3D liver microsphere model of non-alcoholic fatty liver and use thereof

Abstract

Provided are an induction medium of non-alcoholic fatty liver and the use thereof. The provided induction medium of non-alcoholic fatty liver contains a hepatocyte basal medium, monosaccharide, fatty acid and bovine serum albumin. The provided 3D microsphere model of non-alcoholic fatty liver is adapted for the biological activity evaluation of a drug, and can be used to effectively screen a candidate drug that can alleviate non-alcoholic fatty liver disease conditions. Compared with an animal experiment, the cell evaluation model used has a simper experimental procedure and operation, a significantly shortened experiment cycle, and lower cost.

Description

A non-alcoholic fatty liver disease 3D liver microsphere model and its use Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a non-alcoholic fatty liver induction culture medium and application thereof. Background Art

[0002] The liver is a vital and complex organ involved in protein and fat metabolism, bile production, detoxification, and immune regulation. Non-alcoholic fatty liver disease (NAFLD) is now considered one of the most common liver diseases worldwide. It is characterized by excessive fat deposition within hepatocytes (fat accounts for more than 5% of the liver's weight). Simple steatosis is a relatively benign state of liver damage. Fatty liver is susceptible to further damage from oxidative stress, adipocyte apoptosis, and inflammatory cytokines, factors that can lead to the development of non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), and cirrhosis.

[0003] For a long time, the pathogenesis and drug research of NAFLD have relied on in vitro cell lines and in vivo animal models. As a basic research, the state and composition of cells in in vitro cell lines are very different from those in vivo, and they cannot truly simulate the actual state in vivo. The cost of obtaining animal models through drug or dietary intervention is extremely high, and it is difficult to achieve high throughput in a short period of time. With the development of technology, 3D models have solved the above problems to a certain extent in recent years. Liver spheroids are currently the most commonly used 3D culture model. The formation of spheroids is caused by the self-aggregation of hepatocytes, and does not require technical intervention such as non-adhesive surfaces and gravity adhesion.

[0004] NAFLD is the most common chronic liver disease in the world. The global prevalence of NAFLD is 25%, and the incidence in China is 20%. An estimated 200-300 million people in my country suffer from NAFLD and over 30 million suffer from NASH. Obesity, type 2 diabetes, hyperlipidemia, and hypertension are the three major risk factors for NAFLD, with the prevalence of NAFLD in obese individuals reaching as high as 70%. With the increasing prevalence of obesity and the three highs, the prevalence of NAFLD is on the rise. Non-alcoholic fatty liver disease presents with characteristic lesions, ranging from simple NAFL degeneration to NASH, which can then lead to cirrhosis in parts of the liver and further progression to hepatocellular carcinoma (HCC). A US study showed that NAFLD-related HCC accounts for 59% of all HCCs.

[0005] NAFLD has a high incidence and serious consequences, but to date, no drug has been approved by the US Food and Drug Administration or the European Medicines Agency to treat NAFLD. There is still a lack of effective drugs to prevent or reverse disease progression in NASH patients. Studies have shown that lipid accumulation in hepatocytes is a key initiating event in the disease progression from NAFLD to NASH, cirrhosis / liver failure, and HCC. The development of preclinical in vitro models of NAFLD-NASH will pave the way to overcoming these challenges.

[0006] To date, most in vitro studies have utilized two-dimensional (2D) monolayer hepatocyte cultures to elucidate the molecular mechanisms involved in NAFLD. The advantages of 2D monolayer hepatocyte culture technology include mature methods and relatively simple cell selection and culture methods. However, this technology is limited to prolonged cell culture, making it impossible to study changes caused by long-term fat accumulation. More importantly, 2D monolayer cell culture not only fails to represent the complexity of human tissue but also lacks critical hepatocyte-nonparenchymal cell interactions. Summary of the Invention

[0007] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a non-alcoholic fatty liver disease induction culture medium and use thereof, so as to solve the problems in the prior art.

[0008] To achieve the above object and other related objects, the present invention provides a non-alcoholic fatty liver disease induction medium, comprising a hepatocyte basal medium, a monosaccharide, a fatty acid, and BSA, wherein, based on the total volume of the non-alcoholic fatty liver disease induction medium, the molar concentration of the monosaccharide is 200-300 μmol / L; and / or, based on the total volume of the non-alcoholic fatty liver disease induction medium, the molar concentration of the fatty acid is 150-250 μmol / L; and / or, based on the total volume of the non-alcoholic fatty liver disease induction medium, the volume fraction of the BSA is 0.5-1.5%.

[0009] Preferably, the non-alcoholic fatty liver disease induction medium contains lipopolysaccharide.

[0010] The present invention also provides use of the aforementioned non-alcoholic fatty liver disease induction culture medium in preparing a 3D non-alcoholic fatty liver disease microsphere model.

[0011] The present invention also provides a method for constructing a 3D non-alcoholic fatty liver disease microsphere model, which comprises one or more of the following steps:

[0012] 1) Mix HEP and NPC;

[0013] 2) culturing the cells mixed in step 1) in an ultra-low adsorption environment until hepatocyte microspheres are formed;

[0014] 3) Replace the culture medium in step 2) with the aforementioned non-alcoholic fatty liver induction medium and continue culturing until 3D non-alcoholic fatty liver microspheres are formed.

[0015] The present invention also provides 3D non-alcoholic fatty liver microspheres prepared by the aforementioned construction method.

[0016] The present invention also provides use of the aforementioned 3D non-alcoholic fatty liver microspheres in in vitro drug screening.

[0017] The present invention also provides a method for screening drugs for treating non-alcoholic fatty liver disease, the screening method comprising the following steps:

[0018] A) mixing the non-alcoholic fatty liver disease therapeutic drug to be screened with the aforementioned non-alcoholic fatty liver disease induction medium, and culturing the aforementioned 3D non-alcoholic fatty liver disease microspheres;

[0019] B) Screening for non-alcoholic fatty liver disease therapeutic drugs by detecting changes in one or more of the following related factors in the cells of the 3D non-alcoholic fatty liver disease microspheres cultured in step A): Col1a1, Pai1, SMA, Pdk4 or triglycerides.

[0020] As described above, the non-alcoholic fatty liver disease induction medium and its use of the present invention have the following beneficial effects:

[0021] (1) This invention addresses the bottleneck problem of lack of suitable in vitro models for early activity evaluation in drug development. By adopting a multidisciplinary research approach and taking 3D liver microsphere technology as the core, it innovatively constructs a 3D non-alcoholic fatty liver disease microsphere model. Verification shows that this model is suitable for drug bioactivity evaluation and effectively screens candidate drugs that can alleviate non-alcoholic fatty liver disease symptoms.

[0022] (2) Compared with animal experiments, the cell evaluation model used in the present invention has simpler experimental procedures and operations, significantly shortened experimental cycles, and lower costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a schematic diagram showing the key time nodes for the formation of 3D non-alcoholic fatty liver microspheres in the present invention.

[0024] FIG2 shows the image results of the formation process of 3D non-alcoholic fatty liver microspheres and the drug treatment effect in the present invention.

[0025] FIG3 shows the immunofluorescence results of relevant markers after 3D non-alcoholic fatty liver disease microsphere induction and drug treatment in the present invention.

[0026] FIG4 shows the results of the changes in mRNA levels of related factors after 3D non-alcoholic fatty liver disease microsphere induction and drug treatment in the present invention.

[0027] FIG5 shows the results of changes in triglyceride and albumin levels after 3D non-alcoholic fatty liver disease microsphere induction and drug treatment in the present invention.

[0028] FIG6 shows the results of changes in CYP2B and CYP3A enzyme activities after induction of 3D non-alcoholic fatty liver disease microspheres and drug treatment in the present invention. DETAILED DESCRIPTION

[0029] The present invention provides a non-alcoholic fatty liver induction culture medium, which comprises a hepatocyte basal culture medium, monosaccharide, fatty acid and BSA.

[0030] In some embodiments, the non-alcoholic fatty liver disease induction medium may further contain lipopolysaccharide.

[0031] In some specific embodiments, the hepatocyte basal culture medium can be a commercially available hepatocyte culture medium, such as the culture medium with product number 5201 from ScienCell.

[0032] In some embodiments, the monosaccharide may be selected from one or more of glyceraldehyde, erythrose, threose, arabinose, ribose, xylose, lyxose, glucose, mannose, fructose, and galactose. Preferably, the monosaccharide is selected from one or more of glucose or fructose.

[0033] In some embodiments, the fatty acid can be selected from one or more of lauric acid, eleostearic acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, and arachidonic acid. Preferably, the fatty acid is selected from one or more of oleic acid or palmitic acid.

[0034] In some specific embodiments, the molar concentration of the monosaccharide is 200-300 µmol / L, based on the total volume of the non-alcoholic fatty liver disease induction medium. Specifically, the molar concentration of the monosaccharide is 200-220 µmol / L, 220-240 µmol / L, 240-250 µmol / L, 250-260 µmol / L, 260-280 µmol / L, or 280-300 µmol / L. Preferably, the molar concentration of the monosaccharide is 240-260 µmol / L.

[0035] In some specific embodiments, based on the total volume of the non-alcoholic fatty liver disease induction medium, the molar concentration of the fatty acid is 150-250 µmol / L. Specifically, the molar concentration of the fatty acid is 150-170 µmol / L, 170-190 µmol / L, 190-200 µmol / L, 200-210 µmol / L, 210-230 µmol / L, or 230-250 µmol / L. Preferably, the molar concentration of the fatty acid is 190-210 µmol / L.

[0036] In some embodiments, the volume fraction of BSA is 0.5-1.5% based on the total volume of the non-alcoholic fatty liver disease induction medium. Specifically, the volume fraction of BSA is 0.5-0.7%, 0.7-0.9%, 0.9-1%, 1-1.1%, 1.1-1.3%, or 1.3-1.5%. Preferably, the volume fraction of BSA is 0.9-1.1%.

[0037] In some specific embodiments, the concentration of the lipopolysaccharide is 5-15 ng / mL based on the total volume of the non-alcoholic fatty liver disease induction medium. Specifically, the concentration of the lipopolysaccharide is 5-7 ng / mL, 7-9 ng / mL, 9-10 ng / mL, 10-11 ng / mL, 11-13 ng / mL, or 13-15 ng / mL. Preferably, the concentration of the lipopolysaccharide is 9-11 ng / mL.

[0038] The present invention also provides use of the aforementioned non-alcoholic fatty liver disease induction culture medium in preparing a 3D non-alcoholic fatty liver disease microsphere model.

[0039] The present invention also provides a method for constructing a 3D non-alcoholic fatty liver disease microsphere model, which comprises one or more of the following steps:

[0040] 1) Mix HEP and NPC;

[0041] 2) culturing the cells mixed in step 1) in an ultra-low adsorption environment until hepatocyte microspheres are formed;

[0042] 3) The culture medium in step 2) is replaced with the aforementioned non-alcoholic fatty liver disease induction medium and culture is continued until 3D non-alcoholic fatty liver disease microspheres are formed. The ultra-low adsorption environment is an environment in which cells cannot adhere to the wall and grow.

[0043] In some embodiments, the ratio of HEP to NPC in step 1) is (1-3):1. Specifically, the ratio is (1-1.5):1, (1.5-2):1, (2-2.5):1, or (2.5-3):1. Preferably, the ratio is (1.5-2.5):1.

[0044] In some embodiments, based on the volume of HEP and NPC mixed in step 1), the cell density after mixing in step 1) is 20-40 cells / µL. Specifically, the cell density is 20-25 cells / µL, 25-30 cells / µL, 30-35 cells / µL, or 35-40 cells / µL. Preferably, the cell density is 25-35 cells / µL.

[0045] In some embodiments, the non-parenchymal liver cells are selected from one or more of sinusoidal endothelial cells, Kupffer cells, dendritic cells, NK cells, NKT cells, bile duct epithelial cells, or hepatic stellate cells. Preferably, the non-parenchymal liver cells include sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells.

[0046] In some embodiments, the culturing time in step 2) is 4-8 days. Specifically, the culturing time is 4, 5, 6, 7 or 8 days. Preferably, the culturing time is 5-7 days.

[0047] In some specific embodiments, the culturing in step 3) may include two parts: a culture period I and a culture period II, wherein the non-alcoholic fatty liver disease induction medium used in the culture period II contains lipopolysaccharide.

[0048] Furthermore, the culture period I is 5-9 days. Specifically, the culture period I is 5, 6, 7, 8 or 9 days. Preferably, the culture period I is 6-8 days.

[0049] Furthermore, the duration of the culture period II is 1-5 days. Specifically, the duration of the culture period II is 1, 2, 3, 4 or 5 days. Preferably, the duration of the culture period II is 2-4 days.

[0050] The present invention also provides 3D non-alcoholic fatty liver microspheres prepared by the aforementioned construction method.

[0051] The present invention also provides use of the aforementioned 3D non-alcoholic fatty liver microspheres in in vitro drug screening.

[0052] In some embodiments, the drug is selected from one or more of the following:

[0053] 1) Drugs that downregulate Col1a1;

[0054] 2) drugs that downregulate Pai1;

[0055] 3) drugs that downregulate SMA;

[0056] 4) Drugs that downregulate Pdk4;

[0057] 5) Drugs that upregulate Albumin;

[0058] 6) A drug that downregulates triglycerides. The upregulation or downregulation refers to increasing or decreasing the expression or content of the relevant factors in the aforementioned 3D non-alcoholic fatty liver disease microspheres relative to no drug addition.

[0059] The present invention also provides a method for screening drugs for treating non-alcoholic fatty liver disease, the screening method comprising the following steps:

[0060] A) mixing the non-alcoholic fatty liver disease therapeutic drug to be screened with the aforementioned non-alcoholic fatty liver disease induction medium, and culturing the aforementioned 3D non-alcoholic fatty liver disease microspheres;

[0061] B) Screening for non-alcoholic fatty liver disease therapeutic drugs by detecting changes in one or more of the following related factors in the cells of the 3D non-alcoholic fatty liver disease microspheres cultured in step A): Col1a1, Pai1, SMA, Pdk4 or triglycerides.

[0062] In the present invention, liver cells of drug research experimental animals commonly used in the art are applicable to the present invention.

[0063] In the present invention, the sources of HEP and NPC include species such as humans, rats, mice, beagles, and monkeys.

[0064] In the present invention, the 3D non-alcoholic fatty liver disease microspheres have a diameter of 150 to 250 μm, including but not limited to 151 μm, 152 μm, 160 μm, 170 μm, 180 μm, 190 μm, 220 μm, 240 μm or 249 μm.

[0065] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0066] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0067] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0068] Example 1 Construction of 3D non-alcoholic fatty liver microspheres

[0069] In this example, primary mouse hepatocytes were isolated by two-step collagenase perfusion.

[0070] Two-step in situ collagenase perfusion method for rodents

[0071] Primary hepatocytes were isolated from C57 mice (male, 6-8 weeks) and Sprague-Dawley rats (male, 6-8 weeks) using a modified Seglen two-step perfusion method (see Seglen, PO Preparation of isolate drastically liver cells. Methods Cell. Biol. 1976). After anesthesia, the abdominal cavity was opened and a needle was inserted through the portal vein and vena cava. The liver was first perfused with a pre-warmed 37°C optimized buffer containing EDTA, followed by perfusion with a pre-warmed collagenase buffer for enzymatic digestion and isolation of hepatocytes. After enzymatic digestion, the supernatant containing NPCs was collected by filtration (70 µm membrane) and low-speed centrifugation (50g, 2 min) and stored at 4°C for the next step of mouse liver non-parenchymal cell preparation. The cell pellet in the centrifuge tube was resuspended to obtain a mouse HEP suspension.

[0072] Purification and cryopreservation of hepatocytes

[0073] The resulting mouse HEP suspension is purified by density gradient centrifugation of hepatocytes (35% Percoll / 100g / 3min / 4°C for mice). The supernatant is discarded, and the HEP cell pellet is resuspended in liver culture medium containing 3% serum. The HEP cells are washed twice by centrifugation (50g / 3min / 4°C for mice). The supernatant is discarded, and the purified HEP pellet is resuspended in liver mammosphere formation medium to obtain a mouse hepatocyte suspension. The viable HEP cell count and viability (85%-95%) are then determined using a standard trypan blue exclusion method and a cell counter. Purified fresh primary hepatocytes can be used directly for hepatocyte microsphere generation or suspended in freezing medium and aliquoted using an automated cell cryostat for subsequent thawing and subsequent hepatocyte microsphere generation using the same procedure.

[0074] Enrichment, purification, and cryopreservation of non-parenchymal liver cells

[0075] The NPC cell suspension temporarily stored at 4°C was centrifuged twice at low speed (50g / 3min / 4°C for mice). After each centrifugation, the supernatant (NPC) was aspirated and the precipitate (residual HEP) was discarded. The supernatant collected after each centrifugation was then centrifuged at high speed (360g / 8min / 4°C for mice) to collect NPC cells. The cell pellet was resuspended in hepatocyte culture medium containing 3% serum (heat-inactivated) to obtain an NPC cell suspension.

[0076] The obtained NPCs of different species were purified by 2 concentrations of Percoll density gradient centrifugation. 20 mL of Percoll (50%) and 20 mL of Percoll (25%) were added to a centrifuge tube in sequence, and then 10 mL of NPC cell suspension was gently added to the upper surface of the Percoll separation medium. After Percoll density gradient centrifugation (600g / 8min / 4℃ for mice and rats; 1200g / 8min / 4℃ for cynomolgus monkeys), the supernatant containing cell debris and dead cells in the centrifuge tube was aspirated and discarded, and the liquid containing NPCs in the middle of the centrifuge tube was collected. The sediment at the bottom of the centrifuge tube was discarded, and then centrifuged and washed twice with the same culture medium and centrifugal speed as mentioned above. The supernatant was discarded, and the NPC cells were resuspended in conventional hepatocyte culture medium containing 10% serum. The number of viable NPC cells and the viability rate (85%-95%) were determined by conventional trypan blue exclusion method / cell counter.

[0077] Purified fresh primary NPCs can be used directly for the construction of liver microspheres, or suspended in freezing medium and aliquoted using an automated cell cryopreservation instrument to obtain frozen primary NPCs for later recovery and the construction of liver microspheres using the same procedure.

[0078] Following the steps in Figure 1, HEPs and NPCs from C57 mice were suspended in basal culture medium (ScienCell Catalog No. 5201) at a ratio of 2:1. The mixed cell suspension was then plated at 3,000 cells per well of a 96-well ultra-low attachment plate, with 100 µL of cell suspension seeded into each well. The plates were then placed in a 37°C CO2 incubator and incubated statically, minimizing movement. On the third day, the basal culture medium was replaced with half the volume, and the cells were maintained in culture. On the sixth day, after the microspheres stabilized, the old medium was aspirated, and 100 µL of induction medium was slowly added along the walls of the wells. Every two days, 50 µL of the medium was aspirated and the same amount of induction medium was added. After seven days of culture, induction medium containing 10 ng / mL lipopolysaccharide was added for a further three days. The morphology of the 3D non-alcoholic fatty liver microspheres at various time points is shown in Figure 2.

[0079] Example 2 Immunofluorescence analysis of 3D non-alcoholic fatty liver disease microsphere-related markers

[0080] Six microspheres were taken from each experimental group and placed in EP tubes. PFA fixative was added and fixed on ice for 30 min. The fixative was then removed and permeabilization solution was added. The cells were incubated at room temperature for 30 min. The permeabilization solution was removed and the microspheres were rinsed three times with 2% BSA and blocked with 2% BSA at room temperature for 1 h. The cells were incubated with HNF4α primary antibody in the dark at 4°C overnight. The primary antibody was recovered on the second day and the cells were rinsed three times with 2% BSA. The cells were then incubated with secondary antibody in the dark at room temperature for 1 h. The secondary antibody was recovered and the cells were incubated with SMA antibody overnight. On the third day, the SMA antibody was removed and the cells were rinsed three times with 2% BSA. The cells were stained with DPAI for 10 min and then rinsed three times with 1× PBS. The experimental microspheres were observed on a confocal dish and photographed.

[0081] The results of the above experiment are shown in Figure 3. There is no significant difference in the proliferation and differentiation ability of 3D non-alcoholic fatty liver microspheres compared to healthy cell microspheres, but the degree of fibrosis is significantly higher than that of healthy cell microspheres.

[0082] Example 3 Quantitative PCR Analysis of 3D Non-alcoholic Fatty Liver Disease Microsphere-Related Markers

[0083] RNA extraction

[0084] Six pellets were collected from each of the control and NASH groups into labeled 1.5 mL centrifuge tubes. 500 µL of Trizol was added and lysed for 5 minutes. The pellets were then vigorously pipetted and shaken on a shaker to ensure complete lysis. 100 µL of chloroform was added (Trizol:chloroform = 5:1), the tube was capped, and vortexed for 15 seconds. After thorough mixing, the tube was allowed to stand for 2-3 minutes before centrifuging at 12,000 g for 15 minutes at 4°C. Prepare a labeled 1.5 mL centrifuge tube and transfer the supernatant from the centrifugation to the tube. 375 µL of anhydrous ethanol was added and pipetted to mix thoroughly. The supernatant was then transferred to a 2 mL collection tube containing an RNease MinElute column and centrifuged at 12,000 g for 15 seconds. The filtrate was discarded. 500 µL of Buffer RWT was added to the RNease MinElute column and the tube was centrifuged at 12,000 g for 15 seconds. The filtrate was discarded. Add 400 µL of Buffer RPE to the RNease MinElute solution, centrifuge at 12,000 g for 15 seconds, and discard the filtrate from the collection tube. Add 400 µL of 80% RNase-free ethanol to the RNease MinElute solution, centrifuge at 12,000 g for 2 minutes, discard the filtrate from the collection tube, and replace it with a new 2 mL collection tube. Centrifuge at high speed for 5 minutes, discard the collection tube, replace it with a 1.5 mL RNase-free centrifuge tube, add 14 µL of enzyme-free water, and centrifuge at high speed for 1 minute to elute the RNA. Measure the concentration of 1 µL of the extracted RNA sample using a NanoDrop 2000C spectrophotometer; the A260 / 280 ratio should be between 1.8 and 2.0.

[0085] RNA reverse transcription

[0086] Prepare first-strand cDNA synthesis reaction solution

[0087] 2. Prepare the following mixture in an RNase-free centrifuge tube:

[0088] Enzyme-free water to 20 µl 5 × HiScript II Select qRT SuperMix 4 µl 0 Ligo (dT)23VN (10 μM) 1 µl Template RNA Total RNA: 1 pg - 1 µg

[0089] Mix by gently pipetting.

[0090] 3. Perform denaturation annealing reaction on a PCR instrument

[0091] 50℃ for 15 min; 85℃ for 5 s;

[0092] The product can be used immediately for qPCR reactions or stored at -20°C and used within six months. For long-term storage, it is recommended to store the product at -70°C after aliquoting. Avoid repeated freezing and thawing of the cDNA.

[0093] RT‑qPCR measurement of major related gene expression

[0094] SYBR Green Realtime PCR Master Mix kit was used to express the corresponding genes and fluorescence quantitative PCR was configured.

[0095] Reaction system:

[0096] Reagent Volume: Enzyme-free water 8.4 µL cDNA 1 µL Primers 1.6 µL SYBR Green Realtime PCR Master Mix 10 µL

[0097] Add each component to a 96-well plate in the order above. The SYBR Green Realtime PCR Master Mix dye should be protected from light. Three replicates were set for each sample. After sample addition, seal the plate with film and centrifuge at 4°C, 1000 rpm, for 5 minutes. Centrifuge the liquid to the bottom of the 96-well plate, then place the plate in a fluorescent quantitative PCR instrument. The PCR cycle conditions were: 95°C for 30 seconds; 95°C for 10 seconds, and 50°C for 30 seconds for 40 cycles. The default melting curve analysis primer specificity was used.

[0098] Calculation of relative expression: The Control group was used as a control and 2- ΔΔ The relative expression of the corresponding genes was calculated by CT method.

[0099] Δ CT=CT 目的基因 -CT Hprt

[0100] ΔΔ CT= Δ CT NASH - Δ CT Control

[0101] The expression results of the above-mentioned major related genes are shown in Figure 4, which shows that the relative expression levels of fibrosis, smooth muscle, and insulin resistance genes in the NASH group were higher than those in the control group, and the differences were statistically significant. The expression level of the albumin gene was lower in the control group, and the difference was statistically significant.

[0102] Example 4 Analysis of triglyceride and albumin levels in 3D non-alcoholic fatty liver disease microspheres

[0103] Triglycerides:

[0104] Six microspheres from each experimental group were placed in an EP tube. Isopropanol was added to cover the microspheres, and the mixture was manually homogenized and placed in a 60°C water bath for 2 minutes. An appropriate amount of alumina was then added, the tube was capped, and the mixture was rapidly shaken for 2 minutes. The supernatant (i.e., the extract) was collected for later use. 1 ml of protein precipitation solution, 0.5 ml of extract solution, and 0.1 ml of TG alkaline solution were added to the EP tube, mixed, and incubated in a 60°C incubator for 10 minutes. 0.5 ml of TG oxidant and 0.25 ml of TG colorimetric reagent were then added, mixed, and incubated in a 60°C incubator for 20 minutes. After incubation, the centrifuge tube was removed, cooled under running water, and an appropriate amount of the solution was transferred to a 96-well plate. The absorbance of each well was read at 420 nm using a microplate reader.

[0105] albumin:

[0106] Six microspheres were taken from each experimental group and placed in an EP tube. 9 volumes of PBS were added and the mixture was thoroughly homogenized and centrifuged at 2500 g for 10 min. 5 µL of supernatant was added to a 96-well plate. 250 µL of BCG reagent was then added and mixed immediately. The plate was allowed to stand at room temperature for (30 ± 3) s, and the absorbance of each well was read at 628 nm on a microplate reader.

[0107] The results are shown in Figure 5. Compared with healthy cells, the triglyceride and albumin levels in 3D non-alcoholic fatty liver microspheres were significantly increased, and intervention with Selonsertib or obeticholic acid could reduce the triglyceride and albumin levels in 3D non-alcoholic fatty liver microspheres to the levels of healthy cells.

[0108] Example 5 Analysis of changes in CYP metabolic enzyme levels in 3D non-alcoholic fatty liver disease microspheres

[0109] After the formation of HEP / NPC co-cultured 3D liver microspheres and 3D non-alcoholic fatty liver microspheres, the activities of two major P450 metabolic enzymes, CYP2B and CYP3A, were measured. The specific steps are as follows:

[0110] Pre-warm basal medium and prepare substrate (bupropion, testosterone)

[0111] Six pellets were collected in 96-well plates for the Control and NASH groups, respectively.

[0112] The incubation concentration of bupropion substrate was 200 μM, the incubation concentration of testosterone substrate was 75 μM, and the incubation time was 1 h.

[0113] After incubation for 1 h, add 600 μl of acetonitrile precipitant, centrifuge and take the supernatant.

[0114] The results are shown in Figure 6. In contrast, the microsphere model maintained comparable P450 metabolic activity after NASH induction.

[0115] Example 6 Application of 3D non-alcoholic fatty liver disease microspheres in drug screening

[0116] 10 µmol / L inhibitor solutions (Drug A: Selonsertib, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor; Drug B: Obeticholic acid, a farnesoid X receptor (FXR) agonist) were added to each well. The inhibitors were dissolved in DMSO to a 10 mmol / L stock solution for storage and diluted to the appropriate dosing concentration using induction medium. Three days after treatment, changes in 3D non-alcoholic fatty liver disease microsphere-related factors following inhibitor treatment were detected using immunofluorescence staining as described in Example 2, real-time quantitative PCR for albumin as described in Example 3, and a triglyceride assay as described in Example 4. CYP450 enzyme activity was also measured following drug treatment as described in Example 5.

[0117] As can be seen from Figures 3 to 5, after compound treatment, factors related to NASH all underwent significant changes, indicating that the constructed 3D non-alcoholic fatty liver disease microsphere model can be used for the screening of NASH inhibitors.

[0118] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A non-alcoholic fatty liver induction medium, characterized in that The non-alcoholic fatty liver disease induction medium comprises a hepatocyte basal medium, a monosaccharide, a fatty acid, and BSA. Based on the total volume of the non-alcoholic fatty liver disease induction medium, the molar concentration of the monosaccharide is 200-300 μmol / L; and / or, based on the total volume of the non-alcoholic fatty liver disease induction medium, the molar concentration of the fatty acid is 150-250 μmol / L; and / or, based on the total volume of the non-alcoholic fatty liver disease induction medium, the volume fraction of the BSA is 0.5-1.5%.

2. The non-alcoholic fatty liver disease induction medium according to claim 1, characterized in that The non-alcoholic fatty liver disease induction medium contains lipopolysaccharide. Based on the total volume of the non-alcoholic fatty liver disease induction medium, the concentration of the lipopolysaccharide is 5-15 ng / mL.

3. The non-alcoholic fatty liver disease induction medium according to claim 1, characterized in that The monosaccharide is selected from one or more of glyceraldehyde, erythrose, threose, arabinose, ribose, xylose, lyxose, glucose, mannose, fructose, and galactose; and / or the fatty acid is selected from one or more of lauric acid, eleostearic acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, and arachidonic acid; preferably, the monosaccharide is selected from one or more of glucose or fructose; and / or the fatty acid is selected from one or more of oleic acid or palmitic acid.

4. Use of the non-alcoholic fatty liver disease induction medium according to any one of claims 1 to 3 in preparing a non-alcoholic fatty liver disease 3D liver microsphere model.

5. A method for constructing a 3D non-alcoholic fatty liver disease microsphere model, characterized in that: The construction method comprises the following steps: 1) Mixing hepatic parenchymal cells and non-parenchymal hepatic cells; 2) culturing the cells mixed in step 1) in an ultra-low adsorption environment until hepatocyte microspheres are formed; 3) replacing the culture medium in step 2) with the non-alcoholic fatty liver disease induction medium as described in any one of claims 1 to 3 and continuing culturing until 3D non-alcoholic fatty liver disease microspheres are formed.

6. The construction method according to claim 5, characterized in that: The construction method includes one or more of the following features: I) In step 1), the ratio of the number of hepatic parenchymal cells to the number of non-hepatic parenchymal cells is (1-3):1; II) Based on the volume of the mixed hepatic parenchymal cells and non-parenchymal hepatic cells in step 1), the density of the mixed cells in step 1) is 20-40 cells / µL; III) The culturing time in step 2) is 4-8 days; IV) The culture in step 3) comprises two parts: culture period I and culture period II. The culture period I lasts for 5-9 days, and the culture period II lasts for 1-5 days. The non-alcoholic fatty liver induction medium used in culture period II contains lipopolysaccharide.

7. Non-alcoholic fatty liver disease 3D liver microspheres prepared by the construction method according to claim 5 or 6.

8. Use of the non-alcoholic fatty liver disease 3D liver microspheres according to claim 7 in in vitro drug screening.

9. The use according to claim 8, characterized in that The drug is selected from one or more of the following: 1) Drugs that downregulate Col1a1; 2) drugs that downregulate Pai1; 3) drugs that downregulate SMA; 4) Drugs that downregulate Pdk4; 5) Drugs that upregulate Albumin; 6) Drugs that lower triglycerides.

10. A method for screening drugs for treating non-alcoholic fatty liver disease, characterized in that: The screening method comprises the following steps: A) mixing the non-alcoholic fatty liver disease therapeutic drug to be screened with the non-alcoholic fatty liver disease induction medium according to any one of claims 1 to 3, and culturing the non-alcoholic fatty liver disease 3D liver microspheres according to claim 7; B) Screening for drugs for treating non-alcoholic fatty liver disease by detecting changes in one or more of the following related factors in the cells of the non-alcoholic fatty liver disease 3D liver microspheres cultured in step A): Col1a1, Pai1, SMA, Pdk4 or triglycerides.

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