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Method for long-time in-vitro culturing and proliferating hepatic cells and application of method

A technology for in vitro culture and hepatocytes, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc.

Active Publication Date: 2016-02-03
SHANGHAI CELLIVER BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0030] At present, there is no method for long-term in vitro culture and expansion of hepatocytes at home and abroad, especially a method that can induce mature hepatic parenchymal cells into hepatocytes with unlimited proliferation ability in vitro

Method used

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  • Method for long-time in-vitro culturing and proliferating hepatic cells and application of method
  • Method for long-time in-vitro culturing and proliferating hepatic cells and application of method
  • Method for long-time in-vitro culturing and proliferating hepatic cells and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] 1. Long-term culture and expansion of primary hepatocytes

[0095] 1. Pre-coating of cell culture plates.

[0096] The specific coating method of CollagenI is as follows: the entire coating process is performed on ice, taking the 24-well plate coating as an example, put the collagen product (5mg / ml) solution on ice for later use; take a 15ml centrifuge tube and place it on ice , add pre-cooled basal medium, such as: DMEM10ml; take 100ul collagen product (5mg / ml) and add it to the above 10ml DMEM, mix well; the concentration of this solution is 0.05mg / ml; the prepared above Add the collagen solution to the corresponding well plate, add 300 μL to each well of the 24-well plate; place the culture plate on the ultra-clean table, shake gently, so that the collagen solution covers the bottom of the entire well and let stand. Put the well plate in the incubator overnight; take out the culture plate, remove the supernatant, add 500 μL of basal medium to each well to wash twice...

Embodiment 2

[0118] 1. Pre-coating of cell culture plates.

[0119] Use Matrigel for coating, place frozen Matrigel at 4°C overnight to make it liquid, dilute it with pre-cooled serum-free medium (such as DMEM) at 1:80, add it to the culture well, preferably to cover the bottom surface, It can be used after one hour at 37°C.

[0120] 2. The separation of primary human hepatic parenchymal cells is the same as in Example 1.

[0121] 3. Culture and expansion of primary hepatocytes.

[0122] The final concentration of human fibroblast growth factor-2 in the above-mentioned hepatocyte proliferation medium was 50 ng / ml; the final concentration of the small molecule agonist CHIR98014 of the Wnt signaling pathway was 3 μM; the final concentration of the Rho-related protein kinase inhibitor thiazovivin was 1 μM , all the other are with embodiment 1.

[0123] 4. The differentiation and maturation of liver cells and the identification of cell properties are the same as in Example 1, and the identi...

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Abstract

The invention relates to the field of biotechnology and provides a method for long-time in-vitro culturing and proliferating hepatic cells. The method includes that mature hepatic parenchymal cells are induced to be hepatic cells with infinite proliferation capability in vitro. The hepatic cells obtained by the method can be used for toxicological and pharmacological evaluation of compounds and drug, study and diagnosis as well as treatment of hepatitis virus, hepatic cell transplantation therapy and preparation of bioartificial liver.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for long-term in vitro culture and expansion of hepatocytes, which induces mature hepatic parenchymal cells in vitro to become hepatocytes with unlimited proliferation capacity, and the hepatocytes can be used for compounds and Toxicological and pharmacological evaluation of drugs, research and diagnosis and treatment of hepatitis virus, liver cell transplantation treatment, preparation of bioartificial liver and other applications. Background technique [0002] 1. Liver and liver cells [0003] The liver is an important vital organ in animals with metabolic functions as its main function. It regulates a variety of physiological functions, including detoxification, storage of liver glycogen, synthesis of secretory proteins, and production of bile. The liver itself has a strong ability to regenerate. After a normal liver undergoes two-thirds partial hepatectomy, the remainin...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02A61K35/407A61P31/12A61L27/38
Inventor 吴红平
Owner SHANGHAI CELLIVER BIOTECHNOLOGY CO LTD
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