In vitro 3d liver model and enteroliver co-culture model and methods of establishing and using the same

Abstract

The present application relates to a kind of in vitro 3D liver model and enterohepatic co-culture model and its establishment method and application, belong to the technical field of drug evaluation.The method includes the following steps: cell culture: HepaRG cell and human hepatic stellate cell are respectively carried out cell culture, standby;Cell induction: the above-mentioned HepaRG cell is seeded in culture flask and is carried out cell culture, after HepaRG cell adherent growth reaches predetermined quantity, it is replaced with induction medium and is carried out induction culture, and the induced HepaRG cell is obtained, standby;Model construction: the induced HepaRG cell and human hepatic stellate cell are made into mixed cell suspension, seed on predetermined carrier, culture, obtain the microtissue with the three-dimensional structure of liver nodule, i.e. 3D liver model.The model has the three-dimensional structure of liver nodule microtissue of liver, can well simulate the actual physiological condition of in-vivo liver, to accurately predict the liver toxicity of drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug evaluation, in particular to an in vitro 3D liver model and an intestinal-liver co-culture model, a method for establishing the same and an application thereof. BACKGROUND

[0002] Drug-induced liver injury has been the main cause of acute liver injury and withdrawal of marketed drugs. However, due to the difference in liver function between humans and animals, preclinical animal experiments are difficult to accurately predict the metabolism and toxicity of drugs. In recent years, with the development of cell biology, many ex vivo human models have been applied to drug liver toxicity research, such as human liver microsomes, human hepatocytes, primary hepatocytes, etc. Human hepatocytes can be expanded and passaged, and also have the functional characteristics of whole cells, and are widely used in drug toxicity screening, but most immortalized hepatocytes lose part of the liver-specific metabolic function of human hepatocytes, which brings difficulties to accurately evaluate drug liver metabolism. Primary hepatocytes are considered to be the gold standard for studying drug toxicity and metabolism in ex vivo liver models.

[0003] The expression level of liver-specific genes of the isolated primary hepatocytes is high, and the drug metabolism function of the liver is maintained. However, human primary hepatocytes are limited in source, and the sample difference is large, single cell plate culture cannot simulate the actual physiological conditions in vivo, and the liver function is rapidly lost in the standard 2D culture environment in vitro, which brings challenges to the evaluation of drug long-term toxicity and metabolism in vitro. The existing 3D model in vitro also uses human primary hepatocytes, which overcomes the shortcomings of 2D culture, but is also limited by the defects of limited source and large sample difference, which is difficult to promote on a large scale.

[0004] HepaRG is the most popular immortalized human liver cell in the world at present, and studies have shown that HepaRG cells can form 3D cell spheres in vitro or form 3D liver models on scaffolds, which have advantages in metabolism and toxicity evaluation. However, HepaRG cells lack the interaction of non-parenchymal cells in the liver in vivo, which causes a series of problems such as low stability of the 3D model in vitro and inability to form a simulated liver tissue in vivo.

[0005] In recent years, a series of models have appeared to co-culture HepaRG and different liver non-parenchymal cells to better simulate the physiological structure of the liver in vivo, however, there is still a lack of comprehensive and systematic optimization to evaluate the ability of these models to simulate liver function and the application advantages in drug toxicity and new material evaluation.

[0006] In addition, the small intestine absorption and liver metabolism together constitute the first pass barrier of the oral drug into the body, and the absorption and metabolism of the drug in the small intestine have a huge impact on the drug liver toxicity. A large number of studies have shown that the absorption and metabolism of the drug in the small intestine accounts for 15-50% of the liver metabolism, and the small intestine absorption and metabolism of the drug is the first step to accurately study and evaluate the drug in vivo distribution and metabolism. The small intestine absorption model Caco-2 human colon adenocarcinoma cell is an in vitro model widely recognized by the US FDA and the European Medicines Agency EMA to evaluate the absorption and metabolism characteristics of the drug. A large number of studies have shown that Caco-2 has good human correlation in predicting the small intestine permeability of the drug. However, the evaluation of the change of the drug metabolism toxicity in the liver after the small intestine absorption and metabolism still depends on animal experiments.

[0007] Therefore, at present, there is a lack of a liver model which can simulate the actual physiological conditions of the liver in vivo and maintain the function of the liver in vivo for a long time, and a stable combination of the model and the intestinal model, so that there is a lack of an in vitro model which can accurately predict the intestinal absorption and liver toxicity of the drug in high throughput, especially the in vitro toxicity evaluation of multiple and long-term drug administration. SUMMARY

[0008] Therefore, it is necessary to provide an establishment method of an in vitro 3D liver model in view of the above problems. The liver model established by the method can well simulate the actual physiological conditions of the liver in vivo, so as to accurately predict the liver toxicity of the drug.

[0009] An establishment method of an in vitro 3D liver model, comprising the following steps:

[0010] Cell culture: HepaRG cells and human hepatic stellate cells are respectively cultured, and are ready for use;

[0011] Cell induction: the above-mentioned HepaRG cells are inoculated in a culture bottle for cell culture, and after the HepaRG cells are adhered and grown to a predetermined number, an induction medium is replaced for induction culture, so as to obtain induced HepaRG cells, which are ready for use;

[0012] Model construction: the induced HepaRG cells and human hepatic stellate cells are made into a mixed cell suspension, inoculated on a predetermined carrier, and cultured to obtain a microtissue with a liver nodule three-dimensional structure, that is, a 3D liver model.

[0013] The method for establishing the above-mentioned 3D liver model in vitro, by selecting HepaRG cells and human hepatic stellate cells for co-culture, the human hepatic stellate cells can participate in the formation of liver fibrosis by proliferation and secretion of extracellular matrix, and the reconstruction of the intrahepatic structure of the liver parenchymal cells HepaRG cells is strengthened, and a model system simulating the in vivo physiology is constructed by mixing the liver parenchymal cells and non-parenchymal cells, the model has the microtissue of the three-dimensional structure of the liver nodule of the liver, and can well simulate the actual physiological condition of the liver in vivo, so as to accurately predict the liver toxicity of drugs.

[0014] In one embodiment, the culture medium of the HepaRG cells and the human hepatic stellate cells in the cell culture step is complete culture medium containing 2-20% fetal bovine serum. Preferably, the complete culture medium contains 10% fetal bovine serum, more preferably RPMI 1640 complete culture medium containing 2% DMSO and 10% fetal bovine serum. After the inventors' exploration, comparison and screening, the above-mentioned culture medium is selected, and different cell lines are successfully domesticated in a relatively simple culture system, and have the advantage of good growth.

[0015] In one embodiment, the induction culture medium in the cell induction step is complete culture medium containing 0.5-3% DMSO (dimethyl sulfoxide) and 2-20% fetal bovine serum. Preferably, the complete culture medium contains 1.5-2.5% DMSO and 8-12% fetal bovine serum, more preferably RPMI 1640 complete culture medium containing 2% DMSO and 10% fetal bovine serum.

[0016] In one embodiment, the predetermined number in the cell induction step is that the HepaRG cells are normally adherent to grow to cover 75%-95% of the area of the bottle bottom. Preferably, 80%-90%.

[0017] In one embodiment, the cell culture conditions are that the cells are placed in a culture box at 35-39°C, containing 4-6% CO2 and a relative humidity of 85-95%, and the culture medium is replaced every 1-3 days;

[0018] The induction culture conditions are that the HepaRG cells are placed in a culture box at 35-39°C, containing 4-6% CO2 and a relative humidity of 85-95%, and the culture medium is replaced every day, and the induction culture is performed for 12-16 days.

[0019] In one embodiment, the number ratio of the HepaRG cells and the human hepatic stellate cells in the mixed cell suspension in the model construction step is 20:1-5:1. Preferably, 8:1-12:1, more preferably 10:1. The specific ratio can make the human hepatic stellate cells and the HepaRG cells better cooperate with each other, and obtain a better model system simulating the in vivo physiology.

[0020] In one of the embodiments, in the model construction, the predetermined carrier is a hanging drop plate, and the specific method for constructing the hanging drop 3D liver model is as follows:

[0021] Preparation of the mixed cell suspension: the induced HepaRG cells and human hepatic stellate cells are prepared into a mixed cell suspension containing 6x10 4 -10x10 4 HepaRG cells and 0.3x10 4 -2.0x10 4 human hepatic stellate cells per 40 μl;

[0022] Inoculation: the above mixed cell suspension is inoculated into the hanging drop plate at an amount of 30-50 μl / well, and the humidity of the hanging drop plate is controlled to reduce the liquid evaporation of the hanging drop system;

[0023] Culture: the above inoculated hanging drop plate is placed in a culture box at 35-39°C, containing 4-6% CO2 and a relative humidity of 85-95%, for culture until microtissues are formed;

[0024] Model construction: the culture medium is injected into the above hanging drop plate, so that the above microtissues are transferred to a collection plate, and the culture medium is replaced at a regular time, to obtain a hanging drop 3D liver model;

[0025] Alternatively

[0026] The predetermined carrier is a spherical microwell plate, and the specific method for constructing the microwell 3D liver model is as follows:

[0027] Preparation of the mixed cell suspension: the induced HepaRG cells and human hepatic stellate cells are prepared into a mixed cell suspension containing 6x10 4 -10x10 4 HepaRG cells and 0.3x10 4 -2.0x10 4 human hepatic stellate cells per 50 μl;

[0028] Inoculation and culture: the above mixed cell suspension is inoculated into the spherical microwell plate at an amount of 30-70 μl / well, and the above inoculated spherical microwell plate is placed in a culture box at 35-39°C, containing 4-6% CO2 and a relative humidity of 85-95%, for culture until microtissues are formed; preferably, it is inoculated into a 384-well spherical microwell plate at an amount of 40-60 μl / well;

[0029] Model construction: the culture medium in the above spherical microwell plate with microtissues is replaced, and the culture medium is replaced at a regular time, to obtain a microwell plate model;

[0030] Alternatively

[0031] The predetermined carrier is a 3D culture scaffold, and the specific method for constructing the 3D liver model is as follows:

[0032] Preparation of mixed cell suspension: the induced HepaRG cells and human hepatic stellate cells are prepared into a mixed cell suspension containing 4×10 5 -6×10 5 HepaRG and 0.2×10 5 -1.2×10 5 human hepatic stellate cells per 60 μl;

[0033] Inoculation: the above mixed cell suspension is inoculated on the 3D culture scaffold placed in the cell culture plate well;

[0034] Culture: the cell culture plate after inoculation is placed in a culture box at 35-39℃, containing 4-6% CO2 and a relative humidity of 85-95%, and is left to stabilize, and then the culture medium is added to immerse the 3D culture scaffold, and the inoculation is incubated for a predetermined time;

[0035] Model establishment: the 3D culture scaffold after incubation is transferred to another cell culture plate, the culture medium is supplemented, and the culture medium is replaced at a fixed time, and the 3D liver model is obtained.

[0036] In the above model establishment step, the cells that do not successfully adhere to the inside of the scaffold and grow on the culture plate during inoculation are removed by transferring the incubated 3D culture scaffold to a new cell culture plate, so that the influence of the cells on the 3D culture system is eliminated.

[0037] In one embodiment, in the construction of the hanging drop 3D liver model, in the inoculation step, the liquid evaporation of the hanging drop system is reduced by adding water to the surrounding channel of the hanging drop plate;

[0038] In the model establishment step, the culture medium injected into the hanging drop plate is 50-90 μL / well, and after the microtissue is transferred to the collection plate, it is centrifuged at 150-250 r / min for 1-3 min, the supernatant is discarded, 50-90 μL of culture medium is added, and then the culture medium is replaced every 1-3 days to obtain the hanging drop 3D liver model;

[0039] In the construction of the microhole 3D liver model, in the model establishment step, 1 / 3-2 / 3 of the supernatant is removed, and an equal amount of culture medium is added for replacement;

[0040] In the seeding step, the mixed cell suspension is added dropwise to the cell culture plate in an amount of 15-25 μl / well, and then the 3D culture scaffold is placed on the cell suspension, and then the mixed cell suspension is added dropwise to the 3D culture scaffold in an amount of 30-50 μl / well, and the liquid is blown to make the cell seeding on both sides of the 3D culture scaffold uniform; in the culture step, the standing time is 3-5 h, and the incubation time is 12-36 h.

[0041] Through the above specific operation, the 3D liver model can be better established.

[0042] The application further discloses the 3D liver model established by the method for establishing an in-vitro 3D liver model.

[0043] The 3D liver model has a microtissue with a three-dimensional structure of liver nodule, can well simulate the actual physiological condition of the liver in vivo, and thus accurately predicts the liver toxicity of a drug.

[0044] The application further discloses application of the in-vitro 3D liver model in in-vitro liver toxicity evaluation.

[0045] In one embodiment, the in-vitro 3D liver model is applied to preparation of reagents and / or equipment for in-vitro liver toxicity evaluation.

[0046] The application further discloses a method for establishing an in-vitro intestinal-liver co-culture model, comprising the following steps.

[0047] Establishing a small intestine absorption model: a small intestine absorption model is established by Caco-2 cells in a Transwell chamber;

[0048] Establishing an intestinal-liver co-culture model: the 3D liver model is transferred to the receiving pool side of the Transwell chamber, and thus the intestinal-liver co-culture model is obtained.

[0049] The in-vitro intestinal-liver co-culture model established by the method can be stably combined, can maintain the reasonable permeability of the intestinal model and the function of the liver model, and is successfully applied to in-vitro high-throughput accurate prediction of drug intestinal permeability and liver toxicity.

[0050] In one embodiment, in the step of establishing a small intestine absorption model, complete culture medium containing 2-20% fetal bovine serum is added to the receiving pool side of the Transwell chamber, and then Caco-2 cell suspension is added to the supply side of the Transwell chamber, so that the cell density is equivalent to 0.5*10 6 -1.5*10 6 cells / 1.12cm 2, cell culture is carried out until the transmembrane resistance is greater than 180 Ω / cm 2 The small intestine absorption model is obtained.

[0051] In the establishment of the intestinal-liver co-culture model, when the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models are transferred per 1.12 cm 2 In the establishment of the intestinal-liver co-culture model, when the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models are transferred per 1.12 cm 2 In the establishment of the intestinal-liver co-culture model, when the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models are transferred per 1.12 cm 2 In the establishment of the intestinal-liver co-culture model, when the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models are transferred per 1.12 cm

[0052] When different surface area Inserts are used for culture, the number of 3D liver models transferred is increased or decreased in proportion to the change in the area of the small intestine.

[0053] For example, a 24-well culture plate / Insert is used to construct the co-culture model (the surface area of the Insert is 1.12 cm 2 If the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models are transferred per well; if the 3D liver model is a microwell 3D liver model, 15-25 microwell 3D liver models are transferred per well; and if the 3D liver model is a scaffold 3D liver model, 1-2 scaffold 3D liver models are transferred per well.

[0054] The application further discloses an intestinal-liver co-culture model prepared by the method.

[0055] The application further discloses application of the intestinal-liver co-culture model in in-vitro intestinal absorption and liver toxicity evaluation.

[0056] In one embodiment, the intestinal-liver co-culture model is used in preparation of reagents and / or equipment for in-vitro intestinal absorption and liver toxicity evaluation.

[0057] Compared with the prior art, the application has the following beneficial effects:

[0058] The 3D liver model prepared by the method can better simulate in-vivo liver function and liver enzyme activity, thereby accurately predicting metabolic toxicity changes of drugs in the liver, especially in-vitro toxicity evaluation of multiple and long-term administration, and can be used for reasonably evaluating in-vivo absorption and liver toxicity of new dosage forms such as nanoparticles.

[0059] And, the in vitro 3D liver model can be combined with an intestinal model stably, while maintaining the reasonable permeability of the intestinal model and the function of the liver model, and is successfully applied to in vitro high-throughput accurate prediction of drug intestinal permeability and liver toxicity.

[0060] Further, the various cell lines of the above model are successfully mixed and cultured in the same culture medium and maintain good functions respectively, so that the manufacturing cost of the model is low, and the model is conducive to large-scale popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 HepaRG cells (20x) without induction;

[0062] Figure 2 HepaRG cells (20x) after induction for 5 days in culture medium containing 2% DMSO;

[0063] Figure 3 HepaRG cells (40x) after induction for 14 days in culture medium containing 2% DMSO;

[0064] Wherein, H represents hepatocyte-like cells, and EP represents epithelial cells;

[0065] Figure 4 Schematic diagram of hanging drop inoculation;

[0066] Figure 5 Microtissues attached to the collection plate (10x);

[0067] Figure 6 Microtissues covering the bottom of the well (10x);

[0068] Figure 7 Schematic diagram of scaffold inoculation;

[0069] Figure 8 Schematic diagram of scaffold 3D liver model cell mass (40x), wherein A is a Cellusponge scaffold, and B is a Go-Matrix scaffold;

[0070] Figure 9 Expression of P-gp and MCT at different culture times.

[0071] Figure 10 Expression levels of albumin in 2D and 3D models at different culture times: A is a hanging drop model, B is a micro-well model, C is a GO-Matrix scaffold, and D is a CelluSponge scaffold;

[0072] Figure 11Expression levels of urea in 2D and 3D models at different culture times: A for hanging drop model, B for microwell model, C for GO-Matrix scaffold, D for CelluSponge scaffold;

[0073] Figure 12 Expression levels of CYP3A4 in 2D and 3D models at different culture times and expression levels after treatment with inducer and inhibition of CYP3A4 activity: A for hanging drop model, B for microwell model, C for GO-Matrix scaffold, D for CelluSponge scaffold;

[0074] Figure 13 Fold expression levels of CYP3A4, CYP2B6, CYP2C9 and P-gp in 3D models compared to 2D models at different culture times: A for hanging drop model, B for microwell model, C for GO-Matrix scaffold, D for CelluSponge scaffold;

[0075] Figure 14 Expression of albumin in 3D liver models and 3D enteric liver models at different culture times: A for hanging drop model, B for microwell model, C for GO-Matrix scaffold, D for CelluSponge scaffold; wherein the culture time is the culture time after the establishment of the liver-intestine model, not the culture time after the establishment of the liver model;

[0076] Figure 15 Fold expression levels of CYP3A4, CYP2B6, CYP2C9 and P-gp in 3D liver models and 3D enteric liver models compared to 2D models at different culture times: A for hanging drop model, B for microwell model, C for GO-Matrix scaffold, D for CelluSponge scaffold. DETAILED DESCRIPTION

[0077] For the purposes of promoting an understanding of the principles of the application, reference will now be made to the embodiments illustrated in the drawings. It is expressly understood that the drawings are for illustrative purposes and are not a limitation on the scope of the application. In addition, it is to be understood that the specific elements and sequence of steps described or illustrated are intended to be exemplary only. As such, other elements and sequences that are functionally similar can be substituted for those specifically described or illustrated without departing from the spirit and scope of the present application.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this description, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. The term "and / or" includes any and all combinations of one or more of the associated listed items.

[0079] The reagents, cell lines and materials used in the following examples are commercially available.

[0080] Example 1

[0081] Establishment of an in vitro 3D liver model, comprising the following steps:

[0082] I. Cell culture.

[0083] HepaRG cells (source: ATCC, purchased by Guangzhou Jinieer Biological Co., Ltd.) and human hepatic stellate cells (Human Hepatic Stellate Cell, HHSC, source: ScienCell, purchased by Beijing Yuhengfeng Technology Co., Ltd.) were respectively inoculated in RPMI 1640 complete medium (containing 10% FBS) at a concentration of 2 x 10 cells / mL, and the cells grew well and were ready for use.

[0084] The above cells were cultured in a 37°C incubator containing 5% CO 2 2 and a relative humidity of 90%. The culture medium was changed every other day, and the cells were subcultured after 2-3 days of adherent growth, covering 80%-90% of the bottom of the bottle.

[0085] II. Cell induction.

[0086] In a T150 conventional culture bottle, 2 x 10 6 HepaRG cells were inoculated per bottle, and the cells were cultured in 1640 complete medium containing 10% FBS at 37°C in a 5% CO2 and 90% relative humidity incubator. The culture medium was changed every other day. After the cells normally adhered and grew to cover 80%-90% of the bottom of the bottle, fresh RPMI 1640 complete medium containing 2% DMSO and 10% FBS was used for induction culture in the incubator, and the culture medium was changed every day. The induction culture was performed for 14 days.

[0087] The above uninduced HepaRG cells were plump in shape, had good refractive index, and the cytoplasm was clearly visible, with clear cell-cell differentiation, as shown in Figure 1 After 5 days of culture in medium containing 2% DMSO, the cells gradually differentiated into two morphologies, flat and granular, as shown in Figure 2 After 14 days of 2% DMSO induction, the cells became flat and transparent in cytoplasm, as shown in Figure 3 .

[0088] III. Model construction.

[0089] 1. Suspension drop 3D liver model construction.

[0090] 1.1 Preparation of culture materials:

[0091] ① Use tweezers to remove a humidifying pad and immerse it in 20 mL of 0.5×PBS until completely soaked (about 5 min).

[0092] ② Place the fully soaked humidifying pad in the water storage tank of the drip tray.

[0093] 1.2 Preparation of cell suspension:

[0094] After HepaRG cell induction, the cells were digested with trypsin, and the digestion was terminated with culture medium. The cells were then centrifuged at 1000 rpm for 5 min and prepared into cells with a density of 4 × 10⁶ cells / cm² using RPMI 1640 complete medium containing 10% FBS. 6 HepaRG cell suspension at a density of 4 × 10⁶ cells / mL; it is expected that HHSCs (human hepatic stellate cells) will grow to 80%–90% of the culture flask when HepaRG cell induction is just completed, and at the same time, HHSCs will be prepared to a density of 4 × 10⁶ cells / mL using the above method. 6 HHSC cell suspension was prepared at 8 × 10⁻⁶ cells / mL. Then, HepaRG cell suspension and HHSC cell suspension were mixed in equal volumes until homogeneous, achieving a density of (8 × 10⁻⁶ cells / mL). 4 HepaRG+0.8×10 4 A mixed cell suspension of HHSC cells / 40 μl.

[0095] 1.3 Vaccination:

[0096] Gently drop 40 μl of mixed cell suspension per well into the pendant plate to form a hanging drop, as shown below. Figure 4 As shown in the diagram, the suspended semicircle represents the hanging drop. Adding 2 ml of sterile water to the channels around the plate increases humidity, thereby effectively reducing liquid evaporation in the hanging drop system.

[0097] 1.4 Cultivation:

[0098] Cover the container and incubate at 37°C in an incubator with 5% CO2 and 90% relative humidity. Cells aggregate one day after inoculation and grow to form micro-tissues four days later.

[0099] 1.5 Model Establishment:

[0100] Four days after inoculation, gently aspirate 70 μL of fresh RPMI 1640 complete medium containing 10% FBS into each well of the droplet plate, and transfer the microtissue to a low-adsorption collection plate, as follows. Figure 5 As shown, centrifuge at 200 RCF (rpm) for 2 min, discard the supernatant and replace with 70 μL of fresh RPMI 1640 complete medium containing 10% FBS. Continue culturing, changing the medium every other day. By day 5, the culture had filled the bottom wells, and then grew densely with low light transmittance. Figure 6The hanging drop 3D liver model is ready. Note that the tip should be 45° inclined to the hole wall when changing the supernatant, slowly and evenly suck out, do not touch the hole bottom.

[0101] The hanging drop model can be maintained in RPMI 1640 complete medium containing 10% FBS (change every other day) for 30 days without losing the three-dimensional structure of liver nodule and liver-related functions.

[0102] 2. Micro-well 3D liver model construction.

[0103] 2.1 Preparation of cell suspension:

[0104] According to the above method, HepaRG cells and human hepatic stellate cells are prepared into a mixed cell suspension with a density of (8x10 4 HepaRG+0.8x10 4 HHSC) cells / 50 μl.

[0105] 2.2 Inoculation and culture:

[0106] Inoculate 50 μl of mixed cell suspension in each well of a 384-well low-adsorption spherical micro-well plate and place it in a 37°C, 5% CO2 and 90% relative humidity incubator.

[0107] After 3 days of inoculation, replace the medium in the well, specifically: slowly suck out 25 μl of supernatant from each well, discard, and slowly add 25 μl of fresh medium, replace the medium every day.

[0108] The micro-well model can be maintained in RPMI 1640 complete medium containing 10% FBS (change every other day) for 30 days without losing the three-dimensional structure of liver nodule and liver-related functions.

[0109] 3. Scaffold 3D liver model construction.

[0110] 3.1 Preparation of mixed cell suspension:

[0111] According to the above method, HepaRG cells and human hepatic stellate cells are prepared into a mixed cell suspension with a density of (5x10 5 HepaRG+0.5x10 5 HHSC) cells / 60 μl.

[0112] 3.2 Inoculation:

[0113] In the center of each well of the 24-well plate, 20 μl of the cell suspension was added, and the GO-Matrix or CelluSponge scaffold material was placed on top of the cell suspension (without pre-wetting the scaffold), and some time (about 2 min) was allowed for the medium to be fully absorbed by the scaffold. Then, 40 μl of the cell suspension was added to the top of the scaffold, as shown in Figure 7 , and gently pipetted back and forth to ensure even cell seeding on both sides of the scaffold. Care was taken not to blow air bubbles into the scaffold.

[0114] 3.3 Culturing:

[0115] The cell culture plates seeded with cells as described above were incubated in an incubator at 37 °C, 5% CO 2 , and 90% relative humidity for 4 h. (This step removed small air bubbles inside the scaffold and was also beneficial for cell stabilization); then, 500 μl of medium was added to each well to immerse the scaffold. The medium was added slowly from the edge of each well, not directly onto the scaffold. The incubation was then continued overnight.

[0116] 3.4 Model establishment:

[0117] The seeded scaffolds were transferred to new cell culture plates (to prevent cells growing on the cell culture plate from affecting the experimental results) and incubated with fresh medium. The medium was changed every other day, and the cells gradually formed cell clusters in the internal structure of the scaffold as the culture time increased. Very obvious cell clusters were observed after 5 days of culture, and the cell clusters gradually increased as the culture time increased, and the light transmittance under the optical microscope became smaller and smaller. The cell clusters of the 3D liver model of the scaffold are shown in Figure 8 .

[0118] The scaffold model can be maintained in culture for 30 days with 10% FBS-containing RPMI1640 complete medium (changed every other day) without losing the 3D structure and liver-related functions.

[0119] Example 2

[0120] An in vitro intestinal-liver co-culture model was established, including the following steps:

[0121] I. Establishment of a small intestine absorption model.

[0122] 1. Model establishment.

[0123] 1.1. Preparation of a cell suspension: when the MDCKII / Caco-2 cells grew to 80%-90% of the bottle bottom, they were trypsinized, the trypsinization was terminated with culture medium, and centrifuged at 1000 rpm for 5 min. Then, a cell suspension with a density of 2 x 10 6 cells / ml was prepared using 10% FBS-containing RPMI1640 complete medium.

[0124] 1.2, Add fresh 1640 medium containing 10% FBS to each well of the Transwell recipient chamber (bottom well) (keep the recipient and donor chambers level).

[0125] 1.3, Add the prepared cell suspension to each well of the Transwell donor chamber (Inserts) (equivalent to 1 x 10 6 cells / 1.12 cm 2 ).

[0126] 1.4, Incubate in a 37°C, 5% CO2 incubator, and change the medium every other day.

[0127] 1.5, Measure the transmembrane resistance after 15 days of incubation, and then measure it every 2 days.

[0128] 1.6, When the transmembrane resistance is greater than 180 Ω / cm 2 (about 21 days after inoculation), it is considered to form a small intestine absorption model. By measuring the transmembrane resistance of the Transwell, when the transmembrane resistance is stable at 180-220 Ω·cm -2 , it is preliminarily considered that Caco-2 has formed a stable biological membrane on the Inserts membrane; in addition, the apparent permeability coefficient of the membrane can also be detected by the fluorescence penetration detection method (sodium fluorescein) to be 6 x 10 -8 cm·s -1 Hereinafter, the hourly permeation rate is <0.1%, which meets the standard of the small intestine absorption biological membrane for the intercellular space, and the small intestine absorption model is evaluated.

[0129] 2, Model evaluation.

[0130] 2.1 Alkaline phosphatase activity experiment.

[0131] Using the AKP kit, the alkaline phosphatase activity at different time periods was measured by sampling from both sides of the Transwell membrane at 3, 15, 21, and 35 days after inoculation of Caco-2 cells according to the instructions.

[0132] The results show that the alkaline phosphatase activity in the chamber is significantly higher than that outside the chamber from 15 days, with a ratio of 3.7, indicating that the cells have started to polarize. After 22 days of inoculation, the ratio increased to 6.5, and the ratio remained above 6 at 35 days.

[0133] 2.2 Transporter expression experiment.

[0134] The expression of monocarboxylate transporter (MCT) and drug efflux transporter P-gp was detected by real-time fluorescence quantitative PCR (RT-PCR), and the results are shown in Figure 9 .

[0135] The results show that the expression of MCT and P-gp reached a steady state after 21 days of Caco-2 cell seeding and was maintained until day 35.

[0136] II. Establishment of the intestinal-liver co-culture model.

[0137] The above-mentioned hanging drop 3D liver model, microwell 3D liver model, and scaffold 3D liver model formed in Example 1 were transferred to the recipient well.

[0138] The liver microtissues formed by the hanging drop 3D liver model and the microwell 3D liver model were collected and transferred at 20 per well, and the scaffold 3D liver model was transferred at 1 per well.

[0139] The co-culture model can be maintained in RPMI 1640 complete medium containing 10% FBS for 14 days without loss of small intestinal and liver structure and related functions.

[0140] Comparative Example 1

[0141] Establishment of the 2D culture model of liver cells.

[0142] 1. Preparation of cell suspension:

[0143] The mixed cell suspension with a density of (5 x 10 4 HepaRG + 0.5 x 10 4 HHSC) cells / ml was prepared according to the method in 1.2, and was ready for use.

[0144] 2. Seeding:

[0145] 100 μl of the mixed cell suspension was seeded in each well of a 96-well culture plate, and was incubated in a 37°C, 5% CO2, and 90% relative humidity incubator.

[0146] After seeding, the cells were incubated overnight, and adherent growth was observed. Fresh RPMI 1640 complete medium containing 10% FBS or 10% FBS RPMI 1640 complete medium containing a certain concentration of drug was replaced as needed.

[0147] The model was used as a control model for the application of 3D culture liver cell function level detection and toxicity evaluation.

[0148] Experimental Example 1

[0149] Albumin and urea secretion level experiment.

[0150] I. Albumin secretion level.

[0151] The albumin expression levels of the cells in Example 1 and Comparative Example 1 were determined at different time points of cell culture using an albumin kit and high-content fluorescent labeling. The results are shown in Figure 10

[0152] The results show that the albumin secretion of the hanging drop 3D liver model and the scaffold 3D liver model prepared in Example 1 reached a high level after 3 days of culture, and was significantly higher than the 2D culture level (more than 2 times the same number of cells). This level was maintained until 30 days of culture.

[0153] II. Urea secretion level

[0154] The albumin expression levels of the cells were determined at different time points of cell culture using a urea kit, and the results are shown in Figure 11

[0155] The results show that the urea secretion of the hanging drop 3D liver model and the scaffold 3D liver model reached a high level after 5 days of culture, and was significantly higher than the 2D culture level (about 3 times the same number of cells). This level was maintained until 30 days of culture.

[0156] The above results show that the in vitro 3D liver model of the present application can maintain high levels of albumin and urea secretion for a long time, and maintain its liver function.

[0157] Experimental Example 2

[0158] Liver enzyme CYP3A4 activity experiment

[0159] Liver enzyme activity is an important factor affecting drug hepatotoxicity. In order to detect the metabolic capacity of cells under 3D liver model culture, the strengths of liver enzyme expression of cells in Comparative Example 1 and Example 1 under 2D culture and 3D model were detected by CYP3A4 inducer and inhibitor. The chemiluminescence intensity of each well was detected by non-cell lysing CYP3A4 detection reagent (refer to the method provided by Promega product P450-Glo CYP3A4 Assay with Luciferin-IPA kit), and the results are shown in Figure 12

[0160] The results show that after different time of culture, after 24h treatment of inducer (sodium phenytoin with a concentration of 100μM), the CYP3A4 activity induction level of 3D cultured cells was significantly higher than that of 2D culture. At the same time, after 24h treatment of inhibitor (ketoconazole with a concentration of 25μM), the CYP3A4 activity was inhibited, and this induction and inhibition ability was maintained until 30 days of culture.

[0161] Experimental Example 3

[0162] ​​​Expression experiment of major liver drug-metabolizing enzymes and drug-related transporters.

[0163] I. Expression experiment of major liver drug-metabolizing enzymes.

[0164] The expression fold of liver drug-metabolizing enzymes (CYP3A4, CY2B6, CY2C9) in the different liver models of Example 1 and Comparative Example 1 was detected by RealTime-PCR method (refer to Caroline Aninat et al. EXPRESSION OF CYTOCHROMES P450, CONJUGATING ENZYMES AND NUCLEAR RECEPTORS IN HUMAN HEPATOMA HepaRG CELLS. DRUG METABOLISM AND DISPOSITION, Vol. 34, No. 1, 34:75-83, 2006 method). The results are shown in Figure 13

[0165] The results show that the expression of CYP3A4, CY2B6, CY2C9 in the 3D liver model is significantly higher than that of the cell line obtained by 2D planar culture, and can be maintained for 30 days of culture.

[0166] II. Expression experiment of drug-related transporters.

[0167] The expression fold of drug efflux transporter P-gp in the different liver models of Example 1 and Comparative Example 1 was detected by RealTime-PCR method. The results are shown in Figure 13

[0168] The results show that the cell line obtained by 2D culture lacks the expression of transporter P-gp due to the lack of cell polarization. The expression of transporter P-gp reaches a peak after 6 days of culture, and can be maintained for 30 days of culture.

[0169] Experimental Example 4

[0170] After the establishment of the in vitro intestinal-liver co-culture model of Example 2, the above evaluation indexes were further studied at 3, 7, 10, and 14 days after modeling, respectively.

[0171] The experimental results show that the co-culture of the intestinal tranwell model and the liver hanging drop and scaffold model for 14 days has no significant effect on the functions of the two models. The specific results are as follows:

[0172] 1) The results of albumin secretion are shown in Figure 14 The secretion levels of liver albumin and urea in the co-culture model have no significant difference with those in the liver 3D model cultured alone, and can maintain a high level of secretion for 14 days of co-culture.​​

[0173] 2) The results, as shown in Table 2, showed that the expression of liver CYP3A4, CY2B6, CY2C9 and P-gp in co-culture model was not significantly different from that in liver 3D model alone, and the high level of expression could be maintained for 14 days of co-culture. Figure 15

[0174] 3) During the co-culture, the transmembrane electrical resistance of the small intestine was stably maintained at 200 Ω·cm -2 Above, the membrane apparent permeability coefficient was maintained at 6 x 10 -8 cm·s-1or less by using the method of fluorescence penetration detection (sodium fluorescein), and co-culture had no obvious effect on the permeability of the small intestine.

[0175] 4) The results of alkaline phosphatase activity also showed that the small intestine cells remained in a highly polarized state under 14 days of co-culture. The alkaline phosphatase activity inside the chamber was significantly higher than that outside the chamber, and the ratio was maintained at 6 or more.

[0176] 5) The expression of MCT and P-pg was also not significantly different from that in the small intestine alone. The expression ratio of MCT was maintained at 0.9-1.2, and the expression ratio of P-gp was maintained at 0.8-1.2.

[0177] Experimental Example 5

[0178] Application advantages of in vitro 3D liver model.

[0179] The in vitro 3D liver model can simulate the in vivo cell growth environment and liver enzyme expression system, maintain a good liver function state for a long time, and has unique advantages in the new drug development process compared with the in vitro 2D model, and can be used as a screening model for drug liver toxicity in non-clinical safety evaluation of drugs.

[0180] When the liver function of the hanging drop 3D liver model was stable, i.e. on the 7th day after inoculation, the culture medium was replaced with 10% FBS RPMI 1640 complete medium containing different concentrations of liver toxicity positive drugs, and the drug concentrations were as follows:

[0181] Isoniazid and acetaminophen: 100, 200, 400, 600, 800 and 1000 μM;

[0182] Emodin: 0.5, 1, 3.125, 6.25, 12.5 and 25 μg / ml;

[0183] Triptolide: 0.5, 1, 5, 12.5, 32 and 62.5 ng / ml;

[0184] Gallic acid: 50, 80, 100, 120, 150 and 200 μM. ​

[0185] The 3D liver model was co-administered with the same method and drug concentration for 3 times, 48 h each time, and the ATP luminescence intensity was detected after 6 days of co-administration to obtain the IC 50 value. Since the 2D model cannot be cultured for more than 48-72 h while maintaining a good functional level, the drug is only administered once in the 2D model, and the IC50 value is obtained after 24 h of incubation by the same method.

[0186] The results show that, in the 2D model, the administration of isoniazid and acetaminophen to hepatocytes has an IC 50 >1000 μM, and no obvious cytotoxicity is observed. This is because the liver toxicity of these drugs is related to their metabolites, and the 2D model has low drug metabolism ability and cannot be administered for a long time, so the toxicity cannot be significantly evaluated. Therefore, the conventional experiment proves that the liver toxicity of isoniazid and acetaminophen, which are positive drugs in the 2D liver culture, cannot be concluded to have liver toxicity.

[0187] However, the 3D liver model of the present application is administered to obtain an IC 50 of isoniazid = 600.7 μM, an IC 50 of acetaminophen = 539.7 μM. Further systematic evaluation of the liver toxicity of traditional Chinese medicine monomers shows that the IC 50 of emodin = 4.6 μg / mL, the IC 50 of triptolide = 6.6 ng / mL, the IC 50 of gallic acid = 142.9 μM, and the IC 50 of genkwanin = 116.9 μg / mL.

[0188] Experimental Example 6

[0189] Advantages of the in vitro intestinal-liver co-culture model.

[0190] Oral drugs are absorbed and metabolized in the small intestine before entering the liver to cause liver toxicity. This is the main reason for the false positive results of many conventional in vitro liver models in evaluating drug toxicity.

[0191] The in vitro intestinal-liver co-culture model of the present application not only simulates the barrier and metabolic effects of the intestinal tract in vivo, but also combines the 3D liver model to simulate the actual physiological conditions in vivo, and can be used as a screening model for intestinal-liver toxicity of drugs in non-clinical safety evaluation of drugs.

[0192] For example, gallic acid is a common component of traditional Chinese medicine. If only the hepatocyte model is used for evaluation, it will be found to cause certain liver cell damage and apoptosis. The 3D liver model obtains an IC 50= 142 μM. However, the in vitro intestine-liver co-culture model of the present application was used to evaluate the permeability and 3D liver exposure of gallic acid after 24 h incubation with 10, 100, 200, 400, 600, 800 and 1000 μM gallic acid administered in the Transwell chamber.

[0193] It was found that the intestinal absorption of gallic acid was very low (less than 1% in 24 hours), i.e. the 3D liver exposure of gallic acid was less than 10 μM at a concentration of 1000 μM, which was much lower than the IC50value. Therefore, no significant liver toxicity of gallic acid was observed after absorption by the small intestine. This result was similar to most clinical results, i.e. taking Chinese medicine containing gallic acid at a normal dose did not cause significant liver toxicity.

[0194] The technical features of the above-described embodiments can be combined in any manner. In order to make the description simple, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not contradict each other, they should be considered as falling within the scope of the present disclosure.

[0195] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, however, it should not be understood as a limitation on the scope of the patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for establishing an in vitro 3D liver model, characterized in that, Includes the following steps: Cell culture: HepaRG cells and human hepatic stellate cells were cultured separately for later use; Cell induction: The above HepaRG cells were seeded in culture flasks for cell culture. After the HepaRG cells adhered and grew to the predetermined number, the culture medium was replaced with induction medium for induction culture to obtain induced HepaRG cells for later use. Model building: 1) Preparation of mixed cell suspension: induced HepaRG cells and human hepatic stellate cells are prepared to contain 4 x 10 5 -6 x 10 5 individual HepaRG and 0.2 x 10 5 -1.2 x 10 5 individual human hepatic stellate cells per 60 μΐ, the ratio of the number of HepaRG cells and human hepatic stellate cells in the mixed cell suspension being 8:1-12:1; 2) Seeding: Add the mixed cell suspension to the cell culture plate at a rate of 15-25 μl / well, then place the CelluSponge 3D culture scaffold on the cell suspension, and then add the mixed cell suspension to the 3D culture scaffold at a rate of 30-50 μl / well. Wash and blow away the liquid to ensure that the cells are evenly seeded on both sides of the CelluSponge 3D culture scaffold. 3) Culture: Place the cell culture plate after cell seeding in an incubator at 35-39℃ with 4-6% CO2 and 85-95% relative humidity for 3-5 hours to stabilize. Then add culture medium to immerse the 3D culture scaffold and incubate for 12-36 hours. 4) Model establishment: The 3D culture scaffold after incubation was transferred to another cell culture plate, culture medium was added, and the culture medium was changed regularly. After culturing, micro-tissue with the three-dimensional structure of liver glomeruli was obtained, which is the 3D liver model. The culture medium for both HepaRG cells and human hepatic stellate cells was RPMI 1640 complete medium containing 2-20% fetal bovine serum. In the cell induction step, the induction medium is a complete medium containing 0.5-3% DMSO and 2-20% fetal bovine serum.

2. The method of claim 1, wherein the method is performed in vitro. In the cell induction step, the predetermined number is the number of HepaRG cells that normally adhere to the wall and grow to cover 75% to 95% of the bottom of the flask.

3. The method of claim 1, wherein the method is performed in vitro. The cell culture conditions are as follows: cells are cultured in an incubator at 35-39°C with 4-6% CO2 and a relative humidity of 85-95%, and the culture medium is changed every 1-3 days. The induction culture conditions are as follows: HepaRG cells are cultured in an incubator at 35-39°C with 4-6% CO2 and a relative humidity of 85-95%. The culture medium is changed daily, and the induction culture is carried out for a total of 12-16 days.

4. The 3D liver model obtained by the method for establishing an in vitro 3D liver model according to any one of claims 1-3.

5. The application of the in vitro 3D liver model as described in claim 4 in the evaluation of in vitro liver toxicity.

6. Use according to claim 5, characterized in that, Application of the in vitro 3D liver model in the preparation of reagents and / or devices for in vitro liver toxicity evaluation.

7. A method for establishing an in vitro enterosiphon co-culture model, characterized in that, Includes the following steps: Establishment of a small intestinal absorption model: A small intestinal absorption model was established using Caco-2 cells in a Transwell chamber; Establishing a gut-liver co-culture model: The 3D liver model described in claim 5 is transferred to the receiving side of the Transwell chamber to obtain the gut-liver co-culture model.

8. The method for establishing an in vitro gut-liver co-culture model according to claim 7, characterized in that, In the step of establishing the small intestine absorption model, complete culture medium containing 2-20% fetal bovine serum is added to the receiving side of the Transwell chamber, and Caco-2 cell suspension is added to the supply side of the Transwell chamber, so that the cell density is equivalent to 0.5×10 6 -1.5×10 6 cells / 1.12cm 2 , and cell culture is carried out until the transmembrane resistance is greater than 180 Ω / cm 2 , and the small intestine absorption model is obtained. When the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models need to be transferred per 1.12 cm 2 When the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models need to be transferred per 1.12 cm 2 When the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models need to be transferred per 1.12 cm 2 When the 3D liver model is a hanging drop 3D liver model, 15-25 hanging drop 3D liver models need to be transferred per 1.12 cm 9. A method for establishing an in vitro intestinal-liver co-culture model according to any one of claims 7-8, the in vitro intestinal-liver co-culture model being established.

10. Use of the in vitro intestinal-liver co-culture model according to claim 9 for performing in vitro intestinal absorption and liver toxicity evaluation.

11. Use according to claim 10, characterized in that, Use of the in vitro intestinal-liver co-culture model for the manufacture of a reagent and / or equipment for performing in vitro intestinal absorption and liver toxicity evaluation.

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