Method for establishing an in vitro simulated liver disease model and its special three-dimensional medium

An in vitro simulation and culture medium technology, applied in the field of bioengineering, can solve the problems of establishing a three-dimensional liver cell culture model for liver cells, and achieve the effects of easy acquisition, definite chemical composition, and simple operation

Active Publication Date: 2022-04-29
ACADEMY OF MILITARY MEDICAL SCI
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Problems solved by technology

It has been reported in the literature that the 3D co-culture of the human hepatic cell line HepaRG and hepatic stellate cells was used to generate a 3D cell model for toxicity assessment and fibrosis studies (Leite SB, Roosens T, El Taghdouini A et al. Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro. Biomaterials 2016; 78:1-10. Coll M, Perea L, Boon R et al. Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis. Cell StemCell 2018; 23 :101-113e107.), but there is no use of human stem cell-derived hepatocytes to establish a three-dimensional hepatocyte culture model for the study of alcoholic liver disease

Method used

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  • Method for establishing an in vitro simulated liver disease model and its special three-dimensional medium
  • Method for establishing an in vitro simulated liver disease model and its special three-dimensional medium
  • Method for establishing an in vitro simulated liver disease model and its special three-dimensional medium

Examples

Experimental program
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Effect test

Embodiment 1

[0067] Example 1. Establishment of liver organoid culture system and phenotypic identification

[0068] In this embodiment, pluripotent stem cells (cell line H9, human embryonic stem cells (human EmbryonicStem Cells), purchased from Wicell Research Center, USA) are used as starting cells, such as figure 1 As shown, hepatic organoids (hEHOs) were obtained sequentially through the qualitative endoderm stage and the liver stem / progenitor cell stage. Specifically include the following steps:

[0069] 1.1. Culture of cell line H9 cells

[0070] 1) Take Matrigel (Corning) out of the -80°C refrigerator one day in advance, and thaw it in a 4°C refrigerator. After it melts, dilute Matrigel at a ratio of 1:100 with pre-cooled DMEM / F12 medium (Gibco).

[0071] 2) Evenly spread the diluted Matrigel on a six-well plate, 1 mL per well, and incubate for 1 hour in a 37°C incubator for later use.

[0072] 3) Take out the H9 cells to be subcultured from the incubator at 37°C, suck off the wa...

Embodiment 2

[0182] Example 2: Establishment of an in vitro simulated liver disease model

[0183] like figure 1 As shown, this example establishes an in vitro model of alcoholic liver disease based on the co-culture of hEHOs and stromal cells obtained in Example 1. The stromal cells and the added medium components constitute the microenvironment for the differentiation of hEHOs and promote the induction of hEHOs to mature hepatocytes. Additional stromal cells were added as an indicator of whether alcohol-induced injury caused fibrosis. Usually, the fibrotic changes in the pathological injury of alcoholic liver are mainly the activation, proliferation and fibrosis of liver stromal cells. The purpose of adding stromal cells is to simulate the pathological phenomenon of alcoholic liver disease. The stromal cells used in this example can be a commercially available fetal liver stromal cell line (FL 62891 CRL-11005 TM ), commercially available mesenchymal stem cells, derived from isolated...

Embodiment 3

[0209] Example 3: Modeling Alcoholic Liver Disease in Vitro

[0210] like figure 1 As shown, this example uses the hFLMC / hEHO co-culture system established in Example 2 to simulate alcoholic liver disease in vitro by adding alcohol, specifically including the following methods:

[0211] 3.1. Alcohol treatment of hFLMC / hEHO co-culture system

[0212] 1) After hFLMC / hEHO co-cultivation for 14 days, the medium was replaced with HM medium supplemented with 100mM alcohol for 7 days as the alcohol treatment group, and the 96-well plate of the control group was replaced with alcohol-free HM medium for 7 days;

[0213] 2) Change the medium every day, and wrap the 96-well culture plate tightly with parafilm to prevent alcohol from volatilizing.

[0214] 3.2. Quantitative analysis of Dead / Live staining

[0215] 1) Collect the three-dimensional balls of the control group and alcohol-treated group in 3.1 above, discard the supernatant, and wash twice with PBS;

[0216] 2) Add 8 μM cal...

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Abstract

The invention discloses a method for establishing an in vitro model for simulating alcoholic liver disease and a special three-dimensional culture medium thereof, belonging to the technical field of bioengineering. The in vitro simulated alcoholic liver disease model provided by the present invention is obtained by co-culturing liver organoids induced by pluripotent stem cells and stromal cells using the special three-dimensional medium provided by the present invention, which can express enzymes related to alcohol metabolism and can be used for The effects of alcoholic liver disease were mimicked in vitro by the addition of alcohol. The special three-dimensional medium provided contains a variety of small molecules and cytokines, which is more suitable for the mixed co-culture system of stromal cells and liver organoids. It has the characteristics of serum-free, definite chemical composition, simple and easy operation, etc.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for establishing an in vitro simulated liver disease model and its special three-dimensional culture medium, in particular to a method for establishing an in vitro simulated alcoholic liver disease model by using liver organoids induced by pluripotent stem cells And its special three-dimensional culture medium. Background technique [0002] Alcoholic liver disease (ALD) is an important cause of death worldwide, and its progression includes several stages: simple fatty liver, steatohepatitis, liver fibrosis, liver cirrhosis, and hepatocellular carcinoma (Gao , B., and Bataller, R, et al. Alcoholic liver disease: pathogenesis and new therapeutic targets. Gastroenterology 2011 Nov; 141(5):1572-85). However, due to the lack of reliable models to simulate the pathological process of human ALD and elucidate the mechanism of its occurrence, the development of prevention o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0671G01N33/5067C12N2513/00C12N2501/998C12N2500/32C12N2500/60C12N2500/38C12N2501/12C12N2501/727C12N2501/237C12N2501/39C12N2503/02G01N2500/10
Inventor 王韫芳王术勇王璇谭作龙宿钰鑫胡健王勇
Owner ACADEMY OF MILITARY MEDICAL SCI
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