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Method for separating and culturing primary hepatocytes

A technology of primary cells and culture methods, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of hepatocytes without proliferation and activity cannot be maintained

Active Publication Date: 2011-05-04
BEIJING IPHASE PHARMA SERVICES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method for separating and culturing primary liver cells, which can solve the blank in the domestic field of primary liver cells. The cultivation of liver cells adopts the "sandwich glue" interlayer method, which can keep them for a long time The vitality of the liver cells solves the problem that the hepatocytes cannot proliferate like cell lines and cannot maintain their activity

Method used

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  • Method for separating and culturing primary hepatocytes
  • Method for separating and culturing primary hepatocytes
  • Method for separating and culturing primary hepatocytes

Examples

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Effect test

Embodiment 1

[0023] Example 1: Isolation and culture method of dog liver primary cells

[0024] Separation and culture methods: 1) weighing; 2) washing the liver with pre-cooled calcium-free perfusate, on the one hand washing the blood residue in the liver, and on the other hand looking for suitable blood vessels, which can be used as the main blood vessels for washing liver tissue blocks , to prepare for the next step of perfusion with calcium-free and collagenase perfusate; 3) Use sterile gauze to dry the water around the liver tissue block and the cut surface, and insert the yellow tip of the perfusion cannula into the pre-selected blood vessel During the process, the cut surface was wrapped with glue to make it a fluid-tight liver tissue block, and then a small opening was cut at the front end to drain the perfused fluid. Calcium-free and collagenase perfusate were washed successively for 10-15 minutes, and then Stop the perfusion, tear off the rubber, and put it in F containing 5% fet...

Embodiment 2

[0025] Embodiment 2: Monkey liver primary cell isolation and culture method

[0026]Separation and culture methods: 1) weighing; 2) washing the liver with pre-cooled calcium-free perfusate, on the one hand washing the blood residue in the liver, and on the other hand looking for suitable blood vessels, which can be used as the main blood vessels for washing liver tissue blocks , to prepare for the next step of perfusion with calcium-free and collagenase perfusate; 3) Use sterile gauze to dry the water around the liver tissue block and the cut surface, and insert the yellow tip of the perfusion cannula into the pre-selected blood vessel During the process, the cut surface was wrapped with glue to make it a fluid-tight liver tissue block, and then a small opening was cut at the front end to drain the perfused fluid. Calcium-free and collagenase perfusate were washed successively for 10-15 minutes, and then Stop the perfusion, tear off the rubber, and put it in F containing 5% fe...

Embodiment 3

[0027] Embodiment 3: Human liver primary cell isolation method

[0028] Separation and culture methods: 1) weighing; 2) washing the liver with pre-cooled calcium-free perfusate, on the one hand washing the blood residue in the liver, and on the other hand looking for suitable blood vessels, which can be used as the main blood vessels for washing liver tissue blocks , to prepare for the next step of perfusion with calcium-free and collagenase perfusate; 3) Use sterile gauze to dry the water around the liver tissue block and the cut surface, and insert the yellow tip of the perfusion cannula into the pre-selected blood vessel During the process, the cut surface was wrapped with glue to make it a fluid-tight liver tissue block, and then a small opening was cut at the front end to drain the perfused fluid. Calcium-free and collagenase perfusate were washed successively for 10-15 minutes, and then Stop the perfusion, tear off the rubber, and put it in F containing 5% fetal bovine s...

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Abstract

The invention discloses a method for separating primary hepatocytes, which relates to the technical field of biology, and fills in the blank of the field of preparation of the primary hepatocytes in China. The hepatocytes are cultured by a sandwich method, so that the hepatocytes can keep long-time vigor, and the problem that activity cannot be kept because the hepatocytes do not have proliferation property like a cell line is solved.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a method for separating and culturing primary liver cells. Background technique: [0002] The liver is the main place for drug metabolism. After oral or intramuscular injection, most drugs first reach the liver for metabolism and then enter the blood circulation. This is the first-pass effect of the liver. Because the liver is an important metabolic site in the body, most of the drug transformation is completed in the liver, so the initial research and development of new drugs must study the influence of the liver on drug metabolism. Its main content includes the metabolic decomposition rate of the drug in the liver, the main metabolic pathway, the main metabolites and the main liver enzyme system involved in the metabolism of the compound. At the same time, the toxic effects of drugs on the liver also need to be evaluated accordingly, all of which information is of great significa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 刘鸿君沈国林
Owner BEIJING IPHASE PHARMA SERVICES