단할 수 있다.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. In the present invention, like nestin, 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.
뉴런 마커는 뉴런에 존재하는 핵, 세포질, 막 또는 시냅스 주변 제품에서 다양한 발달 단계의 뉴런을 감지할 수 있고, 콜린성, 도파민성, 세로토닌성, GABA성 또는 글루타메이트성 뉴런을 구체적으로 표시하는 것도 가능하다. Pan 뉴런 마커에는 여러 표적 (체세포, 핵, 수지상, 척추 및 축삭 단백질)이 있고 결과적으로 뉴런의 모든 부분에 레이블이 지정될 수 있으며, 뉴런의 특정 영역을 표시하는 특정 마커가 있을 뿐만 아니라 뉴런 형태를 연구하는 데 사용될 수도 있다.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.
본 발명에서, Tuj1 (뉴런 특이적 클래스 III β-튜불린, Neuron-specific Class III β-tubulin)은 새로 생성된 미성숙한 유사분열 후 뉴런 및 분화된 뉴런 및 일부 유사분열 활성 뉴런 전구체에 존재할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, Tuj1 (Neuron-specific Class III β-tubulin) may be present in newly generated immature post-mitotic neurons and differentiated neurons and some mitotically active neuron precursors. , but is not limited to this.
본 발명에서, 뉴런 핵 항원 (NeuN) 또는 Fox-3는 유사분열 후 세포에 존재하는 핵 단백질로, 성숙한 세포로 분화되는 지점에 발현될 수 있으나, 이에 제한되는 것은 아니다. 흑질의 푸르키네 세포 (Purkinje cell), 후각 승모판 세포, 망막 광수용체 및 도파민성 뉴런을 제외한 거의 모든 뉴런 세포 유형에서 발현되므로 이를 감지하기 위한 마커로 활용될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, neuron nuclear antigen (NeuN) 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.
본 발명의 방법에 따라 제조된 뇌 오가노이드에 신경전구세포 마커인 nestin 및 GAFP와, 신경세포 (neuron) 마커인 Tuj1, 및 NeuN가 다량으로 발현되었다는 점에서, 뇌암 환자 유래 유도만능줄기세포로부터 다양한 종류의 신경 관련 세포를 포함하는 뇌 오가노이드, 특히 대뇌 오가노이드가 제조될 수 있으나, 이에 제한되는 것은 아니다.Given that the neural progenitor cell markers nestin and GAFP, and the neuron marker Tuj1 and NeuN were expressed in large amounts in the brain organoid prepared according to the method of the present invention, various types of 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.
본 발명의 일 실시예에 있어서, 상기 암세포는 뇌 오가노이드 당 1개 이상 1×103개 이하일 수 있으나, 이에 제한되는 것은 아니다. 예를 들어, 1×101개 이상 1×103개 이하, 5×101개 이상 1×103개 이하, 1×102개 이상 1×103개 이하, 3×102개 이상 1×103개 이하, 5×102개 이상 1×103개 이하, 7×102개 이상 1×103개 이하, 8×102개 이상 1×103개 이하, 9×102개 이상 1×103개 이하, 9.1×102개 이상 1×103개 이하, 9.2×102개 이상 1×103개 이하, 9.3×102개 이상 1×103개 이하, 9.4×102개 이상 1×103개 이하, 9.5×102개 이상 1×103개 이하, 9.6×102개 이상 1×103개 이하, 9.7×102개 이상 1×103개 이하, 9.8×102개 이상 1×103개 이하, 9.9×102개 이상 1×103개 이하, 또는 1×103개 일수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the number of cancer cells may be from 1 to 1×10 3 per brain organoid, but is not limited thereto. For example, 1 or more 1 ×10 and 3 or fewer 1×10, 1 or more 5×10 but 3 or fewer 1×10, 2 or more 1×10 and 3 or fewer 1×10, 2 or more 3×10 1 3 or less ×10, 2 or more 5×10 3 or fewer 1× 10 , 2 or more 7×10 3 or fewer 1× 10 , 2 or more 8×10 3 or fewer 1× 10 , 2 9× 10 or more 1×10 3 or less, 9.1×10 2 or more 1×10 3 or less, 9.2×10 2 or more 1×10 3 or less, 9.3×10 2 or more 1×10 3 or less, 9.4×10 2 or more 1×10 3 or fewer, 9.5×10 2 or more 1×10 3 or fewer, 9.6×10 2 or more 1×10 3 or fewer, 9.7×10 2 or more 1×10 3 or fewer, 9.8 It may be 2 or more 1×10 and 3 or fewer 1×10, 9.9×10 or more and 3 or fewer 1×10, or 3 1×10, but is not limited thereto.
본 발명의 일 실시예에 따라 제조된 뇌 오가노이드로부터 뇌암 오가노이드, 또는 아바타 모델을 제작하기 위하여, 상기 뇌 오가노이드 유래 환자에서 수술적 치료를 통해 획득한 뇌암 세포 또는 암 오가노이드를 공배양 할 수 있다. 바람직하게는, 1×103개의 뇌암 세포 또는 암오가노이드를 사용하여 공배양할 수 있고, 1×103개를 초과하는 암세포를 배양할 시, 뇌암 오가노이드가 소실되는 경향이 나타날 수 있다.In order to produce a brain cancer organoid or an avatar model from a brain organoid prepared according to an embodiment of the present invention, brain cancer cells or cancer organoids obtained through surgical treatment in a patient derived from the brain organoid are co-cultured. You can. Preferably, 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.
본 발명의 일 실시예에 있어서, 상기 뇌암 오가노이드는 Ki67 또는 Iba1를 발현할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the brain cancer organoid may express Ki67 or Iba1, but is not limited thereto.
본 발명은 하기의 단계를 포함하는 뇌암 치료제 스크리닝 방법을 제공한다: 상기 방법으로 뇌암 오가노이드를 제작하는 단계; 및 상기 뇌암 오가노이드에 뇌암 치료 후보 물질을 처리한 후 치료 효과를 확인하는 단계.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.
본 발명의 일 실시예에 있어서, 상기 방법은 상기 뇌암 치료 후보 물질 처리 후 치료 효과가 나타나는 경우 뇌암 치료제로 선정하는 단계를 더 포함할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, 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.
본 발명에서, “스크리닝”이란, 여러 물질로 이루어진 후보군으로부터 목적으로 하는 어떤 특정한 성질을 갖는 물질을 특정한 조작 또는 평가 방법으로 선별하는 것을 의미할 수 있다. In the present invention, “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.
본 발명의 목적상, 본 발명의 스크리닝 방법은 뇌암 환자에게 가장 우수한 치료 효과를 발생시키는 뇌암 치료제를 확인하기 위하여 본 발명의 뇌암 오가노이드에 후보 물질을 처리하고 이에 대한 뇌암 오가노이드의 치료 반응 및 효과를 확인하는 단계 후, 가장 우수한 치료 효과를 발생시킨 상기 뇌암 치료 물질을 뇌암 치료제로 결정하는 단계를 포함하는 일련의 과정을 의미할 수 있으나, 이에 제한되는 것은 아니다.For the purpose of the present invention, 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.
상기 치료 반응 및 효과를 확인하는 단계는 치료제 후보 물질에 따라 수 회 반복될 수 있고, 치료 반응 및 효과 확인을 위하여 추가적인 물질 또는 단계를 더 추가할 수 있는 등 일반적인 스크리닝 방법으로 당업계에서 사용되는 단계를 추가적으로 더 포함할 수 있으며, 이에 제한되는 것은 아니다.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.
본 발명에서, 상기 “확인”은 “분석”의 의미와 상응할 수 있으나, 이에 제한되는 것은 아니다. 본 발명에서, 분석은 바람직하게는 “측정”을 의미하는 것일 수 있고, 상기 정성분석은 치료 반응을 확인하기 위한 목적하는 물질의 존재 여부를 측정 및 확인하는 것을 의미하는 것일 수 있으며, 상기 정량분석은 목적하는 물질의 존재 수준 (발현 수준) 또는 양의 변화를 측정 및 확인하는 것을 의미하는 것일 수 있다. 본 발명에서 분석 또는 측정은 정성적인 방법과 정량적인 방법을 모두 포함하여 제한 없이 수행될 수 있으며, 정량적인 측정이 수행되는 것일 수 있다.In the present invention, the “confirmation” may correspond to the meaning of “analysis,” but is not limited thereto. In the present invention, 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. In the present invention, analysis or measurement can be performed without limitation, including both qualitative and quantitative methods, and quantitative measurement may be performed.
본 발명에서, 상기 뇌암 치료 후보 물질이란 다양한 뇌암의 치료를 위해 사용될 수 있는 모든 제형, 방법을 모두 포함한다. 예를 들어, 뇌암 치료에 적용되는 항암제일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the brain cancer treatment candidate includes all formulations and methods that can be used for the treatment of various brain cancers. For example, it may be an anticancer agent applied to the treatment of brain cancer, but is not limited thereto.
상기 항암제란 악성종양의 치료를 위하여 사용되는 화학요법제의 총칭이다. 대부분의 항암제는 암세포의 각종 대사경로에 개입하여 주로 핵산의 합성을 억제하거나 항암활성을 나타내는 약제이다. 현재 암치료에 사용되고 있는 항암제는 생화학적인 작용 기전에 따라 6 개의 범주로 분류하고 있다.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.
(1) 알킬화제 (alkylating agents): 어떤 화합물에 알킬기 R-CH2를 도입할 능력을 갖춘, 반응성이 대단히 높은 물질로 세포에 작용시키면 대부분은 DNA의 구아닌의 N7과 반응하여 DNA구조를 변형시키고, 사슬절단을 일으켜 항암효과 및 세포독성 효과를 나타낸다. 여기에 속하는 약물로는, ① 나이트로젠머스터드계: 나이트로젠 머스타드, 클로람부실, 멜팔란, 사이클로포스파마이드 등 ② 에틸렌이민계: 싸이오테파 ③ 알킬설포네이트계: 부설판 ④ 트라이아진계, 하이드라진계: DTIC (다카바진), 프로카바진 ⑤ 나이트로소요소계: BCNU, CCNU, 메틸-CCNU 등이 있다. (1) Alkylating agents: These 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. ② Ethyleneimine: Thiotepa ③ Alkyl sulfonate: Busulfan ④ Triazine, Hydrazine series: DTIC (dacarbazine), procarbazine ⑤ Nitrozourea series: BCNU, CCNU, methyl-CCNU, etc.
(2) 대사길항제 (antimetabolites): 암세포의 증식에 필요한 대사과정을 저해하는 작용을 가진 것으로, ① 엽산유도체: 메소트렉세이트 (MTX) ② 퓨린 유도체: 6-메르캅토퓨린 (6-MP), 6-싸이오구아닌 ③ 피리미딘 유도체: 5-플루오로우라실, 시타라빈 등이 있다. (2) 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.
(3) 항생물질 (antibiotics): 세균에서 생산되는 항생물질 가운데 항암작용을 나타내는 것으로, 아드리아마이신, 다우노루비신, 블레오마이신, 미토마이신-C, 악티노마이신-D 등이 있다. (3) Antibiotics: Among the antibiotics produced by bacteria, those that exhibit anticancer activity include adriamycin, daunorubicin, bleomycin, mitomycin-C, and actinomycin-D.
(4) 유사분열억제제 (vinca alkaloid): 분열시기 특이성 약물로서 유사분열 시기 중 중기 (metaphase)에서 세포분열을 중지시킨다. 빈크리스틴, 빈블라스틴, VP-16-213 및 VM-26이 있다. (4) 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.
(5) 호르몬제: 어떤 종류의 암은 호르몬을 투여함으로써 치료효과를 볼 수 있는데, 남성호르몬을 사용하는 경우는 유방암, 여성호르몬은 전립선암, 프로게스테론은 자궁내막암에 효과가 있으며, 부신피질호르몬은 급성림프성 백혈병이나 림프종 (腫)의 치료에 사용하고 있고, 유방암에 대해서는 항여성 호르몬제인 타목시펜이 쓰이고 있다. 본 발명의 치료 물질로서 항암제는 뇌암에 적용될 수 있는 호르몬제 항암제를 포함할 수 있다. (5) 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.
(6) 기타: 시스플라틴, L-아스파라지네이스, o,p-DDD 등이 있다. 효과적인 항암제 개발에 대한 지속적인 노력에도 불구하고, 현재 선두적인 치료는 수술, 방사선 및 화학요법 등 이 주종을 이룬다. 화학요법적인 접근은 전이성이거나 특별히 공격적인 암을 치료하는데 주로 사용될 수 있다. (6) Others: Cisplatin, L-asparaginase, o,p-DDD, etc. Despite ongoing efforts to develop effective anticancer drugs, the current leading treatments mainly include surgery, radiation, and chemotherapy. Chemotherapeutic approaches can primarily be used to treat metastatic or particularly aggressive cancers.
본 발명에 따른 스크리닝 방법의 적용 대상이 되는 치료 후보 물질은 상기 항암제와 같은 특정 약물 제제뿐만 아니라 방사선 요법, 전기, 전자파 등을 사용하는 요법 또는 화학 요법 등 항암 요법을 모두 대상으로 할 수 있다.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.
본 발명은 상기 스크리닝 방법에서, 상기 치료제를 이를 필요로 하는 개체에서 투여하는 단계를 더 포함하는 뇌암 치료 방법을 제공한다. 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.
본 발명의 일 실시예에 있어서, 상기 뇌암은 교모세포종 (glioblastoma), 성상세포종 (astrocytoma), 상의세포종 (ependymoma), 핍지교종 (oligodendroglioma), 혼합교종 (mixed glioma), 뇌간 교종 (brain stem glioma), 시신경교종 (optic nerve glioma), 뇌하수체 종양 (pituitary adenoma), 두개인두종 (craniopharyngioma), 수모세포종 (medulloblastoma), 원시신경외배엽종양 (primitive neuroectodermal tumors), 송과체 종양 (pineal tumors), 수막종 (meningioma), 신경초종 (schwannoma), 전이성 뇌종양 (metastatic brain tumors), 중추신경 임파종 (CNS lymphoma), 신경섬유종증 (neurofibromatosis), 가성뇌종양 (pseudotumor cerebri), 및 결절성 경화증 (tuberoussclerosis)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, 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.
본 발명에서, “암”이란, 제어되지 않은 성장을 특징으로 하는 세포로, 이러한 비정상적인 세포성장에 의해 종양이라고 불리는 세포 덩어리가 형성되어 주위의 조직으로 침투하고 심한 경우에는 신체의 다른 기관으로 전이되기도 하는 것을 말한다. In the present invention, “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.
본 발명에서, “뇌암”은 뇌조직과 뇌를 싸고 있는 뇌막에서 발생되는 원발성 뇌암과 두개골이나 신체의 다른 부위에서 발생된 암으로부터 전이된 이차성 뇌암을 통칭하는데, 연령, 성별, 인종에 관계없이 발생되는 것을 모두 의미한다. In the present invention, “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.
본 발명은 제1 조성물, 제2 조성물, 및 제3 조성물을 포함하고, 상기 제1 조성물은 상기 유도만능줄기세포 제조용 배지 조성물이고, 상기 제2 조성물은 상기 뇌 오가노이드 제작용 배지 조성물이고, 상기 제3 조성물은 상기 공배양용 배지 조성물이고, 상기 제1 조성물, 상기 제2 조성물, 및 상기 제3 조성물은 각각 순차적으로 사용되는 것인 뇌암 오가노이드 제작용 조성물을 제공한다.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.
본 발명에서, 상기 유도만능줄기세포 제조용 배지는 PBMC로부터 유도만능줄기세포를 제조하기 위한 배지이고, PBMC로부터 유도만능줄기세포를 제조하기 위한 조성물이 포함될 수 있다. 상기 PBMC는 뇌암 환자로부터 분리된 것이므로 일반적으로 PBMC로부터 유도만능줄기세포를 제조하기 위하여 사용되는 성분 외에, 뇌암 환자의 PBMC의 특성에 따라 유도만능줄기세포를 제조하기 위한 성분이 추가적으로 더 포함될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, 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.
본 발명에서, 상기 뇌 오가노이드 제작용 배지는 유도만능줄기세포로부터 뇌 오가노이드를 제작 (또는 제조)하기 위한 배지이고, 유도만능줄기세포로부터 뇌 오가노이드를 제조하기 위한 조성물이 포함될 수 있다. 상기 조성물은 뇌암 환자 유래 유도만능줄기세포로부터 뇌 오가노이드의 제작 효율을 제고하기 위하여 뇌암의 종류, 특성, 뇌암 환자의 특징 등을 고려한 임의의 성분이 더 포함될 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 일 실시예에서는 TEMdiff™ Cerebral Organoid Kit를 사용하여 iPSC를 이용한 오가노이드 분화 프로토콜이 개시된 문헌에 기재된 방법에 따라 뇌 오가노이드를 제조하였으나, 이에 제한되는 것은 아니다.In the present invention, 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. In one embodiment of the present invention, brain organoids were prepared using the TEMdiff™ Cerebral Organoid Kit according to a method described in a literature that discloses an organoid differentiation protocol using iPSC, but is not limited thereto.
본 발명에서, 상기 공배양하는 단계용 배지는 본 발명의 방법에 따라 제조된 뇌 오가노이드와 뇌암 환자에서 분리된 암세포를 공배양하여 뇌암 오가노이드를 제조하는 단계에서 사용되는 배지일 수 있다. 또한, 상기 공배양용 배지는 본 발명의 방법에 따라 제조된 뇌 오가노이드와 뇌암 환자에서 분리된 암세포를 공배양하기 위한 임의의 조성물이 포함된 것일 수 있다. 상기 조성물은 뇌암의 종류, 암의 진행 시기, 기적용된 치료제 또는 치료법의 종류, 적용 기간, 이에 대한 뇌암 환자의 반응 등을 종합적으로 고려하여 적합한 성분이 추가적으로 더 포함될 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 일 실시예에서는 교모세포종 오가노이드 제조를 위한 공배양용 배지로서 상기 뇌 오가노이드 제작용 배지와 DMEM 배지가 1:1의 중량비로 혼합된 배지가 사용되었으나, 이에 제한되는 것은 아니다.In the present invention, 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. Additionally, 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. In one embodiment of the present invention, 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.
본 발명에서, “공배양”이란 어느 한 시기에 서로 다른 세포가 함께 배양되는 것을 의미할 수 있고, 상기 “한 시기”에 “함께 배양”의 의미는 서로 다른 세포가 동시적으로 배양되는 경우, 세포의 특성에 따라 일반적으로 적용되는 시간차에 따라 순차적으로 첨가되어 배양되는 등의 경우를 포함하여 일부 시간 또는 기간동안 함께 배양되는 경우, 및 특정 세포가 먼저 배양된 후 상기 세포의 배양물 (액)에 다른 세포가 첨가될 수 밖에 없으나 앞서 투입된 세포 배양에 영향을 받는 경우를 모두 포함할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, “co-culture” may mean that different cells are cultured together at a certain time, and “co-culture” in the “one period” means when different cells are cultured simultaneously, When 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 Although other cells cannot help but be added, all cases affected by previously introduced cell culture may be included, but are not limited to this.
본 발명은 상기 방법이 기술된 설명서 및 상기 조성물을 포함하는 뇌암 오가노이드 제작용 키트를 제공한다.The present invention provides a kit for preparing brain cancer organoids including instructions describing the method and the composition.
본 발명에서, “키트”는 뇌암 환자의 PBMC로부터 유도만능줄기세포를 제조하기 위한 배지용 조성물, 상기 유도만능줄기세포로부터 뇌암 환자의 뇌 오가노이드를 제조하기 위한 배지용 조성물, 및 뇌 오가노이드와 뇌암 환자 유래 암세포를 공배양에 사용되는 배지용 조성물을 포함하여 뇌암 환자 유래 뇌암 오가노이드를 제작할 수 있도록 하는 제제, 및 이의 제조 방법이 기술되어 있는 설명서를 포함하는 도구를 의미할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, 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.
본 발명의 키트에는 본 발명에 따른 상기 조성물 및 설명서 이외에도 뇌암 오가노이드 제작 방법에 통상적으로 필요한 다른 구성 성분, 조성물, 용액, 장치 등이 포함될 수 있으며, 상기의 물질을 적용하는 선후에는 제한이 없고, 각 물질의 적용은 동시에 진행될 수도 있으며, 미시에 진행될 수도 있다.In addition to the composition and instructions according to the present invention, the 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.
본 발명에서, 상기 키트는 컨테이너 등을 더 포함할 수 있으나, 이에 제한되는 것은 아니다. 상기 컨테이너는 상기 물질을 포장하는 역할을 할 수 있고, 보관 및 고정하는 역할을 할 수도 있다. 상기 컨테이너의 재질은 예컨대, 플라스틱, 유리병 등일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, 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.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.
[실시예][Example]
실시예 1. 뇌암 환자 유래 유도만능줄기세포 제작Example 1. Production of induced pluripotent stem cells derived from brain cancer patients
환자로부터 말초혈액단핵세포 (Peripheral blood mononuclear cells, 이하 PBMC)를 분리하여 PBMC 배지에서 4일 동안 배양한 후 5일째 되는 날 5×105의 PBMC 세포를 8 MOI의 CytoTune-iPS Sendai Reprogramming (Thermo Fisher scientific cat no. A16517)를 이용하여 유도만능줄기세포 (induced pluripotent stem cell, iPS cell/iPSC, 이하 iPSC)로 유도하였다. 약 4일 이후에 유도된 iPSC를 비트로넥틴 (vitronectin)이 코팅된 배지로 이동시켰으며 다음날부터 매일 iPSC 배지인 E8 media 또는 mTesR로 배지를 교체하였다 (도 1).Peripheral blood mononuclear cells (PBMC) were isolated from the patient and cultured in PBMC medium for 4 days. On the 5th day, 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). After about 4 days, 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).
실시예 2. 뇌암 환자 유래 유도만능줄기세포를 이용한 뇌암 환자 유래 대뇌 오가노이드의 제작Example 2. Production of cerebral organoids derived from brain cancer patients using induced pluripotent stem cells derived from brain cancer patients
실시예 1에서 제작된 뇌암 환자 유래 iPSC로부터 뇌암 환자의 대뇌 오가노이드를 제작하였다 (도 2). 실험 방법으로는 iPSC를 이용한 오가노이드 분화 프로토콜이 개시된 문헌 (Lancaster MA et al. Nature, 2013 및 Lancaster MA et al. Science, 2014)을 기반으로 STEMdiff™ Cerebral Organoid Kit (cat no. #08570)를 사용하여 뇌암 환자의 대뇌 오가노이드 (Cerebral Organoid)를 배양 및 제작하였다.A brain cancer patient's cerebral organoid was produced from the brain cancer patient-derived iPSC produced in Example 1 (FIG. 2). As an experimental method, the STEMdiff™ 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.
구체적으로, 유도만능줄기세포 콜로니를 ReLeSR™ (Stem Cell Technology)를 이용하여 작은 세포군집 단위로 분리한 후, Accutase (ThermoFisher)를 사용하여 단일세포로 분리하였다. 배아체 (embryoidbody) 형성을 위해, 9,000 내지 10,000개의 단일세포를 U-bottom low-attachment 96-well plate (Corning)의 각 웰에 분주하였다.Specifically, induced pluripotent stem cell colonies were separated into small cell clusters using ReLeSR™ (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 (STEMCELLTechnologies; 08570)를 이용하여, 신경 유도 배지 (Neural induction media)로 교체하여 2 내지 3일 동안 분화시키고, 파라필름 (parafilm) 위에서 마트리겔 (matrigel) 안에 상기 배아체를 1개씩 심어서 37℃ 5% CO2 인큐베이터에서 20분 동안 고정시켰다. 굳혀진 마트리겔 덩어리를 뇌 분화 배지 (Cerebral differentiation media)에 넣고 약 3일간 지속적으로 신경상피 (Neuroepithelium)를 유도하였다. 이후, 4일째, 오가노이드를 3ml의 성숙 (mature) 배지가 담겨있는 6-웰 플레이트 (low-attachment 6-well plate; Corning)로 옮기고 오비탈 쉐이커 (Orbital shaker, 88 rpm; ThermoFisher)에서 분석 당일까지 오가노이드를 배양하였다. 배지는 3일마다 교체하였다. 총 40일 차까지 성장시켜 뇌 오가노이드를 제조하였다.Using the STEMdiff Cerebral Organoid Kit (STEMCELLTechnologies; 08570), 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.
실시예 3. 뇌암 환자 유래 대뇌 오가노이드의 기능 확인Example 3. Confirmation of function of cerebral organoids derived from brain cancer patients
실시예 2에 따라 제작된 뇌암 환자 유래 대뇌 오가노이드가 대뇌의 기능을 나타내도록 각 분획이 형성되었는지 확인하기 위하여 대뇌의 각 분획에 대한 발현 마커를 분석하였다.In order to confirm whether each fraction of the brain cancer patient-derived cerebral organoid produced according to Example 2 was formed to exhibit cerebral function, expression markers for each fraction of the cerebrum were analyzed.
그 결과, 뇌암 환자 혈액 유래의 iPSC로부터 생성된 오가노이드는 배아체 (EB) 형성 단계로 시작하여 신경 표피가 확장되어, 40일 이상의 기간으로 배양된 성숙한 대뇌 오가노이드에서 신경 전구 세포를 나타내는 Nestin (Santa Cruz Biotechnology Inc.; SC-23927), GFAP (Merck Millipore; AB5804), Tuj1 (Biolegend; 801201), NeuN (ab134014, Abcam)을 형성하는 것으로 나타났고 (도 3), 전자현미경 사진에서 신경연접 부분을 확인하였다. 또한, 심실대 (PAX6+/SOX2+/Ki-67+), 외부 뇌실하대 (Ki-67+/p-Vimentin+), 중간 영역 (TBR2+), 및 피질판 (CTIP2+/MAP2+/TBR1+)을 형성하는 것으로 나타났으며, 이들은 생체 내에서 관찰된 것과 유사한 방향으로 층을 이루고 있는 것으로 확인되었다.As a result, 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. Santa Cruz Biotechnology Inc.; SC-23927), GFAP (Merck Millipore; AB5804), Tuj1 (Biolegend; 801201), and NeuN (ab134014, Abcam) were found to form (Figure 3), and were observed in the neuronal synapse region in electron microscopy images. was confirmed. Additionally, the ventricular zone (PAX6 + /SOX2 + /Ki-67 + ), external subventricular zone (Ki-67 + /p-Vimentin + ), intermediate zone (TBR2 + ), and cortical plate (CTIP2 + /MAP2 + /TBR1 + ), and it was confirmed that they were layered in a direction similar to that observed in vivo.
이에 따르면, 실시예 2에 따라 뇌암 환자 유래 유도만능줄기세포로부터 뇌암 환자의 특성을 나타내는 대뇌 오가노이드를 배양할 수 있고, 40일 이상의 기간으로 배양하는 경우 성숙한 대뇌 오가노이드를 제작할 수 있는 것으로 확인되었다.According to this, according to 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. .
실시예 4. 뇌암 환자 유래 대뇌 오가노이드 및 뇌암 세포를 이용한 뇌암 오가노이드의 제작Example 4. Production of brain cancer organoids using cerebral organoids and brain cancer cells derived from brain cancer patients
뇌암 환자의 수술 과정에서 분리된 환자 뇌 조직을 작은 크기로 chopping한 후 DMEM 배지에서 약 7일 정도 배양하여 환자의 조직 주변으로부터 암 세포를 수득하였다. 계대 배양 후 자란 환자 유래 암세포를 -80℃로 보관하였다. 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.
그 후 실시예 2에서 제작된 대뇌 오가노이드 및 약 1×103개의 뇌암 세포를 준비하였다. 1.5㎖ EP tube에 오가노이드 성숙 배지 및 DMEM 배지를 1:1로 넣고 혼합한 후, 준비된 환자 유래 오가노이드와 1×103개의 뇌 암세포를 넣고 5분 동안 spin down을 진행하였다. 이 때, 약 1×103개를 초과하는 암세포를 공배양 (co-culture)한 결과, 공배양 기간이 지나면 암세포가 증식함에 따라 대뇌 오가노이드 자체가 소실되는 경향이 확인되었다 (도 4). 이 후 incubater에서 약 3일 동안 배양하여 뇌암 환자 유래 오가노이드를 이용한 뇌암 오가노이드 (아바타 모델)를 제작하였다. Afterwards, 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. At this time, as a result of co-culture of more than about 1 × 10 3 cancer cells, it was confirmed that the cerebral organoid itself tended to disappear as the cancer cells proliferated after the co-culture period (FIG. 4). Afterwards, brain cancer organoids (avatar models) were produced using organoids derived from brain cancer patients by culturing them in an incubator for about 3 days.
본 실시예에서 제작된 뇌암 오가노이드와 이종이식 마우스 종양 모델을 비교한 결과, 본 발명에 따른 뇌암 오가노이드가 이종이식 마우스 종양 모델 대비 뇌암에 걸린 환자의 뇌 세포의 구조적 특징과 거의 유사할 뿐만 아니라 종양 내 종양 미세 환경이 잘 유지되어 있는 것으로 확인되었다 (도 5).As a result of comparing the brain cancer organoid produced in this example with the xenograft mouse tumor model, it was found that 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).
실시예 5. 교모세포종 환자 유래 대뇌 오가노이드 및 교모세포종 세포를 이용한 교모세포종 오가노이드의 제작Example 5. Production of glioblastoma organoids using glioblastoma patient-derived cerebral organoids and glioblastoma cells
실시예 5-1. 교모세포종 환자 유래 교모세포종 오가노이드의 제작Example 5-1. Construction of glioblastoma patient-derived glioblastoma organoids
실시예 4와 동일한 방법을 적용하여 뇌암 중 교모세포종 환자 유래 유도만능줄기세포 및 암세포를 이용하여 교모세포종 오가노이드를 제작하였다. 구체적으로, 실시예 1 및 실시예 2의 방법으로 교모세포종 환자로부터 말초혈액단핵세포를 분리하고 유도만능줄기세포로 배양하여 이로부터 대뇌 오가노이드를 제작하였다. 또한, 실시예 4의 방법으로 동일한 교모세포종 환자의 수술에서 암 조직을 분리하였고 이를 상기 대뇌 오가노이드와 공배양하여 교모세포종 모델을 확립하였다 (도 6).Using the same method as in Example 4, glioblastoma organoids were produced using induced pluripotent stem cells and cancer cells derived from patients with glioblastoma, a type of brain cancer. Specifically, 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. In addition, 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).
실시예 5-2. 교모세포종 환자 유래 교모세포종 오가노이드의 우수한 생체 내 특징 반영 수준 확인Example 5-2. Confirmation of excellent reflection of in vivo characteristics of glioblastoma patient-derived glioblastoma organoids
실시예 5-1에서 제작된 교모세포종 오가노이드의 기능을 확인하기 위하여 Ki67 및 Iba1 마커의 발현 수준을 분석하였다. Ki67 (Green)은 세포의 증식능력을 예측하기 위한 표지자로 다양한 암종에서 사용된다. 또한, Iba1 (Red)은 뇌에 상주하는 면역세포인 중추신경계의 미세아교세포에서 특이적으로 발현되기 때문에 미세아교세포 마커로 사용된다. 특히 Iba1는 휴식 중인 미세아교세포에서도 발현되나 활성화된 미세아교세포에서 높은 수준으로 발현되는 것을 특징으로 한다는 점에서 중추신경계 내 상태를 높은 정확도로 확인할 수 있다. 구체적으로, 실험은 교모세포종 오가노이드 (Giloblastoma organoid)와 뇌 오가노이드 (brain organoid)를 공배양 및 PFA 고정한 후 면역형광 염색하여 확인하였다.To confirm the function of the glioblastoma organoid produced in Example 5-1, the expression levels of Ki67 and Iba1 markers were analyzed. Ki67 (Green) is a marker to predict the proliferative ability of cells and is used in various carcinomas. Additionally, Iba1 (Red) 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. In particular, 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. Specifically, the experiment was confirmed by co-culturing glioblastoma organoids and brain organoids, fixing them with PFA, and then immunofluorescently staining them.
그 결과, Ki67은 교모세포종 오가노이드에서 관찰될 뿐만 아니라 뇌 오가노이드로 침윤되는 것이 확인되었다. 또한, 미세아교세포 역시 교모세포종 오가노이드와 뇌 오가노이드 모두에서 관찰되었다 (도 7a 및 도 7b). 이와 같은 결과에 따르면 본 발명의 방법에 따라 제조된 교모세포종 오가노이드는 상기 오가노이드의 유래가 된 환자의 교모세포종의 생체 내 특징을 모두 나타내는 것으로 확인되었다는 점에서, 이를 이용할 경우 환자 개인의 특성을 반영하는 뇌암 오가노이드로서 유용하게 활용될 것으로 기대된다.As a result, it was confirmed that Ki67 was not only observed in glioblastoma organoids but also infiltrated into brain organoids. Additionally, microglia were also observed in both glioblastoma organoids and brain organoids (Figures 7a and 7b). According to these results, 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.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다.The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.
뇌암 환자 유래 유도만능줄기세포로부터 뇌암 오가노이드를 제조하는 방법에 따르면, 환자 유래 유도만능줄기세포 및 암세포를 사용하여 제조 효율이 우수한 뇌 오가노이드를 제작할 수 있다. 본 발명의 제조 방법으로 환자 맞춤형 뇌 오가노이드를 제작할 수 있으므로 환자에게 효과적인 약물을 스크리닝할 수 있을 뿐만 아니라, 다양한 약물에 대한 반응성을 확인할 수 있다는 점에서 뇌와 관련된 암 치료제 스크리닝용 오가노이드로 유용하게 활용될 수 있는 바, 산업상 이용가능성이 인정된다.According to the method for producing brain cancer organoids from induced pluripotent stem cells derived from brain cancer patients, 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.

Claims (14)

  1. 하기의 단계를 포함하는 뇌암 환자 유래 유도만능줄기세포로부터 뇌암 오가노이드를 제작하는 방법:Method for producing brain cancer organoids from brain cancer patient-derived induced pluripotent stem cells comprising the following steps:
    뇌암 환자 유래 유도만능줄기세포 (iPSC)를 제조하는 단계;Preparing induced pluripotent stem cells (iPSC) derived from a brain cancer patient;
    상기 유도만능줄기세포로부터 뇌 오가노이드를 제작하는 단계; 및Creating brain organoids from the induced pluripotent stem cells; and
    상기 뇌 오가노이드 및 뇌암 환자에서 분리된 암세포를 공배양하는 단계.Co-culturing the brain organoid and cancer cells isolated from a brain cancer patient.
  2. 제1항에 있어서,According to paragraph 1,
    상기 뇌 오가노이드는 대뇌 오가노이드 (cerebral organoid)인 것인, 뇌암 오가노이드를 제작하는 방법.A method of producing a brain cancer organoid, wherein the brain organoid is a cerebral organoid.
  3. 제1항에 있어서,According to paragraph 1,
    상기 뇌 오가오이드는 심실대, 외부 뇌실하대, 중간 영역, 및 피질판을 포함하는 것인, 뇌암 오가노이드를 제작하는 방법.A method of producing a brain cancer organoid, wherein the brain organoid includes a ventricular zone, an external subventricular zone, an intermediate zone, and a cortical plate.
  4. 제1항에 있어서,According to paragraph 1,
    상기 뇌 오가노이드는 하기의 (a) 내지 (d)에 해당하는 심실대, 외부 뇌실하대, 중간 영역, 및 피질판 마커 중 어느 하나 이상을 각각 발현하는 것인, 뇌암 오가노이드를 제작하는 방법:A method of producing a brain cancer organoid, wherein the brain organoid expresses one or more of the ventricular zone, external subventricular zone, intermediate zone, and cortical plate markers corresponding to (a) to (d) below:
    (a) PAX6+, SOX2+, 및 Ki-67+; (a) PAX6 + , SOX2 + , and Ki-67 + ;
    (b) Ki-67+ 또는 p-Vimentin+; (b) Ki-67 + or p-Vimentin + ;
    (c) TBR2+; 및 (c) TBR2 + ; and
    (d) CTIP2+, MAP2+, 및 TBR1+. (d) CTIP2 + , MAP2 + , and TBR1 + .
  5. 제1항에 있어서,According to paragraph 1,
    상기 뇌 오가노이드는 Nestin (neuroepithelial stem cell protein), GFAP (Glial fibrillary acidic protein), Tuj1 (Neuron-specific class III beta-tubulin), 및 NeuN (neuronal nuclear protein)으로 이루어진 군으로부터 선택되는 어느 하나 이상을 발현하는 것인, 뇌암 오가노이드를 제작하는 방법.The brain organoid contains at least one selected from the group consisting of Nestin (neuroepithelial stem cell protein), GFAP (Glial fibrillary acidic protein), Tuj1 (Neuron-specific class III beta-tubulin), and NeuN (neuronal nuclear protein). A method of producing brain cancer organoids that express brain cancer.
  6. 제1항에 있어서,According to paragraph 1,
    상기 뇌암 오가노이드는 Ki67 또는 Iba1를 발현하는 것인, 뇌암 오가노이드를 제작하는 방법.A method of producing a brain cancer organoid, wherein the brain cancer organoid expresses Ki67 or Iba1.
  7. 제1항에 있어서,According to paragraph 1,
    상기 뇌암은 교모세포종 (glioblastoma), 성상세포종 (astrocytoma), 상의세포종 (ependymoma), 핍지교종 (oligodendroglioma), 혼합교종 (mixed glioma), 뇌간 교종 (brain stem glioma), 시신경교종 (optic nerve glioma), 뇌하수체 종양 (pituitary adenoma), 두개인두종 (craniopharyngioma), 수모세포종 (medulloblastoma), 원시신경외배엽종양 (primitive neuroectodermal tumors), 송과체 종양 (pineal tumors), 수막종 (meningioma), 신경초종 (schwannoma), 전이성 뇌종양 (metastatic brain tumors), 중추신경 임파종 (CNS lymphoma), 신경섬유종증 (neurofibromatosis), 가성뇌종양 (pseudotumor cerebri), 및 결절성 경화증 (tuberoussclerosis)으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 뇌암 오가노이드를 제작하는 방법.The brain cancers include glioblastoma, astrocytoma, ependymoma, oligodendroglioma, mixed glioma, brain stem glioma, optic nerve glioma, and pituitary gland. Pituitary adenoma, craniopharyngioma, medulloblastoma, primitive neuroectodermal tumors, pineal tumors, meningioma, schwannoma, metastatic brain tumor A method of producing a brain cancer organoid, which is one or more selected from the group consisting of tumors), CNS lymphoma, neurofibromatosis, pseudotumor cerebri, and tuberous sclerosis.
  8. 하기의 단계를 포함하는 뇌암 치료제 스크리닝 방법:Brain cancer therapeutic screening method comprising the following steps:
    제1항의 방법으로 뇌암 오가노이드를 제작하는 단계; 및Producing brain cancer organoids by the method of claim 1; and
    상기 뇌암 오가노이드에 뇌암 치료 후보 물질을 처리한 후 치료 효과를 확인하는 단계.Processing the brain cancer organoid with a brain cancer treatment candidate material and then confirming the treatment effect.
  9. 제8항에 있어서, 상기 방법은 The method of claim 8, wherein the method
    상기 뇌암 치료 후보 물질 처리 후 치료 효과가 나타나는 경우 뇌암 치료제로 선정하는 단계를 더 포함하는 것인, 뇌암 치료제 스크리닝 방법.A screening method for a brain cancer treatment, further comprising selecting a brain cancer treatment if a treatment effect appears after treatment with the brain cancer treatment candidate.
  10. 제8항에 있어서,According to clause 8,
    상기 뇌암은 교모세포종 (glioblastoma), 성상세포종 (astrocytoma), 상의세포종 (ependymoma), 핍지교종 (oligodendroglioma), 혼합교종 (mixed glioma), 뇌간 교종 (brain stem glioma), 시신경교종 (optic nerve glioma), 뇌하수체 종양 (pituitary adenoma), 두개인두종 (craniopharyngioma), 수모세포종 (medulloblastoma), 원시신경외배엽종양 (primitive neuroectodermal tumors), 송과체 종양 (pineal tumors), 수막종 (meningioma), 신경초종 (schwannoma), 전이성 뇌종양 (metastatic brain tumors), 중추신경 임파종 (CNS lymphoma), 신경섬유종증 (neurofibromatosis), 가성뇌종양 (pseudotumor cerebri), 및 결절성 경화증 (tuberoussclerosis)으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 뇌암 치료제 스크리닝 방법.The brain cancers include glioblastoma, astrocytoma, ependymoma, oligodendroglioma, mixed glioma, brain stem glioma, optic nerve glioma, and pituitary gland. Pituitary adenoma, craniopharyngioma, medulloblastoma, primitive neuroectodermal tumors, pineal tumors, meningioma, schwannoma, metastatic brain tumor A screening method for a treatment for brain cancer, which is one or more selected from the group consisting of tumors), CNS lymphoma, neurofibromatosis, pseudotumor cerebri, and tuberous sclerosis.
  11. 제1 조성물, 제2 조성물, 및 제3 조성물을 포함하고, comprising a first composition, a second composition, and a third composition,
    상기 제1 조성물은 제1항의 상기 유도만능줄기세포 제조용 배지 조성물이고, The first composition is the medium composition for producing induced pluripotent stem cells of claim 1,
    상기 제2 조성물은 제1항의 상기 뇌 오가노이드 제작용 배지 조성물이고, The second composition is the medium composition for producing brain organoids of claim 1,
    상기 제3 조성물은 제1항의 상기 공배양용 배지 조성물이고,The third composition is the medium composition for co-culture of claim 1,
    상기 제1 조성물, 상기 제2 조성물, 및 상기 제3 조성물은 각각 순차적으로 사용되는 것인, 뇌암 오가노이드 제작용 조성물.The composition for producing brain cancer organoids, wherein the first composition, the second composition, and the third composition are each used sequentially.
  12. 제1항의 방법이 기술된 설명서 및 제11항의 조성물을 포함하는 뇌암 오가노이드 제작용 키트.A kit for producing brain cancer organoids, comprising an instruction manual describing the method of claim 1 and the composition of claim 11.
  13. 제1항의 방법에 따라 제조된 뇌암 오가노이드의 뇌암 치료제를 스크리닝하기 위한 용도.Use of brain cancer organoids prepared according to the method of claim 1 for screening brain cancer therapeutic agents.
  14. 제11항 조성물 또는 제12항 키트의 뇌암 오가노이드를 제조하기 위한 용도.Use of the composition of claim 11 or the kit of claim 12 for producing brain cancer organoids.
PCT/KR2023/016862 2022-10-27 2023-10-27 Method for producing brain cancer-brain organoid complex by co-culturing brain organoid derived from induced pluripotent stem cells of brain cancer patient with patient brain cancer organoid WO2024091060A1 (en)

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KR20200055673A (en) * 2018-11-13 2020-05-21 한국화학연구원 Organoid using carrier for cell culture and method for evaluating drug toxicity using the same
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KR102296446B1 (en) * 2018-10-17 2021-09-02 코아스템(주) Differentiation method of securing large amount of cells by chopping target cell enriched 3D organoids prepared from human pluripotent stem cells
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