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Hepatocyte in-vitro co-culture system as well as construction method and application thereof

A co-culture system and hepatocyte technology, applied in artificial cell constructs, cell culture active agents, cell culture supports/coatings, etc., can solve the problem of reduced drug metabolism in hepatocytes and loss of normal shape and function of hepatocytes And other issues

Pending Publication Date: 2020-10-23
GUANGDONG PROV MEDICAL INSTR INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to overcome the problem that existing hepatocytes rapidly lose their normal morphology and function in the process of two-dimensional in vitro culture, which leads to the decrease of the drug metabolism function of hepatocytes, the purpose of the first aspect of the present invention is to provide a hepatocyte in vitro Training system

Method used

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  • Hepatocyte in-vitro co-culture system as well as construction method and application thereof
  • Hepatocyte in-vitro co-culture system as well as construction method and application thereof
  • Hepatocyte in-vitro co-culture system as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of 3T3-J2 cell sheet / hepatocyte mass / collagen gel multilayer co-culture system

[0058] The schematic diagram of the construction process of the 3T3-J2 cell sheet / hepatocyte mass / collagen gel multilayer co-culture system is shown in figure 1 As shown, the details are as follows:

[0059] (1) 3T3-J2 fibroblasts (purchased from Kerafast) were taken, cultured according to the method specified in the manual, digested with trypsin, and set aside. Add 2 mL of fetal bovine serum to a 35 mm temperature-sensitive culture dish (purchased from UpCell), coat at 37°C overnight, suck out the fetal bovine serum in the temperature-sensitive culture dish, and place 3T3-J2 fibroblasts in the pre-coated fetal bovine serum Inoculate on temperature-sensitive culture dishes, inoculate 1.5 million to 2 million cells, place at 37°C, 5% CO 2 Cultivate in the incubator for 4 days, so that the cells cover the entire culture dish and produce enough extracellular matrix to ...

Embodiment 2

[0062] Example 2 Construction of Hepatocyte Group / Collagen Gel Individual Culture System

[0063] (1) Paste a silicone ring with an inner diameter of 15.5mm on a 35mm ordinary culture dish, and mix rat tail tendon type Ⅰ collagen stock solution (4mg / mL), 10 times phosphate buffer (pH7.4) and 1N NaOH solution according to 9 The volume ratio of :1.023:0.207 was mixed and prepared on ice to obtain a collagen suspension (the type I collagen stock solution of rat tail tendon was purchased from Corning, and the collagen suspension preparation method refers to the instruction manual). Add 200-300 μL of collagen suspension to the center of the silica gel ring, and solidify into a gel at 37°C for 1h-12h. The hepatocytes were primary human hepatocytes (purchased from Thermo Fisher), which were resuscitated according to the method specified in the instruction manual. The cell viability was detected by trypan blue, and the cell viability reached more than 90%, which was reserved for use. ...

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Abstract

The invention discloses a hepatocyte in-vitro co-culture system as well as a construction method and application thereof. The hepatocyte in-vitro co-culture system comprises an extracellular matrix, hepatocytes and 3T3-J2 fibroblasts, wherein the hepatocytes are planted on the extracellular matrix, and the 3T3-J2 fibroblasts are laid on the surfaces of the hepatocytes. The construction method of the hepatocyte in-vitro co-culture system comprises the following steps: inoculating 3T3-J2 fibroblasts on a temperature-sensitive culture dish, carrying out culturing and stripping to obtain the hepatocyte in-vitro co-culture system. The hepatocyte in-vitro co-culture system provided by the invention provides a three-dimensional form closer to an in-vivo liver tissue microenvironment for human hepatocytes, ensures tight hepatocyte / hepatocyte interconnection and hepatocyte / supporting cell interaction, and can effectively enhance and maintain hepatocyte functions; and the system can be used forlong-term drug evaluation.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to an in vitro co-culture system of hepatocytes and its construction method and application. Background technique [0002] Drug-induced liver injury is an important reason for the failure of drug development and the withdrawal of drugs after marketing. Due to the species differences between animals and humans, animal models cannot guarantee accurate prediction of drug hepatotoxicity in humans. Therefore, it is particularly important to establish a stable and reliable in vitro human primary hepatocyte model and use it to predict the hepatotoxicity of drugs. However, during ordinary two-dimensional in vitro culture of human primary hepatocytes, due to the lack of three-dimensional structure of the liver in vivo, intercellular interactions and growth factor support, the normal morphology and function will be rapidly lost, resulting in drug metabolism in hepatocytes. ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0671G01N33/5014G01N33/5067C12N2502/1323C12N2533/90C12N2533/54C12N2501/39C12N2500/25C12N2501/335C12N2503/02G01N2500/10C12N2513/00
Inventor 高博韬
Owner GUANGDONG PROV MEDICAL INSTR INST
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