Hepatic stellate cell separation and preparation method

A hepatic stellate cell and density gradient separation technology, applied in the field of cell separation, can solve the problems of poor separation effect and single enzymatic hydrolysis, and achieve the effect of promoting enzymatic hydrolysis, high survival rate, and high purity

Inactive Publication Date: 2017-07-14
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 and 40% non-parenchymal cells A large network structure, a single enzymatic hydrolysis can not achieve good results, coupled with the limitations of common buffer systems such as D-Hank's solution, Hank's solution on the enzymatic hydrolysis, making the separation effect is relatively poor, therefore, it is necessary to Improved method for isolation and preparation of hepatic stellate cells in the art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The method for separating and preparing hepatic stellate cells in this embodiment comprises the following steps:

[0041] 1. Preparation of experimental reagents

[0042] Perfusate I: take buffer I;

[0043] Perfusate II: take buffer II, add 0.3 mg pronase E per ml of buffer II;

[0044] Perfusate III: take buffer II, add 0.6mg collagenase D per ml of buffer II;

[0045] Equilibrium solution: take buffer III;

[0046] Dissociation solution: take buffer II, add 0.5 mg pronase E, 0.5 mg collagenase D and 0.02 mg deoxyribonuclease to every milliliter of buffer II;

[0047] Centrifuge solution A: take buffer III, add 0.2g Nycodenz density gradient separation reagent per milliliter of buffer III;

[0048] Centrifuge solution B: take buffer III, and add 0.1 g of Nycodenz density gradient separation reagent per milliliter of buffer III.

[0049] Wherein, every liter of buffer solution I comprises each component of following parts by weight: NaCl 8000 mg, KCl 400 mg, NaH ...

Embodiment 2

[0055] This example is the same as Example 1 except that the components by weight of buffer I, buffer II and buffer III are different.

[0056] Described buffer solution I comprises each component of following parts by weight every liter: NaCl 7500mg, KCl 380mg, NaH 2 PO 4 ·H 2 O 85mg, Na 2 HPO 4 110mg, HEPES 2300mg, NaHCO 3 320mg, EGTA170mg, Glucose 850mg, the rest is H 2 O;

[0057] The buffer II comprises the following components in parts by weight per liter: 7500 mg of NaCl, 380 mg of KCl, NaH 2 PO 4 ·H 2 O85mg, Na 2 HPO 4 110mg, HEPES 2300mg, NaHCO 3 320mg, CaCl 2 2H 2 O 520mg, the rest is H 2 O.

[0058] Described buffer solution III comprises each component of following parts by weight every liter: KCl 345mg, MgCl 6 H 2 O 190mg, MgSO4·7H 2 O 65 mg, Na2 HPO 4 55mg, KH2PO4 27.5mg, Glucose 940mg, NaHCO 3 210mg, CaCl 2 2H 2 O 205mg, the rest is H 2 O.

Embodiment 3

[0060] This example is the same as Example 1 except that the components by weight of buffer I, buffer II and buffer III are different.

[0061] Described buffer solution I comprises each component of following parts by weight every liter: NaCl 8500mg, KCl 420mg, NaH 2 PO 4 ·H 2 O 95mg, Na 2 HPO 4 130mg, HEPES 2500mg, NaHCO 3 380mg, EGTA 220mg, Glucose 950mg, the rest is H 2 O;

[0062] The buffer II comprises the following components in parts by weight per liter: NaCl 8500mg, KCl 420mg, NaH 2 PO 4 ·H 2 O95mg, Na 2 HPO 4 130mg, HEPES 2500mg, NaHCO 3 380mg, CaCl 2 2H 2 O 600mg, the rest is H 2 O.

[0063] Described buffer solution III comprises each component of following parts by weight every liter: KCl 395mg, MgCl 6 H 2 O 230mg, MgSO4·7H 2 O 75 mg, Na 2 HPO 4 65mg, KH2PO4 32.5mg, Glucose 1050mg, NaHCO 3 250 mg, CaCl 2 2H 2 O 245 mg, the rest is H 2 O.

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PUM

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Abstract

The invention discloses a hepatic stellate cell separation and preparation method. The hepatic stellate cell is prepared through the steps of perfusion, stripping, and density gradient separation. The perfusion is carried out successively with a perfusion solution I, a perfusion solution II and a perfusion solution III, wherein the perfusion solution I comprises a buffer solution I; the perfusion solution II is prepared by adding 0.22-0.38 mg of pronase E to 1 ml of a buffer solution II; and the perfusion solution II is prepared by adding 0.52-0.68 mg of collagenase D to 1 ml of the buffer solution II. The invention belongs to the technical field of cell separation, wherein cell surface substrates are completely enzymolyzed successively by means of single enzyme and then compound enzymes matched with the buffer solution II, so that yield of the hepatic stellate cells are increased.

Description

technical field [0001] The invention relates to the technical field of cell separation, in particular to a method for separating and preparing hepatic stellate cells. Background technique [0002] Hepatic stellate cells (HSCs) are located between hepatic primary cells and hepatic sinusoidal endothelial cells. It is generally believed that the main function of hepatic stellate cells is to store nutrients, especially vitamins, and a large loss of vitamin A will lead to stellate cell fibrosis. Under normal circumstances, hepatic stellate cells are in a quiescent state. When the liver is damaged by inflammation or mechanical stimulation, the hepatic stellate cells are activated, and their phenotype changes from quiescent to activated. Activated hepatic stellate cells participate in the formation of liver fibrosis and the reconstruction of intrahepatic structures through proliferation and secretion of extracellular matrix, and on the other hand, increase the internal pressure of...

Claims

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Application Information

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