Method for separating and preparing primary liver cells

A technology of primary cells and hepatocytes, applied in the field of medical biology, can solve problems such as single enzymatic hydrolysis, and achieve high survival rate, increased yield, and high yield

Active Publication Date: 2017-06-13
广州柏赛柯生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The complexity of the liver is that non-parenchymal cells include: stellate cells, sinus endothelium, bile duct epithelium and immune cells (lymphocytes and granulocytes), while the entire liver group is composed of 60% parenchymal cells a

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The invention discloses a method for separating and preparing primary liver cells, which uses compound enzymes to perfuse and separate the liver to obtain primary liver cells. The compound enzyme comprises the following components by weight: 25 parts of collagenase A, 25 parts of collagenase D, and 10 parts of collagenase H.

[0034] The method for separating and preparing primary liver cells in this embodiment includes the following steps:

[0035] 1. Preparation of experimental reagents

[0036] Perfusate I: take 50ml of buffer I for later use;

[0037] Perfusion solution II: Take 100ml buffer II and add 60mg compound enzyme for later use;

[0038] Cleaning solution: take 50ml of buffer II for later use; another 50ml of buffer II for pre-cooling for later use.

[0039] Wherein, every liter of buffer solution I comprises each component of following parts by weight: NaCl 8000 mg, KCl 400 mg, NaH 2 PO 4 ·H 2 O 88.17 mg, Na 2 HPO 4 120.45 mg, HEPES 2380 mg, NaHCO...

Embodiment 2

[0047] This embodiment is the same as embodiment 1 except that the composition of the parts by weight of the components of the complex enzyme is different.

[0048] The compound enzyme comprises the following components by weight: 20 parts of collagenase A, 20 parts of collagenase D, and 5 parts of collagenase H.

Embodiment 3

[0050] This embodiment is the same as embodiment 1 except that the composition of the parts by weight of the components of the complex enzyme is different.

[0051] The compound enzyme comprises the following components by weight: 30 parts of collagenase A, 30 parts of collagenase D and 15 parts of collagenase H.

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PUM

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Abstract

The invention discloses a method for separating and preparing primary liver cells. In the method for separating and preparing the primary liver cells, composite enzymes are used for liver perfusion isolation to obtain the primary liver cells. The method belongs to the technical field of medical biology, the method for separating and preparing the primary liver cells can be used for effective separation of the primary liver cells, by interaction of the composite enzymes, different surface growth matrix of each cell can be fully hydrolyzed so as to obtain a single cell suspension. A suitable environment is provided for action of the composite enzymes by matching of a buffer system, so that enzymolysis is more complete, and the yield of the isolated primary liver cells is improved. A necessary environment is provided for cell life by catalytic properties of the composite enzymes and the buffer system, and the isolated primary liver cells have a higher survival rate.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a method for separating and preparing liver primary cells. Background technique [0002] As an important organ of the body, the liver plays a very important role in metabolism, digestion, detoxification, blood coagulation, immunity, and regulation. The liver is composed of hepatocytes, which perform the main functions of the liver, such as removing toxins, participating in biosynthesis and biotransformation, metabolizing various nutrients, storing glucose, and secreting active substances that promote the growth of liver cells. Isolation of hepatocytes from the body is helpful for in vitro research in pharmacology, toxicology, immunology, cell biology, etc., and with the application of bioartificial liver in the field of liver failure, a large number of highly active primary hepatocytes are increasingly needed as biological material. Therefore, how to isolate and prepare pri...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2509/00
Inventor 兰天罗维
Owner 广州柏赛柯生物技术有限公司
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