Method for isolating and culturing primary liver cells

A primary cell, separation and culture technology, applied in the field of separation and culture of primary liver cells, can solve problems such as troublesome preparation and complex formulation reagents, achieve high adherence rate, simplify reagent preparation steps, and reduce costs.

Inactive Publication Date: 2019-01-08
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can separate and treat a large number of hepatocytes, the formulation reagents such as perfusate used in the operation link are complex and troublesome to prepare.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A method for isolating and culturing primary liver cells, comprising the following steps:

[0027] S1, the isolated liver tissue was stored in the pre-cooled tissue storage solution at 4°C for later use; 1L of the tissue storage solution was prepared according to the following method: HEPES (hydroxyethylpiperazineethanesulfonic acid) 2.4g, 10000U penicillin, 100mg streptomycin, distilled water to 1L, pH7.2;

[0028] S2, take out the liver tissue, and input the perfusate from the hepatic portal vein of the liver tissue, 1L of the perfusate is prepared according to the following method: 8 g of sodium chloride, 0.2 g of potassium chloride, distilled water to 1 L; The input time is 10min, the flow rate of the perfusate is 60mL / min, and the temperature of the perfusate is 37°C.

[0029] S3, after the perfusion is completed, put the liver tissue into another container (sterile petri dish), inject collagenase solution into the liver tissue until turtle-like cracks appear on t...

Embodiment 2

[0035] A method for isolating and culturing primary liver cells, comprising the following steps:

[0036] S1, store the isolated liver tissue in the pre-cooled tissue storage solution at 4°C for later use; prepare 1L of the tissue storage solution according to the following method: HEPES 2.4g, 10000U penicillin, 100mg streptomycin, distilled water to volume to 1L, pH7.4;

[0037] S2, take out the liver tissue, and input the perfusate from the hepatic portal vein of the liver tissue, 1L of the perfusate is prepared according to the following method: 9 g of sodium chloride, 0.4 g of potassium chloride, distilled water to 1 L; The input time is 10min, the flow rate of the perfusate is 60mL / min, and the temperature of the perfusate is 37°C.

[0038]S3, after the perfusion is completed, put the liver tissue into another container (sterile petri dish), inject collagenase solution into the liver tissue until turtle-like cracks appear on the surface of the liver tissue, then cut the ...

Embodiment 3

[0044] A method for isolating and culturing primary liver cells, comprising the following steps:

[0045] S1, store the isolated liver tissue in the pre-cooled tissue storage solution at 4°C for later use; prepare 1L of the tissue storage solution according to the following method: HEPES 2.4g, 10000U penicillin, 100mg streptomycin, distilled water to volume to 1L, pH7.2;

[0046] S2, the liver tissue was taken out, and the perfusion fluid was input from the hepatic portal vein of the liver tissue. 1 L of the perfusion fluid was prepared according to the following method: 8.5 g of sodium chloride, 0.3 g of potassium chloride, and distilled water to 1 L; The input time of the liquid was 10 min, the flow rate of the perfusate was 60 mL / min, and the temperature of the perfusate was 37°C.

[0047] S3, after the perfusion is completed, put the liver tissue into another container (sterile petri dish), inject collagenase solution into the liver tissue until turtle-like cracks appear ...

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PUM

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Abstract

The invention belongs to the technical field of cell culture and particularly relates to a method for isolating and culturing a primary liver cell. The method comprises the following steps: preservingan isolated liver tissue in pre-cooled tissue storage fluid; taking out the liver tissue and inputting a perfusion solution from a hepatic portal vein of the liver tissue; after the completion of perfusion, putting the liver tissue in another container, then injecting a collagenase solution till a turtle-like fissure appears on the surface of the liver tissue, then dicing the liver tissue and soaking in the collagenase solution for 4-8 minutes to obtain a digested mature liver; cleaning the digested mature liver with double distilled water, filtering and collecting liver cell suspension; centrifuging the liver cell suspension, collecting a cell precipitate and performing resuspension culture with a culture medium to obtain the isolated primary liver cell. The method for isolating and culturing the primary liver cell, provided by the invention, has the benefits that a reagent formula of a traditional in-situ two-step perfusion method is simplified, the reagent preparation step is simplified, the cost is reduced, and the survival rate, the vitality and the purity of the isolated cell are improved.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for isolating and culturing primary liver cells. Background technique [0002] The liver is an important organ of the human body. It performs many important functions, such as maintaining glucose homeostasis, detoxification or macromolecular synthesis. Impaired liver function will have a significant impact on health. Viral hepatitis and related liver diseases will seriously endanger human beings. of good health. As an in vitro model, primary cells have outstanding advantages in gene function research because they can simultaneously obtain a large number of cells with uniform characteristics and close to the physiological state in vivo. Hepatocytes are the most important type of parenchymal cells in the liver. They are highly differentiated multifunctional and solid organ cells. In vitro culture systems include culture systems that support their prolifer...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2509/00
Inventor 梁红霞沈德良余祖江张红宇张贤强高晓娟白黎贾乔迪
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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