WO2024091043A1 - Organoïde tubulaire proximal rénal, organoïde de cancer du rein, organoïde de thyroïde ou organoïde de cancer de la thyroïde, procédé de préparation associé et procédé d'évaluation de médicament les utilisant - Google Patents

Organoïde tubulaire proximal rénal, organoïde de cancer du rein, organoïde de thyroïde ou organoïde de cancer de la thyroïde, procédé de préparation associé et procédé d'évaluation de médicament les utilisant 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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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/ko
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Publication of WO2024091043A1 publication Critical patent/WO2024091043A1/fr

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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

La présente invention concerne un organoïde tubulaire proximal rénal, un organoïde de cancer du rein, un organoïde de thyroïde ou un organoïde de cancer de la thyroïde, un procédé de préparation associé et un procédé d'évaluation de médicament les utilisant.
PCT/KR2023/016802 2022-10-26 2023-10-26 Organoïde tubulaire proximal rénal, organoïde de cancer du rein, organoïde de thyroïde ou organoïde de cancer de la thyroïde, procédé de préparation associé et procédé d'évaluation de médicament les utilisant WO2024091043A1 (fr)

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KR1020230144228A KR20240058792A (ko) 2022-10-26 2023-10-25 세포주 기반 갑상선암 오가노이드, 이의 제조방법 및 이를 활용한 약물 평가 방법
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KR1020230144100A KR20240058788A (ko) 2022-10-26 2023-10-25 신장 근위 세뇨관 오가노이드, 이의 제조 방법 및 이를 이용한 약물 평가 방법
KR1020230144099A KR20240058787A (ko) 2022-10-26 2023-10-25 세포주 기반 갑상선 오가노이드, 이의 제조방법 및 이의 용도
KR10-2023-0144228 2023-10-25
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