CN116179470A - High-speed and mass preparation method of liver organoids and method for screening drug efficacy and toxicity by using same - Google Patents

High-speed and mass preparation method of liver organoids and method for screening drug efficacy and toxicity by using same Download PDF

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CN116179470A
CN116179470A CN202211489353.3A CN202211489353A CN116179470A CN 116179470 A CN116179470 A CN 116179470A CN 202211489353 A CN202211489353 A CN 202211489353A CN 116179470 A CN116179470 A CN 116179470A
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liver
differentiation
cell aggregate
dimensional cell
cells
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金南衡
韩东旭
郭浩
姚雪瑞
金哲龙
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Guangdong Ogan Neud Biotechnology Co ltd
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Guangdong Ogan Neud Biotechnology Co ltd
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Abstract

The invention relates to a high-speed mass preparation method of liver organoids, a method for screening liver disease related drugs by using the method, a method for screening in-vitro toxicity of drugs and the like. According to the preparation method, the liver organoids can be prepared in large quantity at high speed by continuously carrying out three-dimensional differentiation process on the same microplate. The liver organoids prepared by the above preparation method comprise hepatocytes (hepatic core parts), cholangiocytes (cholangiocytes), hepatic stellate cells (hepatic stellate cell), cooper cells (kupffer cells) and the like, have cell compositions, structures and functionalities similar to those of actual liver tissues, and can be stably proliferated in vitro. In addition, by utilizing the method for screening the in-vitro toxicity of the liver organoid, the medicine possibly having liver toxicity can be screened in advance, the development time and cost of new medicine are shortened, and the efficiency of the new medicine is improved.

Description

High-speed and mass preparation method of liver organoids and method for screening drug efficacy and toxicity by using same
Technical Field
The invention relates to a high-speed mass preparation method of liver organoids, a screening method of liver related disease drugs, a screening method of drug in-vitro toxicity and the like.
Background
The liver is an organ with multiple functions such as metabolism of substances, synthesis of various proteins such as cholesterol and bile acid, regulation and storage of glycine, and decomposition of toxic substances. Liver disease is one of the 5 leading causes of death among the korean population, and is the second most common disease among men aged 40 to 50 who have the most active social activity, and is the second most common disease. In fact, korea has the highest mortality rate due to liver diseases such as hepatitis, liver cancer, cirrhosis, etc., and has a nursing wage expenditure rate of 3550 billion korea per year, which is about 166 tens of thousands of people in practice, in the economy-co-ordination and development Organization (OECD) country.
The liver is a representative organ with an intrinsic regenerative capacity in the body. Animal models and tumor-derived hepatocytes are widely used in studies such as liver disease mechanism studies and therapeutic drug development, but in vivo-derived primary human hepatocytes (primary human hepatocytes, PHHs) have been evaluated as the most suitable model so far due to human hepatocytes, genetic and physiological differences and limited functionality. However, in vivo derived primary human hepatocytes have various limitations, difficulty in vitro culture, loss of proliferation capacity and functionality upon in vitro culture, and extremely limited availability. Thus, there is a continuing need to create new customized hepatocyte models for human cell-based patients with stable in vitro proliferation capacity and high functionality.
To overcome various limitations of primary human hepatocytes of in vivo origin, studies were conducted using hepatocytes of totipotent stem cell (pluripotent stem Cell, PSC) origin including embryonic stem cells (Embryonic stem cell, ESC), induced pluripotent stem cells (Induced pluripotent stem cell, iPSC). However, hepatocytes (embryonic stem cells or induced stem cell-derived differentiated hepatocytes, cross-differentiated hepatocytes, etc.) prepared by stem cell technology have limited possibilities of use due to limited in vitro proliferation in reverse and functionality different from that of actual liver tissue.
As a recently useful alternative source, liver organoids (organoids) generated from totipotent stem cells (PSCs) are of great interest. Organoids are a novel stem cell differentiation technology which uses the differentiation (differentiation), self-regeneration (self-tissue), and self-tissue (self organization) of stem cells, reproduces the composition and structure of cells similar to that of organs in vivo by three-dimensional culture, and can be used for disease simulation research of various diseases and therapeutic drug screening research by simulating the environment similar to that of organs in vivo. It is known that recently developed liver organoids can be stably cultured in vitro for a long period of time, and exhibit cell composition, structure, functionality at a level similar to that of actual liver tissue, and can be used for in vitro disease modeling research of next-generation liver diseases and new drug development research.
However, to date, liver organoids are mostly prepared using two-dimensional differentiation and three-dimensional differentiation together, and standardized techniques are difficult to develop and liver organoids are difficult to prepare in large quantities, with fatal limitations.
Therefore, the invention optimizes the whole differentiation process of the liver organoid, develops a method for preparing the liver organoid in a three-dimensional way, and determines a method for preparing a large number of liver three-dimensional cell aggregates which can continuously and non-oscillatingly perform the whole process on the same micro-pore plate at high speed based on the method, thereby completing the invention.
Advanced technical literature: patent literature: (patent document 1) KR 10-2107057.
Disclosure of Invention
Problems to be solved:
the technical problem to be achieved by the present invention is to provide a high-efficiency, high-function, high-speed mass production method consisting only of differentiated liver three-dimensional cell aggregates and a liver three-dimensional cell aggregate produced by the above production method.
Another technical problem to be achieved by the present invention is to provide a culture reagent for culturing the above-mentioned liver three-dimensional cell aggregate.
Another technical problem to be solved by the present invention is to provide the above-mentioned liver three-dimensional cell aggregate. The method for screening the liver-related disease drugs by utilizing the liver three-dimensional cell aggregate is provided, and the method for providing information for liver-related disease treatment and the method for screening in-vitro toxicity of the drugs are provided.
However, the technical problems to be achieved by the present invention are not limited to the above-described problems, and other problems not mentioned are clearly understood by those skilled in the art from the following description.
The method for solving the problems comprises the following steps:
in order to solve the above problems, in a first aspect, the present invention provides a high-speed mass production method of a three-dimensional cell aggregate of liver, comprising the steps of:
(1) A step of forming embryoid bodies (embryoid bodies) from stem cells isolated from human beings;
(2) Inducing the embryo body to differentiate into liver tissue to form a liver three-dimensional cell aggregate;
(3) And a step of further differentiating the liver three-dimensional cell aggregate.
Wherein, the steps (1) to (3) are continuous non-shaking culture in the same microplate.
As another example of the present invention, in step (1), the initial starting cell number at which the embryoid body can be formed is 50 to 20,000, preferably 50 to 1,000, preferably 250.
In another embodiment of the present invention, in step (2), the liver three-dimensional cell aggregate is formed within at least 20 to 30 days, and about 20 days.
In another embodiment of the invention, stem cells isolated from humans include totipotent stem cells (pluripotent stem Cell, PSC), adult stem cells (adult stem cells); totipotent stem cells include embryonic stem cells (human embryonic stem cell, hESC), induced pluripotent stem cells (induced pluripotent stem cell, iPSC); adult stem cells include adult stem cells of in vivo origin and adult stem cells prepared by the cross differentiation technique.
In another embodiment of the present invention, after the formation of the liver three-dimensional cell aggregate, further shaking is performed, and long-term culture is possible.
In another embodiment of the invention, the microplate is a multi-well microplate with recesses, preferably 96-well or 384-well microplates.
In another embodiment of the present invention, the step of forming a three-dimensional cell aggregate of the liver includes an endodermal differentiation step or a hepatoblast differentiation step.
In another embodiment of the present invention, the endoderm differentiation step comprises differentiating into any one of the following endoderm: definitive endoderm (definitive endoderm, DE), foregut Endoderm (FE) and hindforegut endoderm (posterior foregut endoderm, PFE).
In another embodiment of the present invention, the step of forming a three-dimensional cell aggregate of the liver may be one or more times of Matrigel (Matrigel) addition in an endodermal differentiation step or a hepatoblast differentiation step. Preferably, matrigel is added to the anterior foregut endoderm differentiation step, the posterior foregut endoderm differentiation step and the hepatoblast differentiation step at a final concentration of 0.1 to 1% by volume, respectively; more specifically, matrigel is added to the culture medium, instead of being embedded (embedding means embedding organoids within matrigel), the final concentration of 0.1-1% means that the matrigel is 0.1-1% by volume in the culture medium.
In another embodiment of the present invention, the embryoid bodies are formed by adding KSR (serum replacement) 20%, FBS (fetal bovine serum) 3%, glutamax (L-glutamine) 1%, P/S (penicillin/streptomycin solution) 1%, Y27632 (ROCK inhibitor) 10. Mu.M and bFGF (basic fibroblast growth factor) 10ng/mL to DMEM/F12 medium.
In another embodiment of the invention, the definitive endoderm differentiation step is 1 day in RPMI1640 medium with 1% B27-VA (vitamin A type B27 additive removed), 1% P/S1%, 1% NEAA (non-essential amino acids), 200ng/mL activin A (activin A), 10ng/mL BMP4 (bone morphogenetic protein 4), 10. Mu.M LY294002 (PI 3K inhibitor) and 3. Mu.M CHIR99021 (GSK 3 inhibitor) and 3 days in RPMI1640 medium containing B27-VA1%, P/S1%, NEAA1% and 200 ng/mL.
In another embodiment of the invention, the foregut endoderm differentiation step is carried out by adding B27-VA1%, P/S1%, NEAA1%, matrigel 0.5-1%, FGF4 (fibroblast growth factor 4) 500ng/mL and 1. Mu.M dorsomorphin (dihydrodeoxymorphine) to RPMI1640 medium for 3 days.
In another embodiment of the present invention, the posterior foregut endoderm differentiation step is 2 days of culture with the addition of B27-VA1%, P/S1%, NEAA1%, matrigel 0.5-1%, FGF4 500ng/mL and Retinoic Acid (RA) 2. Mu.M in RPMI1640 medium.
In another embodiment of the present invention, the step of differentiating the hepatic blast is to add B27-VA1%, P/S1%, NEAA1%, matrigel 0.5-1%, BMP 4ng/mL and bFGF 5ng/mL to RPMI1640 medium for 3 days.
In another embodiment of the present invention, the steps of forming the liver three-dimensional cell aggregate are adding FBS 10%, NEAA1%, glutamax 1%, ITS (insulin-transferrin-selenium medium supplement) 0.1%, dexamethasone (Dexamethasone) 10 mu M, HGF (hepatocyte growth factor) 50ng/mL, OSM (Oncoinhibin M) 20ng/mL, EGF (epidermal growth factor) 50ng/mL, nicotinamide (nicotinamide) 0.5% and A83-01 (TGF-. Beta.inhibitor) 4ng/mL to DMEM/F12 medium for 6-10 days.
In another embodiment of the present invention, the step of further differentiating the liver three-dimensional cell aggregate is performed in a maintenance medium (EM), a differentiation medium (differentiation medium, DM) or a mixed medium thereof, and may be cultured in the differentiation medium for 10 to 15 days.
In another embodiment of the present invention, the maintenance medium (EM) is supplemented with B27% 1, N2 cell culture supplement 1%, glutamax 1%, HGF 25ng/mL, EGF 50ng/mL, N-Acetylcysteine (N-Acetylcysteine) 1mM, gastrin (Gastrin) 10nM, forskolin (Forskolin) 10. Mu. M, A83-01. Mu. M, nicotinamide 10.10 mM, OSM 10ng/mL, bFGF 10ng/mL and ITS 5. Mu.g/mL in DMEM/F12 medium. The Differentiation Medium (DM) was prepared by adding B27.sup.1%, N2 1%, glutamax 1%, HGF 25ng/mL, EGF 50ng/mL, N-acetylcysteine 1mM, gastin 10nM, A83-01.5. Mu. M, DAPT (gamma secretase inhibitor) 10. Mu. M, BMP7 (bone morphogenetic protein 7) 25ng/mL to DMEM/F12 medium.
In another embodiment of the invention, the definitive endoderm differentiation step is treated with 50 to 200ng/mL activin a, preferably 200ng/mL activin a.
In another embodiment of the invention, the foregut endoderm differentiation step is treated with 50 to 200ng/mL noggin, preferably 200ng/mL noggin or 1 μm dorsomorphin for 24 to 72 hours, preferably 72 hours.
In another embodiment of the invention, the posterior foregut endoderm differentiation step is treated with 0.5 to 2 μm of Retinoid Acid (RA), preferably with 2 μm of retinoid acid for 24 to 72 hours, preferably for 48 hours.
In another embodiment of the invention, the hepatoblast differentiation step is treated with 5 to 50ng/mL bFGF, preferably with 5ng/mL; treatment with 10 to 20ng/mL BMP4, preferably 20ng/mL.
Herein, "%" is used to indicate the concentration of a particular substance. Unless otherwise indicated, solids/solids are (weight/weight)%, solids/liquid are (weight/volume)%, and liquid/liquid are (volume/volume)%.
As another embodiment of the present invention, the expression of the genes OCT4, NANOG is decreased and the expression of the genes SOX17, EOMES, CXCR4, FOXA2 may be increased during the definitive endoderm differentiation step. In the foregut endoderm differentiation step, the expression of the gene SOX2 may be increased and the expression of the gene CDX2 may be suppressed. Expression of the genes HNF1B, HNF and oneut 2 may increase during the posterior foregut endoderm differentiation step. In the above-described hepatic cell differentiation step, the expression of the genes CK19, EPCAM, AFP, LGR, ALB, TTR, SOX9, A1AT, HNF4A may be increased.
In a second aspect, the present invention provides a three-dimensional cell aggregate of liver prepared by the high-speed mass production method of the first aspect.
In a third aspect, the present invention provides a culture reagent for culturing the liver three-dimensional cell aggregate according to the second aspect, the culture reagent comprising a concave culture section for culturing the liver three-dimensional cell aggregate, and a cover section for covering the concave culture section. In a specific embodiment of the present invention, the recessed culture part is, for example, a microplate body, and the covering part covering the recessed culture part is, for example, a corresponding microplate cover. Specifically, the depressed culture section and the cover section covering the depressed culture section may be a covered microplate for culturing organoids, preferably 96-well or 384-well covered microplates.
In one embodiment of the present invention, the culture reagent may include a medium for culturing the above-described liver three-dimensional cell aggregate. According to a specific embodiment of the invention, the medium comprises one or more media used in the method of the first aspect described above.
In one embodiment of the invention, the culture reagent may comprise a preservation solution.
In a fourth aspect, the present invention provides a method of screening for a drug related to a liver-related disorder, comprising the steps of:
(1) A step of preparing and culturing a three-dimensional cell aggregate of liver from cells derived from a patient suffering from a liver-related disease using the method according to the first aspect;
(2) Contacting a candidate substance with the liver three-dimensional cell aggregate;
(3) And (2) determining a drug for treating a liver-related disease based on the three-dimensional cell aggregate of the liver contacted with the candidate substance in the step (2).
In one embodiment of the present invention, in the drug screening method of the fourth aspect, step (3) includes detecting cell viability (cell oxygen consumption rate (Oxygen Consumption Rate, OCR), measuring expression level of biomarkers of liver-related diseases, and the like, and judging, screening, evaluating drugs for treating liver-related diseases based on the detection.
In another embodiment of the invention, the liver-related disorder comprises at least any one of the following: hepatitis virus, simple steatosis, non-alcoholic fatty liver disease, liver inflammation, non-alcoholic steatohepatitis (NASH), cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure and liver cancer.
In a fifth aspect, the present invention provides a method for screening for drug toxicity in vitro using a three-dimensional cell aggregate of the liver, comprising the steps of:
(1) In any one or more steps of the method for high-speed mass production of a three-dimensional cell aggregate of liver according to the first aspect, contacting a candidate substance with the three-dimensional cell aggregate;
(2) Comparing the reaction caused by the presence and absence of the candidate substance with the liver three-dimensional cell aggregate in each of the above steps;
(3) Identifying whether or not the cells in the liver three-dimensional cell aggregate die.
In one embodiment of the invention, in the screening method of the fifth aspect, the cells in step (3) include, but are not limited to, one or more of the following: hepatocytes (hepatocytocytocys), cholangiocytes (cholangioytes), hepatic stellate cells (Hepatic stellate cell), and cooper cells (Kupffer cells).
The invention has the advantages that:
the invention relates to a high-speed and mass preparation method of liver organoids, a method for screening liver related disease drugs by using the organoids, a method for screening in-vitro toxicity of drugs and the like. According to the above preparation method, a large number of liver organoids having a cell composition, structure and function similar to those of an actual liver tissue can be prepared at a high speed through a three-dimensional differentiation process on the same microplate, and the prepared liver organoids have hepatocytes (hepatocytes), cholangiocytes (cholangiocytes), hepatic stellate cells (hepatic stellate cell), cooper cells (kupffer cells) and the like. In addition, the in vitro toxicity screening method of liver organoids used in the invention can pre-screen drugs with liver toxicity, so as to shorten the development time and cost of new drugs and improve the efficiency of the new drugs.
Drawings
FIG. 1 shows a preparation method of a liver organoid consisting of only three-dimensional differentiation processes, which includes processes of Embryoid Body (EB) formation, definitive Endoderm (DE) differentiation, foregut Endoderm (FE) differentiation, hindgut endoderm (PFE) differentiation, hepatoblast (HB) differentiation, and hepatocyte maturation, etc., unlike a conventional preparation method of two-dimensional differentiation from human stem cells to hepatocytes, all of which are completed by three-dimensional culture, and shows a high-speed, mass-preparation process of a liver organoid by a continuous non-shaking culture method.
FIG. 2 shows the definitive endoderm differentiation step, with different numbers of starting cells and activin A concentrations resulting in expression of SOX17, EOMES and CXCR4 genes. In fig. 2, hESCs: embryonic stem cells.
FIG. 3 shows SOX2 and CDX2 gene expression at various concentrations of Noggin (NOG) or Dihydrooxymorpholin (DOR) in the foregut endoderm differentiation step for 24, 48, 72 hours. In fig. 3, CHIR: CHIR99021 (GSK 3 inhibitor); IWP2: wnt/β -catenin inhibitors.
FIG. 4 shows the expression of HNF1B, HNF and ONECUT2 genes at different concentrations of Retinoic Acid (RA) for 24, 48 and 72 hours in the posterior foregut endoderm differentiation step. SB431542: TGF-beta/Smad inhibitors.
FIG. 5 shows the expression of CK19, EPCAM, AFP, LGR5, ALB and TTR genes treated with bFGF and BMP4 at different concentrations during the hepatoblast differentiation step.
FIG. 6 shows the expression of specific genes of liver organoids prepared under optimized differentiation conditions according to the present invention in an undifferentiated step, a Definitive Endoderm (DE) differentiation step, a Foregut Endoderm (FE) differentiation step, a Posterior Foregut Endoderm (PFE) differentiation step and a Hepatoblast (HB) differentiation step. In fig. 6, hESC: embryonic stem cells.
FIG. 7 shows the reproducibility results of verification of optimized differentiation conditions using other embryonic stem cell lines H9 and induced stem cell lines hiPSC line 1 (hiPSC line 1), hiPSC line 2 (hiPSC line 2) in addition to embryonic stem cell (hESCs) line H1.
FIG. 8 shows the preparation efficiency of liver organoids prepared under differentiation conditions optimized in the present invention and the preparation ratio of liver organoids of different forms. In fig. 8, LOs: liver organoids (liver organs); cyst LOs: a saccular liver organoid; solid LOs: solid liver organoids.
FIG. 9 compares the gene expression between liver organoids (Cyst) including cys and Solid, liver organoids consisting of Solid only (Solid), and embryonic stem cells (hESCs).
FIG. 10 shows the results of subculture of liver organoids (hereinafter referred to as "cys liver organoids") comprising cys and Solid (cys LOs) and liver organoids consisting of Solid only (Solid LOs).
FIG. 11 shows the results of confirming stable in vitro proliferation of cys liver organoids.
FIG. 12 shows the results of viability assay when cyst liver organoids were frozen and thawed.
FIG. 13 shows the expression of ALB, SOX9, A1AT, HNF4A, EPCAM, LGR5 genes in cys liver organoids. In fig. 13, DAPI:4', 6-diamidino-2-phenylindole; ZO-1: claudin 1 (zonula occludens 1); action: actin.
FIG. 14 shows the gene expression level of mature cyst liver organoids in a Differentiation Medium (DM) by qPCR. In fig. 14, DM: differentiation medium (differentiation medium); EM: the culture medium (Expansion medium) was maintained.
FIG. 15 shows the gene expression of mature cyst liver organoids in Differentiation Medium (DM) by the immunohistochemical staining technique. In fig. 15, DAPI:4', 6-diamidino-2-phenylindole; ZO-1: claudin 1 (zonula occludens 1).
FIG. 16 shows the results of indocyanine green uptake analysis (ICG uptake assay) and PAS staining (snow periodate staining) to determine exogenous compound metabolism and glycogen storage capacity of mature cys liver organoids. In fig. 16, DM: differentiation culture solution; EM: maintaining the culture solution.
FIG. 17 shows the results of ELISA analysis to determine CYP3A4 activity, total bile Acid (ALB) production, albumin (ALB) and urea secretion of mature cys liver organoids.
FIG. 18 shows the expression of specific genes of the capillary membranes such as MRP2 and BSEP in mature cys liver organoids. In fig. 18, DAPI:4', 6-diamidino-2-phenylindole; action: actin.
FIG. 19 shows the results of differentiation step morphology of liver organoids prepared by three-dimensional serial culture in 96-well plates in Foregut Endoderm (FE) and Hepatoblasts (HB).
FIG. 20 shows the results of liver organoid morphological analysis of different starting cell numbers prepared by three-dimensional serial culture in 96-well plates.
FIG. 21 shows the effect of different initial cell numbers on liver organoid preparation efficiency prepared by three-dimensional serial culture in 96-well plates.
FIG. 22 shows the effect of matrigel on liver organoids prepared by three-dimensional serial culture in 96-well plates.
FIG. 23 shows the morphology differences of liver organoids according to the concentration of matrigel when they were prepared by three-dimensional continuous culture in 96-well plates.
FIG. 24 is the results of morphology and gene expression of a large number of prepared cyst liver organoids when cultured in maintenance medium (EM), differentiation Medium (DM) and 1:1 mixed medium of EM and DM, respectively, according to example 2.1.
FIG. 25 is a comparison of ALB, A1AT, TTR, HNF4A, AFP, SOX, CK19 gene expression of mature high-volume Liver organoids (M Liver) prepared according to example 2.2 and mature cys Liver organoids (T Liver), hepatocyte strains HepG2 prepared according to example 1.3. In fig. 25, hESC: embryonic stem cells; t Liver: traditional liver organoids (Typical liver organoid); m Liver: mini liver organoids (Micro liver organoid).
FIG. 26 analyzes the composition of liver organoid cells prepared in large numbers by maturation, and shows cell-specific genes in hepatocytes (hepatoc sections) and cholangiocytes (cytostatic sections).
FIG. 27 shows the results of immunohistochemical staining performed to confirm the presence or absence of hepatic stellate cells (hepatic stellate cell) in the matured and extensively prepared liver organoids. In fig. 27, DAPI:4', 6-diamidino-2-phenylindole; alpha-SMA: alpha-smooth muscle actin.
FIG. 28 shows the morphological changes of two-dimensional (2D) differentiated hepatocytes and cys liver organoids upon treatment with hepatotoxic drugs.
FIG. 29 shows the toxic response caused by hepatotoxic drugs in 2D differentiated hepatocytes and cys liver organoids. Results were validated at the molecular biology level by TUNEL (terminal deoxynucleotidyl transferase mediated dUTP notch end tag, terminal deoxynucleotidyl transferase dUTP nick end labeling) staining.
FIG. 30 compares the response of small, medium, and high doses of hepatotoxic drug treatment in 2D differentiated hepatocytes (2D heps) and cys Liver Organoids (LOs).
Detailed Description
The existing liver organoid preparation technology comprises the following various technical problems: 1) Random and spontaneous (uncontrolled) preparation of two-and three-dimensional differentiation processes of uncontrolled liver organoids; 2) Other culture formats such as transfer from the original 96-well plate to a 6cm dish (dish); 3) Through the process of embedding matrigel; 4) Use of an expensive differentiation-promoting factor R-spondin; 5) Low differentiation efficiency and reproducibility; 6) Without a large number of preparation systems, etc., it is difficult to develop a standardized preparation system.
In order to solve the technical problems, the preparation process of the liver organoid is standardized, and in order to prepare the liver organoid in a large quantity at a high speed, the invention develops the preparation technology of the liver organoid which is only composed of a three-dimensional differentiation process, and further carries out non-shaking culture on a micro-pore plate, so as to prepare the liver organoid in a large quantity at a high speed with the efficiency close to 100%.
More specifically, the full differentiation process from human totipotent stem cells to liver organoids, namely Embryoid Body (EB) formation, definitive endoderm (definitive endoderm, DE) differentiation, foregut Endoderm (FE) differentiation, posterior foregut endoderm (posterior foregut endoderm, PFE) differentiation, liver organoids maturation, etc., was optimized, and on this basis, three-dimensional differentiation process was continuously performed on the same microwell plate to prepare a large amount of liver organoids, and finally completed the present invention (fig. 1).
In the present invention, the "three-dimensional cell aggregate" may be an organoid, and the term "organoid" in the present invention refers to a three-dimensional cell aggregate formed by self-regeneration and self-organization from adult stem cells, embryonic stem cells, induced stem cells, and the like, which are derived from human liver tissue or are prepared by various stem cell techniques such as cross differentiation. Unlike two-dimensional culture, three-dimensional cell culture can be in vitro to grow cells in any direction, and the organoids can be used for simulating in vivo interaction organs, for development of medicines for treating diseases, and the like. Specifically, by constructing organoids from the tissues of a patient, disease modeling based on patient genetic information can be performed, drug screening can be performed by trial and error, and the like.
In the present invention, the term "stem cell" means a cell having the ability to self-replicate and differentiate into two or more cells, and may be classified into a universal stem cell (totipotent stem cell), a totipotent stem cell (pluripotent stem cell), a multi-differentiated stem cell (multipotent stem cell), and the like.
In the present invention, the term "medium" refers to a medium for proliferation, survival and support of differentiation of in vitro (in vitro) liver organoids, including conventional media suitable for liver organoid culture and differentiation used in the art. The kind of the medium and the culture conditions may be appropriately selected according to the kind of the cells. In particular, the medium may generally comprise a Cell Culture Minimal Medium (CCMM) comprising a carbon source, a nitrogen source and trace element components. The minimal medium for cell culture included: DMEM (Dulbecco's Modified Eagle's Medium), F-10, F-12, DMEM/F12, advanced DMEM/F12, alpha-MEM (alpha-Minimal essential Medium), IMDM (Iscove's Modified Dulbecco), BME (Basal Medium Eagle), RPMI1640, etc., but are not limited thereto.
The terminology used in the embodiments is for the purpose of description only and is not to be interpreted in a limiting sense. The singular reference is to be construed to include the singular and the plural unless the context clearly dictates otherwise. The terms "comprises" and "comprising" in this specification are to be taken to specify the presence of stated features, integers, steps, operations, elements, components, or groups thereof, but do not preclude the presence or addition of one or more other features or integers, steps, operations, elements, components, or groups thereof.
Unless defined differently, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one having ordinary knowledge in the technical field to which the embodiments belong. Terms commonly used as previously defined should be construed in the related art to have a consistent meaning and should not be interpreted as being ideal or excessively as formal unless explicitly defined herein.
Hereinafter, embodiments are described in detail with reference to the accompanying drawings. However, various modifications may be made to the embodiments, and thus the scope of the patent application is not limited or restricted by these embodiments. All modifications, equivalents, and alternatives to the embodiments are to be understood as included within the scope of the claims.
In the description with reference to the drawings, the same reference numerals are given to the same components regardless of the graphic symbols, and duplicate descriptions are omitted. In describing the embodiments, if it is considered that a detailed description of the related art will unnecessarily obscure the abstract of the embodiments, a detailed description thereof will be omitted.
Examples
Example 1 liver organoids preparation consisting of three-dimensional differentiation only
1.1 optimizing the conditions of the differentiation steps for the preparation of liver organoids
The optimization of differentiation conditions from human stem cells to liver organoids, namely Embryoid Body (EB) formation, definitive endoderm (definitive endoderm, DE) differentiation, foregut Endoderm (FE) differentiation, posterior foregut endoderm (posterior foregut endoderm, PFE) differentiation and liver organoid maturation, are as follows.
To optimize Embryoid Body (EB) formation and Definitive Endoderm (DE) differentiation conditions, induced differentiation of a number of different starting cell numbers (1X 10) 3 、5×10 3 1X 10 4 ) And activin A (activin A) concentrations (0, 50, 100, 200 ng/mL), and expression of Definitive Endoderm (DE) specific genes SOX17, EOMES, and CXCR4 was confirmed under each condition. The results show that for Definitive Endoderm (DE) differentiation, the number of starting cells is 1X 10 4 In addition, the highest differentiation efficiency was seen in the highest expression level when treated with 200ng/mL of activin A (FIG. 2).
For Foregut Endoderm (FE) differentiation conditions, mid-hindgut differentiation factor BMP inhibitor noggin (noggin) was treated at various concentrations (50, 100, 200 ng/mL) for 24, 48, 72 hours, and expression of the hind foregut endoderm gene CDX2 and foregut endoderm gene SOX2 was confirmed under each condition. As a result, when noggin was treated at 200ng/mL for 72 hours, the amount of SOX2 expressed was the highest, and CDX2 expression was the most inhibited. In addition, the small molecule compound dorsomorphin (dihydrooxymorphine) induced a higher SOX2 expression level than noggin when it was treated instead of noggin, confirming that dorsomorphin could be used instead of noggin during Foregut Endoderm (FE) differentiation, with 1 μm dorsomorphin exhibiting the highest expression level when it was treated (fig. 3).
In order to optimize the conditions for differentiation of the hind foregut endoderm (PFE), retinoic Acid (RA) was treated at various concentrations (0.5, 1, 2 μm) for 24, 48, 72 hours as an important factor for differentiation of the hind foregut endoderm (PFE), and the expression of the hind foregut endoderm (PFE) genes NF1B, NF and oneut 2 was confirmed under each condition. As a result, it was found that the expression level was highest and the differentiation efficiency was highest when 2. Mu.M RA was treated for 48 hours for the purpose of differentiating the postintestinal endoderm (PFE) (FIG. 4).
To optimize the Hepatoblast (HB) differentiation conditions, treatments were performed with varying concentrations of basic fibroblast growth factor (bFGF) (0, 5, 50 ng/mL) and bone morphogenic protein 4 (BMP 4) (0, 10, 20 ng/mL), and the expression of CK19, EPCAM, AFP, LGR5, ALB and TTR genes was determined under each condition. The results showed that the expression level was highest when treated with bFGF of 5ng/mL and BMP4 of 20ng/mL, and the differentiation efficiency was highest (FIG. 5).
After preparing liver organoids according to the above optimized differentiation conditions, the expression of the genes of each step was confirmed by qPCR and immune tissue staining. The results showed that the specific genes were correctly activated or inactivated in each step when the undifferentiated step, definitive Endoderm (DE) differentiation step, foregut Endoderm (FE) differentiation step, hindgut endoderm (PFE) differentiation step, and Hepatoblast (HB) differentiation step were performed (FIG. 6).
To verify reproducibility of the differentiation conditions optimized in the present invention, liver organoids were prepared using other embryonic stem cell lines H9 hESC and induced stem cell lines hiPSC cell line 1, hiPSC cell line 2 in addition to the embryonic stem cell line H1 hESC. As a result, the same was confirmed in other cell lines, which indicates that the optimized differentiation conditions of the present invention are reproducible (FIG. 7).
1.2 characterization of liver organoids prepared according to optimized differentiation conditions
Using the optimized differentiation conditions of the present invention, liver organoids were prepared with 100% efficiency, wherein 90% of the liver organoids were liver organoids (cysts) in the form of packed bile duct cells (hepatomegaids) centered on hepatocytes (hepatomegaids), and 10% were liver organoids (Solid-state liver organoids) consisting of only hepatocytes (hepatomegates) (FIG. 8).
The liver organoids in the form of Cyst packages (hereinafter referred to as "Cyst liver organoids") and the liver organoids composed only of solid did not differ much in gene expression (fig. 9), but the liver organoids composed only of solid were difficult to proliferate at the time of subculture (fig. 10). In contrast, cyst liver organoids were stable to proliferation in vitro and remained stable for more than 6 months (-10 passages) upon physical or chemical subculture (FIG. 11). Therefore, in subsequent experiments, cyst liver organoids were used.
In the case of repeatedly freezing and thawing the cys liver organoid 3 times or more, the cys liver organoid did not lose the ability to proliferate in vitro, remained stable (fig. 12), and the prepared liver organoid was confirmed to express ALB, SOX9, A1AT, HNF4A, EPCAM, LGR and other genes by the tissue immunostaining technique (fig. 13). That is, the liver organoids prepared under the above optimized differentiation conditions exhibit typical liver organoid structural characteristics, can be stably proliferated in vitro, and are useful for large-scale screening of drugs requiring a large number of liver organoids, development of new drugs, and the like.
1.3 liver organoids maturing
The prepared Cyst liver organoids were induced to mature for 1 week in differentiation medium (differential medium, DM) and compared with the gene expression of immature Cyst liver organoids cultured in maintenance medium (expression medium, EM) by qPCR and immunohistochemical staining techniques. The expression level of various hepatocyte, cholangiocyte, and preadipocyte specific genes was higher in the DM-derived cys liver organoids, i.e., mature cys liver organoids, than in the EM-derived immature cys liver organoids (fig. 14 and 15).
To verify the in vitro functionality of mature Cyst liver organoids, ICG uptake assays (xenobiotic metabolism) and PAS staining (glycogen storage) were performed, and CYP3A4 activity, total bile acid (total bile acid) production, albumin (albumin) and urea (urea) secretion were confirmed by ELISA assays. The results showed that mature Cyst liver organoids had improved exogenous metabolism and glycogen storage capacity (fig. 16) and also improved CYP3A4 activity, total bile acid production, albumin and urea secretion capacity (fig. 17) compared to immature Cyst liver organoids. The result shows that the Cyst liver organoid of the invention has mature function and sufficient in vitro functionality.
Bile production is the main function of the liver, and bile produced by hepatocytes is delivered to the bile duct through a thin-walled tubular structure, such as a bile capillary (bile duct). The marker genes of the capillary bile duct membranes such as MRP2, BSEP and the like appear in the mature Cyst liver organoids of the present invention, which shows that the mature Cyst liver organoids of the present invention are similar in structure to actual liver tissues (FIG. 18).
EXAMPLE 2 preparation of liver organoids continuously three-dimensionally differentiated on the same microplate
2.1 method for preparing liver organoids in large quantities
Example 1 was started from an initial 96-well plate, but from the Definitive Endoderm (DE) step, transferred to a 6 cm dish and had to use the matrigel embedding (Matrigel drop Embedding) procedure, which included the limitations of the current organoid technology.
In order to establish a unified full-cycle differentiation technique from organoid preparation to drug screening, it is necessary to establish a continuous culture method without the need for transfer from a multi-well plate such as a 96-well plate to another culture dish or shaking culture method. In addition, matrigel embedding on 96-well plates is difficult to achieve in reality, and therefore development of omitted or alternative methods is required. Recently, some researchers reported a method for preparing various organ-specific organoids using homemade microwell plates and the like. The preparation of organoid technology using existing commercially available microplates suitable for high throughput drug screening and high content imaging is very necessary in the commercialization segment.
Thus, the present invention developed a preparation method that can continuously culture liver organoids in 96-well plates that were initially seeded with cells without shaking culture.
Embryonic stem cells (hESCs) isolated as single cells were seeded in 96-well plates with a starting cell number of 1X 10 2 、2.5×10 2 、1×10 3 、2.5×10 3 5×10 3 And differentiation was induced in steps under optimized conditions in example 1. The morphology of aggregates among the initial cell numbers was observed in the Foregut Endoderm (FE) differentiation step and the Hepatoblast (HB) differentiation step, and the results showed that spherical aggregates were confirmed to be formed in all cases although there was a difference in size (fig. 19).
However, in the liver organogenesis step, very different morphologies are presented depending on the number of starting cells. If 1X 10 is used 3 More than one starting cell, few normal liver organoids with Cyst morphology (1X 10) 3 ) Or not formed at all (2.5X10) 3 、5×10 3 ). In addition, the initial cell number was 1X 10 2 、2.5×10 2 In the case of (a), a typical liver organoid differentiation state occurs (fig. 20).
From the viewpoint of liver organoid preparation efficiency, the starting cell number was 2.5X10 2 When used, the liver organoid preparation efficiency was 100%, and liver organoids of typical Cyst morphology were observed in all wells of the 96-well plate. On the other hand, the efficiency is relatively low under other conditions, at 5×10 3 Under these conditions, no typical morphology of liver organoids was formed (fig. 21).
Unlike the conventional method of performing matrigel embedding, when mass production is performed by continuous non-shaking culture of stem cells, a method of directly adding matrigel to a culture solution is used (fig. 1). Matrigel with final concentration of 1% was treated in the steps of Foregut Endoderm (FE), hindforegut endoderm (PFE), hepatoblast (HB), the concentration of matrigel refers to the final volume percentage concentration of matrigel in the culture medium. A significant increase in liver organoid growth rate and Cyst formation efficiency was observed for all differentiation steps with matrigel treatment compared to untreated populations (fig. 22).
In the three-dimensional continuous non-shaking culture, matrigel (0%, 0.5%, 1%, 2.5%, 5%) at different concentrations was treated in the anterior endoderm (FE), posterior Foreendoderm (PFE), and Hepatoblast (HB) steps, respectively, to confirm the effect of matrigel, and the effects were compared. As a result, liver organogenesis in the form of Cyst was not observed in the case of no matrigel treatment or high concentration (2.5%, 5%), whereas liver organoids in the form of Cyst were efficiently formed in the case of low concentration (0.5%, 1%) (fig. 23). Furthermore, in the case of 0.5% or 1% treatment, formation of liver organoids in the form of cys was observed in the Hepatoblast (HB) step, and it was confirmed that the addition of matrigel at a low concentration directly to the culture broth was more effective in the formation of liver organoids in the form of cys (fig. 23).
2.2 optimization of culture conditions for Mass prepared liver organoids
In example 2.1, in order to optimize the culture conditions of the liver organoids prepared in large quantities, culture was performed in maintenance medium (EM), differentiation Medium (DM), and 1:1 mixed medium of EM and DM, respectively. As a result, in liver organoids cultured in a Differentiation Medium (DM) (hereinafter, simply referred to as "maturing and mass-producing liver organoids"), the expression level of most of specific genes was high (FIG. 24). The above results indicate that functional maturation is possible by culturing a large number of prepared liver organoids in a Differentiation Medium (DM).
2.3 Properties of maturation for Mass production of liver organoids
The gene expression profile of the mature high-volume Liver organoids of example 2.2 (mini Liver organoids, micro Liver organoid, M lever) was compared to that of the mature Cyst Liver organoids of example 1.3 (traditional Liver organoids, typical Liver organoid, T lever) and the hepatocyte strain HepG2 (a 2D Liver cancer cell line, typically compared as a control to 3D Liver organoid functionality). The results demonstrate that most of the specific genes are more highly expressed in the mass production of liver organoids by maturation of example 2.2 (FIG. 25). This shows that the maturation of the present invention produces a greater number of liver organoids than the liver organoids produced by the prior art.
To understand the cellular composition of mature, mass prepared liver organoids, an immune tissue staining analysis was performed. Morphological analysis confirmed that the liver organoids had both hepatocyte Solid (hepatocytocytocytocytocyto) and cholangiocyte cys (cytostic part, cholanophytes). The actual immunohistological staining results confirmed that the hepatocellular fraction of the mature high-volume liver organoids expressed ALB, sodium taurocholate cotransporter (NTCP), etc., whereas the cholangiocyte fraction did not express ALB, NT CP (fig. 26). In addition, the immune tissue staining technique is utilized to verify that non-parenchymal cells, namely hepatic stellate cells (hepatic stellate cell) and cooper cells (kupffer cells), in the actual liver tissue exist in the mature large-scale preparation liver organoids. The results showed that hepatic stellate cell specific gene α -smooth muscle actin (α -SMA), WT1, vimentin (Vimentin) were expressed and the expression of the cooper cell specific gene CD68 was confirmed in the mass preparation of liver organoids 20-40 days of differentiation (fig. 27). The above results indicate that the liver organoids prepared in large quantities by maturation can have a cellular composition (hepatocytes, cholangiocytes, hepatic stellate cells, cooper cells, etc.) similar to that of the actual liver tissue during short-term differentiation.
Example 3 in vitro toxicity screening technique based on liver organoids
Basic in vitro toxicity risk studies were performed using liver organoids of the present invention. For this reason, in two-dimensional (2D) differentiated hepatocytes and the Cyst liver organoids of example 1.2 of the present invention, the low frequency hepatotoxic drugs thioridazine Hydrochloride (HCL) and chloroquine diphosphate (chloroquine diphosphate), cyclosporin A (cyclosporin A), naftoprazone (nefazodone), perhexiline maleate (Rac-Perhexiline mal egene), sulindac (sulindac), troglitazone (troglitazone), tamoxifen (tamoxifen) which were withdrawn from the market due to hepatotoxicity were compared and analyzed for their toxic response. Morphological analysis was performed on 2D differentiated hepatocytes and Csyt liver organs after 24 hours of drug treatment at different concentrations. After treatment with 6 drugs (nefazodone, thioridazine HCL, rac-perhexiline maleate, chloroquine diphosphate, troglitazone, tamoxifen), cell death of 2D differentiated hepatocytes and reduction in the size of the cys liver organoids were observed, and morphological changes were not observed in the cycloporin a and sulindac treated groups (fig. 28).
To verify the toxic response of each drug at the molecular biology level, TUNEL (terminal deoxynucleotidyl transferase mediated dUTP notch end tag, terminal deoxynucleotidyl transferase dUTP nick end labelin g) staining was performed. As a result, most drugs showed more sensitive toxic reactivity in Cyst liver organoids than 2D differentiated hepatocytes (fig. 29). Cyclosporin A showed more sensitive toxic reactivity in liver organoids at all concentrations, nefazodone, thioridazine HCL, rac-Perhexiline maleate, sulindac and chloroquine diphosphate showed more sensitive toxic reactions in liver organoids than two-dimensional differentiated hepatocytes at either low or high concentrations.
Further, no toxicity could be observed if treated with rosiglitazone (rosiglitazone) and buspirone (buspirone), which are structurally similar to troglitazone and nefazodone but non-toxic, indicating that the level of toxicity of the drug could be accurately predicted by the Cyst liver organoids (fig. 28 and 29).
As a result, it was confirmed that Cyst liver organoids had more accurate and more sensitive toxic responses than two-dimensional differentiated hepatocytes (FIG. 30). The above results indicate that not only the Cyst liver organoids of the present invention, which have a cell composition similar to that of actual liver tissue, but also the mass preparation of liver organoids by maturation can be used as an in vitro model for evaluating toxicity of drug candidate substances, and have usability.
As described above, although the embodiments are illustrated by the limited drawings, various technical modifications and variations can be applied on the basis of the above description if a person having ordinary knowledge in the art. For example, the techniques may be performed in a different order than the methods, and elements of the systems, structures, devices, circuits, etc. may be combined or combined in a different manner than the methods, or replaced or substituted with other components or articles to achieve suitable results.
Accordingly, other embodiments and equivalents to the claims are intended to be within the scope of the invention as claimed.

Claims (27)

1. The high-speed mass preparation method of the liver three-dimensional cell aggregate is characterized by comprising the following steps of:
(1) A step of forming embryoid bodies from stem cells isolated from humans;
(2) A step of inducing differentiation from the embryoid body into liver tissue, forming a three-dimensional cell aggregate of the liver for at least 20 to 30 days;
(3) A step of further differentiating the liver three-dimensional cell aggregate;
wherein, the steps (1) to (3) are continuous non-shaking culture on the same microplate.
2. The method of claim 1, wherein in step (1), the formed embryoid bodies are placed in an initial starting cell number of 50 to 1000.
3. The method of claim 1, wherein the human isolated stem cells are totipotent stem cells or adult stem cells.
4. The method according to claim 1, wherein the liver three-dimensional cell aggregate is further subjected to shaking culture after formation, and can be cultured for a long period of time.
5. The method of claim 1, wherein the microplate is a notched multi-microplate.
6. The method of claim 1, wherein the step of forming a three-dimensional cell aggregate of the liver comprises an endodermal differentiation step or a hepatoblast differentiation step.
7. The method of claim 6, wherein the endodermal differentiation step is selected from any one or more endodermal differentiation of definitive endodermal, foregut endodermal, hindgut endodermal.
8. The method according to claim 6, wherein in the step of forming the liver three-dimensional cell aggregate, matrigel is added at a volume ratio of 0.1 to 1% one or more times in the step of endodermal differentiation or hepatoblast differentiation.
9. The method of claim 1, wherein the step of forming the embryoid body is performed on a medium comprising Y27632 and bFGF.
10. The method of claim 7, wherein the definitive endoderm differentiation step is performed on a medium comprising CHIR99021, LY294002, activin a and BMP 4.
11. The method of claim 7, wherein the foregut endoderm differentiation step is carried out on a medium comprising dihydrooxymorphine and FGF 4.
12. The method of claim 7, wherein the posterior foregut endoderm differentiation step is performed on a medium comprising FGF4 and retinoic acid.
13. The method of claim 6, wherein the step of differentiating the hepatic blast is performed on a medium comprising BMP4 and bFGF.
14. The method of claim 1, wherein the step of forming the liver three-dimensional cell aggregate is performed on a medium comprising ITS, dexamethasone, HGF, oncostatin M, EGF, nicotinamide, and a 83-01.
15. The method of claim 1, wherein the step of further differentiating the liver three-dimensional cell aggregate is performed on a medium comprising HGF, EGF, N-acetylcysteine, gastrin, a83-01, DAPT, BMP7, FGF 19.
16. The method of claim 7, wherein in the definitive endoderm differentiation step, 50 to 200ng/mL activin a is treated.
17. The method of claim 7, wherein in the foregut endoderm differentiation step, 50 to 200ng/mL of noggin or 1 μΜ dihydrooxymorphine is treated for 24 to 72 hours.
18. The method of claim 7, wherein in the posterior foregut endoderm differentiation step, 0.5 to 2 μm retinoic acid is treated for 24 to 72 hours.
19. The method of claim 6, wherein in the step of differentiating the liver blast cells, 5 to 50ng/mL bFGF and 10 to 20ng/mL BMP4 are used.
20. A liver three-dimensional cell aggregate, characterized in that the liver three-dimensional cell aggregate is produced according to the high-speed mass production method of a liver three-dimensional cell aggregate according to claim 1.
21. A culture reagent for culturing the liver three-dimensional cell aggregate according to claim 20, comprising a concave culture part of the liver three-dimensional cell aggregate, and a cover part covering the concave culture part.
22. The culture reagent of claim 21, further comprising a preservation solution.
23. A method for screening a drug for liver-related diseases, comprising the steps of:
(1) A step of preparing a three-dimensional cell aggregate of liver from cells derived from a patient suffering from a liver-related disease by the method according to claim 1;
(2) Contacting a candidate substance with the liver three-dimensional cell aggregate;
(3) Screening and evaluating liver related disease drugs according to the condition that the candidate substances contact the liver three-dimensional cell aggregate.
24. The method according to claim 23, wherein in the step (3), the liver-related disease drug is selected and evaluated by detecting cell survival rate, oxygen consumption rate or expression level of liver-related disease biomarker.
25. The method of claim 23, wherein the liver-related disorder is selected from at least one of: hepatitis virus, simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure, and liver cancer.
26. A method for screening for in vitro toxicity of a drug comprising the steps of:
(1) A step of contacting a candidate substance with the three-dimensional cell aggregate in any one or more steps of the method of claim 1;
(2) Comparing the reaction caused by the presence or absence of the candidate substance with the liver three-dimensional cell aggregate in each of the above steps;
(3) And a step of identifying whether cells in the liver three-dimensional cell aggregate die.
27. The method according to claim 26, wherein in the step (3), the cells are selected from any one or more of hepatocytes, cholangiocytes, hepatic stellate cells, and cooper cells.
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