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Method for isolating and culturing liver primary cells

A primary cell, separation and culture technology, applied in the field of cells, can solve the problems of easy damage of liver cells, many operation steps, and low purity, and achieve the effect of maintaining activity and function, reducing the probability of contamination, and clear nuclei.

Inactive Publication Date: 2013-12-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used hepatocyte separation method in the prior art is the perfusion method. The traditional perfusion method has many operating steps, takes a long time, easily damages the liver cells, has low vitality, low efficiency, and low purity.

Method used

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  • Method for isolating and culturing liver primary cells
  • Method for isolating and culturing liver primary cells
  • Method for isolating and culturing liver primary cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Isolation of Primary Hepatocytes

[0041]The liver used in this example is a fresh pig liver collected from a slaughterhouse. Immediately after isolation, pre-cooled 4°C UW solution (purchased from Dupont Critical Care, USA) 3 times the volume of the liver is injected with a syringe through the hepatic portal vein at 80ml / The liver was poured into the liver at a speed of 1 min, so that the temperature of the liver was rapidly and evenly lowered to below 10°C, and then the liver was placed in an ice box (maintained at about 2°C) and transported to the laboratory for subsequent operations within 3 hours.

[0042] Take about 100g of liver, start the peristaltic pump, and start the perfusate from the hepatic portal vein at a constant temperature of 37°C (1L formula: NaCl 9.397g, KCl 0.235g, HEPES 2.383g, distilled water to 1L, pH 7.3) at a flow rate of 60ml / min open perfusion for 12min, then perfuse 0.05% type IV collagenase (purchased from sigma company) soluti...

Embodiment 2

[0044] Example 2 Isolation of Primary Hepatocytes

[0045] The liver used in this example is a fresh animal liver collected from a slaughterhouse. Immediately after isolation, pre-cooled 4°C UW solution (purchased from Dupont Critical Care, USA) 4 times the volume of the liver is injected with a syringe through the hepatic portal vein at 100ml / The liver was poured into the liver at a speed of 1 min, so that the temperature of the liver was rapidly and evenly lowered to below 10°C, and then the liver was placed in an ice box (maintained at about 1°C) and transported to the laboratory for follow-up operations within 3 hours.

[0046] Take about 100g of liver, start the peristaltic pump, and start the perfusion solution (1L formula: NaCl 9g, KCl 0.2g, HEPES 4.8g, distilled water to 1L, pH value 7.2) from the hepatic portal vein at a flow rate of 55ml / Min open perfusion for 15min, then perfuse 0.05% type II collagenase (purchased from sigma company) solution at the same speed, t...

Embodiment 3

[0048] Example 3 Isolation of Primary Hepatocytes

[0049] The experimental suckling pigs in this example are purebred large white pigs from Beijing Shunxinlong Breeding Pig Farm. A 37°C water bath and a peristaltic pump (ALC-B6 constant flow pump, Shanghai Alcott Biotechnology Co., Ltd.) were prepared in advance, and the air bubbles in the pipeline were evacuated with sterile PBS. Collect the liver of suckling pig by the following method, the operating method is: after 5 days old experimental suckling pig fasted 12h, adopt 3% sodium pentobarbital (sterilized normal saline preparation) intraperitoneal injection anesthesia (1.6ml / kg), use Wash the suckling pig skin with warm water, and soak in 75% alcohol for 5 minutes. After Baoding the suckling pigs, the T-shaped port was opened to expose and separate the hepatic portal vein and inferior vena cava, and thread them for later use. Cut a small opening on the portal vein, insert a 16-gauge gavage needle, and fix the arterial cl...

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Abstract

The invention provides a method for isolating and culturing liver primary cells. The method comprises the following steps: (1) inputting perfusate from hepatic portal vein of a liver; (2) inputting collagenase perfusate; (3) soaking the maturely digested liver in the collagenase perfusate; (4) adding a cleaning solution to stop digestion, filtering, and collecting hepatic cell suspension; and (5) centrifuging, discarding supernatant, resuspending with complete medium, and culturing. The operation of the method provided by the invention is simple, is low in cost and is stable and efficient, and the hepatic cells are high in yield, high in vitality and easy for adherence. The method can be generalized in laboratories, and is a conventional method for scientific research.

Description

technical field [0001] The invention relates to the field of cells, in particular to a method for isolating and culturing primary liver cells. Background technique [0002] As an in vitro model, primary cells have outstanding advantages in the study of gene function: a large number of samples with relatively uniform characteristics can be obtained at the same time; it is basically consistent with the situation in vivo, and gene expression can be studied under conditions close to physiological conditions ; Eliminate the interference of many complex factors, such as the interference of nerves and hormones; primary hepatocytes have good reproducibility in vitro experiments, basically maintain the metabolic function of the liver, especially well retain the cytochrome P450 consistent with the body Enzyme (cytochrome P450) level basically retains the original metabolic function and cell differentiation state of the liver. [0003] Hepatocytes are the most important parenchymal ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 方美英董新星陈刚白莹陈洁
Owner CHINA AGRI UNIV
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