Hepatotoxicity three-dimensional cell model and construction method and application thereof

Abstract

The invention relates to a hepatotoxicity three-dimensional cell model as well as a construction method and application thereof, and relates to the field of pharmacology and toxicology. The construction method comprises the following steps: inoculating hepatic cells and hepatic stellate cells into a culture medium containing an extracellular matrix, and carrying out three-dimensional cell co-culture to obtain the three-dimensional cell model. In the early stage of drug research and development, drug compounds with potential hepatotoxicity can be rapidly screened out by using the hepatotoxicity three-dimensional cell model provided by the invention; according to the present invention, whether the drug damages the liver cells or not is determined by observing the changes of the form, the metabolism, the function and the like of the cells under the drug effect, such that the drug candidate possibly having the serious hepatotoxicity can be timely eliminated so as to avoid the problem discovery in the subsequent clinical test, such that the research and development time and the research and development cost can be saved.

Description

Technical Field

[0001] The present invention relates to the fields of pharmacology and toxicology, and particularly to a three-dimensional cell model for hepatotoxicity, a construction method thereof, and an application thereof. Background Art

[0002] Drug-induced liver injury has always been an important cause leading to acute liver failure, seriously threatening human health. The hepatotoxicity model is an important tool for evaluating the toxicity of drugs or chemical substances to the liver. In the process of constructing functional liver tissue in vitro, it is particularly important to simulate the complex microenvironment of hepatocytes in vivo and achieve three-dimensional culture of hepatocytes in vitro to simulate the physiological and pathological states of the liver. In view of this, the present invention provides a three-dimensional cell model for hepatotoxicity, a construction method thereof, and an application thereof. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a three-dimensional cell model for hepatotoxicity, a construction method thereof, and an application thereof. The purpose is to prove that it can be used to screen potential hepatotoxic substances by establishing a three-dimensional cell model for hepatotoxicity and evaluating its drug responses under treatment with different concentrations of alcohol and the strong hepatotoxic drug cisplatin.

[0004] The technical solution of the present invention for solving the above technical problem is as follows:

[0005] In a first aspect, a construction method of a three-dimensional cell model for hepatotoxicity includes the following steps: inoculating hepatocytes and hepatic stellate cells in a culture medium containing an extracellular matrix, and performing three-dimensional cell co-culture to obtain a three-dimensional cell model for hepatotoxicity.

[0006] Among them, the culture medium can adopt a hepatocyte culture medium, etc., and the extracellular matrix can be a fibrous or granular solid extracellular matrix composed of cellulose-L-lysine polymer. This solid extracellular matrix is composed of nanofibers, and the gap between the nanofibers is less than 5 microns; it is insoluble in water, has a low density, allows cell adhesion and free movement on its surface, allows growth on the fiber surface and in the fiber voids, and allows cells to use it to build an extracellular framework structure required for three-dimensional cell growth; at the same time, it establishes and maintains a stable internal environment highly similar to that in vivo for cells; different from existing microcarriers, cells do not completely rely on adhesion to the fiber surface for growth, and will not grow inside the fibers, so it is easier to carry out nutrient and metabolic waste exchange and maintain the consistency of the entire culture system; this extracellular matrix does not adsorb dyes and antibodies and does not produce spontaneous fluorescence, thus not affecting the detection, analysis, and identification of the cultured cells.

[0007] Among them, the hepatocytes include human primary hepatocytes, human hepatocyte lines, animal primary animal hepatocytes, and so on.

[0008] The beneficial effects of the present invention are as follows: The three-dimensional cell model of hepatotoxicity of the present invention selects hepatocytes and hepatic stellate cells, can be cultured for a long time, has high stability, retains the activity of liver metabolic enzymes, can more accurately simulate the physiological and pathological reactions of the human liver, and is more suitable for studying drug metabolism and toxicity reactions; Using the three-dimensional cell model of hepatotoxicity of the present invention, drug compounds with potential hepatotoxicity can be quickly screened out, and drug candidates with serious potential hepatotoxicity can be accurately eliminated in a timely manner, saving R & D time and costs.

[0009] On the basis of the above technical solutions, the present invention can also be improved as follows.

[0010] Further, it includes the following specific steps: inoculate hepatocytes and hepatic stellate cells in a well plate, add a culture medium containing extracellular matrix to the well plate, perform three-dimensional cell co-culture, and obtain a three-dimensional cell model of hepatotoxicity;

[0011] The total cell concentration inoculated in the well plate is 1.0 - 4.0×10 6 cells / mL, and the ratio of the number of hepatocytes inoculated to the number of hepatic stellate cells inoculated is 3:0.5 - 1.5.

[0012] Further, the volume ratio of the extracellular matrix to the culture medium in the culture medium containing extracellular matrix is 0.5 - 1.5:3.

[0013] Further, the conditions for the three-dimensional cell co-culture are: 37°C, 5% CO2, culture for 5 - 7d.

[0014] In the second aspect, a three-dimensional cell model of hepatotoxicity, the three-dimensional cell model of hepatotoxicity is constructed by the construction method described above.

[0015] In the third aspect, an application of a three-dimensional cell model of hepatotoxicity, using the three-dimensional cell model of hepatotoxicity in the evaluation of hepatotoxic substances.

[0016] The beneficial effects of adopting the above solution are as follows: In the early stage of drug R & D, using the three-dimensional cell model of hepatotoxicity of the present invention, drug compounds with potential hepatotoxicity can be quickly screened out. By observing the morphological, metabolic, functional and other changes of cells under the action of drugs, it is judged whether the drugs cause damage to liver cells, so as to timely eliminate drug candidates with serious potential hepatotoxicity, avoid problems being discovered only in subsequent clinical trials, and save R & D time and costs.

[0017] In the fourth aspect, a method for evaluating the hepatotoxicity of a substance using a three-dimensional cell model of hepatotoxicity, including the following steps:

[0018] (1) Cell seeding: Resuspend and count the three-dimensional cell model with a liver cancer cell culture medium to obtain a cell suspension; add extracellular matrix to the cell suspension, mix well and transfer it to a well plate to obtain the well plate for cell seeding.

[0019] (2) Drug hepatotoxicity evaluation: Perform a hepatotoxicity drug addition experiment in the well plate of the cell seeding, and detect cell viability for acute / chronic evaluation of hepatotoxicity caused by substances.

[0020] Further, the density of cells in the cell suspension in step (1) is 0.5 - 2×10 4 cells / mL.

[0021] Further, the volume ratio of the cell suspension to the extracellular matrix in step (1) is 2:0.5 - 1.5.

[0022] Further, any one of the CCK-8 method, MTT CCK-8 method, and CyQUANT method is used for the detection of cell viability.

[0023] Among them, the Cell Counting Kit-8 (CCK-8 method) for cell proliferation / toxicity detection is based on the highly water-soluble tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) for determination. Description of the Drawings

[0024] Figure 1 This is the microscopic image of the co-culture effect of different concentrations of alcohol on THLE-2, THLE-2 and hepatic stellate cells of the present invention;

[0025] Figure 2 This is the cell survival rate graph of the co-culture effect of different concentrations of alcohol on THLE-2, THLE-2 and hepatic stellate cells of the present invention;

[0026] Figure 3 This is the microscopic image of the hepatotoxicity effect of cisplatin on the co-culture of THLE-2, THLE-2 and hepatic stellate cells of the present invention;

[0027] Figure 4 This is the cell survival rate graph of the hepatotoxicity effect of cisplatin on the co-culture of THLE-2, THLE-2 and hepatic stellate cells of the present invention;

[0028] Figure 5 This is the scanning electron microscope image of the fibrous or granular solid extracellular matrix structure composed of cellulose-L-lysine polymer of the present invention. Among them, A-C are the scanning electron microscope images of the dried and dehydrated fibrous or granular solid extracellular matrix at different electron microscope scales. Detailed Embodiments

[0029] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. For those without specific techniques or conditions noted in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be purchased through regular channels.

[0030] 1. Key experimental reagents and materials.

[0031] 1.1. Experimental reagents.

[0032] The sources of the experimental reagents are shown in Table 1:

[0033] Table 1

[0034] Reagent Name Supplier Article Number Batch Number THLE-2 Medium Yiaobang 3.20306 KG202410 Immortalized Hepatocyte Line THLE-2 Yiaobang 06.0759 202409 Hepatic Stellate Cells Scios Bio CL-061h MCB20240821 Cisplatin White Shark Bio BS298 33124562AZ

[0035] In the following examples, the preparation method of the fibrous or granular solid extracellular matrix (i.e., the organoid-based extracellular matrix) composed of cellulose-L-lysine polymer includes the following steps:

[0036] Step 1, selective oxidation:

[0037] Dissolve 1.0 mol of cellulose (size: 250 μm) in 16200 mL of deionized water, stir well to form a suspension, and then add 1.0 mol of sodium periodate. Adjust the pH to 2.0 - 2.5 with 1M HCl. Then, let the mixture react under stirring in the dark at 40 °C for 48 hours. The product is washed with deionized water to remove salts (conductivity below 10 μs).

[0038] Step 2, nucleophilic carbonyl addition reaction to form Schiff base:

[0039] The insoluble 2,3-dialdehyde cellulose after the oxidation reaction is evenly suspended in 2000 mL of deionized water, and the pH is adjusted to 2.0 - 2.5 with 1M HCl. The oxidized cellulose suspension is directly dropped into 1 liter of 2.5M lysine aqueous solution with continuous stirring (300 RPM), pH 4.0. React at 25 °C for 4 hours, and then adjust the pH value of the reaction system to 8.0 with 1M NaOH and react at 25 °C for 4 hours.

[0040] Step 3, reduce the Schiff base (C=N) to secondary amine (C-NH):

[0041] Adjust the pH to 6 with 1M HCl, add 3 mol of sodium triacetoxyborohydride, and continuously react at 25 °C for 36 hours. After adjusting the pH of the reaction system to 8.0 with 1M NaOH, add 1 mol of sodium borohydride and continuously react at 25 °C for 24 hours. The product can be washed with deionized water to remove salts (conductivity below 10 μs). Allow natural precipitation for 24 hours and remove the supernatant. Sterilize by autoclaving.

[0042] As can be seen from Figure 5 A in, the major axis of the dried and dehydrated fibrous or particulate solid extracellular matrix is in the range of 100 - 1000 μm. As Figure 5 shown in B and C in, the fibrous or particulate solid extracellular matrix is formed by cross-linking of nanofibers to form a porous network structure, and the gap between nanofibers is less than 5 μm; it appears as a fibrous or short film-like structure on the surface.

[0043] The selectively oxidized polysaccharide is an insoluble solid fiber or particle, and the fiber length or the major axis of the particle is not less than 1 μm in size. The aldehyde moiety is generated by selectively oxidizing the hydroxyl groups on C2 and C3 of the glucose units, and the oxidation rate of the glucose units contained in the polysaccharide is not more than 80% and the oxidation does not produce more carboxyl groups than aldehyde groups or cause the breakage of the polysaccharide chain. The polypeptide includes one or more of collagen, gelatin, and polylysine; the extracellular matrix composed of cellulose - L-lysine polymer is an insoluble solid fiber or particle with a porous multi-layer network structure.

[0044] 1.2. Instrumentation.

[0045] The description of the instrumentation is shown in Table 2:

[0046] Table 2

[0047] Instrument Name Supplier Model Microplate Reader Beijing PULANG New Technology Co., Ltd. DNM-9602G Carbon Dioxide Incubator Thermo Fisher Scientific 3111

[0048] 2. Three-dimensional cell model of hepatotoxicity.

[0049] A three-dimensional cell model of hepatotoxicity is mainly constructed by the following method: inoculate hepatocytes and hepatic stellate cells in a culture medium containing an extracellular matrix (specifically, a hepatic organoid-based extracellular matrix), such as THLE-2 medium, and perform three-dimensional cell co-culture to obtain a three-dimensional cell model.

[0050] Preferably, this example includes the following specific steps: inoculate hepatocytes and hepatic stellate cells in a well plate, add a culture medium containing an extracellular matrix to the well plate, and perform three-dimensional cell co-culture to obtain a three-dimensional cell model;

[0051] The total cell concentration inoculated in the well plate is 1.0 - 4.0×10 6cells / mL, such as 1.0×10 6 cells / mL, 2×10 6 cells / mL, 4.0×10 6 cells / mL, etc. The inoculation number ratio of the hepatocytes to the hepatic stellate cells is 3:0.5 - 1.5, such as 3:0.5, 3:1, 3:1.5, etc.

[0052] Preferably in this example, the volume ratio of the extracellular matrix to the culture medium in the culture medium containing the extracellular matrix is 0.5 - 1.5:3, such as 0.5:3, 1:3, 1.5:3, etc.

[0053] Preferably in this example, the conditions for the three-dimensional cell co-culture are: 37°C, 5% CO2, and culture for 5 - 7 days.

[0054] 3. Verification experiment.

[0055] 3.1. Cell culture:

[0056] (1) Cell line resuscitation and culture; Take out the cryopreserved THLE-2 cell line and hepatic stellate cell line from liquid nitrogen, quickly put them into a 37°C water bath and gently shake to quickly melt the cells. After melting, transfer them into a 15 mL centrifuge tube, add 10 mL of pre-warmed THLE-2 medium, centrifuge at room temperature, centrifuge at 400 g for 5 minutes, aspirate and discard the supernatant, and perform cell counting. The initial inoculation concentration of the cells is 1.0×10 5 cells per flask. Add 3 mL of medium to resuspend the cells, and then inoculate the cells into a T25 flask and place it in an incubator for standby culture. Regularly observe the cell morphology and growth status. When the cells are in the logarithmic growth phase, perform subculture to maintain cell viability and proliferation ability.

[0057] (2) 3D cell culture: Digest the THLE-2 cell line and hepatic stellate cells in the logarithmic growth phase, count them, and group them into a hepatocyte group and a hepatocyte and hepatic stellate co-culture group (3:1). Adjust the total cell concentration to 2.0×10 6 cells / mL, inoculate the cells into a low-attachment 96-well plate, add 50 μL of extracellular matrix to each well, and supplement the medium to 150 μL.

[0058] 3.2. Detection of different alcoholic liver toxicities:

[0059] (1) Cell seeding: Resuspend the cells with the culture medium and count them. There are approximately 10,000 live cells per well. After thoroughly mixing the extracellular matrix and the cell suspension in a volume ratio of 1:2, use a single-channel pipette to dispense 90 μL of the suspension into each well of a 96-well plate. Set 6 wells as negative controls, and the rest are drug wells. After thoroughly mixing the extracellular matrix and the cell suspension in a volume ratio of 1:1, use a single-channel pipette to dispense 90 μL of the suspension into each well of a 96-well plate as blank control wells, usually set 6 wells. Gently tap the four sides of the well plate to make the cell suspension evenly distributed at the bottom.

[0060] (2) Preparation of different concentrations of alcohol and drug addition: After observing good cell growth under the microscope, conduct the alcohol hepatotoxicity drug addition experiment. The alcohol concentrations are set at 10% and 15%, which are diluted by adding distilled water to 75% alcohol. Observe the status of each well before adding the drug and exclude abnormal wells. The experiment sets a negative control group, a blank group, and an alcohol test group. The blank group has 6 replicates per plate, without adding alcohol, and 10 μL of culture medium is supplemented; the negative control group inoculates organoids, without adding alcohol, and 10 μL of culture medium is supplemented; the alcohol test group inoculates organoids and supplements 10 μL of 10% or 15% alcohol.

[0061] (3) Detection: After drug addition and incubation, conduct the detection. Take out the culture plate, observe under the microscope, exclude abnormal wells, add 10 μL of CCK8, incubate for 4 h, the detection conditions are room temperature (about 25 °) and wavelength 450 nm, shake for 2 min to promote cell lysis, and incubate for 8 min to make the luminescence signal stable. After the detection is completed, export the data, exclude abnormal wells, and calculate the cell survival rate: Survival rate (%) = ((OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)) × 100%; Use GraphPad Prism 8 software to draw the dose-response curve and calculate the EC50 value.

[0062] 3.3. Detection of cisplatin hepatotoxicity:

[0063] (1) Cell seeding: Resuspend the cells with the culture medium and count them. There are approximately 10,000 live cells per well. After thoroughly mixing the extracellular matrix and the cell suspension in a volume ratio of 1:2, use a single-channel pipette to dispense 90 μL of the suspension into each well of a 96-well plate. Set 6 wells as negative controls, and the rest are drug wells. After thoroughly mixing the extracellular matrix and the cell suspension in a volume ratio of 1:1, use a single-channel pipette to dispense 90 μL of the suspension into each well of a 96-well plate as blank control wells, usually set 6 wells. Gently tap the four sides of the well plate to make the cell suspension evenly distributed at the bottom.

[0064] (2) Drug exposure: After observing good cell growth under the microscope, the drug addition experiment was carried out. Before adding the drug, observe the status of each well and exclude abnormal wells. The experiment was set up with a negative control group, a blank group, and a drug test group. The blank group had 6 replicates per plate, without adding drugs, and supplemented with 10 μL of culture medium; the negative control group was seeded with organoids, without adding drugs, and supplemented with 10 μL of culture medium; the drug test group was seeded with organoids and supplemented with 10 μL of the drug solution.

[0065] (3) Preparation of the drug stock solution plate: Dilute the drug stock solution on the drug plate according to the requirements, and then perform gradient dilution. There were a total of 6 concentration gradients. Pipette 10 μL of the drug solution into the cell culture plate. After adding the drug, place it in the incubator and let it stand for 72 h according to the experimental requirements.

[0066] (4) Detection: After drug addition and incubation, the experiment was carried out. Take out the culture plate, observe under the microscope, exclude abnormal wells, add 10 μL of CCK8, incubate for 4 h, the detection conditions were room temperature (about 25 °C) and wavelength 450 nm, shake for 2 min to promote cell lysis, and incubate for 8 min to make the luminescence signal stable. After the detection was completed, export the data, exclude abnormal wells, and calculate the cell survival rate: Survival rate (%) = ((OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)) × 100%. Use GraphPad Prism 8 software to plot the dose-response curve and calculate the EC50 value.

[0067] 3.4. Results of the hepatotoxicity experiment.

[0068] 3.4.1. Results of the effects of different concentrations of alcohol on THLE-2, THLE-2 and hepatic stellate co-culture.

[0069] Figure 1 and Figure 2 The detection results showed that alcohol concentrations of 10% and 15% did not affect the activities of THLE-2 cells, THLE-2 and hepatic stellate cells, confirming that low-concentration alcohol has low toxicity to 3D hepatocytes and good cell tolerance.

[0070] 3.4.2. Results of the hepatotoxicity effects of cisplatin on THLE-2, THLE-2 and hepatic stellate co-culture.

[0071] Table 3

[0072] EC50 (μM) Inhibition Rate at the Highest Concentration (%) Drug Name Cisplatin Cisplatin THLE-2 ＞100 23.91 THLE-2 + Hepatic Stellate ＞100 10.7

[0073] Figure 3 and 4 And the detection results in Table 3 showed that high concentrations of cisplatin, a representative drug for hepatotoxicity, had a certain effect on the activity of THLE-2, but as the concentration decreased, the activity gradually increased. At the same time, the addition of hepatic stellate cells could provide drug resistance for hepatocytes.

[0074] In the early stage of drug research and development, the three-dimensional cell model of hepatotoxicity of the present invention can be used to quickly screen out drug compounds with potential hepatotoxicity. By observing the changes in cell morphology, metabolism, function, etc. under the action of drugs, it is judged whether the drugs cause damage to liver cells, so as to timely eliminate drug candidates that may have serious hepatotoxicity, avoid discovering problems only in subsequent clinical trials, and save research and development time and costs.

[0075] Cisplatin is a widely used chemotherapeutic drug, mainly used to treat various solid tumors (such as ovarian cancer, testicular cancer, lung cancer, etc.). However, the clinical application of cisplatin is limited by its toxic side effects, and hepatotoxicity is one of its important side effects. The present invention proves that THLE-2 cells can be used as a hepatotoxicity-resistant cell model for scientific research by comparing the hepatotoxic effects of different concentrations of alcohol and cisplatin on THLE-2.

[0076] In the subsequent model establishment, the effects of different drug combinations on hepatotoxicity-resistant cells can also be studied to explore the optimal combination drug regimen, so as to enhance the therapeutic effect, reduce the dosage of single drugs, and reduce drug adverse reactions.

[0077] In summary, the three-dimensional cell model of hepatotoxicity of the present invention selects THLE-2 cells and hepatic stellate cells, which can be cultured for a long time, have high stability, retain the activity of liver metabolic enzymes, can more accurately simulate the physiological and pathological reactions of the human liver, and are more suitable for studying drug metabolism and toxicity reactions; using the three-dimensional cell model of hepatotoxicity of the present invention, drug compounds with potential hepatotoxicity can be quickly screened out, and drug candidates that may have serious hepatotoxicity can be timely eliminated, saving research and development time and costs.

[0078] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a three-dimensional cell model of hepatotoxicity, characterized in that, It includes the following steps: inoculating hepatocytes and hepatic stellate cells in a culture medium containing extracellular matrix, performing three-dimensional cell co-culture, and obtaining a three-dimensional cell model of hepatotoxicity.

2. The construction method of a three-dimensional cell model with hepatotoxicity according to claim 1, characterized in that It includes the following specific steps: inoculating hepatocytes and hepatic stellate cells in a well plate, adding a culture medium containing extracellular matrix to the well plate, performing three-dimensional cell co-culture, and obtaining a three-dimensional cell model of hepatotoxicity; The total cell concentration inoculated in the well plate is 1.0 - 4.0×10 6 cells / mL, and the number ratio of the hepatocytes to the hepatic stellate cells inoculated is 3:0.5 - 1.

5.

3. The construction method of a three-dimensional cell model with hepatotoxicity according to claim 1, characterized in that, In the culture medium containing extracellular matrix, the volume ratio of extracellular matrix to the culture medium is 0.5 - 1.5:

3.

4. The construction method of a three-dimensional cell model with hepatotoxicity according to any one of claims 1 to 3, characterized in that, The conditions for the three-dimensional cell co-culture are: 37 °C, 5% CO2, and culturing for 5 - 7 days.

5. A three-dimensional cell model of hepatotoxicity, characterized in that, The three-dimensional cell model of hepatotoxicity is constructed by the construction method described in any one of claims 1 to 4.

6. Use of a three-dimensional cell model of hepatotoxicity, characterized in that, The three-dimensional cell model of hepatotoxicity described in claim 5 is used for the evaluation of hepatotoxic substances.

7. A method for evaluating the hepatotoxicity of a substance using the three-dimensional cell model of hepatotoxicity according to claim 5, characterized in that, It includes the following steps: (1) Cell plating: resuspending and counting the three-dimensional cell model with a hepatocarcinoma cell culture medium to obtain a cell suspension; adding extracellular matrix to the cell suspension, mixing evenly and transferring it to a well plate to obtain a well plate for cell plating; (2) Drug hepatotoxicity evaluation: performing a hepatotoxicity drug addition experiment in the well plate for cell plating, detecting cell viability, and used for the acute / chronic evaluation of hepatotoxicity caused by substances.

8. A method for evaluating the hepatotoxicity of a substance using the three-dimensional cell model of hepatotoxicity according to claim 7, characterized in that The density of the cells in the cell suspension described in step (1) is 0.5 - 2×10 4 cells / mL.

9. A method for evaluating the hepatotoxicity of a substance using the three-dimensional cell model of hepatotoxicity according to claim 7, characterized in that, In step (1), the volume ratio of the cell suspension to the extracellular matrix is 2:0.5 - 1.

5.

10. A method for evaluating the hepatotoxicity of a substance using the three-dimensional cell model of hepatotoxicity according to claim 7, characterized in that, The cell viability detection is performed using any one of the CCK-8 method, MTT CCK-8 method, and CyQUANT method.

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