Culture medium, methods, models and applications of 3D liver models for primary hepatocyte culture

Abstract

The present invention discloses a culture medium, method, model and application of a primary hepatocyte culture 3D liver model, and relates to the field of biotechnology. The culture medium includes: 100 μL of Williams E solution, 200 nmol~2 μmol of apoptosis inhibitor, 1.5~2.5 mmol of amino acid, 0.1~5.2 mg / ml of antibiotic, 3~8 ng / ml of sodium selenite, 3~8 μg / ml of transferrin, 8~12 nmol of steroid compound, 40~60 μmol of adenylate cyclase activator, 8~12 μmol of TGF-β inhibitor, and 8~20 μL of fetal bovine serum. The culture medium can maintain the long-term stability of cell function in vitro.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium, method, model and application of a 3D liver model for culturing primary hepatocytes. Background Art

[0002] The liver is the main organ for drug metabolism. Understanding the metabolic process of candidate drugs in the liver and the possible liver toxicity is one of the most important links in the preclinical drug development process. At the same time, many candidate drugs target liver diseases, such as liver fibrosis, non-alcoholic fatty liver disease, hepatitis, etc. Therefore, in the drug development stage, it is crucial to use a reliable liver model for drug testing.

[0003] In preclinical drug development, testing on liver models is traditionally done through animal models. The construction of in vitro liver models provides a new platform for evaluating the metabolism, toxicity and efficacy of candidate drugs. However, the inventors found that traditional animal models and in vitro liver models have at least the following limitations:

[0004] Regarding animal models, there are differences in the types and expression of liver drug enzymes between human models and animal models, so animal models are not suitable for preclinical drug research on the liver. Related studies have shown that in terms of drug hepatotoxicity prediction, the prediction accuracy of rodents and non-rodents is 43% and 63% respectively, which fully illustrates the differences between animal models and human liver in preclinical drug research on the liver.

[0005] Regarding the in vitro 2D liver model, the in vitro liver models used in related technologies include the 2D hepatocyte model and the 3D hepatocyte model. The 2D hepatocyte model rapidly dedifferentiates in a very short time, that is, it loses the hepatocyte phenotype and function (including the expression of liver markers and the normal metabolism of drugs). Therefore, the application of the in vitro 2D liver model is limited in the long-term study of liver biology and the analysis requiring liver-specific functions, and is not suitable for preclinical drug research in the liver. The liver phenotype and function of the 3D hepatocyte model are maintained for a short and uneven time, and its application is limited, and it is not suitable for related tests of chronic exposure candidate drugs.

[0006] Therefore, providing a 3D hepatocyte model that maintains liver phenotype and function for a long time is a technical problem that needs to be solved urgently by technicians in this field. Summary of the invention

[0007] The present invention discloses a culture medium, method, model and application of a 3D liver model for culturing primary hepatocytes, so as to solve the technical problem in the related art that the liver phenotype and function of the 3D hepatocyte model are maintained for a short time and are uneven.

[0008] In order to solve the above problems, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a culture medium for culturing primary hepatocytes in a 3D liver model.

[0010] The culture medium for culturing a 3D liver model with primary hepatocytes of the present invention comprises the following components:

[0011] 100 μL of Williams E solution,

[0012] 200nmol~2μmol of apoptosis inhibitor,

[0013] 1.5~2.5mmol amino acids,

[0014] 0.1~5.2mg / ml antibiotics,

[0015] 3~8ng / ml sodium selenite,

[0016] 3~8μg / ml transferrin,

[0017] 8~12nmol of steroid compounds,

[0018] 40~60μmol of adenylate cyclase activator,

[0019] 8~12μmol of TGF-β inhibitor,

[0020] 8~20μL of fetal bovine serum.

[0021] According to a preferred embodiment, the adenylate cyclase activator is one or more of forskolin and cyclic adenosine monophosphate, and the dosage of the adenylate cyclase activator is 50 μmol; the TGF-β inhibitor is one or more of A8301 and its salts, SB431542, and the dosage of the TGF-β inhibitor is 10 μmol.

[0022] According to a preferred embodiment, the cell apoptosis inhibitor is zinc sulfate, and the amount of zinc sulfate is 1 μmol.

[0023] According to a preferred embodiment, the amino acid is one or more of L-glutamine, proline, ornithine, N-acetylcysteine, and serine, and the amount of the amino acid is 2 mmol.

[0024] According to a preferred embodiment, the antibiotics include penicillin and streptomycin, wherein the concentration of penicillin is 5.2 mg / ml, and the concentration of streptomycin is 100 μg / ml.

[0025] According to a preferred embodiment, the concentration of sodium selenite is 5 ng / ml; the concentration of transferrin is 5 μg / ml; the steroid compound is selected from one or more of prednisone, methylprednisone, betamethasone, beclomethasone propionate, prednisolone, hydrocortisone, and dexamethasone, and the dosage of the steroid compound is 10 nmol.

[0026] According to a preferred embodiment, the culture medium for culturing the primary hepatocyte 3D liver model includes the following components: 100 μL of Williams E solution, 1 μmol of zinc sulfate, 2 mmol of L-glutamine, 5.2 mg / ml of penicillin, 100 μg / ml of streptomycin, 5 ng / ml of sodium selenite, 5 μg / ml of transferrin, 10 nmol of dexamethasone, 50 μmol of forskolin, 10 μmol of SB431542, and 10 μL of fetal bovine serum.

[0027] The second aspect of the present invention provides a method for culturing a 3D liver model by culturing primary hepatocytes.

[0028] The method for culturing a primary hepatocyte culture 3D liver model of the present invention comprises culturing primary hepatocytes with a culture medium for culturing a primary hepatocyte culture 3D liver model according to any one of the technical solutions of the present invention, and the method comprises at least the following steps:

[0029] Step S100: mixing the culture medium with primary human hepatocytes to prepare a single cell suspension;

[0030] Step S200: by counting cells, 1500 primary hepatocytes are inoculated into a 96-well ultra-low adsorption culture plate per well, the culture plate is centrifuged at a centrifugal force of 80-120 g for 1-3 minutes, and the culture plate is placed in an incubator for static culture;

[0031] Step S300: After the hepatocytes in each well form 3D hepatocyte spheres and / or after 5 days of culture, 40-60% of the original culture medium is replaced with fresh culture medium;

[0032] Step S400: In subsequent culture, 40-60% of the original culture medium is replaced with fresh culture medium at a frequency of once every two days.

[0033] A third aspect of the present invention provides a 3D liver model.

[0034] The 3D liver model of the present invention is based on primary hepatocytes, and the hepatocytes are obtained by culturing the primary hepatocytes according to any technical solution of the present invention. The 3D liver model is spherical, and the phenotype and function of the 3D liver model can be maintained for more than 45 days.

[0035] A fourth aspect of the present invention provides an application of a 3D liver model.

[0036] The application of the 3D liver model of the present invention is that the 3D liver model is used for in vitro research on one or more of the mechanism of hepatocyte death, the mechanism of hepatocyte proliferation, the mechanism of liver disease, hepatocyte therapy, hepatocyte transplantation, preparation of bioartificial liver, liver disease drug screening, candidate drug pharmacology and toxicology evaluation, and candidate drug metabolism research; and when the 3D liver model is used for candidate drug pharmacology and toxicology evaluation and / or candidate drug metabolism research, the candidate drug is a chronic exposure drug.

[0037] The technical solution adopted by the present invention can achieve the following beneficial effects:

[0038] The culture medium for culturing a 3D liver model of the present invention comprises 100 μL of Williams E solution, 200 nmol-2 μmol of apoptosis inhibitor, 1.5-2.5 mmol of amino acid, 0.1-5.2 mg / ml of antibiotic, 3-8 ng / ml of sodium selenite, 3-8 μg / ml of transferrin, 8-12 nmol of steroid compound, 40-60 μmol of adenylate cyclase activator, 8-12 μmol of TGF-β inhibitor, and 8-20 μL of fetal bovine serum. Through the coordination of the components, the culture medium can provide the required energy source and nutrients for the cultivation of primary hepatocytes, reduce the risk of infection during the cultivation process, slow down apoptosis, resist oxidation, support the storage and transport of extracellular iron, maintain the cell differentiation state, and maintain the long-term stability of cell function in vitro.

[0039] The culture medium for culturing a 3D liver model of the present invention is added with 40-60 μmol of an adenylate cyclase activator and 8-12 μmol of a TGF-β inhibitor. Proteins related to epithelial-mesenchymal transition (EMT) have a great effect on the normal function of liver cells. The adenylate cyclase activator can downregulate epithelial-mesenchymal transition marker proteins, thereby maintaining the long-term stability of liver cell function in vitro. During the in vitro liver cell culture process, component proteins in the TGF-β signaling pathway tend to be highly expressed, which is one of the reasons for the dedifferentiation of liver cells in in vitro culture. By adding a TGF-β inhibitor to the culture medium, the TGF-β signal can be inhibited, thereby achieving the purpose of making the in vitro culture phenotype and function of in vitro liver cells more stable. The culture medium of the present invention adds 40-60 μmol of adenylate cyclase activator and 8-12 μmol of TGF-β inhibitor, and the synergistic effect of the two can regulate the phenotype and function of primary hepatocytes in an in vitro 3D model, so that when a 3D liver model is cultured by human primary hepatocytes, the phenotype and function of the hepatocytes in vitro can be maintained for more than 45 days, thereby greatly extending the culture time of the 3D model.

[0040] The 3D liver model cultured using the culture medium of the present invention can maintain its phenotype and function in vitro for more than 45 days. When it is used for candidate drug pharmacology and toxicology evaluation and / or candidate drug metabolism research, it can evaluate the long-term liver toxicity and metabolism time of the candidate drug, which can improve the reliability of evaluating the chronic toxicity of the candidate drug, and avoid the problem that when performing candidate drug pharmacology and toxicology evaluation and / or candidate drug metabolism research, the phenotype and function of the liver model used are maintained for a short time, resulting in the candidate drug being unable to show its toxicity in the in vitro liver model, and thus the pharmacology, toxicology and / or metabolic status of the candidate drug with a long metabolism time cannot be reliably evaluated. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 This is a morphological diagram observed under a microscope during the culture of hepatocytes in Example 1 of the present application;

[0043] Figure 2 This is a graph showing the results of cell viability detection during the hepatocyte culture process in Example 1 of the present application;

[0044] Figure 3 This is a graph showing the results of detecting albumin secretion during the hepatocyte culture process in Example 1 of the present application;

[0045] Figure 4 This is a graph showing the results of detecting urea synthesis during the hepatocyte culture process in Example 1 of the present application;

[0046] Figure 5 This is a graph showing the results of CYP1A2 activity detection during the hepatocyte culture process in Example 1 of the present application;

[0047] Figure 6 This is a graph showing the results of CYP3A4 activity detection during the hepatocyte culture process in Example 1 of the present application;

[0048] Figure 7 This is a graph showing the results of CYP2D6 activity detection during the hepatocyte culture process in Example 1 of the present application;

[0049] Figure 8 This is a graph showing the results of CYP2C8 activity detection during the hepatocyte culture process in Example 1 of the present application;

[0050] Fig. 9 This is a graph showing the results of CYP2C9 activity detection during the hepatocyte culture process in Example 1 of the present application;

[0051] Fig.10 This is a morphological diagram observed under a microscope during the culture of hepatocytes in Example 2 of the present application;

[0052] Fig.11 This is a graph showing the results of cell viability detection during the hepatocyte culture process in Example 2 of the present application;

[0053] Fig.12 This is a graph showing the results of detecting albumin secretion during the hepatocyte culture process in Example 2 of the present application;

[0054] Fig.13 This is a graph showing the detection results of urea synthesis during the hepatocyte culture process in Example 2 of the present application;

[0055] Fig.14 This is a graph showing the results of detecting the CYP1A2 content during the hepatocyte culture process in Example 2 of the present application;

[0056] Fig.15 This is a graph showing the results of CYP3A4 activity detection during the hepatocyte culture process in Example 2 of the present application;

[0057] Fig.16 This is a graph showing the results of CYP2D6 activity detection during the hepatocyte culture process in Example 2 of the present application;

[0058] Fig.17 This is a graph showing the results of CYP2C8 activity detection during the hepatocyte culture process in Example 2 of the present application;

[0059] Fig.18This is a graph showing the results of CYP2C9 activity detection during the hepatocyte culture process in Example 2 of the present application;

[0060] Fig.19 This is a morphological diagram observed under a microscope during the hepatocyte culture process in Example 3 of the present application;

[0061] Fig. 20 This is a graph showing the results of cell viability detection during the hepatocyte culture process in Example 3 of the present application;

[0062] Fig.21 This is a graph showing the results of detecting albumin secretion during the hepatocyte culture process in Example 3 of the present application;

[0063] Fig. 22 This is a graph showing the results of detecting urea synthesis during the hepatocyte culture process in Example 3 of the present application;

[0064] Fig.23 This is a graph showing the results of CYP1A2 activity detection during the hepatocyte culture process in Example 3 of the present application;

[0065] Fig.24 This is a graph showing the results of CYP3A4 activity detection during the hepatocyte culture process in Example 3 of the present application;

[0066] Fig.25 This is a graph showing the results of CYP2D6 activity detection during the hepatocyte culture process in Example 3 of the present application;

[0067] Fig.26 This is a graph showing the results of CYP2C8 activity detection during the hepatocyte culture process in Example 3 of the present application;

[0068] Fig. 27 This is a graph showing the results of CYP2C9 activity detection during the hepatocyte culture process in Example 3 of the present application;

[0069] Fig.28 It is a graph of the cell viability detection results during the hepatocyte culture process of Example 2, Comparative Example 1 and Comparative Example 2 of the present application, as well as a morphological graph observed under a microscope. DETAILED DESCRIPTION

[0070] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0071] In the specification and claims, "and / or" means at least one of the connected objects, and the character " / " generally means that the previously and subsequently associated objects are in an "or" relationship.

[0072] The following is an explanation of the terms involved in this application:

[0073] Human primary hepatocytes: refers to human liver cells that have not undergone any changes. Human primary hepatocytes retain their natural state and characteristics and can be used to study liver physiological functions, drug metabolism, etc. The human primary hepatocytes in the examples of this application can be purchased commercially.

[0074] Stem cells: Stem cells are undifferentiated cells with strong differentiation potential. They can differentiate into many different types of cells. For example, stem cells can differentiate into muscle cells, nerve cells, heart cells, liver cells, etc. They can be widely used in regenerative medicine, tissue engineering and other fields.

[0075] 2D liver model: liver cells in a two-dimensional form. This model is usually cultured on a flat carrier such as a culture dish or a multi-well plate. The liver cells adhere to the surface of the carrier and form a monolayer of cells. It can be used to evaluate drug toxicity, drug metabolism, etc.

[0076] 3D liver model: A three-dimensional liver model, which is usually formed as a sphere or a sphere-like form. It is closer to the morphology and function of a real liver and is more reliable for evaluating drug metabolism and liver function.

[0077] Dedifferentiation of liver model: refers to the mature hepatocytes in the liver model losing their specific mature phenotype and function, and then transforming into primitive or immature cells. During the dedifferentiation process, hepatocytes may lose their typical morphology, surface markers and functions. The dedifferentiated liver model cannot be used to evaluate drug toxicity, drug metabolism, etc.

[0078] Phenotype of liver cells: refers to the morphology, structure and characteristics of liver cells, including cell shape, size, karyotype, organelles in the cytoplasm, etc.

[0079] Functions of liver cells: refers to the biological functions of liver cells, including metabolic function, detoxification function, protein synthesis function, bile secretion function, immune function, fat metabolism, glycogen storage, etc.

[0080] Candidate drug: refers to a compound or molecule that has been screened out during the drug development process and has the potential to be used to treat specific diseases. Candidate drugs must undergo a series of rigorous laboratory and clinical trials to determine their safety, effectiveness, side effects and other characteristics.

[0081] Chronic exposure candidate drugs: refers to candidate drugs whose metabolism, efficacy and / or toxicity take a long time to manifest. Generally, candidate drugs whose metabolism, efficacy and toxicity take more than 14 days to manifest can be called chronic exposure candidate drugs, such as non-rotein drugs.

[0082] Apoptosis Inhibitors: Apoptosis inhibitors are a class of compounds that are used to inhibit or slow down the occurrence of apoptosis (programmed cell death). Apoptosis inhibitors may also be called anti-apoptotic agents.

[0083] Adenylate cyclase activators: Adenylate cyclase activators are a class of compounds that increase the activity of adenylate cyclase.

[0084] TGF-β Inhibitors: TGF-β inhibitors are a class of compounds or drugs used to inhibit the transforming growth factor-β (TGF-β) signaling pathway in the body.

[0085] In the drug development stage, it is crucial to use a reliable liver model for drug testing. In the related art, stem cells can be used to induce differentiation to obtain hepatoblasts, and then the hepatoblasts are cultured to obtain a high proportion of immature hepatocytes, and then a high proportion of immature hepatocytes are cultured to obtain a high proportion of mature hepatocytes. A high proportion of mature hepatocytes is close to the function of the real liver and can be used to evaluate drug metabolism and liver function. However, the inventors found that obtaining mature hepatocytes through stem cells has defects such as uncertainty in stem cell differentiation, complex culture process, and immature hepatocytes in the obtained mature hepatocytes; in addition, the phenotype and function of the obtained mature hepatocytes are maintained for a short time. In the related art, a liver model can also be obtained by culturing primary hepatocytes. However, the inventors found that the current primary hepatocyte culture 3D liver model, the in vitro 3D liver model phenotype and function maintenance time is short and uneven, generally 14 days.

[0086] The liver models obtained in the above-mentioned related technologies all have the problem of short maintenance time of liver model phenotype and function. For chronically exposed candidate drugs (such as non-roside drugs), when evaluating their drug metabolism, toxicity and efficacy, due to the long time required for their functions and toxicity to manifest, misjudgment may occur when evaluating relevant indicators using the existing in vitro 3D liver model, which seriously affects the reliability of the relevant test results of candidate drugs. In other words, the candidate drug has not yet shown its function or toxicity during the period of maintenance of the phenotype and function of the 3D liver model. If the candidate drug tested by the in vitro 3D liver model is used in clinical trials, there may be serious consequences such as the death of the test patient due to the toxicity of the candidate drug. It can be seen that there is an urgent need to provide a 3D liver model that can maintain the phenotype and function of the liver model for a long time for the metabolism, toxicity and efficacy testing of chronically exposed candidate drugs.

[0087] In this regard, the present application provides a culture medium and a culture method for culturing a 3D liver model. Through the culture medium and the culture method, a 3D liver model that can maintain the phenotype and function of the liver model for a long time and stably can be obtained, which can solve the problem that the existing 3D liver model is not suitable for the testing of indicators related to chronic exposure candidate drugs. Specifically, the present application adds adenylate cyclase activator and TGF-β inhibitor to the culture medium formula, and controls the dosage of adenylate cyclase activator and TGF-β inhibitor at the same time. Through the coordinated effect of the two, the long-term stability of liver cell function in vitro can be maintained, thereby greatly extending the culture time of the 3D model.

[0088] In conjunction with the accompanying drawings, the culture medium, method, model and application of the primary hepatocyte culture 3D liver model provided in the embodiment of the present application are described in detail through specific embodiments and their application scenarios.

[0089] The culture medium used for culturing the 3D liver model in this embodiment includes the following components:

[0090] 100 μL of Williams E solution,

[0091] 200nmol~2μmol of apoptosis inhibitor,

[0092] 1.5~2.5mmol amino acids,

[0093] 0.1~5.2mg / ml antibiotics,

[0094] 3~8ng / ml sodium selenite,

[0095] 3~8μg / ml transferrin,

[0096] 8~12nmol of steroid compounds,

[0097] 40~60μmol of adenylate cyclase activator,

[0098] 8~12μmol TGF-β inhibitor, 8~20μL fetal bovine serum.

[0099] Preferably, the dosage of the adenylate cyclase activator is 50 μmol, and the adenylate cyclase activator is one or more of forskolin and cyclic adenosine monophosphate; the dosage of the TGF-β inhibitor is 10 μmol, and the TGF-β inhibitor is one or more of A8301 and its salt, and SB431542. Not limited to this adenylate cyclase activator, it can also be other types of adenylate cyclase activators; the TGF-β inhibitor can also be other types of TGF-β inhibitors. More preferably, the dosage of the adenylate cyclase activator is 50 μmol, and the adenylate cyclase activator is forskolin; the dosage of the TGF-β inhibitor is 10 μmol, and the TGF-β inhibitor is SB431542. Adding 50 μmol of adenylate cyclase activator to the culture medium formula can support the expression of hepatocyte-related genes, and adding 10 μmol of SB431542 can be used to maintain the stability of hepatocytes.

[0100] Adding 40~60μmol of adenylate cyclase activator and 8~12μmol of TGF-β inhibitor to the culture medium formula also has the following effects: proteins related to epithelial-mesenchymal transition (EMT) play a great role in the normal function of hepatocytes, and adenylate cyclase activator can downregulate epithelial-mesenchymal transition marker proteins, thereby maintaining the long-term stability of hepatocyte function in vitro; during in vitro hepatocyte culture, component proteins in the TGF-β signaling pathway tend to be highly expressed, which is one of the reasons for the dedifferentiation of hepatocytes in in vitro culture. By adding TGF-β inhibitors to the culture medium, the TGF-β signal can be inhibited, which can achieve the purpose of making the phenotype and function of in vitro hepatocyte culture more stable. It can be seen that 40-60 μmol of adenylate cyclase activator and 8-12 μmol of TGF-β inhibitor are added to the culture medium of this embodiment. The synergistic effect of the two can regulate the phenotype and function of primary hepatocytes in the in vitro 3D model, so that when the 3D liver model is cultured by human primary hepatocytes, the phenotype and function of the hepatocytes in vitro can be maintained for more than 45 days, thereby greatly extending the culture time of the 3D model.

[0101] Preferably, the cell apoptosis inhibitor is zinc sulfate, and the amount of zinc sulfate is 1 μmol. Adding 1 μmol of zinc sulfate to the culture medium can be used to slow down cell apoptosis; on the other hand, zinc sulfate is selected as a cell apoptosis inhibitor, which can not only slow down cell apoptosis, but also has the effect of supporting liver cell differentiation. Zinc sulfate is synergistic with adenylate cyclase activator and TGF-β inhibitor, which can further maintain the long-term stability of liver cell function in vitro, so as to further extend the 3D model culture time.

[0102] Preferably, the amino acid is one or more of L-glutamine, proline, ornithine, N-acetylcysteine, and serine, and the amount of the amino acid is 2 mmol. Not limited thereto, the amino acid may also be other types of amino acids. The amino acid, Williams E solution, and fetal bovine serum work together to provide the required energy source for in vitro culture of hepatocytes to maintain the growth and function of hepatocytes.

[0103] More preferably, the amount of the amino acid is 2 mmol, and the amino acid is L-glutamine. Adding 2 mmol of L-glutamine to the culture medium can not only provide the energy source required for in vitro culture of hepatocytes, but also play a buffering role in the culture medium and stabilize the pH value of the culture medium. The appropriate pH value is conducive to maintaining the normal growth and function of hepatocytes.

[0104] Preferably, the antibiotics include penicillin and streptomycin. Not limited thereto, the antibiotics may also be other types of antibiotics. Adding penicillin and streptomycin to the culture medium formula can reduce the risk of microbial infection during the hepatocyte culture process.

[0105] More preferably, the antibiotics include 5.2 mg / ml penicillin and 100 μg / ml streptomycin. Penicillin and streptomycin are broad-spectrum antibiotics that have bactericidal effects on more common bacteria. By adding penicillin and streptomycin, the growth and reproduction of bacteria that may exist in the culture medium can be effectively inhibited, reducing bacterial competition on cells and potential bacterial infection risks.

[0106] Preferably, the concentration of sodium selenite is 5 ng / ml. Adding 5 ng / ml of sodium selenite to the culture medium formula can not only stabilize the redox balance in cells and reduce the generation of free radicals, thereby protecting liver cells from oxidative damage; it can also reduce the damage to liver cells caused by inflammatory reactions to maintain the healthy state of liver cells.

[0107] Preferably, the concentration of transferrin is 5 μg / ml. Adding 5 μg / ml of transferrin to the culture medium formula can be used to support the storage and transport of iron outside the liver cells. Iron is a cofactor for many enzymes and is essential for the normal metabolism and function of liver cells. At the same time, iron is also involved in biological processes such as cell redox reactions and DNA synthesis, and plays an important role in maintaining the normal function of liver cells.

[0108] Preferably, steroid compounds refer to various substances derived from steroid compounds of tetracyclic structure. The steroid compounds are selected from one or more of prednisone, methylprednisone, betamethasone, beclomethasone propionate, prednisolone, hydrocortisone, dexamethasone, and the amount of steroid compounds is 10nmol. Not limited to this, steroid compounds can also be steroid compounds of other types. Adding steroid compounds in the culture medium formula can be used to maintain the differentiation state of cells.

[0109] More preferably, the dosage of the steroid compound is 10 nmol, and the steroid compound is dexamethasone. Adding 10 nmol of dexamethasone to the culture medium can not only maintain the differentiation state of the cells, but also has a significant anti-inflammatory effect, and can reduce the inflammatory response during the hepatocyte culture process. On the other hand, the liver is an important organ of the immune system, and the overactivation of immune cells may cause damage to and fibrosis of liver cells. Adding 10 nmol of dexamethasone to the culture medium can also inhibit the activity of the immune system, reduce the production of cytokines and the proliferation of immune cells, reduce the immune response, and help protect the integrity of liver cells.

[0110] The method for culturing the 3D liver model with primary hepatocytes in this embodiment is to culture the primary hepatocytes with the culture medium of any one of the technical solutions in this embodiment.

[0111] The cultivation method of this embodiment comprises at least the following steps:

[0112] Step S100: Mixing the culture medium with the primary human hepatocytes and preparing a single cell suspension. In this step, the culture medium and the primary human hepatocytes can be mixed and prepared into a single cell suspension in an existing manner.

[0113] Step S200: By counting cells, 1500 primary hepatocytes are inoculated into a 96-well ultra-low adsorption culture plate per well, the culture plate is centrifuged at a centrifugal force of 80-120 g for 1-3 min, and the culture plate is placed in an incubator for static culture. Preferably, the culture plate is centrifuged at a centrifugal force of 100 g for 2 min. The culture conditions are: temperature 37°C, 5% CO 2 .

[0114] Step S300: After the hepatocytes in each well form 3D hepatocyte spheres and / or after 5 days of culture, 40-60% of the original culture medium is replaced with fresh culture medium. Preferably, about 50% of the original culture medium is replaced with fresh culture medium.

[0115] Step S400: In subsequent culture, 40-60% of the original culture medium is replaced with fresh culture medium at a frequency of once every two days. Preferably, about 50% of the original culture medium is replaced with fresh culture medium.

[0116] The method for culturing a 3D liver model of primary hepatocytes in this embodiment uses the culture medium of any one of the technical solutions in this embodiment to culture primary hepatocytes, which can provide the required energy source and nutrients for the culture of primary hepatocytes, and can also reduce the risk of infection during the culture process, slow down cell apoptosis, resist oxidation, support the storage and transport of extracellular iron, maintain the cell differentiation state, and maintain the long-term stability of cell function in vitro.

[0117] On the other hand, in the culture method of culturing the 3D liver model of primary hepatocytes in this embodiment, the culture plate is centrifuged at a centrifugal force of 80-120 g for 1-3 minutes, which is beneficial for maintaining a suitable degree of aggregation of the liver cells and facilitating the formation of the 3D model, avoiding excessive centrifugal force or long centrifugation time, which may cause excessive aggregation of liver cells and damage to cell activity, and may also avoid the problem of too small centrifugal force or too short centrifugation time, which may cause the liver cells to be more dispersed and difficult to form a 3D spherical model.

[0118] Thirdly, in the culture method of the primary hepatocyte culture 3D liver model of this embodiment, the culture medium is replaced so that the culture medium can provide various components required to maintain cell growth and function; the culture medium is replaced after the hepatocytes form 3D hepatocyte spheres and / or after 5 days of culture, at which time the 3D liver sphere model has been basically formed, and it is not easy to suck out the liver cells when the culture medium is replaced; when the culture medium is replaced, 40-60% of the original culture medium is replaced with fresh culture medium, which not only ensures that the culture medium can provide various components required to maintain cell growth and function, but also avoids the problem of excessive replacement of culture medium, which causes part of the liver cells to be sucked out when the culture medium is sucked out, resulting in damage to the 3D liver model.

[0119] The 3D liver model of this embodiment is based on primary hepatocytes, and the hepatocytes are obtained by culturing using the culturing method of any one of the technical solutions in this embodiment. The 3D liver model is spherical, and the phenotype and function of the 3D liver model can be maintained for more than 45 days.

[0120] The application of the 3D liver model of this embodiment is that the 3D liver model is used to study in vitro one or more of the mechanisms of hepatocyte death, hepatocyte proliferation, liver disease, hepatocyte therapy, hepatocyte transplantation, preparation of bioartificial liver, liver disease drug screening, candidate drug pharmacology and toxicology evaluation, and candidate drug metabolism research, and when the 3D liver model is used for candidate drug pharmacology and toxicology evaluation and / or candidate drug metabolism research, the candidate drug is a chronic exposure drug. Chronic exposure drugs are, for example, non-roside drugs.

[0121] The culture medium and culture method for culturing a 3D liver model of the present application are described in detail below with specific examples.

[0122] Example 1

[0123] The culture medium used for culturing the 3D liver model in this embodiment includes the following components: 100 μL of Williams E solution, 200 nmol of zinc sulfate, 1.5 mmol of L-glutamine, 0.1 mg / ml of penicillin, 100 μg / ml of streptomycin, 3 ng / ml of sodium selenite, 3 μg / ml of transferrin, 8 nmol of dexamethasone, 40 μmol of cyclic adenosine monophosphate, 8 μmol of SB431542, and 8 μL of fetal bovine serum.

[0124] The method for culturing primary hepatocytes using the above culture medium to obtain a 3D liver model comprises the following steps:

[0125] Step S100: preparing primary human hepatocytes into a single cell suspension using a culture medium;

[0126] Step S200: By counting cells, 1500 primary hepatocytes were inoculated into a 96-well ultra-low adsorption culture plate per well. The culture plate was centrifuged at 100 g for 2 min, and the culture plate was placed in an incubator for static culture. The culture conditions were: temperature 37°C, 5% CO 2 ;

[0127] Step S300: After the hepatocytes in each well form 3D hepatocyte spheres and / or after 5 days of culture, 50% of the original culture medium is replaced with fresh culture medium;

[0128] Step S400: In subsequent culture, 50% of the original culture medium is replaced with fresh culture medium every two days.

[0129] The 3D liver model obtained by the above method can maintain the phenotype and function of the 3D liver model for more than 45 days.

[0130] Example 2

[0131] The culture medium used for culturing the 3D liver model in this embodiment includes the following components: 100 μL of Williams E solution, 1 μmol of zinc sulfate, 2 mmol of L-glutamine, 5.2 mg / ml of penicillin, 100 μg / ml of streptomycin, 5 ng / ml of sodium selenite, 5 μg / ml of transferrin, 10 nmol of dexamethasone, 50 μmol of forskolin, 10 μmol of SB431542, and 10 μL of fetal bovine serum.

[0132] The culture method for culturing primary hepatocytes using the above culture medium to obtain a 3D liver model is the same as in Example 1.

[0133] The 3D liver model obtained by the above method can maintain the phenotype and function of the 3D liver model for more than 45 days.

[0134] Example 3

[0135] The culture medium used for culturing the 3D liver model in this embodiment includes the following components: 100 μL of Williams E solution, 2 μmol of zinc sulfate, 2.5 mmol of L-glutamine, 2.6 mg / ml of penicillin, 100 μg / ml of streptomycin, 8 ng / ml of sodium selenite, 8 μg / ml of transferrin, 12 nmol of dexamethasone, 60 μmol of forskolin, 12 μmol of SB431542, and 20 μL of fetal bovine serum.

[0136] The culture method for culturing primary hepatocytes using the above culture medium to obtain a 3D liver model is the same as in Example 1.

[0137] The 3D liver model obtained by the above method can maintain the phenotype and function of the 3D liver model for more than 45 days.

[0138] The morphology, cell viability, albumin secretion, urea synthesis and liver drug enzyme metabolism data of the 3D hepatocytes obtained in Examples 1 to 3 were tested below.

[0139] Morphology of 3D hepatocytes: On the 5th, 10th, 15th, 20th, 25th, 30th, 35th, 40th, and 45th days of liver cell culture, H&E staining was performed on the liver cells, and the cell morphology was observed under a microscope. The detection results of 3D hepatocytes obtained in Examples 1 to 3 are as follows: Figure 1 , Fig.10 and Fig.19 shown.

[0140] from Figure 1 , Fig.10 and Fig.19 It can be seen that spheroids began to form on the fifth day of liver cell culture, that is, 3D liver cells were formed; in addition, Figure 1 , Fig.10 and Fig.19 It can also be seen that during the culture process from day 5 to day 45, the 3D liver cell spheres always maintained a complete morphology, and the clear cell sphere edges proved that the cells were supported by each other stably. In particular, the cell spheres of Example 2 were slightly transparent from beginning to end, indicating that the cell spheres had good vitality.

[0141] Cell viability of 3D hepatocytes: On the 5th, 10th, 15th, 20th, 25th, 30th, 35th, 40th, and 45th days of liver cell culture, the ATP content of a single 3D hepatocyte spheroid was measured by cell titer-green luminescent cell viability assay (Promega, Sweden), and the cell viability was evaluated by ATP level. The detection results of 3D hepatocytes obtained in Examples 1 to 3 are as follows: Figure 2 , Fig.11 and Fig. 20 shown.

[0142] The cell viability of 3D hepatocyte spheroids is an important criterion for evaluating whether the model can be applied to drug research. Figure 2 , Fig.11 and Fig. 20 It can be seen that during the 45-day culture process, the 3D hepatocyte spheroids as a whole maintained a high activity similar to the initial level.

[0143] It should be noted that Figure 2 , Fig.11 and Fig. 20 The data in the figure are relative values ​​based on the test results on the first day. In the test results on the 15th, 20th and 25th days, the cell viability was slightly higher than 100%, which may be due to a certain deviation in the measurement results on the first day. Figure 3 , Fig.12 , Fig.21 and Fig.28 There is a similar situation in .

[0144] Albumin secretion: On the 5th, 10th, 15th, 20th, 25th, 30th, 35th, 40th and 45th days of liver cell culture, the cell culture medium was collected at the same time point, and the albumin concentration was determined by enzyme-linked immunosorbent assay. The detection results of 3D hepatocytes obtained in Examples 1 to 3 were as follows: Figure 3 , Fig.12 and Fig.21 As shown. Similarly, Figure 3 , Fig.12 and Fig.21 The data obtained are relative values ​​based on the test results on the first day.

[0145] The amount of albumin secretion is an important characteristic of the in vitro hepatocyte model. Figure 3 , Fig.12 and Fig.21It can be seen that during the 45-day culture process, the albumin concentration in the culture medium was always maintained at a level higher than 60%, and the albumin secretion was relatively stable, proving that the primary hepatocytes in the cell spheres performed normal functions. During the culture process, the albumin secretion decreased slightly while remaining relatively stable, which may be due to reasons such as cell quality, culture medium composition, and culture operation.

[0146] Urea synthesis: On the 5th, 10th, 15th, 20th, 25th, 30th, 35th, 40th and 45th days of liver cell culture, the cell culture medium was collected at the same time point, and the urea synthesis was determined by a colorimetric endpoint test. The detection results of the 3D liver cells obtained in Examples 1 to 3 are as follows: Figure 4 , Fig.13 and Fig. 22 As shown. Similarly, Figure 4 , Fig.13 and Fig. 22 The data obtained are relative values ​​based on the test results on the first day.

[0147] Urea synthesis is an important characteristic of in vitro hepatocyte models. Figure 4 , Fig.13 and Fig. 22 It can be seen that during the 45-day culture process, the urea concentration in the culture medium was relatively stable, proving that the primary hepatocytes in the cell spheres performed normal functions. During the culture process, the amount of urea synthesis decreased slightly while remaining relatively stable, which may be due to reasons such as cell quality, culture medium composition, and culture operation.

[0148] Hepatic drug enzyme metabolism data: the amount of acetaminophen generated is used to characterize the activity of CYP1A2 (cytochrome P450 enzyme 1A2); the amount of 1-hydroxymidazolam generated is used to characterize the activity of CYP3A4 (cytochrome P450 enzyme 3A4); the amount of dextrorphan generated is used to characterize the activity of CYP2D6 (cytochrome P450 enzyme 2D6); the amount of amodiaquine generated is used to characterize the activity of CYP2C8 (cytochrome P450 enzyme 2C8); the amount of hydroxy-tolbutamide generated is used to characterize the activity of CYP2C9 (cytochrome P450 enzyme 2C9).

[0149] Liver cells were incubated with the probe substrate mixture, and the metabolites formed by CYP1A2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9 were collected on days 5, 10, 15, 20, 25, 30, 35, 40, and 45 of the liver cell culture. The metabolites (acetaminophen, 1-hydroxymidazolam, dextrorphan, amodiaquine, and hydroxy-tolbutamide) were quantified by LC / MS-MS.

[0150] The test results of CYP1A2, CYP3A4, CYP2D6, CYP2C8 and CYP2C9 activities of 3D hepatocytes obtained in Example 1 are as follows: Figure 5~Figure 9 As shown, the detection results of CYP1A2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9 activities of 3D hepatocytes obtained in Example 2 are as follows: Figure 14~Figure 18 As shown, the detection results of CYP1A2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9 activities of 3D hepatocytes obtained in Example 3 are as follows: Figure 23~Figure 27 shown.

[0151] CYP1A2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9 are important drug-metabolizing enzymes, among which the most important CYP2D6 and CYP3A4 are involved in the first-phase metabolism of almost all drugs. Figure 5~Figure 9 , Figure 14~Figure 18 , Figure 23~Figure 27 It can be seen that hepatocyte spheres can produce similar levels of metabolites during the 45-day culture process, proving that the activity of hepatic drug enzymes in the 3D hepatocyte model is stable and can perform normal metabolic functions.

[0152] Furthermore, the difference between the culture medium of Example 2 and the culture medium in the related art is illustrated by the following two comparative examples.

[0153] Comparative Example 1

[0154] The culture medium used for culturing the 3D liver model in this embodiment includes the following components: 100 μL of Williams E solution, 50 μmol of insulin, 2 mmol of L-glutamine, 5.2 mg / ml of penicillin, 100 μg / ml of streptomycin, 5 ng / ml of sodium selenite, 5 μg / ml of transferrin, 10 nmol of dexamethasone, 50 μmol of forskolin, 10 μmol of SB431542, and 10 μL of fetal bovine serum.

[0155] The primary hepatocytes were cultured using the above culture medium, and the culture method was the same as that in Example 1.

[0156] Comparative Example 2

[0157] The culture medium used for culturing the 3D liver model in this embodiment includes the following components: 100 μL of Williams E solution, 50 μmol of insulin, 2 mmol of L-glutamine, 5.2 mg / ml of penicillin, 100 μg / ml of streptomycin, 5 ng / ml of sodium selenite, 5 μg / ml of transferrin, 10 nmol of dexamethasone, and 10 μL of fetal bovine serum.

[0158] The primary hepatocytes were cultured using the above culture medium, and the culture method was the same as that in Example 1.

[0159] Comparative Example 3

[0160] The culture medium used for culturing the 3D liver model in this embodiment includes the following components: 100 μL of Williams E solution, 1 μmol of zinc sulfate, 2 mmol of L-glutamine, 5.2 mg / ml of penicillin, 100 μg / ml of streptomycin, 5 ng / ml of sodium selenite, 5 μg / ml of transferrin, 10 nmol of dexamethasone, and 10 μL of fetal bovine serum.

[0161] The primary hepatocytes were cultured using the above culture medium, and the culture method was the same as that in Example 1.

[0162] On the 5th, 10th, 15th, 20th, 25th, 30th, 35th, 40th and 45th days of liver cell culture, the ATP content of the single 3D liver cell spheroids in Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3 was measured by cell titer-green luminescent cell viability assay (Promega, Sweden), and the cell viability was evaluated by ATP level. The test results are as follows: Fig.28 shown.

[0163] The viability of 3D hepatocyte spheroids is an important criterion for evaluating whether the model can be applied to drug research. Fig.28 It can be seen that the 3D hepatocyte spheroids of Example 2 maintained high activity similar to the initial level as a whole during the 45-day culture process; after the 30th day, the cell viability of Example 2 was significantly higher than that of Comparative Examples 1, 2 and 3.

[0164] Similarly, in the test results on the 15th, 20th, and 25th days, the cell viability was slightly higher than 100%, which may be due to measurement errors.

[0165] When the liver cells of Example 2 were cultured to the 45th day, the liver cells of Comparative Example 1 were cultured to the 40th day, the liver cells of Comparative Example 2 were cultured to the 40th day, and the liver cells of Comparative Example 3 were cultured to the 30th day, the liver cells were stained with H&E, and the cell morphology was observed under a microscope. The test results are as follows: Fig.28 shown.

[0166] from Fig.28It can be seen that the liver cells of Example 2 were cultured to the 45th day, and the 3D liver cell spheres always maintained a complete morphology. The clear edges of the cell spheres proved that the cells were supported on each other stably, and the cell spheres were slightly transparent from beginning to end, indicating that the cell spheres were active. The liver cells of Comparative Examples 1 and 2 were cultured to the 40th day, and the integrity of the 3D liver cell sphere morphology, the clarity of the cell sphere edges, and the transparency of the cell spheres were slightly lower than those of Example 2. The liver cells of Comparative Example 3 were cultured to the 30th day, and the integrity of the 3D liver cell sphere morphology, the clarity of the cell sphere edges, and the transparency of the cell spheres were slightly lower than those of Example 2, and even lower than the liver cell spheres of Comparative Example 1 on the 40th day.

[0167] From the above comparison, it can be seen that the culture medium obtained in Example 2 of the present application is used to culture primary hepatocytes, and the phenotype and function of the hepatocytes in vitro can be maintained for more than 45 days, which is significantly better than the 3D hepatocyte spheres cultured with the culture medium obtained in Comparative Examples 1, 2 and 3.

[0168] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0169] In addition, it should be noted that the scope of the methods and devices in the embodiments of the present application is not limited to performing functions in the order shown or discussed, and may also include performing functions in a substantially simultaneous manner or in a reverse order according to the functions involved. For example, the described methods may be performed in an order different from that described, and various steps may be added, omitted, or combined. In addition, features described with reference to certain examples may be combined in other examples.

[0170] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A culture medium for culturing primary hepatocytes into a 3D liver model, characterized in that: For culturing primary human hepatocytes, the culture medium comprises the following components: 100 μL of Williams E solution, 200nmol~2μmol of apoptosis inhibitor, 1.5~2.5mmol amino acids, 0.1~5.2mg / ml antibiotics, 3~8ng / ml sodium selenite, 3~8μg / ml transferrin, 8~12nmol of steroid compounds, 40~60μmol of adenylate cyclase activator, 8~12μmol of TGF-β inhibitor, 8-20 μL fetal bovine serum; Wherein, the cell apoptosis inhibitor is zinc sulfate; The amino acid is L-glutamine; The antibiotics are penicillin and streptomycin; The steroid compound is dexamethasone; The adenylate cyclase activator is forskolin or cyclic adenosine monophosphate; The TGF-β inhibitor is SB431542.

2. The culture medium for culturing a 3D liver model with primary hepatocytes according to claim 1, characterized in that: The dosage of the adenylate cyclase activator is 50 μmol; The dosage of the TGF-β inhibitor is 10 μmol.

3. The culture medium for culturing a 3D liver model with primary hepatocytes according to claim 1, characterized in that: The dosage of the zinc sulfate is 1 μmol.

4. The culture medium for culturing a 3D liver model with primary hepatocytes according to claim 1, characterized in that: The amount of the amino acid used is 2 mmol.

5. The culture medium for culturing a 3D liver model with primary hepatocytes according to claim 1, characterized in that: The concentration of penicillin is 5.2 mg / ml, and the concentration of streptomycin is 100 μg / ml.

6. The culture medium for culturing a 3D liver model with primary hepatocytes according to claim 1, characterized in that: The concentration of sodium selenite is 5 ng / ml; The concentration of transferrin is 5 μg / ml; The dosage of the steroid compound is 10 nmol.

7. The culture medium for culturing a 3D liver model with primary hepatocytes according to any one of claims 1 to 6, characterized in that: The composition includes the following formula: 100 μL of Williams E solution, 1 μmol of zinc sulfate, 2 mmol of L-glutamine, 5.2 mg / ml of penicillin, 100 μg / ml of streptomycin, 5 ng / ml of sodium selenite, 5 μg / ml of transferrin, 10 nmol of dexamethasone, 50 μmol of forskolin, 10 μmol of SB431542, and 10 μL of fetal bovine serum.

8. A method for culturing a 3D liver model using primary hepatocytes, characterized in that: The culture method is to culture human primary hepatocytes using the culture medium of the primary hepatocyte culture 3D liver model according to any one of claims 1 to 7, and the culture method comprises at least the following steps: Step S100: mixing the culture medium with primary human hepatocytes to prepare a single cell suspension; Step S200: by counting cells, 1500 primary hepatocytes are inoculated into a 96-well ultra-low adsorption culture plate per well, the culture plate is centrifuged at a centrifugal force of 80-120 g for 1-3 minutes, and the culture plate is placed in an incubator for static culture; Step S300: After the hepatocytes in each well form 3D hepatocyte spheres and / or after 5 days of culture, 40-60% of the original culture medium is replaced with fresh culture medium; Step S400: In subsequent culture, 40-60% of the original culture medium is replaced with fresh culture medium at a frequency of once every two days.

Images