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Method for preparing pig and human liver cell for biological artificial liver

A bioartificial, hepatocyte technology, applied in the field of medical biology, can solve the problems of liver cell membrane damage, unsatisfactory digestion and separation of pig liver cells, etc., and achieve the effect of increasing the number

Inactive Publication Date: 2005-06-29
ZHEJIANG UNIV
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Problems solved by technology

In recent years, the inventor has carried out a large amount of separation experimental research on rats and suckling pig hepatocytes, and found that the two-step perfusion method of Seglen is not very satisfactory to the digestion and separation of pig hepatocytes, so the separation of pig hepatocytes is adopted with collagenase The main related reagents, such as using D-hanks solution containing EDTA and 0.05% type IV collagenase solution, using a self-made extracorporeal circulation perfusion device, and performing two-step perfusion to separate porcine hepatocytes to obtain high-quality porcine hepatocytes, However, affected by collagenase and other factors during the separation process, the liver cell membrane was damaged to varying degrees

Method used

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Embodiment Construction

[0019] In order to further illustrate the content of the present invention, the specific schemes are further described below.

[0020] In this protocol, 80 grams of human embryonic liver obtained from normal pigs, dogs, or adult liver transplantation donors, or legally induced labor sources by surgical methods was taken as an example to separate and prepare hepatocytes.

[0021] First, part of the liver tissue was excised under surgical aseptic conditions, placed in a sterile container pre-cooled to 4°C and containing 500ml of perfusion buffer PB, weighed to about 80 grams, and the excess liver tissue was removed. Then use 300ml of EDTA-containing perfusion solution PBE at room temperature, use a fine dropper to perfuse through a peristaltic pump for 3 minutes, and then continue to perfuse with 700ml EDTA-containing perfusion buffer PBE pre-heated to 39°C for 5 minutes; Perfuse with 500ml of EDTA-free perfusion buffer PB at 39°C for 3 minutes; then discard all the above-mentio...

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Abstract

Disclosed is method to prepare biological artificial pig for liver and liver cell of human, which includes the two courses of pouring outside the body of separating liver cell and washing the liver cell, uses the four-stepped pouring method on the basis of the one with two steps, and designs the relative agent. The invention has increased the quality, living rate and purity of separating the liver cell, it is suitable for acquiring liver from pig, dog or grown-ups, or liver separation from liver of human embryo by legal artificial labor, and the liver cell prepared can be widely used in research and transplantation of liver cell, biological artificial liver and mixed artificial liver.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and relates to a preparation technology of a bioartificial liver, in particular to a method for preparing pig liver and human liver cells used in a bioartificial liver. Background technique [0002] With the maturity of hepatocyte separation, high-density culture and bioreactor technologies, a new generation of modern bioartificial liver (bioartificial liver, BAL) based on cultured hepatocytes - extracorporeal bioartificial liver support system (extracorporeal bioartificial liver support system) , EBLSS) is considered to be the most promising treatment for fulminant hepatic failure (FHF) due to various reasons, and has achieved definite curative effects in animal experiments and clinical applications. The basic principle and structure of BAL is: the cultured exogenous hepatocytes are placed or continued to be cultured in an in vitro bioreactor. When the patient's blood or plasma flows through...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 李兰娟李君
Owner ZHEJIANG UNIV
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