Method for separating and cultivating rat hepatocytes

A technology for separating and culturing liver cells, applied in tissue culture, biochemical equipment and methods, microorganisms, etc., can solve problems such as easy pollution, high technical requirements, and long time required, and achieve low price, large quantity, and good effect Effect

Inactive Publication Date: 2009-09-23
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cost of collagenase and perfusion device used in this method is expensive, the operation links are many, the technical requirements are high, the time required is long, and it is easy to pollute, which limits the application of this method in general laboratories.

Method used

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  • Method for separating and cultivating rat hepatocytes
  • Method for separating and cultivating rat hepatocytes
  • Method for separating and cultivating rat hepatocytes

Examples

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Embodiment

[0020] Fasting SD rats for 24 hours were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (40mg / kg.b.w), anticoagulated by intraperitoneal injection of heparin sodium, fixed on a fixed board, disinfected on the abdominal skin, opened the abdominal cavity, separated and ligated The distal end of the portal vein and the inferior vena cava and the superior vena cava were cannulated from the portal vein with an indwelling needle connected to the infusion set, the proximal end of the inferior vena cava was cut off, the superior vena cava was ligated, and D-Hank's solution at 37°C was used at a flow rate Open perfusion at 25ml / min for about 10min. After the liver turns khaki, use D-Hank's solution containing 0.18% trypsin at 37°C and continue perfusion at a flow rate of 25ml / min for 10min (maintain the temperature of the perfusate during the perfusion process). at 37°C). Stop the perfusion and take out the liver, wash 3 times with PBS containing double-antibody (...

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Abstract

The invention provides a method for separating and cultivating rat hepatocytes. A rat is anaesthetized by Nembutal and is given sodium heparin for anticoagulation; portal catheterization is carried out; two-step reperfusion is carried out for perfusion; a digestive mature liver is taken off; hepatocytes are collected in Hanks liquid containing 1 percent of bovine serum albumin (BSA); hepatocyte suspension is screened by a screen stencil; after the filtrate is centrifugated and deposited, the deposit is collected in Leibovitz's-15(l-15) complete medium; the density of hepatocyte is adjusted to 3 to 6 *10<5>/ml and inoculated on a culture plate coated with rat tail collagen in the environment of temperature of 37 DEG C and 5 percent of CO2; and unattached and dead hepatocytes are removed by changing medium after 4 hours of inoculation, and the hepatocytes can be used for experiment after 20 hours of cultivation. The method is easy, simple and stable, has low cost and high efficiency, can obtain high-activity hepatocytes, is easy to attach, and can be used for pharmacological and toxicity experiments and popularized in common laboratories.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a stable and convenient method for isolating and culturing primary rat hepatocytes. Background technique: [0002] Hepatocytes are widely used in medical related fields. For a long time, many scholars at home and abroad have conducted a lot of research on the separation methods and culture conditions of hepatocytes. However, it is still difficult to obtain hepatocytes with high viability and good function. . In the early days, non-enzymatic methods were mainly used to separate hepatocytes, including mechanical methods (such as homogenization) and chelation methods. However, the mechanical method has great damage to the liver cells and the cell viability is low; the chelation method uses Ca 2+ , Mg 2+ The chelating agent (such as citrate or EDTA) is used to separate hepatocytes, but the effect of chelating agent alone is not good, and it is often used in combination wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06
Inventor 刘宗平刘学忠汪纪仓裔传卉袁燕卞建春
Owner YANGZHOU UNIV
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