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Separation and culture method for primary hepatocytes of sheep

A primary hepatocyte, separation and culture technology, applied in the field of animal cell culture, can solve the problems of cell damage, low viability, easy to cause pollution, etc., and achieve the effect of stable proliferation and typical morphological characteristics

Active Publication Date: 2022-07-15
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current methods for the isolation and culture of primary hepatocytes generally have problems such as low yield and survival rate of the obtained cells, high requirements for operating skills, easy contamination, and low viability of the cells due to perfusion and re-injury.

Method used

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  • Separation and culture method for primary hepatocytes of sheep
  • Separation and culture method for primary hepatocytes of sheep
  • Separation and culture method for primary hepatocytes of sheep

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] (1) After soaking the lamb liver tissue in physiological saline containing 2wt% PS (the dosage ratio of the lamb liver tissue and physiological saline is 1g:5mL), wash with PBS buffer containing 1wt% PS for 3 Second, get lamb liver tissue a;

[0067] (2) Use sterile scissors to cut the lamb liver tissue a into 1mm 3 The tissue block was mixed with the complete medium by pipetting, and a cell sieve with a pore size of 100 μm was used to filter the larger tissue block and impurities, and the filtrate was collected to obtain a hepatocyte suspension;

[0068] (3) discard the supernatant after centrifuging the above-mentioned obtained hepatocyte suspension for 5 min under the condition of 800 rpm to obtain precipitation a;

[0069] (4) Mix the above-mentioned obtained precipitate a and the erythrocyte lysate according to the ratio of 1 g:3 mL, leave it to stand for 10 min at 25° C. and then centrifuge for 5 min under the condition of 800 rpm, discard the supernatant to obta...

Embodiment 2

[0073] (1) After soaking the lamb liver tissue in physiological saline containing 2wt% PS (the dosage ratio of the lamb liver tissue and physiological saline is 1g:5mL), wash with PBS buffer containing 1wt% PS for 3 Second, get lamb liver tissue a;

[0074] (2) Use sterile scissors to cut the lamb liver tissue a into 1mm 3 The tissue block was digested with 5 mL of collagenase IV at a concentration of 1 mg / mL in a water bath at 37°C for 15 min, with constant shaking during the period. After the digestion, complete medium was added to terminate the digestion, and a 100 μm cell sieve was used to filter the larger Tissue blocks and impurities, collect the filtrate to obtain hepatocyte suspension;

[0075] (3) discard the supernatant after centrifuging the above-mentioned obtained hepatocyte suspension for 5 min under the condition of 800 rpm to obtain precipitation a;

[0076] (4) Mix the above-mentioned obtained precipitate a and the erythrocyte lysate according to the ratio o...

Embodiment 3

[0080] (1) After soaking the lamb liver tissue in physiological saline containing 2wt% PS (the dosage ratio of the lamb liver tissue and physiological saline is 1g:5mL), wash with PBS buffer containing 1wt% PS for 3 Second, get lamb liver tissue a;

[0081] (2) Use sterile scissors to cut the lamb liver tissue a into 1mm 3 The tissue blocks were digested with 5 mL of collagenase IV at a concentration of 0.1 mg / mL in a water bath at 37 °C for 15 min, with constant shaking during the period. After the digestion, complete medium was added to terminate the digestion. Large tissue blocks and impurities, collect the filtrate to obtain hepatocyte suspension;

[0082] (3) discard the supernatant after centrifuging the above-mentioned obtained hepatocyte suspension for 5 min under the condition of 800 rpm to obtain precipitation a;

[0083] (4) Mix the above-mentioned obtained precipitate a and the erythrocyte lysate according to the ratio of 1 g:3 mL, leave it to stand for 10 min at...

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Abstract

The invention belongs to the technical field of animal cell culture, and provides a sheep primary hepatocyte separation culture method. The method comprises the following steps: treating liver tissues of the lambs by adopting a direct crushing method or an enzyme digestion method to obtain liver cell suspension; centrifuging the obtained hepatocyte suspension, and discarding the supernatant to obtain a precipitate a; mixing the precipitate a with a red blood cell lysis solution, standing, centrifuging, and discarding supernatant to obtain a precipitate b; mixing the obtained precipitate b with a complete culture medium, and culturing in a rat tail collagen coated 6-pore plate to obtain a liver cell culture; and discarding non-adherent hepatocytes and dead cells in the obtained hepatocyte culture, and mixing the remaining adherent cells with a complete culture medium for culture. The sheep primary hepatocytes prepared by the method disclosed by the invention are stable in proliferation and typical in morphological characteristics, and have passage and proliferation capacities.

Description

technical field [0001] The invention relates to the technical field of animal cell culture, in particular to a method for separating and culturing sheep primary hepatocytes. Background technique [0002] Hepatocytes are used as seed cells for cell transplantation in the treatment of chronic liver failure and as target cells for drug screening. The preparation and culture of large-scale and highly viable hepatocytes are the basic links in these studies. Therefore, the separation and culture technology of hepatocytes is attracting more and more attention and has become a hot spot of current research. The key to culturing hepatocytes in vitro is how to isolate and obtain hepatocytes with complete morphology and high metabolic activity in vitro. However, the current methods for the isolation and culture of primary hepatocytes generally have problems such as low yield and survival rate of the obtained cells, high requirements for operating skills, easy contamination, and low via...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2509/00C12N2509/10C12N2500/32
Inventor 卢曾奎陈博雯窦晓宁李建烨岳耀敬袁超郭婷婷刘建斌杨博辉
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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