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Culture method for eriones unguiculatus primary liver cells

A technology of primary hepatocytes and long-clawed gerbils, applied in the biological field, can solve problems such as the establishment of a perfect culture system, and achieve the effects of firm adhesion, easy operation, and low price

Inactive Publication Date: 2013-03-06
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on the isolated model of primary liver cells of long-clawed gerbils in China, and a perfect culture system has not yet been established abroad.

Method used

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  • Culture method for eriones unguiculatus primary liver cells
  • Culture method for eriones unguiculatus primary liver cells
  • Culture method for eriones unguiculatus primary liver cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Take long-clawed gerbils fasted for 16 hours, anesthetize with Sumianxin (1mL / kg) intramuscular injection, intraperitoneally inject 0.1mL (12500IU / mL) heparin sodium anticoagulant, place on the dissecting board and fix, chest and abdomen are disinfected and laparotomy, intravenous The portal vein cannula was fixed with an indwelling needle, and the calcium-free perfusion solution preheated in a 37°C water bath was used to open and perfuse until the liver was grayish white, and 20ml of perfusion was perfused. Then perfuse the type IV collagenase solution with a calcium concentration of 0.025%, slowly open the perfusion at the beginning, clamp the inferior vena cava after 2 minutes, continue to perfuse the collagenase solution to fill the liver, and wait until the liver is soft and collapses, leaving marks after pressure Stop perfusion. The total time of collagenase digestion is 8min, and the total volume is 10ml. Separate the liver, place the liver in a petri dish contai...

Embodiment 2

[0040] Take long-clawed gerbils fasted for 16 hours, anesthetize with Sumianxin (1mL / kg) intramuscular injection, intraperitoneally inject 0.1mL (12500IU / mL) heparin sodium anticoagulant, place on the dissecting board and fix, chest and abdomen are disinfected and laparotomy, intravenous The portal vein cannula was fixed with an indwelling needle, and the calcium-free perfusion solution preheated in a 37°C water bath was used to open and perfuse until the liver was grayish white, and 20ml of perfusion was perfused. Then perfuse the type IV collagenase solution with a calcium concentration of 0.025%, slowly open the perfusion at the beginning, clamp the inferior vena cava after 2 minutes, continue to perfuse the collagenase solution to fill the liver, and wait until the liver is soft and collapses, leaving marks after pressure Stop perfusion. The total time of collagenase digestion is 8min, and the total volume is 10ml. Separate the liver, place the liver in a petri dish conta...

Embodiment 3

[0044] Take long-clawed gerbils fasted for 16 hours, anesthetize with Sumianxin (1mL / kg) intramuscular injection, intraperitoneally inject 0.1mL (12500IU / mL) heparin sodium anticoagulant, place on the dissecting board and fix, chest and abdomen are disinfected and laparotomy, intravenous The portal vein cannula was fixed with an indwelling needle, and the calcium-free perfusion solution preheated in a 37°C water bath was used to open and perfuse until the liver was grayish white, and 20ml of perfusion was perfused. Then perfuse the type IV collagenase solution with a calcium concentration of 0.025%, slowly open the perfusion at the beginning, clamp the inferior vena cava after 2 minutes, continue to perfuse the collagenase solution to fill the liver, and wait until the liver is soft and collapses, leaving marks after pressure Stop perfusion. The total time of collagenase digestion is 8min, and the total volume is 10ml. Separate the liver, place the liver in a petri dish conta...

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Abstract

The present invention discloses a culture method for eriones unguiculatus primary liver cells. The method comprises: (1) collecting a liver cell primary suspension from digested and matured isolated eriones unguiculatus liver, filtering, removing impurities from a filtrate, adding 5% calf serum-containing HBSS, repeatedly carrying out centrifugation washing, collecting cell precipitate and adding to a 20% new born calf serum-containing DMEM complete culture medium, and adjusting a liver cell concentration to 3.0*10<5>-5.0*10<5> / mL to prepare a liver cell suspension; and (2) inoculating the liver cell suspension in a culture plate coated with rat tail collagen, culturing at a temperature of 37 DEG C in a 5% CO2 environment, removing impurities after 4 hours, changing the culture medium into a maintenance culture medium containing cell factors, and carrying out cell adherence overnight to obtain the eriones unguiculatus primary liver cells. The method has characteristics of low cost, economy, practicality, and simple and easy-performing operation, wherein the obtained liver cells have characteristics of strong adherence, high vitality, high survival rate, stability and reliability, and the method is suitable for mass production.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a simple and practical method for culturing primary liver cells of long-clawed gerbils. Background technique [0002] The experimental animalization research on long-clawed gerbils in my country began in 1978. At present, there are two main groups of long-clawed gerbils in China, which are kept in Zhejiang Experimental Animal Center and Capital Medical University respectively. Gerbils are sensitive to fat, vitamin E, and cholesterol in food, and can be used as an animal model to study food-derived cholesterol-induced hypercholesterolemia. Low-density lipoprotein receptor expression in gerbil liver was significantly correlated with food-derived protein, whereas human hepatoma cell expression of low-density lipoprotein receptor was not significantly changed, suggesting that when studying cell surface receptors in primary hepatocytes Advantages over neoplastic hepat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 陈立青郭红刚李长龙卢领群萨晓婴戴方伟宋晓明
Owner ZHEJIANG ACAD OF MEDICAL SCI
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