Methods for constructing liver organoids and their applications

Abstract

The present invention discloses a method for constructing liver organoids and their application. The liver organoids constructed by the present invention contain hepatocytes and non-parenchymal cells, can maintain typical liver functions, have the ability to transplant, survive and repair damaged liver tissue in mice, can well simulate fatty degeneration and inflammatory response, and simulate the pathological process and mechanism of ALD, and have broad application prospects in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for constructing a liver organoid and its application. Background Art

[0002] The human liver is an important organ that provides many important metabolic functions for life, such as lipid metabolism, ammonium and bile production, coagulation, and detoxification of exogenous compounds. However, related liver diseases such as viral hepatitis, alcoholic fatty liver, liver cancer, etc. have caused a heavy social burden and seriously threatened human life and health. Therefore, in order to reveal the mechanism of occurrence and development of liver diseases and develop treatment methods for liver diseases, it is of great significance to establish a liver research model that is highly relevant to the human body.

[0003] Liver organoids are an emerging model for liver research that share key features of the human liver, such as cell composition and functional features such as albumin synthesis, glycogen synthesis, and lipid accumulation. They are widely used in basic research, drug development, and other fields. Compared with traditional animal models, organoid models do not have species differences and have apparent metabolic capabilities that are highly relevant to the human body. Compared with two-dimensional culture models, they have advantages such as a cell microenvironment similar to that in vivo, liver tissue-specific cell populations, and the ability to simulate the interaction between parenchymal cells and non-parenchymal cells.

[0004] At present, organoid construction mostly uses the method of directly embedding large pieces of hydrogel. This method affects the supply of nutrients and oxygen, resulting in problems such as low organoid production efficiency and poor uniformity. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides a method for constructing a liver organoid and its application.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a hAHOs liver organoid culture medium, which comprises a basal culture medium, a serum replacement, FGF, N-acetylcysteine, a Wnt agonist, nicotinamide, gastrin, a mitogen-activated protein kinase (MAPK), HGF, and forskolin.

[0008] Furthermore, the basal culture medium is selected from Advanced DMEM / F12.

[0009] Furthermore, the serum replacement includes B27 and / or N2.

[0010] Furthermore, the FGF is selected from FGF10.

[0011] Furthermore, the Wnt agonist includes Wnt protein and R-spo.

[0012] Furthermore, the Wnt agonist is selected from R-spo.

[0013] Furthermore, the R-spo is selected from R-spo-1.

[0014] Furthermore, the mitogenic proliferation factors include EGF, BDNF, and KGF.

[0015] Furthermore, the mitogenic proliferation factor is selected from EGF.

[0016] Furthermore, the concentration of B27 is 2%.

[0017] Furthermore, the concentration of N2 is 1%.

[0018] Furthermore, the concentration of FGF10 is 100 ng / ml.

[0019] Furthermore, the concentration of the N-acetylcysteine ​​is 1.25 mM.

[0020] Furthermore, the concentration of R-spo-1 is 20 ng / ml.

[0021] Furthermore, the concentration of nicotinamide is 10 mM.

[0022] Furthermore, the concentration of gastrin is 10 nM.

[0023] Furthermore, the concentration of EGF is 50 ng / ml.

[0024] Furthermore, the concentration of HGF is 25 ng / ml.

[0025] Furthermore, the concentration of forskolin is 10 μM.

[0026] The second aspect of the present invention provides a method for constructing hAHOs liver organoids, the method comprising culturing HLCs using the culture medium described in the first aspect of the present invention.

[0027] Furthermore, the HLCs are induced from stem cells.

[0028] Further, the culture medium for inducing stem cells to form HLCs includes: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, a fifth culture medium, and a sixth culture medium.

[0029] The first culture medium comprises: a basal culture medium, SP,

[0030] The second culture medium comprises: basal culture medium, Activin, BSA, SP,

[0031] The third culture medium comprises: basal culture medium, Activin, BSA, SP, ITS,

[0032] The fourth culture medium comprises: BSA, ITS, BMP, FGF, SP,

[0033] The fifth culture medium comprises: BSA, ITS, HGF, SP,

[0034] The sixth culture medium includes: BSA, ITS, IL-6 family cytokines, DEX, HGF, and SP.

[0035] Furthermore, the basal culture medium is selected from Advanced DMEM / F12.

[0036] Furthermore, the Activin is selected from ActivinA.

[0037] Furthermore, the BMP is selected from BMP2.

[0038] Furthermore, the FGF is selected from FGF4.

[0039] Furthermore, the IL-6 family cytokine is selected from OSM.

[0040] Furthermore, the stem cells are selected from mesenchymal stem cells.

[0041] Furthermore, the mesenchymal stem cells are selected from adipose-derived mesenchymal stem cells.

[0042] Furthermore, the concentration of the SP is 1%.

[0043] Furthermore, the concentration of Activin A is 100 ng / ml.

[0044] Furthermore, the BSA is 10% BSA 0.5 mg / ml.

[0045] Furthermore, the concentration of the ITS is 1%.

[0046] Furthermore, the concentration of BMP2 is 20 ng / ml.

[0047] Furthermore, the concentration of FGF4 is 30 ng / ml.

[0048] Furthermore, the concentration of HGF is 20 ng / mL.

[0049] Furthermore, the concentration of OSM is 10 ng / mL.

[0050] Furthermore, the concentration of DEX is 1 μM.

[0051] Furthermore, the method of producing hALOs liver organoids comprises:

[0052] a) culturing the stem cells using a first culture medium,

[0053] b) the cells obtained in step a) are transferred to a second culture medium for culture, and the cells are induced to develop into endoderm,

[0054] c) the endoderm cells obtained in step b) are transferred to a third culture medium for induction into ventral foregut endoderm,

[0055] d) the ventral foregut endoderm cells obtained in step c) are transferred into a fourth culture medium and differentiated into liver precursors,

[0056] e) transferring the liver progenitor cells obtained in step d) into the fifth culture medium to induce hepatocytes,

[0057] f) the hepatocytes obtained in step e) are transferred to the sixth culture medium for culture, and mature hepatocytes are induced to obtain HLCs,

[0058] g) After digestion, the HLCs obtained in step f) are transferred to the hAHOs hepatic organoid culture medium described in the first aspect of the present invention for culture.

[0059] Furthermore, the method further comprises co-culturing the cells with the ECM after step g).

[0060] Furthermore, the culturing time of steps a) and c) is 48 hours.

[0061] Furthermore, the culturing time in step b) is 24 hours.

[0062] Furthermore, the culturing time of steps d), e) and f) is 5 days.

[0063] Furthermore, the culturing time in step g) is 24 hours.

[0064] Furthermore, in step g), trypsin is used to digest the HLCs.

[0065] Furthermore, the ECM includes BME and Matrigel.

[0066] Further, the ECM is selected from Matrigel.

[0067] Furthermore, the culture conditions were 37°C, 5% CO2, and 95% air.

[0068] The third aspect of the present invention provides a hALOs liver organoid culture medium, the culture medium comprising: culture medium I and culture medium II,

[0069] The culture medium I comprises: a basic culture medium, a serum replacement, RA,

[0070] The culture medium II includes: HCM, HGF, Dex, and IL-6 family cytokines.

[0071] Furthermore, the basal culture medium is selected from Advanced DMEM / F12.

[0072] Furthermore, the serum replacement includes B27 and / or N2.

[0073] Furthermore, the IL-6 family cytokine is selected from OSM.

[0074] Furthermore, the concentration of RA is 2 μM.

[0075] The fourth aspect of the present invention provides a method for constructing hALOs liver organoids, the method comprising culturing hEPCs using the culture medium described in the third aspect of the present invention.

[0076] Furthermore, the hEPCs are induced from stem cells.

[0077] Furthermore, the culture medium for inducing stem cells to form hEPCs includes the first culture medium, the second culture medium, and the third culture medium described in the second aspect of the present invention.

[0078] Furthermore, the stem cells are selected from mesenchymal stem cells.

[0079] Furthermore, the mesenchymal stem cells are selected from adipose-derived mesenchymal stem cells.

[0080] Furthermore, the method for constructing hALOs liver organoids comprises:

[0081] a) culturing the stem cells using a first culture medium,

[0082] b) the cells obtained in step a) are transferred to a second culture medium for culture, and the cells are induced to develop into endoderm,

[0083] c) the endoderm cells obtained in step b) are transferred to a third culture medium for culture, and induced toward the ventral foregut endoderm to obtain hEPCs,

[0084] d) hEPCs are digested and cultured using the medium I described in the third aspect of the present invention,

[0085] e) Add medium II for culture.

[0086] Furthermore, the culturing time of steps a) and c) is 48 hours.

[0087] Furthermore, the culturing time in step b) is 24 hours.

[0088] Furthermore, the culturing time of steps d) and e) is 5 days.

[0089] Furthermore, in step d), hEPCs are digested using trypsin.

[0090] The fifth aspect of the present invention provides a liver organoid, which is constructed by the method described in the second aspect of the present invention or the fourth aspect of the present invention.

[0091] The sixth aspect of the present invention provides a transplant material, which comprises the liver organoid described in the fifth aspect of the present invention.

[0092] The seventh aspect of the present invention provides a method for evaluating the pharmacokinetics or toxicity of a test substance using the liver organoid described in the fifth aspect of the present invention.

[0093] Furthermore, the method includes: contacting the test substance with the liver organoid, and measuring or evaluating the metabolism, absorbability, membrane permeability, drug interaction, induction of drug metabolizing enzymes or induction of drug transporters, or toxicity of the test substance.

[0094] The eighth aspect of the present invention provides any one of the following applications:

[0095] (1) Use of the liver organoids described in the fifth aspect of the present invention in constructing a liver disease model;

[0096] (2) Use of the liver organoids described in the fifth aspect of the present invention in the study of the pathogenesis or pathogenic factors of liver disease;

[0097] (3) Use of the liver organoids described in the fifth aspect of the present invention in screening drugs for preventing and / or treating liver disease or improving at least one symptom or pathological manifestation of liver disease;

[0098] (4) Application of the liver organoids described in the fifth aspect of the present invention in regenerative medicine.

[0099] Furthermore, the liver disease model described in (1) is an ALD model.

[0100] Advantages and beneficial effects of the present invention:

[0101] The liver organoids constructed by the present invention contain hepatocytes and liver matrix lineages, can maintain typical liver functions, have the ability to colonize and reconstruct damaged liver tissue in mice, can well simulate fatty liver degeneration and inflammatory response, and simulate the pathological process and mechanism of ALD, and have broad application prospects in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1Figure 1 is a diagram of the establishment of the hAHOs system, wherein 1A is a phase contrast microscopy image of hAHOs at different stages (left), scale: 20 μm, a line graph measuring the size of hAHOs (right), 1B is a transmission electron microscopy image of hAHOs on day 10, 1C is a graph of the relative mRNA levels of ALB, HNF4α, CYP3A4 and CK19 in hAHOs, 1D is an immunofluorescence staining image of ALB, CEBPα, CYP2A6, GSTA2 and CK19 in hAHOs on day 10, 1E is a graph of PAS staining, LDL uptake and ICG uptake and release of hAHOs on day 10, 1F is a graph of albumin levels, CYP3A4 activity and urea production of hASCs, hHLCs and hAHOs on day 10;

[0103] Figure 2 Figure 2 is the in vivo transplantation and function diagram of hAHOs, where 2A is the phase contrast and fluorescence micrograph of EGFP-hASCs and EGFP-hAHOs, 2B is the IVIS spectrum of EGFP-hAHOs implanted in mice two weeks later, 2C is the liver observation diagram of hAHOs transplanted mice, 2D is the HE staining diagram of liver sections of hAHOs transplanted mice, 2E is the expression diagram of ALB, HNF4α, CK19 and EGFP in liver tissue of hAHOs mice, and 2F is the drug metabolism activity diagram of hAHOs in mouse liver on the 14th day after implantation;

[0104] Figure 3 Figure 3 is a diagram of the establishment of the hAHOs culture system of the disease model, wherein 3A is a diagram of the lipogenesis level of hAHOs treated with control and ethanol using BODIPY 558 / 568C12, 3B is a diagram of the lipid droplet level of hAHOs in the control and ethanol-treated groups measured by HCS LipidTOX green neutral lipid staining, 3C is a diagram of the cell activity of hAHOs treated with control and ethanol using Dead / Live staining on the third day of ethanol treatment, 3D is a diagram of the relative mRNA levels of FASN, SCD, COLLI, ACTA2, IL-6, TNF-α, ACOX1 and CPT1A in hAHOs treated with control and ethanol, 3E is a diagram of the staining of COLLI and α-SMA in hAHOs treated with control and ethanol;

[0105] Figure 4is a diagram of the establishment of the hALOs system, wherein 4A is a micrograph of hALOs at different stages, 4B is a diagram of the relative levels of ALB, HNF4α, CYP3A4 and CK19 in hALOs, 4C is a diagram of immunofluorescence staining of ALB, GSTA2, CK19, DES, CD68, COLLI and CEBPα in hALOs on day 10, 4D is a transmission electron micrograph of hALOs on day 10, 4E is a diagram of LDL uptake, ICG uptake and release, and Rho-123 transport function of hALOs on day 10, 4F is a diagram of albumin concentration of hALOs on day 10, and 4G is a diagram of urea production of hALOs on day 10;

[0106] Figure 5 Figure 5 is a diagram of the establishment of the disease model hALOs system, wherein 5A is a diagram showing the relative levels of FASN, SCD, COLLI, ACTA2, IL-6, TNF-α, ACOX1, CPT1A, ADH1B and ALDH1B1 mRNA in control and ethanol-treated hALOs, 5B is a diagram showing the secretion of IL-6 in control and ethanol-treated hALOs, 5C is a diagram showing the lipogenesis level in control and ethanol-treated hALOs analyzed by BODIPY 558 / 568C12, and 5D is a diagram showing the lipid accumulation level in control and ethanol-treated hALOs measured by HCS Lipid TOX green neutral lipid staining;

[0107] Figure 6 is the immunofluorescence staining of α-SMA and TXNDC5 in the control group and ethanol-treated hALOs;

[0108] Figure 7 7A is a representative picture of Sirius red staining, Masson staining and Coli staining in hALOs of the control group and ethanol-treated group, 7B is an immunofluorescence staining picture of α-SMA and TXNDC5 in hALOs of the control group and ethanol-treated group, 7C is a picture of extracellular matrix deposition, α-SMA positivity and TXNDC5 level in liver tissues of the normal control group and ALD patients, 7D is a transmission electron microscopy picture of hALOs of the control group and hALOs of the ethanol-treated group;

[0109] Figure 88A is a diagram showing the establishment of the disease model hALOs system, wherein 8A is a diagram showing the expression levels of ADH1B and ALDH1B in the liver tissues of the normal control group and ALD patients, 8B is a diagram showing the expression of ADH1B and ALDH1B in the control and ethanol-treated hALOs, 8C is a diagram showing the relative mRNA levels of ADH1B and ALDH1B in the control and ethanol-treated hALOs, 8D is a diagram showing the cellular ROS levels in the control and ethanol-treated hALOs detected by the DCFDA / H_2DCFDA-cell reactive oxygen species detection kit, and 8E is a diagram showing the cell activity determination of the control and ethanol-treated hALOs. DETAILED DESCRIPTION

[0110] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0111] The invention provides a hAHOs liver organoid culture medium, which comprises a basal culture medium, a serum substitute, FGF, N-acetylcysteine, a Wnt agonist, nicotinamide, gastrin, a mitogenic proliferation factor, HGF and forskolin.

[0112] In one embodiment of the present invention, the basal culture medium includes DMEM (Dulbecco's Modified Eagle Medium), DMEM / Nutrient Mixture F-12 (DMEM / F-12), Advanced DMEM / F-12, RPMI 1640, IMDM (Iskoff's Modified Dulbecco's Medium), MEM (Minimum Essential Medium), BME (Basal Medium Eagle), KnockOut DMEM, KnockOut DMEM / F12, Advanced DMEM / F12, neurobasal. In another embodiment, the basal culture medium includes a combination of the above exemplary basal culture media in various proportions.

[0113] In a specific embodiment of the present invention, the basal culture medium is selected from Advanced DMEM / F12.

[0114] In one embodiment of the present invention, the serum substitute in the culture medium has a meaning well known to those skilled in the art, which refers to a composition or formulation used as a serum substitute in the process of culturing pluripotent stem cells while maintaining an undifferentiated state. That is, the serum substitute can support the growth of embryonic stem cells or undifferentiated pluripotent stem cells without the need for serum supplementation. In certain exemplary embodiments, the serum substitute comprises: one or more amino acids, one or more vitamins, one or more trace metal elements. In some cases, the serum substitute may further comprise one or more components selected from the following: albumin, reduced glutathione, transferrin, insulin, etc. Non-limiting examples of serum substitutes include, but are not limited to, KnockOutTM SR (abbreviated as KOSR), KOSR CTS, N2 additive (N2), CTS N2 additive, B27 additive (B27), Physiologix TM XF SR, StemSure TM SerumSubstituteSupplement, Knockout TM One or more of SR.

[0115] In a specific embodiment of the present invention, the serum replacement includes B27 and N2.

[0116] In an embodiment of the present invention, the concentration of B27 is 2%, but not limited thereto.

[0117] In an embodiment of the present invention, the concentration of N2 is 1%, but not limited thereto.

[0118] In one embodiment of the invention, FGF (fibroblast growth factor) is a family of growth factors involved in angiogenesis, wound healing and embryonic development. FGF is a heparin binding protein, and the interaction of the heparan sulfate proteoglycan associated with the cell surface is essential for FGF signal transduction. Those of ordinary skill in the art will readily understand suitable FGF pathway activators. FGF pathway activators include but are not limited to FGF1, FGF2, FGF3, FGF4, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23.

[0119] In a preferred embodiment of the present invention, FGF is selected from FGF7 and FGF10.

[0120] In a specific embodiment of the present invention, the FGF is selected from FGF10.

[0121] In an embodiment of the present invention, the concentration of FGF10 is 100 ng / ml, but is not limited thereto.

[0122] In an embodiment of the present invention, the concentration of N-acetylcysteine ​​is 1.25 mM, but is not limited thereto.

[0123] In one embodiment of the present invention, a Wnt agonist refers to an agent that activates T cell factor (TCF) / lymphoid enhancer factor (LEF)-mediated transcription in cells. Wnt agonists are not limited to Wnt family proteins, but also include Wnt agonists that bind to members of the frizzled receptor family to activate, intracellular β-catenin and TCF / LEF activating substances. Wnt agonists are preferably at least one of Wnt proteins, R-spondin (R-spondin) and GSK-3β inhibitors.

[0124] Among them, Wnt proteins can be used that are derived from various organisms. Among them, Wnt proteins derived from mammals are preferred. As mammals, for example, humans, mice, rats, cattle, pigs, and rabbits can be cited. Wnt proteins as mammals include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.

[0125] GSK-3β inhibitors include CHIR-99021, CHIR-98014 (Sigma-Aldrich), lithium (Sigma), kemperolone (Biomol International, Leost, M. et al. (2000) Eur J Biochem 267, 5983-5994), 6-bromoindirubin-30-acetone oxime (Meyer, L et al., (2003) Chem. Biol. 10, 1255-1266), SB 216763 and SB 415286 (Sigma-Aldrich), as well as FRAT family members and peptides derived from FRAT that prevent the interaction of GSK-3 with Axin.

[0126] R-spo (R-spondin, R-spongyrin) includes R-spo-1 (R-spondin 1, R-spongyrin 1), R-spo-2 (R-spondin 2, R-spongyrin 2), R-spo-3 (R-spondin 3, R-spongyrin 3) and R-spo-4 (R-spondin 4, R-spongyrin 4). R-spondin can be used in various combinations.

[0127] In a preferred embodiment of the present invention, the Wnt agonist is selected from R-spo (R-spondin, R-spongodin).

[0128] In a specific embodiment of the present invention, R-spo (R-spondin, R-sponglin) is selected from R-spo-1 (R-spondin 1, R-sponglin 1).

[0129] In an embodiment of the present invention, the concentration of R-spo-1 is 20 ng / ml, but is not limited thereto.

[0130] In an embodiment of the present invention, the concentration of nicotinamide is 10 mM, but is not limited thereto.

[0131] In an embodiment of the present invention, the concentration of gastrin is 10 nM, but not limited thereto.

[0132] In an embodiment of the present invention, the concentration of EGF is 50 ng / ml, but is not limited thereto.

[0133] In an embodiment of the present invention, the concentration of HGF is 25 ng / ml, but is not limited thereto.

[0134] In an embodiment of the present invention, the concentration of forskolin is 10 μM, but is not limited thereto.

[0135] The present invention provides a method for constructing hAHOs liver organoids, which comprises culturing HLCs using the above-mentioned culture medium.

[0136] The HLCs are induced from stem cells.

[0137] The culture medium for inducing stem cells to form HLCs includes: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, a fifth culture medium, and a sixth culture medium.

[0138] The second culture medium includes: basal culture medium, Activin, BSA, and SP.

[0139] The third culture medium includes: basal culture medium, Activin, BSA, SP, and ITS.

[0140] The fourth culture medium includes: BSA, ITS, BMP, FGF, and SP.

[0141] The fifth culture medium includes: BSA, ITS, HGF, and SP.

[0142] The sixth culture medium includes: BSA, ITS, IL-6 family cytokines, DEX, HGF, and SP.

[0143] In one embodiment of the present invention, examples of BMP (bone morphogenetic protein) include BMP2, BMP4, BMP7 and Growth Differentiation Factor (GDF)7.

[0144] In a specific embodiment of the present invention, the BMP is selected from BMP2.

[0145] In an embodiment of the present invention, the concentration of BMP2 is 20 ng / ml, but is not limited thereto.

[0146] In one embodiment of the present invention, IL-6 family cytokines include, for example, interleukin-6 (IL-6), interleukin-11 (IL-11), OSM (oncostatin M), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1) and ciliary neurotrophic factor (CNTF).

[0147] In a specific embodiment of the present invention, the IL-6 family cytokine is selected from OSM (Oncostatin M).

[0148] In an embodiment of the present invention, the concentration of OSM is 10 ng / mL, but is not limited thereto.

[0149] In an embodiment of the present invention, the concentration of the SP (Penicillin-Streptomycin Liquid) is 1%, but not limited thereto.

[0150] In an embodiment of the present invention, the concentration of Activin A is 100 ng / ml, but is not limited thereto.

[0151] In an embodiment of the present invention, the BSA (bovine serum albumin) is 10% BSA 0.5 mg / ml, but is not limited thereto.

[0152] In an embodiment of the present invention, the concentration of ITS (Insulin-Transferrin-Selenium) is 1%, but not limited thereto.

[0153] In an embodiment of the present invention, the concentration of FGF4 is 30 ng / ml, but is not limited thereto.

[0154] In an embodiment of the present invention, the concentration of HGF (hepatocyte growth factor) is 20 ng / mL, but is not limited thereto.

[0155] In an embodiment of the present invention, the concentration of DEX (dexamethasone) is 1 μM, but is not limited thereto.

[0156] The method further comprises the step of transferring the obtained HLCs into the hAHOs hepatic organoid culture medium for culture after digestion.

[0157] The method further comprises co-culturing the cells with the ECM.

[0158] In one embodiment of the present invention, the ECM (extracellular matrix) is a three-dimensional matrix including BME (basement membrane extract) and Matrigel.

[0159] In a specific embodiment of the present invention, the ECM is selected from Matrigel.

[0160] The present invention provides a hALOs liver organoid culture medium, which comprises: culture medium I and culture medium II, wherein the culture medium I comprises: basal culture medium, serum replacement, and RA, and the culture medium II comprises: HCM (hepatocyte culture medium), HGF, Dex, and IL-6 family cytokines.

[0161] In an embodiment of the present invention, the concentration of RA is 2 μM, but is not limited thereto.

[0162] The present invention provides a transplant material, which comprises the liver organoid.

[0163] In one embodiment of the present invention, the transplant material or liver organoid of the present invention can be applied to the treatment of various liver diseases (such as alcoholic liver disease). In particular, it is envisioned to be used as a material for the regeneration or reconstruction of damaged (including dysfunctional) liver tissue. That is, it can contribute to regenerative medicine. The transplant material of the present invention can be used as a transplant material directly or after being treated with matrix gel, collagen gel embedding, etc. In addition, it is also envisioned that it can be used as a screening of candidate compounds for therapeutic drugs in various liver disease pathological models and the elucidation of pathological mechanisms. In order to protect cells, dimethyl sulfoxide (DMSO), serum albumin, etc. can be contained in the transplant material of the present invention. In order to prevent the mixing of bacteria, antibiotics, etc. can be contained in the transplant material of the present invention. For the activation, proliferation or differentiation induction of cells, etc., various components (vitamins, cytokines, growth factors, steroids, etc.) can be contained in the transplant material of the present invention. In addition, the transplant material of the present invention may also contain other components allowed in the preparation (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, painless agents, stabilizers, preservatives, preservatives, saline, etc.).

[0164] The transplant material of the present invention can also be used to construct an in vivo experimental system. For example, a transplant material containing liver organoids made using human pluripotent stem cells can be transplanted into experimental animals such as mice, rats, guinea pigs, hamsters, pigs, cynomolgus monkeys, macaques, chimpanzees, etc. to produce humanized animals. Such humanized animals are particularly useful for experiments such as pharmacokinetics and toxicity tests, and can be expected to contribute to the study of the effects of first-pass effects on oral drugs, drug-induced liver disease, etc.

[0165] The present invention provides a method for evaluating the pharmacokinetics or toxicity of a test substance using the liver organoid.

[0166] In one embodiment of the present invention, the liver organoids are useful for evaluating the pharmacokinetics (absorption, metabolism, etc.) and toxicity of the liver. In other words, the liver organoids of the present invention can be used in evaluating the pharmacokinetics and toxicity of compounds.

[0167] Specifically, the liver organoids of the present invention can be used to test the metabolism, absorbability, membrane permeability, drug interaction, induction of drug metabolizing enzymes, induction of drug transporters, toxicity, etc. of the test substance. That is, as one of the uses of liver organoids, the present invention provides a method for evaluating the metabolism, absorbability, membrane permeability, drug interaction, induction of drug metabolizing enzymes, induction of drug transporters, toxicity, etc. of the test substance. In this method, the following steps are performed: (I) a step of contacting the test substance with the liver organoid obtained by the preparation method of the present invention, and (II) a step of measuring or evaluating the metabolism, absorbability, membrane permeability, drug interaction, induction of drug metabolizing enzymes or induction of drug transporters, or toxicity of the test substance.

[0168] The contact in step (I) is typically performed by adding the test substance to the culture medium. The timing of adding the test substance is not particularly limited. Therefore, the test substance may be added at a certain time after starting the culture in a culture medium that does not contain the test substance, or the culture may be started in a culture medium that previously contains the test substance.

[0169] As the test substance, organic compounds or inorganic compounds of various molecular sizes can be used. As examples of organic compounds, nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycoglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) can be exemplified. Existing ingredients or candidate ingredients such as pharmaceuticals, nutritional foods, food additives, pesticides, and perfumes (cosmetics) are also preferred test substances. Plant extracts, cell extracts, culture supernatants, etc. can also be used as test substances. By adding two or more test substances at the same time, interactions, synergistic effects, etc. between the test substances can be investigated. The test substance can be of natural origin, or it can be a substance obtained by synthesis. In the latter case, for example, a combinatorial synthesis method can be used to construct an effective analysis system.

[0170] The contact time of the test substance can be set arbitrarily. The contact time is, for example, 10 minutes to 3 days, preferably 1 hour to 1 day. The contact can also be performed in multiple times.

[0171] After step (I), the metabolism, absorbability, membrane permeability, drug interaction, induction of drug metabolizing enzymes, induction of drug transporters, or toxicity of the test substance are measured or evaluated (step (II)). Metabolism, etc. can be measured or evaluated immediately after step (I), that is, after the test substance is contacted, without a substantial time interval, or metabolism, etc. can be measured or evaluated after a certain period of time (e.g., 10 minutes to 5 hours). The measurement of metabolism can be carried out, for example, by the detection of metabolites. At this time, the culture fluid after step (I) is usually used as a sample to perform qualitative or quantitative measurement of the expected metabolites. The measurement method only needs to select an appropriate measurement method according to the metabolites, for example, mass spectrometry, liquid chromatography, immunological methods (such as fluorescent immunoassay (FIA method), enzyme immunoassay (EIA method)), etc.

[0172] Typically, when a metabolite of the test substance is detected, it is determined or evaluated that the test substance is metabolized. In addition, the metabolic amount of the test substance can be evaluated based on the amount of the metabolite. The metabolic efficiency of the test substance can be calculated based on the detection results of the metabolite and the amount of the test substance used (typically, the amount added to the culture medium).

[0173] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0174] Example 1 Method for constructing liver organoids

[0175] 1 Experimental Materials

[0176] hASCs and hASCs were primarily extracted from adipose tissue of parturients in Fuxing Hospital, Capital Medical University.

[0177] 2 Experimental methods

[0178] 2.1 Construction of hAHOs organoids

[0179] First, human adipose-derived mesenchymal stem cells (hASCs) were differentiated into hepatocyte-like cells (HLCs) by:

[0180] D0 day: aspirate and discard hASCs cell proliferation medium (DMEM-F12 plus 10% FBS (fetal bovine serum) plus 1% SP), wash twice with PBS, add DMEM-F12 medium (1% SP), and keep for 48 hours.

[0181] Day D2: discard the old solution, wash once with PBS, add DMEM-F12 medium (Activin A 100 ng / ml, 10% BSA 0.5 mg / ml, 1% SP), and induce endoderm for 24 hours.

[0182] Day D3: 1% ITS was added to each dish and induced into ventral foregut endoderm for 48 hours.

[0183] Day D5: discard the old solution, wash once with PBS, add MEM-NEAA medium (10% BSA 0.5 mg / ml, 1% ITS, BMP2 20 ng / ml, FGF4 30 ng / ml, 1% SP), and differentiate into directed liver precursors for 5 days.

[0184] D7 day: discard the old solution, wash once with PBS, and add MEM-NEAA medium (10% BSA 0.5 mg / ml, 1% ITS, BMP2 20 ng / ml, FGF4 30 ng / ml, 1% SP).

[0185] D10 day: discard the old solution, wash once with PBS, add MEM-NEAA medium (10% BSA 0.5 mg / ml, 1% ITS, HGF 20 ng / mL, 1% SP), and induce hepatocytes for 5 days.

[0186] D12 day: discard the old solution, wash once with PBS, and add MEM-NEAA medium (10% BSA 0.5 mg / ml, 1% ITS, HGF 20 ng / mL, 1% SP).

[0187] D15 day: discard the old solution, wash once with PBS, add MEM-NEAA medium (10% BSA 0.5 mg / ml, 1% ITS, OSM 10 ng / mL, DEX 1 μM, HGF 20 ng / mL, 1% SP), and induce mature hepatocytes for 5 days.

[0188] D17 day: discard the old solution, wash once with PBS, and add MEM-NEAA medium (10% BSA 0.5 mg / ml, 1% ITS, OSM 10 ng / mL, DEX 1 μM, HGF 20 ng / mL, 1% SP).

[0189] D20: HLCs induction is completed.

[0190] Then, HLCs were collected and trypsinized with 0.05% Trypsin-EDTA (Invitrogen) for 3-4 min at 37° C. The separated cells were washed with PBS, and the cells were seeded at a ratio of 2500 cells / well into CellCarrier-96 Spheroid ULA / CS (PerkinElmer Medical Diagnostics Shanghai Co., Ltd., Waltham, MA, USA), and 100 μL of expansion medium (EM) was added. EM consists of Advanced DMEM / F-12 (Invitrogen) + 20ng / ml R-SPO-1 conditioned medium (Peprotech), 2% B27 (Gibco), 1% N2 (Gibco), 50ng / ml EGF (Peprotech), 1.25mM N-Acetylcysteine ​​(Sigma), 10nM gastrin (Sigma), 25ng / ml HGF (Peprotech), 100ng / ml FGF10 (Peprotech), 10mM nicotinamide (Sigma), 10μM Forskolin (TOCRIS). Then centrifuge at 1000rpm for 5 minutes and incubate at 37°C and 5% CO2. After 24h, spheroids appear on the plate. Remove the old supernatant and add 50μL Matrigel (CORNING), 50μL per well. After the Matrigel gel solidified, 100 μL of EM was added. The culture conditions were 37°C, 5% CO2, and 95% air. The medium was changed every 2-3 days for a total of 10 days to form hAHOs. To analyze the properties of hAHOs, organoids were separated from Matrigel by scratching and pipetting.

[0191] 2.2 Construction of hALOs organoids

[0192] First, human adipose-derived stem cells (hASCs) were differentiated into endoderm progenitor cells (hEPCs). hEPCs were cultured in three dimensions, including 5 days of RA treatment and 5 days of differentiation induction and maturation. hASCs-derived liver organoids containing hepatocytes and non-parenchymal cells were formed.

[0193] First, human adipose-derived mesenchymal stem cells (hASCs) are cultured and induced to differentiate into endoderm progenitor cells (hEPCs), and the specific method includes:

[0194] Day D0: discard the old solution, wash twice with PBS, add DMEM-F12 medium (1% SP), and keep for 48 hours.

[0195] Day D2: discard the old solution, wash once with PBS, add DMEM-F12 medium (Activin A 100 ng / ml, 10% BSA 0.5 mg / ml, 1% SP), and induce endoderm for 24 hours.

[0196] Day D3: 1% ITS was added to each dish and induced into ventral foregut endoderm for 48 hours.

[0197] D5 day: hEPCs induction is completed.

[0198] Then, hEPCs were collected and trypsinized with 0.05% Trypsin-EDTA (Invitrogen) at 37°C for 3-4 minutes. Free cells were washed with PBS, and the cells were seeded on CellCarrier-96Spheroid ULA / CS (PerkinElmer Medical Diagnostic Products Shanghai Co., Ltd.) at a ratio of 2500 cells per well, and 100 μL of culture medium containing Advanced DMEM / F-12 (Invitrogen) plus 2 μM retinoic acid (RA; Sigma-Aldrich), 2% B27 (Gibco), and 1% N2 (Gibco). Centrifuge at 1000 rpm for 5 minutes and incubate at 37°C, 5% CO2. After 24 hours, spheroids appeared on the plate and the old supernatant was removed. 200 μL of new culture medium was added to each well, and the culture conditions were 37°C, 5% CO2 and 95% air. The medium was changed once every 2 days for a total of 4 days. After 5 days, the medium was discarded and new medium was added. The new medium composition included: HCM (Lonza) plus 20ng / ml HGF (Peprotech), 10ng / ml OSM (Peprotech) and 1μM dexamethasone (Dex; Sigma-Aldrich). The culture conditions were 37°C, 5% CO2 and 95% air. The medium was changed every 3 days for 5 consecutive days to generate hALOs. To analyze hALOs, organoids were isolated by scratching and pipetting.

[0199] Example 2 Characteristics and Applications of Liver Organoids

[0200] 1 hAHOs show characteristics of proliferative hepatocyte organelles

[0201] To investigate whether hHLC-derived spheres have the ability to grow in suspension in Matrigel, an optimal growth medium for generating hAHOs was designed. During 3 days of culture, Matrigel-encapsulated HLCs appeared as small organoids under the culture conditions. Organoids grew to a diameter of 150 μm ( Figure 1 A).

[0202] To gain a more comprehensive understanding of the hepatocyte structure, ultrastructural analysis was performed using transmission electron microscopy (TEM). hAHOs at day 10 had typical hepatocyte structures, including abundant mitochondria, endoplasmic reticulum, Golgi apparatus, intercellular junctions, microvilli, and bile canaliculi ( Figure 1 B).

[0203] The results of quantitative RT-PCR evaluation of the changes in gene expression over time in hAHOs from the three donors showed that the expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4α), cytochrome P450 family 3 subfamily A member 4 (CYP3A4), and keratin 19 (CK19, a bile duct marker) in organoids on days 5 and 10 was significantly higher than that in HLCs (day 0). The expression level of CK19 in organoids on day 10 was lower than that in organoids on day 5 ( Figure 1 C). hAHOs were then analyzed by immunofluorescence staining. hAHOs showed strong expression of ALB, CCAAT enhancer binding protein α (CEBPα), CYP2A6, and glutathione S-transferase α2 (GSTA2) ( Figure 1 D).

[0204] Functionally, hAHOs showed strong periodic acid Schiff (PAS) staining, indicating glycogen accumulation in hAHOs. Low-density lipoprotein (LDL) uptake was easily observed using fluorescent probes. hAHOs showed indocyanine green (ICG) uptake and release, suggesting an interaction between sinusoidal uptake and biliary excretion in determining hepatic ICG clearance in hAHOs ( Figure 1 E). Compared with undifferentiated hASCs and hHLCs, hAHOs showed significantly increased albumin secretion levels, CYP3A4 metabolic activity, and urea production ( Figure 1 F). These data suggest that hHLCs-derived hepatocyte organoids can grow and differentiate into mature hepatic functions.

[0205] 2 hAHOs have the ability to transplant, survive, and repair damaged liver tissue in mice

[0206] To investigate whether hAHOs are able to engraft and repopulate damaged liver tissue, enhanced green fluorescent protein (EGFP-hAHOs) were generated from hASCs-derived EGFP-hHLCs stably transduced with EGFP ( Figure 2 A). hAHOs were subsequently implanted into the right upper lobe edge of the damaged liver of CCL4-induced athymic nude BALB / C mice.

[0207] Two weeks after implantation, the distribution of EGFP-hAHOs in the mouse liver was imaged using the IVIS Spectrum Imaging System ( Figure 2 B). Through a stereo microscope ( Figure 2 C) and H&E staining analysis ( Figure 2 D) Confirmation of the initial colonization of the small clusters in the liver. Immunofluorescence analysis showed that ALB, HNF4α, and CK19-positive cells could be detected in the grafts and co-localized with cells expressing EGFP ( Figure 2 E).

[0208] The functional maturation of hAHOs in the implants was evaluated by measuring the metabolic activity of ketoprofen, which is known to be metabolized differently in mice and humans. Figure 2 F, upper panel). However, 1-hydroxyketoprofen (mouse metabolite) was detected in the urine of all hAHOs ( Figure 2 F, lower panel). These data indicate that hAHOs can successfully repair damaged livers after transplantation and exhibit metabolic function.

[0209] 3. Ethanol treatment to simulate hepatic steatosis and inflammation in hAHOs

[0210] To evaluate whether ethanol treatment induces steatosis and leads to inflammatory response and fibrosis, hAHOs from three donors were treated with 100 mM ethanol for 72 h. Figure 3 A) and HCS LipidTOX TM Neutral lipid staining ( Figure 3 B) Analysis of lipogenesis and lipid accumulation in hAHOs. The results showed that the levels of lipogenesis and lipid accumulation in ethanol-treated hAHOs were significantly higher than those in the control group. Ethanol-treated hAHOs showed decreased cell viability, as indicated by an increased dead / live cell content ratio ( Figure 3 C).

[0211] Quantitative RT-PCR analysis showed that the expression levels of fat synthesis-related enzymes fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD), and acyl-CoA oxidase 1 (ACOX1) in ethanol-treated hAHOs were significantly higher than those in the control group. In addition, the mRNA levels of inflammatory response factors IL-6 and TNF-α were upregulated in ethanol-treated hAHOs and were significantly higher than those in the control group ( Figure 3 D).

[0212] To further verify whether hAHOs can show fibrotic response to ethanol, Sirius red staining, Masson staining, and immunohistochemical staining of COLLI and smooth muscle actin (α-SMA) were performed. The results showed that the expression levels of COLLI and α-SMA were similar in hAHOs treated with ethanol and hAHOs treated with control ( Figure 3 E) These data suggest that hAHOs can recapitulate the initial symptoms of ALD, including steatosis and inflammation.

[0213] 4. Generation of liver organoids from hASCs

[0214] First, human adipose-derived stem cells (hASCs) were differentiated into endoderm progenitor cells (hEPCs). hEPCs were cultured in three dimensions, including 5 days of RA treatment and 5 days of differentiation and maturation. hASCs-derived liver organoids containing hepatocytes and non-parenchymal cells were formed. They were named hALOs ( Figure 4 A).

[0215] The mRNA levels of hepatocyte markers (ALB, HNF4α, CYP3A4) and bile duct marker (CK19) were significantly upregulated in hALOs on day 10 compared with hEPCs (day 0) and hALOs (day 5). Figure 4 B). Cell types in hALOs were identified using immunofluorescence staining for hepatocyte markers ALB, CEBPα and GSTA2, cholangiocyte markers CK19, stellate cell markers desmin and COLLI, and Kupffer cell marker CD68 ( Figure 4 C). Ultrastructural analysis showed that hALOs( Figure 4 D, left panel) contains hepatoid cells, Kupffer-like cells, fat-storing cells, and endothelial-like cells. Typical liver ultrastructure includes microvilli, cell junction complexes, lipid droplets (LDs), endoplasmic reticulum, mitochondria, microvessels, and extracellular matrix (ECM) ( Figure 4 D, right).

[0216] Functionally, hALOs have liver-specific LDL uptake function, significantly high-level ICG uptake and release function, and Rho-123 transport function ( Figure 4 E). The ALB secretion and urea production levels of hALOs were significantly higher than those of hHLCs and hAHOs ( Figure 4 F, 4G). These data indicate that hALOs contain both hepatocytes and non-parenchymal cells and are able to maintain typical liver functions.

[0217] 5 Establishing ALD Model Using hALOs by Ethanol Treatment

[0218] To investigate the effect of ethanol treatment on gene expression in hALOs, quantitative RT-PCR was performed. The results showed that the mRNA levels of FASN and SCD, which are associated with lipogenesis, IL-6 and TNF-α, which are associated with inflammation, and ACOX1 and CPT1A, which are associated with oxidative phosphorylation, were upregulated in hALOs treated with ethanol. This finding was similar to the response of hAHO to ethanol treatment. However, the mRNA levels of COLLI and ACTA2, which are associated with fibrosis, were upregulated in hALOs treated with ethanol ( Figure 5 A).

[0219] To verify whether ALD-related pathological changes could be detected in ethanol-treated hALOs, the level of steatosis was measured. BODIPY 558 / 568C12 and HCS LipidTOX TM Neutral lipid staining and IL-6 secretion analysis showed that compared with the control group ( Figure 5 Compared with 5B, 5C, and 5D), the lipid accumulation and inflammatory response levels of hALOs treated with ethanol were significantly increased.

[0220] To evaluate whether hALOs exhibit a fibrotic response to ethanol treatment, changes in ECM components were assessed according to clinical parameters of fibrosis. Sirius red and masson staining analysis showed that ECM components were significantly increased in ethanol-treated hALOs compared with control hALOs. Immunohistochemical staining demonstrated that the increased ECM deposition after ethanol treatment was type I collagen a. Levels of endoplasmic reticulum proteins thioredoxin domain-containing protein 5 (TXNDC5) and smooth muscle actin (α-SMA) were increased in ethanol-treated hALOs ( Figure 6 , Figure 7 A, 7B). TXNDC5 is a member of the protein disulfide isomerase family and plays an important role in the formation of fibrosis in various tissues. α-SMA is often used to mark smooth muscle cells and affects the deposition of extracellular matrix. The deposited matrix and the expression of TXNDC5 and α-SMA were further confirmed in the liver tissue of ALD patients ( Figure 7 C).

[0221] To understand the changes in the structure of hALOs after ethanol treatment, TEM was used for ultrastructural analysis. After ethanol treatment, the mitochondria of hALOs became significantly shorter and smaller, the endoplasmic reticulum became fragmented, and the cavities became larger. These results indicate that the function of mitochondria and endoplasmic reticulum was impaired. Importantly, a large number of fibers ( Figure 7 D).

[0222] Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) are the main alcohol dehydrogenases in ethanol metabolism. The expression of ADH1B and ALDH1B was further confirmed in the liver tissue of ALD patients ( Figure 8 In addition, immunohistochemical staining showed that the expression of ADH1B and ALDH1B was upregulated in ethanol-treated hALOs compared with control hALOs ( Figure 8 B). The mRNA levels of ADH1B and ALDH1B were upregulated in ethanol-treated hALOs ( Figure 8 C). ADH metabolizes ethanol to toxic acetaldehyde while generating reactive oxygen species (ROS), which further induces oxidative stress and steatosis in ALD. The oxidative stress level of hALOs was examined. It was found that ethanol treatment led to increased oxidative stress ( Figure 8 D). Further studies found that hALOs showed liver damage, as evidenced by decreased cell survival and increased dead / live cell content ratio ( Figure 8 E). These data confirm that hALOs mimic the pathological processes and mechanisms of ALD.

[0223] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing hALOs liver organoids, characterized in that: The method comprises a) The stem cells are cultured using a first culture medium, b) the cells obtained in step a) are transferred to a second culture medium for culture, and the cells are induced to develop into endoderm, c) The endoderm cells obtained in step b) are transferred to the third culture medium for culture and induced to the ventral foregut endoderm to obtain hEPCs, d) hEPCs were digested and cultured in medium I. e) adding medium II for culturing; the medium for inducing stem cells to form hEPCs includes a first medium, a second medium, and a third medium; The first culture medium includes: DMEM / F12, SP; The second culture medium comprises: DMEM / F12, Activin A, BSA, SP; The third culture medium comprises: DMEM / F12, Activin A, BSA, SP, and ITS; The culture medium for culturing hEPCs includes: culture medium I and culture medium II; The culture medium I includes: DMEM / F-12, B27, N2, RA; The culture medium II comprises: HCM, HGF, Dex, and OSM; The concentration of the SP is 1%; The concentration of Activin A is 100 ng / ml; The BSA is 10% BSA 0.5 mg / ml; The concentration of the ITS is 1%; The concentration of B27 is 2%; The concentration of N2 is 1%; The concentration of RA was 2 μM; The concentration of HGF is 20 ng / ml; The concentration of Dex is 1 μM; The concentration of OSM is 10 ng / ml; The culturing time of steps a) and c) is 48 hours; The culturing time of step b) is 24 hours; The culture time of steps d) and e) is 5 days; Step d) digesting hEPCs using trypsin; The stem cells are adipose-derived mesenchymal stem cells.

2. A liver organoid, characterized in that: The liver organoid is constructed by the method of claim 1.

3. A transplant material, characterized in that: The transplant material comprises the liver organoid according to claim 2.

4. Use of the liver organoid according to claim 2 in constructing a liver disease model; The liver disease is ALD.

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