WO2024091043A1 - Renal proximal tubule organoid, kidney cancer organoid, thyroid organoid or thyroid cancer organoid, preparation method therefor, and drug evaluation method using same - Google Patents

Renal proximal tubule organoid, kidney cancer organoid, thyroid organoid or thyroid cancer organoid, preparation method therefor, and drug evaluation method using same Download PDF

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WO2024091043A1
WO2024091043A1 PCT/KR2023/016802 KR2023016802W WO2024091043A1 WO 2024091043 A1 WO2024091043 A1 WO 2024091043A1 KR 2023016802 W KR2023016802 W KR 2023016802W WO 2024091043 A1 WO2024091043 A1 WO 2024091043A1
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organoids
thyroid
drug
toxicity
cancer
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PCT/KR2023/016802
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French (fr)
Korean (ko)
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이병석
유민희
김윤희
조재연
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한국화학연구원
오알지 주식회사
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Priority claimed from KR1020230144228A external-priority patent/KR20240058792A/en
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Publication of WO2024091043A1 publication Critical patent/WO2024091043A1/en

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    • 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

Definitions

  • the present invention relates to an organoid having a structure and function similar to that of a living kidney proximal tubule, and more specifically, to an organoid that includes a lumen and an epithelial cell layer surrounding the lumen, has a round spherical three-dimensional shape, and is used in renal proximal tubule epithelial cell lines and human bodies. It relates to extracellular matrix-based renal proximal tubule organoids, methods for producing them, and drug evaluation methods using them.
  • the present invention relates to cell line-derived kidney cancer organoids, methods for producing the same, and techniques for evaluating the efficacy or toxicity of drugs using the same.
  • the present invention relates to cell line-based thyroid cancer organoids, methods for producing the same, and techniques for evaluating the efficacy or toxicity of test substances using the same.
  • the present invention relates to cell line-based thyroid organoids, methods for producing the same, and techniques for evaluating the efficacy or toxicity of test substances using the same.
  • Organoids are attracting attention as a new human body simulation model. Organoids are formed by growing stem cells into specific cells to form three-dimensional structures such as organs. Organoids, unlike two-dimensional cell-based models, are cultured in a three-dimensional environment and can be cultured for a longer period of time. In addition, organoids are only small in size, but their constituent cells and structure are similar to actual organs. Accordingly, organoids are evaluated as the optimal test object for examining the efficacy and stability of drugs in the process of developing new drugs. Furthermore, the organoid-related field is a field with high potential that can be used not only for drug toxicity and efficacy evaluation in new drug development, but also for disease models, cancer research, personalized medicine, and regenerative treatments.
  • kidney organoids have been successfully developed, including stomach, intestine, early liver, thyroid, lung, and brain.
  • maturity and characteristics of the kidney organoids developed to date are immature compared to kidney cells in the body. More specifically, the cells that make up a conventional kidney organoid may have different biomarker expression than kidney cells in the body and thus have different properties than the kidney in the body.
  • conventional kidney organoids have different characteristics from organs in the body as described above, they have limitations in that they cannot represent living kidneys in drug and toxicity evaluations as organoids.
  • kidney organoids that have similar or identical expression of biomarkers to living kidneys and can mimic structurally and morphologically.
  • kidney organoids formed by stem cell-derived or iPSC-derived cells have been provided.
  • stem cells have become an ethical issue, their use can only be done after obtaining approval from the Research Ethics Committee (IRB), which has the disadvantage of requiring a lot of time.
  • IRB Research Ethics Committee
  • stem cells each have variations, it was difficult to guarantee similarity in results.
  • organoid production using stem cells takes a lot of time to produce cells, and the quality of the cells produced can be very different depending on the skill of the experimenter.
  • differentiating stem cells into specific lineages requires more time and materials than general cell culture, resulting in high costs.
  • materials such as Matrigel or hydrogel are used to produce organoids using stem cells.
  • Matrigel and hydrogel are used, cell culture and spheroid (organoid) formation are carried out. It takes a lot of time.
  • organoids is very sensitive to the environment and composition of the culture, and organoids with very different shapes and levels of maturity have conventionally been provided depending on the skill level and environment of the fabricator and experimenter.
  • kidney organoids Accordingly, various experiments using kidney organoids have very low reliability and have not been used to evaluate the effectiveness of substances such as actual drugs and toxicity tests. Furthermore, with conventional kidney organoids, it was difficult to secure organoids of uniform homogeneity, and the cultivation and provision period was very long, making smooth distribution difficult.
  • kidney proximal tubule organoids that are similar in structure and characteristics (biomarkers) to living kidneys, thyroid organoids that are similar to living thyroid glands, kidney cancer organoids or thyroid cancers that well mimic the structure and characteristics of cancer. Organoids were developed.
  • the problem to be solved by the present invention is to develop a medium (Org 3D culture solution) containing ECM for 3D cell culture that can be applied regardless of species such as all cell lines, stem cells, and organs, and to ECM (extracellular matrix) was developed in a form containing nanofibers from fibroblasts of human skin and has the same form as the in vivo extracellular matrix, providing a spheroid or organoid culture environment with excellent in vivo mimicry.
  • the aim is to provide a method for manufacturing organoids using this, and through this, to provide kidney proximal tubule organoids with high mimicry of living kidneys and a drug evaluation method accordingly.
  • kidney cancer organoid for evaluating the efficacy or toxicity of a drug derived from a kidney cancer cell line that highly mimics living kidney cancer, and a drug evaluation method using the same.
  • it provides thyroid cancer organoids that highly mimic living thyroid cancer and a drug evaluation method using them.
  • it provides a thyroid organoid that highly mimics the living thyroid gland and a drug evaluation method accordingly.
  • the present invention is a renal proximal tubule organoid based on a renal proximal tubule epithelial cell line and human extracellular matrix (ECM), which contains a lumen, and Provided are kidney proximal tubular organoids, which contain a layer of epithelial cells lining the lumen and have a round, spherical three-dimensional shape.
  • ECM human extracellular matrix
  • the epithelial cell layer may include at least one of Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin, but is not limited thereto, and may contain various biomarkers. More may be included.
  • OAT may include at least one of OAT1, OAT2, OAT3, OAT4, OAT5, and URAT1, but is not limited thereto.
  • Na+/K+ ATPase and E-cadherin may be translocated to the cell membrane or cytosol of epithelial cells by drug treatment.
  • the ECM may be used for three-dimensional cell culture.
  • ECM can be obtained after treating a fibroblast patch with a proteolytic enzyme and decellularizing it, but is not limited thereto.
  • organoids may be used to evaluate the efficacy or toxicity of drugs, but are not limited thereto, and may also be used for various drugs such as regenerative medicine.
  • the present invention includes the steps of treating a drug in a kidney proximal tubule organoid, Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, and Vimentin of the drug-treated organoid. and determining whether the drug is effective or toxic based on the expression level for at least one biomarker of F-actin.
  • the determining step may include comparing the biomarker expression level with the biomarker expression level in a drug-untreated group or control group, but is not limited thereto.
  • the determining step may further include determining whether the drug is effective or toxic based on the expression location of Na+/K+ ATPase or E-cadherin in the organoid treated with the drug.
  • the drug is effective or toxic based on the expression location of Na+/K+ ATPase or E-cadherin in the organoid treated with the drug.
  • it is not limited to this.
  • the present invention includes treating the kidney proximal tubule organoid with at least one drug, Na+/K+ ATPase, OAT, E-cadherin,
  • a drug screening method using kidney proximal tubule organoids comprising the step of determining drug candidates based on the expression level for at least one biomarker among 8-OHdG, Vimentin, and F-actin.
  • the determining step may include comparing the biomarker expression level with the biomarker expression level in a drug-untreated group or control group, but is not limited thereto.
  • the determining step may further include determining drug candidates based on the expression location of Na+/K+ ATPase or E-cadherin in the organoid treated with the drug. It is not limited.
  • the present invention relates to a manufacturing method for forming a kidney proximal tubule organoid including a lumen and an epithelial cell layer, wherein the kidney proximal tubule organoid is formed.
  • ECM human extracellular matrix
  • Provided is a method for producing kidney proximal tubule organoids.
  • the first culturing step may be performed for at least one period of about 1 hour to 3 days, but is not limited thereto.
  • ECM can be obtained after treating a fibroblast patch with a proteolytic enzyme and decellularizing it, but is not limited thereto.
  • the renal proximal tubule epithelial cell line and the first culture medium may be included in a 1:1 ratio, but are not limited thereto.
  • the second culturing step may be performed for at least one period of about 3 to 50 days, but is not limited thereto.
  • the second culturing step may further include, but is not limited to, the step of treating a drug.
  • the step of treating the drug may be performed for the first time within 7 days of culture, but is not limited thereto.
  • the present invention provides kidney cancer organoids for evaluating the efficacy or toxicity of drugs derived from kidney cancer cell lines.
  • kidney cancer organoids may express at least one selected from the group consisting of Na+/K+ ATPase, E-cadherin, and Vimentin.
  • kidney cancer organoids can mimic the cancer microenvironment by treatment with cancer activators.
  • the cancer microenvironment may have increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
  • EMT epithelial to mesenchymal transition
  • F-actin abnormality F-actin abnormality
  • the present invention includes treating kidney cancer organoids with a drug for evaluating drug efficacy or toxicity; Including measuring the level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin for the organoid treated with the drug.
  • a biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin for the organoid treated with the drug.
  • the above-described drug evaluation method may further include the step of confirming the expression site of Na+/K+ ATPase or E-cadherin.
  • the level of F-actin abnormality or Vimentin is reduced or the level of Na+/K+ ATPase or E-cadherin is decreased in the organoid treated with the drug compared to the untreated group or positive control group. If it increases in the cell membrane, the drug can be judged to be effective.
  • the determination may further include a case where the level of Na+/K+ ATPase or E-cadherin is decreased in the cytosol.
  • F-actin abnormality or Vimentin levels are increased or Na+/K+ ATPase or E-cadherin levels are increased in the cell membrane compared to the untreated group or positive control group. If there is a decrease in , the drug can be judged to be toxic.
  • the determination may further include a case where the level of Na+/K+ ATPase or E-cadherin is increased in the cytosol.
  • the present invention includes the steps of treating a kidney cancer organoid for evaluating drug efficacy or toxicity with an anticancer candidate material;
  • the above-described screening method may further include the step of confirming the expression site of Na+/K+ ATPase or E-cadherin.
  • the present invention includes the steps of first culturing a medium containing a kidney cancer cell line and human extracellular matrix (ECM) to form cancer organoids; and a second culture step of mixing the cancer organoids and the growth medium; It provides a method for producing kidney cancer organoids for evaluating drug efficacy or toxicity, including.
  • ECM extracellular matrix
  • the human extracellular matrix may be obtained from human-derived fibroblasts.
  • the human extracellular matrix may be an ECM for three-dimensional cell culture obtained after treating a patch of human-derived fibroblasts with proteolytic enzymes and decellularizing them.
  • the medium (Org 3D culture solution) containing the ECM for 3D cell culture of the present invention can be cultured simply by mixing with stem cells, cell lines, organs, etc. without adding matrigel, hydrogel, growth factors, etc. required for organoid growth. Provides a culture environment in which a sufficient amount of organoids with consistent quality can be obtained. Accordingly, it can be produced using only low-cost chemical elements and has the advantage of being continuously supplied from fibroblast culture in vitro.
  • a medium containing a kidney cancer cell line and human extracellular matrix (ECM) may be included in a 1:1 ratio.
  • organoids having a size of 50um to 200um can be formed in the first culturing step.
  • the first culturing step may be performed for at least one period of about 1 hour to 3 days.
  • the second culturing step may be performed for at least one period of about 3 to 50 days.
  • treating the drug in the second culturing step may further include.
  • the step of treating the drug can be performed for the first time within 7 days of culture.
  • the present inventors used the above-described human-derived 3D cell culture ECM (hECM, human extracellular matrix) and applied it to human kidney cancer cell lines that can be easily cultured. As a result, kidney function and kidney cancer characteristics were improved in a short period of time compared to conventional technology.
  • the quality of the organoid does not vary depending on the deformation of the stem cells or the skill of the experimenter, and homogeneous organoids can be produced.
  • the inventors of the present invention suggest that it is possible to evaluate drugs or evaluate the effect of chemical substances on cancer growth in vitro using human kidney cancer organoids according to the present invention, which are excellent at replicating cancer characteristics in the process of developing new drugs. . As a result, the selection stage for drugs to be applied to clinical trials is shortened, thereby shortening the development period for new drugs.
  • the present invention provides a composition for culturing kidney cancer organoids simulating a cancer microenvironment, comprising a growth medium containing a kidney cancer cell line, human extracellular matrix (ECM), and a cancer activator.
  • a growth medium containing a kidney cancer cell line comprising a growth medium containing a kidney cancer cell line, human extracellular matrix (ECM), and a cancer activator.
  • ECM extracellular matrix
  • the human extracellular matrix may be obtained from human-derived fibroblasts.
  • the volume ratio of human extracellular matrix and growth medium may be 1:4 to 1:6.
  • the cancer microenvironment may have increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
  • EMT epithelial to mesenchymal transition
  • F-actin abnormality F-actin abnormality
  • the present invention provides a kidney cancer organoid simulating a cancer microenvironment prepared with the composition described above.
  • epithelial-mesenchymal transition is activated in kidney cancer organoids treated with the cancer activator PFOA, resulting in changes in EMT-related proteins such as E-cadherin and Vimentin, and dynamic reorganization of F-actin. Cancer characteristics such as expression were acquired. Accordingly, according to the present invention, by providing a kidney cancer organoid that simulates a cancer microenvironment with increased epithelial-mesenchymal transition (EMT) or F-actin abnormality, drug evaluation and the cancer proliferation effect of chemicals are evaluated. It can be used as a tool.
  • the present invention provides an animal model in which kidney cancer organoids simulating a cancer microenvironment are xenografted.
  • Kidney cancer organoids according to the present invention can be used as xenografts in animal models.
  • the “animal model” refers to a disease animal model. Specifically, the animal model may be an animal model created to suffer from a disease similar to a human disease or to be born with the disease. Animals that can be used as animal models are mammals other than humans, for example, selected from the group consisting of rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep and goats. It can be at least one thing.
  • the present invention provides thyroid cancer organoids for evaluating the efficacy or toxicity of drugs derived from thyroid cancer cell lines.
  • the thyroid cancer organoid may express at least one selected from the group consisting of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin.
  • TSHR thyroid-stimulating hormone receptor
  • Tg thyroglobulin
  • TPO thyroperoxidase
  • E-cadherin E-cadherin
  • thyroid cancer organoids can simulate a cancer microenvironment by treatment with a cancer activator.
  • the cancer microenvironment may have increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
  • EMT epithelial to mesenchymal transition
  • F-actin abnormality F-actin abnormality
  • the present invention includes the steps of treating thyroid cancer organoids with a drug for evaluating drug efficacy or toxicity;
  • a drug for evaluating drug efficacy or toxicity selected from the group consisting of F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin.
  • TSHR thyroid stimulating hormone receptor
  • Tg thyroglobulin
  • TPO thyroperoxidase
  • E-cadherin E-cadherin.
  • the above-described drug evaluation method may further include the step of confirming the expression location of thyroid-stimulating hormone receptor (TSHR) or E-cadherin.
  • TSHR thyroid-stimulating hormone receptor
  • E-cadherin E-cadherin
  • F-actin abnormality, thyroid stimulating hormone receptor (TSHR), and thyroperoxidase (TPO) were detected in organoids treated with the drug compared to the untreated group or the positive control group.
  • the drug can be judged to be effective when the level of at least one selected from the group is reduced or the level of thyroglobulin (Tg) or E-cadherin is increased.
  • Tg thyroglobulin
  • E-cadherin E-cadherin
  • the level of thyroid stimulating hormone receptor (TSHR) may be decreased in intracellular vesicles or the level of E-cadherin may be increased in the cell membrane.
  • F-actin abnormality, thyroid stimulating hormone receptor (TSHR), and thyroperoxidase (TPO) were detected in organoids treated with the drug compared to the untreated group or the positive control group.
  • the drug can be judged to be toxic if the level of at least one selected from the group is increased or the level of thyroglobulin (Tg) or E-cadherin is decreased.
  • Tg thyroglobulin
  • E-cadherin E-cadherin
  • the level of thyroid stimulating hormone receptor (TSHR) may be increased in intracellular vesicles or the level of E-cadherin may be decreased in the cell membrane.
  • the present invention includes the steps of treating thyroid cancer organoids for evaluating drug efficacy or toxicity with an anticancer candidate material; F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO) and E Comparing the level of at least one biomarker selected from the group consisting of -cadherin; Provides an anticancer drug screening method using thyroid cancer organoids, including.
  • TSHR thyroid stimulating hormone receptor
  • Tg thyroglobulin
  • TPO thyroperoxidase
  • E Comparing the level of at least one biomarker selected from the group consisting of -cadherin
  • the above-described screening method may further include the step of confirming the expression location of thyroid-stimulating hormone receptor (TSHR) or E-cadherin.
  • TSHR thyroid-stimulating hormone receptor
  • E-cadherin E-cadherin
  • the present invention includes the steps of first culturing a medium containing a thyroid cancer cell line and human extracellular matrix (ECM) to form cancer organoids; and a second culture step of mixing the cancer organoids and the growth medium; It provides a method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity, including.
  • ECM human extracellular matrix
  • the human extracellular matrix may be obtained from human-derived fibroblasts.
  • the human extracellular matrix may be an ECM for three-dimensional cell culture obtained after treating a patch of human-derived fibroblasts with proteolytic enzymes and decellularizing them.
  • a medium containing a thyroid cancer cell line and human extracellular matrix (ECM) may be included in a 1:1 ratio.
  • organoids having a size of 50um to 200um can be formed in the first culturing step.
  • the first culturing step may be performed for at least one period of about 1 hour to 3 days.
  • the second culturing step may be performed for at least one period of about 3 to 50 days.
  • treating the drug in the second culturing step may further include.
  • the step of treating the drug can be performed for the first time within 7 days of culture.
  • the present invention provides a composition for culturing thyroid cancer organoids simulating a cancer microenvironment, comprising a thyroid cancer cell line, human extracellular matrix (ECM), and a growth medium containing a cancer activator.
  • a composition for culturing thyroid cancer organoids simulating a cancer microenvironment comprising a thyroid cancer cell line, human extracellular matrix (ECM), and a growth medium containing a cancer activator.
  • the human extracellular matrix may be obtained from human-derived fibroblasts.
  • the volume ratio of human extracellular matrix and growth medium may be 1:4 to 1:6.
  • the cancer microenvironment may have increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
  • EMT epithelial to mesenchymal transition
  • F-actin abnormality F-actin abnormality
  • the present invention provides a thyroid cancer organoid simulating a cancer microenvironment prepared with the composition described above.
  • the present invention provides an animal model in which thyroid cancer organoids simulating a cancer microenvironment are xenografted.
  • the present invention provides thyroid organoids for evaluating the efficacy or toxicity of test substances prepared based on thyroid cell lines.
  • the thyroid organoid for evaluating the efficacy or toxicity of a test substance is one selected from the group consisting of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. More than one species may appear.
  • TSHR thyroid-stimulating hormone receptor
  • Tg thyroglobulin
  • TPO thyroperoxidase
  • E-cadherin More than one species may appear.
  • the thyroid organoid for evaluating the efficacy or toxicity of the test substance of the present invention can secrete thyroid hormones.
  • changes in thyroid hormone levels due to test substance treatment can be simulated.
  • the inventors of the present invention suggest that it is possible to evaluate the efficacy or toxicity of test substances in vitro using the human thyroid organoid according to the present invention, which has excellent biomimeticity, during the process of developing a new drug. As a result, the selection stage for drugs to be applied to clinical trials is shortened, thereby shortening the development period for new drugs.
  • the inventors of the present invention used the human thyroid organoid according to the present invention, which has excellent biomimeticity, to evaluate the toxicity and analyze the effect on the thyroid gland in vitro for various environmentally exposed chemicals such as hormone disruptors. We suggest that this is possible.
  • thyroid hormone analysis in animals which is performed when evaluating the toxicity of drugs or chemicals, is possible in an in vitro environment, so it can be replaced with animal testing in the future.
  • the present invention includes the steps of first culturing a medium containing a thyroid cell line and human extracellular matrix (ECM) to form an organoid; and secondly culturing the organoids by mixing them with a thyroid organoid culture medium containing Thyroid-Stimulating Hormone and Potassium Iodide; Provides a method for producing thyroid organoids for evaluating the efficacy or toxicity of test substances, including.
  • ECM extracellular matrix
  • the first culturing step may be performed for at least one period of about 1 hour to 3 days.
  • the second culturing step may be performed for at least one period of about 3 to 50 days.
  • treating the test substance in the second culturing step may further include.
  • the step of treating the test substance can be performed for the first time within 7 days of culture.
  • a medium containing a thyroid cell line and human extracellular matrix (ECM) may be included in a 1:1 ratio.
  • the human extracellular matrix may be an ECM for 3D cell culture.
  • the medium containing the ECM for 3D cell culture of the present invention (Org 3D culture solution) can be cultured simply by mixing with stem cells, cell lines, organs, etc. without adding Matrigel, hydrogel, growth factors, etc. required for organoid growth.
  • Org 3D culture solution can be cultured simply by mixing with stem cells, cell lines, organs, etc. without adding Matrigel, hydrogel, growth factors, etc. required for organoid growth.
  • a culture environment in which a sufficient amount of organoids with consistent quality can be obtained. Accordingly, it can be produced using only low-cost chemical elements and has the advantage of being continuously supplied from fibroblast culture in vitro.
  • the present inventors used the above-described human-derived ECM (hECM) for 3D cell culture and applied it to a human thyroid cell line that anyone can easily culture, producing a human thyroid organoid that secretes thyroid hormones and expresses biomarkers related to thyroid function. succeeded in doing so.
  • hECM human-derived ECM
  • organoids were formed within 3 days, which is a shorter period of time compared to the prior art, it was possible to culture organoids by exposing them to test substances within 7 days of culture. Accordingly, it was confirmed that the period for producing organoids for evaluating the efficacy or toxicity of the target test substance can be significantly shortened.
  • the quality of the organoid does not vary depending on the deformation of the stem cells or the skill of the experimenter, and homogeneous organoids can be produced.
  • the ECM for 3D cell culture may be an extracellular matrix obtained after treating a human-derived fibroblast patch with proteolytic enzymes and decellularizing it.
  • thyroid-stimulating hormone in the second culturing step, may be included at a concentration of 0.01 to 1 mU/mL based on the total volume of the thyroid organoid culture medium.
  • potassium iodide in the second culturing step, may be included at a concentration of 1 to 20 nM based on the total volume of the thyroid organoid culture medium.
  • the present invention provides a test comprising a thyroid cell line, a medium containing human extracellular matrix (ECM), Thyroid-Stimulating Hormone, and Potassium Iodide.
  • ECM extracellular matrix
  • Thyroid-Stimulating Hormone Thyroid-Stimulating Hormone
  • Potassium Iodide A culture composition for producing thyroid organoids for evaluating the efficacy or toxicity of a substance is provided.
  • the culture composition for producing thyroid organoids for evaluating the efficacy or toxicity of a test substance may be used for producing thyroid organoids that secrete thyroid hormones in vitro.
  • the culture composition for preparing thyroid organoids for evaluating the efficacy or toxicity of test substances is composed of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. It may be used for producing thyroid organoids expressing one or more species selected from the group.
  • TSHR thyroid-stimulating hormone receptor
  • Tg thyroglobulin
  • TPO thyroperoxidase
  • E-cadherin E-cadherin. It may be used for producing thyroid organoids expressing one or more species selected from the group.
  • the present invention includes the steps of processing a test substance of interest into a thyroid organoid for evaluating the efficacy or toxicity of the test substance; And the group treated with the test substance was compared with the untreated group or positive control group to show higher levels of triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyroglobulin.
  • T3 triiodothyronine
  • T4 tetraiodothyronine
  • TSHR thyroid-stimulating hormone receptor
  • Tg thyroglobulin
  • thyroglobulin thyroglobulin
  • Determining the efficacy of the test substance according to the increase or decrease of one or more biomarkers selected from the group consisting of peroxidase (TPO) and E-cadherin; Provides a method for evaluating the efficacy of test substances, including.
  • the present invention includes the steps of processing a test substance of interest into a thyroid organoid for evaluating the efficacy or toxicity of the test substance; And the group treated with the test substance was compared with the untreated group or positive control group to show higher levels of triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyroglobulin.
  • T3 triiodothyronine
  • T4 tetraiodothyronine
  • TSHR thyroid-stimulating hormone receptor
  • Tg thyroglobulin
  • thyroglobulin thyroglobulin
  • Determining the toxicity of the test substance according to the increase or decrease of one or more biomarkers selected from the group consisting of peroxidase (TPO) and E-cadherin; Provides a method for evaluating the toxicity of test substances, including.
  • the present invention provides a kidney proximal tubule organoid, a kidney cancer organoid, a thyroid organoid, or a thyroid cancer organoid, a manufacturing method thereof, and a drug evaluation method using the same, to verify the drug in the development of new drugs, that is, effectiveness, side effects, and toxicity. There is an effect that can be evaluated.
  • biomarker analysis in animals which is performed when evaluating drug toxicity, is possible in an in vitro environment, it has the effect of being able to replace animal testing in the future.
  • the organoid of the present invention can be used for screening drug candidates in the development of new drugs, dramatically reducing the cost and time required, and can be used for physiological research and clinical trials of diseases related to kidney cancer dysfunction.
  • Figure 1 exemplarily shows the procedure of a method for producing kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 2a is a fluorescence image for biomarkers of kidney proximal tubule organoids according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 2b is a fluorescence image of the structure of kidney proximal tubule organoids according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 3a exemplarily illustrates the procedure of a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 3b is a flowchart of a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 4 is a microscopic image of drug evaluation based on OAT1 expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 5 is a microscopic image of drug evaluation based on F-actine expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 6 is a microscopic image of drug evaluation based on Na+/K+ ATPase expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 7 is a microscopic image of drug evaluation based on E-cadherin expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 8 is a microscopic image of drug evaluation based on 8-OHdG expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 9 is a microscopic image of drug evaluation based on vimentin expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • Figure 10 exemplarily illustrates a method for producing kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
  • Figure 11 exemplarily illustrates a first culture method for producing kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
  • Figure 12 exemplarily shows a second culture method for producing kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
  • Figure 13 shows H&E staining microscopic images of kidney cancer organoids and drug-treated kidney cancer organoids according to an embodiment of the present invention.
  • Figure 14 shows F-actin staining microscopy images and quantification data of kidney cancer organoids and drug-treated kidney cancer organoids according to an embodiment of the present invention.
  • Figure 15 shows changes in Na+/K+ ATPase in kidney cancer organoids and kidney cancer organoids exposed to drugs according to an embodiment of the present invention.
  • Figure 16 shows changes in E-cadherin expression in kidney cancer organoids and drug-exposed kidney cancer organoids according to an embodiment of the present invention.
  • Figure 17 shows changes in Vimentin expression in kidney cancer organoids and kidney cancer organoids exposed to drugs according to an embodiment of the present invention.
  • Figure 18 is a flowchart of a drug evaluation method using kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
  • Figure 19 exemplarily illustrates a method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
  • Figure 20 shows a microscopic image of a thyroid cancer organoid produced by a production method according to an embodiment of the present invention.
  • Figure 21 exemplarily illustrates a method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention, further including drug treatment.
  • Figure 22 shows H&E staining of thyroid cancer organoids according to an embodiment of the present invention. It shows a microscope image.
  • Figure 23 shows a Hoechst33342 staining microscope image of a thyroid cancer organoid according to an embodiment of the present invention.
  • Figure 24 shows a microscopic image of F-actin staining using a phalloidin staining protocol for thyroid cancer organoids according to an embodiment of the present invention.
  • FIGS. 25a and 25b show changes in thyroid stimulating hormone receptor (TSHR) expression in thyroid cancer organoids for evaluating the efficacy or toxicity of a drug prepared by a manufacturing method according to an embodiment of the present invention.
  • TSHR thyroid stimulating hormone receptor
  • Figure 26 shows changes in thyroglobulin (Tg) expression in thyroid cancer organoids for evaluating the efficacy or toxicity of a drug prepared by the production method according to an embodiment of the present invention.
  • FIG. 27 shows changes in the expression of thyroperoxidase (TPO) in thyroid cancer organoids for evaluating the efficacy or toxicity of a drug prepared by the production method according to an embodiment of the present invention.
  • TPO thyroperoxidase
  • Figures 28a and 28b show changes in E-cadherin expression in thyroid cancer organoids for evaluating the efficacy or toxicity of a drug prepared by the production method according to an embodiment of the present invention.
  • Figure 29 is a flowchart of a drug evaluation method using thyroid cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
  • Figure 30 exemplarily shows a method for producing thyroid organoids for evaluating the efficacy or toxicity of a test substance according to an embodiment of the present invention.
  • Figure 31 shows a microscope image of a thyroid organoid prepared by a manufacturing method according to an embodiment of the present invention.
  • Figure 32 exemplarily shows a method of manufacturing thyroid organoids for evaluating the efficacy or toxicity of a test substance according to an embodiment of the present invention, further including treatment of the test substance.
  • Figure 33 shows a microscopic image of a thyroid organoid for evaluating the efficacy or toxicity of a test substance prepared by a production method according to an embodiment of the present invention and a thyroid organoid prepared further including test substance treatment.
  • Figure 34 shows a comparison of thyroid hormone changes in thyroid organoids prepared without exposure to BHA or BPA and thyroid organoids prepared with low-concentration-long-term exposure to BHA or BPA.
  • Figure 34a shows the results for thyroid organoids derived from Nthy-ori3-1 cells
  • Figure 34b shows the results for thyroid organoids derived from H6040 cells.
  • FIG 35 shows the expression of thyroid stimulating hormone receptor (TSHR) in thyroid organoids for evaluating the efficacy or toxicity of test substances prepared by the production method according to an embodiment of the present invention.
  • TSHR thyroid stimulating hormone receptor
  • Figure 36 shows thyroglobulin (Tg) expression in thyroid organoids for evaluating the efficacy or toxicity of test substances prepared by the production method according to an embodiment of the present invention.
  • FIG 37 shows the expression of thyroperoxidase (TPO) in thyroid organoids for evaluating the efficacy or toxicity of test substances prepared by the production method according to an embodiment of the present invention.
  • TPO thyroperoxidase
  • Figure 38 shows E-cadherin expression in thyroid organoids for evaluating the efficacy or toxicity of test substances prepared by the production method according to an embodiment of the present invention.
  • Figure 39 is a flowchart of a method for evaluating the efficacy or toxicity of a test substance using thyroid organoids for evaluating the efficacy or toxicity of the test substance according to an embodiment of the present invention.
  • the term “about” refers to the normal error range for each value, which is readily known to those skilled in the art. Reference herein to “about” a value or parameter includes instances of the value or parameter itself. Furthermore, the term “about” refers to a range of values that falls within 10% in either direction (above or below) of the stated reference value, unless otherwise stated or apparent from the context.
  • the term “patient or subject” is used interchangeably and refers to any single animal in need of treatment, more preferably a mammal (such as a non-human animal, e.g., cat, dog, (including horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates). Patients referred to in various embodiments herein may be humans.
  • a mammal such as a non-human animal, e.g., cat, dog, (including horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates).
  • Patients referred to in various embodiments herein may be humans.
  • differentiation refers to the development of cells to the level of a specific cell or tissue complex or entity with a special function.
  • organoids refers to a small culture that recapitulates both the form and function of a tissue or organ. More specifically, organoids must contain one or more cell types among the various types of cells that make up an organ or tissue, must be able to reproduce the special functions of each organ, and must be able to reproduce the special functions of each organ, and the cells must cluster together to form an organ spatially. It should be organized in a similar form. Organoids differ from spheroids in that they form a system rather than a simple collection of cells, and can be used as a patient-specific model for new drug development, artificial organs, disease treatments, and disease treatment.
  • medium refers to the growth of various cells in vitro, containing essential elements for cell growth and proliferation, such as sugars, amino acids, various nutrients, serum, growth factors, and minerals. and mixtures for propagation.
  • extracellular matrix refers to a three-dimensional tissue that plays an important role in providing signals that affect various cellular metabolic pathways such as cell proliferation, differentiation, and death.
  • the extracellular matrix stores and supplies the biochemical factors necessary for cell growth and differentiation, while also providing a physical environment that cells can recognize.
  • the extracellular matrix is a product produced by the cells that make up each tissue as needed, including structural proteins such as collagen and elastin, polysaccharides such as GAG (glycosaminoglycan), and other substances that help cells adhere. Contains adhesive proteins and growth factors.
  • This extracellular matrix is composed of different components depending on the tissue and cells from which it is derived, and has special physical properties.
  • Extracellular matrices typically used for organoid culture include Matrigel and Hydrogel, but they have the problem of requiring the addition of other growth factors and taking a lot of time.
  • the inventors of the present invention have developed an extracellular matrix for organoid culture that overcomes this, and can be understood by referring to the patent applications of KR10-2021-0145017 and KR10-2022-0152904.
  • the 'human extracellular matrix' is an extracellular matrix (ECM) for organoid culture developed by the inventors of the present invention, and may be an ECM for three-dimensional cell culture.
  • ECM extracellular matrix
  • the description of the ECM for 3D cell culture can be shared with all the contents described in the above-mentioned patent application.
  • ECM for 3D cell culture is an extracellular matrix (ECM) obtained by culturing fibroblasts from the dermis of human skin to obtain a patch, treating the obtained human-derived fibroblast patch with proteolytic enzymes, and then decellularizing it. It can be.
  • the obtained extracellular matrix contains collagen, actinin, and actin-binding-like protein (filamin-C), and may be in the form of tangled nanofibers. That is, going beyond the conventional method of culturing cells with a powder-type extracellular matrix coated on the surface of a culture plate, the fiber-type extracellular matrix according to the present invention covers the entire surface area of floating cells and is cultured. Accordingly, it can stably simulate the in vivo environment as closely as possible and be obtained in sufficient quantities within a short period of time.
  • the ECM for 3D cell culture of the present invention includes the steps of culturing fibroblasts in a stimulation medium so that a fibroblast patch containing fibroblasts and extracellular matrix is formed; Processing the formed fibroblast patch with a proteolytic enzyme, freezing the protease-treated fibroblast patch, and thawing the frozen fibroblast patch so that the fibroblasts in the fibroblast patch are decellularized. and obtaining extracellular matrix from the decellularized fibroblast patch.
  • the stimulation medium may contain 0.01 to 2 mM ascorbic acid.
  • ascorbic acid is an antioxidant that is involved in procollagen synthesis and is a cofactor associated with increased type 1 collagen production. Ascorbic acid can stimulate and regulate the proliferation of various cells such as adipocytes, osteoblasts, and chondrocytes in vitro. Furthermore, when ascorbic acid is added at a certain concentration, it acts as a cell growth promoter, increases cell proliferation, and even promotes DNA synthesis. However, if the concentration of ascorbic acid is not appropriate, it may inhibit the proliferation of cells and be cytotoxic, causing apoptosis.
  • the appropriate concentration of ascorbic acid that can improve the proliferation of cells, that is, the synthesis and excretion of extracellular matrix may be 0.01 to 1 mM, but is not limited thereto, and a more preferable concentration of ascorbic acid is 0.1 to 1 mM. It may be 1mM.
  • the stimulation medium is a medium containing ascorbic acid
  • it contains the basic medium as a basis.
  • basic medium is a mixture containing sugars, amino acids, and water necessary for cells to live, excluding serum, nutrients, and various growth factors.
  • the basic medium of the present invention can be artificially synthesized and used, or a commercially produced medium can be used.
  • commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F-12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, ⁇ - May include, but are not limited to, MEM ( ⁇ -Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium, and FBS (Fetal bovine serum), and are preferably DMEM/F. It could be -12.
  • the stimulation medium may further include 0.0001 to 0.001% of acetic acid, but is not limited thereto.
  • the culture period may be at least one period of 3 to 20 weeks, but is not limited thereto, and preferably may be at least one period of 6 to 12 weeks. .
  • fibroblasts can be stimulated by ascorbic acid to produce and release extracellular matrix, and a fibroblast patch containing fibroblasts and extracellular matrix can be formed. there is.
  • fibroblasts which produce and release extracellular matrix, are a type of biological cell that synthesizes extracellular matrix and collagen, and are the most common connective tissue that creates the structural framework in animal tissue, and can be obtained from various tissues such as heart tissue. You can.
  • fibroblasts can be found in at least one of tendon, ligament, muscle, skin, periodontium, cornea, cartilage, bone, liver, blood vessel, heart, small intestine, large intestine, and intervertebral disc. It may be derived from, but is not limited to.
  • a decellularization process may be performed to extract only the extracellular matrix from the fibroblast patch, and the decellularization process includes treatment with proteolytic enzymes and freezing the fibroblast patch. It may include the step of thawing the fibroblast patch and the step of thawing the fibroblast patch.
  • the extracellular matrix to be used for cell culture that is, as a biomaterial, must have as many three-dimensional structures and bioactive substances as possible in order for specific cells to maintain their physiological characteristics. Accordingly, as antigens that can cause immune rejection must be removed, a decellularization process that removes everything except cells that serve as structures may be essential.
  • proteolytic enzyme may be 0.01 to 1% trypsin, but is preferably 0.25% trypsin.
  • proteolytic enzymes are not limited to this and may include all proteolytic enzymes capable of decomposing binding proteins between fibroblasts and extracellular matrix.
  • proteolytic enzymes include Collagenase, Elastase, Dispase, Protease, Pepsin, Rennin, Chymotrypsin, and Erepsin. (Erepsin), Enterokinase, Peptidase, Proteinase, etc.
  • a step of freezing the proteolytic enzyme-treated fibroblast patch may be performed.
  • the freezing step may be performed at a temperature of -10°C or lower, but is not limited thereto.
  • a step may then be performed to thaw the frozen fibroblast patch, such that the fibroblasts in the fibroblast patch are decellularized.
  • the thawing step may be performed at room temperature for 2 hours or more.
  • fibroblasts are exuded from the fibroblast patch, and only extracellular matrix that does not contain fibroblasts and other cells can be obtained.
  • the decellularization process can be performed even if it does not include the above-described freezing and thawing.
  • the method for producing an extracellular matrix according to an embodiment of the present invention may further include the step of treating a decellularization buffer after treating a proteolytic enzyme as a decellularization process.
  • the decellularization buffer may include Triton-X or EDTA.
  • the composition of the decellularization buffer is not limited to the above-described Triton-X or EDTA, and may include all nonionic surfactant ingredients commercially used in the field of the present invention.
  • the method for producing an extracellular matrix may further include the step of treating the thawed fibroblast patch with a protease if decellularization is not completely achieved after the thawing step. there is.
  • the proteolytic enzyme may be under the same conditions as the proteolytic enzyme described above, and then decellularization may be achieved through the same process as the above-mentioned process.
  • the step of treating the thawed fibroblast patch with a proteolytic enzyme does not involve freezing and thawing, but rather involves treating the fibroblast patch with a proteolytic enzyme and a protease in a constant temperature water bath at 37°C. This can be done with agitation by adding PBS.
  • the PBS may contain 3% triton-X and 0.05% EDTA, but is not limited thereto.
  • the method for producing an extracellular matrix according to an embodiment of the present invention may further include, but is not limited to, the step of freeze-drying the obtained extracellular matrix after the obtaining step. Through these steps, distribution and supply of the obtained extracellular matrix can be facilitated.
  • the method for producing an extracellular matrix can produce an extracellular matrix with only a simple process of adding ascorbic acid and freezing and thawing, compared to the conventional extracellular matrix. It can be more economical than the substrate production method.
  • the method for producing thyroid organoids according to an embodiment of the present invention can achieve the production of cell line-derived thyroid organoids at low cost and high efficiency based on the above-described human extracellular matrix and easily available thyroid cell lines.
  • drug may include any substance used to change or modify a physiological system or disease state for the benefit of an organism. It can also include any substance used to affect body structure or function. For example, vitamins, hormones, metal salts, vaccines, antiserum agents, antibiotics, anticancer agents, chemotherapy agents, cardiotonic agents, blood pressure regulators, antihistamines, steroids, antidotes, contrast media, drug-like compounds, hit compounds, etc. , may be lead substances, new drug candidates, or various types of chemical substances, but are not limited thereto.
  • Kidney cancer organoids or thyroid cancer organoids may exhibit characteristics that mimic the cancer microenvironment. Specifically, it can simulate cancer characteristics with increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
  • EMT epithelial to mesenchymal transition
  • F-actin abnormality EMT
  • epithelial-mesenchymal transition used in this specification is a process in which epithelial cells are transformed into cells with metastatic and invasive abilities, which can affect cancer progression, metastasis, resistance to anti-cancer treatment, and cancerous lines. It plays an important role in the process of acquiring air cell characteristics. It was confirmed that the kidney cancer organoid or thyroid cancer organoid according to the present invention was caused by a change in the pathological characteristics of epithelial cells due to an increase in epithelial-mesenchymal transition (EMT), thereby mimicking the characteristics of the cancer microenvironment. .
  • EMT epithelial-mesenchymal transition
  • F-actin abnormality is an early biomarker of cancer based on reports of abnormal actin isoform expression in many cancers.
  • F-actin is a family of globular multifunctional proteins that form fine filaments in the cytoskeleton and thin filaments in muscle fibrils and are present in all eukaryotic cells.
  • the cytoskeleton is an organelle within the cytoplasm and is composed of a fluid structure, which not only plays an important role in maintaining cell shape but also enables cell movement. This cytoskeleton is known to change more dynamically in cancer cells than in normal cells. Dynamic changes in F-actin in cancer cells play a major role in the division, growth, and migration ability of cancer cells, contributing to the development, progression, and metastasis of cancer.
  • Na+/K+ ATPase used herein is located in the basement membrane and maintains intracellular Na and K balance. More specifically, the proximal duct of the kidney is responsible for the transport of uric acid to the kidney and is where uric acid reabsorption primarily occurs.
  • Proximal Renal Tubular Epithelial Cells PTECs are rich in mitochondria and lysosomes, as they excrete uric acid and express ion and lactic acid transport channels. At this time, the driving force of uric acid transporter in renal proximal tubular epithelial cells is generated from Na+/K+ ATPase present at the tubule epithelium basolateral.
  • Na+/K+ ATPase As the main function of Na+/K+ ATPase is to control electrolyte and fluid homeostasis in the kidney, changes in the expression of Na+/K+ ATPase in kidney cancer organoids determine the efficacy of drugs using kidney cancer organoids according to the present invention. Alternatively, it can be an important biomarker in toxicity evaluation.
  • Na+/K+ ATPase in kidney cancer organoids, but also its location may be very important. More specifically, Na+/K+ ATPase must be present in the cell membrane (basement membrane) as it must maintain Na+/K+ equilibrium with the tubules and interstitial fluid, and its location change may affect the pathophysiology of the renal proximal tubules due to drugs and diseases. It can represent change.
  • E-cadherin used herein is a cadherin-family molecule that maintains adhesion between cells. It is expressed in most epithelial cells and is an important protein that stably maintains connections between epithelial cells. In particular, it is one of the proteins robustly expressed in normal tissues, playing an important role in cell adhesion and kidney tissue structure.
  • EMT epithelial-mesenchymal transition
  • the term “Vimentin” refers to a medium-sized fibrous protein and is mainly found in cells of mesenchymal and nervous tissue.
  • EMT which converts epithelial cells into mobile mesenchymal cells
  • vimentin increases.
  • kidney disease especially neuropathy or fibrosis
  • vimentin is upregulated. Accordingly, as changes (increases) in vimentin expression cause pathological phenomena or are caused by pathological phenomena, pathophysiological changes in the kidney can be predicted based on this.
  • Kidney cancer organoids according to an embodiment of the present invention were confirmed to express at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin. . Therefore, by using the kidney cancer organoid according to an embodiment of the present invention, the efficacy and toxicity of the target drug can be predicted using the above-described biomarkers.
  • the present invention includes the steps of treating a drug or anticancer candidate material to kidney cancer organoids for evaluating drug efficacy or toxicity; Measure the level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin for organoids treated with the drug or anticancer candidate material. It is possible to provide a drug evaluation method or an anticancer drug screening method using kidney cancer organoids, including the step of:
  • the above-described method may further include the step of confirming the expression location of Na+/K+ ATPase or E-cadherin.
  • F-actin abnormality or Vimentin levels were decreased or Na+/K+ ATPase or E-cadherin levels were decreased compared to the drug-untreated group or positive control group. If it increases in the cell membrane, the drug can be judged to be effective.
  • the determination may further include a case where the level of Na+/K+ ATPase or E-cadherin is decreased in the cytosol.
  • F-actin abnormality or Vimentin levels were increased or Na+/K+ ATPase or E-cadherin levels were increased compared to the drug-untreated group or positive control group. If it decreases in the cell membrane, the drug can be judged to be toxic.
  • the determination may further include a case where the level of Na+/K+ ATPase or E-cadherin is increased in the cytosol.
  • thyroid cancer-stimulating hormone receptor used herein is a G protein-coupled receptor located on the cell membrane surface of thyroid cancer cells, which is a receptor for thyroid cancer-stimulating hormone secreted by the pituitary.
  • thyroid cancer-stimulating hormone combines with the thyroid cancer-stimulating hormone receptor, thyroid cancer growth, differentiation of thyroid cancer cells, and synthesis of thyroid cancer hormones occur.
  • the pituitary gland is stimulated and thyroid cancer-stimulating hormone increases or decreases, which affects the receptor for thyroid cancer-stimulating hormone. Therefore, it can be an important biomarker in evaluating the efficacy or toxicity of drugs using the thyroid cancer organoid according to the present invention.
  • Thyroglobulin (Tg) used herein is a major protein involved in thyroid cancer hormone synthesis. Since thyroglobulin is secreted only in normal thyroid cancer tissue and thyroid cancer tissue, it can be a thyroid cancer-specific biomarker and an indicator for evaluating the biosimilarity of the thyroid cancer organoid according to the present invention. Additionally, the thyroid cancer organoid according to the present invention can be used as an important biomarker in evaluating the efficacy or toxicity of a drug.
  • TPO thyroperoxidase
  • Thyroid organoid consists of F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. Expression of at least one biomarker selected from the group was confirmed. Therefore, by using the thyroid cancer organoid according to an embodiment of the present invention, the efficacy and toxicity of the target drug can be predicted using the above-described biomarker as an indicator.
  • the present invention includes the steps of treating a drug or anticancer candidate material to a thyroid cancer organoid for evaluating drug efficacy or toxicity;
  • a drug or anticancer candidate material for organoids treated with the above drugs or anticancer candidates, F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin were detected.
  • TSHR thyroid stimulating hormone receptor
  • Tg thyroglobulin
  • TPO thyroperoxidase
  • E-cadherin E-cadherin
  • a drug evaluation method or an anticancer drug screening method using thyroid cancer organoids can be provided, including the step of measuring the level of at least one biomarker selected from the group consisting of.
  • the above-described method may further include the step of confirming the expression location of thyroid-stimulating hormone receptor (TSHR) or E-cadherin.
  • TSHR thyroid-stimulating hormone receptor
  • E-cadherin E-cadherin
  • organoids treated with drugs or anticancer candidates were compared to the drug-untreated group or the positive control group to determine F-actin abnormality, thyroid-stimulating hormone receptor (TSHR), and thyroperoxidase (TPO).
  • THR thyroid-stimulating hormone receptor
  • TPO thyroperoxidase
  • organoids treated with drugs or anticancer candidates were compared to the drug-untreated group or the positive control group to determine F-actin abnormality, thyroid-stimulating hormone receptor (TSHR), and thyroperoxidase (TPO). If the level of at least one selected from the group consisting of ) increases or the level of thyroglobulin (Tg) or E-cadherin decreases, the drug may be judged to be toxic or have no anticancer activity.
  • the above-mentioned thyroid stimulating hormone receptor (TSHR) level may mean an increase in intracellular vesicles or a decrease in the level of E-cadherin in the cell membrane.
  • test substance may refer to a drug or chemical substance for use in activity or toxicity testing.
  • drug compounds such as butylhydroxyanisole (BHA), which is widely used in foods and cosmetics as an antioxidant, drug-like compounds, hit compounds, lead substances, new drug candidates, or bisphenol A, which is an endocrine disruptor that affects thyroid hormones,
  • BHA butylhydroxyanisole
  • these may be environmentally exposed chemicals such as perfluorinated compounds, but are not limited thereto.
  • environmentally exposed chemicals are chemicals that are widely used in living environments, industries, etc. and can refer to substances that affect the environment and animals and plants.
  • it may be an endocrine disrupting chemical (EDC) or a perfluoroalkyl substance (PFAS), but is not limited thereto.
  • endocrine disrupting chemicals include polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBB), dioxins, furans, pesticides, perfluorinated compounds, phthalates, bisphenol-A (BPA), UV filters, triclosan, It may be, but is not limited to, perchlorate, paraben, or butylhydroxytoluene (BHT).
  • the thyroid organoid manufacturing method according to the present invention expresses thyroid biomarkers and provides a large amount of thyroid organoids with excellent biomimetic properties in a short period of time, thereby providing a thyroid organoid that can evaluate the efficacy or toxicity of test substances in vitro. can do.
  • the thyroid organoid contains triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and Expression of at least one biomarker selected from the group consisting of E-cadherin was confirmed.
  • T3 triiodothyronine
  • T4 thyroid-stimulating hormone receptor
  • Tg thyroglobulin
  • TPO thyroperoxidase
  • the efficacy and toxicity of the target test substance can be predicted using the above-described biomarker as an indicator.
  • T3 triiodothyronine
  • T4 thyroid-stimulating hormone receptor
  • Tg thyroglobulin
  • Tg thyroglobulin
  • Tg thyroglobulin
  • Kidney proximal tubule organoids method of producing the same, and method of drug evaluation using the same
  • kidney proximal tubule organoid according to an embodiment of the present invention, a method for producing the same, and a drug evaluation method using the same will be described in detail.
  • Figure 1 exemplarily shows the procedure of a method for producing kidney proximal tubule organoids according to an embodiment of the present invention. At this time, for convenience of explanation, description will be made with reference to FIGS. 2A to 3B.
  • the method for producing kidney proximal tubule organoids is a manufacturing method for forming three-dimensional kidney proximal tubule organoids based on a three-dimensional cell culture method. It may include a first culturing step to form and a second culturing step to grow the formed organoid.
  • the first culturing step is a step of culturing the renal proximal tubular epithelial cell line in a first culture medium containing human extracellular matrix (ECM) to form renal proximal tubular organoids. More specifically, in the first culture step, the proximal tubular epithelial cells (main) and the human extracellular matrix fuse together, the distinction between cells disappears, tight junctions occur, and self-organization (self-organization) occurs. This may be the stage in which organoids are formed as organization occurs.
  • ECM extracellular matrix
  • the renal proximal tubular epithelial cell line is the commercially available RPTEC/TERT1 cell line, which may mean, but is not limited to, a human renal proximal tubular epithelial cell line, and is not limited to all commercially available renal proximal tubular epithelial cell lines. can be used. Additionally, renal proximal tubular epithelial cell lines, as well as commercially available cell lines, can be used as renal proximal tubular epithelial cell lines obtained directly from living kidneys.
  • the first culture medium may refer to a medium that essentially contains human extracellular matrix (ECM). More specifically, the first culture medium may be a basic culture medium containing at least one of extracellular matrix, amino acids, acetic acid, glutamax, ascorbic acid, B27, and IWR-1, and may include the above-mentioned composition and a commercially available culture medium. It may be a medium containing ORG 3D solution used, but is not limited thereto.
  • ECM extracellular matrix
  • the extracellular matrix of the first culture medium is an extracellular matrix produced from fibroblasts derived from human skin dermis.
  • a patch is obtained by culturing fibroblasts derived from human skin dermis, and a proteolytic enzyme is added to the obtained patch. It can be obtained after treatment and decellularization.
  • the obtained extracellular matrix contains collagen, actinin, and actin-binding-like protein (filamin-C), and may be in the form of tangled nanofibers.
  • the extracellular matrix in the form of nanofibers is rapidly mixed with kidney proximal tubule epithelial cells (mainly), and as it covers the entire surface area of the cells and is cultured, it stably mimics the in vivo environment to produce kidney proximal tubule organoids in a short period of time. can be formed within.
  • the method for producing kidney proximal tubule organoids according to an embodiment of the present invention includes the above-described extracellular matrix, and thus can form and produce kidney proximal tubule organoids in a short period of time at low cost and high efficiency.
  • the basic culture medium of the first culture medium is BME (Basal medium Eagle's), MEM (Minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199, and It may include at least one of RPMI 1640, but is not limited thereto, and various commercially available basic culture media can be used.
  • the kidney proximal tubule epithelial cell line and the first culture medium may be included in a 1:1 ratio, but are not limited to this, and preferably, based on 1 mL of the first culture medium, the kidney proximal tubule epithelial cell line is 1:1. It may include, but is not limited to, x 10 5 to 1 x 10 7 cells.
  • the first culturing step may be performed for at least one period of about 1 hour to 3 days, but is not limited thereto.
  • conventional organoid formation takes several weeks or more.
  • the method for producing kidney proximal tubule organoids according to an embodiment of the present invention overcomes the limitations of the conventional method for producing kidney proximal tubule organoids in which kidney proximal tubule organoids can be formed quickly within a short time of less than 3 days. That could be one way.
  • a kidney proximal tubule organoid that expresses the same biomarkers as the kidney in vivo and can mimic the pathophysiological function and response of the kidney in vivo is created. It can be formed (created).
  • the organoids formed by the first culturing step may have a size of less than 100 ⁇ m, and this size is not sufficient to observe the reaction of the organoids under a microscope in experimental or clinical drug (compound) evaluation. It may not be possible.
  • the method for producing renal proximal tubule organoids according to an embodiment of the present invention produces organoids with homogeneous characteristics, that is, In order to form organoids that are uniform in size and contain the same biomarker expression, a second culture step may be included.
  • the second culturing step is a step of culturing the organoids in the second culture medium so that the formed kidney proximal tubule organoids grow (mature).
  • the second culture medium may be the basic culture medium of the first culture medium.
  • the second culture medium is a basic culture medium, such as BME (Basal medium Eagle's), MEM (Minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), DMEM/F12, HAM'S F-10, HAM'S F-12, It may include at least one of MEDIUM 199 and RPMI 1640, but is not limited thereto, and various commercially available basic culture media can be used as the second culture medium.
  • the second culture medium may be replaced once every three days (once/3 days).
  • the second culturing step may be performed for at least one period of about 3 to 50 days, but is not limited thereto.
  • organoids grown by the second culturing step of the present invention may have a size (diameter) of about 100 ⁇ m or more around day 7 (about 168 hours). That is, uniform and highly reproducible kidney proximal tubule organoids can be formed (produced) over a culture period of about 7 days or more. Accordingly, the period of the second culture step for commercial use of the kidney proximal tubule organoid of the present invention may be about 7 days or more, but is not limited thereto.
  • kidney proximal tubule organoids on the 7th day or more after the second culturing step of the present invention had homogeneous characteristics, that is, expressed the same biomarkers.
  • kidney proximal tubule organoids according to a method of producing kidney proximal tubule organoids according to one embodiment of the present invention is shown.
  • the kidney proximal tubule organoid according to an embodiment of the present invention observed under a microscope is an organoid of about 21 days.
  • Kidney proximal tubule organoids may include OAT, F-actin, Na+/K+ ATPase, E-cadherin, 8-OHdG, and Vimentin.
  • OAT is an organic anion transporter (OAT), which plays a role in excreting endogenous or exogenous organic anions out of the body. In addition to excreting organic anions, it is also used as a clinically important organic anion drug, anti-HIV treatment drug, and anticancer drug. , various drugs such as antibiotics, antihypertensive agents, and anti-inflammatory drugs, or metabolites such as uremic toxin can be transported and excreted from the body.
  • OAT organic anion transporter
  • various drugs such as antibiotics, antihypertensive agents, and anti-inflammatory drugs, or metabolites such as uremic toxin can be transported and excreted from the body.
  • OAT includes the following subtypes (OAT family): OAT1, OAT2, OAT3, OAT4, OAT5, and URAT1. Their expression distribution may differ depending on the tissue and cell, but OAT subtypes are mainly expressed in the kidney and some It is observed in the liver, brain, and placenta.
  • OAT1 is mainly expressed in the basolateral membrane of the proximal tubule, and as a PAH transporter and OA/dicarboxylate exchanger, it transports OA from the blood into the epithelial cells of the proximal tubule. More specifically, OAT1 uses a tertiary transport mechanism in the kidney to move organic anions through the basolateral membrane and then excrete them with urine.
  • OAT1 is expressed only in the proximal tubules of the urinary tubules and is involved in the transport of over 100 substances, including dicarboxylates such as alpha-ketoglutarate, cyclic nucleotides, prostaglandins, urate, and various drugs.
  • the expression of OAT1 may be very important to simulate the transport and excretion of various metabolites, including drugs in the body, which are the main functions of the kidney.
  • kidney epithelial cells lose the expression of OAT1 and the OAT family including it, and their characteristics for kidney tissue are lost.
  • kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention, OAT1 is expressed and maintained without inducing overexpression of OAT1 and the OAT family including it.
  • the kidney proximal tubule organoid of the present invention does not lose the characteristics of the kidney proximal tubules during the organoid culture process using kidney cell lines, but maintains and improves them, so it can represent kidney tissue (organ) in vivo, and has a high degree of mimicry. This may mean that it is an organoid.
  • the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention shows that OAT1 is present in the lateral membrane as in the kidney in vivo. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on not only the expression amount of OAT1 but also the expression location.
  • the kidney proximal tubule organoid appears to express F-actin.
  • F-actin is a cytoskeletal element that can participate in the structure formation of cells as they form tissues.
  • F-actin connects podocytes in the kidney to the glomerular basement membrane, and a series of connecting proteins can attach podocytes to the slit membrane. Accordingly, the expression of F-actin is very important in the structural formation of kidney tissue, and various changes in kidney function can be observed depending on its expression pattern.
  • the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention contains F-actin in areas such as epithelial cells, endothelial cells, and glomerular basement membrane, as in the kidney in vivo. appears to exist. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on not only the expression amount of F-actin but also the expression location.
  • the kidney proximal tubule organoid appears to express Na+/K+ ATPase.
  • Na+/K+ ATPase is located in the basement membrane and maintains Na and K in the cell. More specifically, the proximal duct of the kidney is responsible for the transport of uric acid to the kidney and is where uric acid reabsorption primarily occurs.
  • Proximal Renal Tubular Epithelial Cells are rich in mitochondria and lysosomes, as they excrete uric acid and express ion and lactic acid transport channels.
  • the driving force of uric acid transporter in renal proximal tubular epithelial cells is generated from Na+/K+ ATPase present at the tubule epithelium basolateral.
  • Na+/K+ ATPase As the main function of Na+/K+ ATPase is to control electrolyte and fluid homeostasis in the kidney, expression of Na+/K+ ATPase in kidney organoids may be very important.
  • Na+/K+ ATPase in kidney organoids, but also its location may be very important. More specifically, Na+/K+ ATPase must be present in the epithelial cell membrane (basement membrane) as it must maintain Na+/K+ equilibrium with the tubule and interstitial fluid, and its location change is associated with the pathophysiology of the renal proximal tubule due to drugs and diseases. It can represent academic change.
  • kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention, the expression of Na+/K+ ATPase is present in the epithelial cell membrane (basolateral) surrounding the lumen. It appears that it does. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based not only on the expression level of Na+/K+ ATPase but also on the expression location.
  • E-cadherin is a cadherin family molecule that maintains adhesion between cells.
  • it is an important protein that is mainly expressed in the proximal tubular epithelial cells of the kidney and stably maintains connections between epithelial cells.
  • EMT epithelial-mesenchymal transition
  • E-cadherin As changes in E-cadherin occur due to pathological phenomena, changes in function and structure of renal proximal tubular epithelial cells can be predicted based on this.
  • the expression of E-cadherin is an important biomarker that can predict pathophysiological functions caused by drugs and diseases in the renal proximal tubule.
  • the expression of E-cadherin appears to be present in the epithelial cells surrounding the lumen. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on the expression of E-cadherin.
  • 8-OHdG (8-Hydroxy-2'-deoxyguanosine) is one of the major oxidative modification products of DNA damage and is a pathological marker for oxidative stress caused by radicals.
  • Increased 8-OHdG expression can be associated with various diseases, including cellular aging and cancer, and increased 8-OHdG expression in the renal proximal tubules increases oxidative stress in the kidney, leading to loss of kidney function.
  • Various diseases can result. Accordingly, changes (increases) in 8-OHdG expression cause pathological phenomena or are caused by pathological phenomena, and based on this, pathophysiological changes in the renal proximal tubules can be predicted.
  • kidney proximal tubule organoid As shown in the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoid according to an embodiment of the present invention, the expression of 8-OHdG appears to be present in the epithelial cells surrounding the lumen. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on the expression of 8-OHdG.
  • Vimentin is a medium-sized fibrous protein found mainly in cells of mesenchymal and nervous tissue.
  • EMT which converts epithelial cells into mobile mesenchymal cells
  • vimentin increases.
  • kidney disease especially neuropathy or fibrosis
  • EMT in the renal proximal tubules is activated, and vimentin is upregulated in the renal proximal tubular epithelial cells. Accordingly, as changes (increases) in vimentin expression cause pathological phenomena or are caused by pathological phenomena, pathophysiological changes in the renal proximal tubules can be predicted based on this.
  • the expression of vimentin appears to be present in the epithelial cells surrounding the lumen. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on the expression of vimentin.
  • the kidney proximal tubule organoid of the present invention contains Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin, as described above, and thus has the same properties (biomarkers) as the kidney in vivo. expression) can be simulated, making it possible to predict pathophysiological changes in the kidney caused by various drugs.
  • kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention not only expresses biomarkers but also has a structure similar to that of a living kidney.
  • FIG. 2B a fluorescence image of the structure of a kidney proximal tubule organoid according to a method of producing kidney proximal tubule organoids according to an embodiment of the present invention is shown.
  • the renal proximal tubular organoid according to the method for producing renal proximal tubular organoid according to an embodiment of the present invention includes a lumen and an epithelial cell layer surrounding the lumen, and appears to have a round spherical three-dimensional shape.
  • the kidney proximal tubule organoid of the present invention can mimic the same structure as the kidney in vivo, making it possible to more easily predict pathophysiological changes in the kidney caused by various drugs.
  • the kidney proximal tubule organoid of the present invention can be used as a biosimilar model with high biocorrespondence.
  • the kidney proximal tubule organoids in the second culture step contain biomarkers and structures that can evaluate the efficacy and toxicity of the drug.
  • the culturing step may further include the step of treating the drug. That is, the present invention may include a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention.
  • FIG. 3A an exemplary diagram showing the procedure of a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention is shown.
  • the drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention can be performed simultaneously by being included in the second culture step of the method for producing kidney proximal tubule organoids according to an embodiment of the present invention.
  • organoids can be sorted into one or more different containers to perform a second culture, and at the same time, drugs can be added to a specific container and cultured.
  • T0 in Figure 3a may be the kidney proximal tubule organoid on day 7 in the second culture, but is not limited thereto, and organoids in the second culture for various periods of time may be used depending on the user's experimental purpose and plan. It can be. For example, if evaluation of the efficacy and toxicity of a drug during the development of the kidney proximal tubules is desired, kidney proximal tubule organoids from day 0 of the second culture can be used at T0.
  • the drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention can be performed by treating the organoids with a drug for 21 days, and the drug treatment period is not limited to 21 days. It can be set in various ways depending on the user's experiment purpose and plan.
  • kidney proximal tubule organoids of the present invention that underwent drug treatment for 21 days can be subjected to morphological and protein expression analysis.
  • Morphological analysis may include, but is not limited to, microscopic structural analysis.
  • protein expression analysis analysis based on the expression level of at least one biomarker among Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin can be used, as well as the amount and location of expression. Depending on this, the efficacy or toxicity of the drug can be determined.
  • the efficacy or toxicity of a drug can be determined by comparing the biomarker expression level of the drug-treated group (treatment, Che-1, Che-2) with the biomarker expression level of the drug-untreated group (control, CTL).
  • FIG. 3b a flow chart of a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention is shown, and drug evaluation using kidney proximal tubule organoids according to an embodiment of the present invention.
  • the method may include treating a kidney proximal tubule organoid with a drug (S310) and determining whether the drug is effective or toxic based on the expression level for a biomarker in the drug-treated organoid (S320). there is.
  • the determining step (S320) may include comparing the biomarker expression level of the drug-treated organoid with the biomarker expression level in the drug-untreated group or control group, and the comparison result may include: Based on this, the efficacy or toxicity of the drug can be determined.
  • the kidney proximal tubule organoid can express specific biomarkers at the same location in the living kidney organ. Accordingly, the determining step (S320) may further include determining the efficacy or toxicity of the drug based on the expression location of Na+/K+ ATPase or E-cadherin in the organoid treated with the drug.
  • the present invention may include a drug screening method using kidney proximal tubule organoids according to an embodiment of the present invention, which includes the same steps as the drug evaluation method according to an embodiment of the present invention.
  • the method for producing kidney proximal tubule organoids according to an embodiment of the present invention can provide kidney proximal tubule organoids that can reproduce living kidneys with a high degree of similarity, which can be used in various drug evaluation and screening methods.
  • Example 1 Drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention
  • kidney proximal tubule organoids according to an embodiment of the present invention, that is, a drug evaluation method based on biomarker expression in organoids, will be described in detail.
  • the drug evaluation in FIGS. 4 to 9 used the kidney proximal tubule organoid of the present invention with a diameter of about 100 ⁇ m at about 7 days, and drug treatment was performed for 21 days. Furthermore, the medium was changed every three days during the drug treatment and culture period.
  • drug may include any substance used to change or modify a physiological system or disease state for the benefit of an organism. More specifically, it may include at least one of the group consisting of vitamins, hormones, metal salts, vaccines, antiserum agents, antibiotics, chemotherapy agents, cardiotonic agents, blood pressure regulators, antihistamines, steroids, antidotes, and contrast agents. It is not limited.
  • Figure 4 is a microscopic image of drug evaluation based on OAT1 expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • the drug treated with the organoid was bisphenol A, and it was treated at a concentration of 10 ⁇ M.
  • Both the control group (vehicle CTL) and the treatment group (bisphenol A) appear to express OAT1.
  • this may mean that the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses OAT1, thereby mimicking the pathophysiological function of the kidney's organic anion transport process.
  • the experimental results of the present invention show that OAT1 is expressed in the epithelial cell layer in both the control group (vehicle CTL) and the treatment group (bisphenol A), similar to the living kidney.
  • the bisphenol A used in this experiment did not show any difference in the expression of OAT, including OAT1, in kidney proximal tubule organoids compared to the control group.
  • the renal proximal tubule organoid of the present invention it is possible to present the possibility of evaluating drugs that affect OAT, including OAT1, and how the target drug affects organic anion excretion and metabolism during kidney function through changes in this. The impact can be predicted.
  • Figure 5 is a microscopic image of drug evaluation based on F-actine expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • the drugs treated with the organoids were PFOA and PFDA, which are perfluorinated compounds that were widely used in waterproofing and coating products in the past, but are currently banned due to their toxicity, and were treated at 10 ⁇ M each for 21 days.
  • both the control group (vehicle CTL) and the treatment group (PFOA, PFDA) appear to express F-actin.
  • the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses F-actine, which means that it can represent (simulate) structural changes in the kidney caused by disease and drugs.
  • F-actine in the control group (vehicle CTL) appears to be clearly expressed in the epithelial cell layer, similar to the living kidney.
  • F-actine in the treatment group (PFOA, PFDA) was confirmed to be expressed even in the lumen of the organoid, which indirectly proves that the lumen of the renal proximal tubule in the treatment group was narrowed, making it difficult to function normally.
  • the kidney proximal tubule organoid of the present invention appears to have differences in the expression of F-actine depending on drug treatment, it is possible to predict how the target drug affects the function and metabolism of structural cells of the kidney through this change. You can.
  • Figure 6 is a microscopic image of drug evaluation based on Na+/K+ ATPase expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • the drugs treated with the organoids were PFOA and PFDA, each treated at 10 ⁇ M for 21 days.
  • Both the control group (vehicle CTL) and the treatment group (PFOA, PFDA) appear to express Na+/K+ ATPase.
  • the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses Na+/K+ ATPase, which means that it can represent (simulate) the pathophysiological function of electrolyte and liquid homeostasis in the kidney. .
  • Na+/K+ ATPase in the control group (vehicle CTL) appears to be expressed on the epithelial cell basement membrane (basolateral), similar to the living kidney.
  • Na+/K+ ATPase in the treatment groups (PFOA, PFDA) appears to be expressed in the cytosol of the organoids.
  • PFDA a long chain PFAC and is known to be more toxic than PFOA
  • kidney proximal tubule organoid of the present invention appears to have differences in the expression of Na+/K+ ATPase due to drug treatment, and a change in the translocation of Na+/K+ ATPase from the membrane to the cytosol. It can also be observed.
  • the kidney proximal tubule organoid of the present invention can predict how a target drug affects electrolyte and liquid homeostasis in the kidney during kidney function through changes in Na+/K+ ATPase.
  • Figure 7 is a microscopic image of drug evaluation based on E-cadherin expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • the drugs treated with the organoids were PFOA and PFDA, each treated at 10 ⁇ M for 21 days.
  • both the control group (vehicle CTL) and the treatment group (PFOA, PFDA) appear to express E-cadherin.
  • the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses E-cadherin, which means that it can mimic (mimicking) the pathophysiological functions related to epithelial cells in the kidney and their EMT. .
  • E-cadherin in the control group (vehicle CTL) appears to be expressed in the epithelial cell layer as in the living kidney.
  • the amount of E-cadherin expression in PFOA was reduced compared to the control group, and among the treatment groups, PFDA was clearly expressed in the cytosol of the organoid, clearly showing that the function of E-cadherin was lost.
  • the amount of E-cadherin expressed on the cell membrane of normal epithelial cells is reduced by drug treatment, or the amount of E-cadherin expressed on the cell membrane is reduced or translocated from the cell membrane to the cytosol.
  • kidney proximal tubule organoid of the present invention can predict how a target drug affects structural support (adhesion) within kidney epithelial cells and subsequent functions during kidney function through changes in E-cadherin.
  • Figure 8 is a microscopic image of drug evaluation based on 8-OHdG expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • the drug treated with the organoids was PFOA, one of the banned perfluorinated compounds, and was treated at 10 ⁇ M for 21 days.
  • kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses 8-OHdG, thereby mimicking the pathophysiological functions related to oxidative stress in the kidney.
  • 8-OHdG in the control group (vehicle CTL) appears to be expressed in a very small amount in the epithelial cell layer, as in living kidneys.
  • 8-OHdG in the treatment group (PFOA) was expressed in various locations in the organoid, and its expression level appeared to be increased. That is, the kidney proximal tubule organoids of the present invention appear to have differences in the expression of 8-OHdG depending on drug treatment.
  • the kidney proximal tubule organoid of the present invention can predict (evaluate) the toxicity of a target drug to the kidney through changes in 8-OHdG.
  • Figure 9 is a microscopic image of drug evaluation based on vimentin expression in kidney proximal tubule organoids according to an embodiment of the present invention.
  • the drugs treated with the organoids were PFOA and PFDA, each treated at 10 ⁇ M for 21 days.
  • both the control group (vehicle CTL) and the treatment group (PFOA, PFDA) appear to express vimentin.
  • this may mean that the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses vimentin, thereby mimicking the pathological state of the kidney.
  • Vimentin in the control group (vehicle CTL) appears to be expressed in a very small amount in the epithelial cell layer, similar to the living kidney.
  • vimentin in the treatment groups (PFOA, PFDA) was expressed in various locations in the organoids, and its expression level appeared to be increased. That is, the kidney proximal tubule organoids of the present invention appear to have differences in vimentin expression depending on drug treatment.
  • the kidney proximal tubule organoid of the present invention can predict (evaluate) whether a target drug is toxic to the kidney and the resulting pathological state of the kidney through changes in vimentin.
  • the kidney proximal tubule organoid according to an embodiment of the present invention is not only a structural form but also the characteristics, that is, the expression of biomarkers, are very similar to living kidneys, making it a bio-mimetic model with a higher biocorrespondence. It can be used as.
  • the kidney proximal tubule organoid according to an embodiment of the present invention can be used as a human model to evaluate side effects, toxicity, and effectiveness of drugs, and to determine the safety and effectiveness of candidate substances for toxicity in the process of developing new drugs. It can also be used in experiments. For example, after the kidney proximal tubule organoid of the present invention is reacted with a drug in a plate (container), its condition can be observed microscopically, or changes in it can be confirmed through protein analysis, etc.
  • kidney cancer organoid according to an embodiment of the present invention and a kidney cancer organoid resulting therefrom will be described in detail.
  • Figure 10 exemplarily illustrates a method for producing kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
  • the manufacturing method includes first culturing a medium containing a kidney cancer cell line and human extracellular matrix (ECM) to form a cancer organoid; and a second culture step of mixing the cancer organoids and the growth medium; may include.
  • ECM extracellular matrix
  • medium containing a kidney cancer cell line and human extracellular matrix may be included in a 1:1 ratio.
  • the number of kidney cancer cell lines may be 1 to 5 x 10 5 to 1 to 5 x 10 7 based on 1 mL of medium containing human extracellular matrix, which is ECM for 3D cell culture, but is not limited thereto.
  • a renal cancer cell line is, for example, Caki-1 cell, which is a representative RCC (human clear cell renal cell carcinoma) showing proximal tubular epithelial morphology, but is not limited thereto.
  • the first culturing step may be performed by mixing a medium containing a kidney cancer cell line and a human extracellular matrix.
  • kidney cancer cells Caki-1 were mixed at 1 x 10 6 cells in 10% FBS-DMEM medium containing 1 mL of human extracellular matrix and mixed so that the cells could be distributed. Seed the plate.
  • FBS-DMEM medium containing 1 mL of human extracellular matrix
  • seed the plate According to the characteristics of the human extracellular matrix according to the present invention, floating cells fuse with each other, tight junctions and self-organization occur, and organoids are formed within 3 days of culture. Replace the medium with fresh medium once every 3 days until the cancer organoids become homogeneous at 100 um (about 7 days).
  • the first culturing step may be performed for at least one period of about 1 hour to 3 days, but is not limited thereto.
  • conventional organoid formation takes several weeks or more.
  • the method for producing kidney cancer organoids according to an embodiment of the present invention provides the formation of kidney cancer organoids within a short time of less than 3 days.
  • the organoids formed by the first culturing step may have a size of 50 - 200 ⁇ m and each size and maturity may be different, so in experimental or clinical drug (compound) evaluation, the organoids
  • the medium can be replaced once every 3 days for a second culture within 50 days.
  • the second culturing step may be performed for at least one period of about 3 to 50 days, but is not limited thereto.
  • the first culturing step and the second culturing step may be performed sequentially or simultaneously.
  • the culture medium used in the first or second culturing step may be a growth medium. It can be artificially synthesized and used, or a commercially prepared medium can be used.
  • medium, DMEM), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199, and RPMI 1640 may include at least one of various serum-free media and variants thereof.
  • the growth medium may additionally include at least one of the following amino acids: ascorbic acid, acetic acid, FBS, B27, and IWR-1.
  • Amino acids necessary for cell growth may further include, but are not limited to, L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, or L-glutamine. .
  • L-Glutamine can be replaced with Glutamax.
  • the growth medium may additionally include antibiotics.
  • the volume ratio of human extracellular matrix and growth medium may be 1:4 to 1:6, preferably 1:5, but is not limited thereto.
  • the second culturing step may further include treating the drug.
  • the organoids can be sorted into one or more different containers to perform a second culture, and at the same time, the organoids can be cultured by adding a drug to a specific container. At this time, it may be desirable to perform drug treatment (treatment group) on the 7th day of culture.
  • T0 in FIG. 12 may be a kidney cancer organoid on day 7 in the second culture, but is not limited thereto.
  • Organoids in secondary culture can be used for various periods of time depending on the user's experimental purpose and plan. For example, if evaluation of drug efficacy and toxicity during the development of kidney cancer is required, kidney cancer organoids from day 0 of the second culture can be used at T0.
  • the efficacy or toxicity of a drug may be evaluated by comparative analysis of morphological and protein expression for a drug-untreated group (CTL) and a drug-treated group (PFOA, PFDA).
  • Morphological analysis may include, but is not limited to, microscopic structural analysis.
  • Protein expression analysis may be, but is not limited to, analysis based on the level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin.
  • EMT epithelial-mesenchymal transition
  • the biomarker level may be analyzed based on expression level and expression location.
  • treatment with the drug may be performed for the first time within 7 days of culturing the organoid, including the first culture. Specifically, from the time when the organoid size became uniform (around 7 days), kidney cancer organoids were grown by mixing the medium containing 10 ⁇ M PFOA or PFDA and exposing it to the medium for 21 days, replacing it every 3 days. Afterwards, it was stored in an LN2 tank after a freezing process so that it could be thawed and used.
  • Hematoxylin & Eosin staining is the most commonly used staining technique for microscopic observation of tissue or cell samples. This technique is widely used in pathology and is used to visualize the morphology and structure of organoids.
  • Figure 13 shows H&E staining of kidney cancer organoids and drug-treated kidney cancer organoids according to an embodiment of the present invention. It shows a microscope image. Specifically, referring to Figure 13, the structure of the kidney cancer organoid can be confirmed through paraffin sectioning and H&E staining.
  • the efficacy or toxicity of the drug can be evaluated by checking the change in the shape of the organoid according to the drug.
  • F-actin staining which is present in all cells, changes in cytoskeleton morphology of kidney cancer organoids treated with the control group and kidney cancer organoids treated with test substances can be analyzed.
  • the role of F-actin in cancer tissue is very important. Dynamic reorganization of F-actin found in cancer cells plays an important role in cancer cell migration, invasion, and metastasis.
  • Figure 14 shows F-actin staining microscopy images and quantification data using a phalloidin staining protocol for kidney cancer organoids according to an embodiment of the present invention.
  • kidney cancer organoids (CTL) treated with vehicle control and kidney cancer organoids (PFOA) kidney cancer organoids
  • CTL kidney cancer organoids
  • PFOA kidney cancer organoids
  • the cytoskeleton of kidney cancer organoids treated with vehicle control was expressed as a normal straight skeleton, but in kidney cancer organoids treated with test drug (PFOA), F-actin was abnormal.
  • An increase in spots in the cytoskeleton can be observed due to development.
  • kidney cancer organoids treated with PFDA the amount of cytoskeletal expression was significantly reduced compared to the vehicle control. This shows that the cytoskeleton, which plays an important role in cells, is a household chemical and that low-concentration and long-term exposure to perfluorinated compounds, which are known to have the potential to cause kidney cancer, affects the skeleton of cancer cells.
  • Na+/K+ ATPase analysis was performed on the kidney cancer organoid prepared in Example 2 using the phalloidin staining protocol.
  • Figure 15 shows changes in Na+/K+ ATPase in kidney cancer organoids and kidney cancer organoids exposed to drugs according to an embodiment of the present invention.
  • Na+/K+ ATPase one of the important biomarkers in the kidney, is expressed in kidney cancer organoids (PFOA, PFDA) treated with cancer activators, but Na+/K+ ATPase expressed in normal kidneys It can be confirmed that it is not mainly expressed in the cell membrane, which is the location of , but is expressed in a form translocated to the cytosol. This phenomenon is especially noticeable when cancer metastasis is actively occurring.
  • kidney cancer organoids As a result of analyzing Na+/K+ ATPase expressed in kidney cancer organoids, depending on the drug (PFOA, PFDA) treated at 10 ⁇ M for 21 days, the expression location of Na+/K+ ATPase changes from the cell membrane to the cytoplasm or the expression level itself decreases. You can get results that work. In particular, a decrease in the expression level or a change in the location of Na+/K+ ATPase in the kidney increases the metastasis of kidney cancer, so the kidney cancer organoid of the present invention can be used as a tool to evaluate the efficacy of drugs and the effect of chemicals on cancer growth. there is.
  • E-cadherin immunostaining was performed on the kidney cancer organoid prepared in Example 2 to analyze the expression of E-cadherin, one of the kidney cancer biomarkers.
  • Caki-1 an RCC-derived kidney cancer cell line
  • Caki-1 cell-derived kidney cancer organoids characteristically express E-cadherin, which is normally expressed in epithelial cells.
  • E-cadherin is one of the important biomarkers expressed in most epithelial cells.
  • EMT epithelial to mesenchymal transition
  • kidney cancer organoids PFOA
  • PFOA kidney cancer organoids
  • the expression location was relocated from the cell membrane to the cytoplasm. Accordingly, by using the kidney cancer organoid of the present invention to confirm the increase/decrease and translocation expression of E-cadherin due to drug treatment, the effect on the drug or cancer growth can be evaluated.
  • EMT-related protein expression analysis including Vimentin expression, was performed on the kidney cancer organoid prepared in Example 2.
  • Vimentin is one of the important biomarkers that is increased when epithelial-mesenchymal transition (EMT) is activated. In particular, Vimentin expression increases when cancer metastasis is activated.
  • EMT epithelial-mesenchymal transition
  • RCC-derived kidney cancer organoids increase Vimentin expression when exposed to the cancer activator (PFOA) at low concentration and for a long period of time. Accordingly, the effect of drugs or chemicals on cancer growth can be evaluated using the kidney cancer organoid according to the present invention through analysis of the expression of EMT-related proteins, including Vimentin.
  • PFOA cancer activator
  • Figure 19 exemplarily illustrates a method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
  • the manufacturing method includes first culturing a mixture of a medium containing a thyroid cancer cell line and human extracellular matrix (ECM) to form an organoid; and a second culture step of mixing the organoids and growth medium; may include.
  • ECM human extracellular matrix
  • thyroid cancer cell lines and human extracellular matrix may be included in a 1:1 ratio.
  • the thyroid cancer cell line may be included in the number of 1 to 5 x 10 5 to 1 to 5 x 10 7 cells, but is not limited thereto.
  • the route such as obtaining the thyroid cancer cell line directly by separating it from a patient-derived thyroid cancer tissue or tumor or purchasing a commercial cancer cell line.
  • the thyroid cancer cell line may be, for example, but is not limited to TBP-3868, T238, WRO, FTC133, BCPAP, TPC1, K1, 8505C, HTH7, C643, SW1736, MDA-T41 or SNU-790 cell lines. Any thyroid cancer cell line used in the art can be used.
  • the first culturing step may be performed by mixing a medium containing a thyroid cancer cell line and a human extracellular matrix.
  • the first culturing step is to mix medium containing 1 mL of human extracellular matrix and thyroid cancer cells SNU-790 at a cell count of 1 x 10 6 and distribute the cells in 10% FBS-RPMI. Mix well and seed on the plate.
  • the human extracellular matrix according to the present invention, it fuses with floating cells, tight junctions and self-organization occur, and organoids are formed within 3 days of culture. Replace the medium with fresh medium once every 3 days until the cancer organoids become homogeneous at 100 um (about 7 days).
  • the first culturing step may be performed for at least one period of about 1 hour to 3 days, but is not limited thereto.
  • conventional organoid formation takes several weeks or more.
  • the method for producing thyroid cancer organoids according to an embodiment of the present invention provides the formation of thyroid cancer organoids within a short time of less than 3 days.
  • the organoids formed by the first culturing step may have a size of 50 - 200 ⁇ m and each size and maturity may be different, so in experimental or clinical drug (compound) evaluation, the organoids
  • a second culture can be performed by replacing the medium once every 3 days for a period of 50 days.
  • the second culturing step may be performed for at least one period of about 3 to 50 days, but is not limited thereto.
  • the first culturing step and the second culturing step may be performed sequentially or simultaneously.
  • the culture medium used in the first or second culturing step may be a growth medium. It can be artificially synthesized and used, or a commercially prepared medium can be used. For example, basal medium Eagle's (BME), Minimal essential medium (MEM), Eagle's MEM, Dulbecco's modified Eagle's medium, without insulin. It may include at least one of various serum-free media such as DMEM), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199, and RPMI 1640, and their variants.
  • BME basal medium Eagle's
  • MEM Minimal essential medium
  • Eagle's MEM Dulbecco's modified Eagle's medium
  • DMEM serum-free media
  • DMEM/F12 DMEM/F12
  • HAM'S F-10 HAM'S F-10
  • HAM'S F-12 MEDIUM 199
  • RPMI 1640 RPMI 1640
  • the growth medium may additionally include at least one of the following amino acids: ascorbic acid, acetic acid, FBS, B27, and IWR-1.
  • Amino acids necessary for cell growth may further include, but are not limited to, L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, or L-glutamine. .
  • L-Glutamine can be replaced with Glutamax.
  • the growth medium may additionally include antibiotics.
  • the volume ratio of human extracellular matrix and growth medium may be 1:4 to 1:6, preferably 1:5, but is not limited thereto.
  • the second culturing step may further include treating the drug.
  • the organoids can be sorted into one or more different containers to perform a second culture, and at the same time, the organoids can be cultured by adding a drug to a specific container. At this time, it may be desirable to perform drug treatment (treatment group) on the 7th day of culture.
  • T0 in FIG. 21 may be a thyroid cancer organoid on day 7 in the second culture, but is not limited thereto.
  • Organoids in secondary culture can be used for various periods of time depending on the user's experimental purpose and plan. For example, if evaluation of drug efficacy and toxicity during the development of thyroid cancer is required, thyroid cancer organoids from day 0 of the second culture can be used at T0.
  • the efficacy or toxicity of a drug may be evaluated by comparative analysis of morphological and protein expression for a drug-untreated group (CTL) and a drug-treated group (PFOA, PFDA).
  • Morphological analysis may include, but is not limited to, microscopic structural analysis.
  • Protein expression analysis includes at least one biomarker selected from the group consisting of F-actin abnormality, thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. The analysis may be based on the marker level, that is, the expression level and expression location of the biomarker, but is not limited thereto.
  • drug treatment may be performed for the first time within 7 days of culturing the organoid, including the first culture. Specifically, from the time when the organoid size becomes uniform (around 7 days), PFOA or PFDA, which has been used as a coating for waterproofing for a long time but is currently banned due to its proven toxicity, is mixed with new medium at a concentration of 10 ⁇ M and then once every 3 days. Thyroid cancer organoids were maintained by replacing them and exposing them to the compound for 21 days.
  • thyroid cancer organoids exposed to various compounds for a long period of time were fixed with 4% paraformaldehyde for morphology and biomarker analysis, and slides were made using paraffin. For quantitative analysis, the remaining part was transferred to an RNAase free tube, went through a freezing process, and was stored in a -80 1 ⁇ 4 C deepfreezer.
  • Hematoxylin & Eosin staining is one of the most commonly used staining techniques for microscopic observation of tissue or cell samples. This technique is widely used in pathology and is used to visualize the morphology and structure of organoids.
  • Figure 22 shows H&E staining of thyroid cancer organoids according to an embodiment of the present invention. It shows a microscope image. Specifically, referring to Figure 22, the lumen of the thyroid cancer organoid can be confirmed through paraffin sectioning and H&E staining.
  • Staining nuclei using Hoechst33342 in cancer organoid research has various advantages.
  • Hoechst33342 is a fluorescent dye, it can clearly identify and visualize the nucleus of a cell through a microscope, which is useful for evaluating the structure and arrangement of the cell, nuclear division, etc.
  • Hoechst33342 staining allows assessing the state of the nucleus at each stage of the cell cycle, which is especially important in studies related to cell division.
  • the number of cells within cancer organoids can be quantitatively analyzed using Hoechst 33342. This allows us to evaluate how certain drug treatments or conditions affect the growth of cancer organoids.
  • cell death can be assessed by identifying abnormal nuclear morphology or organization through Hoechst 33342 staining.
  • Figure 23 shows a Hoechst33342 staining microscope image of a thyroid cancer organoid according to an embodiment of the present invention.
  • thyroid cancer organoids CTL
  • PFOA PFOA
  • PFDA PFDA
  • Figure 23 is a Z-stack image of the shape of a thyroid cancer organoid stained with Hoechst33342 using a confocal microscope. Therefore, the effect of drugs on thyroid cancer can be analyzed through analysis of the shape changes and nuclear shape changes of thyroid cancer that can be observed in these images. Impact can be assessed.
  • F-actin staining which is present in all cells, morphological changes in thyroid cancer organoids treated with vehicle control and thyroid cancer organoids treated with drugs can be analyzed.
  • the role of F-actin in cancer tissue is very important. Dynamic reorganization of F-actin found in cancer cells plays an important role in cancer cell migration, invasion, and metastasis.
  • Figure 24 shows a microscopic image of F-actin staining using a phalloidin staining protocol for thyroid cancer organoids according to an embodiment of the present invention.
  • the cytoskeletal form is confirmed through the above-mentioned F-actin abnormality analysis, and the thyroid cancer organoid model according to the present invention can be used to determine the efficacy of new drugs and Toxicity can be assessed.
  • Thyroid stimulating hormone receptor (THSR) expression analysis was performed on the thyroid cancer organoid prepared in Example 7 using the TSHR immunostaining protocol.
  • FIGS 25a and 25b show changes in thyroid stimulating hormone receptor (TSHR) in thyroid cancer organoids according to an embodiment of the present invention.
  • TSHR thyroid stimulating hormone receptor
  • PFOA or PFDA was mixed with new medium at a concentration of 10 ⁇ M and then changed once every 3 days and exposed to the compound for 21 days. Changes in the expression of TSHR can be confirmed. Because the expression of TSHR is related to the progression of thyroid cancer, when TSHR expression is reduced, cancer progression accelerates and thyroid cancer metastases easily. Based on this, the present invention analyzed the difference in TSHR expression when thyroid cancer organoids were exposed to a control drug and a perfluorinated compound at low concentration and for a long period of time. As a result, TSHR expression was decreased in thyroid cancer organoids exposed to perfluorinated compounds, revealing that perfluorinated compounds affect the proliferation and metastasis of thyroid cancer.
  • CTL thyroid cancer organoids
  • the effect of the drug on cancer can be evaluated.
  • thyroid tumor-stimulating hormone receptor (TSHR) is normally expressed in the basal portion of thyroid follicular epithelial cells.
  • PFOA or PFDA was mixed with new medium at a concentration of 10 ⁇ M, replaced once every 3 days, and exposed to the compound for 21 days. After that, the TSHR expression site was translocated to intracellular vesicles. You can check that. In particular, the changes were evident in thyroid cancer organoids exposed to the banned compound PFOA.
  • the effect of the drug on cancer can be evaluated by confirming the location of TSHR expression by drug treatment using the thyroid cancer organoid of the present invention in which TSHR is expressed.
  • Tg Thyroglobulin
  • Thyroglobulin (Tg) expression analysis one of the thyroid cancer biomarkers, was performed on the thyroid cancer organoid prepared in Example 7 using the Tg immunostaining protocol.
  • Thyroglobulin is a protein secreted only from normal thyroid tissue and thyroid cancer tissue and is one of the thyroid and thyroid cancer specific biomarkers.
  • the thyroid cancer organoid according to the present invention clearly expressed Tg (thyroglobulin), mimicking the characteristics of human thyroid cancer.
  • Tg expression in thyroid cancer organoids was increased as PFOA or PFDA was mixed in new medium at a concentration of 10 ⁇ M and exposed to the compound for 21 days, replaced once every 3 days, which was found to be involved in activating hormone secretion in thyroid cancer. This suggests that there is a possibility of causing secondary cell proliferation.
  • the activity or inhibition of thyroid cancer for the test drug can be predicted by analyzing the increase or decrease in Tg expression level in the thyroid cancer organoid of the present invention.
  • TPO Tyroperoxidase
  • Thyroperoxidase (TPO) expression one of the thyroid cancer biomarkers, was analyzed for the thyroid cancer organoid prepared in Example 7 using the TPO immunostaining protocol.
  • Thyroperoxidase is an enzyme involved in thyroid hormone synthesis. It catalyzes the oxidation of iodide from the tyrosine residue of thyroglobulin to synthesize T3 (Triiodothyronine) and T4 (Tetraiodothyronine; Thyroxine). Increased expression of TPO in the thyroid gland can indirectly predict the possibility of promoting thyroid cancer.
  • TPO expression was clearly observed in the thyroid cancer organoid according to the present invention, mimicking the characteristics of human thyroid cancer.
  • TPO expression in thyroid cancer organoids increased as PFOA or PFDA, which are hazardous substances in daily life, were mixed into new medium at a concentration of 10 ⁇ M, replaced every three days, and exposed to the compounds for 21 days.
  • PFOA or PFDA which are hazardous substances in daily life
  • E-cadherin immunostaining was performed on the thyroid cancer organoid prepared in Example 7 to analyze the expression of E-cadherin, one of the thyroid cancer biomarkers.
  • E-cadherin is one of the important biomarkers expressed in most epithelial cells.
  • epithelial-mesenchymal transition EMT
  • pathological phenomena are observed. These changes are mainly found during fibrosis or cancer invasion and metastasis, and a decrease in E-cadherin can be expected to indicate that these pathological phenomena occur and changes in the function and structure of thyroid epithelial cells occur.
  • the thyroid cancer organoid of the present invention was exposed to the compound for 21 days by mixing PFOA or PFDA, a household hazardous substance, in a new medium at a concentration of 10 ⁇ M and replacing it every 3 days. Afterwards, the level of E-cadherin expression was analyzed through immunostaining, and a decrease in E-cadherin expression was confirmed in thyroid cancer organoids exposed to low concentration and long-term exposure to PFOA, which is banned as a harmful substance.
  • the effect of the drug on cancer can be evaluated.
  • E-cadherin normally exists in the cell membrane and performs its function, but when signals such as cancer metastasis are strengthened, E-cadherin expression is transferred from the cell membrane to the cytosol.
  • FIG 28b it can be seen that when treated with PFOA or PFDA, which are hazardous substances in life, at a concentration of 10 ⁇ M, the E-cadherin expression location is translocated to the cytoplasm or cell membrane.
  • PFOA or PFDA a household hazardous substance, is mixed into new medium at a concentration of 10 ⁇ M, replaced once every 3 days, and E-cadherin expression by exposure to the compound for 21 days.
  • the effect of the drug on cancer can be evaluated by confirming the change in the expression location of E-cadherin due to drug treatment using the thyroid cancer organoid of the present invention.
  • Figure 30 illustrates an exemplary method for producing thyroid organoids for evaluating the efficacy or toxicity of test substances according to an embodiment of the present invention.
  • the manufacturing method includes first culturing a mixture of a medium containing a thyroid cell line and human extracellular matrix (ECM) to form an organoid; and secondly culturing the organoids by mixing them with a thyroid organoid culture medium containing Thyroid-Stimulating Hormone and Potassium Iodide; may include.
  • ECM human extracellular matrix
  • thyroid cells fuse with each other and the human extracellular matrix, which is an ECM (Org 3D culture solution) for three-dimensional cell culture developed by the inventors of the present invention, forming tight junctions while the distinction between cells disappears. ) occurs, and self-organization occurs, forming organoids.
  • ECM Organic 3D culture solution
  • organoids that most closely mimic the in vivo environment can be stably cultured.
  • the first culturing step may be performed for at least one period of about 1 hour to 3 days, and the second culturing step may be performed for at least one of about 3 days to 50 days. .
  • the first culturing step and the second culturing step may be performed sequentially or simultaneously.
  • thyroid organoids were formed with a size of about 50-200 um within 3 days. Therefore, to evaluate the efficacy or toxicity of a test substance using thyroid organoids, treatment of the test substance can be performed for the first time within 7 days of organoid culture. Cultivation of thyroid organoids treated with test substances will be described later with reference to FIG. 32.
  • thyroid cell lines may be normal thyroid cells or normal thyroid epithelial cells.
  • medium containing a thyroid cell line and human extracellular matrix may be included in a 1:1 ratio.
  • the thyroid cell line may be included in the number of 1 to 5 x 10 5 to 1 to 5 x 10 7 cells, but is not limited thereto.
  • thyroid organoids 1 mL of medium containing human extracellular matrix was mixed with thyroid cells H6040 or Nthy-ori3-1 at a cell number of 1 x 10 6 .
  • thyroid cells self-organized for a short period of time within 3 days to form organoids.
  • the organoids formed through the first culture may be cultured a second time by mixing them with a thyroid organoid culture medium containing thyroid-stimulating hormone and potassium iodide to increase the degree of mimicry of the thyroid gland, which is an endocrine organ.
  • the thyroid organoid culture medium can be replaced every three days during the culture period.
  • the medium or culture solution used in the first and second culturing steps may be artificially synthesized or commercially produced.
  • basal medium Eagle's (BME) without insulin Minimal essential medium (MEM), Eagle's MEM, Dulbecco's modified Eagle's medium. , DMEM), Ham's F12, SF 12, and RPMI 1640, and may include at least one of various serum-free media and their variants. It may also further include FBS (Fetal bovine serum). Preferably, it may include FBS-RPMI, FBS-DMEM, and epithelial cell medium (EpiCM), but is not limited thereto.
  • FBS Fetal bovine serum
  • the thyroid cell line culture medium may additionally include, but is not limited to, at least one of amino acids, acetic acid, glutamax, and ascorbic acid.
  • Amino acids necessary for cell growth may further include L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, or L-glutamine.
  • Thyroid-Stimulating Hormone (TSH) contained in the thyroid organoid culture medium may be included in an amount of 0.01 to 1 mU/mL, preferably 0.05 to 0.5 mU/mL, based on the total amount of the thyroid organoid culture medium. mU/mL may be included.
  • TSH Thyroid-Stimulating Hormone
  • potassium iodide (PI) contained in the thyroid organoid culture medium may be included in an amount of 1 to 20 nM, preferably 5 to 15 nM, in the total amount of the thyroid organoid culture medium.
  • PI potassium iodide
  • the H6040 cell-derived organoids formed in the first culturing step were cultured a second time for 30 days in epithelial cell medium supplemented with 0.1 mU/mL TSH and 10 nM concentration of PI.
  • Nthy-ori3-1 cell-derived organoids were cultured a second time for 30 days in 10% FBS-RPM medium supplemented with 0.1 mU/mL TSH and 10 nM concentration of PI.
  • the human thyroid organoid after producing a human thyroid organoid, the human thyroid organoid was planted in a paraffin block and a section was made with a thickness of 4 ⁇ m, followed by Hematoxylin & Eosin (H&E) staining.
  • H&E staining is one of the most common methods used for histological analysis of various organs.
  • FIG 31 shows a microscope image of a thyroid organoid prepared by a manufacturing method according to an embodiment of the present invention.
  • the thyroid organoid of the present invention appears to have a round spherical three-dimensional form like a follicle containing a colloid and an epithelial cell layer surrounding the colloid. Since it has a structure similar to that of human thyroid tissue, it has been verified to be morphologically similar to living organisms.
  • a method of producing thyroid organoids for evaluating the efficacy or toxicity of a drug includes processing the drug in a second culturing step; It may further include. Additionally, the drug treatment step can be performed for the first time within 7 days of organoid culture.
  • the organoids formed in the first culturing step can be treated with the desired drug in the second culturing step where the organoids are mixed with the thyroid organoid culture medium.
  • the mixture of thyroid organoid culture medium and drug was replaced every three days during the culture period, and the old medium exposed to the organoid was collected to analyze changes in thyroid hormones according to the drug and stored at -70°C or below. keep it.
  • Thyroid organoids exposed to low concentrations of endocrine disrupting chemicals (EDC) for a long period of time were prepared by culturing for 30 days, changing the culture medium once every three days. Specifically, during long-term exposure to low concentrations of EDC, new EDC was added to fresh media once every three days, and then all old media was removed and fresh media was added, resulting in long-term exposure.
  • EDC endocrine disrupting chemicals
  • the thyroid organoid culture medium treated with BHA or BPA was collected and replaced every three days, stored at -70°C or lower, and then slowly thawed at 4°C. Afterwards, to remove possible cell debris, the tube was spun down at 1000 rpm for 1 minute, and only the supernatant was transferred to a new tube, and thyroid hormone analysis was performed using ELISA.
  • Figure 34 shows a comparison of thyroid hormone changes in thyroid organoids prepared without exposure to drugs and thyroid organoids prepared with low-concentration-long-term exposure to BHA or BPA.
  • Figure 34a shows the results for thyroid organoids derived from Nthy-ori3-1 cells
  • Figure 34b shows the results for thyroid organoids derived from H6040 cells.
  • thyroid organoids derived from Nthy-ori3-1 cells prepared by exposure to 1 ⁇ BHA or 1 ⁇ M BPA have higher levels of thyroid hormones T3 and T4 compared to thyroid organoids (Vehicle CTL) prepared without exposure to drugs. an increase was observed.
  • human thyroid organoids exposed to the drug increase thyroid hormone secretion, which suggests that the EDC drug used in the present invention is likely to cause endocrine disruption and induce human hyperthyroidism. This is a case proven using . Therefore, using these reactions, it is possible to develop new drugs or conduct validation studies on many compounds that have been proven in animals but have not been completely proven to disrupt thyroid hormones in humans.
  • BPA is well known as a representative endocrine disruptor, and it has been reported that it can cause an increase in thyroid hormones in humans.
  • this as a positive control drug and exposing the human thyroid organoid of the present invention to a low concentration for a long period of time it was concluded that thyroid hormone secretion was increased in the human thyroid organoid.
  • BHA which is widely used as an antioxidant used in the present invention, has been reported to inhibit thyroid hormone secretion in rodents, but is a substance that has not yet been clearly proven in humans.
  • thyroid hormone secretion was statistically significantly reduced, proving that it can directly affect the thyroid gland in humans as in rodents. In this way, it is presented as a tool to test in advance what effect a compound or new drug may have on the human thyroid gland.
  • Thyroid-stimulating hormone receptor (THSR) analysis was performed on thyroid organoids prepared by treating perfluorinated compounds using the method shown in Figures 30 and 32, and TSHR analysis, one of the important biomarkers of the thyroid gland, was performed.
  • THSR thyroid-stimulating hormone receptor
  • FIG 35 is an analysis of thyroid-stimulating hormone receptor (TSHR) expression in thyroid organoids prepared by a production method according to another embodiment of the present invention.
  • THSR is one of the important receptors present in the thyroid gland, and is the receptor for Thyroid-stimulating hormone (TSH) secreted by the pituitary.
  • TSH thyroid-stimulating hormone
  • THSR is one of the important receptors present in the thyroid gland, and is the receptor for Thyroid-stimulating hormone (TSH) secreted by the pituitary.
  • TSH combines with TSHR, thyorid growth, thyrocyte differentiation, and thyroid hormone synthesis occur.
  • the present invention can be used to evaluate how chemicals affect the increase or decrease of thyroid hormones.
  • Thyroglobulin one of the thyroid biomarkers, was immunostained to analyze Tg expression on thyroid organoids prepared by treating perfluorinated compounds using the method shown in Figures 30 and 3.
  • Thyroglobulin is a protein secreted only from normal thyroid tissue and thyroid cancer tissue and is a thyroid-specific biomarker.
  • the thyroid organoid according to the present invention clearly showed expression of Tg (thyroglobulin), confirming its similarity to the human thyroid gland. Therefore, it is a tool that can predict whether chemicals or new drugs activate or inhibit human thyroid function by measuring changes in Tg expression after exposing various endocrine disruptors or new drugs to the human thyroid organiode of the present invention. You can utilize it.
  • Tg thyroglobulin
  • TPO Tyroperoxidase
  • Thyroperoxidase (TPO) immunostaining one of the thyroid biomarkers, was performed on thyroid organoids prepared by treating perfluorinated compounds using the method shown in Figures 30 and 32 to analyze TPO expression.
  • Thyroperoxidase is an enzyme involved in thyroid hormone synthesis. It catalyzes the oxidation of iodide from the tyrosine residue of thyroglobulin to synthesize T3 (Triiodothyronine) and T4 (Tetraiodothyronine; Thyroxine). It is known that increased expression of TPO in the thyroid gland is an indicator that abnormalities in thyroid function have occurred.
  • TPO expression was confirmed in the thyroid organoid according to the present invention, it was confirmed that there was similarity to the human thyroid gland.
  • TPO expression in the prepared thyroid organoids was increased or decreased as a result of exposing human thyroid organoids at low concentrations and for a long period of time to perfluorinated compounds, which were used as coating agents for waterproofing but were banned for use due to confirmed toxicity. Accordingly, the present invention Using thyroid organoids, test substances that activate or inhibit thyroid function can be predicted.
  • E-cadherin immunostaining was performed on thyroid organoids prepared by treating perfluorinated compounds using the method shown in Figures 30 and 32 to analyze the expression of E-cadherin, one of the thyroid biomarkers.
  • E-cadherin is one of the important biomarkers expressed in most epithelial cells. In particular, it is expressed in thyroid follicles and is one of the proteins robustly expressed in normal tissues, playing an important role in thyroid cell adhesion and tissue structure. It is known that the expression pattern of E-cadherin changes when pathological conditions such as thyroid disease, especially cancer, occur.

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Abstract

The present invention relates to a renal proximal tubule organoid, a kidney cancer organoid, a thyroid organoid or a thyroid cancer organoid, a preparation method therefor, and a drug evaluation method using same.

Description

신장 근위 세뇨관 오가노이드, 신장암 오가노이드, 갑상선 오가노이드 또는 갑상선암 오가노이드, 이의 제조방법 및 이를 이용한 약물 평가 방법Kidney proximal tubule organoid, kidney cancer organoid, thyroid organoid or thyroid cancer organoid, method for producing the same, and drug evaluation method using the same
본 발명은 생체 신장 근위 세뇨관과 유사한 구조 및 기능을 가지는 오가노이드에 관한 것으로, 보다 구체적으로, 내강 및 내강을 감싸고 있는 상피세포층을 포함하고 둥근 구상의 입체적 형태를 가지고, 신장 근위 세뇨관 상피세포주 및 인체 세포외기질 기반의 신장 근위 세뇨관 오가노이드, 이의 제조 방법 및 이를 이용한 약물 평가 방법에 관한 것이다.The present invention relates to an organoid having a structure and function similar to that of a living kidney proximal tubule, and more specifically, to an organoid that includes a lumen and an epithelial cell layer surrounding the lumen, has a round spherical three-dimensional shape, and is used in renal proximal tubule epithelial cell lines and human bodies. It relates to extracellular matrix-based renal proximal tubule organoids, methods for producing them, and drug evaluation methods using them.
본 발명은 세포주 유래 신장암 오가노이드, 이의 제조 방법 및 이를 활용한 약물의 효능 또는 독성 평가기술에 관한 것이다. The present invention relates to cell line-derived kidney cancer organoids, methods for producing the same, and techniques for evaluating the efficacy or toxicity of drugs using the same.
본 발명은 세포주 기반 갑상선암 오가노이드, 이의 제조 방법 및 이를 활용한 시험물질의 효능 또는 독성 평가기술에 관한 것이다. The present invention relates to cell line-based thyroid cancer organoids, methods for producing the same, and techniques for evaluating the efficacy or toxicity of test substances using the same.
본 발명은 세포주 기반 갑상선 오가노이드, 이의 제조 방법 및 이를 활용한 시험물질의 효능 또는 독성 평가기술에 관한 것이다. The present invention relates to cell line-based thyroid organoids, methods for producing the same, and techniques for evaluating the efficacy or toxicity of test substances using the same.
신약개발의 초기 단계에서는 정확한 독성 및 유효성 예측을 평가하는 모델이 필요하다. 현 기술로는 동물 모델이 신약의 독성 및 유효성에 대하여 가장 유사하게 모사할 수 있다. 그러나, 동물 실험은 시간, 금전적으로 부담이 크며 종 간의 유전적, 생화학적 및 대사과정의 차이로 인하여 인간의 생체 내 환경을 완전히 반영하기 어렵다. 또한, 기술적으로도 동물 내부에서 일어나는 일을 모니터링하는 것이 어렵고 윤리적으로도 문제가 될 수 있다. In the early stages of new drug development, a model is needed to accurately predict toxicity and efficacy. With current technology, animal models can most closely simulate the toxicity and effectiveness of new drugs. However, animal experiments are burdensome in terms of time and money, and it is difficult to completely reflect the human in vivo environment due to differences in genetic, biochemical, and metabolic processes between species. Additionally, it can be technically difficult and ethically problematic to monitor what is happening inside the animal.
이에, 인간의 조직에서 직접 분리하여 체외 배양된 일차 배양 세포가 표준 모델로 사용되고 있지만, 조직을 얻기에도 어렵고, 조직 세포가 체외에서 증식하지 못하는 실험적 한계가 있다. 나아가, 2차원 세포 기반 모델(cell based in vitro model)은 비용 및 노동력 측면에서 인간의 조직으로부터 유래된 일차 배양 세포보다 효율적이기 때문에 약물 독성 및 유효성 평가에 많이 사용되고 있다. 그러나, 2차원 세포 기반 모델은 세포-세포 및 세포-세포외 기질의 상호 작용으로 발생하는 생리 기능과 조직 복합성을 구현하기엔 불충분하다.Accordingly, primary cultured cells directly isolated from human tissues and cultured in vitro are used as a standard model, but there are experimental limitations in that it is difficult to obtain tissues and tissue cells cannot proliferate in vitro. Furthermore, two-dimensional cell-based in vitro models are widely used to evaluate drug toxicity and effectiveness because they are more efficient than primary cultured cells derived from human tissues in terms of cost and labor. However, two-dimensional cell-based models are insufficient to embody the physiological functions and tissue complexity that arise from cell-cell and cell-extracellular matrix interactions.
한편, 새로운 인체 모사 모델로 오가노이드가 주목을 받고 있다. 오가노이드란 줄기세포를 특정 세포로 자라게 하여 장기와 같은 입체 구조물로 형성된다. 오가노이드는 2차원의 세포 기반 모델과는 다르게 3차원의 환경에서 배양되어, 보다 장기간으로 배양될 수 있다. 또한, 오가노이드는 크기가 작을 뿐 구성 세포나 구조가 실제 장기와 흡사하다. 이에, 오가노이드는 신약 개발 과정에서 약물의 효능과 안정성을 알아보기에 최적인 실험체로 평가되고 있다. 나아가, 오가노이드 관련 분야는 신약 개발의 약물 독성 및 유효성 평가뿐만 아니라, 질병모델, 암 연구, 맞춤의학 및 재생 치료제 등에도 이용될 수 있는 잠재력이 높은 분야이다. Meanwhile, organoids are attracting attention as a new human body simulation model. Organoids are formed by growing stem cells into specific cells to form three-dimensional structures such as organs. Organoids, unlike two-dimensional cell-based models, are cultured in a three-dimensional environment and can be cultured for a longer period of time. In addition, organoids are only small in size, but their constituent cells and structure are similar to actual organs. Accordingly, organoids are evaluated as the optimal test object for examining the efficacy and stability of drugs in the process of developing new drugs. Furthermore, the organoid-related field is a field with high potential that can be used not only for drug toxicity and efficacy evaluation in new drug development, but also for disease models, cancer research, personalized medicine, and regenerative treatments.
현재까지 위, 장, 초기 간, 갑상선, 폐, 뇌 등의 다양한 오가노이드가 성공적으로 개발되었다. 그러나, 현재까지 개발된 신장 오가노이드는 이를 구성하는 세포의 성숙도 및 특성이 신체 내 신장 세포와 비교하여 미성숙한 상태이다. 보다 구체적으로, 종래의 신장 오가노이드를 구성하는 세포들은, 신체 내 신장 세포와 상이한 바이오 마커 발현을 가지며 이에 따라, 신체 내 신장과 다른 특성을 가질 수 있다. 즉, 종래의 신장 오가노이드는 전술한 바와 같이 신체 내 기관과 상이한 특성을 가짐에 따라, 오가노이드로써 약물 및 독성 평가 등에 생체 신장을 대변하지 못하는 한계를 가진다. To date, various organoids have been successfully developed, including stomach, intestine, early liver, thyroid, lung, and brain. However, the maturity and characteristics of the kidney organoids developed to date are immature compared to kidney cells in the body. More specifically, the cells that make up a conventional kidney organoid may have different biomarker expression than kidney cells in the body and thus have different properties than the kidney in the body. In other words, since conventional kidney organoids have different characteristics from organs in the body as described above, they have limitations in that they cannot represent living kidneys in drug and toxicity evaluations as organoids.
결국, 생체 신장과 유사 또는 동일한 바이오마커의 발현을 가지고, 구조 및 형태학적으로도 유사하게 모사할 수 있는 신장 오가노이드의 개발이 필요한 실정이다. Ultimately, there is a need to develop kidney organoids that have similar or identical expression of biomarkers to living kidneys and can mimic structurally and morphologically.
발명의 배경이 되는 기술은 본 발명에 대한 이해를 보다 용이하게 하기 위해 작성되었다. 발명의 배경이 되는 기술에 기재된 사항들이 선행기술로 존재한다고 인정하는 것으로 이해되어서는 안 된다. The technology behind the invention has been written to facilitate easier understanding of the invention. It should not be understood as an admission that matters described in the technology underlying the invention exist as prior art.
종래에는 줄기세포 유래 또는 iPSC 유래의 세포들에 의하여 형성된 신장 오가노이드들이 제공되어 왔다. 그러나, 줄기세포는 윤리적으로 이슈가 되고 있음에 따라, 이를 활용하기 위해서는 연구윤리위원회(IRB)의 승인을 받은 후 활용이 가능하여 많은 시간이 소요되는 단점이 있다. 나아가, 줄기세포는 줄기세포 각각의 차이(variation)이 있음에 따라, 결과에 대한 유사성 보장이 어려웠다. Conventionally, kidney organoids formed by stem cell-derived or iPSC-derived cells have been provided. However, as stem cells have become an ethical issue, their use can only be done after obtaining approval from the Research Ethics Committee (IRB), which has the disadvantage of requiring a lot of time. Furthermore, as stem cells each have variations, it was difficult to guarantee similarity in results.
더 나아가, 줄기세포(iPSC 유래의 세포)를 통한 오가노이드 제작은 세포 생산의 시간이 많이 소요되며, 실험자의 숙련도에 따라 생산되는 세포의 질(quality)가 매우 다를 수 있다. 또한, 줄기세포를 특정 계통으로 분화시키기 위해서는 일반적인 세포 배양보다 많은 시간 및 물질이 요구됨에 따라, 이에 따른 비용이 많이 요구된다. 예를 들어, 줄기세포를 통한 오가노이즈 제작에는 마트리겔(Matrigel)이나 하이드로겔(hydrogel)과 같은 물질이 이용되는데, 마트리겔 및 하이드로겔이 이용될 경우, 세포 배양 및 스페로이드(오가노이드) 형성에 많은 시간이 소요된다. Furthermore, organoid production using stem cells (iPSC-derived cells) takes a lot of time to produce cells, and the quality of the cells produced can be very different depending on the skill of the experimenter. In addition, differentiating stem cells into specific lineages requires more time and materials than general cell culture, resulting in high costs. For example, materials such as Matrigel or hydrogel are used to produce organoids using stem cells. When Matrigel and hydrogel are used, cell culture and spheroid (organoid) formation are carried out. It takes a lot of time.
나아가, 오가노이드의 형성은 배양의 환경 및 조성에 매우 예민하여, 종래에는 제작 및 실험자의 숙련도 및 환경에 따라, 모양 및 성숙도가 매우 상이한 오가노이드가 제공되어 왔다. Furthermore, the formation of organoids is very sensitive to the environment and composition of the culture, and organoids with very different shapes and levels of maturity have conventionally been provided depending on the skill level and environment of the fabricator and experimenter.
즉, 종래에는 오가노이드의 기초가 되는 재료(줄기세포)의 민감성 및 이에 따른 배양 기간에 의하여 동질성의 균일한 오가노이드 제공이 어려움에 따라, 신장 오가노이드를 통한 약물 효능 및 독성 시험 또한, 동일한 실험 결과를 도출하기 어려웠다. That is, in the past, it was difficult to provide homogeneous and uniform organoids due to the sensitivity of the basic material (stem cells) of organoids and the resulting culture period, so drug efficacy and toxicity tests using kidney organoids were also conducted in the same experiment. It was difficult to derive results.
이에, 신장 오가노이드를 통한 다양한 실험은 매우 낮은 신뢰도를 가지며, 실제 약물 및 독성 테스트 등의 물질 유효성 평가에는 이용되지 못하였다. 나아가, 종래의 신장 오가노이드는 균일한 동질성의 오가노이드 확보가 어려움에 따라, 이의 배양 및 제공 기간이 매우 길어 원활한 유통이 어려웠다. Accordingly, various experiments using kidney organoids have very low reliability and have not been used to evaluate the effectiveness of substances such as actual drugs and toxicity tests. Furthermore, with conventional kidney organoids, it was difficult to secure organoids of uniform homogeneity, and the cultivation and provision period was very long, making smooth distribution difficult.
한편, 본 발명의 발명자들은 줄기세포가 아닌 시중의 일반적인 상피세포주를 특정 3차원 배양 환경에서 배양할 경우, 단시간 내에 균일한 오가노이드를 형성할 수 있다는 것을 발견하였으며, 그 결과, 본 발명의 발명자들은 전술한 배양 조건을 통하여, 구조 및 특성(바이오마커)적으로 생체 신장과 유사한 신장 근위 세뇨관 오가노이드, 생체 갑상선과 유사한 갑상선 오가노이드, 암의 구조 및 암 특성을 잘 모사하는 신장암 오가노이드 또는 갑상선암 오가노이드를 개발하였다. Meanwhile, the inventors of the present invention discovered that when culturing commercially available epithelial cell lines, rather than stem cells, in a specific three-dimensional culture environment, uniform organoids can be formed within a short period of time. As a result, the inventors of the present invention discovered that Through the above-mentioned culture conditions, kidney proximal tubule organoids that are similar in structure and characteristics (biomarkers) to living kidneys, thyroid organoids that are similar to living thyroid glands, kidney cancer organoids or thyroid cancers that well mimic the structure and characteristics of cancer. Organoids were developed.
이에, 본 발명이 해결하고자 하는 과제는, 모든 세포주, 줄기세포, 장기 등 종류(species)에 상관없이 적용 가능한 3차원 세포배양용 ECM을 포함하는 배지 (Org 3D culture solution)을 개발하고, 상술한 ECM(세포외기질)은 인체 피부의 섬유아세포로부터 나노 섬유를 포함하는 형태로 개발되어 생체 내 세포외기질과 동일한 형태를 가짐에 따라 생체 내 모사가 뛰어난 스페로이드 또는 오가노이드 배양 환경을 제공함으로써, 이를 활용한 오가노이드 제조 방법을 제공하고, 이를 통하여 생체 신장의 모사도가 높은 신장 근위 세뇨관 오가노이드 및 이에 따른 약물 평가 방법을 제공하는 것이다. 또한 생체 신장암의 모사도가 높은 신장암 세포주 유래 약물의 효능 또는 독성 평가용 신장암 오가노이드 및 이를 활용한 약물 평가 방법을 제공하는 것이다. 또한 생체 갑상선암의 모사도가 높은 갑상선암 오가노이드 및 이를 활용한 약물 평가 방법을 제공하는 것이다. 또한 생체 갑상선의 모사도가 높은 갑상선 오가노이드 및 이에 따른 약물 평가 방법을 제공하는 것이다. Accordingly, the problem to be solved by the present invention is to develop a medium (Org 3D culture solution) containing ECM for 3D cell culture that can be applied regardless of species such as all cell lines, stem cells, and organs, and to ECM (extracellular matrix) was developed in a form containing nanofibers from fibroblasts of human skin and has the same form as the in vivo extracellular matrix, providing a spheroid or organoid culture environment with excellent in vivo mimicry. The aim is to provide a method for manufacturing organoids using this, and through this, to provide kidney proximal tubule organoids with high mimicry of living kidneys and a drug evaluation method accordingly. In addition, we provide a kidney cancer organoid for evaluating the efficacy or toxicity of a drug derived from a kidney cancer cell line that highly mimics living kidney cancer, and a drug evaluation method using the same. In addition, it provides thyroid cancer organoids that highly mimic living thyroid cancer and a drug evaluation method using them. In addition, it provides a thyroid organoid that highly mimics the living thyroid gland and a drug evaluation method accordingly.
본 발명의 과제들은 이상에서 언급한 과제들로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다. The problems of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
신장 근위 세뇨관 오가노이드Kidney proximal tubule organoids
전술한 바와 같은 과제를 해결하기 위해, 본 발명은 신장 근위 세뇨관 상피세포주 및 인체 세포외기질(extracellular matrix, ECM) 기반의 신장 근위 세뇨관 오가노이드(renal proximal tubule organoid)로써, 내강(lumen), 및 내강을 감싸고 있는 상피세포층을 포함하고, 둥근 구상의 입체적 형태인, 신장 근위 세뇨관 오가노이드를 제공한다.In order to solve the problems described above, the present invention is a renal proximal tubule organoid based on a renal proximal tubule epithelial cell line and human extracellular matrix (ECM), which contains a lumen, and Provided are kidney proximal tubular organoids, which contain a layer of epithelial cells lining the lumen and have a round, spherical three-dimensional shape.
본 발명의 특징에 따르면, 상피세포층은, Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin 및 F-actin 중 적어도 하나를 포함할 수 있으나, 이에 제한되는 것은 아니며, 다양한 바이오마커를 더 포함할 수 있다.According to the features of the present invention, the epithelial cell layer may include at least one of Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin, but is not limited thereto, and may contain various biomarkers. More may be included.
본 발명의 다른 특징에 따르면, OAT는, OAT1, OAT2, OAT3, OAT4, OAT5 및 URAT1 중 적어도 하나를 포함할 수 있으나, 이에 제한되는 것은 아니다. According to another feature of the present invention, OAT may include at least one of OAT1, OAT2, OAT3, OAT4, OAT5, and URAT1, but is not limited thereto.
본 발명의 또 다른 특징에 따르면, Na+/K+ ATPase 및 E-cadherin은 약물 처리에 의하여 상피세포의 세포막 또는 세포기질(cytosol)로 이동(translocation) 가능할 수 있다.According to another feature of the present invention, Na+/K+ ATPase and E-cadherin may be translocated to the cell membrane or cytosol of epithelial cells by drug treatment.
본 발명의 또 다른 특징에 따르면, ECM은, 3차원 세포 배양용일 수 있다.According to another feature of the present invention, the ECM may be used for three-dimensional cell culture.
본 발명의 또 다른 특징에 따르면, ECM은, 섬유아세포 패치에 단백질 분해효소를 처리하고, 탈세포화한 후 수득될 수 있으나, 이에 제한되는 것은 아니다. According to another feature of the present invention, ECM can be obtained after treating a fibroblast patch with a proteolytic enzyme and decellularizing it, but is not limited thereto.
본 발명의 또 다른 특징에 따르면, 오가노이드는, 약물의 효능 또는 독성 평가용일 수 있으나, 이에 제한되는 것은 아니며, 재생 의학 등 다양한 약물로도 이용될 수 있다. According to another feature of the present invention, organoids may be used to evaluate the efficacy or toxicity of drugs, but are not limited thereto, and may also be used for various drugs such as regenerative medicine.
나아가, 전술한 바와 같은 과제를 해결하기 위하여 본 발명은, 신장 근위 세뇨관 오가노이드에 약물을 처리하는 단계, 약물이 처리된 오가노이드의 Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin 및 F-actin 중 적어도 하나의 바이오마커에 대한 발현 수준에 기초하여 약물의 효능 또는 독성 여부를 결정하는 단계를 포함하는, 신장 근위 세뇨관 오가노이드를 이용한 약물의 평가 방법을 제공한다.Furthermore, in order to solve the problems described above, the present invention includes the steps of treating a drug in a kidney proximal tubule organoid, Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, and Vimentin of the drug-treated organoid. and determining whether the drug is effective or toxic based on the expression level for at least one biomarker of F-actin.
본 발명의 특징에 따르면, 결정하는 단계는, 바이오마커 발현 수준을 약물 미처리군 또는 대조군(control)에서의 바이오마커 발현 수준과 비교하는 단계를 포함할 수 있으나, 이에 제한되는 것은 아니다.According to a feature of the present invention, the determining step may include comparing the biomarker expression level with the biomarker expression level in a drug-untreated group or control group, but is not limited thereto.
본 발명의 다른 특징에 따르면, 결정하는 단계는, 약물이 처리된 오가노이드의 Na+/K+ ATPase 또는 E-cadherin의 발현 위치에 기초하여, 약물의 효능 또는 독성 여부를 결정하는 단계를 더 포함할 수 있으나, 이에 제한되는 것은 아니다.According to another feature of the present invention, the determining step may further include determining whether the drug is effective or toxic based on the expression location of Na+/K+ ATPase or E-cadherin in the organoid treated with the drug. However, it is not limited to this.
더 나아가, 전술한 바와 같은 과제를 해결하기 위해, 본 발며응ㄴ 신장 근위 세뇨관 오가노이드에 적어도 하나 이상의 약물을 처리하는 단계, 약물이 처리된 오가노이드의 Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin 및 F-actin 중 적어도 하나의 바이오마커에 대한 발현 수준에 기초하여 약물 후보군을 결정하는 단계를 포함하는, 신장 근위 세뇨관 오가노이드를 이용한 약물 스크리닝 방법을 제공한다. Furthermore, in order to solve the above-described problem, the present invention includes treating the kidney proximal tubule organoid with at least one drug, Na+/K+ ATPase, OAT, E-cadherin, Provided is a drug screening method using kidney proximal tubule organoids, comprising the step of determining drug candidates based on the expression level for at least one biomarker among 8-OHdG, Vimentin, and F-actin.
본 발명의 특징에 따르면, 결정하는 단계는, 바이오마커 발현 수준을 약물 미처리군 또는 대조군(control)에서의 바이오마커 발현 수준과 비교하는 단계를 포함할 수 있으나, 이에 제한되는 것은 아니다.According to a feature of the present invention, the determining step may include comparing the biomarker expression level with the biomarker expression level in a drug-untreated group or control group, but is not limited thereto.
본 발명의 다른 특징에 따르면, 결정하는 단계는, 약물이 처리된 오가노이드의 Na+/K+ ATPase 또는 E-cadherin의 발현 위치에 기초하여, 약물의 후보군을 결정하는 단계를 더 포함할 수 있으나, 이에 제한되는 것은 아니다.According to another feature of the present invention, the determining step may further include determining drug candidates based on the expression location of Na+/K+ ATPase or E-cadherin in the organoid treated with the drug. It is not limited.
더 나아가, 전술한 바와 같은 과제를 해결하기 위해, 본 발명은 내강(lumen) 및 상피세포층을 포함하는 신장 근위 세뇨관 오가노이드를 형성하기 위한 제조 방법에 있어서, 신장 근위 세뇨관 오가노이드가 형성되도록 신장 근위 세뇨관 상피세포주를 인체 세포외기질(ECM)을 포함하는 제 1 배양 배지에서 제 1 배양하는 단계, 및 형성된 신장 근위 세뇨관 오가노이드가 성장하도록 오가노이드를 제 2 배양 배지에서 제 2 배양하는 단계를 포함하는, 신장 근위 세뇨관 오가노이드 제조 방법을 제공한다.Furthermore, in order to solve the problems described above, the present invention relates to a manufacturing method for forming a kidney proximal tubule organoid including a lumen and an epithelial cell layer, wherein the kidney proximal tubule organoid is formed. First culturing the tubular epithelial cell line in a first culture medium comprising human extracellular matrix (ECM), and second culturing the organoids in a second culture medium to grow the formed renal proximal tubule organoids. Provided is a method for producing kidney proximal tubule organoids.
본 발명의 특징에 따르면, 제 1 배양하는 단계는, 약 1시간 내지 3일 중 적어도 하나의 기간 동안 배양할 수 있으나, 이에 제한되는 것은 아니다.According to the characteristics of the present invention, the first culturing step may be performed for at least one period of about 1 hour to 3 days, but is not limited thereto.
본 발명의 다른 특징에 따르면, ECM은, 섬유아세포 패치에 단백질 분해효소를 처리하고, 탈세포화한 후 수득될 수 있으나, 이에 제한되는 것은 아니다.According to another feature of the present invention, ECM can be obtained after treating a fibroblast patch with a proteolytic enzyme and decellularizing it, but is not limited thereto.
본 발명의 또 다른 특징에 따르면, 신장 근위 세뇨관 상피세포주 및 제 1 배양 배지는, 1:1 비율로 포함될 수 있으나, 이에 제한되는 것은 아니다.According to another feature of the present invention, the renal proximal tubule epithelial cell line and the first culture medium may be included in a 1:1 ratio, but are not limited thereto.
본 발명의 또 다른 특징에 따르면, 제 2 배양하는 단계는, 약 3일 내지 50일 중 적어도 하나의 기간동안 배양할 수 있으나, 이에 제한되는 것은 아니다. According to another feature of the present invention, the second culturing step may be performed for at least one period of about 3 to 50 days, but is not limited thereto.
본 발명의 또 다른 특징에 따르면, 제 2 배양하는 단계는, 약물을 처리하는 단계를 더 포함할 수 있으나, 이에 제한되는 것은 아니다.According to another feature of the present invention, the second culturing step may further include, but is not limited to, the step of treating a drug.
본 발명의 또 다른 특징에 따르면, 약물을 처리하는 단계는, 배양 7일 이내 처음 수행될 수 있으나, 이에 제한되는 것은 아니다. According to another feature of the present invention, the step of treating the drug may be performed for the first time within 7 days of culture, but is not limited thereto.
신장암 오가노이드Kidney cancer organoids
전술한 바와 같은 과제를 해결하기 위해, 본 발명은 신장암 세포주 유래 약물의 효능 또는 독성 평가용 신장암 오가노이드를 제공한다.In order to solve the problems described above, the present invention provides kidney cancer organoids for evaluating the efficacy or toxicity of drugs derived from kidney cancer cell lines.
본 발명의 특징에 따르면, 신장암 오가노이드는 Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나 이상을 발현할 수 있다.According to features of the present invention, kidney cancer organoids may express at least one selected from the group consisting of Na+/K+ ATPase, E-cadherin, and Vimentin.
본 발명의 특징에 따르면, 신장암 오가노이드는 암 활성제 처리에 의해 암 미세환경을 모사할 수 있다.According to features of the present invention, kidney cancer organoids can mimic the cancer microenvironment by treatment with cancer activators.
본 발명의 특징에 따르면, 암 미세환경은 상피중간엽전이(EMT; epithelial to mesenchymal transition) 또는 F-액틴 비정상(F-actin abnormality)이 증가된 것일 수 있다.According to the features of the present invention, the cancer microenvironment may have increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 약물의 효능 또는 독성 평가용 신장암 오가노이드에 약물을 처리하는 단계; 상기 약물이 처리된 오가노이드에 대해 F-액틴 비정상(F-actin abnormality), Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 측정하는 단계를 포함하는, 신장암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법을 제공한다.In order to solve other problems as described above, the present invention includes treating kidney cancer organoids with a drug for evaluating drug efficacy or toxicity; Including measuring the level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin for the organoid treated with the drug. Provides a method for evaluating the efficacy or toxicity of a drug using kidney cancer organoids.
본 발명의 특징에 따르면, 상술한 약물 평가 방법은 Na+/K+ ATPase 또는 E-cadherin 발현 위치를 확인하는 단계를 더 포함할 수 있다.According to a feature of the present invention, the above-described drug evaluation method may further include the step of confirming the expression site of Na+/K+ ATPase or E-cadherin.
본 발명의 특징에 따르면, 약물이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality) 또는 Vimentin 수준이 감소하거나 Na+/K+ ATPase 또는 E-cadherin 수준이 세포막에서 증가한 경우 약물이 효능을 보이는 것으로 판단할 수 있다.According to a feature of the present invention, the level of F-actin abnormality or Vimentin is reduced or the level of Na+/K+ ATPase or E-cadherin is decreased in the organoid treated with the drug compared to the untreated group or positive control group. If it increases in the cell membrane, the drug can be judged to be effective.
본 발명의 특징에 따르면, 상기 판단은 Na+/K+ ATPase 또는 E-cadherin의 수준이 세포질(cytosol)에서 감소한 경우를 더 포함할 수 있다.According to the features of the present invention, the determination may further include a case where the level of Na+/K+ ATPase or E-cadherin is decreased in the cytosol.
본 명의 특징에 따르면, 약물이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality) 또는 Vimentin 수준이 증가하거나 Na+/K+ ATPase 또는 E-cadherin 수준이 세포막에서 감소한 경우 약물이 독성을 보이는 것으로 판단할 수 있다.According to the present invention, in drug-treated organoids, F-actin abnormality or Vimentin levels are increased or Na+/K+ ATPase or E-cadherin levels are increased in the cell membrane compared to the untreated group or positive control group. If there is a decrease in , the drug can be judged to be toxic.
본 발명의 특징에 따르면, 상기 판단은 Na+/K+ ATPase 또는 E-cadherin의 수준이 세포질(cytosol)에서 증가한 경우를 더 포함할 수 있다.According to the features of the present invention, the determination may further include a case where the level of Na+/K+ ATPase or E-cadherin is increased in the cytosol.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 약물의 효능 또는 독성 평가용 신장암 오가노이드에 항암 후보물질을 처리하는 단계; 상기 항암 후보물질이 처리된 오가노이드 및 항암 후보물질 미처리군에서 F-액틴 비정상(F-actin abnormality), Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 비교하는 단계; 를 포함하는, 신장암 오가노이드를 이용한 항암제 스크리닝 방법을 제공한다.In order to solve other problems as described above, the present invention includes the steps of treating a kidney cancer organoid for evaluating drug efficacy or toxicity with an anticancer candidate material; The level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin in the organoid treated with the anticancer candidate material and the group untreated with the anticancer candidate material. ) comparing; It provides an anticancer drug screening method using kidney cancer organoids, including.
본 발명의 특징에 따르면, 상술한 스크리닝 방법은 Na+/K+ ATPase 또는 E-cadherin 발현 위치를 확인하는 단계를 더 포함할 수 있다.According to a feature of the present invention, the above-described screening method may further include the step of confirming the expression site of Na+/K+ ATPase or E-cadherin.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 암오가노이드가 형성되도록 신장암 세포주 및 인체 세포외기질(ECM)을 포함하는 배지를 혼합하여 제1 배양하는 단계; 및 상기 암오가노이드 및 성장 배지를 혼합하여 제2 배양하는 단계; 를 포함하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법을 제공한다.In order to solve other problems as described above, the present invention includes the steps of first culturing a medium containing a kidney cancer cell line and human extracellular matrix (ECM) to form cancer organoids; and a second culture step of mixing the cancer organoids and the growth medium; It provides a method for producing kidney cancer organoids for evaluating drug efficacy or toxicity, including.
본 발명의 특징에 따르면, 인체 세포외기질은 인체 유래 섬유아세포로부터 수득된 것일 수 있다.According to the features of the present invention, the human extracellular matrix may be obtained from human-derived fibroblasts.
본 발명의 특징에 따르면, 인체 세포외기질은 인체 유래 섬유아세포 패치에 단백질 분해효소를 처리하고 탈세포화한 후 수득된 3차원 세포배양용 ECM일 수 있다.According to the features of the present invention, the human extracellular matrix may be an ECM for three-dimensional cell culture obtained after treating a patch of human-derived fibroblasts with proteolytic enzymes and decellularizing them.
본 발명의 3차원 세포배양용 ECM을 포함하는 배지(Org 3D culture solution)는 오가노이드 성장에 필요한 마트리겔, 하이드로겔, 성장인자 등을 첨가하지 않고도 줄기세포, 세포주, 장기 등과 혼합 배양하는 것만으로 일관적인 퀄리티를 갖는 충분한 양의 오가노이드를 수득할 수 있는 배양 환경을 제공한다. 이에 저비용의 화학적 요소만으로 생산 가능하며 시험관내에서 섬유아세포의 배양으로부터 지속적으로 공급될 수 있는 장점이 있다.The medium (Org 3D culture solution) containing the ECM for 3D cell culture of the present invention can be cultured simply by mixing with stem cells, cell lines, organs, etc. without adding matrigel, hydrogel, growth factors, etc. required for organoid growth. Provides a culture environment in which a sufficient amount of organoids with consistent quality can be obtained. Accordingly, it can be produced using only low-cost chemical elements and has the advantage of being continuously supplied from fibroblast culture in vitro.
본 발명의 특징에 따르면, 제1 배양하는 단계에서, 신장암 세포주 및 인체 세포외기질(ECM)을 포함하는 배지는 1:1 비율로 포함될 수 있다.According to a feature of the present invention, in the first culturing step, a medium containing a kidney cancer cell line and human extracellular matrix (ECM) may be included in a 1:1 ratio.
본 발명의 특징에 따르면, 제1 배양하는 단계에서 50um 내지 200um 크기를 갖는 오가노이드가 형성될 수 있다.According to a feature of the present invention, organoids having a size of 50um to 200um can be formed in the first culturing step.
본 발명의 특징에 따르면, 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간 동안 수행될 수 있다.According to features of the present invention, the first culturing step may be performed for at least one period of about 1 hour to 3 days.
본 발명의 특징에 따르면, 제2 배양하는 단계는 약 3일 내지 50일 중 적어도 하나의 기간동안 수행될 수 있다.According to features of the present invention, the second culturing step may be performed for at least one period of about 3 to 50 days.
본 발명의 특징에 따르면, 제2 배양하는 단계에서 약물을 처리하는 단계; 를 더 포함할 수 있다.According to a feature of the present invention, treating the drug in the second culturing step; It may further include.
본 발명의 특징에 따르면, 약물을 처리하는 단계는 배양 7일 이내 처음 수행될 수 있다.According to the features of the present invention, the step of treating the drug can be performed for the first time within 7 days of culture.
본 발명자들은 상술한 인체 유래 3차원 세포배양용 ECM (hECM, 인체 세포외기질)을 이용하여 누구나 손쉽게 배양할 수 있는 인간 신장암 세포주에 적용한 결과 종래의 기술 대비 짧은 시간내에 신장 기능 및 신장암 특성 관련 바이오마커를 발현하는 인간 신장암 오가노이드를 제작하는데 성공하였다. 구체적으로 배양 3일 이내 오가노이드가 형성됨에 따라 배양 7일 이내 목적하는 약물에 노출시켜 오가노이드를 배양하는 것이 가능하였다. 이에 목적하는 약물의 효능 또는 독성 평가용 오가노이드를 제작하는 기간을 현저히 단축시킬 수 있음을 확인하였다. 또한 종래 줄기세포 기반의 오가노이드 기술에서처럼 줄기세포의 변형이나 실험자의 숙련도에 따라 오가노이드의 퀄리티가 다르지 않고 균질한 오가노이드를 제작할 수 있다.The present inventors used the above-described human-derived 3D cell culture ECM (hECM, human extracellular matrix) and applied it to human kidney cancer cell lines that can be easily cultured. As a result, kidney function and kidney cancer characteristics were improved in a short period of time compared to conventional technology. We succeeded in producing human kidney cancer organoids that express relevant biomarkers. Specifically, as organoids were formed within 3 days of culture, it was possible to culture the organoids by exposing them to the desired drug within 7 days of culture. Accordingly, it was confirmed that the period for producing organoids for evaluating the efficacy or toxicity of the target drug can be significantly shortened. In addition, as in conventional stem cell-based organoid technology, the quality of the organoid does not vary depending on the deformation of the stem cells or the skill of the experimenter, and homogeneous organoids can be produced.
본 발명의 발명자들은 신약 개발 과정에서 암 특성의 모사가 뛰어난 본 발명에 따른 인간 신장암 오가노이드를 이용하여 시험관내(in vitro)에서, 약물 평가 또는 화학물질의 암 증식 영향 평가가 가능함을 제시한다. 이에 임상시험에 적용하기 위한 약물의 선정 단계를 단축시키므로 신약 개발 기간을 단축시킬 수 있다.The inventors of the present invention suggest that it is possible to evaluate drugs or evaluate the effect of chemical substances on cancer growth in vitro using human kidney cancer organoids according to the present invention, which are excellent at replicating cancer characteristics in the process of developing new drugs. . As a result, the selection stage for drugs to be applied to clinical trials is shortened, thereby shortening the development period for new drugs.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 신장암 세포주, 인체 세포외기질(ECM) 및 암 활성제 함유 성장 배지를 포함하는, 암 미세환경 모사 신장암 오가노이드 배양용 조성물을 제공한다.In order to solve other problems as described above, the present invention provides a composition for culturing kidney cancer organoids simulating a cancer microenvironment, comprising a growth medium containing a kidney cancer cell line, human extracellular matrix (ECM), and a cancer activator.
본 발명의 특징에 따르면, 인체 세포외기질은 인체 유래 섬유아세포로부터 수득된 것 일 수 있다.According to the features of the present invention, the human extracellular matrix may be obtained from human-derived fibroblasts.
본 발명의 특징에 따르면, 인체 세포외기질 및 성장 배지의 부피비는 1 : 4 내지 1 : 6일 수 있다.According to the features of the present invention, the volume ratio of human extracellular matrix and growth medium may be 1:4 to 1:6.
본 발명의 특징에 따르면, 암 미세환경은 상피중간엽전이(EMT; epithelial to mesenchymal transition) 또는 F-액틴 비정상(F-actin abnormality)이 증가된 것일 수 있다.According to the features of the present invention, the cancer microenvironment may have increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 상술한 조성물로 제조된 암 미세환경 모사 신장암 오가노이드를 제공한다.In order to solve other problems as described above, the present invention provides a kidney cancer organoid simulating a cancer microenvironment prepared with the composition described above.
본 발명의 일 실시예에 따르면 암 활성제 PFOA를 처리한 신장암 오가노이드는 상피중간엽전이(EMT)가 활성화되어 E-cadherin, Vimentin 등의 EMT 관련 단백질의 변화가 발생하고 F-액틴의 동적 재구성이 발현되는 등의 암 특성을 획득하였다. 이에 본 발명에 따르면 상피중간엽전이(EMT) 또는 F-액틴 비정상(F-actin abnormality)이 증가된 암 미세환경을 모사하는 신장암 오가노이드를 제공함에 따라 약물 평가 및 화학물질의 암 증식 영향 평가의 도구로 활용할 수 있다.According to one embodiment of the present invention, epithelial-mesenchymal transition (EMT) is activated in kidney cancer organoids treated with the cancer activator PFOA, resulting in changes in EMT-related proteins such as E-cadherin and Vimentin, and dynamic reorganization of F-actin. Cancer characteristics such as expression were acquired. Accordingly, according to the present invention, by providing a kidney cancer organoid that simulates a cancer microenvironment with increased epithelial-mesenchymal transition (EMT) or F-actin abnormality, drug evaluation and the cancer proliferation effect of chemicals are evaluated. It can be used as a tool.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 암 미세환경 모사 신장암 오가노이드가 이종 이식된 동물 모델을 제공한다.In order to solve other problems as described above, the present invention provides an animal model in which kidney cancer organoids simulating a cancer microenvironment are xenografted.
본 발명에 따른 신장암 오가노이드는 동물모델에 이종 이식되어 이용될 수 있다. 상기 "동물 모델"은 질환 동물 모델을 의미한다. 구체적으로, 동물 모델은 인간의 질병과 유사한 상태의 질병에 걸리거나 선천적으로 그 질병에 걸리도록 만들어낸 동물 모델일 수 있다. 동물 모델로 이용될 수 있는 동물은 인간을 제외한 포유 동물로, 예를 들면, 래트, 마우스, 모르모트, 햄스터, 토끼, 원숭이, 개, 고양이, 소, 말, 돼지, 양 및 염소로 구성된 군으로부터 선택되는 적어도 하나일 수 있다.Kidney cancer organoids according to the present invention can be used as xenografts in animal models. The “animal model” refers to a disease animal model. Specifically, the animal model may be an animal model created to suffer from a disease similar to a human disease or to be born with the disease. Animals that can be used as animal models are mammals other than humans, for example, selected from the group consisting of rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep and goats. It can be at least one thing.
갑상선암 오가노이드Thyroid cancer organoids
전술한 바와 같은 과제를 해결하기 위해, 본 발명은 갑상선암 세포주 유래 약물의 효능 또는 독성 평가용 갑상선암 오가노이드를 제공한다.In order to solve the problems described above, the present invention provides thyroid cancer organoids for evaluating the efficacy or toxicity of drugs derived from thyroid cancer cell lines.
본 발명의 특징에 따르면, 갑상선암 오가노이드는 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나를 발현할 수 있다.According to a feature of the present invention, the thyroid cancer organoid may express at least one selected from the group consisting of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin.
본 발명의 특징에 따르면, 갑상선암 오가노이드는 암 활성제 처리에 의해 암 미세환경을 모사할 수 있다.According to features of the present invention, thyroid cancer organoids can simulate a cancer microenvironment by treatment with a cancer activator.
본 발명의 특징에 따르면, 암 미세환경은 상피중간엽전이(EMT; epithelial to mesenchymal transition) 또는 F-액틴 비정상(F-actin abnormality)이 증가한 것일 수 있다.According to the features of the present invention, the cancer microenvironment may have increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 약물의 효능 또는 독성 평가용 갑상선암 오가노이드에 약물을 처리하는 단계; 상기 약물이 처리된 오가노이드에 대해 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 측정하는 단계를 포함하는, 갑상선암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법을 제공한다.In order to solve other problems as described above, the present invention includes the steps of treating thyroid cancer organoids with a drug for evaluating drug efficacy or toxicity; For organoids treated with the above drug, selected from the group consisting of F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. A method for evaluating the efficacy or toxicity of a drug using thyroid cancer organoids is provided, including measuring the level of at least one biomarker.
본 발명의 특징에 따르면, 상술한 약물 평가 방법은 갑상선 자극 호르몬 수용체(TSHR) 또는 E-cadherin 발현 위치를 확인하는 단계를 더 포함할 수 있다.According to a feature of the present invention, the above-described drug evaluation method may further include the step of confirming the expression location of thyroid-stimulating hormone receptor (TSHR) or E-cadherin.
본 발명의 특징에 따르면, 약물이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 및 티로페록시다제(TPO)로 이루어진 군에서 선택된 적어도 하나의 수준이 감소하거나 티로글로불린(Tg) 또는 E-cadherin의 수준이 증가한 경우 약물이 효능을 보이는 것으로 판단할 수 있다. 이 때 갑상선 자극 호르몬 수용체(TSHR) 수준이 세포내소포(intracellular vesicle)에서 감소 또는 E-cadherin의 수준이 세포막(membrane)에서 증가한 것일 수 있다.According to a feature of the present invention, F-actin abnormality, thyroid stimulating hormone receptor (TSHR), and thyroperoxidase (TPO) were detected in organoids treated with the drug compared to the untreated group or the positive control group. ), the drug can be judged to be effective when the level of at least one selected from the group is reduced or the level of thyroglobulin (Tg) or E-cadherin is increased. At this time, the level of thyroid stimulating hormone receptor (TSHR) may be decreased in intracellular vesicles or the level of E-cadherin may be increased in the cell membrane.
본 발명의 특징에 따르면, 약물이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 및 티로페록시다제(TPO)로 이루어진 군에서 선택된 적어도 하나의 수준이 증가하거나 티로글로불린(Tg) 또는 E-cadherin의 수준이 감소한 경우 약물이 독성을 보이는 것으로 판단할 수 있다. 이 때 갑상선 자극 호르몬 수용체(TSHR) 수준이 세포내소포(intracellular vesicle)에서 증가 또는 E-cadherin의 수준이 세포막(membrane)에서 감소한 것일 수 있다.According to a feature of the present invention, F-actin abnormality, thyroid stimulating hormone receptor (TSHR), and thyroperoxidase (TPO) were detected in organoids treated with the drug compared to the untreated group or the positive control group. ), the drug can be judged to be toxic if the level of at least one selected from the group is increased or the level of thyroglobulin (Tg) or E-cadherin is decreased. At this time, the level of thyroid stimulating hormone receptor (TSHR) may be increased in intracellular vesicles or the level of E-cadherin may be decreased in the cell membrane.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 약물의 효능 또는 독성 평가용 갑상선암 오가노이드에 항암 후보물질을 처리하는 단계; 상기 항암 후보물질이 처리된 오가노이드 및 항암 후보물질 미처리군에서 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 비교하는 단계; 를 포함하는, 갑상선암 오가노이드를 이용한 항암제 스크리닝 방법을 제공한다.In order to solve other problems as described above, the present invention includes the steps of treating thyroid cancer organoids for evaluating drug efficacy or toxicity with an anticancer candidate material; F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO) and E Comparing the level of at least one biomarker selected from the group consisting of -cadherin; Provides an anticancer drug screening method using thyroid cancer organoids, including.
본 발명의 특징에 따르면, 상술한 스크리닝 방법은 갑상선 자극 호르몬 수용체(TSHR) 또는 E-cadherin 발현 위치를 확인하는 단계를 더 포함할 수 있다.According to a feature of the present invention, the above-described screening method may further include the step of confirming the expression location of thyroid-stimulating hormone receptor (TSHR) or E-cadherin.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 암오가노이드가 형성되도록 갑상선암 세포주 및 인체 세포외기질(ECM)을 포함하는 배지를 혼합하여 제1 배양하는 단계; 및 상기 암오가노이드 및 성장 배지를 혼합하여 제2 배양하는 단계; 를 포함하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법을 제공한다.In order to solve other problems as described above, the present invention includes the steps of first culturing a medium containing a thyroid cancer cell line and human extracellular matrix (ECM) to form cancer organoids; and a second culture step of mixing the cancer organoids and the growth medium; It provides a method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity, including.
본 발명의 특징에 따르면, 인체 세포외기질은 인체 유래 섬유아세포로부터 수득된 것일 수 있다.According to the features of the present invention, the human extracellular matrix may be obtained from human-derived fibroblasts.
본 발명의 특징에 따르면, 인체 세포외기질은 인체 유래 섬유아세포 패치에 단백질 분해효소를 처리하고 탈세포화한 후 수득된 3차원 세포배양용 ECM일 수 있다.According to the features of the present invention, the human extracellular matrix may be an ECM for three-dimensional cell culture obtained after treating a patch of human-derived fibroblasts with proteolytic enzymes and decellularizing them.
본 발명의 특징에 따르면, 제1 배양하는 단계에서, 갑상선암 세포주 및 인체 세포외기질 (extracellular matrix, ECM)을 포함하는 배지는 1:1 비율로 포함될 수 있다.According to a feature of the present invention, in the first culturing step, a medium containing a thyroid cancer cell line and human extracellular matrix (ECM) may be included in a 1:1 ratio.
본 발명의 특징에 따르면, 제1 배양하는 단계에서 50um 내지 200um 크기를 갖는 오가노이드가 형성될 수 있다.According to a feature of the present invention, organoids having a size of 50um to 200um can be formed in the first culturing step.
본 발명의 특징에 따르면, 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간 동안 수행될 수 있다.According to features of the present invention, the first culturing step may be performed for at least one period of about 1 hour to 3 days.
본 발명의 특징에 따르면, 제2 배양하는 단계는 약 3일 내지 50일 중 적어도 하나의 기간동안 수행될 수 있다.According to features of the present invention, the second culturing step may be performed for at least one period of about 3 to 50 days.
본 발명의 특징에 따르면, 제2 배양하는 단계에서 약물을 처리하는 단계; 를 더 포함할 수 있다.According to a feature of the present invention, treating the drug in the second culturing step; It may further include.
본 발명의 특징에 따르면, 약물을 처리하는 단계는 배양 7일 이내 처음 수행될 수 있다.According to the features of the present invention, the step of treating the drug can be performed for the first time within 7 days of culture.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 갑상선암 세포주, 인체 세포외기질(ECM) 및 암 활성제 함유 성장 배지를 포함하는, 암 미세환경 모사 갑상선암 오가노이드 배양용 조성물을 제공한다.In order to solve other problems as described above, the present invention provides a composition for culturing thyroid cancer organoids simulating a cancer microenvironment, comprising a thyroid cancer cell line, human extracellular matrix (ECM), and a growth medium containing a cancer activator.
본 발명의 특징에 따르면, 인체 세포외기질은 인체 유래 섬유아세포로부터 수득된 것 일 수 있다.According to the features of the present invention, the human extracellular matrix may be obtained from human-derived fibroblasts.
본 발명의 특징에 따르면, 인체 세포외기질 및 성장 배지의 부피비는 1 : 4 내지 1 : 6일 수 있다.According to the features of the present invention, the volume ratio of human extracellular matrix and growth medium may be 1:4 to 1:6.
본 발명의 특징에 따르면, 암 미세환경은 상피중간엽전이(EMT; epithelial to mesenchymal transition) 또는 F-액틴 비정상(F-actin abnormality)이 증가된 것일 수 있다.According to the features of the present invention, the cancer microenvironment may have increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 상술한 조성물로 제조된 암 미세환경 모사 갑상선암 오가노이드를 제공한다.In order to solve other problems as described above, the present invention provides a thyroid cancer organoid simulating a cancer microenvironment prepared with the composition described above.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 암 미세환경 모사 갑상선암 오가노이드가 이종 이식된 동물 모델을 제공한다.In order to solve other problems as described above, the present invention provides an animal model in which thyroid cancer organoids simulating a cancer microenvironment are xenografted.
갑상선 오가노이드thyroid organoids
전술한 바와 같은 과제를 해결하기 위해, 본 발명은 갑상선 세포주를 기반으로 제조된 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드를 제공한다.In order to solve the problems described above, the present invention provides thyroid organoids for evaluating the efficacy or toxicity of test substances prepared based on thyroid cell lines.
본 발명의 특징에 따르면, 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드는 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상을 발현할 수 있다.According to the features of the present invention, the thyroid organoid for evaluating the efficacy or toxicity of a test substance is one selected from the group consisting of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. More than one species may appear.
본 발명의 특징에 따르면, 본 발명의 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드는 갑상선 호르몬을 분비할 수 있다. 특히, 시험물질 처리에 의한 갑상선 호르몬의 수치 변화를 모사할 수 있다.According to the features of the present invention, the thyroid organoid for evaluating the efficacy or toxicity of the test substance of the present invention can secrete thyroid hormones. In particular, changes in thyroid hormone levels due to test substance treatment can be simulated.
본 발명의 발명자들은 신약 개발 과정에서 생체 모사도가 뛰어난 본 발명에 따른 인간 갑상선 오가노이드를 이용하여 시험관내(in vitro)에서, 시험물질의 효능 또는 독성 평가가 가능함을 제시한다. 이에 임상시험에 적용하기 위한 약물의 선정 단계를 단축시키므로 신약 개발 기간을 단축시킬 수 있다.The inventors of the present invention suggest that it is possible to evaluate the efficacy or toxicity of test substances in vitro using the human thyroid organoid according to the present invention, which has excellent biomimeticity, during the process of developing a new drug. As a result, the selection stage for drugs to be applied to clinical trials is shortened, thereby shortening the development period for new drugs.
또한, 본 발명의 발명자들은 생체 모사도가 뛰어난 본 발명에 따른 인간 갑상선 오가노이드를 이용하여 호르몬 교란물질 등 다양한 환경 노출 화학물질에 대해 시험관내(in vitro)에서, 독성 평가 및 갑상선에 미치는 영향의 분석이 가능함을 제시한다. In addition, the inventors of the present invention used the human thyroid organoid according to the present invention, which has excellent biomimeticity, to evaluate the toxicity and analyze the effect on the thyroid gland in vitro for various environmentally exposed chemicals such as hormone disruptors. We suggest that this is possible.
나아가, 약물 또는 화학물질의 독성 평가 시 수행되는 동물에서의 갑상선 호르몬 분석을 시험관내 환경에서 가능하기에, 추후 동물 실험 대체가 가능함을 확인하였다. Furthermore, it was confirmed that thyroid hormone analysis in animals, which is performed when evaluating the toxicity of drugs or chemicals, is possible in an in vitro environment, so it can be replaced with animal testing in the future.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 오가노이드를 형성하도록 갑상선 세포주 및 인체 세포외기질(ECM)을 포함하는 배지를 혼합하여 제1 배양하는 단계; 및 상기 오가노이드를 갑상선 자극 호르몬(Thyroid-Stimulating Hormone) 및 요오드화 칼륨(Potassium Iodide)을 포함하는 갑상선 오가노이드 배양액과 혼합하여 제2 배양하는 단계; 를 포함하는, 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법을 제공한다.In order to solve other problems as described above, the present invention includes the steps of first culturing a medium containing a thyroid cell line and human extracellular matrix (ECM) to form an organoid; and secondly culturing the organoids by mixing them with a thyroid organoid culture medium containing Thyroid-Stimulating Hormone and Potassium Iodide; Provides a method for producing thyroid organoids for evaluating the efficacy or toxicity of test substances, including.
본 발명의 특징에 따르면, 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간 동안 수행될 수 있다.According to features of the present invention, the first culturing step may be performed for at least one period of about 1 hour to 3 days.
본 발명의 특징에 따르면, 제2 배양하는 단계는 약 3일 내지 50일 중 적어도 하나의 기간동안 수행될 수 있다.According to features of the present invention, the second culturing step may be performed for at least one period of about 3 to 50 days.
본 발명의 특징에 따르면, 제2 배양하는 단계에서 시험물질을 처리하는 단계; 를 더 포함할 수 있다.According to a feature of the present invention, treating the test substance in the second culturing step; It may further include.
본 발명의 특징에 따르면, 시험물질을 처리하는 단계는 배양 7일 이내 처음 수행될 수 있다.According to the features of the present invention, the step of treating the test substance can be performed for the first time within 7 days of culture.
본 발명의 특징에 따르면, 제1 배양하는 단계에서, 갑상선 세포주 및 인체 세포외기질(ECM)을 포함하는 배지는 1:1 비율로 포함될 수 있다.According to a feature of the present invention, in the first culturing step, a medium containing a thyroid cell line and human extracellular matrix (ECM) may be included in a 1:1 ratio.
본 발명의 특징에 따르면, 인체 세포외기질은 3차원 세포배양용 ECM일 수 있다.According to the features of the present invention, the human extracellular matrix may be an ECM for 3D cell culture.
본 발명의 3차원 세포배양용 ECM을 포함하는 배지 (Org 3D culture solution)는 오가노이드 성장에 필요한 마트리겔, 하이드로겔, 성장인자 등을 첨가하지 않고도 줄기세포, 세포주, 장기 등과 혼합 배양하는 것만으로 일관적인 퀄리티를 갖는 충분한 양의 오가노이드를 수득할 수 있는 배양 환경을 제공한다. 이에 저비용의 화학적 요소만으로 생산 가능하며 시험관내에서 섬유아세포의 배양으로부터 지속적으로 공급될 수 있는 장점이 있다.The medium containing the ECM for 3D cell culture of the present invention (Org 3D culture solution) can be cultured simply by mixing with stem cells, cell lines, organs, etc. without adding Matrigel, hydrogel, growth factors, etc. required for organoid growth. Provides a culture environment in which a sufficient amount of organoids with consistent quality can be obtained. Accordingly, it can be produced using only low-cost chemical elements and has the advantage of being continuously supplied from fibroblast culture in vitro.
본 발명자들은 상술한 인체 유래 3차원 세포배양용 ECM (hECM)를 이용하여 누구나 손쉽게 배양할 수 있는 인간 갑상선 세포주에 적용한 결과 갑상선 호르몬을 분비 및 갑상선 기능 관련 바이오마커를 발현하는 인간 갑상선 오가노이드를 제작하는데 성공하였다.The present inventors used the above-described human-derived ECM (hECM) for 3D cell culture and applied it to a human thyroid cell line that anyone can easily culture, producing a human thyroid organoid that secretes thyroid hormones and expresses biomarkers related to thyroid function. succeeded in doing so.
아울러 종래 기술 대비 짧은 기간인 3일 이내 오가노이드가 형성됨에 따라 배양 7일 이내 시험물질에 노출시켜 오가노이드를 배양하는 것이 가능하였다. 이에 목적하는 시험물질의 효능 또는 독성 평가용 오가노이드를 제작하는 기간을 현저히 단축시킬 수 있음을 확인하였다. 또한 종래 줄기세포 기반의 오가노이드 기술에서처럼 줄기세포의 변형이나 실험자의 숙련도에 따라 오가노이드의 퀄리티가 다르지 않고 균질한 오가노이드를 제작할 수 있다.In addition, as organoids were formed within 3 days, which is a shorter period of time compared to the prior art, it was possible to culture organoids by exposing them to test substances within 7 days of culture. Accordingly, it was confirmed that the period for producing organoids for evaluating the efficacy or toxicity of the target test substance can be significantly shortened. In addition, as in conventional stem cell-based organoid technology, the quality of the organoid does not vary depending on the deformation of the stem cells or the skill of the experimenter, and homogeneous organoids can be produced.
본 발명의 특징에 따르면, 3차원 세포배양용 ECM은 인체 유래 섬유아세포 패치에 단백질 분해효소를 처리하고 탈세포화한 후 수득된 세포외기질일 수 있다.According to the features of the present invention, the ECM for 3D cell culture may be an extracellular matrix obtained after treating a human-derived fibroblast patch with proteolytic enzymes and decellularizing it.
본 발명의 특징에 따르면, 제2 배양하는 단계에서, 갑상선 자극 호르몬은 상기 갑상선 오가노이드 배양액 총 부피에 대하여 0.01 내지 1 mU/mL 농도로 포함될 수 있다.According to a feature of the present invention, in the second culturing step, thyroid-stimulating hormone may be included at a concentration of 0.01 to 1 mU/mL based on the total volume of the thyroid organoid culture medium.
본 발명의 특징에 따르면, 제2 배양하는 단계에서, 요오드화 칼륨은 상기 갑상선 오가노이드 배양액 총 부피에 대하여 1 내지 20nM 농도로 포함될 수 있다.According to a feature of the present invention, in the second culturing step, potassium iodide may be included at a concentration of 1 to 20 nM based on the total volume of the thyroid organoid culture medium.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 갑상선 세포주, 인체 세포외기질(ECM)을 포함하는 배지, 갑상선 자극 호르몬(Thyroid-Stimulating Hormone) 및 요오드화 칼륨(Potassium Iodide)을 포함하는, 시험물질의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물을 제공한다.In order to solve other problems as described above, the present invention provides a test comprising a thyroid cell line, a medium containing human extracellular matrix (ECM), Thyroid-Stimulating Hormone, and Potassium Iodide. A culture composition for producing thyroid organoids for evaluating the efficacy or toxicity of a substance is provided.
본 발명의 특징에 따르면, 시험물질의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물은 시험관내에서 갑상선 호르몬을 분비하는 갑상선 오가노이드 제조용일 수 있다.According to the features of the present invention, the culture composition for producing thyroid organoids for evaluating the efficacy or toxicity of a test substance may be used for producing thyroid organoids that secrete thyroid hormones in vitro.
본 발명의 특징에 따르면, 시험물질의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물은 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상을 발현하는 갑상선 오가노이드 제조용일 수 있다.According to the features of the present invention, the culture composition for preparing thyroid organoids for evaluating the efficacy or toxicity of test substances is composed of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. It may be used for producing thyroid organoids expressing one or more species selected from the group.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드에 목적하는 시험물질을 처리하는 단계; 및 상기 시험물질이 처리된 군이 시험물질 미처리군 또는 양성 대조군과 비교하여 트리오요오드티로닌(T3), 테트라요오드티로닌(T4), 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상의 바이오마커의 증감에 따라 상기 시험물질의 효능을 판단하는 단계; 를 포함하는, 시험물질의 효능 평가 방법을 제공한다.In order to solve other problems as described above, the present invention includes the steps of processing a test substance of interest into a thyroid organoid for evaluating the efficacy or toxicity of the test substance; And the group treated with the test substance was compared with the untreated group or positive control group to show higher levels of triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyroglobulin. Determining the efficacy of the test substance according to the increase or decrease of one or more biomarkers selected from the group consisting of peroxidase (TPO) and E-cadherin; Provides a method for evaluating the efficacy of test substances, including.
전술한 바와 같은 다른 과제를 해결하기 위해, 본 발명은 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드에 목적하는 시험물질을 처리하는 단계; 및 상기 시험물질이 처리된 군이 시험물질 미처리군 또는 양성 대조군과 비교하여 트리오요오드티로닌(T3), 테트라요오드티로닌(T4), 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상의 바이오마커 증감에 따라 상기 시험물질의 독성을 판단하는 단계; 를 포함하는, 시험물질의 독성 평가 방법을 제공한다. In order to solve other problems as described above, the present invention includes the steps of processing a test substance of interest into a thyroid organoid for evaluating the efficacy or toxicity of the test substance; And the group treated with the test substance was compared with the untreated group or positive control group to show higher levels of triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyroglobulin. Determining the toxicity of the test substance according to the increase or decrease of one or more biomarkers selected from the group consisting of peroxidase (TPO) and E-cadherin; Provides a method for evaluating the toxicity of test substances, including.
본 발명은 신장 근위 세뇨관 오가노이드, 신장암 오가노이드, 갑상선 오가노이드 또는 갑상선암 오가노이드, 이의 제조방법 및 이를 이용한 약물 평가 방법을 제공하여, 신약 개발에 있어 약물에 대한 검증 즉, 유효성, 부작용 및 독성을 평가할 수 있는 효과가 있다. The present invention provides a kidney proximal tubule organoid, a kidney cancer organoid, a thyroid organoid, or a thyroid cancer organoid, a manufacturing method thereof, and a drug evaluation method using the same, to verify the drug in the development of new drugs, that is, effectiveness, side effects, and toxicity. There is an effect that can be evaluated.
또한 약물의 독성 평가 시 수행되는 동물에서의 바이오마커 분석을 시험관내 환경에서 가능하기에, 추후 동물 실험 대체가 가능한 효과가 있다.In addition, since biomarker analysis in animals, which is performed when evaluating drug toxicity, is possible in an in vitro environment, it has the effect of being able to replace animal testing in the future.
또한 목적하는 오가노이드를 제작하는 비용 및 기간을 현저히 단축시킬 수 있는 효과가 있다. 이에 본 발명의 오가노이드는 신약 개발에 있어 약물 후보 물질 스크리닝에 활용되어 소요되는 비용 및 시간을 획기적으로 줄일 수 있으며, 신장암 기능이상 관련 질환의 생리학적 연구 및 임상시험에 활용될 수 있다. In addition, it has the effect of significantly shortening the cost and period of producing the desired organoid. Accordingly, the organoid of the present invention can be used for screening drug candidates in the development of new drugs, dramatically reducing the cost and time required, and can be used for physiological research and clinical trials of diseases related to kidney cancer dysfunction.
본 발명에 따른 효과는 이상에서 예시된 내용에 의해 제한되지 않으며, 더욱 다양한 효과들이 본 명세서 내에 포함되어 있다. The effects according to the present invention are not limited to the contents exemplified above, and further various effects are included in the present specification.
도 1은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 제조 방법의 절차를 예시적으로 도시한 것이다.Figure 1 exemplarily shows the procedure of a method for producing kidney proximal tubule organoids according to an embodiment of the present invention.
도 2a는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드의 바이오마커에 대한 형광 이미지이다. Figure 2a is a fluorescence image for biomarkers of kidney proximal tubule organoids according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention.
도 2b는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드의 구조에 대한 형광 이미지이다.Figure 2b is a fluorescence image of the structure of kidney proximal tubule organoids according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention.
도 3a는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법의 절차를 예시적으로 도시한 것이다.Figure 3a exemplarily illustrates the procedure of a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention.
도 3b는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법에 대한 흐름도이다.Figure 3b is a flowchart of a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention.
도 4는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 OAT1 발현에 기초한 약물 평가의 현미경 이미지이다.Figure 4 is a microscopic image of drug evaluation based on OAT1 expression in kidney proximal tubule organoids according to an embodiment of the present invention.
도 5는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 F-actine 발현에 기초한 약물 평가의 현미경 이미지이다.Figure 5 is a microscopic image of drug evaluation based on F-actine expression in kidney proximal tubule organoids according to an embodiment of the present invention.
도 6은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 Na+/K+ ATPase 발현에 기초한 약물 평가의 현미경 이미지이다.Figure 6 is a microscopic image of drug evaluation based on Na+/K+ ATPase expression in kidney proximal tubule organoids according to an embodiment of the present invention.
도 7은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 E-cadherin 발현에 기초한 약물 평가의 현미경 이미지이다.Figure 7 is a microscopic image of drug evaluation based on E-cadherin expression in kidney proximal tubule organoids according to an embodiment of the present invention.
도 8은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 8-OHdG 발현에 기초한 약물 평가의 현미경 이미지이다.Figure 8 is a microscopic image of drug evaluation based on 8-OHdG expression in kidney proximal tubule organoids according to an embodiment of the present invention.
도 9는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 vimentin 발현에 기초한 약물 평가의 현미경 이미지이다.Figure 9 is a microscopic image of drug evaluation based on vimentin expression in kidney proximal tubule organoids according to an embodiment of the present invention.
도 10은 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법을 예시적으로 도시한 것이다.Figure 10 exemplarily illustrates a method for producing kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
도 11는 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 신장암 오가노이드를 제조하기 위한 제1 배양 방법을 예시적으로 도시한 것이다.Figure 11 exemplarily illustrates a first culture method for producing kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
도 12은 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 신장암 오가노이드를 제조하기 위한 제2 배양 방법을 예시적으로 도시한 것이다.Figure 12 exemplarily shows a second culture method for producing kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
도 13는 본 발명의 일 실시예에 따른 신장암 오가노이드 및 약물 처리 신장암 오가노이드의 H&E 염색 현미경 이미지를 도시한 것이다.Figure 13 shows H&E staining microscopic images of kidney cancer organoids and drug-treated kidney cancer organoids according to an embodiment of the present invention.
도 14는 본 발명의 일 실시예에 따른 신장암 오가노이드 및 약물 처리 신장암 오가노이드의 F-액틴 염색 현미경 이미지 및 정량화 데이터를 도시한 것이다.Figure 14 shows F-actin staining microscopy images and quantification data of kidney cancer organoids and drug-treated kidney cancer organoids according to an embodiment of the present invention.
도 15은 본 발명의 일 실시예에 따른 신장암 오가노이드 및 약물에 노출된 신장암 오가노이드의 Na+/K+ ATPase 변화를 도시한 것이다. Figure 15 shows changes in Na+/K+ ATPase in kidney cancer organoids and kidney cancer organoids exposed to drugs according to an embodiment of the present invention.
도 16은 본 발명의 일 실시예에 따른 신장암 오가노이드 및 약물에 노출된 신장암 오가노이드의 E-cadherin 발현 변화를 도시한 것이다.Figure 16 shows changes in E-cadherin expression in kidney cancer organoids and drug-exposed kidney cancer organoids according to an embodiment of the present invention.
도 17은 본 발명의 일 실시예에 따른 신장암 오가노이드 및 약물에 노출된 신장암 오가노이드의 Vimentin 발현 변화를 도시한 것이다.Figure 17 shows changes in Vimentin expression in kidney cancer organoids and kidney cancer organoids exposed to drugs according to an embodiment of the present invention.
도 18는 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 신장암 오가노이드를 이용한 약물의 평가 방법에 대한 흐름도이다.Figure 18 is a flowchart of a drug evaluation method using kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
도 19은 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 갑상선암 오가노이드를 제조하기 위한 방법을 예시적으로 도시한 것이다.Figure 19 exemplarily illustrates a method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
도 20는 본 발명의 일 실시예에 따른 제조방법으로 제조된 갑상선암 오가노이드의 현미경 이미지를 도시한 것이다.Figure 20 shows a microscopic image of a thyroid cancer organoid produced by a production method according to an embodiment of the present invention.
도 21은 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 갑상선암 오가노이드를 제조하기 위해 약물 처리를 더 포함하여 제조하는 방법을 예시적으로 도시한 것이다.Figure 21 exemplarily illustrates a method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention, further including drug treatment.
도 22는 본 발명의 일 실시예에 따른 갑상선암 오가노이드의 H&E 염색 현미경 이미지를 도시한 것이다. Figure 22 shows H&E staining of thyroid cancer organoids according to an embodiment of the present invention. It shows a microscope image.
도 23는 본 발명의 일 실시예에 따른 갑상선암 오가노이드의 Hoechst33342 염색 현미경 이미지를 도시한 것이다.Figure 23 shows a Hoechst33342 staining microscope image of a thyroid cancer organoid according to an embodiment of the present invention.
도 24은 본 발명의 일 실시예에 따른 갑상선암 오가노이드에 대해 팔로이딘(phalloidin) 염색 프로토콜을 이용한 F-액틴 염색 현미경 이미지를 도시한 것이다.Figure 24 shows a microscopic image of F-actin staining using a phalloidin staining protocol for thyroid cancer organoids according to an embodiment of the present invention.
도 25a 및 도 25b는 본 발명의 일 실시예에 따른 제조방법으로 제조된 약물의 효능 또는 독성 평가용 갑상선암 오가노이드의 갑상선 자극 호르몬 수용체(TSHR) 발현 변화를 도시한 것이다.Figures 25a and 25b show changes in thyroid stimulating hormone receptor (TSHR) expression in thyroid cancer organoids for evaluating the efficacy or toxicity of a drug prepared by a manufacturing method according to an embodiment of the present invention.
도 26은 본 발명의 일 실시예에 따른 제조방법으로 제조된 약물의 효능 또는 독성 평가용 갑상선암 오가노이드의 티로글로불린(Tg) 발현 변화를 도시한 것이다.Figure 26 shows changes in thyroglobulin (Tg) expression in thyroid cancer organoids for evaluating the efficacy or toxicity of a drug prepared by the production method according to an embodiment of the present invention.
도 27는 본 발명의 일 실시예에 따른 제조방법으로 제조된 약물의 효능 또는 독성 평가용 갑상선암 오가노이드의 티로페록시다제(TPO) 발현 변화를 도시한 것이다.Figure 27 shows changes in the expression of thyroperoxidase (TPO) in thyroid cancer organoids for evaluating the efficacy or toxicity of a drug prepared by the production method according to an embodiment of the present invention.
도 28a 및 28b는 본 발명의 일 실시예에 따른 제조방법으로 제조된 약물의 효능 또는 독성 평가용 갑상선암 오가노이드의 E-cadherin 발현 변화를 도시한 것이다.Figures 28a and 28b show changes in E-cadherin expression in thyroid cancer organoids for evaluating the efficacy or toxicity of a drug prepared by the production method according to an embodiment of the present invention.
도 29은 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 갑상선암 오가노이드를 이용한 약물의 평가 방법에 대한 흐름도이다.Figure 29 is a flowchart of a drug evaluation method using thyroid cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
도 30은 본 발명의 일 실시예에 따른 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드를 제조하기 위한 방법을 예시적으로 도시한 것이다.Figure 30 exemplarily shows a method for producing thyroid organoids for evaluating the efficacy or toxicity of a test substance according to an embodiment of the present invention.
도 31는 본 발명의 일 실시예에 따른 제조방법으로 제조된 갑상선 오가노이드의 현미경 이미지를 도시한 것이다.Figure 31 shows a microscope image of a thyroid organoid prepared by a manufacturing method according to an embodiment of the present invention.
도 32은 본 발명의 일 실시예에 따른 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드를 제조하기 위해 시험물질 처리를 더 포함하여 제조하는 방법을 예시적으로 도시한 것이다.Figure 32 exemplarily shows a method of manufacturing thyroid organoids for evaluating the efficacy or toxicity of a test substance according to an embodiment of the present invention, further including treatment of the test substance.
도 33는 본 발명의 일 실시예에 따른 제조방법으로 제조된 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드 및 시험물질 처리를 더 포함하여 제조된 갑상선 오가노이드의 현미경 이미지를 도시한 것이다.Figure 33 shows a microscopic image of a thyroid organoid for evaluating the efficacy or toxicity of a test substance prepared by a production method according to an embodiment of the present invention and a thyroid organoid prepared further including test substance treatment.
도 34는 BHA 또는 BPA에 노출되지 않고 제조된 갑상선 오가노이드 및 BHA 또는 BPA에 저농도-장기간 노출되어 제조된 갑상선 오가노이드의 갑상선 호르몬 변화를 비교하여 도시한 것이다. 도 34a는 Nthy-ori3-1 세포 유래 갑상선 오가노이드, 도 34b는 H6040 세포 유래 갑상선 오가노이드에 대한 결과이다.Figure 34 shows a comparison of thyroid hormone changes in thyroid organoids prepared without exposure to BHA or BPA and thyroid organoids prepared with low-concentration-long-term exposure to BHA or BPA. Figure 34a shows the results for thyroid organoids derived from Nthy-ori3-1 cells, and Figure 34b shows the results for thyroid organoids derived from H6040 cells.
도 35은 본 발명의 일 실시예에 따른 제조방법으로 제조된 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드의 갑상선자극호르몬수용체(TSHR) 발현을 도시한 것이다.Figure 35 shows the expression of thyroid stimulating hormone receptor (TSHR) in thyroid organoids for evaluating the efficacy or toxicity of test substances prepared by the production method according to an embodiment of the present invention.
도 36은 본 발명의 일 실시예에 따른 제조방법으로 제조된 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드의 티로글로불린(Tg) 발현을 도시한 것이다.Figure 36 shows thyroglobulin (Tg) expression in thyroid organoids for evaluating the efficacy or toxicity of test substances prepared by the production method according to an embodiment of the present invention.
도 37은 본 발명의 일 실시예에 따른 제조방법으로 제조된 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드의 티로페록시다제(TPO) 발현을 도시한 것이다.Figure 37 shows the expression of thyroperoxidase (TPO) in thyroid organoids for evaluating the efficacy or toxicity of test substances prepared by the production method according to an embodiment of the present invention.
도 38는 본 발명의 일 실시예에 따른 제조방법으로 제조된 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드의 E-cadherin 발현을 도시한 것이다.Figure 38 shows E-cadherin expression in thyroid organoids for evaluating the efficacy or toxicity of test substances prepared by the production method according to an embodiment of the present invention.
도 39은 본 발명의 일 실시예에 따른 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드를 이용한 시험물질의 효능 또는 독성 평가 방법에 대한 흐름도이다.Figure 39 is a flowchart of a method for evaluating the efficacy or toxicity of a test substance using thyroid organoids for evaluating the efficacy or toxicity of the test substance according to an embodiment of the present invention.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 첨부되는 도면과 함께 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나, 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다. The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments only serve to ensure that the disclosure of the present invention is complete and are within the scope of common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.
본 명세서에서 사용되는 용어 "또는"은 달리 언급하지 않는 한 "및/또는"을 의미한다As used herein, the term “or” means “and/or” unless otherwise stated.
본 명세서에서 사용되는 용어 "약"은, 당업자에게 용이하게 알려진 각각의 값에 대한 보통의 오차 범위를 지칭한다. 본 명세서에서 "약" 값 또는 파라미터 지칭은 그 값 또는 파라미터 자체에 관한 실시예를 포함한다. 나아가, 용어 "약" 은 문맥으로부터 달리 언급되거나 달리 명백하지 않는 한 언급된 참조 값의 어느 한 방향(초과 또는 미만)으로 10 % 내에 해당하는 값의 범위를 나타낸다.As used herein, the term “about” refers to the normal error range for each value, which is readily known to those skilled in the art. Reference herein to “about” a value or parameter includes instances of the value or parameter itself. Furthermore, the term “about” refers to a range of values that falls within 10% in either direction (above or below) of the stated reference value, unless otherwise stated or apparent from the context.
본 명세서에서 사용되는 용어 "환자 또는 개체"는, 상호교환적으로 사용되고 치료가 요구되는 임의의 단일 동물, 더 바람직하게는 포유동물 (그와 같은 비-인간 동물, 예를 들어, 고양이, 개, 말, 토끼, 동물원 동물, 소, 돼지, 양, 및 비-인간 영장류 포함)을 지칭한다. 본 명세서의 다양한 실시예에서 지칭하는 환자는 인간일 수 있다.As used herein, the term “patient or subject” is used interchangeably and refers to any single animal in need of treatment, more preferably a mammal (such as a non-human animal, e.g., cat, dog, (including horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates). Patients referred to in various embodiments herein may be humans.
본 명세서에서 사용되는 용어 "분화(Differentiation)"는 세포가 특별한 기능을 갖는 특정한 세포나 조직의 복합체 또는 개체의 수준으로 발달하는 것을 의미한다.As used herein, the term “differentiation” refers to the development of cells to the level of a specific cell or tissue complex or entity with a special function.
본 명세서 내에서 사용되는 용어, "오가노이드"는 조직 또는 기관의 형태와 기능 모두를 재현하는 작은 배양체를 의미한다. 보다 구체적으로, 오가노이드는 기관 또는 조직을 구성하는 여러 종류의 세포들 중 하나 이상의 세포 종류를 포함하고 있어야 하며, 각 기관이 갖는 특수한 기능을 재현할 수 있어야 하며, 세포들끼리 서로 뭉쳐서 공간적으로 기관과 유사한 형태로 조직되어야 한다. 이러한, 오가노이드는 단순한 세포들의 집합체가 아닌 계통을 이루고 있다는 점에서 스페로이드(speroid)와 차이를 가지며, 신약 개발, 인공 장기, 질병 치료제 및 질병 치료를 위한 환자 별 모델로 이용될 수 있다. As used herein, the term “organoid” refers to a small culture that recapitulates both the form and function of a tissue or organ. More specifically, organoids must contain one or more cell types among the various types of cells that make up an organ or tissue, must be able to reproduce the special functions of each organ, and must be able to reproduce the special functions of each organ, and the cells must cluster together to form an organ spatially. It should be organized in a similar form. Organoids differ from spheroids in that they form a system rather than a simple collection of cells, and can be used as a patient-specific model for new drug development, artificial organs, disease treatments, and disease treatment.
본 명세서 내에서 사용되는 용어 "배지"는, 당, 아미노산, 각종 영양물질, 혈청, 성장인자, 무기질 등의 세포의 성장 및 증식 등에 필수적인 요소를 포함하는 생체 외(In vitro)에서 다양한 세포의 성장 및 증식을 위한 혼합물을 의미한다.The term "medium" used within this specification refers to the growth of various cells in vitro, containing essential elements for cell growth and proliferation, such as sugars, amino acids, various nutrients, serum, growth factors, and minerals. and mixtures for propagation.
이때, 본 명세서에서 사용되는 용어 "세포외 기질(extracellular matrix, ECM) "은 세포의 증식, 분화 및 사멸 등의 여러가지 세포 대사경로에서 영향을 미치는 신호를 제공하는데 중요한 역할을 하는 3차원 구조의 조직의 발달에 지지체를 의미한다. 세포외 기질은 세포가 성장하고 분화하는데 필요한 화학적 인자(biochemical factors)들을 저장하고, 공급해줌과 동시에 세포가 인식할 수 있는 물리적 환경을 제공할 수 있다. 세포외 기질은 각 조직을 구성하는 세포들이 필요에 의해서 만들어낸 산물로서, 콜라겐(collagen) 및 엘라스틴(elastin)과 같은 구조체 단백질(structural protein), GAG(glycosaminoglycan)과 같은 다당류, 세포의 부착을 돕는 부착 단백질(adhesive proteins) 및 성장인자들을 포함하고 있다. 이러한, 세포외 기질은 유래되는 조직 및 세포에 따라 서로 다른 성분으로 구성되어 있으며, 특수한 물리적 성질을 지니고 있다. At this time, the term “extracellular matrix (ECM)” used in this specification refers to a three-dimensional tissue that plays an important role in providing signals that affect various cellular metabolic pathways such as cell proliferation, differentiation, and death. stands for support in the development of The extracellular matrix stores and supplies the biochemical factors necessary for cell growth and differentiation, while also providing a physical environment that cells can recognize. The extracellular matrix is a product produced by the cells that make up each tissue as needed, including structural proteins such as collagen and elastin, polysaccharides such as GAG (glycosaminoglycan), and other substances that help cells adhere. Contains adhesive proteins and growth factors. This extracellular matrix is composed of different components depending on the tissue and cells from which it is derived, and has special physical properties.
오가노이드 배양에 대표적으로 이용되는 세포외기질은 마트리겔(Matrigel), 하이드로겔(Hidrogel) 등이 있으나 다른 성장인자의 첨가가 필요하고 시간이 많이 소요되는 문제점이 있다. 본 발명의 발명자들은 이를 극복한 오가노이드 배양용 세포외기질을 개발하였으며 KR10-2021-0145017, KR10-2022-0152904 의 특허출원을 참고하여 이해될 수 있다.Extracellular matrices typically used for organoid culture include Matrigel and Hydrogel, but they have the problem of requiring the addition of other growth factors and taking a lot of time. The inventors of the present invention have developed an extracellular matrix for organoid culture that overcomes this, and can be understood by referring to the patent applications of KR10-2021-0145017 and KR10-2022-0152904.
본 발명의 일 실시예에 따른 '인체 세포외기질'은 본 발명의 발명자들이 개발한 오가노이드 배양용 세포외기질(ECM)로서 3차원 세포배양용 ECM일 수 있다. 3차원 세포배양용 ECM에 관한 설명은 상술한 특허출원에 기재된 모든 내용을 공유할 수 있다. The 'human extracellular matrix' according to an embodiment of the present invention is an extracellular matrix (ECM) for organoid culture developed by the inventors of the present invention, and may be an ECM for three-dimensional cell culture. The description of the ECM for 3D cell culture can be shared with all the contents described in the above-mentioned patent application.
구체적으로 3차원 세포배양용 ECM은 인체 피부 진피의 섬유아세포를 배양하여 패치를 획득하고, 획득된 인체 유래 섬유아세포 패치에 단백질 분해효소를 처리한 다음 탈세포화한 후 수득된 세포외기질(ECM)일 수 있다. 수득된 세포외기질은 콜라겐(collagne), 액티닌(actinin) 및 액틴-결합 유사 단백질(filamin-C)을 포함하고, 나노 섬유(nano fiber)가 뒤엉킨 형태일 수 있다. 즉, 종래의 파우더 형태의 세포외기질이 배양 플레이트 표면에 코팅된 상태에서 세포를 배양하는 방식에서 나아가, 본 발명에 따른 섬유 형태의 세포외기질은 부유성을 갖는 세포의 모든 표면적을 감싸 배양됨에 따라 안정적으로 생체 내 환경을 가장 유사하게 모사하고, 단시간 내에 충분한 양으로 획득될 수 있다. Specifically, ECM for 3D cell culture is an extracellular matrix (ECM) obtained by culturing fibroblasts from the dermis of human skin to obtain a patch, treating the obtained human-derived fibroblast patch with proteolytic enzymes, and then decellularizing it. It can be. The obtained extracellular matrix contains collagen, actinin, and actin-binding-like protein (filamin-C), and may be in the form of tangled nanofibers. That is, going beyond the conventional method of culturing cells with a powder-type extracellular matrix coated on the surface of a culture plate, the fiber-type extracellular matrix according to the present invention covers the entire surface area of floating cells and is cultured. Accordingly, it can stably simulate the in vivo environment as closely as possible and be obtained in sufficient quantities within a short period of time.
더욱 구체적으로, 본원발명의 3차원 세포배양용 ECM은 섬유아세포(fibroblast) 및 세포외기질을 포함하는 섬유아세포 패치(patch)가 형성되도록, 섬유아세포를 자극 배지(stimulation medium)에서 배양하는 단계, 형성된 섬유아세포 패치에 단백질 분해효소를 처리하는 단계, 단백질 분해효소가 처리된 섬유아세포 패치를 냉동시키는 단계, 섬유아세포 패치에서 섬유아세포가 탈세포화(decellularization)되도록, 냉동된 섬유아세포 패치를 해동하는 단계 및 탈세포화된 섬유아세포 패치로부터 세포외 기질을 수득하는 단계를 포함한다. 이때, 자극 배지에서 배양하는 단계에서, 자극 배지는 0.01 내지 2 mM의 아스코르빅 산(ascorvic acid)을 포함하고 있을 수 있다.More specifically, the ECM for 3D cell culture of the present invention includes the steps of culturing fibroblasts in a stimulation medium so that a fibroblast patch containing fibroblasts and extracellular matrix is formed; Processing the formed fibroblast patch with a proteolytic enzyme, freezing the protease-treated fibroblast patch, and thawing the frozen fibroblast patch so that the fibroblasts in the fibroblast patch are decellularized. and obtaining extracellular matrix from the decellularized fibroblast patch. At this time, in the step of culturing in the stimulation medium, the stimulation medium may contain 0.01 to 2 mM ascorbic acid.
보다 구체적으로, 아스코르빅산(Ascorbic acid)은 산화방지제로 procollagen 합성에 관여하며, type 1 콜라겐생산의 증가와 관련된 보조인자(Cofactor)이다. 아스코르빅산은 in vitro에서 지방세포, 조골세포, 연골세포와 같은 다양한 세포 증식을 자극 및 조절할 수 있다. 나아가, 특정 농도의 아스코르빅산을 첨가할 경우, 세포 성장 촉진제로 작용하여 세포의 증식력이 증가되고, DNA의 합성까지 촉진시킬 수 있다. 그러나, 아스코르빅산의 농도가 적절하지 못할 경우, 오히려 세포의 증식력을 억제시키고, 세포 독성(Cytotoxic)을 가짐으로 세포 자살(Apoptosis)를 일으킬 수 있다. 이에, 세포의 증식력 즉, 세포외 기질의 합성 및 배출을 향상시킬 수 있는 적정 아스코르빅산의 농도는 0.01 내지 1 mM일 수 있으나, 이에 제한되는 것은 아니며, 보다 바람직한 아스코르빅산의 농도는 0.1 내지 1 mM일 수 있다.More specifically, ascorbic acid is an antioxidant that is involved in procollagen synthesis and is a cofactor associated with increased type 1 collagen production. Ascorbic acid can stimulate and regulate the proliferation of various cells such as adipocytes, osteoblasts, and chondrocytes in vitro. Furthermore, when ascorbic acid is added at a certain concentration, it acts as a cell growth promoter, increases cell proliferation, and even promotes DNA synthesis. However, if the concentration of ascorbic acid is not appropriate, it may inhibit the proliferation of cells and be cytotoxic, causing apoptosis. Accordingly, the appropriate concentration of ascorbic acid that can improve the proliferation of cells, that is, the synthesis and excretion of extracellular matrix, may be 0.01 to 1 mM, but is not limited thereto, and a more preferable concentration of ascorbic acid is 0.1 to 1 mM. It may be 1mM.
나아가, 자극 배지는 아스코르빅산을 포함하는 배지임에 따라, 기초가 되는 기본 배지를 포함하고 있다. 예를들어, 기본 배지는 세포가 살아가기 위해 필요한, 당, 아미노산 및 물 등이 포함되어 있는 혼합물로서, 혈청, 영양 물질 및 각종 성장인자를 제외한 혼합물이다. 본 발명의 기본 배지는 인위적으로 합성하여 제조하여 사용하거나 상업적으로 제조된 배지를 사용할 수 있다. 예를 들어, 상업적으로 제조된 배지는 DMEM(Dulbecco's Modified Eagle's Medium), DMEM/F-12, MEM(Minimal Essential Medium), BME(Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM(α-Minimal Essential Medium), G-MEM(Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium 및 FBS (Fetal bovine serum) 등을 포함할 수 있으나, 이에 제한되는 것은 아니며, 바람직하게는 DMEM/F-12일 수 있다.Furthermore, since the stimulation medium is a medium containing ascorbic acid, it contains the basic medium as a basis. For example, basic medium is a mixture containing sugars, amino acids, and water necessary for cells to live, excluding serum, nutrients, and various growth factors. The basic medium of the present invention can be artificially synthesized and used, or a commercially produced medium can be used. For example, commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F-12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, α -May include, but are not limited to, MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium, and FBS (Fetal bovine serum), and are preferably DMEM/F. It could be -12.
나아가, 자극 배지는, 0.0001 내지 0.001 %의 아세틱산(acetic acid)을 더 포함할 수 있으나, 이에 제한되는 것은 아니다.Furthermore, the stimulation medium may further include 0.0001 to 0.001% of acetic acid, but is not limited thereto.
또한, 자극 배지에서 배양하는 단계에서, 배양 기간은 3 내지 20주(weeks) 중 적어도 하나의 기간일 수 있으나, 이에 제한되는 것은 아니며, 바람직하게는 6 내지 12주 중 적어도 하나의 기간일 수 있다.Additionally, in the step of culturing in a stimulation medium, the culture period may be at least one period of 3 to 20 weeks, but is not limited thereto, and preferably may be at least one period of 6 to 12 weeks. .
이에, 전술한 배양하는 단계를 통하여, 섬유아세포는 아스코르빅산에 의하여 자극(stimulation)되어 세포외 기질을 생산 및 방출할 수 있으며, 섬유아세포 및 세포외 기질을 포함하는 섬유아세포 패치가 형성될 수 있다.Accordingly, through the above-described culturing step, fibroblasts can be stimulated by ascorbic acid to produce and release extracellular matrix, and a fibroblast patch containing fibroblasts and extracellular matrix can be formed. there is.
이때, 세포외 기질을 생산 및 방출하는 섬유아세포는 세포외 기질과 콜라겐을 합성하는 생물학적 세포의 한 종류로서, 동물조직에서 구조적 틀을 만드는 가장 흔합 결합조직이며, 심장 조직과 같은 다양한 조직으로부터 수득될 수 있다. 예를 들어, 섬유아세포는 힘줄(tendon), 인대(ligament), 근육, 피부, 치주, 각막, 연골(cartilage), 뼈, 간, 혈관, 심장, 소장, 대장 및 추간판(intervertebral disc) 중 적어도 하나로부터 유래될수 있으나, 이에 제한되는 것은 아니다.At this time, fibroblasts, which produce and release extracellular matrix, are a type of biological cell that synthesizes extracellular matrix and collagen, and are the most common connective tissue that creates the structural framework in animal tissue, and can be obtained from various tissues such as heart tissue. You can. For example, fibroblasts can be found in at least one of tendon, ligament, muscle, skin, periodontium, cornea, cartilage, bone, liver, blood vessel, heart, small intestine, large intestine, and intervertebral disc. It may be derived from, but is not limited to.
다시, 배양하는 단계 이후, 섬유아세포 패치로부터 세포외 기질만을 추출하기 위한, 탈세포화(decellularization) 과정이 수행될 수 있으며, 탈세포화를 위한 과정으론 단백질 분해효소를 처리하는 단계, 섬유아세포 패치를 냉동시키는 단계 및 섬유아세포 패치를 해동하는 단계를 포함할 수 있다. 나아가, 세포 배양 즉 생체소재로 사용될 세포외 기질은 특정 세포가 생리학적 특성을 유지하기 위하여, 세포외 기질의 3차원적 구조와 생리활성 물질들을 최대한 많이 보유하여야 한다. 이에, 면역 거부반응을 일으킬 수 있는 항원성이 제거되어야 함에 따라, 구조체 역할을 세포를 제외한 나머지는 모두 제거하는 탈세포화 과정이 필수적일 수 있다.Again, after the culturing step, a decellularization process may be performed to extract only the extracellular matrix from the fibroblast patch, and the decellularization process includes treatment with proteolytic enzymes and freezing the fibroblast patch. It may include the step of thawing the fibroblast patch and the step of thawing the fibroblast patch. Furthermore, the extracellular matrix to be used for cell culture, that is, as a biomaterial, must have as many three-dimensional structures and bioactive substances as possible in order for specific cells to maintain their physiological characteristics. Accordingly, as antigens that can cause immune rejection must be removed, a decellularization process that removes everything except cells that serve as structures may be essential.
이에, 형성된 섬유아세포 패치에 단백질 분해효소를 처리하는 단계가 수행될 수 있다. 이때, 단백질 분해효소는 0.01 내지 1 %의 트립신(trypsin)일 수 있으나, 바람직하게는, 0.25 %의 트립신일 수 있다. 그러나, 단백질 분해효소는 이에 제한되는 것은 아니며, 섬유아세포 및 세포외 기질간의 결합 단백질을 분해할 수 있는 단백질 분해효소를 모두 포함할 수 있다. 예를 들어, 단백질 분해효소는 콜라제네이즈(Collagenase), 엘라스테이즈(Elastase), 디스페이즈(Dispase), 프로테아제(Protease), 펩신(Pepsin), 레닌(Rennin), 키모트립신(Chymotrypsin), 에렙신(Erepsin), 엔테로키네이즈(Enterokinase), 펩티데이스(Peptidase) 및 프로티네이즈(Proteinase) 등을 포함할 수 있다.Accordingly, a step of treating the formed fibroblast patch with a proteolytic enzyme may be performed. At this time, the proteolytic enzyme may be 0.01 to 1% trypsin, but is preferably 0.25% trypsin. However, proteolytic enzymes are not limited to this and may include all proteolytic enzymes capable of decomposing binding proteins between fibroblasts and extracellular matrix. For example, proteolytic enzymes include Collagenase, Elastase, Dispase, Protease, Pepsin, Rennin, Chymotrypsin, and Erepsin. (Erepsin), Enterokinase, Peptidase, Proteinase, etc.
그 다음, 단백질 분해효소가 처리된 섬유아세포 패치를 냉동시키는 단계가 수행될 수 있다. 이때, 냉동시키는 단계는 -10 ℃이하의 온도에서 수행될 수 있으나, 이에 제한되는 것은 아니다.Next, a step of freezing the proteolytic enzyme-treated fibroblast patch may be performed. At this time, the freezing step may be performed at a temperature of -10°C or lower, but is not limited thereto.
그 다음, 섬유아세포 패치에서 섬유아세포가 탈세포화되도록, 냉동된 섬유아세포 패치를 해동하는 단계가 수행될 수 있다. 이때, 해동하는 단계는, 2시간 이상 동안 실온(room temperature)에서 수행될 수 있다.A step may then be performed to thaw the frozen fibroblast patch, such that the fibroblasts in the fibroblast patch are decellularized. At this time, the thawing step may be performed at room temperature for 2 hours or more.
이에, 전술한 탈세포화(decellularization) 과정을 통하여, 섬유아세포 패치로부터 섬유아세포가 유출되어, 섬유아세포 및 기타 세포들을 포함하지 않는 세포외 기질만이 수득할 수 있다.Accordingly, through the above-described decellularization process, fibroblasts are exuded from the fibroblast patch, and only extracellular matrix that does not contain fibroblasts and other cells can be obtained.
한편, 탈세포화(decellularization) 과정은 전술한 냉동 및 해동을 포함하지 않더라도, 수행될 수 있다. 보다 구체적으로, 본 발명의 일 실시예에 따른 세포외 기질의 제조 방법은 탈세포화를 위한 과정으로 단백질 분해효소를 처리하는 단계 이후, 탈세포화 버퍼(decellularization buffer)를 처리하는 단계를 더 포함할 수 있으며, 이때, 탈세포화 버퍼는 Triton-X 또는 EDTA를 포함할 수 있다. 그러나, 탈세포화 버퍼의 구성은 전술한 Triton-X 또는 EDTA에 제한되는 것은 아니며, 본 발명의 분야에서 상업적으로 이용되는 비이온성 계면 활성 성분이 모두 포함될 수 있다.Meanwhile, the decellularization process can be performed even if it does not include the above-described freezing and thawing. More specifically, the method for producing an extracellular matrix according to an embodiment of the present invention may further include the step of treating a decellularization buffer after treating a proteolytic enzyme as a decellularization process. In this case, the decellularization buffer may include Triton-X or EDTA. However, the composition of the decellularization buffer is not limited to the above-described Triton-X or EDTA, and may include all nonionic surfactant ingredients commercially used in the field of the present invention.
나아가, 본 발명의 일 실시예에 따른 세포외 기질의 제조 방법은, 해동하는 단계 이후, 탈세포화가 완전히 이루어지지 않을 경우, 해동된 섬유아세포 패치에 단백질 분해효소를 처리하는 단계를 더 포함할 수 있다.Furthermore, the method for producing an extracellular matrix according to an embodiment of the present invention may further include the step of treating the thawed fibroblast patch with a protease if decellularization is not completely achieved after the thawing step. there is.
이때, 해동된 섬유아세포 패치에 단백질 분해효소를 처리하는 단계에서 단백질 분해효소는 전술한 단백질 분해효소와 동일한 조건일 수 있으며, 이후 전술한 과정과 동일한 과정을 통하여 탈세포화가 이루어질 수 있다. 그러나, 이에 제한되는 것은 아니며, 예를 들어, 해동된 섬유아세포 패치에 단백질 분해효소를 처리하는 단계는 냉동 및 해동하는 단계가 수행되지 않고, 37 ℃의 항온수조에서 섬유아세포 패치에 단백질 분해효소 및 PBS를 첨가하여 교반과 함께 수행될 수 있다. 나아가, 이때, PBS에는 3 %의 triton-X 및 0.05 %의 EDTA가 포함될 수 있으나, 이에 제한되는 것은 아니다.At this time, in the step of treating the thawed fibroblast patch with a proteolytic enzyme, the proteolytic enzyme may be under the same conditions as the proteolytic enzyme described above, and then decellularization may be achieved through the same process as the above-mentioned process. However, it is not limited to this, and for example, the step of treating the thawed fibroblast patch with a proteolytic enzyme does not involve freezing and thawing, but rather involves treating the fibroblast patch with a proteolytic enzyme and a protease in a constant temperature water bath at 37°C. This can be done with agitation by adding PBS. Furthermore, at this time, the PBS may contain 3% triton-X and 0.05% EDTA, but is not limited thereto.
더 나아가, 본 발명의 일 실시예에 따른 세포외 기질의 제조 방법은, 수득하는 단계 이후, 수득된 세포외기질을 동결건조하는 단계를 더 포함할 수 있으나, 이에 제한되는 것은 아니며, 동결건조하는 단계를 통하여, 수득된 세포외 기질의 유통 및 공급이 용이할 수 있다.Furthermore, the method for producing an extracellular matrix according to an embodiment of the present invention may further include, but is not limited to, the step of freeze-drying the obtained extracellular matrix after the obtaining step. Through these steps, distribution and supply of the obtained extracellular matrix can be facilitated.
이에, 본 발명의 일 실시예에 따른 세포외 기질의 제조 방법은, 아스코르빅산 단 한종류의 화학 물질 첨가와 냉동 및 해동이라는 간단한 과정만으로 세포외 기질을 생산할 수 있음에 따라, 종래의 세포외 기질의 생산 방법보다 경제적일 수 있다.Accordingly, the method for producing an extracellular matrix according to an embodiment of the present invention can produce an extracellular matrix with only a simple process of adding ascorbic acid and freezing and thawing, compared to the conventional extracellular matrix. It can be more economical than the substrate production method.
본 발명의 일 실시예에 따른 갑상선 오가노이드 제조방법은 전술한 인체 세포외기질 및 손쉽게 구할 수 있는 갑상선 세포주를 기반으로 세포주 유래 갑상선 오가노이드의 생산을 저비용 및 고효율로 달성시킬 수 있다. The method for producing thyroid organoids according to an embodiment of the present invention can achieve the production of cell line-derived thyroid organoids at low cost and high efficiency based on the above-described human extracellular matrix and easily available thyroid cell lines.
본 명세서에서 사용되는 용어 “약물”은 생물의 이익을 위해 생리적 시스템 또는 질병 상태를 변화시키거나 검토하기 위해서 사용되는 모든 물질을 포함할 수 있다. 또한 신체 구조 또는 기능에 영향을 미치기 위해 사용하는 모든 물질을 포함할 수 있다. 예를 들어, 비타민제, 호르몬제, 금속염류, 백신, 항혈청제, 항생제, 항암제, 화학요법제제, 강심제, 혈압조절제, 항히스타민제, 스테로이드제, 해독제, 조영제 등과 같은 약물화합물, 약물 유사 화합물, 히트 화합물, 선도 물질, 신약 후보물질 또는 다양한 종류의화학물질일 수 있으나, 이에 제한되는 것은 아니다.As used herein, the term “drug” may include any substance used to change or modify a physiological system or disease state for the benefit of an organism. It can also include any substance used to affect body structure or function. For example, vitamins, hormones, metal salts, vaccines, antiserum agents, antibiotics, anticancer agents, chemotherapy agents, cardiotonic agents, blood pressure regulators, antihistamines, steroids, antidotes, contrast media, drug-like compounds, hit compounds, etc. , may be lead substances, new drug candidates, or various types of chemical substances, but are not limited thereto.
본 발명의 일 실시예에 따른 신장암 오가노이드 또는 갑상선암 오가노이드는 암 미세환경을 모사하는 특성을 나타낼 수 있다. 구체적으로 상피중간엽전이(EMT; epithelial to mesenchymal transition) 또는 F-액틴 비정상(F-actin abnormality)이 증가된 암 특성을 모사할 수 있다.Kidney cancer organoids or thyroid cancer organoids according to an embodiment of the present invention may exhibit characteristics that mimic the cancer microenvironment. Specifically, it can simulate cancer characteristics with increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
본 명세서에서 사용되는 용어 “상피중간엽전이(EMT; epithelial- mesenchymal transition)”은 상피세포가 전이능력과 침윤능력을 가지는 세포로 변환하는 과정으로 암의 진행, 전이, 항암치료에 대한 내성 및 암줄기세포 특성을 획득하는 과정에서 중요한 역할을 한다. 본 발명에 따른 신장암 오가노이드 또는 갑상선암 오가노이드는 상피중간엽전이(EMT) 증가에 따른 상피세포의 병리적 특성 변화에 의한 현상이 발생된 것으로 확인되어 암 미세환경 특성을 모사하고 있음을 확인하였다.The term “epithelial-mesenchymal transition (EMT)” used in this specification is a process in which epithelial cells are transformed into cells with metastatic and invasive abilities, which can affect cancer progression, metastasis, resistance to anti-cancer treatment, and cancerous lines. It plays an important role in the process of acquiring air cell characteristics. It was confirmed that the kidney cancer organoid or thyroid cancer organoid according to the present invention was caused by a change in the pathological characteristics of epithelial cells due to an increase in epithelial-mesenchymal transition (EMT), thereby mimicking the characteristics of the cancer microenvironment. .
본 명세서에서 사용되는 용어 “F-액틴 비정상(F-actin abnormality)”은 많은 암에서 비정상적인 액틴 이소형 발현이 보고되는 것에 기초한 암의 초기 바이오마커이다. F-액틴은 세포골격에서 미세 필라멘트를 형성하고 근육 원섬유에서 얇은 필라멘트를 형성하는 구형 다기능 단백질 계열로서 모든 진핵세포에 존재한다. 세포골격은 세포질 내 소기관이며 유동적인 구조로 이루어져 있어 세포 형태를 유지하는 중요한 역할을 할 뿐만 아니라 세포이동을 가능하게 하는데 이러한 세포골격은 정상세포보다 암세포에서 더 역동적으로 변화하는 것으로 알려져 있다. 암세포에서 F-액틴의 역동적인 변화는 암세포의 분열과 성장, 이동 능력에 주요 역할을 하므로 암의 발전과 진행, 전이 등에 기여하게 된다. 이와 같이 세포에 존재하는 F-액틴이 암조직에 미치는 다양한 영향을 기반으로 본 발명에 따른 신장암 오가노이드 또는 갑상선암 오가노이드에서 F-액틴 비정상(F-actin abnormality)에 따른 형태 변화를 분석함으로써 약물의 효능 및 독성 평가에 이용할 수 있다.As used herein, the term “F-actin abnormality” is an early biomarker of cancer based on reports of abnormal actin isoform expression in many cancers. F-actin is a family of globular multifunctional proteins that form fine filaments in the cytoskeleton and thin filaments in muscle fibrils and are present in all eukaryotic cells. The cytoskeleton is an organelle within the cytoplasm and is composed of a fluid structure, which not only plays an important role in maintaining cell shape but also enables cell movement. This cytoskeleton is known to change more dynamically in cancer cells than in normal cells. Dynamic changes in F-actin in cancer cells play a major role in the division, growth, and migration ability of cancer cells, contributing to the development, progression, and metastasis of cancer. In this way, based on the various effects of F-actin present in cells on cancer tissue, morphological changes due to F-actin abnormality in kidney cancer organoids or thyroid cancer organoids according to the present invention are analyzed to determine drug efficacy. It can be used to evaluate the efficacy and toxicity of
본 명세서에서 사용되는 용어 “Na+/K+ ATPase”는 기저막에 위치하여 세포 내 Na 및 K 균형을 유지시킨다. 보다 구체적으로, 신장의 근위관은 신장의 요산(uric acid) 운송을 담당하며, 요산의 재흡수가 주로 일어나는 곳이다. 근위 신장 관상피세포(Proximal Renal Tubular Epithelial Cells, PTECs)는 요산을 배출하고, 이온 및 유산 운송 채널을 발현함에 따라, 미토콘드리아 및 리소좀이 풍부하게 포함되어 있다. 이때, 신장 근위 세뇨관 상피세포에서의 요산 수송(uric acid transporter)의 추진력은 세관 상피 기저(tubule epithelium basolateral)에 존재하는 Na+/K+ ATPase에서 발생된다. Na+/K+ ATPase의 주요 기능은 신장 내 전해질 및 액체의 항상성을 제어하는 것임에 따라, 신장암 오가노이드에서의 Na+/K+ ATPase의 발현 변화는 본 발명에 따른 신장암 오가노이드를 활용한 약물의 효능 또는 독성 평가에서 중요한 바이오마커가 될 수 있다.The term “Na+/K+ ATPase” used herein is located in the basement membrane and maintains intracellular Na and K balance. More specifically, the proximal duct of the kidney is responsible for the transport of uric acid to the kidney and is where uric acid reabsorption primarily occurs. Proximal Renal Tubular Epithelial Cells (PTECs) are rich in mitochondria and lysosomes, as they excrete uric acid and express ion and lactic acid transport channels. At this time, the driving force of uric acid transporter in renal proximal tubular epithelial cells is generated from Na+/K+ ATPase present at the tubule epithelium basolateral. As the main function of Na+/K+ ATPase is to control electrolyte and fluid homeostasis in the kidney, changes in the expression of Na+/K+ ATPase in kidney cancer organoids determine the efficacy of drugs using kidney cancer organoids according to the present invention. Alternatively, it can be an important biomarker in toxicity evaluation.
나아가, 신장암 오가노이드에서의 Na+/K+ ATPase는 발현 유무뿐만 아니라, 이의 위치 또한 매우 중요할 수 있다. 보다 구체적으로, Na+/K+ ATPase는 세뇨관 및 간질액과의 Na+/K+ 평형을 유지해야함에 따라, 세포막(기저막)에 존재해야 하며, 이의 위치 변경은 약물 및 질병에 의한 신장 근위 세뇨관의 병태생리학적 변화를 대변할 수 있다.Furthermore, not only the expression of Na+/K+ ATPase in kidney cancer organoids, but also its location may be very important. More specifically, Na+/K+ ATPase must be present in the cell membrane (basement membrane) as it must maintain Na+/K+ equilibrium with the tubules and interstitial fluid, and its location change may affect the pathophysiology of the renal proximal tubules due to drugs and diseases. It can represent change.
본 명세서에서 사용되는 용어 “E-cadherin”은 세포 간의 접착을 유지하는 cadherin 계열의 분자로 대부분의 상피세포에서 발현이 되며 상피세포간 연결을 안정적으로 유지시키는 중요한 단백질이다. 특히, 세포 접착(adhesion) 및 신장 조직 구조(tissue structure)에 중요한 역할을 하는, 정상 조직에서 견고히 발현되는 단백질 중 하나이다. 상피세포의 특성이 변하게 되면 상피-중간엽전이(Epithelial-mesenchymal transition; EMT)가 활성화되어 병리적 현상이 관찰된다. 따라서 본 발명에 따른 오가노이드를 활용한 약물의 효능 또는 독성 평가에서 E-cadherin 발현 변화는 중요한 바이오마커로 이용될 수 있다.The term “E-cadherin” used herein is a cadherin-family molecule that maintains adhesion between cells. It is expressed in most epithelial cells and is an important protein that stably maintains connections between epithelial cells. In particular, it is one of the proteins robustly expressed in normal tissues, playing an important role in cell adhesion and kidney tissue structure. When the characteristics of epithelial cells change, epithelial-mesenchymal transition (EMT) is activated and pathological phenomena are observed. Therefore, changes in E-cadherin expression can be used as an important biomarker in evaluating the efficacy or toxicity of drugs using the organoid according to the present invention.
본 명세서에서 사용되는 용어 “Vimentin”은 중간 사이즈의 섬유 단백질로써, 주로 중간엽 및 신경 조직의 세포에서 발견된다. 질병 및 약물에 의하여 상피세포가 손실되고, 이에, 상피세포가 이동성을 가진 중간엽 세포로 변환되는 EMT가 활성화되면 vimentin이 증가된다. 이와 관련하여, 생체 내 신장이 질환 특히, 신경병증이나 섬유화가 발생하였을 경우, EMT가 활성화되어 vimentin이 상향조절(upregulation)된다. 이에, vimentin 발현의 변화(증가)는 병리적 현상을 초래하거나, 병리적 현상에 의하여 발생됨에 따라, 이에 기초하여 신장의 병태생리학적 변화를 예측할 수 있다. As used herein, the term “Vimentin” refers to a medium-sized fibrous protein and is mainly found in cells of mesenchymal and nervous tissue. When epithelial cells are lost due to disease or drugs, and EMT, which converts epithelial cells into mobile mesenchymal cells, is activated, vimentin increases. In this regard, when kidney disease, especially neuropathy or fibrosis, occurs in vivo, EMT is activated and vimentin is upregulated. Accordingly, as changes (increases) in vimentin expression cause pathological phenomena or are caused by pathological phenomena, pathophysiological changes in the kidney can be predicted based on this.
본 발명의 일 실시예에 따른 신장암 오가노이드는 F-액틴 비정상(F-actin abnormality), Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커를 발현하는 것을 확인하였다. 따라서 본 발명의 일 실시예에 따른 신장암 오가노이드를 활용하면 상술한 바이오마커를 지표로 목적하는 약물의 효능 및 독성을 예측할 수 있다. Kidney cancer organoids according to an embodiment of the present invention were confirmed to express at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin. . Therefore, by using the kidney cancer organoid according to an embodiment of the present invention, the efficacy and toxicity of the target drug can be predicted using the above-described biomarkers.
따라서 본 발명은 약물의 효능 또는 독성 평가용 신장암 오가노이드에 약물 또는 항암 후보물질을 처리하는 단계; 상기 약물 또는 항암 후보물질이 처리된 오가노이드에 대해 F-액틴 비정상(F-actin abnormality), Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 측정하는 단계를 포함하는, 신장암 오가노이드를 이용한 약물의 평가 방법 또는 항암제 스크리닝 방법을 제공할 수 있다.Therefore, the present invention includes the steps of treating a drug or anticancer candidate material to kidney cancer organoids for evaluating drug efficacy or toxicity; Measure the level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin for organoids treated with the drug or anticancer candidate material. It is possible to provide a drug evaluation method or an anticancer drug screening method using kidney cancer organoids, including the step of:
본 발명의 특징에 따르면, 상술한 방법은 Na+/K+ ATPase 또는 E-cadherin 발현 위치를 확인하는 단계를 더 포함할 수 있다.According to a feature of the present invention, the above-described method may further include the step of confirming the expression location of Na+/K+ ATPase or E-cadherin.
구체적으로, 약물 또는 항암 후보물질이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality) 또는 Vimentin 수준이 감소하거나 Na+/K+ ATPase 또는 E-cadherin 수준이 세포막에서 증가한 경우 약물이 효능을 보이는 것으로 판단할 수 있다. 상기 판단은 Na+/K+ ATPase 또는 E-cadherin의 수준이 세포질(cytosol)에서 감소한 경우를 더 포함할 수 있다.Specifically, for organoids treated with drugs or anticancer candidates, F-actin abnormality or Vimentin levels were decreased or Na+/K+ ATPase or E-cadherin levels were decreased compared to the drug-untreated group or positive control group. If it increases in the cell membrane, the drug can be judged to be effective. The determination may further include a case where the level of Na+/K+ ATPase or E-cadherin is decreased in the cytosol.
구체적으로, 약물 또는 항암 후보물질이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality) 또는 Vimentin 수준이 증가하거나 Na+/K+ ATPase 또는 E-cadherin 수준이 세포막에서 감소한 경우 약물이 독성을 보이는 것으로 판단할 수 있다. 상기 판단은 Na+/K+ ATPase 또는 E-cadherin의 수준이 세포질(cytosol)에서 증가한 경우를 더 포함할 수 있다.Specifically, for organoids treated with drugs or anticancer candidates, F-actin abnormality or Vimentin levels were increased or Na+/K+ ATPase or E-cadherin levels were increased compared to the drug-untreated group or positive control group. If it decreases in the cell membrane, the drug can be judged to be toxic. The determination may further include a case where the level of Na+/K+ ATPase or E-cadherin is increased in the cytosol.
본 명세서에서 사용되는 용어 “갑상선암 자극 호르몬 수용체(THSR)”는 갑상선암 세포의 세포막 표면에 위치한 G 단백질 결합 수용체로서, 이는 뇌하수체(pituitary)에서 분비하는 갑상선암 자극 호르몬의 수용체이다. 갑상선암 자극 호르몬이 갑상선암 자극 호르몬 수용체와 결합이 되면 갑상선암 성장, 갑상선암 세포의 분화, 갑상선암 호르몬의 합성 등이 일어난다. 약물에 포함되는 화학물질 또는 환경에서 노출되는 화학물질 등의 다양한 화학물질에 생체가 노출되었을 때 뇌하수체가 자극되어 갑상선암 자극 호르몬이 증가 또는 감소하고, 이는 갑상선암 자극 호르몬의 수용체에 영향을 주게 된다. 따라서 본 발명에 따른 갑상선암 오가노이드를 활용한 약물의 효능 또는 독성 평가에서 중요한 바이오마커가 될 수 있다.The term “thyroid cancer-stimulating hormone receptor (THSR)” used herein is a G protein-coupled receptor located on the cell membrane surface of thyroid cancer cells, which is a receptor for thyroid cancer-stimulating hormone secreted by the pituitary. When thyroid cancer-stimulating hormone combines with the thyroid cancer-stimulating hormone receptor, thyroid cancer growth, differentiation of thyroid cancer cells, and synthesis of thyroid cancer hormones occur. When the body is exposed to various chemicals, such as chemicals contained in drugs or chemicals exposed in the environment, the pituitary gland is stimulated and thyroid cancer-stimulating hormone increases or decreases, which affects the receptor for thyroid cancer-stimulating hormone. Therefore, it can be an important biomarker in evaluating the efficacy or toxicity of drugs using the thyroid cancer organoid according to the present invention.
본 명세서에서 사용되는 용어 “티로글로불린(Thyroglobulin; Tg)”은 갑상선암 호르몬 합성에 관여하는 주요 단백질이다. 티로글로불린은 정상 갑상선암 조직 및 갑상선암 조직에서만 분비되므로 갑상선암 특이 바이오마커로서 본 발명에 따른 갑상선암 오가노이드의 생체 유사도를 평가하는 지표가 될 수 있다. 또한 본 발명에 따른 갑상선암 오가노이드를 활용한 약물의 효능 또는 독성 평가에서 중요한 바이오마커로 이용될 수 있다.The term “Thyroglobulin (Tg)” used herein is a major protein involved in thyroid cancer hormone synthesis. Since thyroglobulin is secreted only in normal thyroid cancer tissue and thyroid cancer tissue, it can be a thyroid cancer-specific biomarker and an indicator for evaluating the biosimilarity of the thyroid cancer organoid according to the present invention. Additionally, the thyroid cancer organoid according to the present invention can be used as an important biomarker in evaluating the efficacy or toxicity of a drug.
본 명세서에서 사용되는 용어 “티로페록시다제(Thyroperoxidase; TPO)”는 갑상선암 호르몬 합성에 관여하는 효소로 티로글로불린의 티로신 잔기에서 요오드화물(iodide)의 산화를 촉매하여 T3(Triiodothyronine)와 T4(Tetraiodothyronine; Thyroxine)을 합성에 필요한 단계를 촉진하는 역할을 한다. 갑상선암에서 TPO의 발현 증가는 갑상선암에 이상이 생겼음을 간접적으로 예측하는 지표임이 알려져 있다. 따라서 본 발명에 따른 갑상선암 오가노이드를 활용한 약물의 효능 또는 독성 평가에서 중요한 바이오마커로 이용될 수 있다.The term “thyroperoxidase (TPO)” used herein is an enzyme involved in thyroid cancer hormone synthesis that catalyzes the oxidation of iodide from the tyrosine residue of thyroglobulin to produce T3 (Triiodothyronine) and T4 (Tetraiodothyronine). It plays a role in promoting the steps necessary for synthesizing Thyroxine. It is known that increased expression of TPO in thyroid cancer is an indicator that indirectly predicts abnormalities in thyroid cancer. Therefore, it can be used as an important biomarker in evaluating the efficacy or toxicity of drugs using the thyroid cancer organoid according to the present invention.
본 발명의 일 실시예에 따른 갑상선 오가노이드는 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커를 발현하는 것을 확인하였다. 따라서 본 발명의 일 실시예에 따른 갑상선암 오가노이드를 활용하면 상술한 바이오마커를 지표로 목적하는 약물의 효능 및 독성을 예측할 수 있다. Thyroid organoid according to an embodiment of the present invention consists of F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. Expression of at least one biomarker selected from the group was confirmed. Therefore, by using the thyroid cancer organoid according to an embodiment of the present invention, the efficacy and toxicity of the target drug can be predicted using the above-described biomarker as an indicator.
따라서 본 발명은 약물의 효능 또는 독성 평가용 갑상선암 오가노이드에 약물 또는 항암 후보물질을 처리하는 단계; 상기 약물 또는 항암 후보물질이 처리된 오가노이드에 대해 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 측정하는 단계를 포함하는, 갑상선암 오가노이드를 이용한 약물의 평가 방법 또는 항암제 스크리닝 방법을 제공할 수 있다.Therefore, the present invention includes the steps of treating a drug or anticancer candidate material to a thyroid cancer organoid for evaluating drug efficacy or toxicity; For organoids treated with the above drugs or anticancer candidates, F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin were detected. A drug evaluation method or an anticancer drug screening method using thyroid cancer organoids can be provided, including the step of measuring the level of at least one biomarker selected from the group consisting of.
본 발명의 특징에 따르면, 상술한 방법은 갑상선 자극 호르몬 수용체(TSHR) 또는 E-cadherin 발현 위치를 확인하는 단계를 더 포함할 수 있다.According to a feature of the present invention, the above-described method may further include the step of confirming the expression location of thyroid-stimulating hormone receptor (TSHR) or E-cadherin.
구체적으로, 약물 또는 항암 후보물질이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 및 티로페록시다제(TPO)로 이루어진 군에서 선택된 적어도 하나의 수준이 감소하거나 티로글로불린(Tg) 또는 E-cadherin의 수준이 증가한 경우 약물이 효능을 보이거나 항암 활성을 보이는 것으로 판단할 수 있다. 상술한 갑상선 자극 호르몬 수용체(TSHR) 수준은 세포내소포(intracellular vesicle)에서 감소 또는 E-cadherin의 수준은 세포막(membrane)에서 증가한 것을 의미할 수 있다.Specifically, organoids treated with drugs or anticancer candidates were compared to the drug-untreated group or the positive control group to determine F-actin abnormality, thyroid-stimulating hormone receptor (TSHR), and thyroperoxidase (TPO). ) If the level of at least one selected from the group consisting of is decreased or the level of thyroglobulin (Tg) or E-cadherin is increased, the drug may be judged to be effective or show anticancer activity. The above-mentioned thyroid stimulating hormone receptor (TSHR) level may mean a decrease in intracellular vesicles or an increase in the level of E-cadherin in the cell membrane.
구체적으로, 약물 또는 항암 후보물질이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 및 티로페록시다제(TPO)로 이루어진 군에서 선택된 적어도 하나의 수준이 증가하거나 티로글로불린(Tg) 또는 E-cadherin의 수준이 감소한 경우 약물이 독성을 보이거나 항암 활성이 없는 것으로 판단할 수 있다. 상술한 갑상선 자극 호르몬 수용체(TSHR) 수준은 세포내소포(intracellular vesicle)에서 증가 또는 E-cadherin의 수준은 세포막(membrane)에서 감소한 것을 의미할 수 있다.Specifically, organoids treated with drugs or anticancer candidates were compared to the drug-untreated group or the positive control group to determine F-actin abnormality, thyroid-stimulating hormone receptor (TSHR), and thyroperoxidase (TPO). If the level of at least one selected from the group consisting of ) increases or the level of thyroglobulin (Tg) or E-cadherin decreases, the drug may be judged to be toxic or have no anticancer activity. The above-mentioned thyroid stimulating hormone receptor (TSHR) level may mean an increase in intracellular vesicles or a decrease in the level of E-cadherin in the cell membrane.
본 명세서에서 사용되는 용어 “시험물질”은 활성 또는 독성 시험에 사용하기 위한 약물 또는 화학물질을 의미할 수 있다. 바람직하게는 항산화제로써 식품, 화장품 등에 다수 사용되는 butylhydroxyanisole (BHA) 등과 같은 약물화합물, 약물 유사 화합물, 히트 화합물, 선도 물질, 신약 후보물질 또는 갑상선 호르몬에 영향을 주는 내분비계 교란물질인 bisphenol A와 과불화화합물 등과 같은 환경 노출 화학물질일 수 있으나 이에 제한되는 것은 아니다.As used herein, the term “test substance” may refer to a drug or chemical substance for use in activity or toxicity testing. Preferably, drug compounds such as butylhydroxyanisole (BHA), which is widely used in foods and cosmetics as an antioxidant, drug-like compounds, hit compounds, lead substances, new drug candidates, or bisphenol A, which is an endocrine disruptor that affects thyroid hormones, These may be environmentally exposed chemicals such as perfluorinated compounds, but are not limited thereto.
위에서도 언급했듯이 환경 노출 화학물질은 생활 환경, 산업체 등에서 광범위하게 사용되는 화학물질로서 환경과 동식물에 영향을 주는 물질을 의미할 수 있다. 바람직하게는 내분비계 교란 화학물질(endocrine disrupting chemical, EDC) 또는 과불화 알킬 물질(PFAS)일 수 있으나 이에 제한되는 것은 아니다. 예를 들어, 내분비계 교란 화학물질은 폴리염화비페닐(PCBs), 폴리브롬화비페닐(PBB), 다이옥신, 푸란, 살충제, 과불화화합물, 프탈레이트, 비스페놀-A(BPA), UV필터, 트리클로산, 과염소산염, 파라벤 또는 부틸하이드록시톨루엔(BHT)일 수 있으나 이에 제한되는 것은 아니다. As mentioned above, environmentally exposed chemicals are chemicals that are widely used in living environments, industries, etc. and can refer to substances that affect the environment and animals and plants. Preferably, it may be an endocrine disrupting chemical (EDC) or a perfluoroalkyl substance (PFAS), but is not limited thereto. For example, endocrine disrupting chemicals include polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBB), dioxins, furans, pesticides, perfluorinated compounds, phthalates, bisphenol-A (BPA), UV filters, triclosan, It may be, but is not limited to, perchlorate, paraben, or butylhydroxytoluene (BHT).
본 발명에 따른 갑상선 오가노이드 제조방법은 갑상선 바이오마커를 발현하여 생체 모사가 뛰어난 갑상선 오가노이드를 짧은 기간에 다량 제공함에 따라 시험물질의 효능 또는 독성 평가를 시험관내에서 진행할 수 있는 갑상선 오가노이드를 제공할 수 있다. The thyroid organoid manufacturing method according to the present invention expresses thyroid biomarkers and provides a large amount of thyroid organoids with excellent biomimetic properties in a short period of time, thereby providing a thyroid organoid that can evaluate the efficacy or toxicity of test substances in vitro. can do.
본 발명의 일 실시예에 따른 갑상선 오가노이드는 트리이오드티로닌(T3), 테트라이오드티로닌(T4), 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나 이상의 바이오마커를 발현하는 것을 확인하였다.The thyroid organoid according to an embodiment of the present invention contains triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and Expression of at least one biomarker selected from the group consisting of E-cadherin was confirmed.
따라서 본 발명의 일 실시예에 따른 갑상선 오가노이드를 활용하면 상술한 바이오마커를 지표로 목적하는 시험물질의 효능 및 독성을 예측할 수 있다. Therefore, by using the thyroid organoid according to an embodiment of the present invention, the efficacy and toxicity of the target test substance can be predicted using the above-described biomarker as an indicator.
구체적으로 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드에 목적하는 시험물질을 처리하는 단계; 및 상기 시험물질이 처리된 군을 시험물질 미처리군 또는 양성 대조군과 비교하여 트리이오드티로닌(T3), 테트라이오드티로닌(T4), 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상의 바이오마커 증감에 따라 시험물질의 효능을 판단하는 단계; 를 포함하는, 시험물질의 효능 평가 방법을 제공할 수 있다Specifically, processing the test substance of interest into a thyroid organoid for evaluating the efficacy or toxicity of the test substance; And by comparing the group treated with the test substance with the untreated group or positive control group, the levels of triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyroglobulin (Tg) were increased. Determining the efficacy of the test substance according to the increase or decrease of one or more biomarkers selected from the group consisting of peroxidase (TPO) and E-cadherin; Can provide a method for evaluating the efficacy of test substances, including
구체적으로 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드에 목적하는 시험물질을 처리하는 단계; 및 상기 시험물질이 처리된 군을 약물 미처리군 또는 양성 대조군과 비교하여 트리이요오드티로닌(T3), 테트라이오드티로닌(T4), 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상의 바이오마커 증감에 따라 시험물질의 독성을 판단하는 단계; 를 포함하는, 시험물질의 독성 평가 방법을 제공할 수 있다.Specifically, processing the test substance of interest into a thyroid organoid for evaluating the efficacy or toxicity of the test substance; And the group treated with the test substance was compared with the drug-untreated group or the positive control group to produce triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyrophe. Determining the toxicity of the test substance according to the increase or decrease of one or more biomarkers selected from the group consisting of roxidase (TPO) and E-cadherin; It is possible to provide a method for evaluating the toxicity of test substances, including:
이하, 실시예를 통하여 본 발명을 보다 상세히 설명한다. 다만, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것에 불과하므로 본 발명의 범위가 이들 실시예에 의해 한정되는 것으로 해석되어서는 아니된다. Hereinafter, the present invention will be described in more detail through examples. However, since these examples are only for illustrative purposes of the present invention, the scope of the present invention should not be construed as being limited by these examples.
신장 근위 세뇨관 오가노이드Kidney proximal tubule organoids
신장 근위 세뇨관 오가노이드, 이의 제조 방법 및 이를 이용한 약물 평가 방법Kidney proximal tubule organoids, method of producing the same, and method of drug evaluation using the same
이하에서는 도 1 내지 3b를 참조하여, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드, 이의 제조 방법 및 이를 이용한 약물 평가 방법에 대하여 구체적으로 설명한다.Hereinafter, with reference to FIGS. 1 to 3B, a kidney proximal tubule organoid according to an embodiment of the present invention, a method for producing the same, and a drug evaluation method using the same will be described in detail.
도 1은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 제조 방법의 절차를 예시적으로 도시한 것이다. 이때, 설명의 편의를 위하여, 도 2a 내지 3b를 참조하여 설명하도록 한다. Figure 1 exemplarily shows the procedure of a method for producing kidney proximal tubule organoids according to an embodiment of the present invention. At this time, for convenience of explanation, description will be made with reference to FIGS. 2A to 3B.
도 1을 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 제조 방법은 3차원 세포 배양 방법에 기초하여 입체적인 신장 근위 세뇨관 오가노이드를 형성하기 위한 제조 방법으로써, 신장 근위 세뇨관 오가노이드를 형성하기 위한 제 1 배양하는 단계 및 형성된 오가노이드를 성장시키기 위한 제 2 배양하는 단계를 포함할 수 있다.Referring to Figure 1, the method for producing kidney proximal tubule organoids according to an embodiment of the present invention is a manufacturing method for forming three-dimensional kidney proximal tubule organoids based on a three-dimensional cell culture method. It may include a first culturing step to form and a second culturing step to grow the formed organoid.
제 1 배양하는 단계는 신장 근위 세뇨관 오가노이드가 형성되도록 신장 근위 세뇨관 상피세포주를 인체 세포외기질(ECM)을 포함하는 제 1 배양 배지에서 배양하는 단계이다. 보다 구체적으로, 제 1 배양하는 단계는 근위 세뇨관 상피세포(주)들과 인체 세포외기질이 함께 서로 융합되어 세포들 사이에 구분이 사라지면서 밀착 연접(tight junction)이 일어나고, 자가 조직화(self-organization)가 일어나면서 오가노이드가 형성되는 단계일 수 있다. The first culturing step is a step of culturing the renal proximal tubular epithelial cell line in a first culture medium containing human extracellular matrix (ECM) to form renal proximal tubular organoids. More specifically, in the first culture step, the proximal tubular epithelial cells (main) and the human extracellular matrix fuse together, the distinction between cells disappears, tight junctions occur, and self-organization (self-organization) occurs. This may be the stage in which organoids are formed as organization occurs.
이때, 신장 근위 세뇨관 상피세포주는 상업적으로 이용되는 RPTEC/TERT1 세포주(cell line)로, 인간 신장 근위 세뇨관 상피세포주를 의미할 수 있으나, 이에 제한되는 것은 아니며, 상업적으로 이용가능한 모든 신장 근위 세뇨관 상피세포주가 이용될 수 있다. 또한, 신장 근위 세뇨관 상피세포주는 상업적으로 이용되는 세포주 뿐만 아니라, 생체 신장으로부터 직접 수득된 신장 근위 세뇨관 상피세포주가 이용될 수 있다.At this time, the renal proximal tubular epithelial cell line is the commercially available RPTEC/TERT1 cell line, which may mean, but is not limited to, a human renal proximal tubular epithelial cell line, and is not limited to all commercially available renal proximal tubular epithelial cell lines. can be used. Additionally, renal proximal tubular epithelial cell lines, as well as commercially available cell lines, can be used as renal proximal tubular epithelial cell lines obtained directly from living kidneys.
제 1 배양 배지는 인체 세포외기질(ECM)을 필수적으로 포함하는 배지를 의미할 수 있다. 보다 구체적으로, 제 1 배양 배지는 세포외기질, 아미노산, 아세트산, 글루타맥스, 아스코르빅산, B27 및 IWR-1 중 적어도 하나를 포함하는 기본 배양 배지일 수 있으며, 전술한 구성을 포함하며 상업적으로 이용되는 ORG 3D solution을 포함하는 배지를 일 수 있으나, 이에 제한되는 것은 아니다. The first culture medium may refer to a medium that essentially contains human extracellular matrix (ECM). More specifically, the first culture medium may be a basic culture medium containing at least one of extracellular matrix, amino acids, acetic acid, glutamax, ascorbic acid, B27, and IWR-1, and may include the above-mentioned composition and a commercially available culture medium. It may be a medium containing ORG 3D solution used, but is not limited thereto.
이때, 제 1 배양 배지의 세포외기질은 인체 피부 진피에서 유래한 섬유아세포로부터 생산된 세포외기질로써, 인체 피부 진피에서 유래한 섬유아세포를 배양하여 패치를 획득하고, 획득된 패치에 단백질 분해효소를 처리하여 탈세포화한 후 수득될 수 있다. 나아가, 수득된 세포외기질은 콜라겐(collagne), 액티닌(actinin) 및 액틴-결합 유사 단백질(filamin-C)을 포함하고, 나노 섬유(nano fiber)가 뒤엉킨 형태일 수 있다. 즉, 나노 섬유 형태의 세포외기질은 신장 근위 세뇨관 상피세포(주)와 빠르게 혼합되고, 세포의 모든 표면적을 감싸 배양됨에 따라 안정적으로 생체 내 환경을 가장 유사하게 모사하여 신장 근위 세뇨관 오가노이드를 단기간 내에 형성할 수 있다. 이에, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법은 전술한 세포외기질을 포함함에 따라, 저비용 및 고효율로 단시간 내에 신장 근위 세뇨관 오가노이드를 형성 및 생산할 수 있다.At this time, the extracellular matrix of the first culture medium is an extracellular matrix produced from fibroblasts derived from human skin dermis. A patch is obtained by culturing fibroblasts derived from human skin dermis, and a proteolytic enzyme is added to the obtained patch. It can be obtained after treatment and decellularization. Furthermore, the obtained extracellular matrix contains collagen, actinin, and actin-binding-like protein (filamin-C), and may be in the form of tangled nanofibers. In other words, the extracellular matrix in the form of nanofibers is rapidly mixed with kidney proximal tubule epithelial cells (mainly), and as it covers the entire surface area of the cells and is cultured, it stably mimics the in vivo environment to produce kidney proximal tubule organoids in a short period of time. can be formed within. Accordingly, the method for producing kidney proximal tubule organoids according to an embodiment of the present invention includes the above-described extracellular matrix, and thus can form and produce kidney proximal tubule organoids in a short period of time at low cost and high efficiency.
나아가, 제 1 배양 배지의 기본 배양 배지는 BME(Basal medium Eagle's), MEM(Minimum essential medium), DMEM(Dulbecco's modified Eagle's medium), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199 및 RPMI 1640 중 적어도 하나를 포함할 수 있으나, 이에 제한되는 것은 아니며, 상업적으로 이용되는 다양한 기본 배양 배지가 이용될 수 있다. Furthermore, the basic culture medium of the first culture medium is BME (Basal medium Eagle's), MEM (Minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199, and It may include at least one of RPMI 1640, but is not limited thereto, and various commercially available basic culture media can be used.
제 1 배양하는 단계에서 신장 근위 세뇨관 상피세포주 및 제 1 배양 배지는 1 : 1 비율로 포함될 수 있으나, 이에 제한되는 것은 아니며, 바람직하게는 제 1 배양 배지 1mL을 기준으로 신장 근위 세뇨관 상피세포주는 1 x 105 내지 1 x 107 세포수로 포함될 수 있으나 이에 제한되는 것은 아니다. In the first culture step, the kidney proximal tubule epithelial cell line and the first culture medium may be included in a 1:1 ratio, but are not limited to this, and preferably, based on 1 mL of the first culture medium, the kidney proximal tubule epithelial cell line is 1:1. It may include, but is not limited to, x 10 5 to 1 x 10 7 cells.
제 1 배양하는 단계는 약 1 시간 내지 3일 중 적어도 하나의 기간 동안 수행될 수 있으나, 이에 제한되는 것은 아니다. 이와 관련하여, 종래의 오가노이드 형성은 수주 이상의 기간이 소요되었다. 그러나, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법은 3일 이내의 단 시간 내에 빠르게 신장 근위 세뇨관 오가노이드가 형성될 수 있는 종래의 신장 근위 세뇨관 오가노이드의 제조 방법의 한계를 극복한 방법일 수 있다. The first culturing step may be performed for at least one period of about 1 hour to 3 days, but is not limited thereto. In this regard, conventional organoid formation takes several weeks or more. However, the method for producing kidney proximal tubule organoids according to an embodiment of the present invention overcomes the limitations of the conventional method for producing kidney proximal tubule organoids in which kidney proximal tubule organoids can be formed quickly within a short time of less than 3 days. That could be one way.
나아가, 제 1 배양하는 단계가 수행된 후 3일(약 72시간) 이내에, 생체 내 신장과 동일한 바이오마커가 발현되어 생체 신장의 병태생리학적 기능 및 반응을 모사할 수 있는 신장 근위 세뇨관 오가노이드가 형성(생성)될 수 있다. Furthermore, within 3 days (about 72 hours) after the first culturing step is performed, a kidney proximal tubule organoid that expresses the same biomarkers as the kidney in vivo and can mimic the pathophysiological function and response of the kidney in vivo is created. It can be formed (created).
한편, 제 1 배양하는 단계에 의하여 형성된 오가노이드들은 100 μm 미만의 크기를 가질 수 있으며, 이의 크기는 실험 또는 임상적인 약물(화합물) 평가에 있어, 오가노이드의 반응을 현미경으로 관찰하기에 충분하지 않을 수 있다. 나아가, 제 1 배양하는 단계에 의하여 형성된 오가노이드들은 각각의 크기 및 성숙도가 상이할 수 있음에 따라, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법은 균질한 특성을 가진 오가노이드 즉, 크기가 균일하고 동일한 바이오마커 발현을 포함하는 오가노이드 형성을 위하여, 제 2 배양하는 단계를 포함할 수 있다. Meanwhile, the organoids formed by the first culturing step may have a size of less than 100 μm, and this size is not sufficient to observe the reaction of the organoids under a microscope in experimental or clinical drug (compound) evaluation. It may not be possible. Furthermore, as the organoids formed by the first culturing step may have different sizes and degrees of maturity, the method for producing renal proximal tubule organoids according to an embodiment of the present invention produces organoids with homogeneous characteristics, that is, In order to form organoids that are uniform in size and contain the same biomarker expression, a second culture step may be included.
즉, 제 2 배양하는 단계는 형성된 신장 근위 세뇨관 오가노이드가 성장(성숙)하도록, 오가노이드를 제 2 배양 배지에서 배양하는 단계이다. That is, the second culturing step is a step of culturing the organoids in the second culture medium so that the formed kidney proximal tubule organoids grow (mature).
이때, 제 2 배양 배지는 제 1 배양 배지의 기본 배양 배지일 수 있다. 예를 들어, 제 2 배양 배지는 기본 배양 배지로써, BME(Basal medium Eagle's), MEM(Minimum essential medium), DMEM(Dulbecco's modified Eagle's medium), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199 및 RPMI 1640 중 적어도 하나를 포함할 수 있으나, 이에 제한되는 것은 아니며, 상업적으로 이용되는 다양한 기본 배양 배지가 제 2 배양 배지로써 이용될 수 있다. At this time, the second culture medium may be the basic culture medium of the first culture medium. For example, the second culture medium is a basic culture medium, such as BME (Basal medium Eagle's), MEM (Minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), DMEM/F12, HAM'S F-10, HAM'S F-12, It may include at least one of MEDIUM 199 and RPMI 1640, but is not limited thereto, and various commercially available basic culture media can be used as the second culture medium.
제 2 배양하는 단계의 기간 동안 제 2 배양 배지는 3일에 한번씩(1회/3일) 교체될 수 있다. During the period of the second culturing step, the second culture medium may be replaced once every three days (once/3 days).
제 2 배양하는 단계는 약 3일 내지 50일 중 적어도 하나의 기간 동안 수행될 수 있으나, 이에 제한되는 것은 아니다. The second culturing step may be performed for at least one period of about 3 to 50 days, but is not limited thereto.
한편, 본 발명의 제 2 배양하는 단계에 의하여 성장되는 오가노이드들은 약 7일차(약 168 시간) 전후로 약 100 μm 이상의 크기(지름)를 가질 수 있다. 즉, 균일하고 높은 재현성을 가지는 신장 근위 세뇨관 오가노이드는 약 7일 이상의 배양 기간에 의하여 형성(생산)될 수 있다. 이에, 본 발명의 신장 근위 세뇨관 오가노이드가 상업적으로 이용되기 위한 제 2 배양하는 단계의 기간은 약 7일 이상일 수 있으나, 이에 제한되는 것은 아니다.Meanwhile, organoids grown by the second culturing step of the present invention may have a size (diameter) of about 100 μm or more around day 7 (about 168 hours). That is, uniform and highly reproducible kidney proximal tubule organoids can be formed (produced) over a culture period of about 7 days or more. Accordingly, the period of the second culture step for commercial use of the kidney proximal tubule organoid of the present invention may be about 7 days or more, but is not limited thereto.
나아가, 본 발명의 제 2 배양하는 단계가 수행된 7일차 이상의 신장 근위 세뇨관 오가노이드은 모두 균질한 특성 즉, 동일한 바이오마커를 발현하고 있다.Furthermore, all kidney proximal tubule organoids on the 7th day or more after the second culturing step of the present invention were performed had homogeneous characteristics, that is, expressed the same biomarkers.
보다 구체적으로, 도 2a를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드의 바이오마커에 대한 형광 이미지가 도시된다. 이때, 현미경 관찰된 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 약 21일 차의 오가노이드이다. More specifically, referring to Figure 2A, a fluorescence image for biomarkers of kidney proximal tubule organoids according to a method of producing kidney proximal tubule organoids according to one embodiment of the present invention is shown. At this time, the kidney proximal tubule organoid according to an embodiment of the present invention observed under a microscope is an organoid of about 21 days.
본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 OAT, F-actin, Na+/K+ ATPase, E-cadherin, 8-OHdG 및 Vimentin 포함할 수 있다.Kidney proximal tubule organoids according to an embodiment of the present invention may include OAT, F-actin, Na+/K+ ATPase, E-cadherin, 8-OHdG, and Vimentin.
먼저, 도 2a의 (a)를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 OAT1을 발현하는 것으로 나타난다. OAT은 유기 음이온 운반체(organic anion transporter, OAT)로써, 내인성 혹은 외인성 유기 음이온을 체외로 배출하는 역할을 하며, 유기 음이온의 배출뿐만 아니라, 임상적으로 중요한 유기 음이온 약물, 항-HIV 치료 약물, 항암제, 항생제, 항고혈압 제제 및 항염증성 약물 등 다양한 약물이나 uremic toxin과 같은 대사물질을 체내에서 수송 및 배출할 수 있다. First, referring to (a) of FIG. 2A, the kidney proximal tubule organoid according to an embodiment of the present invention appears to express OAT1. OAT is an organic anion transporter (OAT), which plays a role in excreting endogenous or exogenous organic anions out of the body. In addition to excreting organic anions, it is also used as a clinically important organic anion drug, anti-HIV treatment drug, and anticancer drug. , various drugs such as antibiotics, antihypertensive agents, and anti-inflammatory drugs, or metabolites such as uremic toxin can be transported and excreted from the body.
이러한, OAT는 OAT1, OAT2, OAT3, OAT4, OAT5 및 URAT1의 아형(OAT family)을 포함하고 있으며, 이들의 발현 분포는 조직 및 세포에 따라 상이할 수 있으나, OAT 아형은 주로 신장에서 발현되며 일부 간, 뇌, 및 태반에서 관찰된다. OAT family 중 OAT1은 근위 세뇨관 기저외측막에서 주로 발현되며, PAH 수송체 및 OA/dicarboxylate 교환체로서 OA를 혈액에서 근위 세뇨관 상피세포 내로 수송하고 있다. 보다 구체적으로, OAT1은 신장에서 3차 수송 기작(tertiary transport mechanism)을 사용하여 유기 음이온들을 기저측세포막(basolateral membrane)을 통해 이동시킨 후, 소변과 함께 배출하게 한다. OAT includes the following subtypes (OAT family): OAT1, OAT2, OAT3, OAT4, OAT5, and URAT1. Their expression distribution may differ depending on the tissue and cell, but OAT subtypes are mainly expressed in the kidney and some It is observed in the liver, brain, and placenta. Among the OAT family, OAT1 is mainly expressed in the basolateral membrane of the proximal tubule, and as a PAH transporter and OA/dicarboxylate exchanger, it transports OA from the blood into the epithelial cells of the proximal tubule. More specifically, OAT1 uses a tertiary transport mechanism in the kidney to move organic anions through the basolateral membrane and then excrete them with urine.
나아가, OAT1은 요세관에서 근위 세뇨관에서만 발현되며, alpha-ketoglutarate와 같은 dicarboxylate, cyclic nucleotide, prostaglandin, urate, 및 여 러 약제 등 100가지가 넘는 물질의 수송에 관여하고 있다. 즉, 신장의 주요 기능인 체내 약물을 포함하는 다양한 대사물질 수송 및 배출을 모사하기 위해선, OAT1의 발현이 매우 중요할 수 있다. 일반적으로 신장 상피세포는 배양 과정에서 이러한 OAT1 및 이를 포함하는 OAT family의 발현이 감소되며, 신장 조직에 대한 특성이 상실된다. 이와 마찬가지로, 종래의 신장 오가노이드 또한, OAT1 및 이를 포함하는 OAT family의 발현이 감소되어, 신장 근위 세뇨관의 병태생리학적 특성을 대변할 수 없었다. 이에, 종래에서는 인위적으로 OAT1 및 이를 포함하는 OAT family를 과발현시킨 세포주가 이용되어 왔다. Furthermore, OAT1 is expressed only in the proximal tubules of the urinary tubules and is involved in the transport of over 100 substances, including dicarboxylates such as alpha-ketoglutarate, cyclic nucleotides, prostaglandins, urate, and various drugs. In other words, the expression of OAT1 may be very important to simulate the transport and excretion of various metabolites, including drugs in the body, which are the main functions of the kidney. Generally, during the culture process, kidney epithelial cells lose the expression of OAT1 and the OAT family including it, and their characteristics for kidney tissue are lost. Likewise, conventional kidney organoids also had reduced expression of OAT1 and the OAT family including it, so they could not represent the pathophysiological characteristics of the renal proximal tubules. Accordingly, conventionally, cell lines that artificially overexpress OAT1 and the OAT family including it have been used.
그러나, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 OAT1 및 이를 포함하는 OAT family의 과발현 유도 없이, OAT1가 발현 및 유지되는 것으로 나타난다. 즉, 본 발명의 신장 근위 세뇨관 오가노이드는 신장 세포주를 통한 오가노이드 배양 과정에서 신장 근위 세뇨관의 특성이 상실되지 않고, 유지 및 향상되어 생체 내 신장 조직(기관)을 대변할 수 있는, 모사도 높은 오가노이드임을 의미할 수 있다. However, in the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention, OAT1 is expressed and maintained without inducing overexpression of OAT1 and the OAT family including it. In other words, the kidney proximal tubule organoid of the present invention does not lose the characteristics of the kidney proximal tubules during the organoid culture process using kidney cell lines, but maintains and improves them, so it can represent kidney tissue (organ) in vivo, and has a high degree of mimicry. This may mean that it is an organoid.
나아가, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 도시된 바와 같이, OAT1가 생체 내 신장에서와 같이 외측막(lateral)에 존재하는 것을 나타난다. 이에, 본 발명의 신장 근위 세뇨관 오가노이드는 OAT1의 발현양 뿐만 아니라 발현 위치에 기초하여 다양한 약물의 효능 및 독성 평가를 수행할 수 있다. Furthermore, as shown, the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention shows that OAT1 is present in the lateral membrane as in the kidney in vivo. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on not only the expression amount of OAT1 but also the expression location.
그 다음, 도 2a의 (b)를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 F-actin을 발현하는 것으로 나타난다. F-actin은 세포골격 요소로써, 세포가 조직으로 형성됨에 있어 이의 구조 형성 등에 관여할 수 있다. 예를 들어, F-actin은 신장 내 족세포(podocyte)를 사구체 기저막에 연결시키며, 일련의 연결 연결 단백질을 통해 족세포를 세극막에 부착시킬 수 있다. 이에, F-actin의 발현은 신장 조직의 구조 형성에 있어 매우 중요하며, 이의 발현 양상에 의하여 신장 기능의 다양한 변화를 관찰할 수 있다. Next, referring to (b) of FIG. 2A, the kidney proximal tubule organoid according to one embodiment of the present invention appears to express F-actin. F-actin is a cytoskeletal element that can participate in the structure formation of cells as they form tissues. For example, F-actin connects podocytes in the kidney to the glomerular basement membrane, and a series of connecting proteins can attach podocytes to the slit membrane. Accordingly, the expression of F-actin is very important in the structural formation of kidney tissue, and various changes in kidney function can be observed depending on its expression pattern.
본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 도시된 바와 같이, F-actin이 생체 내 신장에서와 같이 상피세포, 내피세포 및 사구체 기저막과 같은 영역에 존재하는 것으로 나타난다. 이에, 본 발명의 신장 근위 세뇨관 오가노이드는 F-actin의 발현양 뿐만 아니라 발현 위치에 기초하여 다양한 약물의 효능 및 독성 평가를 수행할 수 있다. As shown, the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention contains F-actin in areas such as epithelial cells, endothelial cells, and glomerular basement membrane, as in the kidney in vivo. appears to exist. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on not only the expression amount of F-actin but also the expression location.
그 다음, 도 2a의 (c)를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 Na+/K+ ATPase를 발현하는 것으로 나타난다. Na+/K+ ATPase는 기저막에 위치하여 세포 내 Na 및 K를 유지시킨다. 보다 구체적으로, 신장의 근위관은 신장의 요산(uric acid) 운송을 담당하며, 요산의 재흡수가 주로 일어하는 곳이다. 근위 신장 관상피세포(Proximal Renal Tubular Epithelial Cells, PTECs)는 요산을 배출하고, 이온 및 유산 운송 채널을 발현함에 따라, 미토콘드리아 및 리소좀이 풍부하게 포함되어 있다. 이때, 신장 근위 세뇨관 상피세포에서의 요산 수송(uric acid transporter)의 추진력은 세관 상피 기저(tubule epithelium basolateral)에 존재하는 Na+/K+ ATPase에서 발생된다. Na+/K+ ATPase의 주요 기능은 신장 내 전해질 및 액체의 항상성을 제어하는 것임에 따라, 신장 오가노이드에서의 Na+/K+ ATPase의 발현은 매우 중요할 수 있다. Next, referring to (c) of FIG. 2A, the kidney proximal tubule organoid according to one embodiment of the present invention appears to express Na+/K+ ATPase. Na+/K+ ATPase is located in the basement membrane and maintains Na and K in the cell. More specifically, the proximal duct of the kidney is responsible for the transport of uric acid to the kidney and is where uric acid reabsorption primarily occurs. Proximal Renal Tubular Epithelial Cells (PTECs) are rich in mitochondria and lysosomes, as they excrete uric acid and express ion and lactic acid transport channels. At this time, the driving force of uric acid transporter in renal proximal tubular epithelial cells is generated from Na+/K+ ATPase present at the tubule epithelium basolateral. As the main function of Na+/K+ ATPase is to control electrolyte and fluid homeostasis in the kidney, expression of Na+/K+ ATPase in kidney organoids may be very important.
나아가, 신장 오가노이드에서의 Na+/K+ ATPase는 발현 유무뿐만 아니라, 이의 위치 또한 매우 중요할 수 있다. 보다 구체적으로, Na+/K+ ATPase는 세뇨관 및 간질액과의 Na+/K+ 평형을 유지해야함에 따라, 상피세포막(기저막)에 존재해야 하며, 이의 위치 변경은 약물 및 질병에 의한 신장 근위 세뇨관의 병태생리학적 변화를 대변할 수 있다. Furthermore, not only the presence or absence of expression of Na+/K+ ATPase in kidney organoids, but also its location may be very important. More specifically, Na+/K+ ATPase must be present in the epithelial cell membrane (basement membrane) as it must maintain Na+/K+ equilibrium with the tubule and interstitial fluid, and its location change is associated with the pathophysiology of the renal proximal tubule due to drugs and diseases. It can represent academic change.
이와 관련하여, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 도시된 바와 같이, Na+/K+ ATPase의 발현이 내강을 감싸고 있는 상피세포막(basolateral)에 존재하는 것으로 나타난다. 이에, 본 발명의 신장 근위 세뇨관 오가노이드는 Na+/K+ ATPase의 발현양 뿐만 아니라 발현 위치에 기초하여 다양한 약물의 효능 및 독성 평가를 수행할 수 있다. In this regard, as shown in the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention, the expression of Na+/K+ ATPase is present in the epithelial cell membrane (basolateral) surrounding the lumen. It appears that it does. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based not only on the expression level of Na+/K+ ATPase but also on the expression location.
그 다음, 도 2a의 (d)를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 E-cadherin을 발현하는 것으로 나타난다. E-cadherin은 세포 간의 첩착을 유지하는 cadherin 계열의 분자로써, 특히, 신장의 근위 세뇨관 상피세포에서 주로 발현되어 상피세포간 연결을 안정적으로 유지하는 중요한 단백질이다. 약물 또는 질병에 의하여 신장 근위 세뇨관 상피세포의 특성이 변화되면, 상피-중간엽 전이(epithelial-mesenchymal transition, EMT)가 활성화되어, 섬유화, 암의 침윤 및 전이와 같은 병리적 변화 현상이 관찰될 수 있다. 이에, E-cadherin의 변화는 병리적 현상에 의하여 발생됨에 따라, 이에 기초하여 신장 근위 세뇨관 상피 세포의 기능 및 구조 변화를 예측할 수 있다. 즉, E-cadherin의 발현은 신장 근위 세뇨관에서 약물 및 질병에 의한 병태생리학적 기능을 예측할 수 있는 중요한 바이오마커이다. Next, referring to (d) of FIG. 2A, the kidney proximal tubule organoid according to one embodiment of the present invention appears to express E-cadherin. E-cadherin is a cadherin family molecule that maintains adhesion between cells. In particular, it is an important protein that is mainly expressed in the proximal tubular epithelial cells of the kidney and stably maintains connections between epithelial cells. When the characteristics of kidney proximal tubular epithelial cells are changed by drugs or diseases, epithelial-mesenchymal transition (EMT) is activated, and pathological changes such as fibrosis, cancer invasion, and metastasis can be observed. there is. Accordingly, as changes in E-cadherin occur due to pathological phenomena, changes in function and structure of renal proximal tubular epithelial cells can be predicted based on this. In other words, the expression of E-cadherin is an important biomarker that can predict pathophysiological functions caused by drugs and diseases in the renal proximal tubule.
이와 관련하여, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 도시된 바와 같이, E-cadherin의 발현이 내강을 감싸고 있는 상피세포에 존재하는 것으로 나타난다. 이에, 본 발명의 신장 근위 세뇨관 오가노이드는 E-cadherin의 발현에 기초하여 다양한 약물의 효능 및 독성 평가를 수행할 수 있다. In this regard, as shown in the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention, the expression of E-cadherin appears to be present in the epithelial cells surrounding the lumen. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on the expression of E-cadherin.
그 다음, 도 2a의 (e)를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 8-OHdG를 발현하는 것으로 나타난다. 8-OHdG(8-Hydroxy-2'-deoxyguanosine)는 DNA 손상의 주요 산화적 변형 제품 중 하나로써, 라디칼(radical)에 의한 산화적 스트레스(oxidative stress)에 대한 병리학적 마커이다. 8-OHdG 발현의 증가는 세포의 노화 및 암 등을 포함하는 다양한 질환과 연관될 수 있으며, 신장 근위 세뇨관에서의 8-OHdG 발현의 증가는 신장의 산화적 스트레스가 증가되어 신장의 기능 상실과 같은 다양한 질병이 초래될 수 있다. 이에, 8-OHdG 발현의 변화(증가)는 병리적 현상을 초래하거나, 병리적 현상에 의하여 발생됨에 따라, 이에 기초하여 신장 근위 세뇨관의 병태생리학적 변화를 예측할 수 있다. Next, referring to (e) of FIG. 2A, it appears that the renal proximal tubules express 8-OHdG according to one embodiment of the present invention. 8-OHdG (8-Hydroxy-2'-deoxyguanosine) is one of the major oxidative modification products of DNA damage and is a pathological marker for oxidative stress caused by radicals. Increased 8-OHdG expression can be associated with various diseases, including cellular aging and cancer, and increased 8-OHdG expression in the renal proximal tubules increases oxidative stress in the kidney, leading to loss of kidney function. Various diseases can result. Accordingly, changes (increases) in 8-OHdG expression cause pathological phenomena or are caused by pathological phenomena, and based on this, pathophysiological changes in the renal proximal tubules can be predicted.
이와 관련하여, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 도시된 바와 같이, 8-OHdG의 발현이 내강을 감싸고 있는 상피세포에 존재하는 것으로 나타난다. 이에, 본 발명의 신장 근위 세뇨관 오가노이드는 8-OHdG의 발현에 기초하여 다양한 약물의 효능 및 독성 평가를 수행할 수 있다. In this regard, as shown in the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoid according to an embodiment of the present invention, the expression of 8-OHdG appears to be present in the epithelial cells surrounding the lumen. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on the expression of 8-OHdG.
그 다음, 도 2a의 (f)를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 Vimentin을 발현하는 것으로 나타난다. Vimentin은 중간 사이즈의 섬유 단백질로써, 주로 중간엽 및 신경 조직의 세포에서 발견된다. 질병 및 약물에 의하여 상피세포가 손실되고, 이에, 상피세포가 이동성을 가진 중간엽 세포로 변환되는 EMT가 활성화되면 vimentin이 증가된다. 이와 관련하여, 생체 내 신장이 질환 특히, 신경병증이나 섬유화가 발생하였을 경우, 신장 근위 세뇨관의 EMT가 활성화되어 신장 근위 세뇨관 상피세포에서도 vimentin이 상향조절(upregulation)된다. 이에, vimentin 발현의 변화(증가)는 병리적 현상을 초래하거나, 병리적 현상에 의하여 발생됨에 따라, 이에 기초하여 신장 근위 세뇨관의 병태생리학적 변화를 예측할 수 있다. Next, referring to (f) of FIG. 2A, it appears that the kidney proximal tubules express Vimentin according to an embodiment of the present invention. Vimentin is a medium-sized fibrous protein found mainly in cells of mesenchymal and nervous tissue. When epithelial cells are lost due to disease or drugs, and EMT, which converts epithelial cells into mobile mesenchymal cells, is activated, vimentin increases. In this regard, when kidney disease, especially neuropathy or fibrosis, occurs in vivo, EMT in the renal proximal tubules is activated, and vimentin is upregulated in the renal proximal tubular epithelial cells. Accordingly, as changes (increases) in vimentin expression cause pathological phenomena or are caused by pathological phenomena, pathophysiological changes in the renal proximal tubules can be predicted based on this.
이와 관련하여, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 도시된 바와 같이, vimentin의 발현이 내강을 감싸고 있는 상피세포에 존재하는 것으로 나타난다. 이에, 본 발명의 신장 근위 세뇨관 오가노이드는 vimentin의 발현에 기초하여 다양한 약물의 효능 및 독성 평가를 수행할 수 있다. In this regard, as shown in the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention, the expression of vimentin appears to be present in the epithelial cells surrounding the lumen. Accordingly, the kidney proximal tubule organoid of the present invention can evaluate the efficacy and toxicity of various drugs based on the expression of vimentin.
결국, 본 발명의 신장 근위 세뇨관 오가노이드는 전술한 바와 같이, Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin 및 F-actin을 포함함에 따라, 생체 내 신장과 동일한 특성(바이오마커 발현)을 모사할 수 있어 다양한 약물에 의한 신장의 병태생리학적 변화를 예측할 수 있다. Ultimately, the kidney proximal tubule organoid of the present invention contains Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin, as described above, and thus has the same properties (biomarkers) as the kidney in vivo. expression) can be simulated, making it possible to predict pathophysiological changes in the kidney caused by various drugs.
나아가, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 바이오마커의 발현뿐만 아니라, 생체 신장과 유사한 구조를 가진다.Furthermore, the kidney proximal tubule organoid according to the method for producing kidney proximal tubule organoids according to an embodiment of the present invention not only expresses biomarkers but also has a structure similar to that of a living kidney.
보다 구체적으로, 도 2b를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드의 구조에 대한 형광 이미지가 도시된다. More specifically, referring to Figure 2B, a fluorescence image of the structure of a kidney proximal tubule organoid according to a method of producing kidney proximal tubule organoids according to an embodiment of the present invention is shown.
본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법에 따른 신장 근위 세뇨관 오가노이드는 내강(lumen), 및 내강을 감싸고 있는 상피세포층을 포함하고, 둥근 구상의 입체적 형태인 것으로 나타난다.The renal proximal tubular organoid according to the method for producing renal proximal tubular organoid according to an embodiment of the present invention includes a lumen and an epithelial cell layer surrounding the lumen, and appears to have a round spherical three-dimensional shape.
결국, 본 발명의 신장 근위 세뇨관 오가노이드는 생체 내 신장과 동일한 구조를 모사할 수 있어 다양한 약물에 의한 신장의 병태생리학적 변화를 보다 용이하게 예측할 수 있다. 즉, 본 발명의 신장 근위 세뇨관 오가노이드는 생체 대응성이 높은 생체 유사 모델로 이용될 수 있다.Ultimately, the kidney proximal tubule organoid of the present invention can mimic the same structure as the kidney in vivo, making it possible to more easily predict pathophysiological changes in the kidney caused by various drugs. In other words, the kidney proximal tubule organoid of the present invention can be used as a biosimilar model with high biocorrespondence.
다시 도 1을 참조하면, 앞서 도 2a 및 2b에서 전술된 바와 같이, 제 2 배양하는 단계에서의 신장 근위 세뇨관 오가노이드는 약물의 효능 및 독성을 평가할 수 있는 바이오마커 및 구조를 포함함에 따라, 제 2 배양하는 단계는 약물을 처리하는 단계를 더 포함할 수 있다. 즉, 본 발명은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법을 포함할 수 있다.Referring again to FIG. 1, as previously described in FIGS. 2A and 2B, the kidney proximal tubule organoids in the second culture step contain biomarkers and structures that can evaluate the efficacy and toxicity of the drug. 2 The culturing step may further include the step of treating the drug. That is, the present invention may include a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention.
보다 구체적으로, 도 3a을 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법의 절차를 예시도가 도시된다. More specifically, referring to FIG. 3A, an exemplary diagram showing the procedure of a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention is shown.
본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 제조 방법의 제 2 배양 단계에 포함되어 동시에 수행될 수 있다.The drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention can be performed simultaneously by being included in the second culture step of the method for producing kidney proximal tubule organoids according to an embodiment of the present invention.
즉, 일부 오가노이드들을 하나 이상의 다른 용기에 분류하여 제 2 배양을 수행함과 동시에, 특정 용기에 약물을 추가하며 배양될 수 있다. That is, some organoids can be sorted into one or more different containers to perform a second culture, and at the same time, drugs can be added to a specific container and cultured.
이때, 약물의 처리(treatment group, 처리군)는 배양 7일차에 수행되는 것이 바람직할 수 있다. 이에, 도 3a에서의 T0은 제 2 배양에서 7일차의 신장 근위 세뇨관 오가노이드일 수 있으나, 이에 제한되는 것은 아니며, 사용자의 실험 목적 및 계획에 따라 다양한 기간의 제 2 배양에서의 오가노이드가 이용될 수 있다. 예를 들어, 신장 근위 세뇨관의 발달 과정에서의 약물의 효능 및 독성 평가가 요구되는 경우, 제 2 배양의 0일차의 신장 근위 세뇨관 오가노이드가 T0에서 이용될 수 있다.At this time, it may be desirable to perform drug treatment (treatment group) on the 7th day of culture. Accordingly, T0 in Figure 3a may be the kidney proximal tubule organoid on day 7 in the second culture, but is not limited thereto, and organoids in the second culture for various periods of time may be used depending on the user's experimental purpose and plan. It can be. For example, if evaluation of the efficacy and toxicity of a drug during the development of the kidney proximal tubules is desired, kidney proximal tubule organoids from day 0 of the second culture can be used at T0.
본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법은 도시된 바와 같이, 21일간 오가노이드에 약물이 처리되어 수행될 수 있으며, 약물 처리 기간은 21일에 제한되는 것은 아니며, 사용자의 실험 목적 및 계획에 따라 다양하게 설정될 수 있다. As shown, the drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention can be performed by treating the organoids with a drug for 21 days, and the drug treatment period is not limited to 21 days. It can be set in various ways depending on the user's experiment purpose and plan.
21일일 간의 약물 처리가 수행된 본 발명의 신장 근위 세뇨관 오가노이드는 형태학적 및 단백질 발현 분석이 수행될 수 있다. The kidney proximal tubule organoids of the present invention that underwent drug treatment for 21 days can be subjected to morphological and protein expression analysis.
형태학적 분석으로는 현미경적 구조 분석이 될 수 있으나, 이에 제한되는 것은 아니다.Morphological analysis may include, but is not limited to, microscopic structural analysis.
단백질 발현 분석으로는 Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin 및 F-actin 중 적어도 하나의 바이오마커의 발현 수준에 기초한 분석이 이용될 수 있으며, 이의 발현양 및 발현 위치 여부에 따라 약물의 효능 또는 독성 여부가 결정될 수 있다. As protein expression analysis, analysis based on the expression level of at least one biomarker among Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin can be used, as well as the amount and location of expression. Depending on this, the efficacy or toxicity of the drug can be determined.
즉, 약물 처리군(treatment, Che-1, Che-2)의 바이오마커 발현 수준을 약물 미처리군(control, CTL)의 바이오마커 발현 수준과 비교하여, 약물을 효능 또는 독성 여부가 결정될 수 있다. In other words, the efficacy or toxicity of a drug can be determined by comparing the biomarker expression level of the drug-treated group (treatment, Che-1, Che-2) with the biomarker expression level of the drug-untreated group (control, CTL).
이에, 도 3b를 참조하면, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법에 대한 흐름도가 도시되며, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법은 신장 근위 세뇨관 오가노이드에 약물을 처리하는 단계(S310) 및 약물이 처리된 오가노이드의 바이오마커에 대한 발현 수준에 기초하여 약물의 효능 또는 독성 여부를 결정하는 단계(S320)을 포함할 수 있다.Accordingly, referring to Figure 3b, a flow chart of a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention is shown, and drug evaluation using kidney proximal tubule organoids according to an embodiment of the present invention. The method may include treating a kidney proximal tubule organoid with a drug (S310) and determining whether the drug is effective or toxic based on the expression level for a biomarker in the drug-treated organoid (S320). there is.
보다 구체적으로, 결정하는 단계(S320)는 약물이 처리된 오가노이드의 바이오마커 발현 수준을 약물 미처리군 또는 대조군(control)에서의 바이오마커 발현 수준과 비교하는 단계를 포함할 수 있으며, 비교 결과에 기초하여 약물의 효능 또는 독성 여부가 결정될 수 있다.More specifically, the determining step (S320) may include comparing the biomarker expression level of the drug-treated organoid with the biomarker expression level in the drug-untreated group or control group, and the comparison result may include: Based on this, the efficacy or toxicity of the drug can be determined.
나아가, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 특정 바이오마커들이 생체 신장 기관에서의 동일한 위치에 발현될 수 있다. 이에, 결정하는 단계(S320)는 약물이 처리된 오가노이드의 Na+/K+ ATPase 또는 E-cadherin의 발현 위치에 기초하여, 약물의 효능 또는 독성 여부를 결정하는 단계를 더 포함할 수 있다. Furthermore, the kidney proximal tubule organoid according to one embodiment of the present invention can express specific biomarkers at the same location in the living kidney organ. Accordingly, the determining step (S320) may further include determining the efficacy or toxicity of the drug based on the expression location of Na+/K+ ATPase or E-cadherin in the organoid treated with the drug.
더 나아가, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법에 기초하여, 약물 스크리닝이 가능할 수 있다. 이에, 본 발명은 본 발명의 일 실시예에 따른 약물 평가 방법과 동일한 단계를 포함하는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 스크리닝 방법이 포함될 수 있다. Furthermore, drug screening may be possible based on the drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention. Accordingly, the present invention may include a drug screening method using kidney proximal tubule organoids according to an embodiment of the present invention, which includes the same steps as the drug evaluation method according to an embodiment of the present invention.
따라서, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드 제조 방법은 생체 신장을 높은 유사도로 재현가능한 신장 근위 세뇨관 오가노이드를 제공할 수 있으며, 이는 다양한 약물 평가 및 스크리닝 방법에 이용가능할 수 있다. Therefore, the method for producing kidney proximal tubule organoids according to an embodiment of the present invention can provide kidney proximal tubule organoids that can reproduce living kidneys with a high degree of similarity, which can be used in various drug evaluation and screening methods.
실시예 1. 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법Example 1. Drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention
이하에서는, 도 4 내지 9를 참조하여, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드를 이용한 약물 평가 방법 즉, 오가노이드의 바이오마커 발현에 기초한 약물 평가 방법에 대하여 구체적으로 설명하도록 한다. Hereinafter, with reference to FIGS. 4 to 9, a drug evaluation method using kidney proximal tubule organoids according to an embodiment of the present invention, that is, a drug evaluation method based on biomarker expression in organoids, will be described in detail.
이때, 도 4 내지 9의 약물 평가는 약 7일차의 약 100 μm의 지름의 크기를 가지는 본 발명의 신장 근위 세뇨관 오가노이드가 이용되었으며, 약물의 처리는 21일간 수행되었다. 나아가, 약물이 처리되어 배양되는 기간 동안 3일에 한번씩 배지가 교체되었다. At this time, the drug evaluation in FIGS. 4 to 9 used the kidney proximal tubule organoid of the present invention with a diameter of about 100 μm at about 7 days, and drug treatment was performed for 21 days. Furthermore, the medium was changed every three days during the drug treatment and culture period.
나아가, 본 명세서에서 사용되는 용어 "약물"은, 생물의 이익을 위해 생리적 시스템 또는 질병 상태를 변화시키거나 검토하기 위해서 사용되는 모든 물질을 포함할 수 있다. 보다 구체적으로, 비타민제, 호르몬제, 금속염류, 백신, 항혈청제, 항생제, 화학요법제제, 강심제, 혈압조절제, 항히스타민제, 스테로이드제, 해독제 및 조영제로 이루어진 그룹 중 적어도 하나를 포함할 수 있으나, 이에 제한되는 것은 아니다.Furthermore, the term “drug” as used herein may include any substance used to change or modify a physiological system or disease state for the benefit of an organism. More specifically, it may include at least one of the group consisting of vitamins, hormones, metal salts, vaccines, antiserum agents, antibiotics, chemotherapy agents, cardiotonic agents, blood pressure regulators, antihistamines, steroids, antidotes, and contrast agents. It is not limited.
도 4는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 OAT1 발현에 기초한 약물 평가의 현미경 이미지이다. 이때, 오가노이드에 처리된 약물은 bisphenol A이며, 10 νM의 농도로 처리되었다. Figure 4 is a microscopic image of drug evaluation based on OAT1 expression in kidney proximal tubule organoids according to an embodiment of the present invention. At this time, the drug treated with the organoid was bisphenol A, and it was treated at a concentration of 10 νM.
대조군(vehicle CTL) 및 처리군(bisphenol A)은 모두 OAT1를 발현하는 것으로 나타난다. 즉, 독성 평가에 이용된 본 발명의 신장 근위 세뇨관 오가노이드는 OAT1를 발현함에 따라, 신장의 유기 음이온 수송 과정의 병태생리학적 기능을 대변(모사)할 수 있다는 것을 의미할 수 있다.Both the control group (vehicle CTL) and the treatment group (bisphenol A) appear to express OAT1. In other words, this may mean that the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses OAT1, thereby mimicking the pathophysiological function of the kidney's organic anion transport process.
본 발명에서의 실험결과는 대조군(vehicle CTL)과 처리군(bisphenol A)에서 모두 OAT1 발현이 생체 신장과 마찬가지로 상피세포층에 발현되는 것으로 나타난다. 본 실험에서 사용된 bisphenol A는 신장 근위 세뇨관 오가노이드에서 OAT1을 포함한 OAT의 발현이 대조군과 차이를 나타내지 않았다. 하지만, 본 발명인 신장근위세뇨관 오가노이드를 활용하여 OAT1을 포함한 OAT에 영향을 주는 약물에 대한 평가 가능성을 제시할 수 있고, 이의 변화를 통하여 표적 약물이 신장 기능 중 유기 음이온 배출 및 대사에 어떠한 영향을 미치는지 예측할 수 있다. The experimental results of the present invention show that OAT1 is expressed in the epithelial cell layer in both the control group (vehicle CTL) and the treatment group (bisphenol A), similar to the living kidney. The bisphenol A used in this experiment did not show any difference in the expression of OAT, including OAT1, in kidney proximal tubule organoids compared to the control group. However, by using the renal proximal tubule organoid of the present invention, it is possible to present the possibility of evaluating drugs that affect OAT, including OAT1, and how the target drug affects organic anion excretion and metabolism during kidney function through changes in this. The impact can be predicted.
도 5는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 F-actine 발현에 기초한 약물 평가의 현미경 이미지이다. 이때, 오가노이드에 처리된 약물은 과거 방수 및 코팅을 위한 제품에 널리 사용되었으나, 독성으로 인하여 현재 사용이 금지된 과불화화합물 중 PFOA 와 PFDA이며, 각각 10 μM로 21일간 처리되었다. Figure 5 is a microscopic image of drug evaluation based on F-actine expression in kidney proximal tubule organoids according to an embodiment of the present invention. At this time, the drugs treated with the organoids were PFOA and PFDA, which are perfluorinated compounds that were widely used in waterproofing and coating products in the past, but are currently banned due to their toxicity, and were treated at 10 μM each for 21 days.
대조군(vehicle CTL) 및 처리군(PFOA, PFDA)은 모두 F-actin을 발현하는 것으로 나타난다. 즉, 독성 평가에 이용된 본 발명의 신장 근위 세뇨관 오가노이드는 F-actine를 발현함에 따라, 질병 및 약물에 의한 신장의 구조적 변화를 대변(모사)할 수 있다는 것을 의미할 수 있다.Both the control group (vehicle CTL) and the treatment group (PFOA, PFDA) appear to express F-actin. In other words, the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses F-actine, which means that it can represent (simulate) structural changes in the kidney caused by disease and drugs.
대조군(vehicle CTL)의 F-actine은 생체 신장과 마찬가지로 상피세포층에 명확히 발현되는 것으로 나타난다. 그러나, 처리군(PFOA, PFDA)의 F-actine은 오가노이드의 내강까지 발현되는 것으로 확인이 되며, 이는 처리군에서 신장근위세뇨관의 내강이 좁아져 정상적 기능을 하기 어렵다는 것을 간접적으로 증명한다. 즉, 본 발명의 신장 근위 세뇨관 오가노이드는 F-actine의 발현이 약물 처리에 의하여 차이를 가지는 것으로 나타남에 따라, 이의 변화를 통하여 표적 약물이 신장의 구조적 세포의 기능 및 대사에 어떠한 영향을 미치는지 예측할 수 있다.F-actine in the control group (vehicle CTL) appears to be clearly expressed in the epithelial cell layer, similar to the living kidney. However, F-actine in the treatment group (PFOA, PFDA) was confirmed to be expressed even in the lumen of the organoid, which indirectly proves that the lumen of the renal proximal tubule in the treatment group was narrowed, making it difficult to function normally. In other words, as the kidney proximal tubule organoid of the present invention appears to have differences in the expression of F-actine depending on drug treatment, it is possible to predict how the target drug affects the function and metabolism of structural cells of the kidney through this change. You can.
도 6은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 Na+/K+ ATPase 발현에 기초한 약물 평가의 현미경 이미지이다. 이때, 오가노이드에 처리한 약물은 PFOA 와 PFDA이며, 각각 10 μM로 21일간 처리되었다. Figure 6 is a microscopic image of drug evaluation based on Na+/K+ ATPase expression in kidney proximal tubule organoids according to an embodiment of the present invention. At this time, the drugs treated with the organoids were PFOA and PFDA, each treated at 10 μM for 21 days.
대조군(vehicle CTL) 및 처리군(PFOA, PFDA)은 모두 Na+/K+ ATPase를 발현하는 것으로 나타난다. 즉, 독성 평가에 이용된 본 발명의 신장 근위 세뇨관 오가노이드는 Na+/K+ ATPase를 발현함에 따라, 신장 내 전해질 및 액체의 항상성의 병태생리학적 기능을 대변(모사)할 수 있다는 것을 의미할 수 있다.Both the control group (vehicle CTL) and the treatment group (PFOA, PFDA) appear to express Na+/K+ ATPase. In other words, the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses Na+/K+ ATPase, which means that it can represent (simulate) the pathophysiological function of electrolyte and liquid homeostasis in the kidney. .
대조군(vehicle CTL)의 Na+/K+ ATPase는 생체 신장과 마찬가지로 상피세포 기저막(basolateral)에 발현되는 것으로 나타난다. 그러나, 처리군(PFOA, PFDA)의 Na+/K+ ATPase는 오가노이드의 세포질(cytosol)에 발현되는 것으로 나타난다. 특히, long chain PFAC로서 PFOA 보다 독성이 더 심하다고 알려진 PFDA를 처리한 오가노이드에서 Na+/K+ ATPase 발현은 대조군과 현격한 차이를 보인다. 즉, 본 발명의 신장 근위 세뇨관 오가노이드는 Na+/K+ ATPase의 발현이 약물 처리에 의하여 차이를 가지는 것으로 나타나며, Na+/K+ ATPase가 막(membrane)에서 세포기질(cytosol)로 이동(translocation)되는 변화 또한 관찰될 수 있다. 결국, 본 발명의 신장 근위 세뇨관 오가노이드는 Na+/K+ ATPase 변화를 통하여 표적 약물이 신장 기능 중 신장 내 전해질 및 액체의 항상성에 어떠한 영향을 미치는지 예측할 수 있다. Na+/K+ ATPase in the control group (vehicle CTL) appears to be expressed on the epithelial cell basement membrane (basolateral), similar to the living kidney. However, Na+/K+ ATPase in the treatment groups (PFOA, PFDA) appears to be expressed in the cytosol of the organoids. In particular, the expression of Na+/K+ ATPase in organoids treated with PFDA, which is a long chain PFAC and is known to be more toxic than PFOA, shows a significant difference from the control group. That is, the kidney proximal tubule organoid of the present invention appears to have differences in the expression of Na+/K+ ATPase due to drug treatment, and a change in the translocation of Na+/K+ ATPase from the membrane to the cytosol. It can also be observed. Ultimately, the kidney proximal tubule organoid of the present invention can predict how a target drug affects electrolyte and liquid homeostasis in the kidney during kidney function through changes in Na+/K+ ATPase.
도 7은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 E-cadherin 발현에 기초한 약물 평가의 현미경 이미지이다. 이때, 오가노이드에 처리된 약물은 PFOA 와 PFDA이며, 각각 10 μM로 21일간 처리되었다. Figure 7 is a microscopic image of drug evaluation based on E-cadherin expression in kidney proximal tubule organoids according to an embodiment of the present invention. At this time, the drugs treated with the organoids were PFOA and PFDA, each treated at 10 μM for 21 days.
대조군(vehicle CTL) 및 처리군(PFOA, PFDA)은 모두 E-cadherin를 발현하는 것으로 나타난다. 즉, 독성 평가에 이용된 본 발명의 신장 근위 세뇨관 오가노이드는 E-cadherin를 발현함에 따라, 신장 내 상피세포 및 이의 EMT와 관련된 병태생리학적 기능을 대변(모사)할 수 있다는 것을 의미할 수 있다.Both the control group (vehicle CTL) and the treatment group (PFOA, PFDA) appear to express E-cadherin. In other words, the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses E-cadherin, which means that it can mimic (mimicking) the pathophysiological functions related to epithelial cells in the kidney and their EMT. .
대조군(vehicle CTL)의 E-cadherin은 생체 신장과 마찬가지로 상피세포층에 발현되는 것으로 나타난다. 그러나, 처리군 중 PFOA는 E-cadherin 발현의 양이 대조군에 비하여 감소되었으며, 처리군 중 PFDA는 오가노이드의 세포기질(cytosol)에 발현이 뚜렷하여 E-cadherin의 기능을 상실하였음을 명백히 보여주고 있다. 즉, 본 발명의 신장 근위 세뇨관 오가노이드는 정상 상피세포의 세포막에 발현되는 E-cadherin이 약물 처리에 의하여 세포막에 발현되는 양이 감소하거나, 세포막(membrane)에서 세포기질(cytosol)로 이동(translocation)되는 변화 또한 관찰될 수 있다. 결국, 본 발명의 신장 근위 세뇨관 오가노이드는 E-cadherin 변화를 통하여 표적 약물이 신장 기능 중 신장의 상피세포 내 구조적 지지(접착) 및 이에 따른 기능에 어떠한 영향을 미치는지 예측할 수 있다. E-cadherin in the control group (vehicle CTL) appears to be expressed in the epithelial cell layer as in the living kidney. However, among the treatment groups, the amount of E-cadherin expression in PFOA was reduced compared to the control group, and among the treatment groups, PFDA was clearly expressed in the cytosol of the organoid, clearly showing that the function of E-cadherin was lost. there is. That is, in the kidney proximal tubule organoid of the present invention, the amount of E-cadherin expressed on the cell membrane of normal epithelial cells is reduced by drug treatment, or the amount of E-cadherin expressed on the cell membrane is reduced or translocated from the cell membrane to the cytosol. ) changes can also be observed. Ultimately, the kidney proximal tubule organoid of the present invention can predict how a target drug affects structural support (adhesion) within kidney epithelial cells and subsequent functions during kidney function through changes in E-cadherin.
도 8은 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 8-OHdG 발현에 기초한 약물 평가의 현미경 이미지이다. 이때, 오가노이드에 처리된 약물은 과불화화합물 중 사용이 금지된 약물중 하나인 PFOA이며, 10 μM로 21일간 처리되었다. Figure 8 is a microscopic image of drug evaluation based on 8-OHdG expression in kidney proximal tubule organoids according to an embodiment of the present invention. At this time, the drug treated with the organoids was PFOA, one of the banned perfluorinated compounds, and was treated at 10 μM for 21 days.
대조군(vehicle CTL) 및 처리군(PFOA)은 모두 8-OHdG를 발현하는 것으로 나타났으나, 처리군에서 그 발현이 증가되었음을 관찰할 수 있다. 즉, 독성 평가에 이용된 본 발명의 신장 근위 세뇨관 오가노이드는 8-OHdG를 발현함에 따라, 신장의 산화적 스트레스 와 관련된 병태생리학적 기능을 대변(모사)할 수 있다는 것을 의미할 수 있다.Both the control group (vehicle CTL) and the treatment group (PFOA) were found to express 8-OHdG, but its expression was observed to be increased in the treatment group. In other words, this may mean that the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses 8-OHdG, thereby mimicking the pathophysiological functions related to oxidative stress in the kidney.
대조군(vehicle CTL)의 8-OHdG은 생체 신장과 마찬가지로 상피세포층에 매우 적은 양으로 발현되는 것으로 나타난다. 그러나, 처리군(PFOA)의 8-OHdG은 오가노이드의 다양한 위치에 발현되며, 이의 발현양이 증가한 것으로 나타난다. 즉, 본 발명의 신장 근위 세뇨관 오가노이드는 8-OHdG의 발현이 약물 처리에 의하여 차이를 가지는 것으로 나타난다. 결국, 본 발명의 신장 근위 세뇨관 오가노이드는 8-OHdG 변화를 통하여 표적 약물의 신장에 대한 독성 여부를 예측(평가)할 수 있다. 8-OHdG in the control group (vehicle CTL) appears to be expressed in a very small amount in the epithelial cell layer, as in living kidneys. However, 8-OHdG in the treatment group (PFOA) was expressed in various locations in the organoid, and its expression level appeared to be increased. That is, the kidney proximal tubule organoids of the present invention appear to have differences in the expression of 8-OHdG depending on drug treatment. Ultimately, the kidney proximal tubule organoid of the present invention can predict (evaluate) the toxicity of a target drug to the kidney through changes in 8-OHdG.
도 9는 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드의 vimentin 발현에 기초한 약물 평가의 현미경 이미지이다. 이때, 오가노이드에 처리된 약물은 PFOA 와 PFDA이며, 각각 10 μM로 21일간 처리되었다.Figure 9 is a microscopic image of drug evaluation based on vimentin expression in kidney proximal tubule organoids according to an embodiment of the present invention. At this time, the drugs treated with the organoids were PFOA and PFDA, each treated at 10 μM for 21 days.
대조군(vehicle CTL) 및 처리군(PFOA, PFDA)은 모두 vimentin을 발현하는 것으로 나타난다. 즉, 독성 평가에 이용된 본 발명의 신장 근위 세뇨관 오가노이드는 vimentin를 발현함에 따라, 신장의 병적 상태를 대변(모사)할 수 있다는 것을 의미할 수 있다.Both the control group (vehicle CTL) and the treatment group (PFOA, PFDA) appear to express vimentin. In other words, this may mean that the kidney proximal tubule organoid of the present invention used for toxicity evaluation expresses vimentin, thereby mimicking the pathological state of the kidney.
대조군(vehicle CTL)의 vimentin은 생체 신장과 마찬가지로 상피세포층에 매우 적은 양으로 발현되는 것으로 나타난다. 그러나, 처리군(PFOA, PFDA)의 vimentin은 오가노이드의 다양한 위치에 발현되며, 이의 발현양이 증가한 것으로 나타난다. 즉, 본 발명의 신장 근위 세뇨관 오가노이드는 vimentin의 발현이 약물 처리에 의하여 차이를 가지는 것으로 나타난다. 결국, 본 발명의 신장 근위 세뇨관 오가노이드는 vimentin 변화를 통하여 표적 약물의 신장에 대한 독성 여부 및 이에 따른 신장의 병적 상태를 예측(평가)할 수 있다. Vimentin in the control group (vehicle CTL) appears to be expressed in a very small amount in the epithelial cell layer, similar to the living kidney. However, vimentin in the treatment groups (PFOA, PFDA) was expressed in various locations in the organoids, and its expression level appeared to be increased. That is, the kidney proximal tubule organoids of the present invention appear to have differences in vimentin expression depending on drug treatment. Ultimately, the kidney proximal tubule organoid of the present invention can predict (evaluate) whether a target drug is toxic to the kidney and the resulting pathological state of the kidney through changes in vimentin.
이상의 결과에 따라, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 구조적인 형태뿐만 아니라, 특성 즉, 바이오마커의 발현 또한 생체 신장과 매우 유사함에 따라, 보다 생체 대응성이 높은 생체 유사 모델로 이용될 수 있다.According to the above results, the kidney proximal tubule organoid according to an embodiment of the present invention is not only a structural form but also the characteristics, that is, the expression of biomarkers, are very similar to living kidneys, making it a bio-mimetic model with a higher biocorrespondence. It can be used as.
이에, 본 발명의 일 실시예에 따른 신장 근위 세뇨관 오가노이드는 약물에 대한 부작용, 독성 및 유효성 평가에 있어 인체 모사 모델로 이용될 수 있으며, 신약 개발 과정에서 후보물질의 독성에 대한 안정성 및 효과 판별 실험에도 사용될 수 있다. 예를 들어, 본 발명의 신장 근위 세뇨관 오가노이드를 약물과 함께 플레이트(용기)에서 반응시킨 뒤, 이의 상태를 현미경적 방법으로 관찰하거나, 단백질 분석 등을 통하여 이의 변화를 확인할 수 있다.Accordingly, the kidney proximal tubule organoid according to an embodiment of the present invention can be used as a human model to evaluate side effects, toxicity, and effectiveness of drugs, and to determine the safety and effectiveness of candidate substances for toxicity in the process of developing new drugs. It can also be used in experiments. For example, after the kidney proximal tubule organoid of the present invention is reacted with a drug in a plate (container), its condition can be observed microscopically, or changes in it can be confirmed through protein analysis, etc.
신장암 오가노이드Kidney cancer organoids
실시예 2. 신장암 세포주 기반 인간 신장암 오가노이드 제조Example 2. Preparation of human kidney cancer organoids based on kidney cancer cell lines
이하에서는 도 10 내지 도 12을 참조하여, 본 발명의 일 실시예에 따른 신장암 오가노이드 제조방법 및 이에 따른 신장암 오가노이드를 구체적으로 설명한다.Hereinafter, with reference to FIGS. 10 to 12, a method for producing a kidney cancer organoid according to an embodiment of the present invention and a kidney cancer organoid resulting therefrom will be described in detail.
도 10은 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법을 예시적으로 도시한 것이다.Figure 10 exemplarily illustrates a method for producing kidney cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
도 10을 참조하면, 제조방법은 암오가노이드를 형성하도록 신장암 세포주 및 인체 세포외기질(ECM)을 포함하는 배지를 혼합하여 제1 배양하는 단계; 및 상기 암오가노이드 및 성장 배지를 혼합하여 제2 배양하는 단계; 를 포함할 수 있다. Referring to Figure 10, the manufacturing method includes first culturing a medium containing a kidney cancer cell line and human extracellular matrix (ECM) to form a cancer organoid; and a second culture step of mixing the cancer organoids and the growth medium; may include.
다양한 실시예에서, 신장암 세포주 및 인체 세포외기질을 포함하는 배지는 1:1 비율로 포함될 수 있다. 바람직하게는 3차원 세포배양용 ECM인 인체 세포외기질을 포함하는 배지 1mL 기준으로 신장암 세포주는 1~5 x 105 내지 1~5 x 107 세포수로 포함될 수 있으나 이에 제한되는 것은 아니다. In various embodiments, medium containing a kidney cancer cell line and human extracellular matrix may be included in a 1:1 ratio. Preferably, the number of kidney cancer cell lines may be 1 to 5 x 10 5 to 1 to 5 x 10 7 based on 1 mL of medium containing human extracellular matrix, which is ECM for 3D cell culture, but is not limited thereto.
다양한 실시예에서, 신장암 세포주는 환자 유래 신장암 조직 또는 종양으로부터 분리하여 직접 수득하거나 상업용 암세포주를 구입하는 등의 경로에 있어 제한이 없다. 신장암 세포주는 예를 들어 Caki-1 cell 이며, 이는 근위관 상피형태(proximal tubular epithelial morphology)를 보이는 대표적인 RCC(human clear cell renal cell carcinoma)이나 이에 제한되는 것은 아니다.In various embodiments, there are no restrictions on the route, such as obtaining renal cancer cell lines directly by isolating them from patient-derived renal cancer tissues or tumors or purchasing commercial cancer cell lines. A renal cancer cell line is, for example, Caki-1 cell, which is a representative RCC (human clear cell renal cell carcinoma) showing proximal tubular epithelial morphology, but is not limited thereto.
제1 배양하는 단계는 구체적으로, 도 11를 참조하면, 신장암 세포주와 인체 세포외기질을 포함하는 배지를 혼합하여 배양될 수 있다.Specifically, referring to FIG. 11, the first culturing step may be performed by mixing a medium containing a kidney cancer cell line and a human extracellular matrix.
본 발명의 일 실시예에 따르면, 1mL의 인체 세포외기질을 포함하는 10% FBS-DMEM 배지에 신장암 세포 Caki-1을 1 x 106 세포수로 혼합하여 세포들이 분포할 수 있도록 섞어준 후 플레이트에 시딩한다. 본 발명에 따른 인체 세포외기질의 특성에 따라 부유성을 갖는 세포들과 서로 융합되어 밀착 연접 및 자가 조직화가 일어나면서 배양 3일 이내 오가노이드가 형성된다. 암 오가노이드가 100 um로 균질해지는 시기까지(약 7일) 신선한 배지로 3일에 한번씩 교체해준다. According to one embodiment of the present invention, kidney cancer cells Caki-1 were mixed at 1 x 10 6 cells in 10% FBS-DMEM medium containing 1 mL of human extracellular matrix and mixed so that the cells could be distributed. Seed the plate. According to the characteristics of the human extracellular matrix according to the present invention, floating cells fuse with each other, tight junctions and self-organization occur, and organoids are formed within 3 days of culture. Replace the medium with fresh medium once every 3 days until the cancer organoids become homogeneous at 100 um (about 7 days).
다양한 실시예에서, 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간 동안 수행될 수 있으나 이에 제한되는 것은 아니다. 이와 관련하여, 종래의 오가노이드 형성은 수주 이상의 기간이 소요되었다. 그러나, 본 발명의 일 실시예에 따른 신장암 오가노이드 제조 방법은 3일 이내의 단 시간 내에 신장암 오가노이드의 형성을 제공한다. In various embodiments, the first culturing step may be performed for at least one period of about 1 hour to 3 days, but is not limited thereto. In this regard, conventional organoid formation takes several weeks or more. However, the method for producing kidney cancer organoids according to an embodiment of the present invention provides the formation of kidney cancer organoids within a short time of less than 3 days.
한편, 제1 배양하는 단계에 의하여 형성된 오가노이드들은 50 - 200 μm 크기를 가질 수 있으며 각각의 크기 및 성숙도가 상이할 수 있음에 따라, 실험 또는 임상적인 약물(화합물) 평가에 있어, 오가노이드의 반응을 현미경으로 관찰하기에 충분하도록 균질한 크기 및 신장암의 특성을 발현하는 오가노이드 성장을 위하여 50일내의 기간동안 배지를 3일에 한번 교체하며 제2 배양할 수 있다. On the other hand, the organoids formed by the first culturing step may have a size of 50 - 200 μm and each size and maturity may be different, so in experimental or clinical drug (compound) evaluation, the organoids In order to grow organoids that have a uniform size and express the characteristics of kidney cancer enough to observe the reaction under a microscope, the medium can be replaced once every 3 days for a second culture within 50 days.
제2 배양하는 단계는 구체적으로 도 12을 참고하면, 약 3일 내지 50일 중 적어도 하나의 기간 동안 수행될 수 있으나 이에 제한되는 것은 아니다. 제1 배양하는 단계 및 제2 배양하는 단계는 순차적으로 또는 동시에 수행될 수 있다.Referring specifically to FIG. 12, the second culturing step may be performed for at least one period of about 3 to 50 days, but is not limited thereto. The first culturing step and the second culturing step may be performed sequentially or simultaneously.
다양한 실시예에서, 제1 배양하는 단계 또는 제2 배양하는 단계에서 사용되는 배양 배지는 성장 배지일 수 있다. 인위적으로 합성하여 제조하여 사용하거나 상업적으로 제조된 배지를 사용할 수 있다. 예를 들어, 인슐린을 포함하지 않는, 이글 기본 배지(Basal medium Eagle's, BME), 최소 필수 배지(Minimal essential medium, MEM), 이글 최소 필수 배지(Eagle's MEM), 둘베코 수정 이글 배지(Dulbecco's modified Eagle's medium, DMEM), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199 및 RPMI 1640 등과 같은 다양한 무혈청 배지 및 이들의 변이형 중 적어도 하나를 포함할 수 있다. In various embodiments, the culture medium used in the first or second culturing step may be a growth medium. It can be artificially synthesized and used, or a commercially prepared medium can be used. For example, basal medium Eagle's (BME), Minimal essential medium (MEM), Eagle's MEM, Dulbecco's modified Eagle's medium, without insulin. medium, DMEM), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199, and RPMI 1640, and may include at least one of various serum-free media and variants thereof.
다양한 실시예에서, 성장 배지는 추가적으로 아미노산, 아스코르빅산, 아세트산, FBS, B27 및 IWR-1 중 적어도 하나를 포함할 수 있다. 세포의 성장에 필요한 아미노산으로서 L-글라이신, L-알라닌, L-아스파라긴, L-아스파틱산, L-글루탐산, L-프롤린, L-세린 또는 L-글루타민을 더 포함할 수 있으나 이에 제한되는 것은 아니다. L-글루타민은 글루타맥스(Glutamax)로 대체될 수 있다.In various embodiments, the growth medium may additionally include at least one of the following amino acids: ascorbic acid, acetic acid, FBS, B27, and IWR-1. Amino acids necessary for cell growth may further include, but are not limited to, L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, or L-glutamine. . L-Glutamine can be replaced with Glutamax.
다양한 실시예에서, 성장 배지는 추가적으로 항생제를 포함할 수 있다. In various embodiments, the growth medium may additionally include antibiotics.
다양한 실시예에서, 인체 세포외기질 및 성장 배지의 부피비는 1:4 내지 1:6일 수 있으며, 바람직하게는 1:5의 비율일 수 있으나 이에 제한되는 것은 아니다.In various embodiments, the volume ratio of human extracellular matrix and growth medium may be 1:4 to 1:6, preferably 1:5, but is not limited thereto.
다양한 실시예에서, 제2 배양하는 단계는 약물을 처리하는 단계를 더 포함할 수 있다. 다시 도 12을 참조하면, 오가노이드들을 하나 이상의 다른 용기에 분류하여 제2 배양을 수행함과 동시에, 특정 용기에 약물을 추가하여 배양될 수 있다. 이때, 약물의 처리(treatment group, 처리군)는 배양 7일차에 수행되는 것이 바람직할 수 있다. 이에, 도 12에서의 T0은 제2 배양에서 7일차의 신장암 오가노이드일 수 있으나, 이에 제한되는 것은 아니다. 사용자의 실험 목적 및 계획에 따라 다양한 기간의 제2 배양에서의 오가노이드가 이용될 수 있다. 예를 들어, 신장암의 발달 과정에서의 약물의 효능 및 독성 평가가 요구되는 경우, 제2 배양의 0일차의 신장암 오가노이드가 T0에서 이용될 수 있다.In various embodiments, the second culturing step may further include treating the drug. Referring again to FIG. 12, the organoids can be sorted into one or more different containers to perform a second culture, and at the same time, the organoids can be cultured by adding a drug to a specific container. At this time, it may be desirable to perform drug treatment (treatment group) on the 7th day of culture. Accordingly, T0 in FIG. 12 may be a kidney cancer organoid on day 7 in the second culture, but is not limited thereto. Organoids in secondary culture can be used for various periods of time depending on the user's experimental purpose and plan. For example, if evaluation of drug efficacy and toxicity during the development of kidney cancer is required, kidney cancer organoids from day 0 of the second culture can be used at T0.
다양한 실시예에서, 약물 미처리군(CTL) 및 약물 처리군(PFOA, PFDA)에 대해 형태학적 및 단백질 발현을 비교 분석하는 방법으로 약물의 효능 또는 독성 평가를 수행할 수 있다. 형태학적 분석으로는 현미경적 구조 분석이 될 수 있으나, 이에 제한되는 것은 아니다. 단백질 발현 분석으로는 F-액틴 비정상(F-actin abnormality), Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준에 기초한 분석일 수 있으나 이에 제한되는 것은 아니다. 예를 들어 상피중간엽전이(EMT) 활성 관련 바이오마커라면 모두 이용될 수 있다. 또한 바이오마커 수준은 발현량 및 발현 위치에 기초하여 분석된 것일 수 있다.In various embodiments, the efficacy or toxicity of a drug may be evaluated by comparative analysis of morphological and protein expression for a drug-untreated group (CTL) and a drug-treated group (PFOA, PFDA). Morphological analysis may include, but is not limited to, microscopic structural analysis. Protein expression analysis may be, but is not limited to, analysis based on the level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin. For example, any biomarker related to epithelial-mesenchymal transition (EMT) activity can be used. Additionally, the biomarker level may be analyzed based on expression level and expression location.
본 발명의 일 실시예에 따르면 신장암 오가노이드를 이용한 약물의 효능 또는 독성 평가를 위해 약물의 처리는 실질적으로 제1 배양을 포함하는 오가노이드의 배양 7일 이내 처음 수행될 수 있다. 구체적으로 오가노이드 크기가 균질해지는 시기(7일 전후)부터 10 νM PFOA 또는 PFDA 함유된 배지에 섞은 후 3일에 한번씩 교체하며 21일 동안 노출시켜 신장암 오가노이드를 성장시켰다. 이후 해동하여 사용할 수 있도록, 동결과정을 거쳐 LN2 tank에 보관해두었다.According to one embodiment of the present invention, in order to evaluate the efficacy or toxicity of a drug using kidney cancer organoids, treatment with the drug may be performed for the first time within 7 days of culturing the organoid, including the first culture. Specifically, from the time when the organoid size became uniform (around 7 days), kidney cancer organoids were grown by mixing the medium containing 10 νM PFOA or PFDA and exposing it to the medium for 21 days, replacing it every 3 days. Afterwards, it was stored in an LN2 tank after a freezing process so that it could be thawed and used.
실시예 3: 신장암 오가노이드의 형태 분석Example 3: Morphological analysis of kidney cancer organoids
3-1. H&E 염색을 통한 신장암 오가노이드의 구조 확인3-1. Confirmation of the structure of kidney cancer organoids through H&E staining
Hematoxylin & Eosin 염색은 조직이나 세포 샘플을 현미경으로 관찰하기 위한 가장 일반적으로 사용되는 염색 기술이다. 이 기법은 병리학에서 널리 사용되며, 오가노이드의 형태와 구조를 시각화를 위하여 사용된다.Hematoxylin & Eosin staining is the most commonly used staining technique for microscopic observation of tissue or cell samples. This technique is widely used in pathology and is used to visualize the morphology and structure of organoids.
도 13는 본 발명의 일 실시예에 따른 신장암 오가노이드 및 약물 처리 신장암 오가노이드의 H&E 염색 현미경 이미지를 도시한 것이다. 구체적으로, 도 13를 참조하면, 신장암 오가노이드의 파라핀 절편 및 H&E 염색을 통해 신장암 오가노이드의 구조를 확인할 수 있다.Figure 13 shows H&E staining of kidney cancer organoids and drug-treated kidney cancer organoids according to an embodiment of the present invention. It shows a microscope image. Specifically, referring to Figure 13, the structure of the kidney cancer organoid can be confirmed through paraffin sectioning and H&E staining.
이에 본 발명에 따른 신장암 오가노이드에 시험하고자 하는 약물을 처리 시, 약물에 따른 오가노이드의 형태 변화를 확인함으로써 약물의 효능 또는 독성 여부를 평가할 수 있다.Accordingly, when treating a kidney cancer organoid according to the present invention with a drug to be tested, the efficacy or toxicity of the drug can be evaluated by checking the change in the shape of the organoid according to the drug.
3-2. F-actin 염색을 통한 세포골격 이상유무 확인3-2. Confirmation of cytoskeletal abnormalities through F-actin staining
모든 세포에 존재하는 F-액틴 염색을 통하여, 대조군을 처리한 신장암 오가노이드와 시험물질을 처리한 신장암 오가노이드의 세포골격 형태(Cytoskeleton morphology) 변화를 분석할 수 있다. 특히 암조직에서의 F-액틴의 역할은 매우 중요하다. 암세포에서 발견되는 F-액틴의 동적 재구성은 암세포의 이동과 침입, 그리고 전이에 중요한 역할을 한다.Through F-actin staining, which is present in all cells, changes in cytoskeleton morphology of kidney cancer organoids treated with the control group and kidney cancer organoids treated with test substances can be analyzed. In particular, the role of F-actin in cancer tissue is very important. Dynamic reorganization of F-actin found in cancer cells plays an important role in cancer cell migration, invasion, and metastasis.
도 14는 본 발명의 일 실시예에 따른 신장암 오가노이드에 대해 팔로이딘(phalloidin) 염색 프로토콜을 이용한 F-액틴 염색 현미경 이미지 및 정량화 데이터를 도시한 것이다. Figure 14 shows F-actin staining microscopy images and quantification data using a phalloidin staining protocol for kidney cancer organoids according to an embodiment of the present invention.
구체적으로, 도 14를 참조하면, vehicle control을 처리한 신장암 오가노이드(CTL)와 약물을 21일간 지속적으로 노출(3일에 한번씩 새로운 배지와 약물로 교체)을 시킨 신장암 오가노이드(PFOA, PFDA)의 세포골격을 비교한 결과, vehicle control을 처리한 신장암 오가노이드의 세포골격은 정상적인 모양인 직선형 골격으로 발현이 되나, 시험약물(PFOA)를 처리한 신장암 오가노이드에서는 F-액틴 비정상 발생에 의해 세포골격에서의 spot이 증가된 현상을 관찰할 수 있다. 또한, PFDA를 처리한 신장암 오가노이드에서는 vehicle control 대비 세포골격 발현량이 현저히 감소되었다. 이는 세포에서 중요한 역할을 하는 세포골격이 생활화학물질이며 신장암을 유발시킬 가능성이 있다고 알려진 과불화화합물에 저농도-장기간 노출시 암세포의 골격에 영향을 주는 결과를 얻은 것이다. Specifically, referring to Figure 14, kidney cancer organoids (CTL) treated with vehicle control and kidney cancer organoids (PFOA, As a result of comparing the cytoskeleton of kidney cancer organoids treated with vehicle control, the cytoskeleton of kidney cancer organoids treated with vehicle control was expressed as a normal straight skeleton, but in kidney cancer organoids treated with test drug (PFOA), F-actin was abnormal. An increase in spots in the cytoskeleton can be observed due to development. Additionally, in kidney cancer organoids treated with PFDA, the amount of cytoskeletal expression was significantly reduced compared to the vehicle control. This shows that the cytoskeleton, which plays an important role in cells, is a household chemical and that low-concentration and long-term exposure to perfluorinated compounds, which are known to have the potential to cause kidney cancer, affects the skeleton of cancer cells.
따라서, 신장암 오가노이드의 F-액틴 비정상(F-actin abnormality) 분석을 통해 세포골격 모양을 확인함으로써 신장암을 타겟으로 하는 신규 약물이나 본 발명에서 수행한 독성물질에 대한 영향을 평가할 수 있다.Therefore, by confirming the cytoskeleton shape through F-actin abnormality analysis of kidney cancer organoids, the impact of new drugs targeting kidney cancer or toxic substances performed in the present invention can be evaluated.
실시예 4: 신장암 오가노이드의 Na+/K+ ATPase 분석Example 4: Na+/K+ ATPase analysis of kidney cancer organoids
실시예 2에서 제조된 신장암 오가노이드에 대해 팔로이딘(phalloidin) 염색 프로토콜을 이용하여 Na+/K+ ATPase 분석을 수행하였다.Na+/K+ ATPase analysis was performed on the kidney cancer organoid prepared in Example 2 using the phalloidin staining protocol.
도 15은 본 발명의 일 실시예에 따른 신장암 오가노이드 및 약물에 노출된 신장암 오가노이드의 Na+/K+ ATPase 변화를 도시한 것이다. Figure 15 shows changes in Na+/K+ ATPase in kidney cancer organoids and kidney cancer organoids exposed to drugs according to an embodiment of the present invention.
구체적으로, 도 15을 참조하면, 신장에서 중요한 바이오마커 중 하나인 Na+/K+ ATPase는 암 활성제를 처리한 신장암 오가노이드(PFOA, PFDA)에서 발현이 되나, 정상 신장에서 발현되는 Na+/K+ ATPase의 위치인 세포막(membrane)에서 주로 발현이 되는 것이 아니라, 세포질(cytosol)로 전위된 형태로 발현되는 것을 확인할 수 있다. 특히 암의 전이가 활발히 발생되는 경우 이러한 현상은 두드러진다.Specifically, referring to Figure 15, Na+/K+ ATPase, one of the important biomarkers in the kidney, is expressed in kidney cancer organoids (PFOA, PFDA) treated with cancer activators, but Na+/K+ ATPase expressed in normal kidneys It can be confirmed that it is not mainly expressed in the cell membrane, which is the location of , but is expressed in a form translocated to the cytosol. This phenomenon is especially noticeable when cancer metastasis is actively occurring.
신장암 오가노이드에서 발현되는 Na+/K+ ATPase를 분석한 결과, 10 νM 로 21일간 처리하는 약물(PFOA, PFDA)에 따라 Na+/K+ ATPase의 발현 위치가 세포막에서 세포질로 변화하거나 발현양 자체가 감소되는 결과를 얻을 수 있다. 특히 신장에서 Na+/K+ ATPase의 발현양 감소나 위치 변화는 신장암의 전이를 증가시키기 때문에 본 발명의 신장암 오가노이드를 활용하여 약물의 효능 및 화학물질의 암증식 영향평가의 도구로 활용될 수 있다.As a result of analyzing Na+/K+ ATPase expressed in kidney cancer organoids, depending on the drug (PFOA, PFDA) treated at 10 νM for 21 days, the expression location of Na+/K+ ATPase changes from the cell membrane to the cytoplasm or the expression level itself decreases. You can get results that work. In particular, a decrease in the expression level or a change in the location of Na+/K+ ATPase in the kidney increases the metastasis of kidney cancer, so the kidney cancer organoid of the present invention can be used as a tool to evaluate the efficacy of drugs and the effect of chemicals on cancer growth. there is.
실시예 5: 신장암 오가노이드의 E-cadherin 분석Example 5: E-cadherin analysis of kidney cancer organoids
실시예 2에서 제조된 신장암 오가노이드에 대해 E-cadherin 면역염색을 수행하여 신장암 바이오마커 중 하나인 E-cadherin 발현 분석을 수행하였다.E-cadherin immunostaining was performed on the kidney cancer organoid prepared in Example 2 to analyze the expression of E-cadherin, one of the kidney cancer biomarkers.
RCC 유래 신장암 세포주인 Caki-1은 세뇨관 상피(tubular epithelium)의 특징을 가지고 있기에, Caki-1 cell 유래 신장암 오가노이드는 상피세포에서 정상적으로 발현되는 E-cadherin이 특징적으로 발현된다. Since Caki-1, an RCC-derived kidney cancer cell line, has the characteristics of tubular epithelium, Caki-1 cell-derived kidney cancer organoids characteristically express E-cadherin, which is normally expressed in epithelial cells.
E-cadherin은 대부분의 상피세포에서 발현이 되는 중요한 바이오마커 중 하나이다. 신장 상피세포의 특성이 변하게 되면, 상피중간엽전이(EMT; epithelial to mesenchymal transition)이 활성화되어 병리적 현상이 관찰된다. 이러한 변화는 주로 섬유화나 암의 침윤과 전이 시 발견되며, E-cadherin의 감소는 이러한 병리적 현상이 발생되고, 신장 상피세포의 기능 및 구조 변화가 생겼다는 것을 예상할 수 있다. E-cadherin is one of the important biomarkers expressed in most epithelial cells. When the characteristics of kidney epithelial cells change, epithelial to mesenchymal transition (EMT) is activated and pathological phenomena are observed. These changes are mainly found during fibrosis or cancer invasion and metastasis, and a decrease in E-cadherin can be expected to indicate that these pathological phenomena occur and changes in the function and structure of renal epithelial cells occur.
도 16을 참조하면, 암 활성제를 처리한 신장암 오가노이드(PFOA)에서 E-cadherin 발현이 감소되고 특히 발현 위치가 세포막에서 세포질로 전위된 것을 확인할 수 있다. 이에 본 발명의 신장암 오가노이드를 활용하여 약물 처리에 의한 E-cadherin의 증감 및 전위 발현을 확임함으로써, 약물 또는 암 증식에 대한 영향을 평가할 수 있다.Referring to Figure 16, it can be seen that E-cadherin expression was reduced in kidney cancer organoids (PFOA) treated with a cancer activator, and in particular, the expression location was relocated from the cell membrane to the cytoplasm. Accordingly, by using the kidney cancer organoid of the present invention to confirm the increase/decrease and translocation expression of E-cadherin due to drug treatment, the effect on the drug or cancer growth can be evaluated.
실시예 6: 신장암 오가노이드의 Vimentin 분석Example 6: Vimentin analysis of kidney cancer organoids
실시예 2에서 제조된 신장암 오가노이드에 대해 Vimentin 발현을 포함하는 EMT 관련 단백질 발현 분석을 수행하였다.EMT-related protein expression analysis, including Vimentin expression, was performed on the kidney cancer organoid prepared in Example 2.
Vimentin은 상피-중간엽전이(EMT)가 활성화되었을 때 증가되는 중요한 바이오마커 중 하나이며, 특히, 암의 전이가 활성화되는 경우에 Vimentin 발현이 증가한다. Vimentin is one of the important biomarkers that is increased when epithelial-mesenchymal transition (EMT) is activated. In particular, Vimentin expression increases when cancer metastasis is activated.
도 17을 참조하면, RCC 유래 신장암 오가노이드는 암 활성제(PFOA)에 저농도-장기간 노출시, Vimentin 발현 증가를 확인할 수 있다. 이에 Vimentin을 포함하는 EMT 관련 단백질 발현 분석을 통해 본 발명에 따른 신장암 오가노이드를 이용하여 약물 평가 또는 화학물질의 암 증식에 대한 영향을 평가할 수 있다.Referring to Figure 17, it can be seen that RCC-derived kidney cancer organoids increase Vimentin expression when exposed to the cancer activator (PFOA) at low concentration and for a long period of time. Accordingly, the effect of drugs or chemicals on cancer growth can be evaluated using the kidney cancer organoid according to the present invention through analysis of the expression of EMT-related proteins, including Vimentin.
갑상선암 오가노이드Thyroid cancer organoids
실시예 7. 갑상선암 세포주 기반 인간 갑상선암 오가노이드 제조Example 7. Preparation of human thyroid cancer organoids based on thyroid cancer cell lines
이하에서는 도 19 내지 도 21을 참조하여, 본 발명의 일 실시예에 따른 갑상선암 오가노이드 제조방법 및 이에 따른 갑상선암 오가노이드를 구체적으로 설명한다.Hereinafter, with reference to FIGS. 19 to 21, a method for producing a thyroid cancer organoid according to an embodiment of the present invention and a thyroid cancer organoid resulting therefrom will be described in detail.
도 19은 본 발명의 일 실시예에 따른 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법을 예시적으로 도시한 것이다.Figure 19 exemplarily illustrates a method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity according to an embodiment of the present invention.
도 19을 참조하면, 제조방법은 오가노이드를 형성하도록 갑상선암 세포주 및 인체 세포외기질(ECM)을 포함하는 배지를 혼합하여 제1 배양하는 단계; 및 상기 오가노이드 및 성장 배지를 혼합하여 제2 배양하는 단계; 를 포함할 수 있다. Referring to Figure 19, the manufacturing method includes first culturing a mixture of a medium containing a thyroid cancer cell line and human extracellular matrix (ECM) to form an organoid; and a second culture step of mixing the organoids and growth medium; may include.
다양한 실시예에서, 갑상선암 세포주 및 인체 세포외기질은 1:1 비율로 포함될 수 있다. 바람직하게는 인체 세포외기질을 포함하는 배지 1mL 기준으로 갑상선암 세포주는 1~5 x 105 내지 1~5 x 107 세포수로 포함될 수 있으나 이에 제한되는 것은 아니다. In various embodiments, thyroid cancer cell lines and human extracellular matrix may be included in a 1:1 ratio. Preferably, based on 1 mL of medium containing human extracellular matrix, the thyroid cancer cell line may be included in the number of 1 to 5 x 10 5 to 1 to 5 x 10 7 cells, but is not limited thereto.
다양한 실시예에서, 갑상선암 세포주는 환자 유래 갑상선암 조직 또는 종양으로부터 분리하여 직접 수득하거나 상업용 암세포주를 구입하는 등의 경로에 있어 제한이 없다. 갑상선암 세포주는 예를 들어 TBP-3868, T238, WRO, FTC133, BCPAP, TPC1, K1, 8505C, HTH7, C643, SW1736, MDA-T41 또는 SNU-790 세포주일 수 있으나 이에 제한되는 것은 아니다. 당해 기술분야에서 사용되는 갑상선암 세포주라면 모두 이용될 수 있다.In various embodiments, there are no restrictions on the route, such as obtaining the thyroid cancer cell line directly by separating it from a patient-derived thyroid cancer tissue or tumor or purchasing a commercial cancer cell line. The thyroid cancer cell line may be, for example, but is not limited to TBP-3868, T238, WRO, FTC133, BCPAP, TPC1, K1, 8505C, HTH7, C643, SW1736, MDA-T41 or SNU-790 cell lines. Any thyroid cancer cell line used in the art can be used.
제 1배양하는 단계는 구체적으로 도 20를 참조하면, 갑상선암 세포주와 인체 세포외기질을 포함하는 배지를 혼합하여 배양될 수 있다.Referring specifically to FIG. 20, the first culturing step may be performed by mixing a medium containing a thyroid cancer cell line and a human extracellular matrix.
본 발명의 일 실시예에 따르면, 제1 배양하는 단계는 1mL의 인체 세포외기질을 포함하는 배지와 갑상선암 세포 SNU-790을 1 x 106 세포수로 혼합하여 10% FBS-RPMI에 세포들이 분포할 수 있도록 섞어준 후 플레이트에 시딩한다. 본 발명에 따른 인체 세포외기질의 특성에 따라 부유하는 세포들과 서로 융합되어 밀착 연접 및 자가 조직화가 일어나면서 배양 3일 이내 오가노이드가 형성된다. 암 오가노이드가 100 um로 균질해지는 시기까지(약 7일) 신선한 배지로 3일에 한번씩 교체해준다. According to one embodiment of the present invention, the first culturing step is to mix medium containing 1 mL of human extracellular matrix and thyroid cancer cells SNU-790 at a cell count of 1 x 10 6 and distribute the cells in 10% FBS-RPMI. Mix well and seed on the plate. According to the characteristics of the human extracellular matrix according to the present invention, it fuses with floating cells, tight junctions and self-organization occur, and organoids are formed within 3 days of culture. Replace the medium with fresh medium once every 3 days until the cancer organoids become homogeneous at 100 um (about 7 days).
다양한 실시예에서, 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간 동안 수행될 수 있으나 이에 제한되는 것은 아니다. 이와 관련하여, 종래의 오가노이드 형성은 수주 이상의 기간이 소요되었다. 그러나, 본 발명의 일 실시예에 따른 갑상선암 오가노이드 제조 방법은 3일 이내의 단 시간 내에 갑상선암 오가노이드의 형성을 제공한다. In various embodiments, the first culturing step may be performed for at least one period of about 1 hour to 3 days, but is not limited thereto. In this regard, conventional organoid formation takes several weeks or more. However, the method for producing thyroid cancer organoids according to an embodiment of the present invention provides the formation of thyroid cancer organoids within a short time of less than 3 days.
한편, 제 1 배양하는 단계에 의하여 형성된 오가노이드들은 50 - 200 μm 크기를 가질 수 있으며 각각의 크기 및 성숙도가 상이할 수 있음에 따라, 실험 또는 임상적인 약물(화합물) 평가에 있어, 오가노이드의 반응을 현미경으로 관찰하기에 충분하도록 균질한 크기 및 갑상선암의 특성을 발현하는 오가노이드 성장을 위하여 50일내의 기간동안 배지를 3일에 한번 교체하며 제2 배양할 수 있다. On the other hand, the organoids formed by the first culturing step may have a size of 50 - 200 μm and each size and maturity may be different, so in experimental or clinical drug (compound) evaluation, the organoids In order to grow organoids with a uniform size sufficient to observe the reaction under a microscope and expressing the characteristics of thyroid cancer, a second culture can be performed by replacing the medium once every 3 days for a period of 50 days.
제2 배양하는 단계는 구체적으로 도 21을 참고하면, 약 3일 내지 50일 중 적어도 하나의 기간 동안 수행될 수 있으나 이에 제한되는 것은 아니다. 제1 배양하는 단계 및 제2 배양하는 단계는 순차적으로 또는 동시에 수행될 수 있다.Referring specifically to FIG. 21, the second culturing step may be performed for at least one period of about 3 to 50 days, but is not limited thereto. The first culturing step and the second culturing step may be performed sequentially or simultaneously.
다양한 실시예에서, 제1 배양하는 단계 또는 제2 배양하는 단계에서 사용되는 배양 배지는 성장 배지일 수 있다. 인위적으로 합성하여 제조하여 사용하거나 상업적으로 제조된 배지를 사용할 수 있다. 예를 들어, 인슐린을 포함하지 않는, 이글 기본 배지(Basal medium Eagle's, BME), 최소 필수 배지(Minimal essential medium, MEM), 이글 최소 필수 배지(Eagle's MEM), 둘베코 수정 이글 배지(Dulbecco's modified Eagle's medium, DMEM), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199 및 RPMI 1640 등과 같은 다양한 무혈청 배지 및 이들의 변이형 중 적어도 하나를 포함할 수 있다. In various embodiments, the culture medium used in the first or second culturing step may be a growth medium. It can be artificially synthesized and used, or a commercially prepared medium can be used. For example, basal medium Eagle's (BME), Minimal essential medium (MEM), Eagle's MEM, Dulbecco's modified Eagle's medium, without insulin. It may include at least one of various serum-free media such as DMEM), DMEM/F12, HAM'S F-10, HAM'S F-12, MEDIUM 199, and RPMI 1640, and their variants.
다양한 실시예에서, 성장 배지는 추가적으로 아미노산, 아스코르빅산, 아세트산, FBS, B27 및 IWR-1 중 적어도 하나를 포함할 수 있다. 세포의 성장에 필요한 아미노산으로서 L-글라이신, L-알라닌, L-아스파라긴, L-아스파틱산, L-글루탐산, L-프롤린, L-세린 또는 L-글루타민을 더 포함할 수 있으나 이에 제한되는 것은 아니다. L-글루타민은 글루타맥스(Glutamax)로 대체될 수 있다.In various embodiments, the growth medium may additionally include at least one of the following amino acids: ascorbic acid, acetic acid, FBS, B27, and IWR-1. Amino acids necessary for cell growth may further include, but are not limited to, L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, or L-glutamine. . L-Glutamine can be replaced with Glutamax.
다양한 실시예에서, 성장 배지는 추가적으로 항생제를 포함할 수 있다. In various embodiments, the growth medium may additionally include antibiotics.
다양한 실시예에서, 인체 세포외기질 및 성장 배지의 부피비는 1:4 내지 1:6일 수 있으며, 바람직하게는 1:5의 비율일 수 있으나 이에 제한되는 것은 아니다.In various embodiments, the volume ratio of human extracellular matrix and growth medium may be 1:4 to 1:6, preferably 1:5, but is not limited thereto.
다양한 실시예에서, 제2 배양하는 단계는 약물을 처리하는 단계를 더 포함할 수 있다. 다시 도 21을 참조하면, 오가노이드들을 하나 이상의 다른 용기에 분류하여 제2 배양을 수행함과 동시에, 특정 용기에 약물을 추가하여 배양될 수 있다. 이때, 약물의 처리(treatment group, 처리군)는 배양 7일차에 수행되는 것이 바람직할 수 있다. 이에, 도 21에서의 T0은 제 2 배양에서 7일차의 갑상선암 오가노이드일 수 있으나, 이에 제한되는 것은 아니다. 사용자의 실험 목적 및 계획에 따라 다양한 기간의 제2 배양에서의 오가노이드가 이용될 수 있다. 예를 들어, 갑상선암의 발달 과정에서의 약물의 효능 및 독성 평가가 요구되는 경우, 제2 배양의 0일차의 갑상선암 오가노이드가 T0에서 이용될 수 있다.In various embodiments, the second culturing step may further include treating the drug. Referring again to FIG. 21, the organoids can be sorted into one or more different containers to perform a second culture, and at the same time, the organoids can be cultured by adding a drug to a specific container. At this time, it may be desirable to perform drug treatment (treatment group) on the 7th day of culture. Accordingly, T0 in FIG. 21 may be a thyroid cancer organoid on day 7 in the second culture, but is not limited thereto. Organoids in secondary culture can be used for various periods of time depending on the user's experimental purpose and plan. For example, if evaluation of drug efficacy and toxicity during the development of thyroid cancer is required, thyroid cancer organoids from day 0 of the second culture can be used at T0.
다양한 실시예에서, 약물 미처리군(CTL) 및 약물 처리군(PFOA, PFDA)에 대해 형태학적 및 단백질 발현을 비교 분석하는 방법으로 약물의 효능 또는 독성 평가를 수행할 수 있다. 형태학적 분석으로는 현미경적 구조 분석이 될 수 있으나, 이에 제한되는 것은 아니다. 단백질 발현 분석으로는 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준 즉, 바이오마커의 발현량 및 발현 위치에 기초한 분석일 수 있으나 이에 제한되는 것은 아니다.In various embodiments, the efficacy or toxicity of a drug may be evaluated by comparative analysis of morphological and protein expression for a drug-untreated group (CTL) and a drug-treated group (PFOA, PFDA). Morphological analysis may include, but is not limited to, microscopic structural analysis. Protein expression analysis includes at least one biomarker selected from the group consisting of F-actin abnormality, thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. The analysis may be based on the marker level, that is, the expression level and expression location of the biomarker, but is not limited thereto.
본 발명의 일 실시예에 따르면 갑상선암 오가노이드를 이용한 약물의 효능 또는 독성 평가를 위해 약물의 처리는 실질적으로 제1 배양을 포함하는 오가노이드의 배양 7일 이내 처음 수행될 수 있다. 구체적으로 오가노이드 크기가 균질해지는 시기(7일 전후)부터 방수를 위한 코팅제로 오랫동안 사용되었으나 독성이 검증되어 현재 사용이 금지된 PFOA 또는 PFDA를 10 νM 의 농도로 새로운 배지에 섞은 후 3일에 한번씩 교체하며 21일동안 화합물에 노출시켜 갑상선암 오가노이드를 유지시켰다. 여러가지 화합물에 장기간 노출된 갑상선 암 오가노이드의 일부는 형태 및 바이오마커 분석을 위하여 4% 파라포름알데하이드로 고정을 한 후, 파라핀을 이용하여 슬라이드 제작을 하였다. 나머지 일부는 정량적 분석을 위하여 RNAase free tube에 옮긴 후 동결과정을 거쳐 -80 ¼C deepfreezer에 보관해 두었다. According to one embodiment of the present invention, in order to evaluate the efficacy or toxicity of a drug using thyroid cancer organoids, drug treatment may be performed for the first time within 7 days of culturing the organoid, including the first culture. Specifically, from the time when the organoid size becomes uniform (around 7 days), PFOA or PFDA, which has been used as a coating for waterproofing for a long time but is currently banned due to its proven toxicity, is mixed with new medium at a concentration of 10 νM and then once every 3 days. Thyroid cancer organoids were maintained by replacing them and exposing them to the compound for 21 days. Some of the thyroid cancer organoids exposed to various compounds for a long period of time were fixed with 4% paraformaldehyde for morphology and biomarker analysis, and slides were made using paraffin. For quantitative analysis, the remaining part was transferred to an RNAase free tube, went through a freezing process, and was stored in a -80 ¼ C deepfreezer.
실시예 8: 갑상선암 오가노이드의 형태 분석Example 8: Morphological analysis of thyroid cancer organoids
8-1. H&E 염색을 통한 갑상선암 오가노이드의 구조 8-1. Structure of thyroid cancer organoids through H&E staining
Hematoxylin & Eosin 염색은 조직이나 세포 샘플을 현미경으로 관찰하기 위한 가장 일반적으로 사용되는 염색 기술 중 하나이다. 이 기법은 병리학에서 널리 사용되며, 오가노이드의 형태와 구조를 시각화를 위하여 사용된다.Hematoxylin & Eosin staining is one of the most commonly used staining techniques for microscopic observation of tissue or cell samples. This technique is widely used in pathology and is used to visualize the morphology and structure of organoids.
도 22는 본 발명의 일 실시예에 따른 갑상선암 오가노이드의 H&E 염색 현미경 이미지를 도시한 것이다. 구체적으로, 도 22를 참조하면, 갑상선암 오가노이드의 파라핀 절편 및 H&E 염색을 통해 내강(lumen)을 확인할 수 있다.Figure 22 shows H&E staining of thyroid cancer organoids according to an embodiment of the present invention. It shows a microscope image. Specifically, referring to Figure 22, the lumen of the thyroid cancer organoid can be confirmed through paraffin sectioning and H&E staining.
이에 본 발명에 따른 갑상선암 오가노이드에 시험하고자 하는 방수를 위한 코팅제로 오랫동안 사용되었으나 독성이 검증되어 현재 사용이 금지된 PFOA 또는 PFDA를 10 νM을 처리 후, 오가노이드의 형태 변화를 확인함으로써 시험물질의 효능 또는 독성 여부를 평가할 수 있다. 도 22와 같이 과불화화합물에 저농도-장기간 노출된 갑상선암 오가노이드에서 갑상선암 세포수가 증가된 것을 육안으로 확인할 수 있기에, 과불화화합물이 갑상선암의 증식에 영향을 미칠 수 있다는 결론을 낼 수 있다. Accordingly, after treatment with 10 νM of PFOA or PFDA, which has been used for a long time as a coating agent for waterproofing to be tested on the thyroid cancer organoid according to the present invention, but is currently banned due to its toxicity, the test substance was confirmed by confirming the change in the shape of the organoid. Efficacy or toxicity can be evaluated. As shown in Figure 22, an increase in the number of thyroid cancer cells can be visually confirmed in thyroid cancer organoids exposed to perfluorinated compounds at low concentration and for a long period of time, so it can be concluded that perfluorinated compounds can affect the proliferation of thyroid cancer.
8-2. Hoechst33342 염색을 통한 갑상선암 형태 및 핵 형태 분석8-2. Thyroid cancer morphology and nuclear morphology analysis through Hoechst33342 staining
암 오가노이드 연구에서 Hoechst33342를 사용하여 핵을 염색하는 것은 다양한 장점을 가지고 있다. 첫째, Hoechst33342는 형광 염료이므로, 현미경을 통해 세포의 핵을 명확하게 식별하고 시각화할 수 있으며, 이는 세포의 구조와 배치, 핵 분할 등을 평가하는데 유용하다. 둘째, Hoechst33342 염색을 통해 세포 주기의 각 단계에서 핵의 상태를 평가할 수 있으며, 이는 세포 분열과 관련된 연구에서 특히 중요하다. 셋째, Hoechst 33342를 사용하여 암 오가노이드 내의 세포 수를 정량적으로 분석할 수 있다. 이를 통해 특정 약물의 처리나 조건이 암 오가노이드의 성장에 어떻게 영향을 미치는지 평가할 수 있다. 넷째, Hoechst 33342 염색을 통해 비정상적인 핵 형태나 조직을 식별함으로써 세포 사멸을 평가할 수 있다.Staining nuclei using Hoechst33342 in cancer organoid research has various advantages. First, because Hoechst33342 is a fluorescent dye, it can clearly identify and visualize the nucleus of a cell through a microscope, which is useful for evaluating the structure and arrangement of the cell, nuclear division, etc. Second, Hoechst33342 staining allows assessing the state of the nucleus at each stage of the cell cycle, which is especially important in studies related to cell division. Third, the number of cells within cancer organoids can be quantitatively analyzed using Hoechst 33342. This allows us to evaluate how certain drug treatments or conditions affect the growth of cancer organoids. Fourth, cell death can be assessed by identifying abnormal nuclear morphology or organization through Hoechst 33342 staining.
도 23는 본 발명의 일 실시예에 따른 갑상선암 오가노이드의 Hoechst33342 염색 현미경 이미지를 도시한 것이다. 구체적으로, 도 23를 참조하면, vehicle control을 처리한 갑상선암 오가노이드(CTL)와 방수를 위한 코팅제로 오랫동안 사용되었으나 독성이 검증되어 현재 사용이 금지된 PFOA 또는 PFDA를 10 νM로 21일간 처리한 갑상선암 오가노이드의 형태 및 염색된 핵의 형태를 비교함에 따라 시험물질이 갑상선암 형태 및 핵에 미치는 영향을 평가할 수 있다. 도 23는 공초점 현미경을 활용하여 Hoechst33342 염색된 갑상선암 오가노이드의 모양을 Z-stack으로 이미지를 확보한 것이므로 이러한 이미지에서 관찰될 수 있는 갑상선암의 모양 변화 및 핵 형태 변화 분석을 통하여 약물이 갑상선암에 미치는 영향을 평가할 수 있다. Figure 23 shows a Hoechst33342 staining microscope image of a thyroid cancer organoid according to an embodiment of the present invention. Specifically, referring to Figure 23, thyroid cancer organoids (CTL) treated with vehicle control and thyroid cancer treated with PFOA or PFDA, which have been used for a long time as a coating agent for waterproofing but are currently banned due to proven toxicity, at 10 νM for 21 days. By comparing the shape of the organoid and the shape of the stained nucleus, the effect of the test substance on the shape and nucleus of thyroid cancer can be evaluated. Figure 23 is a Z-stack image of the shape of a thyroid cancer organoid stained with Hoechst33342 using a confocal microscope. Therefore, the effect of drugs on thyroid cancer can be analyzed through analysis of the shape changes and nuclear shape changes of thyroid cancer that can be observed in these images. Impact can be assessed.
8-3. F-actin 염색을 통한 갑상선암 오가노이드 세포골격 형태 분석8-3. Analysis of thyroid cancer organoid cytoskeleton morphology through F-actin staining
모든 세포에 존재하는 F-액틴 염색을 통하여, vehicle control을 처리한 갑상선암 오가노이드와 약물을 처리한 갑상선암 오가노이드의 형태 변화를 분석할 수 있다. 특히 암조직에서의 F-액틴의 역할은 매우 중요하다. 암세포에서 발견되는 F-액틴의 동적 재구성은 암세포의 이동과 침입, 그리고 전이에 중요한 역할을 한다.Through F-actin staining, which is present in all cells, morphological changes in thyroid cancer organoids treated with vehicle control and thyroid cancer organoids treated with drugs can be analyzed. In particular, the role of F-actin in cancer tissue is very important. Dynamic reorganization of F-actin found in cancer cells plays an important role in cancer cell migration, invasion, and metastasis.
도 24은 본 발명의 일 실시예에 따른 갑상선암 오가노이드에 대해 팔로이딘(phalloidin) 염색 프로토콜을 이용한 F-액틴 염색 현미경 이미지를 도시한 것이다. Figure 24 shows a microscopic image of F-actin staining using a phalloidin staining protocol for thyroid cancer organoids according to an embodiment of the present invention.
구체적으로, 도 24을 참조하면, vehicle control을 처리한 갑상선암 오가노이드(CTL)와 PFOA 또는 PFDA를 10 νM로 21일간 노출된 갑상선암 오가노이드의 F-액틴 비정상에 의한 세포골격의 변화를 비교하여 약물의 갑상선암 오가노이드 세포골격에 미치는 영향을 평가할 수 있다. 세포 골격은 세포를 지지하는 역할을 하기 때문에 암세포에서의 세포 골격은 정상세포의 세포골격과 형태적으로 차이를 나타낸다. 특히 암 전이가 증가되는 경우 세포골격에서 spot의 증가가 관찰되며 전이와 연관있는 대표적인 세포골격의 형태 변화 중 하나이다. 이러한 이유로 항암제의 효능 또는 생활유해물질의 독성을 평가할 때 상술한 F-액틴 비정상(F-actin abnormality) 분석을 통해 세포골격 형태를 확인함으로써 본 발명에 따른 갑상선암 오가노이드 모델을 활용해 신약의 효능 및 독성을 평가할 수 있다.Specifically, referring to Figure 24, changes in the cytoskeleton due to F-actin abnormality in thyroid cancer organoids (CTL) treated with vehicle control and thyroid cancer organoids exposed to PFOA or PFDA at 10 νM for 21 days were compared. The effect on the thyroid cancer organoid cytoskeleton can be evaluated. Because the cytoskeleton plays a role in supporting cells, the cytoskeleton in cancer cells is morphologically different from the cytoskeleton in normal cells. In particular, when cancer metastasis increases, an increase in spots is observed in the cytoskeleton and is one of the representative cytoskeleton morphological changes associated with metastasis. For this reason, when evaluating the efficacy of anticancer drugs or the toxicity of household harmful substances, the cytoskeletal form is confirmed through the above-mentioned F-actin abnormality analysis, and the thyroid cancer organoid model according to the present invention can be used to determine the efficacy of new drugs and Toxicity can be assessed.
실시예 9: 갑상선암 오가노이드의 갑상선 자극 호르몬 수용체(TSHR) 분석Example 9: Thyroid-stimulating hormone receptor (TSHR) analysis of thyroid cancer organoids
실시예 7에서 제조된 갑상선암 오가노이드에 대해 TSHR 면역염색 프로토콜을 이용하여 갑상선 자극 호르몬 수용체(THSR) 발현 분석을 수행하였다.Thyroid stimulating hormone receptor (THSR) expression analysis was performed on the thyroid cancer organoid prepared in Example 7 using the TSHR immunostaining protocol.
도 25a 및 도 25b는 본 발명의 일 실시예에 따른 갑상선암 오가노이드의 갑상선 자극 호르몬 수용체(TSHR) 변화를 도시한 것이다. Figures 25a and 25b show changes in thyroid stimulating hormone receptor (TSHR) in thyroid cancer organoids according to an embodiment of the present invention.
구체적으로, 도 25a를 참조하면, vehicle control을 처리한 갑상선암 오가노이드(CTL)와 비교하여 PFOA 또는 PFDA를 10 νM의 농도로 새로운 배지에 섞은 후 3일에 한번씩 교체하며 21일동안 화합물에 노출시켜 TSHR의 발현의 변화를 확인할 수 있다. TSHR의 발현과 갑상선암 진행과 관련이 있기 때문에, TSHR 발현이 감소되면 암진행이 가속화되고 갑상선암의 전이가 쉽게 되는 특징이 있다. 이를 기반으로 본 발명에서는 갑상선암 오가노이드에 대조약물과 과불화화합물을 저농도-장기간 노출시 TSHR 발현의 차이를 분석한 것이다. 그 결과 과불화화합물에 노출된 갑상선암 오가노이드에서 TSHR 발현이 감소되어 과불화화합물이 갑상선암의 증식 및 전이에 영향을 미친다는 것을 밝혔다. Specifically, referring to Figure 25a, compared to thyroid cancer organoids (CTL) treated with vehicle control, PFOA or PFDA was mixed with new medium at a concentration of 10 νM and then changed once every 3 days and exposed to the compound for 21 days. Changes in the expression of TSHR can be confirmed. Because the expression of TSHR is related to the progression of thyroid cancer, when TSHR expression is reduced, cancer progression accelerates and thyroid cancer metastases easily. Based on this, the present invention analyzed the difference in TSHR expression when thyroid cancer organoids were exposed to a control drug and a perfluorinated compound at low concentration and for a long period of time. As a result, TSHR expression was decreased in thyroid cancer organoids exposed to perfluorinated compounds, revealing that perfluorinated compounds affect the proliferation and metastasis of thyroid cancer.
이에 TSHR가 발현되어 있는 본 발명의 갑상선암 오가노이드를 활용하여 약물 처리에 의한 TSHR의 증감을 확임함으로써, 약물의 암에 대한 영향을 평가할 수 있다. Accordingly, by confirming the increase or decrease in TSHR due to drug treatment using the thyroid cancer organoid of the present invention in which TSHR is expressed, the effect of the drug on cancer can be evaluated.
한편, 갑상선암 자극 호르몬 수용체(TSHR)는 정상적으로 갑상선 여포 상피세포의 기저(basal)부분에서 발현한다. 하지만 도 25b를 참조하면, PFOA 또는 PFDA를 10 νM의 농도로 새로운 배지에 섞은 후 3일에 한번씩 교체하며 21일동안 화합물에 노출시킨 후, TSHR 발현 위치가 세포내소포(intracellular vesicle)로 전위되는 것을 확인할 수 있다. 특히 금지 화합물인 PFOA에 노출된 갑상선암 오가노이드에서 그 변화가 뚜렷하게 나타났다. Meanwhile, thyroid tumor-stimulating hormone receptor (TSHR) is normally expressed in the basal portion of thyroid follicular epithelial cells. However, referring to Figure 25b, PFOA or PFDA was mixed with new medium at a concentration of 10 νM, replaced once every 3 days, and exposed to the compound for 21 days. After that, the TSHR expression site was translocated to intracellular vesicles. You can check that. In particular, the changes were evident in thyroid cancer organoids exposed to the banned compound PFOA.
이에 TSHR가 발현되어 있는 본 발명의 갑상선암 오가노이드를 활용하여 약물 처리에 의한 TSHR 발현의 위치를 확인함으로써, 약물의 암에 대한 영향을 평가할 수 있다. Accordingly, the effect of the drug on cancer can be evaluated by confirming the location of TSHR expression by drug treatment using the thyroid cancer organoid of the present invention in which TSHR is expressed.
실시예 10: 갑상선암 오가노이드의 티로글로불린(Thyroglobulin, Tg) 분석Example 10: Thyroglobulin (Tg) analysis of thyroid cancer organoids
실시예 7에서 제조된 갑상선암 오가노이드에 대해 Tg 면역염색 프로토콜을 이용하여 갑상선암 바이오마커 중 하나인 티로글로불린(Tg) 발현 분석을 수행하였다.Thyroglobulin (Tg) expression analysis, one of the thyroid cancer biomarkers, was performed on the thyroid cancer organoid prepared in Example 7 using the Tg immunostaining protocol.
티로글로불린은 정상 갑상선 조직 및 갑상선암 조직에서만 분비되는 단백질로 갑상선 및 갑상선암 특이 바이오마커 중 하나이다. Thyroglobulin is a protein secreted only from normal thyroid tissue and thyroid cancer tissue and is one of the thyroid and thyroid cancer specific biomarkers.
도 26을 참조하면 본 발명에 따른 갑상선암 오가노이드는 Tg(티로글로불린)의 발현이 명확하게 나타나 인체 갑상선암의 특성을 모사함을 확인하였다. 또한 PFOA 또는 PFDA를 10 νM의 농도로 새로운 배지에 섞은 후 3일에 한번씩 교체하며 21일동안 화합물에 노출시킴에 따라 갑상선암 오가노이드의 Tg 발현이 증가되는 것을 확인하였고, 이는 갑상선암에서 호르몬 분비 활성화에 따른 2차 세포증식을 유발할 가능성이 있음을 시사한다. Referring to Figure 26, it was confirmed that the thyroid cancer organoid according to the present invention clearly expressed Tg (thyroglobulin), mimicking the characteristics of human thyroid cancer. In addition, it was confirmed that Tg expression in thyroid cancer organoids was increased as PFOA or PFDA was mixed in new medium at a concentration of 10 νM and exposed to the compound for 21 days, replaced once every 3 days, which was found to be involved in activating hormone secretion in thyroid cancer. This suggests that there is a possibility of causing secondary cell proliferation.
따라서, 신규 항암제 및 유해물질에 대한 효능 및 독성평가 시 본 발명인 갑상선암 오가노이드에서 Tg 발현량 증감을 분석함으로써 시험약물에 대한 갑상선암의 활성 또는 억제를 예측할 수 있다.Therefore, when evaluating the efficacy and toxicity of new anticancer drugs and harmful substances, the activity or inhibition of thyroid cancer for the test drug can be predicted by analyzing the increase or decrease in Tg expression level in the thyroid cancer organoid of the present invention.
실시예 11: 갑상선암 오가노이드의 티로페록시다제(TPO) 분석Example 11: Tyroperoxidase (TPO) analysis of thyroid cancer organoids
실시예 7에서 제조된 갑상선암 오가노이드에 대해 TPO 면역염색 프로토콜을 이용하여 갑상선암 바이오마커 중 하나인 티로페록시다제(Thyroperoxidase; TPO) 발현 분석을 수행하였다.Thyroperoxidase (TPO) expression, one of the thyroid cancer biomarkers, was analyzed for the thyroid cancer organoid prepared in Example 7 using the TPO immunostaining protocol.
티로페록시다제는 갑상선 호르몬 합성에 관여하는 효소로 티로글로불린의 티로신 잔기에서 요오드화물(iodide)의 산화를 촉매하여 T3(Triiodothyronine)와 T4(Tetraiodothyronine; Thyroxine)을 합성하는 역할을 한다. 갑상선에서 TPO의 발현 증가는 갑상선암이 촉진될 가능성이 있음을 간접적으로 예측할 수 있다.Thyroperoxidase is an enzyme involved in thyroid hormone synthesis. It catalyzes the oxidation of iodide from the tyrosine residue of thyroglobulin to synthesize T3 (Triiodothyronine) and T4 (Tetraiodothyronine; Thyroxine). Increased expression of TPO in the thyroid gland can indirectly predict the possibility of promoting thyroid cancer.
도 27를 참조하면 본 발명에 따른 갑상선암 오가노이드에서 TPO 발현이 명확하게 나타나 인체 갑상선암의 특성을 모사하고 있음을 확인하였다. 또한 생활유해물질인 PFOA 또는 PFDA를 10 νM의 농도로 새로운 배지에 섞은 후 3일에 한번씩 교체하며 21일동안 화합물에 노출시킴에 따라 갑상선암 오가노이드의 TPO 발현이 증가되는 것을 확인하였다. 특히 본 발명을 활용하여 갑상선암 오가노이드에 과불화화합물을 저농도-장기간 노출이 TPO 발현을 증가시켜 갑상선암을 촉진시킨다는 결론을 지을 수 있게 하였다. 이에 본 발명의 갑상선암 오가노이드를 이용하면 이와 같이 갑상선암을 활성 또는 억제시키는 약물을 예측할 수 있다.Referring to Figure 27, it was confirmed that TPO expression was clearly observed in the thyroid cancer organoid according to the present invention, mimicking the characteristics of human thyroid cancer. In addition, it was confirmed that TPO expression in thyroid cancer organoids increased as PFOA or PFDA, which are hazardous substances in daily life, were mixed into new medium at a concentration of 10 νM, replaced every three days, and exposed to the compounds for 21 days. In particular, using the present invention, it was possible to conclude that low-concentration and long-term exposure to perfluorinated compounds in thyroid cancer organoids increases TPO expression and promotes thyroid cancer. Accordingly, using the thyroid cancer organoid of the present invention, drugs that activate or inhibit thyroid cancer can be predicted.
실시예 12: 갑상선암 오가노이드의 E-cadherin 분석Example 12: E-cadherin analysis of thyroid cancer organoids
실시예 7에서 제조된 갑상선암 오가노이드에 대해 E-cadherin 면역염색을 수행하여 갑상선암 바이오마커 중 하나인 E-cadherin 발현 분석을 수행하였다.E-cadherin immunostaining was performed on the thyroid cancer organoid prepared in Example 7 to analyze the expression of E-cadherin, one of the thyroid cancer biomarkers.
E-cadherin은 대부분의 상피세포에서 발현이 되는 중요한 바이오마커 중 하나이다. 갑상선 상피세포의 특성이 변하게 되면, 상피중간엽전이(EMT)가 활성화되어 병리적 현상이 관찰된다. 이러한 변화는 주로 섬유화나 암의 침윤과 전이 시 발견되며, E-cadherin의 감소는 이러한 병리적 현상이 발생되고, 갑상선 상피세포의 기능 및 구조 변화가 생겼다는 것을 예상할 수 있다. E-cadherin is one of the important biomarkers expressed in most epithelial cells. When the characteristics of thyroid epithelial cells change, epithelial-mesenchymal transition (EMT) is activated and pathological phenomena are observed. These changes are mainly found during fibrosis or cancer invasion and metastasis, and a decrease in E-cadherin can be expected to indicate that these pathological phenomena occur and changes in the function and structure of thyroid epithelial cells occur.
도 28a를 참조하면, 본 발명인 갑상선암 오가노이드를 생활유해물질인 PFOA 또는 PFDA를 10 νM의 농도로 새로운 배지에 섞은 후 3일에 한번씩 교체하며 21일동안 화합물에 노출시켰다. 그후 E-cadherin 발현이 정도를 면역염색을 통하여 분석한 결과, 유해물질로써 사용이 금지된 PFOA에 저농도-장기간 노출된 갑상선암 오가노이드에서 E-cadherin 발현 감소를 확인하였다. Referring to Figure 28a, the thyroid cancer organoid of the present invention was exposed to the compound for 21 days by mixing PFOA or PFDA, a household hazardous substance, in a new medium at a concentration of 10 νM and replacing it every 3 days. Afterwards, the level of E-cadherin expression was analyzed through immunostaining, and a decrease in E-cadherin expression was confirmed in thyroid cancer organoids exposed to low concentration and long-term exposure to PFOA, which is banned as a harmful substance.
이에 E-cadherin가 발현되어 있는 본 발명의 갑상선암 오가노이드를 활용하여 약물 처리에 의한 E-cadherin의 증감을 확임함으로써, 약물의 암에 대한 영향을 평가할 수 있다. Accordingly, by confirming the increase or decrease of E-cadherin due to drug treatment using the thyroid cancer organoid of the present invention in which E-cadherin is expressed, the effect of the drug on cancer can be evaluated.
한편, E-cadherin는 정상적으로 세포막에 존재하여 그 기능을 수행하지만, 암 전이 등의 신호가 강화되면 E-cadherin 발현은 세포막(membrane)에서 세포질(cytosol)로 전위된다. 도 28b를 참조하면, 생활유해물질인 PFOA 또는 PFDA를 10 νM의 농도 처리 시, E-cadherin 발현 위치가 세포질 또는 세포막으로 전위되는 것을 확인할 수 있다. 보다 자세히 설명하면, 본 발명의 갑상선암 오가노이드를 활용하여 생활유해물질인 PFOA 또는 PFDA를 10 νM의 농도로 새로운 배지에 섞은 후 3일에 한번씩 교체하며 21일동안 화합물에 노출에 의한 E-cadherin 발현의 위치를 확인함으로써, 약물의 암에 대한 영향을 평가할 수 있다. 특히 유해물질로 인정되어 사용이 금지된 PFOA에 저농도-장기간 노출된 갑상선암 오가노이드에서 E-cadherin 발현이 세포질로 이동한 것이 명확히 검증되었다. Meanwhile, E-cadherin normally exists in the cell membrane and performs its function, but when signals such as cancer metastasis are strengthened, E-cadherin expression is transferred from the cell membrane to the cytosol. Referring to Figure 28b, it can be seen that when treated with PFOA or PFDA, which are hazardous substances in life, at a concentration of 10 νM, the E-cadherin expression location is translocated to the cytoplasm or cell membrane. In more detail, using the thyroid cancer organoid of the present invention, PFOA or PFDA, a household hazardous substance, is mixed into new medium at a concentration of 10 νM, replaced once every 3 days, and E-cadherin expression by exposure to the compound for 21 days. By confirming the location of , the effect of the drug on cancer can be evaluated. In particular, it was clearly verified that E-cadherin expression moved to the cytoplasm in thyroid cancer organoids exposed to low concentration and long-term exposure to PFOA, which is recognized as a hazardous substance and is banned from use.
따라서, 본 발명의 갑상선암 오가노이드를 활용하여 약물 처리에 의한 E-cadherin의 발현 위치의 변화를 확임함으로써, 약물의 암에 대한 영향을 평가할 수 있다. Therefore, the effect of the drug on cancer can be evaluated by confirming the change in the expression location of E-cadherin due to drug treatment using the thyroid cancer organoid of the present invention.
갑상선 오가노이드thyroid organoids
실시예 13. 갑상선 세포주 기반 인간 갑상선 오가노이드 제조Example 13. Preparation of human thyroid organoids based on thyroid cell lines
*이하에서는 도 30 내지 도 33를 참조하여, 본 발명의 일 실시예에 따른 갑상선 오가노이드 제조방법 및 이에 따른 갑상선 오가노이드를 구체적으로 설명한다.*Hereinafter, with reference to FIGS. 30 to 33, the thyroid organoid manufacturing method and the resulting thyroid organoid according to an embodiment of the present invention will be described in detail.
도 30은 본 발명의 일 실시예에 따른 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법을 예시적으로 도시한 것이다.Figure 30 illustrates an exemplary method for producing thyroid organoids for evaluating the efficacy or toxicity of test substances according to an embodiment of the present invention.
도 30을 참조하면, 제조방법은 오가노이드를 형성하도록 갑상선 세포주 및 인체 세포외기질(ECM)을 포함한느 배지를 혼합하여 제1 배양하는 단계; 및 상기 오가노이드를 갑상선 자극 호르몬(Thyroid-Stimulating Hormone) 및 요오드화 칼륨(Potassium Iodide)을 포함하는 갑상선 오가노이드 배양액과 혼합하여 제2 배양하는 단계; 를 포함할 수 있다.Referring to Figure 30, the manufacturing method includes first culturing a mixture of a medium containing a thyroid cell line and human extracellular matrix (ECM) to form an organoid; and secondly culturing the organoids by mixing them with a thyroid organoid culture medium containing Thyroid-Stimulating Hormone and Potassium Iodide; may include.
다양한 실시예에서, 갑상선 세포들은 본 발명의 발명자들이 개발한 3차원 세포배양용 ECM(Org 3D culture solution)인 인체 세포외기질과 함께 서로 융합되어 세포들 사이의 구분이 사라지면서 밀착 연접(tight junction)이 일어나고, 자가 조직화(self-organization)가 일어나면서 오가노이드가 형성된다. 본 발명에 따른 세포외기질은 세포의 모든 표면적을 감싸 배양됨에 따라 안정적으로 생체 내 환경을 가장 유사하게 모사하는 오가노이드를 배양할 수 있다.In various embodiments, thyroid cells fuse with each other and the human extracellular matrix, which is an ECM (Org 3D culture solution) for three-dimensional cell culture developed by the inventors of the present invention, forming tight junctions while the distinction between cells disappears. ) occurs, and self-organization occurs, forming organoids. As the extracellular matrix according to the present invention surrounds the entire surface area of cells and is cultured, organoids that most closely mimic the in vivo environment can be stably cultured.
다양한 실시예에서, 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간 동안 배양할 수 있고, 제2 배양하는 단계는 약 3일 내지 50일 중 적어도 하나의 기간 동안 배양할 수 있다. 제1 배양하는 단계 및 제2 배양하는 단계는 순차적으로 또는 동시에 수행될 수 있다.In various embodiments, the first culturing step may be performed for at least one period of about 1 hour to 3 days, and the second culturing step may be performed for at least one of about 3 days to 50 days. . The first culturing step and the second culturing step may be performed sequentially or simultaneously.
본 발명의 일 실시예에 따르면, 본 발명에 따른 인체 세포외기질을 포함하여 갑상선 세포를 배양한 결과 3일 이내에 50-200 um 정도의 사이즈로 갑상선 오가노이드가 형성되었다. 이에 갑상선 오가노이드를 이용한 시험물질의 효능 또는 독성 평가를 위해 시험물질의 처리는 실질적으로 오가노이드 배양 7일 이내 처음 수행될 수 있다. 시험물질이 처리된 갑상선 오가노이드의 배양은 도 32을 참조하여 후술하기로 한다.According to one embodiment of the present invention, as a result of culturing thyroid cells including human extracellular matrix according to the present invention, thyroid organoids were formed with a size of about 50-200 um within 3 days. Therefore, to evaluate the efficacy or toxicity of a test substance using thyroid organoids, treatment of the test substance can be performed for the first time within 7 days of organoid culture. Cultivation of thyroid organoids treated with test substances will be described later with reference to FIG. 32.
다양한 실시예에서, 갑상선 세포주는 갑상선 조직으로부터 분리하여 수득하거나 상업용 세포주를 구입하는 등의 경로에 있어 제한이 없다. 갑상선 세포주는 갑상선 정상세포 또는 갑상선 정상 상피세포일 수 있다. In various embodiments, there are no restrictions on the route, such as obtaining the thyroid cell line by isolating it from thyroid tissue or purchasing a commercial cell line. Thyroid cell lines may be normal thyroid cells or normal thyroid epithelial cells.
다양한 실시예에서, 갑상선 세포주 및 인체 세포외기질을 포함하는 배지는 1:1 비율로 포함될 수 있다. 바람직하게는 인체 세포외기질을 포함하는 배지 1mL 기준으로 갑상선 세포주는 1~5 x 105 내지 1~5 x 107 세포수로 포함될 수 있으나 이에 제한되는 것은 아니다. In various embodiments, medium containing a thyroid cell line and human extracellular matrix may be included in a 1:1 ratio. Preferably, based on 1 mL of medium containing human extracellular matrix, the thyroid cell line may be included in the number of 1 to 5 x 10 5 to 1 to 5 x 10 7 cells, but is not limited thereto.
본 발명의 일 실시예에 따르면, 갑상선 오가노이드 형성을 위해 인체 세포외기질을 포함하는 배지 1mL에 갑상선 세포 H6040 또는 Nthy-ori3-1를 1 x 106 세포수로 혼합하였다. 본 발명의 발명자들이 개발한 3차원 세포배양용 ECM과의 융합으로 갑상선 세포는 3일 이내 짧은 기간동안 자가조직화되어 오가노이드를 형성하였다.According to one embodiment of the present invention, to form thyroid organoids, 1 mL of medium containing human extracellular matrix was mixed with thyroid cells H6040 or Nthy-ori3-1 at a cell number of 1 x 10 6 . By fusion with the ECM for 3D cell culture developed by the inventors of the present invention, thyroid cells self-organized for a short period of time within 3 days to form organoids.
다양한 실시예에서, 제1 배양을 통해 형성된 오가노이드는 내분비 기관인 갑상선의 모사도를 높이기 위해 갑상선 자극 호르몬 및 요오드화 칼륨을 포함하는 갑상선 오가노이드 배양액과 혼합하여 제2 배양될 수 있다. 이 때 갑상선 오가노이드 배양액은 배양 기간동안 3일에 한 번씩 교체해줄 수 있다.In various embodiments, the organoids formed through the first culture may be cultured a second time by mixing them with a thyroid organoid culture medium containing thyroid-stimulating hormone and potassium iodide to increase the degree of mimicry of the thyroid gland, which is an endocrine organ. At this time, the thyroid organoid culture medium can be replaced every three days during the culture period.
다양한 실시예에서, 제1 배양하는 단계 및 제2 배양하는 단계에서 사용되는 배지 또는 배양액은 인위적으로 합성하여 제조하여 사용하거나 상업적으로 제조된 것을 사용할 수 있다. 예를 들어, 인슐린을 포함하지 않는 이글 기본 배지(Basal medium Eagle's, BME), 최소 필수 배지(Minimal essential medium, MEM), 이글 최소 필수 배지(Eagle's MEM), 둘베코 수정 이글 배지(Dulbecco's modified Eagle's medium, DMEM), Ham's F12, SF 12 및 RPMI 1640 등과 같은 다양한 무혈청 배지 및 이들의 변이형 중 적어도 하나를 포함할 수 있다. 또한 FBS (Fetal bovine serum) 등을 더 포함할 수 있다. 바람직하게는 FBS-RPMI, FBS-DMEM, 상피세포배지(EpiCM)를 포함할 수 있으나 이에 제한되는 것은 아니다.In various embodiments, the medium or culture solution used in the first and second culturing steps may be artificially synthesized or commercially produced. For example, basal medium Eagle's (BME) without insulin, Minimal essential medium (MEM), Eagle's MEM, Dulbecco's modified Eagle's medium. , DMEM), Ham's F12, SF 12, and RPMI 1640, and may include at least one of various serum-free media and their variants. It may also further include FBS (Fetal bovine serum). Preferably, it may include FBS-RPMI, FBS-DMEM, and epithelial cell medium (EpiCM), but is not limited thereto.
다양한 실시예에서, 갑상선 세포주 배양액은 추가적으로 아미노산, 아세트산, 글루타맥스 및 아스코르빅산 중 적어도 하나를 포함할 수 있으나 이에 제한되는 것은 아니다. 세포의 성장에 필요한 아미노산으로서 L-글라이신, L-알라닌, L-아스파라긴, L-아스파틱산, L-글루탐산, L-프롤린, L-세린 또는 L-글루타민을 더 포함할 수 있다.In various embodiments, the thyroid cell line culture medium may additionally include, but is not limited to, at least one of amino acids, acetic acid, glutamax, and ascorbic acid. Amino acids necessary for cell growth may further include L-glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, or L-glutamine.
다양한 실시예에서, 갑상선 오가노이드 배양액에 포함되는 갑상선 자극 호르몬(Thyroid-Stimulating Hormone, 이하 TSH)은 갑상선 오가노이드 배양액 총량에 대에 0.01 내지 1 mU/mL로 포함될 수 있고, 바람직하게는 0.05 내지 0.5 mU/mL 포함될 수 있다. 본 발명에서 제시한 농도보다 낮은 농도로 갑상선 오가노이드가 노출되었을때는 약물에 대한 반응이 낮게 나타내며, 보다 높은 농도로 노출되었을때는 약물처리를 안한 vehicle control 자체에서 갑상선호르몬 분비가 증가되는 작용으로 인하여 약물에 대한 반응을 쉽게 판별하기 어려울 가능성이 높다. In various embodiments, Thyroid-Stimulating Hormone (TSH) contained in the thyroid organoid culture medium may be included in an amount of 0.01 to 1 mU/mL, preferably 0.05 to 0.5 mU/mL, based on the total amount of the thyroid organoid culture medium. mU/mL may be included. When thyroid organoids are exposed to a lower concentration than that suggested in the present invention, the response to the drug is low, and when exposed to a higher concentration, the drug increases due to the action of increasing thyroid hormone secretion in the untreated vehicle control itself. It is highly likely that it will be difficult to easily determine the reaction to .
다양한 실시예에서, 갑상선 오가노이드 배양액에 포함되는 요오드화 칼륨 (Potassium Iodide, 이하 PI) 은 갑상선 오가노이드 배양액 총량에 1 내지 20nM 로 포함될 수 있고 바람직하게는 5 내지 15nM 로 포함될 수 있다. 본 발명에서 제시하는 농도보다 낮은 PI의 농도가 배양액에 갑상선 오가노이드가 노출되었을때는 약물 노출에 의하여 만들어지는 갑상선 호르몬 자체의 양이 적기 때문에 배지에 분비된 호르몬 분석에 어려움이 있을 수 있으며, 높은 농도의 PI에 노출시 갑상선 오가노이드 자체에 미미하지만 독성을 유발시킬 수 있기에 본 발명에서 제시하는 농도 사용을 권장한다.In various embodiments, potassium iodide (PI) contained in the thyroid organoid culture medium may be included in an amount of 1 to 20 nM, preferably 5 to 15 nM, in the total amount of the thyroid organoid culture medium. When thyroid organoids are exposed to a culture medium with a concentration of PI lower than that suggested in the present invention, there may be difficulties in analyzing the hormones secreted in the medium because the amount of thyroid hormone itself produced by drug exposure is small, and high concentrations Exposure to PI may cause slight but toxic effects on the thyroid organoid itself, so it is recommended to use the concentration suggested in the present invention.
본 발명의 일 실시예에 따르면, 제1 배양하는 단계에서 형성된, H6040 세포 유래 오가노이드를 0.1 mU/mL TSH 및 10 nM 농도의 PI가 첨가된 상피세포 배지에서 30일 동안 제2 배양하였다. 또한 Nthy-ori3-1 세포 유래 오가노이드를 0.1 mU/mL TSH 및 10 nM 농도의 PI가 첨가된 10% FBS-RPM 배지에서 30일 동안 제2 배양하였다.According to one embodiment of the present invention, the H6040 cell-derived organoids formed in the first culturing step were cultured a second time for 30 days in epithelial cell medium supplemented with 0.1 mU/mL TSH and 10 nM concentration of PI. In addition, Nthy-ori3-1 cell-derived organoids were cultured a second time for 30 days in 10% FBS-RPM medium supplemented with 0.1 mU/mL TSH and 10 nM concentration of PI.
본 발명의 일 실시예에 따르면, 인간 갑상선 오가노이드 제작 후, paraffin block에 인간 갑상선 오가노이드를 심어 4um의 두께로 절편을 만든 후, Hematoxylin & Eosin (H&E) 염색을 하였다. H&E 염색은 여러장기의 조직학적 분석에 활용되는 가장 보편적인 방법 중에 하나이다. According to one embodiment of the present invention, after producing a human thyroid organoid, the human thyroid organoid was planted in a paraffin block and a section was made with a thickness of 4 μm, followed by Hematoxylin & Eosin (H&E) staining. H&E staining is one of the most common methods used for histological analysis of various organs.
도 31는 본 발명의 일 실시예에 따른 제조방법으로 제조된 갑상선 오가노이드의 현미경 이미지를 도시한 것이다. 도 31를 참조하면, 본 발명인 갑상선 오가노이드는 colloid 및 colloid를 감싸고 있는 상피세포층을 포함하는 여포와 같은 둥근 구상의 입체적 형태인 것으로 나타난다. 이는 인간 갑상선 조직에서와 비슷한 구조를 가지고 있기에 형태학적으로 생체유사도가 있음이 검증되었다.Figure 31 shows a microscope image of a thyroid organoid prepared by a manufacturing method according to an embodiment of the present invention. Referring to Figure 31, the thyroid organoid of the present invention appears to have a round spherical three-dimensional form like a follicle containing a colloid and an epithelial cell layer surrounding the colloid. Since it has a structure similar to that of human thyroid tissue, it has been verified to be morphologically similar to living organisms.
다양한 실시예에서, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법은 제2 배양하는 단계에서 약물을 처리하는 단계; 를 더 포함할 수 있다. 또한 약물을 처리하는 단계는 오가노이드 배양 7일 이내 처음 수행될 수 있다. In various embodiments, a method of producing thyroid organoids for evaluating the efficacy or toxicity of a drug includes processing the drug in a second culturing step; It may further include. Additionally, the drug treatment step can be performed for the first time within 7 days of organoid culture.
도 32을 참조하면, 제1 배양하는 단계에서 형성된 오가노이드를 갑상선 오가노이드 배양액과 혼합하는 제2 배양하는 단계에서 목적하는 약물을 처리할 수 있다. 이 때 갑상선 오가노이드 배양액 및 약물의 혼합물은 배양 기간동안 3일에 한 번씩 교체해주고, 오가노이드에 노출되었던 old medium은 약물에 따른 갑상선 호르몬의 변화를 분석하기 위해 모두 수집한 후 -70℃ 이하에서 보관한다.Referring to FIG. 32, the organoids formed in the first culturing step can be treated with the desired drug in the second culturing step where the organoids are mixed with the thyroid organoid culture medium. At this time, the mixture of thyroid organoid culture medium and drug was replaced every three days during the culture period, and the old medium exposed to the organoid was collected to analyze changes in thyroid hormones according to the drug and stored at -70°C or below. keep it.
본 발명의 일 실시예에 따르면, Nthy-ori3-1 세포 유래 오가노이드 및 H6040 세포 유래 오가노이드의 제2 배양 과정에서 BHA(부틸하이드록시아니솔) 또는 BPA(비스페놀-A)를 1uM씩 처리한 배양액을 3일에 한 번씩 교체하며 30일 동안 배양하여 내분비계 교란 화학물질(EDC)에 저농도로 장기간 노출된 갑상선 오가노이드를 제조하였다. 구체적으로, EDC를 저농도 장기간 노출시 3일에 한번씩 fresh media에 EDC를 새로 추가한 후, old media를 모두 빼낸 다음 fresh media를 추가시킴으로써 장기간 노출을 시켰다.According to one embodiment of the present invention, during the second culture of Nthy-ori3-1 cell-derived organoids and H6040 cell-derived organoids, 1uM each of BHA (butylhydroxyanisole) or BPA (bisphenol-A) was treated. Thyroid organoids exposed to low concentrations of endocrine disrupting chemicals (EDC) for a long period of time were prepared by culturing for 30 days, changing the culture medium once every three days. Specifically, during long-term exposure to low concentrations of EDC, new EDC was added to fresh media once every three days, and then all old media was removed and fresh media was added, resulting in long-term exposure.
도 33를 참조하면 본 발명의 일 실시예에 따른 제조방법으로 제조된 갑상선 오가노이드를 vehicle control 처리한 오가노이드(CTL), 부틸하이드록시아니솔을 처리하여 제조된 갑상선 오가노이드(EDC-1) 및 비스페놀-A를 처리하여 제조된 갑상선 오가노이드(EDC-2)의 형태를 관찰할 수 있다. Referring to Figure 33, an organoid (CTL) produced by vehicle control treatment of a thyroid organoid produced by a production method according to an embodiment of the present invention, and a thyroid organoid produced by treatment of butylhydroxyanisole (EDC-1). And the shape of the thyroid organoid (EDC-2) prepared by treatment with bisphenol-A can be observed.
이하, 본 발명의 일 실시예에 따른 갑상선 오가노이드를 이용하여 약물 처리에 의한 갑상선 호르몬 및 다양한 갑상선 바이오마커의 변화를 다음 실시예에서 기술하였다Hereinafter, changes in thyroid hormones and various thyroid biomarkers due to drug treatment using thyroid organoids according to an embodiment of the present invention are described in the following examples.
실시예 14: 갑상선 오가노이드의 갑상선 호르몬(T3, T4) 분석Example 14: Analysis of thyroid hormones (T3, T4) in thyroid organoids
도 30 및 도 32에 도시된 방법으로, BHA 또는 BPA을 처리한 갑상선 오가노이드 배양액을 3일에 한 번씩 교체하며 수집하고 -70℃ 이하에서 보관해 두었다가 4℃에서 서서히 해동하였다. 이후 섞여 있을 가능성이 있는 세포 잔해를 제거하기 위해 1000 rpm으로 1분간 spin-down한 후 상층액만 새로운 튜브로 옮기고 ELISA를 이용해 갑상선 호르몬 분석을 진행하였다. By the method shown in Figures 30 and 32, the thyroid organoid culture medium treated with BHA or BPA was collected and replaced every three days, stored at -70°C or lower, and then slowly thawed at 4°C. Afterwards, to remove possible cell debris, the tube was spun down at 1000 rpm for 1 minute, and only the supernatant was transferred to a new tube, and thyroid hormone analysis was performed using ELISA.
도 34는 약물에 노출되지 않고 제조된 갑상선 오가노이드 및 BHA 또는 BPA에 저농도-장기간 노출되어 제조된 갑상선 오가노이드의 갑상선 호르몬 변화를 비교하여 도시한 것이다. 도 34a는 Nthy-ori3-1 세포 유래 갑상선 오가노이드, 도 34b는 H6040 세포 유래 갑상선 오가노이드에 대한 결과이다.Figure 34 shows a comparison of thyroid hormone changes in thyroid organoids prepared without exposure to drugs and thyroid organoids prepared with low-concentration-long-term exposure to BHA or BPA. Figure 34a shows the results for thyroid organoids derived from Nthy-ori3-1 cells, and Figure 34b shows the results for thyroid organoids derived from H6040 cells.
도 34a를 참조하면, 1νΝ BHA 또는 1νM BPA에 노출되어 제조된 Nthy-ori3-1 세포 유래 갑상선 오가노이드는 약물에 노출되지 않고 제조된 갑상선 오가노이드(Vehicle CTL)와 비교하여 갑상선 호르몬 T3, T4의 증가가 나타났다. 이는 약물에 노출된 인간 갑상선 오가노이드가 갑상선 호르몬 분비를 증가시킨다는 것을 의미하며, 이는 본 발명에서 사용되었던 EDC 약물이 내분비계 교란을 유발시켜 인간 갑상선기능 항진을 유발시킬 가능성이 있음을 인간 갑상선 오가노이드를 활용하여 증명한 사례이다. 따라서, 이러한 반응을 활용하여 새로운 약물 개발이나, 동물에서는 증명되었지만 인간에서는 갑상선 호르몬 교란 등에 대한 증명이 완벽하게 되지 않은 많은 화합물들에 대한 검증 연구가 가능하다.Referring to Figure 34a, thyroid organoids derived from Nthy-ori3-1 cells prepared by exposure to 1νΝ BHA or 1νM BPA have higher levels of thyroid hormones T3 and T4 compared to thyroid organoids (Vehicle CTL) prepared without exposure to drugs. an increase was observed. This means that human thyroid organoids exposed to the drug increase thyroid hormone secretion, which suggests that the EDC drug used in the present invention is likely to cause endocrine disruption and induce human hyperthyroidism. This is a case proven using . Therefore, using these reactions, it is possible to develop new drugs or conduct validation studies on many compounds that have been proven in animals but have not been completely proven to disrupt thyroid hormones in humans.
도 34b를 참조하면, 1M BPA에 노출되어 제조된 H6040 세포 유래 갑상선 오가노이드는 갑상선 호르몬 T3, T4의 증가가 통계적으로 유의하게 나타났다(p=0.035). 1νM BHA에 노출되어 제조된 H6040 세포 유래 갑상선 오가노이드의 경우 갑상선 호르몬 T3, T4의 감소가 통계적으로 유의하게 나타났다(각각 p=0.0001, p=0.0096). BPA는 대표적인 내분비계 교란물질로 잘 알려져 있고, 사람에서도 갑상선 호르몬 증가를 유발시킬 수 있다고 보고되어 있다. 이를 양성대조약물로 사용하여 본 발명품인 인간 갑상선 오가노이드에 저농도 장기간 노출시킨 결과, 인간 갑상선 오가노이드에서 갑상선 호르몬 분비를 증가시키는 결론을 얻었다. Referring to Figure 34b, thyroid organoids derived from H6040 cells prepared by exposure to 1M BPA showed a statistically significant increase in thyroid hormones T3 and T4 ( p=0.035 ). In the case of H6040 cell-derived thyroid organoids prepared by exposure to 1νM BHA, there was a statistically significant decrease in thyroid hormones T3 and T4 ( p=0.0001 and p=0.0096, respectively). BPA is well known as a representative endocrine disruptor, and it has been reported that it can cause an increase in thyroid hormones in humans. As a result of using this as a positive control drug and exposing the human thyroid organoid of the present invention to a low concentration for a long period of time, it was concluded that thyroid hormone secretion was increased in the human thyroid organoid.
본 발명에서 사용된 항산화제로 널리 사용되고 있는 BHA는 설치류에서는 갑상선 호르몬 분비를 억제한다는 보고는 있으나, 사람에서는 아직 명확히 증명되지 않은 물질이다. 이를 본 발명품인 인간 갑상선 오가노이드에 저농도-장기간 노출시킨 결과, 갑상선 호르몬 분비가 통계적으로 유의하게 감소되는 것을 증명하여, 사람에서도 설치류와 마찬가지로 갑상선에 직접적인 영향을 줄 수 있다는 것을 증명한 것이다. 이와 같이 화합물이나 신규약물이 인간 갑상선에 어떠한 영향을 미칠 수 있는지 미리 시험할 수 있는 도구로 제시한다.BHA, which is widely used as an antioxidant used in the present invention, has been reported to inhibit thyroid hormone secretion in rodents, but is a substance that has not yet been clearly proven in humans. As a result of low-concentration and long-term exposure to the human thyroid organoid of the present invention, it was demonstrated that thyroid hormone secretion was statistically significantly reduced, proving that it can directly affect the thyroid gland in humans as in rodents. In this way, it is presented as a tool to test in advance what effect a compound or new drug may have on the human thyroid gland.
실시예 15: 갑상선 오가노이드의 갑상선 자극 호르몬 수용체(TSHR) 분석Example 15: Thyroid-stimulating hormone receptor (TSHR) analysis of thyroid organoids
도 30 및 도 32에 도시된 방법으로 과불화화합물을 처리하여 제조된 갑상선 오가노이드에 대해 갑상선 자극 호르몬 수용체(THSR) 분석을 면역염색하여 갑상선의 중요한 바이오마커 중 하나인 TSHR 분석을 수행하였다. Thyroid-stimulating hormone receptor (THSR) analysis was performed on thyroid organoids prepared by treating perfluorinated compounds using the method shown in Figures 30 and 32, and TSHR analysis, one of the important biomarkers of the thyroid gland, was performed.
도 35은 본 발명의 다른 실시예에 따른 제조방법으로 제조된 갑상선 오가노이드의 갑상선 자극 호르몬 수용체(TSHR) 발현을 분석한 것이다. THSR은 갑상선에 존재하는 중요한 receptor 중 하나이며, 이는 pituitary에서 분비하는 Thyroid-stimulating hormone (TSH)의 수용체이다. TSH 가 TSHR과 결합이 되면 thyorid growth, thyrocyte differentiation, thyroid hormone synthesis 등이 일어난다. THSR은 갑상선에 존재하는 중요한 receptor 중 하나이며, 이는 pituitary에서 분비하는 Thyroid-stimulating hormone (TSH)의 수용체이다. TSH 가 TSHR과 결합이 되면 thyorid growth, thyrocyte differentiation, thyroid hormone synthesis 등이 일어난다. 이러한 화학물질들을 인간 갑상선 오가노이드에 노출시, TSHR의 expression변화를 유도하거나. TSHR 발현 변화 없이 T3 및 T4 분비의 증가를 유도할 수 있다. 즉, 화학물질들이 갑상선 호르몬 증감에 어떠한 영향을 미치는지에 평가하기 위하여 본 발명품 활용이 가능하다. Figure 35 is an analysis of thyroid-stimulating hormone receptor (TSHR) expression in thyroid organoids prepared by a production method according to another embodiment of the present invention. THSR is one of the important receptors present in the thyroid gland, and is the receptor for Thyroid-stimulating hormone (TSH) secreted by the pituitary. When TSH combines with TSHR, thyorid growth, thyrocyte differentiation, and thyroid hormone synthesis occur. THSR is one of the important receptors present in the thyroid gland, and is the receptor for Thyroid-stimulating hormone (TSH) secreted by the pituitary. When TSH combines with TSHR, thyorid growth, thyrocyte differentiation, and thyroid hormone synthesis occur. When these chemicals are exposed to human thyroid organoids, they induce changes in the expression of TSHR. It can induce an increase in T3 and T4 secretion without changing TSHR expression. In other words, the present invention can be used to evaluate how chemicals affect the increase or decrease of thyroid hormones.
도 35에서는 생활용품의 코팅제로 많이 사용되었던 화학물질인 과불화화합물인 PFOA와 PFDA를 저농도-장기간 동안 본 발명품에 노출시킨 결과, TSHR의 발현이 vehicle control에 비하여 현저히 감소되는 것을 증명하였으며, 이는 과불화화합물이 인체에 노출되면 TSHR 발현을 감소시켜 갑상선 기능 이상을 유발한다는 것을 증명한 것이다. In Figure 35, it was demonstrated that the expression of TSHR was significantly reduced compared to the vehicle control as a result of exposing the perfluorinated compounds PFOA and PFDA, which are chemicals widely used as coating agents for household goods, to the present invention at low concentration for a long period of time, which is It has been proven that when the chemical compound is exposed to the human body, it reduces TSHR expression and causes thyroid dysfunction.
실시예 16: 갑상선 오가노이드의 티로글로불린(Thyroglobulin) 분석Example 16: Thyroglobulin analysis of thyroid organoids
도 30 및 도 3에 도시된 방법으로 과불화화합물을 처리하여 제조된 갑상선 오가노이드에 대해 갑상선 바이오마커 중 하나인 티로글로불린(Thyroglobulin) 면역염색을 수행하여 Tg 발현 분석을 수행하였다. 티로글로불린은 정상 갑상선 조직 및 갑상선암 조직에서만 분비되는 단백질로 갑상선 특이 바이오마커이다. Thyroglobulin, one of the thyroid biomarkers, was immunostained to analyze Tg expression on thyroid organoids prepared by treating perfluorinated compounds using the method shown in Figures 30 and 3. Thyroglobulin is a protein secreted only from normal thyroid tissue and thyroid cancer tissue and is a thyroid-specific biomarker.
도 36을 참조하면 본 발명에 따른 갑상선 오가노이드는 Tg (티로글로불린)의 발현이 명확하게 나타나 인체 갑상선에 대한 유사도가 있음을 확인하였다. 따라서, 다양한 내분비계 교란물질이나 신규약물을 본 발명품인 인간 갑상선 오가니오드에 노출시킨 후 Tg 발현 변화를 측정함으로써 화학물질이나 신규약물들이 인간 갑상선 기능을 활성 또는 억제시키는지 여부를 예측할 수 있는 tool 로 활용할 수 있다. Referring to Figure 36, the thyroid organoid according to the present invention clearly showed expression of Tg (thyroglobulin), confirming its similarity to the human thyroid gland. Therefore, it is a tool that can predict whether chemicals or new drugs activate or inhibit human thyroid function by measuring changes in Tg expression after exposing various endocrine disruptors or new drugs to the human thyroid organiode of the present invention. You can utilize it.
도 36에서 사용된 화학물질은 도 35에서와 마찬가지로, 생활에서 방수를 위하여 코팅제로 사용되었던 과불화화합물이 인간 갑상선에 어떠한 영향을 미치는 지를 Tg 면역염색 후 분석하였다. 도 36에서와 같이 과불화화합물에 저농도 장기간 노출된 인간 갑상선에서 Tg 발현이 증가되는 것을 확인하였고, 이는 과불화화합물이 인간 갑상선에 직접적으로 영향을 미치는 것을 증명한 것이다. The chemicals used in Figure 36 were analyzed after Tg immunostaining to determine the effect of perfluorinated compounds used as coating agents for waterproofing on the human thyroid gland, as in Figure 35. As shown in Figure 36, it was confirmed that Tg expression was increased in the human thyroid gland exposed to low concentrations of perfluorinated compounds for a long period of time, proving that perfluorinated compounds directly affect the human thyroid gland.
실시예 17: 갑상선 오가노이드의 티로페록시다제(TPO) 분석Example 17: Tyroperoxidase (TPO) analysis of thyroid organoids
도 30 및 도 32에 도시된 방법으로 과불화화합물을 처리하여 제조된 갑상선 오가노이드에 대해 갑상선 바이오마커 중 하나인 티로페록시다제(Thyroperoxidase; TPO) 면역염색을 수행하여 TPO 발현 분석을 수행하였다.Thyroperoxidase (TPO) immunostaining, one of the thyroid biomarkers, was performed on thyroid organoids prepared by treating perfluorinated compounds using the method shown in Figures 30 and 32 to analyze TPO expression.
티로페록시다제는 갑상선 호르몬 합성에 관여하는 효소로 티로글로불린의 티로신 잔기에서 요오드화물(iodide)의 산화를 촉매하여 T3(Triiodothyronine)와 T4(Tetraiodothyronine; Thyroxine)을 합성하는 역할을 한다. 갑상선에서 TPO의 발현 증가는 갑상선 기능에 이상이 생겼음을 확인하는 지표임이 알려져 있다.Thyroperoxidase is an enzyme involved in thyroid hormone synthesis. It catalyzes the oxidation of iodide from the tyrosine residue of thyroglobulin to synthesize T3 (Triiodothyronine) and T4 (Tetraiodothyronine; Thyroxine). It is known that increased expression of TPO in the thyroid gland is an indicator that abnormalities in thyroid function have occurred.
도 37을 참조하면 본 발명에 따른 갑상선 오가노이드에서 TPO 발현을 확인함에 따라 인체 갑상선에 대한 유사도가 있음을 확인하였다. 또한 방수를 위한 코팅제로 사용되었으나 독성이 확인되어 사용이 금지된 과불화화합물을 저농도-장기간 인간 갑상선 오가노이드에 노출시킨 결과, 제조된 갑상선 오가노이드의 TPO 발현이 증감되는 것을 확인함에 따라, 본 발명의 갑상선 오가노이드를 이용하면 갑상선 기능을 활성 또는 억제시키는 시험물질을 예측할 수 있다. Referring to Figure 37, as TPO expression was confirmed in the thyroid organoid according to the present invention, it was confirmed that there was similarity to the human thyroid gland. In addition, it was confirmed that TPO expression in the prepared thyroid organoids was increased or decreased as a result of exposing human thyroid organoids at low concentrations and for a long period of time to perfluorinated compounds, which were used as coating agents for waterproofing but were banned for use due to confirmed toxicity. Accordingly, the present invention Using thyroid organoids, test substances that activate or inhibit thyroid function can be predicted.
실시예 18: 갑상선 오가노이드의 E-cadherin 분석Example 18: E-cadherin analysis of thyroid organoids
도 30 및 도 32에 도시된 방법으로 과불화화합물을 처리하여 제조된 갑상선 오가노이드에 대해 E-cadherin 면역염색을 수행하여 갑상선 바이오마커 중 하나인 E-cadherin 발현 분석하였다.E-cadherin immunostaining was performed on thyroid organoids prepared by treating perfluorinated compounds using the method shown in Figures 30 and 32 to analyze the expression of E-cadherin, one of the thyroid biomarkers.
E-cadherin은 대부분의 상피세포에서 발현이 되는 중요한 바이오마커 중 하나이다. 특히, 갑상선 여포에서 발현되며, 갑상선의 세포 접착(adhesion) 및 조직 구조(tissue structure)에 중요한 역할을 하는, 정상조직에서 견고히 발현되는 단백질 중 하나이다. 갑상선 질환, 특히 암과 같은 병리학적 상태가 되었을 때, E-cadherin의 발현 패턴이 변화하는 것으로 알려져 있다.E-cadherin is one of the important biomarkers expressed in most epithelial cells. In particular, it is expressed in thyroid follicles and is one of the proteins robustly expressed in normal tissues, playing an important role in thyroid cell adhesion and tissue structure. It is known that the expression pattern of E-cadherin changes when pathological conditions such as thyroid disease, especially cancer, occur.
도 38를 참조하면, 인간 갑상선 오가노이드에서 E-cadherin 발현을 확인함에 따라 인체 갑상선에 대한 유사도가 있음을 확인하고, 특히 갑성선 여포를 정상적으로 모사하고 있음을 확인하였다. 특히 여러 제품의 코팅제로 사용되었으나 독성이 검증되어 사용이 금지된 과불화화합물중 PFOA를 10 νM로 21일간 노출시킨 결과, 그림과 같이 E-cadherin의 발현이 세포막에서 세포기질로 전위되는 것이 확인되었고, long chain PFAC로 알려진 PFDA에 같은 농도로 21일간 노출시킨 결과, E-cadherin 발현이 대조군보다 감소되는 것을 확인하였다. E-cadherin의 발현의 감소 및 전위 모두 E-cadherin 기능을 상실하였기에 본 바이오마커 분석을 통하여 갑상선의 기능을 평가하는 도구로 사용가능함을 증명하였다.Referring to Figure 38, as the expression of E-cadherin was confirmed in the human thyroid organoid, it was confirmed that there was similarity to the human thyroid gland, and in particular, it was confirmed that thyroid follicles were normally simulated. In particular, as a result of exposure to PFOA at 10 νM for 21 days, which is a perfluorinated compound used as a coating agent in several products but banned due to its toxicity, it was confirmed that the expression of E-cadherin was translocated from the cell membrane to the cytosol, as shown in the figure. , As a result of exposure to PFDA, known as long chain PFAC, at the same concentration for 21 days, it was confirmed that E-cadherin expression was reduced compared to the control group. Since both the decrease and translocation of E-cadherin expression result in loss of E-cadherin function, this biomarker analysis proved that it can be used as a tool to evaluate thyroid function.
이상 첨부된 도면을 참조하여 본 발명의 실시 예들을 더욱 상세하게 설명하였으나, 본 발명은 반드시 이러한 실시 예로 국한되는 것은 아니고, 본 발명의 기술사상을 벗어나지 않는 범위 내에서 다양하게 변형 실시될 수 있다. 따라서, 본 발명에 개시된 실시 예들은 본 발명의 기술 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시 예에 의하여 본 발명의 기술 사상의 범위가 한정되는 것은 아니다. 그러므로, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 본 발명의 보호 범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리범위에 포함되는 것으로 해석되어야 할 것이다.Although embodiments of the present invention have been described in more detail with reference to the accompanying drawings, the present invention is not necessarily limited to these embodiments, and may be modified and implemented in various ways without departing from the technical spirit of the present invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but rather to explain it, and the scope of the technical idea of the present invention is not limited by these embodiments. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of protection of the present invention should be interpreted in accordance with the claims below, and all technical ideas within the equivalent scope should be construed as being included in the scope of rights of the present invention.
[과제고유번호] 1711195885[Assignment number] 1711195885
[과제번호] KK-2307[Assignment number] KK-2307
[부처명] 과학기술정보통신부[Ministry Name] Ministry of Science and ICT
[과제관리(전문)기관명] 안전성평가연구소[Name of project management (professional) organization] Safety Evaluation Research Institute
[연구사업명] 안전성평가연구소연구운영비지원(주요사업비)[Research Project Name] Safety Evaluation Research Institute Research Operation Cost Support (Main Project Expenses)
[연구과제명] 생활환경 유해물질 대체 친환경 신소재 개발 및 플랫폼 구축[Research project name] Development of new eco-friendly materials to replace hazardous substances in the living environment and establishment of a platform
[기여율] 1/ 1[Contribution rate] 1/ 1
[과제수행기관명] 한국화학연구원 부설 안전성평가연구소[Name of project carrying out organization] Safety Evaluation Research Institute affiliated with Korea Research Institute of Chemical Technology
[연구기간] 2023.01.01 ~ 2023.12.31[Research period] 2023.01.01 ~ 2023.12.31

Claims (97)

  1. 신장 근위 세뇨관 상피세포주를 기반으로 제조된, 약물의 효능 또는 독성 평가용 신장 근위 세뇨관 오가노이드(renal proximal tubule organoid)로써, A renal proximal tubule organoid manufactured based on a renal proximal tubule epithelial cell line for evaluating drug efficacy or toxicity.
    상기 오가노이드는 내강(lumen), 및The organoid has a lumen, and
    상기 내강을 감싸고 있는 상피세포층을 포함하고,It includes an epithelial cell layer surrounding the lumen,
    둥근 구상의 입체적 형태인, 신장 근위 세뇨관 오가노이드.Kidney proximal tubular organoids, which are round and spherical in shape.
  2. 제 1항에 있어서,According to clause 1,
    상기 상피세포층은,The epithelial cell layer is,
    Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin 및 F-actin 중 적어도 하나를 포함하는, 신장 근위 세뇨관 오가노이드. A renal proximal tubular organoid containing at least one of Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin.
  3. 제 2항에 있어서,According to clause 2,
    상기 OAT는,The OAT is,
    OAT1, OAT2, OAT3, OAT4, OAT5 및 URAT1 중 적어도 하나를 포함하는, 신장 근위 세뇨관 오가노이드.A kidney proximal tubule organoid comprising at least one of OAT1, OAT2, OAT3, OAT4, OAT5 and URAT1.
  4. 제 2항에 있어서,According to clause 2,
    상기 Na+/K+ ATPase 및 E-cadherin은The Na+/K+ ATPase and E-cadherin are
    약물 처리에 의하여 상기 상피세포의 세포막 또는 세포기질(cytosol)로 이동(translocation) 가능한, 신장 근위 세뇨관 오가노이드. Kidney proximal tubular organoids capable of translocation to the cell membrane or cytosol of the epithelial cells by drug treatment.
  5. 제 1항 내지 제 4항 중 어느 한 항에 기재된 신장 근위 세뇨관 오가노이드에 약물을 처리하는 단계;Treating the kidney proximal tubule organoid according to any one of claims 1 to 4 with a drug;
    상기 약물이 처리된 오가노이드의 Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin 및 F-actin 중 적어도 하나의 바이오마커에 대한 발현 수준에 기초하여 약물의 효능 또는 독성 여부를 결정하는 단계를 포함하는, 신장 근위 세뇨관 오가노이드를 이용한 약물의 평가 방법. Determining the efficacy or toxicity of the drug based on the expression level of at least one biomarker among Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin in the organoid treated with the drug. A method for evaluating a drug using kidney proximal tubule organoids, comprising the steps:
  6. 제 5항에 있어서,According to clause 5,
    상기 결정하는 단계는,The determining step is,
    상기 바이오마커 발현 수준을 약물 미처리군 또는 대조군(control)에서의 바이오마커 발현 수준과 비교하는 단계를 포함하는, 신장 근위 세뇨관 오가노이드를 이용한 약물의 평가 방법. A method for evaluating a drug using kidney proximal tubule organoids, comprising comparing the biomarker expression level with the biomarker expression level in a drug-untreated group or control group.
  7. 제 5항에 있어서,According to clause 5,
    상기 결정하는 단계는,The determining step is,
    상기 약물이 처리된 오가노이드의 상기 Na+/K+ ATPase 또는 E-cadherin의 발현 위치에 기초하여,Based on the expression location of the Na+/K+ ATPase or E-cadherin in the drug-treated organoid,
    약물의 효능 또는 독성 여부를 결정하는 단계를 더 포함하는, 신장 근위 세뇨관 오가노이드를 이용한 약물의 평가 방법.A method for evaluating a drug using kidney proximal tubule organoids, further comprising determining whether the drug is effective or toxic.
  8. 제 1항 내지 제 4항 중 어느 한 항에 기재된 신장 근위 세뇨관 오가노이드에 적어도 하나 이상의 약물을 처리하는 단계;Treating the kidney proximal tubule organoid according to any one of claims 1 to 4 with at least one drug;
    상기 약물이 처리된 오가노이드의 Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin 및 F-actin 중 적어도 하나의 바이오마커에 대한 발현 수준에 기초하여 약물 후보군을 결정하는 단계를 포함하는, 신장 근위 세뇨관 오가노이드를 이용한 약물 스크리닝 방법. Comprising the step of determining drug candidates based on the expression level of at least one biomarker among Na+/K+ ATPase, OAT, E-cadherin, 8-OHdG, Vimentin, and F-actin in the organoid treated with the drug. , Drug screening method using kidney proximal tubule organoids.
  9. 제 8항에 있어서,According to clause 8,
    상기 결정하는 단계는,The determining step is,
    상기 바이오마커 발현 수준을 약물 미처리군 또는 대조군(control)에서의 바이오마커 발현 수준과 비교하는 단계를 포함하는, 신장 근위 세뇨관 오가노이드를 이용한 약물 스크리닝 방법.A drug screening method using kidney proximal tubule organoids, comprising comparing the biomarker expression level with the biomarker expression level in a drug-untreated group or control group.
  10. 제 8항에 있어서,According to clause 8,
    상기 결정하는 단계는,The determining step is,
    상기 약물이 처리된 오가노이드의 상기 Na+/K+ ATPase 또는 E-cadherin의 발현 위치에 기초하여,Based on the expression location of the Na+/K+ ATPase or E-cadherin in the drug-treated organoid,
    약물의 후보군을 결정하는 단계를 더 포함하는, 신장 근위 세뇨관 오가노이드를 이용한 약물 스크리닝 방법.A drug screening method using kidney proximal tubule organoids, further comprising determining drug candidates.
  11. 내강(lumen) 및 상피세포층을 포함하는 신장 근위 세뇨관 오가노이드를 형성하기 위한 제조 방법에 있어서,In the manufacturing method for forming renal proximal tubular organoids comprising a lumen and an epithelial cell layer,
    신장 근위 세뇨관 오가노이드가 형성되도록 신장 근위 세뇨관 상피세포주를 인체 세포외기질(ECM)을 포함하는 제 1 배양 배지에서 제 1 배양하는 단계, 및first culturing the renal proximal tubular epithelial cell line in a first culture medium containing human extracellular matrix (ECM) to form renal proximal tubular organoids, and
    형성된 상기 신장 근위 세뇨관 오가노이드가 성장하도록 상기 오가노이드를 제 2 배양 배지에서 제 2 배양하는 단계를 포함하는, 약물의 효능 또는 독성 평가용 신장 근위 세뇨관 오가노이드 제조 방법.A method of producing kidney proximal tubule organoids for evaluating the efficacy or toxicity of a drug, comprising the step of second culturing the formed kidney proximal tubule organoids in a second culture medium to allow growth of the kidney proximal tubule organoids.
  12. 제 11항에 있어서,According to clause 11,
    상기 ECM은,The ECM is,
    3차원 세포 배양용인, 약물의 효능 또는 독성 평가용 신장 근위 세뇨관 오가노이드 제조 방법.Method for producing kidney proximal tubule organoids for 3D cell culture and for evaluating drug efficacy or toxicity.
  13. 제 11항에 있어서,According to claim 11,
    상기 ECM은,The ECM is,
    섬유아세포 패치에 단백질 분해효소를 처리하고,Treat the fibroblast patch with proteolytic enzymes,
    탈세포화한 후 수득된, 약물의 효능 또는 독성 평가용 신장 근위 세뇨관 오가노이드 제조 방법. Method for preparing kidney proximal tubule organoids obtained after decellularization for evaluating drug efficacy or toxicity.
  14. 제 11항에 있어서, According to clause 11,
    상기 제 1 배양하는 단계는,The first culturing step is,
    약 1시간 내지 3일 중 적어도 하나의 기간 동안 배양하는, 신장 근위 세뇨관 오가노이드 제조 방법.A method of producing kidney proximal tubule organoids, comprising culturing for at least one of about 1 hour to 3 days.
  15. 제 11항에 있어서,According to clause 11,
    상기 ECM은,The ECM is,
    섬유아세포 패치에 단백질 분해효소를 처리하고,Treat the fibroblast patch with proteolytic enzymes,
    탈세포화한 후 수득된, 신장 근위 세뇨관 오가노이드 제조 방법.Method for producing renal proximal tubule organoids obtained after decellularization.
  16. 제 11항에 있어서,According to clause 11,
    상기 신장 근위 세뇨관 상피세포주 및 제 1 배양 배지는,The renal proximal tubule epithelial cell line and the first culture medium,
    1:1 비율로 포함되는, 신장 근위 세뇨관 오가노이드 제조 방법. Method for producing kidney proximal tubule organoids, comprising a 1:1 ratio.
  17. 제 11항에 있어서,According to claim 11,
    상기 제 2 배양하는 단계는,The second culturing step is,
    약 3일 내지 50일 중 적어도 하나의 기간동안 배양하는, 신장 근위 세뇨관 오가노이드 제조 방법.A method of producing kidney proximal tubule organoids, comprising culturing for at least one of about 3 to 50 days.
  18. 제 11항에 있어서,According to claim 11,
    상기 제 2 배양하는 단계는,The second culturing step is,
    약물을 처리하는 단계를 더 포함하는, 신장 근위 세뇨관 오가노이드 제조 방법.A method of producing kidney proximal tubule organoids, further comprising processing a drug.
  19. 제 18항에 있어서,According to clause 18,
    상기 약물을 처리하는 단계는,The step of processing the drug is,
    배양 7일 이내 처음 수행되는, 신장 근위 세뇨관 오가노이드 제조 방법.A method for producing renal proximal tubule organoids, first performed within 7 days of culture.
  20. 신장암 세포주 유래 약물의 효능 또는 독성 평가용 신장암 오가노이드.Kidney cancer organoids for evaluating efficacy or toxicity of drugs derived from kidney cancer cell lines.
  21. 제20항에 있어서,According to clause 20,
    상기 신장암 오가노이드는 Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나 이상을 발현하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드.The kidney cancer organoid is a kidney cancer organoid for evaluating drug efficacy or toxicity, expressing at least one selected from the group consisting of Na+/K+ ATPase, E-cadherin, and Vimentin.
  22. 제20항에 있어서,According to clause 20,
    상기 신장암 오가노이드는 암 활성제 처리에 의해 암 미세환경을 모사하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드.The kidney cancer organoid is a kidney cancer organoid for evaluating drug efficacy or toxicity, characterized in that it simulates a cancer microenvironment by treatment with a cancer activator.
  23. 제22항에 있어서,According to clause 22,
    상기 암 미세환경은 상피중간엽전이(EMT; epithelial to mesenchymal transition) 증가 또는 F-액틴 비정상(F-actin abnormality)인 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드.Kidney cancer organoid for evaluating drug efficacy or toxicity, wherein the cancer microenvironment is characterized by increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
  24. 제20항 내지 제23항 중 어느 한 항에 기재된 약물의 효능 또는 독성 평가용 신장암 오가노이드에 약물을 처리하는 단계; 및Treating a drug to kidney cancer organoids for evaluating the efficacy or toxicity of the drug according to any one of claims 20 to 23; and
    상기 약물이 처리된 오가노이드에 대해 F-액틴 비정상(F-actin abnormality), Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 측정하는 단계;를 포함하는, 신장암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.Measuring the level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin for the organoid treated with the drug; Including, a method for evaluating the efficacy or toxicity of a drug using kidney cancer organoids.
  25. 제24항에 있어서,According to clause 24,
    상기 방법은, Na+/K+ ATPase 또는 E-cadherin 발현 위치를 확인하는 단계;를 더 포함하는, 신장암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.The method further includes the step of confirming the expression location of Na+/K+ ATPase or E-cadherin.
  26. 제24항에 있어서,According to clause 24,
    상기 약물이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality) 또는 Vimentin 수준이 감소하거나 Na+/K+ ATPase 또는 E-cadherin 수준이 세포막(membrane)에서 증가한 경우 약물이 효능을 보이는 것으로 판단하는, 신장암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.For organoids treated with the drug, F-actin abnormality or Vimentin levels were decreased or Na+/K+ ATPase or E-cadherin levels were increased in the cell membrane compared to the untreated group or positive control group. A method of evaluating the efficacy or toxicity of a drug using kidney cancer organoids to determine if the drug is effective.
  27. 제26항에 있어서,According to clause 26,
    상기 판단은 Na+/K+ ATPase 또는 E-cadherin의 수준이 세포질(cytosol)에서 감소한 경우를 더 포함하는 것인, 신장암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.The method of evaluating the efficacy or toxicity of a drug using kidney cancer organoids, wherein the determination further includes a case where the level of Na+/K+ ATPase or E-cadherin is reduced in the cytosol.
  28. 제24항에 있어서,According to clause 24,
    상기 약물이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality) 또는 Vimentin 수준이 증가하거나 Na+/K+ ATPase 또는 E-cadherin 수준이 세포막(membrane)에서 감소한 경우 약물이 독성을 보이는 것으로 판단하는, 신장암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.For organoids treated with the drug, F-actin abnormality or Vimentin levels were increased or Na+/K+ ATPase or E-cadherin levels were decreased in the cell membrane compared to the untreated group or positive control group. A method of evaluating the efficacy or toxicity of a drug using kidney cancer organoids, in which case the drug is judged to be toxic.
  29. 제28항에 있어서,According to clause 28,
    상기 판단은 Na+/K+ ATPase 또는 E-cadherin의 수준이 세포질(cytosol)에서 증가한 경우를 더 포함하는 것인, 신장암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.The method of evaluating the efficacy or toxicity of a drug using kidney cancer organoids, wherein the determination further includes a case where the level of Na+/K+ ATPase or E-cadherin is increased in the cytosol.
  30. 제20항 내지 제23항 중 어느 한 항에 기재된 약물의 효능 또는 독성 평가용 신장암 오가노이드에 항암 후보물질을 처리하는 단계; 및Treating an anti-cancer candidate substance to a kidney cancer organoid for evaluating the efficacy or toxicity of the drug according to any one of claims 20 to 23; and
    상기 항암 후보물질이 처리된 오가노이드 및 항암 후보물질 미처리군에 서 F-액틴 비정상(F-actin abnormality), Na+/K+ ATPase, E-cadherin 및 Vimentin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 비교하는 단계; 를 포함하는, 신장암 오가노이드를 이용한 항암제 스크리닝 방법.The level of at least one biomarker selected from the group consisting of F-actin abnormality, Na+/K+ ATPase, E-cadherin, and Vimentin in the organoid treated with the anticancer candidate material and the group untreated with the anticancer candidate material ( Comparing level); Including, anticancer drug screening method using kidney cancer organoids.
  31. 제30항에 있어서,According to clause 30,
    상기 방법은, Na+/K+ ATPase 또는 E-cadherin 발현 위치를 확인하는 단계;를 더 포함하는, 신장암 오가노이드를 이용한 항암제 스크리닝 방법.The method further includes the step of confirming the expression location of Na+/K+ ATPase or E-cadherin. An anticancer drug screening method using kidney cancer organoids.
  32. 암오가노이드가 형성되도록 신장암 세포주 및 인체 세포외기질(ECM)을 포함하는 배지를 혼합하여 제1 배양하는 단계; 및A first culture step of mixing a medium containing a kidney cancer cell line and human extracellular matrix (ECM) to form cancer organoids; and
    상기 암오가노이드 및 성장 배지를 혼합하여 제2 배양하는 단계;를 포함하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법.A method for producing kidney cancer organoids for evaluating drug efficacy or toxicity, comprising: mixing the cancer organoids and the growth medium and performing a second culture.
  33. 제32항에 있어서,According to clause 32,
    상기 인체 세포외기질은 인체 유래 섬유아세포로부터 수득된 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법.A method for producing kidney cancer organoids for evaluating drug efficacy or toxicity, wherein the human extracellular matrix is obtained from human-derived fibroblasts.
  34. 제32항에 있어서,According to clause 32,
    상기 인체 세포외기질은 인체 유래 섬유아세포 패치에 단백질 분해효소를 처리하고 탈세포화한 후 수득된 3차원 세포배양용 ECM인 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드.The human extracellular matrix is a kidney cancer organoid for evaluating the efficacy or toxicity of a drug, characterized in that the human extracellular matrix is an ECM for three-dimensional cell culture obtained after treating a patch of human-derived fibroblasts with proteolytic enzymes and decellularizing them.
  35. 제32항에 있어서,According to clause 32,
    상기 신장암 세포주 및 인체 세포외기질을 포함하는 배지는 1:1 비율로 포함되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법.A method for producing kidney cancer organoids for evaluating drug efficacy or toxicity, characterized in that the medium containing the kidney cancer cell line and human extracellular matrix is contained in a 1:1 ratio.
  36. 제32항에 있어서, According to clause 32,
    상기 제1 배양하는 단계에서 50um 내지 200um 크기를 갖는 오가노이드가 형성되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법.A method of producing kidney cancer organoids for evaluating drug efficacy or toxicity, characterized in that organoids having a size of 50um to 200um are formed in the first culturing step.
  37. 제32항에 있어서, According to clause 32,
    상기 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간동안 수행되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법.A method of producing kidney cancer organoids for evaluating drug efficacy or toxicity, wherein the first culturing step is performed for at least one of about 1 hour to 3 days.
  38. 제32항에 있어서, According to clause 32,
    상기 제2 배양하는 단계는 약 3일 내지 50일 중 적어도 하나의 기간동안 수행되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법.A method of producing kidney cancer organoids for evaluating drug efficacy or toxicity, wherein the second culturing step is performed for at least one period of about 3 to 50 days.
  39. 제32항에 있어서, According to clause 32,
    상기 제2 배양하는 단계는 약물을 처리하는 단계; 를 더 포함하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법.The second culturing step includes treating a drug; A method for producing kidney cancer organoids for evaluating drug efficacy or toxicity, further comprising:
  40. 제39항에 있어서, According to clause 39,
    상기 약물을 처리하는 단계는 배양 7일 이내 처음 수행되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드 제조방법.A method for producing kidney cancer organoids for evaluating drug efficacy or toxicity, wherein the step of treating the drug is performed for the first time within 7 days of culture.
  41. 신장암 세포주, 인체 세포외기질(ECM) 및 암 활성제 함유 성장 배지를 포함하는, 암 미세환경 모사 신장암 오가노이드 배양용 조성물.A composition for culturing kidney cancer organoids simulating a cancer microenvironment, comprising a kidney cancer cell line, human extracellular matrix (ECM), and a growth medium containing a cancer activator.
  42. 제41항에 있어서In paragraph 41
    상기 인체 세포외기질은 인체 유래 섬유아세포로부터 수득된 것을 특징으로 하는, 암 미세환경 모사 신장암 오가노이드 배양용 조성물.A composition for culturing kidney cancer organoids simulating a cancer microenvironment, wherein the human extracellular matrix is obtained from human-derived fibroblasts.
  43. 제41항에 있어서,According to clause 41,
    상기 인체 세포외기질 및 성장 배지의 부피비는 1 : 4 내지 1 : 6인 것을 특징으로 하는, 암 미세환경 모사 신장암 오가노이드 배양용 조성물.A composition for culturing kidney cancer organoids simulating a cancer microenvironment, characterized in that the volume ratio of the human extracellular matrix and the growth medium is 1:4 to 1:6.
  44. 제41항에 있어서,According to clause 41,
    상기 암 미세환경은 상피중간엽전이(EMT; epithelial to mesenchymal transition) 또는 F-액틴 비정상(F-actin abnormality)이 증가한 것을 특징으로 하는, 암 미세환경 모사 신장암 오가노이드 배양용 조성물.A composition for culturing kidney cancer organoids simulating a cancer microenvironment, wherein the cancer microenvironment is characterized by increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
  45. 제41항 내지 제44항 중 어느 한 항의 조성물로 제조된 암 미세환경 모사 신장암 오가노이드.A cancer microenvironment simulating kidney cancer organoid prepared from the composition of any one of claims 41 to 44.
  46. 제45항의 암 미세환경 모사 신장암 오가노이드가 이종 이식된 동물 모델.An animal model in which the cancer microenvironment-mimicking kidney cancer organoid of item 45 is xenografted.
  47. 갑상선암 세포주 유래 약물의 효능 또는 독성 평가용 갑상선암 오가노이드.Thyroid cancer organoids for evaluating the efficacy or toxicity of drugs derived from thyroid cancer cell lines.
  48. 제47항에 있어서,According to clause 47,
    상기 갑상선암 오가노이드는 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나를 발현하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드.The thyroid cancer organoid is a thyroid cancer organoid for evaluating drug efficacy or toxicity, expressing at least one selected from the group consisting of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. Noid.
  49. 제47항에 있어서,According to clause 47,
    상기 갑상선암 오가노이드는 암 활성제 처리에 의해 암 미세환경을 모사하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드.The thyroid cancer organoid is a thyroid cancer organoid for evaluating drug efficacy or toxicity, characterized in that it simulates a cancer microenvironment by treatment with a cancer activator.
  50. 제49항에 있어서,According to clause 49,
    상기 암 미세환경은 상피중간엽전이(EMT; epithelial to mesenchymal transition) 또는 F-액틴 비정상(F-actin abnormality)이 증가된 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드.A thyroid cancer organoid for evaluating drug efficacy or toxicity, wherein the cancer microenvironment is characterized by increased epithelial to mesenchymal transition (EMT) or F-actin abnormality.
  51. 제47항 내지 제50항 중 어느 한 항에 기재된 약물의 효능 또는 독성 평가용 갑상선암 오가노이드에 약물을 처리하는 단계; Treating a drug to a thyroid cancer organoid for evaluating the efficacy or toxicity of the drug according to any one of claims 47 to 50;
    상기 약물이 처리된 오가노이드에 대해 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 측정하는 단계를 포함하는, 갑상선암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.For organoids treated with the above drug, selected from the group consisting of F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. A method for evaluating the efficacy or toxicity of a drug using thyroid cancer organoids, comprising measuring the level of at least one biomarker.
  52. 제51항에 있어서,According to clause 51,
    상기 방법은, 갑상선 자극 호르몬 수용체(TSHR) 또는 E-cadherin 발현 위치를 확인하는 단계를 더 포함하는, 갑상선암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.The method further includes the step of confirming the expression location of thyroid stimulating hormone receptor (TSHR) or E-cadherin. A method of evaluating drug efficacy or toxicity using thyroid cancer organoids.
  53. 제51항에 있어서,According to clause 51,
    상기 약물이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 및 티로페록시다제(TPO)로 이루어진 군에서 선택된 적어도 하나의 수준이 감소하거나 티로글로불린(Tg) 또는 E-cadherin의 수준이 증가한 경우 약물이 효능을 보이는 것으로 판단하는, 갑상선암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.Organoids treated with the above drugs were selected from the group consisting of F-actin abnormality, thyroid stimulating hormone receptor (TSHR), and thyroperoxidase (TPO) compared to the untreated group or the positive control group. A method for evaluating the efficacy or toxicity of a drug using thyroid cancer organoids, where the drug is judged to be effective when the level of at least one decreases or the level of thyroglobulin (Tg) or E-cadherin increases.
  54. 제53항에 있어서,According to clause 53,
    상기 갑상선 자극 호르몬 수용체(TSHR) 수준이 세포내소포(intracellular vesicle)에서 감소 또는 E-cadherin의 수준이 세포막(membrane)에서 증가한 것인, 갑상선암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.A method for evaluating the efficacy or toxicity of a drug using thyroid cancer organoids, wherein the level of thyroid stimulating hormone receptor (TSHR) is decreased in intracellular vesicles or the level of E-cadherin is increased in the cell membrane.
  55. 제51항에 있어서,According to clause 51,
    상기 약물이 처리된 오가노이드에 대해 약물 미처리군 또는 양성 대조군과 비교하여 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 및 티로페록시다제(TPO)로 이루어진 군에서 선택된 적어도 하나의 수준이 증가하거나 티로글로불린(Tg) 또는 E-cadherin의 수준이 감소한 경우 약물이 독성을 보이는 것으로 판단하는, 갑상선암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.Organoids treated with the above drugs were selected from the group consisting of F-actin abnormality, thyroid stimulating hormone receptor (TSHR), and thyroperoxidase (TPO) compared to the untreated group or the positive control group. A method for evaluating the efficacy or toxicity of a drug using thyroid cancer organoids, where the drug is judged to be toxic when the level of at least one is increased or the level of thyroglobulin (Tg) or E-cadherin is decreased.
  56. 제55항에 있어서,According to clause 55,
    상기 갑상선 자극 호르몬 수용체(TSHR) 수준이 세포내소포(intracellular vesicle)에서 증가 또는 E-cadherin의 수준이 세포막(membrane)에서 감소한 것인, 갑상선암 오가노이드를 이용한 약물의 효능 또는 독성 평가 방법.A method for evaluating the efficacy or toxicity of a drug using thyroid cancer organoids, wherein the level of thyroid stimulating hormone receptor (TSHR) is increased in intracellular vesicles or the level of E-cadherin is decreased in the cell membrane.
  57. 제47항 내지 제50항 중 어느 한 항에 기재된 약물의 효능 또는 독성 평가용 갑상선암 오가노이드에 항암 후보물질을 처리하는 단계; Treating thyroid cancer organoids for evaluating the efficacy or toxicity of the drug according to any one of claims 47 to 50 with an anticancer candidate material;
    상기 항암 후보물질이 처리된 오가노이드 및 항암 후보물질 미처리군에 서 F-액틴 비정상(F-actin abnormality), 갑상선 자극 호르몬 수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 적어도 하나의 바이오마커 수준(level)을 비교하는 단계; 를 포함하는, 갑상선암 오가노이드를 이용한 항암제 스크리닝 방법.In the organoids treated with the above anticancer candidate substances and the anticancer candidate untreated group, F-actin abnormality, thyroid stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO) and Comparing the level of at least one biomarker selected from the group consisting of E-cadherin; Including, anticancer drug screening method using thyroid cancer organoids.
  58. 제57항에 있어서,According to clause 57,
    상기 방법은, 갑상선 자극 호르몬 수용체(TSHR) 또는 E-cadherin 발현 위치를 확인하는 단계를 더 포함하는, 갑상선암 오가노이드를 이용한 항암제 스크리닝 방법.The method further includes the step of confirming the expression location of thyroid stimulating hormone receptor (TSHR) or E-cadherin.
  59. 암오가노이드가 형성되도록 갑상선암 세포주 및 인체 세포외기질(ECM)을 포함하는 배지를 혼합하여 제1 배양하는 단계; 및A first culture step of mixing a medium containing a thyroid cancer cell line and human extracellular matrix (ECM) to form cancer organoids; and
    상기 암오가노이드 및 성장 배지를 혼합하여 제2 배양하는 단계; 를 포함하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법.A second culture step of mixing the cancer organoids and growth medium; Method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity, including.
  60. 제59항에 있어서,According to clause 59,
    상기 인체 세포외기질은 인체 유래 섬유아세포로부터 수득된 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법.A method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity, wherein the human extracellular matrix is obtained from human-derived fibroblasts.
  61. 제59항에 있어서,According to clause 59,
    상기 인체 세포외기질은 인체 유래 섬유아세포 패치에 단백질 분해효소를 처리하고 탈세포화한 후 수득된 3차원 세포배양용 ECM인 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 신장암 오가노이드.The human extracellular matrix is a kidney cancer organoid for evaluating the efficacy or toxicity of a drug, characterized in that the human extracellular matrix is an ECM for three-dimensional cell culture obtained after treating a patch of human-derived fibroblasts with proteolytic enzymes and decellularizing them.
  62. 제59항에 있어서,According to clause 59,
    상기 갑상선암 세포주 및 인체 세포외기질을 포함하는 배지는 1:1 비율로 포함되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법.A method of producing thyroid cancer organoids for evaluating drug efficacy or toxicity, characterized in that the medium containing the thyroid cancer cell line and human extracellular matrix is contained in a 1:1 ratio.
  63. 제59항에 있어서, According to clause 59,
    상기 제1 배양하는 단계에서 50um 내지 200um 크기를 갖는 오가노이드가 형성되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법.A method of producing thyroid cancer organoids for evaluating drug efficacy or toxicity, characterized in that organoids having a size of 50um to 200um are formed in the first culturing step.
  64. 제59항에 있어서, According to clause 59,
    상기 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간동안 수행되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법.A method of producing thyroid cancer organoids for evaluating drug efficacy or toxicity, wherein the first culturing step is performed for at least one of about 1 hour to 3 days.
  65. 제59항에 있어서, According to clause 59,
    상기 제2 배양하는 단계는 약 3일 내지 50일 중 적어도 하나의 기간동안 수행되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법.A method of producing thyroid cancer organoids for evaluating drug efficacy or toxicity, wherein the second culturing step is performed for at least one period of about 3 to 50 days.
  66. 제59항에 있어서, According to clause 59,
    상기 제2 배양하는 단계는 약물을 처리하는 단계; 를 더 포함하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법.The second culturing step includes treating a drug; A method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity, further comprising:
  67. 제66항에 있어서, According to clause 66,
    상기 약물을 처리하는 단계는 배양 7일 이내 처음 수행되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선암 오가노이드 제조방법.A method for producing thyroid cancer organoids for evaluating drug efficacy or toxicity, wherein the drug treatment step is performed for the first time within 7 days of culture.
  68. 갑상선암 세포주, 인체 세포외기질(ECM) 및 암 활성제 함유 성장 배지를 포함하는, 암 미세환경 모사 갑상선암 오가노이드 배양용 조성물.A composition for culturing thyroid cancer organoids simulating a cancer microenvironment, comprising a thyroid cancer cell line, human extracellular matrix (ECM), and a growth medium containing a cancer activator.
  69. 제68항에 있어서In paragraph 68
    상기 인체 세포외기질은 인체 유래 섬유아세포로부터 수득된 것을 특징으로 하는, 암 미세환경 모사 갑상선암 오가노이드 배양용 조성물.A composition for culturing thyroid cancer organoids simulating a cancer microenvironment, wherein the human extracellular matrix is obtained from human-derived fibroblasts.
  70. 제68항에 있어서,According to clause 68,
    상기 인체 세포외기질 및 성장 배지의 부피비는 1 : 4 내지 1 : 6인 것을 특징으로 하는, 암 미세환경 모사 갑상선암 오가노이드 배양용 조성물.A composition for culturing thyroid cancer organoids simulating a cancer microenvironment, characterized in that the volume ratio of the human extracellular matrix and the growth medium is 1:4 to 1:6.
  71. 제68항에 있어서,According to clause 68,
    상기 암 미세환경은 상피중간엽전이(EMT; epithelial to mesenchymal transition) 또는 F-액틴 비정상(F-actin abnormality)이 증가한 것을 특징으로 하는, 암 미세환경 모사 갑상선암 오가노이드 배양용 조성물.The cancer microenvironment is characterized by increased epithelial to mesenchymal transition (EMT) or F-actin abnormality. A composition for thyroid cancer organoid culture simulating a cancer microenvironment.
  72. 제68항 내지 제71항 중 어느 한 항의 조성물로 제조된 암 미세환경 모사 갑상선암 오가노이드.A cancer microenvironment simulating thyroid cancer organoid prepared from the composition of any one of claims 68 to 71.
  73. 제72항의 암 미세환경 모사 갑상선암 오가노이드가 이종 이식된 동물 모델.An animal model in which the cancer microenvironment-mimicking thyroid cancer organoid of item 72 is xenografted.
  74. 갑상선 세포주를 기반으로 제조된, 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드.Thyroid organoids manufactured based on thyroid cell lines for evaluating the efficacy or toxicity of test substances.
  75. 제74항에 있어서,According to clause 74,
    상기 갑상선 오가노이드는 갑상선 호르몬을 분비하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드.The thyroid organoid is a thyroid organoid for evaluating drug efficacy or toxicity, characterized in that the thyroid organoid secretes thyroid hormones.
  76. 제74항에 있어서, According to clause 74,
    상기 갑상선 오가노이드는 약물에 의한 갑상선 호르몬의 수치 변화를 모사한 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드.The thyroid organoid is a thyroid organoid for evaluating drug efficacy or toxicity, characterized in that it simulates changes in thyroid hormone levels caused by drugs.
  77. 제74항에 있어서,According to clause 74,
    상기 갑상선 오가노이드는 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상을 발현하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드.The thyroid organoid is characterized in that it expresses one or more selected from the group consisting of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin, the efficacy of the drug or Thyroid organoids for toxicity assessment.
  78. 오가노이드를 형성하도록 갑상선 세포주 및 인체 세포외기질(ECM)을 포함하는 배지를 혼합하여 제1 배양하는 단계; 및 A first culture step of mixing a medium containing a thyroid cell line and human extracellular matrix (ECM) to form organoids; and
    상기 오가노이드를 갑상선 자극 호르몬(Thyroid-Stimulating Hormone) 및 요오드화 칼륨(Potassium Iodide)을 포함하는 갑상선 오가노이드 배양액과 혼합하여 제2 배양하는 단계; 를 포함하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.A second culture step of mixing the organoid with a thyroid organoid culture medium containing Thyroid-Stimulating Hormone and Potassium Iodide; Method for producing thyroid organoids for evaluating drug efficacy or toxicity, including.
  79. 제78항에 있어서,According to clause 78,
    상기 인체 세포외기질은 3차원 세포배양용 ECM인 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.A method of producing thyroid organoids for evaluating drug efficacy or toxicity, wherein the human extracellular matrix is ECM for three-dimensional cell culture.
  80. 제79항에 있어서,According to clause 79,
    상기 3차원 세포배양용 ECM은 인체 유래 섬유아세포 패치에 단백질 분해효소를 처리하고 탈세포화한 후 수득된 세포외기질(ECM)인 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.The ECM for 3D cell culture is an extracellular matrix (ECM) obtained after treating a patch of human-derived fibroblasts with proteolytic enzymes and decellularizing them. A method of producing thyroid organoids for evaluating drug efficacy or toxicity. .
  81. 제78항에 있어서, According to clause 78,
    상기 제1 배양하는 단계는 약 1시간 내지 3일 중 적어도 하나의 기간 동안 배양하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.The first culturing step is characterized in that culturing for at least one period of about 1 hour to 3 days, a method of producing thyroid organoids for evaluating drug efficacy or toxicity.
  82. 제78항에 있어서In paragraph 78
    상기 제2 배양하는 단계는 약 3일 내지 50일 중 적어도 하나의 기간동안 배양하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.The second culturing step is a method of producing thyroid organoids for evaluating drug efficacy or toxicity, characterized in that culturing for at least one period of about 3 to 50 days.
  83. 제78항에 있어서In paragraph 78
    상기 제2 배양하는 단계에서 약물을 처리하는 단계; 를 더 포함하는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.treating a drug in the second culturing step; A method for producing thyroid organoids for evaluating drug efficacy or toxicity, further comprising:
  84. 제83항에 있어서In paragraph 83
    상기 약물을 처리하는 단계는 배양 7일 이내 처음 수행되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.A method of producing thyroid organoids for evaluating drug efficacy or toxicity, wherein the drug treatment step is performed for the first time within 7 days of culture.
  85. 제78항에 있어서,According to clause 78,
    상기 인체 세포외기질은 3차원 세포배양용 ECM인 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.A method of producing thyroid organoids for evaluating drug efficacy or toxicity, wherein the human extracellular matrix is ECM for three-dimensional cell culture.
  86. 제78항에 있어서,According to clause 78,
    상기 갑상선 세포주 및 인체 세포외기질을 포함하는 배지는 1:1 비율로 포함되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.A method of producing thyroid organoids for evaluating drug efficacy or toxicity, characterized in that the medium containing the thyroid cell line and human extracellular matrix is contained in a 1:1 ratio.
  87. 제78항에 있어서,According to clause 78,
    상기 갑상선 자극 호르몬은 상기 갑상선 오가노이드 배양액 총 부피에 대하여 0.01 내지 1 mU/mL 농도로 포함되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.A method of producing a thyroid organoid for evaluating drug efficacy or toxicity, wherein the thyroid stimulating hormone is contained at a concentration of 0.01 to 1 mU/mL based on the total volume of the thyroid organoid culture medium.
  88. 제78항에 있어서,According to clause 78,
    상기 요오드화 칼륨은 상기 갑상선 오가노이드 배양액 총 부피에 대하여 1 내지 20nM 농도로 포함되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가용 갑상선 오가노이드 제조방법.A method of producing a thyroid organoid for evaluating drug efficacy or toxicity, wherein the potassium iodide is contained at a concentration of 1 to 20 nM relative to the total volume of the thyroid organoid culture medium.
  89. 갑상선 세포주, 인체 세포외기질(ECM)를 포함하는 배지, 갑상선 자극 호르몬(Thyroid-Stimulating Hormone) 및 요오드화 칼륨(Potassium Iodide)을 포함하는, 약물의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물.A culture composition for producing thyroid organoids for evaluating the efficacy or toxicity of a drug, comprising a thyroid cell line, a medium containing human extracellular matrix (ECM), Thyroid-Stimulating Hormone, and Potassium Iodide.
  90. 제89항에 있어서,According to clause 89,
    상기 갑상선 세포주 및 인체 세포외기질을 포함하는 배지는 1:1 비율로 포함되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물.A culture composition for producing thyroid organoids for evaluating drug efficacy or toxicity, characterized in that the medium containing the thyroid cell line and human extracellular matrix is contained in a 1:1 ratio.
  91. 제89항에 있어서,According to clause 89,
    상기 인체 세포외기질(ECM)은 3차원 세포배양용 ECM인 것을 특징으로 하는, 약물의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물.The human extracellular matrix (ECM) is a culture composition for producing thyroid organoids for evaluating drug efficacy or toxicity, wherein the human extracellular matrix (ECM) is ECM for three-dimensional cell culture.
  92. 제89항에 있어서In paragraph 89
    상기 갑상선 자극 호르몬은 상기 조성물 총 부피에 대하여 0.01 내지 1 mU/mL 농도로 포함되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물.A culture composition for producing thyroid organoids for evaluating drug efficacy or toxicity, wherein the thyroid-stimulating hormone is contained at a concentration of 0.01 to 1 mU/mL based on the total volume of the composition.
  93. 제89항에 있어서,According to clause 89,
    상기 요오드화 칼륨은 상기 조성물 총 부피에 대하여 1 내지 20nM 농도로 포함되는 것을 특징으로 하는, 약물의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물.A culture composition for producing thyroid organoids for evaluating drug efficacy or toxicity, wherein the potassium iodide is contained at a concentration of 1 to 20 nM based on the total volume of the composition.
  94. 제89항에 있어서,According to clause 89,
    상기 조성물은 시험관내에서 갑상선 호르몬을 분비하는 갑상선 오가노이드 제조용인 것을 특징으로 하는, 약물의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물.A culture composition for producing thyroid organoids for evaluating the efficacy or toxicity of a drug, characterized in that the composition is used for producing thyroid organoids that secrete thyroid hormones in vitro.
  95. 제89항에 있어서,According to clause 89,
    상기 조성물은 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상을 발현하는 갑상선 오가노이드 제조용인 것을 특징으로 하는, 약물의 효능 또는 독성 평가를 위한 갑상선 오가노이드 제조용 배양 조성물.The composition is a drug for producing thyroid organoids expressing at least one selected from the group consisting of thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), thyroperoxidase (TPO), and E-cadherin. Culture composition for producing thyroid organoids for evaluating efficacy or toxicity.
  96. 제89항 내지 제95항 중 어느 한 항의 조성물로 제조된 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드에 목적하는 시험물질을 처리하는 단계; 및Processing the test substance of interest to a thyroid organoid for evaluating the efficacy or toxicity of the test substance prepared with the composition of any one of claims 89 to 95; and
    상기 시험물질이 처리된 군을 시험물질 미처리군 또는 양성 대조군과 비교하여 트리이오드티로닌(T3), 테트라이오드티로닌(T4), 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상의 바이오마커 증감에 따라 시험물질의 효능을 판단하는 단계; 를 포함하는, 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드를 이용한 시험물질의 효능 평가 방법.Triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyrophe were compared with the group treated with the test substance or the positive control group. Determining the efficacy of the test substance according to the increase or decrease of one or more biomarkers selected from the group consisting of oxidase (TPO) and E-cadherin; A method for evaluating the efficacy of a test substance using thyroid organoids for evaluating the efficacy or toxicity of the test substance, including.
  97. 제89항 내지 제95항 중 어느 한 항의 조성물로 제조된 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드에 목적하는 시험물질을 처리하는 단계; 및Processing the test substance of interest to a thyroid organoid for evaluating the efficacy or toxicity of the test substance prepared with the composition of any one of claims 89 to 95; and
    상기 시험물질이 처리된 군을 시험물질 미처리군 또는 양성 대조군과 비교하여 트리이오드티로닌(T3), 테트라이오드티로닌(T4), 갑상선자극호르몬수용체(TSHR), 티로글로불린(Tg), 티로페록시다제(TPO) 및 E-cadherin으로 이루어진 군에서 선택된 1종 이상의 바이오마커 증감에 따라 시험물질의 독성을 판단하는 단계; 를 포함하는, 시험물질의 효능 또는 독성 평가용 갑상선 오가노이드를 이용한 시험물질의 독성 평가 방법.Triiodothyronine (T3), tetraiodothyronine (T4), thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyrophe were compared with the group treated with the test substance or the positive control group. Determining the toxicity of the test substance according to the increase or decrease of one or more biomarkers selected from the group consisting of roxidase (TPO) and E-cadherin; A method for evaluating the toxicity of a test substance using thyroid organoids for evaluating the efficacy or toxicity of the test substance, including.
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