In-vitro construction method of liver fibrosis organ model

A liver fibrosis and construction method technology, applied in the direction of artificial cell constructs, vertebrate cells, animal cells, etc., can solve the problems of not being able to simulate the microenvironment of the liver well, lack of cell-cells, etc., and achieve rapid construction, small size The effect of uniform structure and uniform model shape

Active Publication Date: 2019-06-14
AFFILIATED HOSPITAL OF NANTONG UNIV
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Problems solved by technology

Although it has shown certain reference value for the study of fibrosis mechanism and drug screening, ordinary monolayer culture cannot well simulate the m

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  • In-vitro construction method of liver fibrosis organ model
  • In-vitro construction method of liver fibrosis organ model
  • In-vitro construction method of liver fibrosis organ model

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Embodiment Construction

[0028] see figure 1 , figure 1 The process flow diagram for the preparation of liver fibrosis organoids provided by the present invention.

[0029] Step 1: Primary sorting of various liver component cells, and cryopreservation in liquid nitrogen; or direct purchase of various frozen liver component cells. Liver cells, hepatic stellate cells, Kupffer cells, and hepatic sinusoidal endothelial cells were carefully recovered using an automatic cell recovery system, centrifuged at 800rps, resuspended in medium A, and counted using a cell counter. According to the ratio of 7:1.5:0.5:1, 1200 cells per well / 100 µL were carefully and evenly planted on ultra-low adsorption round-bottom cell culture plates using a row gun, and cultured for 3 days. Medium A components include: 2 µg / mL rat tail collagen type I, 10% fetal bovine serum, 8 µg / mL bovine insulin, 10 ng / mL hydrocortisone, 2 µmol / mL glutamine (GlutaMAX), 2 µmol / mL 4 -Hydroxyethylpiperazineethanesulfonic acid (HEPES), William's...

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Abstract

The invention discloses an in-vitro construction method of a liver fibrosis organ model. According to the invention, hepatocytes, hepatic stellate cells, Kupffer cells and liver sinusoidal endothelialcells are suspended in a medium A and cultured, and then is cultured by changing a medium B at the fourth day; and from the tenth day, the medium B is changed to a medium C for continuous culture forsix days. The method is based on the proportion of cellular components in the liver, and rapidly constructs the in-vitro model of 3D liver fibrosis organs with uniform size and structure and obviousfibrosis symptom. The liver fibrosis organ model constructed by the invention has the advantages of clear principle, stable structure, simple method, rapid construction and strong operability, is suitable for the study of a generation and development mechanism of liver fibrosis as well as high-throughput screening of anti-fibrotic drugs, and has industrial significance.

Description

technical field [0001] The invention relates to an in vitro construction method of a liver fibrosis organoid model. Background technique [0002] Liver fibrosis is a complex inflammatory response caused by long-term chronic liver damage, and there is a great risk of developing liver cirrhosis and even liver cancer. Etiology shows that many factors are related to liver fibrosis, such as hepatitis virus infection, excessive drinking, autoimmune diseases, obesity and so on. Liver fibrosis can be defined as a generalized scarring process accompanied by changes in internal composition and function. Among them, the abnormal secretion of extracellular matrix caused by the activation of hepatic stellate cells plays an extremely important role in liver fibrosis. Therefore, previous in vitro models of liver fibrosis were often based on plain 2D cultures of hepatic stellate cells. Although it has shown certain reference value for the study of fibrosis mechanism and drug screening, o...

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Application Information

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IPC IPC(8): C12N5/071
Inventor 郑文杰顾志峰郭益冰姚登福赵伟新陈飞郭悦华杨君伶
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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