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Human primary hepatocyte separation and culture method

A technology of primary hepatocytes and a culture method, which is applied in the field of separation and culture of human primary hepatocytes, can solve the problems of inability to meet the needs of human hepatocytes, large volume requirements of liver tissue blocks, and inapplicability, and achieves the goal of achieving cell growth. The effect of good growth condition, low cost and easier preservation

Inactive Publication Date: 2018-05-25
JIANGYIN CHI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires a large volume of liver tissue, and is not suitable for surgically resected irregular small pieces of human liver tissue, and cannot meet the needs of human hepatocytes in basic research.

Method used

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  • Human primary hepatocyte separation and culture method
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Embodiment Construction

[0020] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0021] The present invention will be described in detail below in conjunction with the accompanying drawings and examples, and the protection content of the present invention is not limited to the following examples.

[0022] In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

[0023] The experimental instruments and reagents used in this experiment are as follows:

[0024] A set of surgical instruments, CCK-8 cell counting box (Sigma, USA), inverted microscope (XDS-1A, Shanghai), fluorescence microscope (Leica, USA), cryogenic centrifuge (TD24B-WS, Shanghai), ultra-low temperature refrigerator ( Zhongke Meiling), pipette gun (Eppendorf, USA), electronic analytical balance (Sartorius, USA), ultra-clean bench (HJ-CJ-1D, Shanghai), CO2 cell incubat...

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Abstract

The invention provides a human primary hepatocyte separation and culture method. The method comprises the following steps of (1) cutting a fresh human hepatocyte under an aseptic condition, sealing at4 DEG C and then carrying back to a lab; (2) placing the tissue into a precooled sterile PBS buffer liquid containing double antibodies, and washing for multiple times to remove blood and connectivetissues; (3) irrigating through a preheated PBS buffer liquid through a needle-punching method; (4) irrigating through a preheated HBSS buffer liquid through a needle-punching method; (5) irrigating through a preheated GBSS mixed enzyme liquid through a needle-punching method; (6) cutting up the tissue, and using the GBSS mixed enzyme liquid for digesting, centrifuging, and removing a supernatant;(7) using trypan-blue for dyeing a cell filter liquor to detect cell activity; (8) after carrying out cell counting, resuspending a complete medium, inoculating into a coated culture flask, and culturing at 37 DEG C in an environment with 5 percent of CO2; (9) identifying cell morphology; (10) detecting through an ELISA (Enzyme-linked Immuno Sorbent Assay) method; (11) detecting through RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The invention provides the human primary hepatocyte separation and culture method which is simple to operate, the number of the obtained cells is large, the survival rate is high, the method is the ideal human primary hepatocyte separation and culture method, and a reliable cell resource is provided for experiments.

Description

technical field [0001] The invention belongs to the technical field of cell culture of modern biotechnology, and specifically relates to a method for separating and culturing primary human hepatocytes. Background technique [0002] Hepatocytes are used as seed cells for cell transplantation to treat chronic liver failure and as target cells for drug screening. The preparation and cultivation of large-scale, highly viable hepatocytes is the most basic link in these studies. Therefore, the technology of separation and culture of hepatocytes is attracting increasing attention and has become a hot spot in current research. [0003] At present, the separation methods of hepatocytes include mechanical separation, chelation and enzymatic digestion. The mechanical method is simple to operate, but the number of isolated hepatocytes is small and their activity is poor. Howard created the collagenase perfusion method, and Seglen further developed this method into a two-step perfusion...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0068C12N5/067C12N2509/00
Inventor 不公告发明人
Owner JIANGYIN CHI SCI
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