Application and method of stem cell population of hepatogenous expression NG2 (neuron-glial antigen 2) as seed cells in in-vitro 3D (three-dimensional) culture and reconstruction of artificial liver

A technology of seed cells and stem cells, applied in the field of biomedicine, can solve the problems of inability to realize liver function, replacement, lack of seed cells, etc., and achieve the effect of saving one week, low cost and simple operation

Active Publication Date: 2015-09-09
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, artificial liver methods such as temporarily assisting or replacing the liver in vitro, such as biological artificial liver (Bioartificial liver, BAL) and physical artificial liver, can remove various harmful substances produced or increased due to liver failure, and supplement liver synthesis BAL can improve the patient's internal environment such as water, electrolyte and acid-base balance, but BAL can only temporarily improve the symptoms of patients with advanced liver disease, and has never been able to fully replace the liver function
Although progress has been made in the development of natural decellularized liver scaffolds in recent years, due to the lack of effective seed cells and the microenvironment to induce them to generate functional whole liver organs, it is difficult to imitate the original liver organs to the greatest extent to achieve engineered new ones. The purpose of organs so far the world's scientists have failed to achieve

Method used

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  • Application and method of stem cell population of hepatogenous expression NG2 (neuron-glial antigen 2) as seed cells in in-vitro 3D (three-dimensional) culture and reconstruction of artificial liver
  • Application and method of stem cell population of hepatogenous expression NG2 (neuron-glial antigen 2) as seed cells in in-vitro 3D (three-dimensional) culture and reconstruction of artificial liver
  • Application and method of stem cell population of hepatogenous expression NG2 (neuron-glial antigen 2) as seed cells in in-vitro 3D (three-dimensional) culture and reconstruction of artificial liver

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Obtaining of Natural Whole Liver Decellularized Biological Scaffold (WDLS) of Adult Mouse

[0047] The main materials and reagents used in this embodiment are as follows:

[0048] Perfusion pump, ultra-clean table, infusion needle, 0.9% normal saline, 1% sodium dodecyl sulfate (SDS), 0.005% trypsin, 0.002% EDTA mixture (0.25% trypsin: 0.02% EDTA = 1:1 , Hyclone, cat.no.J110831), deionized sterile double distilled water (ddH 2 O), 1×PBS phosphate buffered saline (0.01M PBS, Beijing Zhongshan Jinqiao Biotechnology Company, cat.no.ZLI-9062).

[0049] The WDLS was obtained by in vitro perfusion, and the specific steps were as follows:

[0050] (1) Take the isolated mouse liver, and then simultaneously infuse 600 mL of 0.9% normal saline into the portal vein and hepatic artery at a speed of 5 mL / min to wash out the red blood cells, and the perfusion time is about 0.5 hours;

[0051] (2) Perfuse the liver with sterile double distilled water (ddH 2 0) to begin t...

Embodiment 2

[0055] Embodiment 2, separate culture seed cell (NG2 + HSC)

[0056] At present, there is no ideal seed cell that can reconstruct the liver organ with structure and function. Although the academic community uses adult hepatocytes and the most promising mesenchymal stem cells (Mesenchymal stem cells, MSCs), the long-term culture of hepatocytes in vitro has been proved to be poor survival and MSCs lack the potential to differentiate into functional hepatocytes in large quantities, making these Cell applications are limited. The present invention chooses NG2 + HSC is used as a seed cell, and the separation method is described in Chinese Patent Publication No. 102899291A. The research found that the NG2 + Under the induction of conditioned medium, HSC can not only rapidly (about 1 h) differentiate into a large number of functional hepatocytes ( image 3 ), which can also differentiate into endothelial cells ( figure 2 ) and liver supporting cells such as astrocytes. The re...

Embodiment 3

[0057] Embodiment 3, conditioned medium (culture microenvironment) induces NG2 + HSCs generate an artificial liver with organ architecture and functionality

[0058] The kit materials used in this embodiment are as follows: 24-well culture plate (Therom, cat.no.142475), shaker (40~240rmp / min), 37 ℃, 5% CO 2 Incubator, conditioned medium (CM1, CM2, CM3), 100 U / mL penicillin, 100 μg / mL streptomycin (Cellgro, cat.no.30-001-CI), 5000 U / mL penicillin, 5000 μg / mL streptomycin , DMEM / F12 (Gibco-BRL, cat.no.11330), ECGS (Endothelial cell growth supplements) (Pepnotech, cat.no.100-20C), HGF (R&D, cat.no.294-HG-005 / CF ), bFGF (Pepnotech, cat.no.100-188), bovine insulin (Sigma, cat.no.I6634), human transferrin (Sigma, cat.no.T1147), levothyroxine (Sigma, cat.no .T6397), sodium selenite (Sigma, cat.no.S9133), putrescine (Sigma, cat.no.P5780), progesterone (Sigma, cat.no.7556), Oncostatin M, dexamethasone, non Essential Amino Acid, L-Glutamine.

[0059] Establish a "culture microenviro...

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Abstract

The invention discloses application and a method of a stem cell population (NG2+HSC (hematopoietic stem cell)) of hepatogenous expression NG2 (neuron-glial antigen 2) as seed cells in in-vitro 3D (three-dimensional) culture and reconstruction of an artificial liver. The method includes: injecting the NG2+HSC into a decellularized liver natural biological scaffold, adding a medium allowing the NG2+HSC to be reconstructed into the artificial liver, and simulating a microenvironment for the development and formation of in-vivo liver tissues to perform in-vitro 3D culture and reconstruction of the liver. The application and the method have the advantages that the method is simple, the cost is low, the culture time is short, the complete liver outline can form in 21 days, the artificial liver cultured has a hepatic lobule structure similar to normal liver tissues, hepatic functional proteins increase over culture time, the artificial liver has the function of repairing the hardened liver and acute hepatic failure and has the function of functional recovery, and the artificial liver can serve as a liver implant and is of great significance to clinic treatment of end-stage liver diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a liver-derived expression of neuroglial antigen 2 positive cell population (NG2 + HSC) were used as seed cells to induce NG2 in 3-D culture in vitro by conditioned medium microenvironment + The application of HSC in the reconstruction of liver organoids also involves the use of NG2 + A method for HSC reconstruction of liver organoids. Background technique [0002] End-stage liver disease (ESLD), including liver cirrhosis and acute liver failure, has a high mortality rate and seriously endangers human health. At present, the only effective treatment is liver transplantation, but liver transplantation is difficult to be widely used due to the shortage of donors. Therefore, finding effective liver function replacement technology is the key to solve this problem. At present, artificial liver methods such as temporarily assisting or replacing the liver in vitro, such as biological arti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797A61L27/38
Inventor 白莲花帅领赖洁娟张玉君赖向东张宏宇别平
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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