Hepatocyte 3D culture model containing liver microsome and construction method thereof

Abstract

The invention relates to the field of in-vitro drug metabolism evaluation, and particularly discloses a liver microsome-containing liver cell 3D culture model and a construction method thereof. According to the invention, primary hepatocytes are used as a cell biological material, and a three-dimensional hepatocyte model is prepared through a three-dimensional bio-printer in combination with Matrigen glue, rat tail glue and calcium ions; the liver microsome hydrogel containing metabolic enzyme is combined in the middle of the system, and the liver microsome-containing liver cell 3D culture model is jointly formed and used for evaluating drug metabolism and drug interaction in vitro, revealing the treatment effect of liver tissue in the liver on drugs and predicting reasonable application of the drugs. The model is simple in structure and relatively mature in preparation process, is expected to be further applied industrially, and has relatively high popularization value.

Description

Technical Field

[0001] The present invention belongs to the field of biomanufacturing and tissue engineering, and specifically relates to a 3D hepatocyte culture model containing liver microsomes and a construction method thereof, which are used to simulate liver metabolic function and to study in vitro liver drug metabolism. Background Art

[0002] Hepatocyte models are mainly used in the study of drug metabolism, especially to better understand and predict drug metabolism and toxicity in the human body. The following are some key features of these models: (1) Three-dimensional (3D) co-culture liver model: Compared with two-dimensional (2D) or single-type 3D culture, 3D co-culture models (such as the hepatoma cell and fibroblast co-culture model constructed by Al Hrout et al.) can better reflect the development characteristics of hepatocellular carcinoma in vivo and more accurately evaluate the effects of anticancer drugs. This type of model cannot evaluate drug metabolism-related studies because it does not have specific highly expressed metabolic enzymes. HepaRG cells exhibit higher CYP450 activity / metabolic capacity, significantly better than standard HepG2 / C3A cells, and are suitable for pharmaceutical and therapeutic applications. Literature reports (Mazzocchi, A.; Devarasetty, M.; Huntwork, R.; Soker, S.; Skardal, A. Optimization of collagen type I-hyaluronan hybrid bioink for 3D bioprinted liver microenvironments. Biofabrication 2018, 11, 015003.) improved the in vitro culture conditions of PHHs by using a 3D culture environment composed of Matrigel or a 3D bioprinting model to maintain its morphology and function for several months and enhance its sensitivity to hepatotoxic substances. Although this model can simulate the morphology and function of liver tissue, it has not been able to achieve research on its drug metabolism.

[0003] Primary hepatocytes (PHs) are widely considered the gold standard for constructing in vitro liver models because of their secretory properties and metabolic enzyme activities similar to those of in vivo hepatocytes. 3D co-culture liver models and their improved culture conditions, the application of multiple hepatocyte cell lines, and stem cell-derived organoids provide a more precise and practical platform for drug development, helping to solve problems in traditional models, such as lack of physiological relevance or inability to fully represent complex cell-cell interactions. The literature reports (Development of an alcoholic liver disease model for drug evaluation from human induced pluripotent stem cell-derived liver organoids [J]. Acta BiochimBiophys Sin, 2024, 56 (10): 1460–1472) that an alcoholic liver disease model was established using liver organoids derived from human induced pluripotent stem cells. Serum substitutes, activins, recombinant human fibroblast growth factor, retinoic acid, etc. were used to induce stem cells to differentiate into hepatocyte models. The fraction of organoids can simulate liver structure and function, providing a new tool for studying the disease. However, it has not been used for the study of drug metabolism.

[0004] Currently, existing 3D hepatocyte models focus more on cell survival and basic functions (such as albumin secretion), but lack effective integration of drug-metabolizing enzyme systems (such as CYP3A and P-gp). Traditional co-culture models often simply mix hepatocytes and microsomes, resulting in rapid inactivation of enzyme activity due to uneven spatial distribution or inappropriate microenvironment. In addition, a single hydrogel layer is difficult to simulate the compartmentalized distribution characteristics of metabolic enzymes in the liver. Therefore, there is an urgent need for a 3D culture system that can stably maintain the synergistic function of hepatocytes and microsomes. Summary of the invention

[0005] In view of the problems of low drug metabolism efficiency, low expression of drug metabolizing enzymes and poor preparation repeatability in existing three-dimensional hepatocyte models, the present invention provides a hepatocyte 3D culture model containing liver microsomes and a construction method thereof.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is: Technical Theme 1 A 3D culture model of hepatocytes containing liver microsomes, including at least two layers: from bottom to top, the first layer contains primary hepatocytes combined with rat tail glue and Matrigen glue, in a culture medium containing calcium ions, so that they form a liver plate-like structure; from bottom to top, the second layer is a hydrogel layer containing liver microsomes; wherein the liver microsomes contain CYP3A4 and / or CYP2C9 and / or CYP2D6 and / or P-gp metabolic enzymes. The hydrogel forms an integrated structure through physical or chemical cross-linking.

[0007] Furthermore, the hydrogel comprises the following components: Pluronic, acrylamide chloride, acrylamide; Methacryloylated gelatin coupled to hyaluronic acid (GelMA-HA); Polyethylene glycol diacrylate (PEG-DA); extracellular matrix (dECM); Photoinitiator (LAP or Ingacure 2929); The liver microsome concentration is 0.1-1 mg / mL; The concentration of primary hepatocytes was 3×10 6 -8×10 6 Pieces / mL.

[0008] Compared with the prior art, the present invention constructs liver microsomes with an innovative hydrogel system, which can maintain the activity of liver microsomes for a long time on the one hand, and fix the microsomes in a three-dimensional hydrogel system on the other hand, so that the liver microsomes can play a role in a three-dimensional space, which is closer to the real liver tissue environment. The hydrogel system in the present invention can be printed with a three-dimensional bioprinter, and the shape can be fixed after light exposure. The preparation method is simple and mature, and it is convenient for large-scale development. The present invention uses a hydrogel system to layer liver cells in the wells of a culture plate using a three-dimensional printer, and uses instantaneous light to form a spatial structure for cell growth, and grow cells therein. The liver microsome hydrogel is then loaded therein, and then the liver cell hydrogel is printed thereon to form a sandwich structure, forming a three-dimensional model of the symbiosis of metabolic enzymes and cells. The present invention designs a sandwich structure hydrogel model, combines primary hepatocytes with hydrogels containing liver microsomes in layers, and realizes spatially controllable distribution of metabolic enzyme systems. The hydrogel components are optimized to provide biocompatibility for GelMA-HA, PEG-DA regulates hardness (2-10 kPa), dECM simulates liver matrix, and photoinitiator achieves rapid cross-linking. Liver microsomes are evenly dispersed in the form of liquid suspension to avoid enzyme inactivation. This model shows significant advantages in drug metabolism, toxicity testing and disease modeling.

[0009] Furthermore, the hydrogel liver microsome system includes Pluronic F127, acrylamide chloride, and ammonium persulfate.

[0010] Furthermore, the hydrogel liver microsome system comprises the following components in parts by mass: 10 parts of Pluronic F127, 1 part of acrylamide chloride, and 0.01 to 0.03 parts of ammonium persulfate; Furthermore, the hydrogel hepatocyte system also includes rat tail glue, Matrigen glue, and a culture medium containing calcium ions.

[0011] Furthermore, the hydrogel hepatocyte component includes the following components in mass fractions: number of cells: 100,000 parts, 1 part of rat tail glue, 0.5 parts to 1.5 parts of Matrigen glue, and 0.01 parts to 0.05 parts of culture medium containing calcium ions; Technical Theme 2 The preparation method of the model described in the technical theme 1 is as follows: a. Preparation of primary hepatocyte hydrogel: primary hepatocytes were mixed with a hydrogel precursor containing GelMA-HA, PEG-DA, extracellular matrix (dECM), and a photoinitiator in weight proportions of 3-8 parts: 0.04-0.1 parts: 0.08-0.5 parts: 0.01-0.04 parts: 0.001-0.003 parts; b. Preparation of liver microsome hydrogel: The liver microsomes are mixed with a hydrogel containing Pluronic, acrylamide chloride, and acrylamide, and then loaded with liver microsomes, and the weight ratios are: 0.1-0.5 parts: 4000-12000 parts: 10-500 parts: 100-300 parts; c. Printing the hydrogel in step a by a 3D bioprinter; d. Blue light cross-linking (wavelength 460-480 nm) for 10-30 seconds to solidify the model; e. Place a layer of plywood with a thickness of 2-4 mm on top of the hydrogel printed in step c, and repeat step c on the plywood to form a sandwich 3D liver cell model; f. After the liver cell model has grown, remove the splint and place the hydrogel containing liver microsomes in the gap between the splints to form a 3D culture model of liver cells containing liver microsomes.

[0012] Furthermore, the temperature of the printing platform was 5-15°C and the humidity of the printing environment was 60-80% to maintain cell activity.

[0013] Technical Theme 3 Technical Topic 1: Application of the model in drug metabolism toxicity testing or liver disease simulation.

[0014] The prepared 3D culture model of hepatocytes containing liver microsomes has excellent activity, long-term stability, and a three-dimensional liver microsome and hepatocyte co-incubation system, which effectively solves the disadvantage that the hepatocyte model cannot predict drug metabolism and interactions. It can realize the in vitro evaluation of drug metabolism and interactions, and provide a reference for animal experiments and clinical applications. The preparation process is simple, reproducible, and easy to realize large-scale industrial production, and has a high value for promotion and application. DETAILED DESCRIPTION

[0015] Example 1: Basic metabolic 3D liver model system of sandwich model Hydrogel preparation: 1) Constructing hydrogel. 12g of Pluronic (F127) was dissolved in dichloromethane (dehydrated with sodium metal and stored in a desiccator), and appropriate amounts of 0.5g of acrylamide chloride and triethylamine were added respectively, and stirred overnight (N2 protection). The obtained product was taken out, washed with ice ether three times, and vacuum dried to obtain Pluronic acrylamide (F127ACR).

[0016] Prepare F127ACR into a 30% aqueous solution and stir to dissolve in an ice bath. Take the F127ACR solution, add 35mL of PBS, 0.6mL of 30% acrylamide solution, 0.6mL of 10% ammonium persulfate, and 0.3mL of tetramethylethylenediamine in sequence, vortex to mix, and obtain the solution. Take the above solution and add it to a 96-well plate, 300 μL per well, place the 96-well plate in a 37°C water bath, and after solidification, take it out, take out the hydrogel in each well, wash it 5 times with 5% mannitol, place it in a decolorizing shaker to remove impurities, and freeze-dry the hydrogel in a freeze dryer for use at 4°C.

[0017] Preparation of hydrogel containing liver microsomes: Take a hydrogel block (obtained from 50 μL of liquid) and add 1 mL of 0.1 mg / mL human liver microsome solution, soak for 48 h, and the obtained liver microsome hydrogel is stored at -80°C.

[0018] Preparation of hepatocyte-containing hydrogel: GelMA-HA 40 g / L, PEG-DA 80 g / L, dECM 10 g / L, LAP 1 g / L, primary mouse hepatocytes 3×10 6 / mL, calcium ion 1mmol / L, and rat tail glue, Matrigen glue.

[0019] Construction method: Use a bioprinter to print the liver cell hydrogel mixture, blue light crosslink for 20 seconds, print one layer, and then print the second layer. Leave a 2-4mm space between the first and second layers. After 7 days of culture, the activity of liver cells is 85%±5% of the in vivo level. Then add the constructed liver microsomes into the space between the first and second layers to form a sandwich hydrogel liver cell culture system.

[0020] Incubation with atorvastatin: Atorvastatin (200 μM) was incubated with the hydrogel liver microsome (CYP3A4) 3D liver model in a Franz cell at a flow rate of 20 mL / min. The changes in the atorvastatin content were determined by UPLC-MS / MS analysis.

[0021] Example 2: High Metabolic 3D Liver Model System in Sandwich Model Hydrogel preparation: 1) Constructing hydrogel. 4 g of Pluronic (F127) was dissolved in dichloromethane (dehydrated with sodium metal and stored in a desiccator), and 0.01 g of acrylamide chloride and triethylamine were added respectively, and stirred overnight (N2 protection). The obtained product was taken out, washed with ice ether three times, and vacuum dried to obtain F127ACR.

[0022] Prepare F127ACR into a 10% aqueous solution and stir to dissolve in an ice bath. Take the F127ACR solution, add 35mL of PBS, 0.033mL of 30% acrylamide solution, 0.2mL of 10% ammonium persulfate, and 0.1mL of tetramethylethylenediamine in sequence, vortex and mix well to obtain a solution. Take the above solution and add it to a 96-well plate, 300 μL per well, place the 96-well plate in a 37°C water bath, and after solidification, take it out, take out the hydrogel in each well, wash it 5 times with 5% mannitol, place it in a decolorizing shaker to remove impurities, and freeze-dry the hydrogel in a freeze dryer for use at 4°C.

[0023] Preparation of hydrogel containing liver microsomes: Take a hydrogel block (obtained from 50 μL of liquid) and add 1 mL of 0.5 mg / mL human liver microsome solution, soak for 48 h, and the obtained liver microsome hydrogel is stored at -80°C.

[0024] Preparation of hepatocyte-containing hydrogel: GelMA-HA 100 g / L, PEG-DA 500 g / L, dECM 40 g / L, LAP3 g / L, primary mouse hepatocytes 8×10 6 Pieces / mL. Construction method: Use a bioprinter to print the liver cell hydrogel mixture, blue light crosslinking for 20 seconds, hardness 6 kPa, and quickly print the second layer after printing one layer, leaving a 2-4mm space between the first and second layers. After 7 days of culture, the activity of liver cells is 85%±5% of the in vivo level. Then, the constructed liver microsomes are added to the space between the first and second layers to form a sandwich hydrogel liver cell culture system.

[0025] Incubation with atorvastatin: Atorvastatin (200 μM) was incubated with the hydrogel liver microsome liver model (CYP 2D6 or CYP2C9, etc.) in a Franz cell at a flow rate of 20 mL / min. The changes in the atorvastatin content were determined by UPLC-MS / MS analysis.

[0026] Example 3: Medium Metabolic 3D Liver Model System of Sandwich Model Hydrogel preparation: 1) Constructing hydrogel. 8 g of Pluronic (F127) was dissolved in dichloromethane (dehydrated with sodium metal and stored in a desiccator), and 0.025 g of acrylamide chloride and triethylamine were added respectively, and stirred overnight (N2 protection). The obtained product was taken out, washed with ice ether three times, and vacuum dried to obtain F127ACR.

[0027] Prepare F127ACR into a 10% aqueous solution and stir to dissolve in an ice bath. Take the F127ACR solution, add 35mL of PBS, 0.4mL of 30% acrylamide solution, 0.4mL of 10% ammonium persulfate, and 0.2mL of tetramethylethylenediamine in sequence, vortex and mix well to obtain a solution. Take the above solution and add it to a 96-well plate, 300 μL per well, place the 96-well plate in a 37°C water bath, and after solidification, take it out, take out the hydrogel in each well, wash it 5 times with 5% mannitol, place it in a decolorizing shaker to remove impurities, and freeze-dry the hydrogel in a freeze dryer for use at 4°C.

[0028] Preparation of hydrogel containing liver microsomes: Take a hydrogel block (obtained from 50 μL of liquid) and add 1 mL of 0.25 mg / mL human liver microsome solution, soak for 48 h, and the obtained hydrogel containing liver microsomes is stored at -80°C.

[0029] Preparation of hepatocyte-containing hydrogel: GelMA-HA 70 g / L, PEG-DA 290 g / L, dECM 30 g / L, LAP 2 g / L, primary mouse hepatocytes 5×10 6 Pieces / mL. Construction method: Use a bioprinter to print the liver cell hydrogel mixture, blue light crosslinking for 20 seconds, hardness 6 kPa, and quickly print the second layer after printing one layer, leaving a 2-4mm space between the first and second layers. After 7 days of culture, the activity of liver cells is 85%±5% of the in vivo level. Then, the constructed liver microsomes are added to the space between the first and second layers to form a sandwich hydrogel liver cell culture system.

[0030] Incubation with atorvastatin: Atorvastatin (200 μM) was incubated with the hydrogel liver microsome liver model (CYP 2D6 or CYP2C9, etc.) in a Franz cell at a flow rate of 20 mL / min. The changes in the atorvastatin content were determined by UPLC-MS / MS analysis.

[0031] Example 4 Liver microsome activity test in a hepatocyte model containing liver microsomes The metabolic enzyme activity of liver microsomes in the 3D hepatocyte culture model containing liver microsomes was tested to verify its drug metabolism function.

[0032] The CYP3A4 enzyme activity test was performed using the model constructed in Example 3 and midazolam as a substrate. High performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

[0033] Remove the 3D model of hepatocytes containing liver microsomes from the incubator and gently wash it three times with PBS buffer to remove the residual culture medium. Place the model in a preheated culture plate, add an appropriate amount of PBS buffer (37°C), and equilibrate for 10 min. Prepare the substrate solution: For CYP3A4 enzyme activity test: Dissolve midazolam in PBS buffer to a final concentration of 10 μM. Add the substrate solution to the model to ensure that the surface of the model is completely covered. Add the NADPH regeneration system (final concentration of 1 mM) to start the metabolic reaction. Incubate in an incubator at 37°C and 5% CO2 for different times. After the incubation, remove the model and collect the reaction solution. Add an equal volume of acetonitrile to terminate the reaction and mix thoroughly. Centrifuge the sample at 4°C and 12,000 rpm for 10 min, and take the supernatant for metabolite detection.

[0034] Determination of CYP3A4 enzyme activity: The metabolite of midazolam, 1'-hydroxymidazolam, was detected by HPLC-MS / MS.

[0035] Chromatographic conditions: Chromatographic column: C 18 Reversed Phase Column Mobile phase: acetonitrile-water (containing 0.1% formic acid) Flow rate: 0.3 mL / min Detection wavelength: 254 nm (midazolam) Mass spectrometry conditions: Ion source: electrospray ionization (ESI).

[0036] Detection mode: positive ion mode.

[0037] Evaluation indicators: The activity of liver microsomes was evaluated by comparing with the control group (a model without liver microsomes).

[0038] Example 5 Experiments on drug transport using a hepatocyte model containing liver microsomes The model construction experiment is the same as that of the corresponding Example 3.

[0039] Add Ca-containing 2+ and Ca-free 2+ Hanks solution was pre-incubated for 10 min; the fluorescent substrate CFDA was added to detect the bile duct formation status. After the model was successfully established, the hepatocyte uptake experiment was carried out. The cells were cultured for 7 days and Ca-containing 2+ and Ca-free 2+ The cells were pre-incubated with Hanks solution for 10 min, and the reaction was initiated after adding atorvastatin and different concentrations of transporter inhibitor cyclosporine A, and the intracellular drug accumulation content was detected. The bile duct efflux fraction (BEI) was calculated using the following formula.

[0040] Comparative Example 1: Ordinary Liver Microparticle Liver Model System Instead of constructing a liver microsome hydrogel system, only a 3D model of liver cells was constructed, and the liver microsomes were added into the cavity of the middle layer of the liver microsomes.

[0041] Preparation of liver microsomes: Prepare 1 mL of 0.25 mg / mL human liver microsome solution.

[0042] Preparation of hepatocyte-containing hydrogel: GelMA-HA 70 g / L, PEG-DA 290 g / L, dECM 30 g / L, LAP 2 g / L, primary mouse hepatocytes 5×10 6 Pieces / mL.

[0043] Construction method: Use a bioprinter to print the liver cell hydrogel mixture, blue light cross-link for 20 seconds, hardness 6 kPa, and quickly print the second layer after printing one layer, leaving a 2-4mm space between the first and second layers. After 7 days of culture, the activity of liver cells is 85%±5% of the in vivo level. Then add the liver microsome liquid to the space between the first and second layers, so that it can be spread out in one layer to form a sandwich liver cell culture system.

[0044] Incubation with atorvastatin: Atorvastatin (200 μM) was incubated with the hydrogel liver model (CYP 2D6 or CYP2C9, etc.) in a Franz cell at a flow rate of 20 mL / min. The changes in the atorvastatin content were determined by UPLC-MS / MS analysis.

[0045] Comparative Example 2: Hydrogel liver microparticles and liver model form a double-layer model Hydrogel preparation: 1) Constructing hydrogel. 8 g of Pluronic (F127) was dissolved in dichloromethane (dehydrated with sodium metal and stored in a desiccator), and 0.025 g of acrylamide chloride and triethylamine were added respectively, and stirred overnight (N2 protection). The obtained product was taken out, washed with ice ether three times, and vacuum dried to obtain F127ACR.

[0046] Prepare F127ACR into a 10% aqueous solution and stir to dissolve in an ice bath. Take the F127ACR solution, add 35mL of PBS, 0.4mL of 30% acrylamide solution, 0.4mL of 10% ammonium persulfate, and 0.2mL of tetramethylethylenediamine in sequence, vortex and mix well to obtain a solution. Take the above solution and add it to a 96-well plate, 300 μL per well, place the 96-well plate in a 37°C water bath, and after solidification, take it out, take out the hydrogel in each well, wash it 5 times with 5% mannitol, place it in a decolorizing shaker to remove impurities, and freeze-dry the hydrogel in a freeze dryer for use at 4°C.

[0047] Preparation of hydrogel containing liver microsomes: Take a hydrogel block (obtained from 50 μL of liquid) and add 1 mL of 0.25 mg / mL human liver microsome solution, soak for 48 h, and the obtained hydrogel containing liver microsomes is stored at -80°C.

[0048] Preparation of hepatocyte-containing hydrogel: GelMA-HA 70 g / L, PEG-DA 290 g / L, dECM 30 g / L, LAP 2 g / L, primary mouse hepatocytes 5×10 6 Pieces / mL. Construction method: Use a bioprinter to print a liver cell hydrogel mixture, blue light crosslinking for 20 seconds, hardness 6 kPa, after printing a layer, after 7 days of culture, the liver cell activity is 85%±5% of the in vivo level. Then add the constructed liver microsome small particles to the above liver cell gel layer to form a double-layer hydrogel liver cell culture system.

[0049] Incubation with atorvastatin: Atorvastatin (200 μM) was incubated with the hydrogel liver microsome liver model (CYP 2D6 or CYP2C9, etc.) in a Franz cell at a flow rate of 20 mL / min. The changes in the atorvastatin content were determined by UPLC-MS / MS analysis.

[0050] Comparative Example 3: Liver Model Transport Experiment The model construction corresponds to Example 1, and the transport experiment corresponds to Example 5.

[0051] Comparative Example 4: Liver Model Transport Experiment The model construction corresponds to Example 2, and the transport experiment corresponds to Example 5.

[0052] Table 1 Metabolic enzyme activity investigation of different examples Table 2 Investigation of transport capacity of hepatocyte models in different embodiments

Claims

1. A sandwich 3D culture model of hepatocytes containing liver microsomes, characterized in that: Including three-layer sandwich structure: The first layer contains primary hepatocytes combined with rat tail glue and Matrigen glue in a culture medium containing calcium ions to form a liver plate-like structure; The second layer is a hydrogel layer containing liver microsomes; The third layer has primary liver plate-like structures; Wherein, the liver microsomes contain CYP3A4 and / or CYP2C9 and / or CYP2D6 metabolic enzymes, and the hydrogel forms an integrated structure through physical or chemical cross-linking.

2. The model according to claim 1, characterized in that The hydrogel comprises the following components: Pluronic, acrylamide chloride, acrylamide; Methacryloylated gelatin coupled to hyaluronic acid (GelMA-HA); Polyethylene glycol diacrylate (PEG-DA); extracellular matrix (dECM); Photoinitiator (LAP or Ingacure 2929); The liver microsome concentration is 0.1-1 mg / mL; The concentration of primary hepatocytes was 3×10 6 -8×10 6 Pieces / mL.

3. The model according to claim 2, characterized in that The liver microsomes are derived from human, pig or mouse livers and are dispersed in the hydrogel in the form of particles with a particle size of ≤ 0.9 μm after freeze-drying.

4. The model according to claim 1, characterized in that The sandwich three-dimensional structure is an alternating multi-layer structure, comprising 3-5 layers of primary hepatocyte hydrogel layers and liver microsome hydrogel layers.

5. A method for constructing a model according to any one of claims 1 to 4, characterized in that: The following steps are involved: a. Preparation of primary hepatocyte hydrogel: primary hepatocytes were mixed with a hydrogel precursor containing GelMA-HA, PEG-DA, extracellular matrix (dECM), and a photoinitiator in weight proportions of 3-8 parts: 0.04-0.1 parts: 0.08-0.5 parts: 0.01-0.04 parts: 0.001-0.003 parts; b. Preparation of liver microsome hydrogel: The liver microsomes are mixed with a hydrogel containing Pluronic, acrylamide chloride, and acrylamide, and then loaded with liver microsomes, and the weight ratios are: 0.1-0.5 parts: 4000-12000 parts: 10-500 parts: 100-300 parts; c. Printing the hydrogel in step a by a 3D bioprinter; d. Blue light cross-linking (wavelength 460-480 nm) for 10-30 seconds to solidify the model; e. Place a layer of plywood with a thickness of 2-4 mm on top of the hydrogel printed in step c, and repeat step c on the plywood to form a sandwich 3D liver cell model; f. After the liver cell model has grown, remove the splint and place the hydrogel containing liver microsomes in the gap between the splints to form a 3D culture model of liver cells containing liver microsomes.

6. The method according to claim 5, characterized in that The printing platform temperature is 5-15°C and the printing environment humidity is 60-80% to maintain cell activity.

7. Use of the model according to claim 1 in drug metabolism toxicity testing or liver disease simulation.

Images