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Separation and culture method for primary pig liver cells

A culture method and technology of pig liver, applied in the field of cell culture of modern biotechnology, can solve the problems of poor activity and low number of liver cells, and achieve the effects of stable quality, high cell viability and good activity.

Inactive Publication Date: 2018-12-18
JIANGYIN CHI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The mechanical method is simple to operate, but the number of isolated hepatocytes is small and the activity is poor

Method used

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  • Separation and culture method for primary pig liver cells
  • Separation and culture method for primary pig liver cells

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Embodiment Construction

[0024] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0025] 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.

[0026] In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

[0027] The experimental instruments and reagents used in this experiment are as follows:

[0028] 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), CO 2 Cell cultu...

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Abstract

The invention provides a separation and culture method for primary pig liver cells. The separation and culture method comprises: (1) selecting a 3-4-week-old male Chinese experimental minipig with thebody weight of 2.5-4 kg, carrying out absolute diet and water deprivation at the 12th h before surgery, and carrying out intraperitoneal injection of sodium pentobarbital and heparin; (2) anesthetizing the experimental pig, fixing on a plate, and cutting the abdominal cavity under aseptic conditions, wherein the abdomen surface is upward; (3) carrying out venous cannula, and perfusing with a pre-heated HBSS buffer liquid by using a constant flow pump; (4) perfusing with a pre-heated GBSS mixed enzyme solution by using the constant flow pump; (5) shearing the tissue, carrying out shaking digestion with a GBSS mixed enzyme solution, filtering with a sieve mesh, carrying out centrifugation, and discarding the supernatant; (6) taking the cell filtrate, and detecting the viability of cells byusing Trypan Blue staining; (7) after cell counting, re-suspending with a complete culture medium, inoculating into a coated culture flask, and culturing in a 37 DEG C 5% CO2 environment; (8) carryingout cell morphology identification; (9) carrying out ELISA detection; and (10) carrying out RT-PCR detection. According to the present invention, with the separation and culture method, the operationis simple, a large number of the cells can be obtained, and the survival rate is high; and the method is the ideal method for separating and culturing primary pig liver cells so as to provide the reliable cell resources for experiments.

Description

technical field [0001] The invention belongs to the technical field of cell culture of modern biotechnology, and in particular relates to a method for separating and culturing primary pig hepatocytes. Background technique [0002] Hepatocytes are responsible for most of the main and complex functions of the normal liver, including regulating the biosynthesis of sugar, fat and protein, detoxifying and metabolizing endogenous and exogenous abnormal substances, and maintaining normal liver function. plays a key role. The physiological and metabolic functions of pig hepatocytes are the closest to those of human hepatocytes. At the same time, the production of semipermeable membranes and transgenic pigs can effectively reduce the immune response, making pig hepatocytes the most widely used liver cells in bioartificial liver (BAL) research. [0003] Hepatocytes are used as seed cells for cell transplantation to treat chronic liver failure and as target cells for drug screening. T...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2509/00C12N2509/10C12N2533/32
Inventor 不公告发明人
Owner JIANGYIN CHI SCI
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