Method for extracting, separating and culturing consubstantial xenogeneic liver primary cell

A primary cell and heterogeneous technology, applied in the field of cell culture, can solve the problems of difficult separation and culture of various non-parenchymal cells, low extraction activity, and low purity, and achieve low cost, reduced cell damage, and high purity.

Inactive Publication Date: 2019-02-26
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method solves the problems of low extraction activity and purity of primary liver cells, difficulty in separating and culturing various non-parenchymal cells, etc.

Method used

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  • Method for extracting, separating and culturing consubstantial xenogeneic liver primary cell
  • Method for extracting, separating and culturing consubstantial xenogeneic liver primary cell
  • Method for extracting, separating and culturing consubstantial xenogeneic liver primary cell

Examples

Experimental program
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Effect test

Embodiment 1

[0022] After anesthetizing 150g male SD rats, open the abdomen, use a 0.7×19mm 24G indwelling needle to intubate the hepatic portal vein, and then inject 10mM EGTA and 20mM HEPES without Ca 2+ , Mg 2+ The HBSS perfusate was perfused at a constant temperature of 37°C for 10 min through the hepatic portal vein, and the perfusion rate was controlled at 10 mL / min, and no air bubbles should be generated during the perfusion process; replace the perfusate with a concentration of 50 mg / L CaCl 2 , 50mM HEPES, 100mg / L type Ⅳ collagenase, 100U / mL penicillin, 100U / mL streptomycin, DMEM-high glucose digestion solution was perfused at a constant temperature of 37°C for 10min through the hepatic portal vein, and the perfusion rate was controlled at 10mL / min; The digested liver was carefully taken out and placed in a vessel containing DMEM-high sugar with a concentration of 5 μM dexamethasone and 10% FBS, and the capsule on the surface of the liver was carefully scratched with tweezers, blun...

Embodiment 2

[0024] After anesthetizing 100g male Wistar rats, open the abdomen, use a 0.7×19mm 24G indwelling needle to intubate the hepatic portal vein, and then inject D-Hanks perfusate with a concentration of 2mM EGTA and 10mM HEPES through the hepatic portal vein at a constant temperature of 37°C in situ Perfuse for 15 minutes, the perfusion speed is controlled at 15mL / min, and no air bubbles should be generated during the perfusion process; replace the perfusate with a concentration of 50mg / L CaCl 2 , 50mM HEPES, 100mg / L type Ⅱ collagenase, 100U / mL penicillin, 100U / mL streptomycin, DMEM-high glucose digestion solution was in situ perfused at a constant temperature of 37°C for 10min through the hepatic portal vein, and the perfusion rate was controlled at 10mL / min; The digested liver was carefully taken out and placed in a vessel containing DMEM-high sugar with a concentration of 5 μM dexamethasone and 10% FBS, and the capsule on the surface of the liver was carefully scratched with tw...

Embodiment 3

[0026] After anesthetizing 20g male C3H / He mice, open the abdomen, use a 0.7×19mm 24G indwelling needle to intubate the hepatic portal vein, and then inject D-Hanks perfusate with a concentration of 5mM EGTA and 20mM HEPES through the hepatic portal vein in situ37 ℃ constant temperature perfusion for 10min, the perfusion rate is controlled at 5mL / min, and no air bubbles should be generated during the perfusion process; replace the perfusate with a concentration of 50mg / L CaCl 2 , 50mM HEPES, 100mg / L type Ⅱ collagenase, 100U / mL penicillin, 100U / mL streptomycin, DMEM-high glucose digestion solution was in situ perfused at a constant temperature of 37°C for 10min through the hepatic portal vein, and the perfusion rate was controlled at 10mL / min; The digested liver was carefully taken out and placed in a vessel containing DMEM-high sugar with a concentration of 5 μM dexamethasone and 10% FBS, and the capsule on the surface of the liver was carefully scratched with tweezers, bluntly...

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Abstract

The invention belongs to the technical field of the cell culture, and discloses a method for extracting, separating and culturing consubstantial xenogeneic liver primary cells. The method comprises the following steps: perfusate is in in-situ perfusion through hepatic portal vein; (2) digestive juice is in in-situ perfusion through the hepatic portal vein; (3) the liver is in vitro, the pathologyis torn so as to acquire the cell suspension containing multiple liver primaries through screening; (4), the acquired cell suspension is conventionally centrifuged to acquire parenchyma cell and collected supernatant; (5) the supernatant separates sternzellen and residual submucosa cell suspension through density gradient centrifuging; (6) the residual submucosa cell suspension is incubated with immunomagnetic beads connected with hepatic sinusoidal endothelial cell and kupffer cell specificity, and acquires the hepatic sinusoidal endothelial cell and the kupffer cell through magnetic field sorting. After being separated, the liver primary cell is immediately resuspended in a corresponding and specifically prepared culture medium to culture. The cell yield is high, the separation effect isgood, and a stable cell source is provided for in-vitro construction of a multi-cell liver model.

Description

technical field [0001] The invention relates to a method for extracting, separating and cultivating allogenic heterogeneous liver primary cells, belonging to the technical field of cell culture. Background technique [0002] The liver is a large internal organ unique to vertebrates and plays an important role in maintaining the homeostasis of the internal environment. The liver participates in many life activities, such as metabolizing toxic foreign substances, synthesizing various plasma proteins, maintaining blood sugar stability, hormone production and bile secretion, etc. The liver also plays a very important role in regulating biochemical reactions in the body, such as ammonia metabolism, immune regulation, etc. In addition, the liver is also the main site of drug metabolism and one of the target organs of drug toxicity. Drug hepatotoxicity is one of the biggest obstacles to the actual clinical use of drugs. The study of the liver is of great importance to life science...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2500/32C12N2501/165C12N2501/39C12N2509/00
Inventor 罗勇邓九高志刚赵伟杰林炳承
Owner DALIAN UNIV OF TECH
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