In vitro culture-amplified human liver progenitor cell and preparation thereof

A technology of hepatic progenitor cells and in vitro culture, which is applied in the field of in vitro expansion and preparation of human hepatic progenitor cells, and can solve the problem of inability to overcome animal protein pollution, human hepatic stem/progenitor cells difficult to satisfy, and human fetal liver non-hepatic parenchymal cell technology. Difficulty in quality control of indicators

Inactive Publication Date: 2008-10-01
芦银雪
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, animal (mouse)-derived protein and potential animal cell and virus contamination seriously affect its clinical application
For example, human liver stem / progenitor cells were cultured in However, it still cannot overcome the pollution of the above-mentioned animal protein and even tumor factors
For example, co-culture of human hepatic stem cells and unpurified human fetal liver non-hepatic parenchymal cells, although overcoming the above-mentioned factors of animal protein and cell contamination, seems to restore purified human liver stem / progenitor cells to the unpurified state, and human Only limited improvement in self-renewal and clonal growth capacity of hepatic stem / progenitor cells
Another disadvantage is that the technical indicators of different batches of unpurified human fetal liver non-hepatic parenchymal cells are difficult to quality control
[0013] Although the patent publication No. CN1742082A mentions the separation and subculture of human liver stem / progenitor cells, however, its "colony formation efficiency after passage from plastic to STO feeder layer is very low, ... may be due to the need to Because the cells were subjected to lengthy collagenase digestion to obtain a single-cell suspension” (see page 51 of the manual), it is still difficult to obtain a large number of purified human liver stem / progenitor cells suitable for clinical applications satisfactory

Method used

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  • In vitro culture-amplified human liver progenitor cell and preparation thereof
  • In vitro culture-amplified human liver progenitor cell and preparation thereof
  • In vitro culture-amplified human liver progenitor cell and preparation thereof

Examples

Experimental program
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Effect test

example 1

[0056] The preparation of example 1 human fibrin glue

[0057] Thrombin (Shanghai Xinxing Pharmaceutical, China) was added to the M199 medium containing 3-5 mg / ml human fibrinogen (Shanghai Xinxing Pharmaceutical, China) to a final concentration of 0.02 units / ml. Quickly add 0.5 to 2 ml of the above solution to one well of a six-well plate to cover the entire well. The six-well plate was placed in a cell culture incubator at 37°C. After human fibrinogen was degraded for at least two hours, aprotinin (Hangzhou Aoya, China) was added to serum-free medium to a final concentration of 20 units / ml to inhibit thrombin activity for at least two hours. The prepared human fibrin glue can be sealed and stored at 4° C. for long-term storage. When human fibrin glue is used together with other extracellular matrices (such as laminin, fibronectin or both), it can be added to the M199 medium of human fibrinogen first, and then added to thrombin for degradation. If an acidic solution contai...

example 2

[0059] Example 2 Co-culture of human fetal liver progenitor cells and human umbilical cord vein endothelial cells on human fibrin glue

[0060] According to the method reported in the literature, the fetal liver or adult liver of 16-22 weeks of gestation was preheated with 0.2 mg / ml deoxyribonuclease I and 0.1% collagenase H or 120 μg / ml highly purified collagenase liberase Blendzyme 3 (Roche Applied Sciences, USA) Hank's buffer (containing Ca 2+ and Mg 2+ Ionic Hank's Balanced Salt Solution) was degraded and prepared as a human liver single cell suspension. Rich in EpCAM + or / and CD44 + but CD45 - 、NCAM - 、HLA - / low Human fetal liver progenitor cells can be isolated and purified by FACS or immunomagnetic bead sorting using the method described by Schmelzer et al. (16) , or with Dan et al. (17) , Herraza, etc. (18) , Turner, etc. (19) The described methods are enriched. In order to increase the purity of human fetal liver progenitor cells, the human fetal liver prog...

example 3

[0061] Example 3 Preparation of human liver progenitor cells from the co-culture of human fetal liver progenitor cells and human vascular endothelial cells

[0062] After washing the co-culture of human fetal liver progenitor cells and human vascular endothelial cells twice with 1× phosphate (PBS) buffer, add 0.5 ml containing 0.1% collagenase H or 120 μg / ml highly purified collagenase liberase Blendzyme 3 ( Roche Applied Sciences, the United States) Hank's buffer (containing Ca 2+ and Mg 2+ Ionic Hank's Balanced Salt Solution) was added to each well, degraded at 37°C for 1-4 minutes with slight shaking, until most of the culture was detached from the surface of human fibrin glue, and another 2ml of Hank's buffer was added to From each well, collect the culture into a sterile tube and centrifuge at low speed (100 xg) for 10 minutes to pellet the cell culture. The cell pellet was suspended in a Ca-free 2+ and Mg 2+Add an equal volume of trypsin-EDTA to the ionic Hank's buff...

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Abstract

The present invention provides a preparing method of a human hepatic progenitor cell which is amplified and cultivated in vitro, including: a. separating the human hepatic progenitor cell; b. co-cultivating a feeder cell and the human hepatic progenitor cell separated from the step a by a medium having no serum on an extracellular matrix containing human fibrin sealant or other analogues, obtaining a human hepatic progenitor cell colony by amplification. The human hepatic progenitor cell colony is easy to separate from the surface of human fibrin sealant by the simple gelatinolytic band process, purified or / and subcultured by further single cell preparation technology process such as enzymatic degradation etc. The human hepatic progenitor cell prepared by the method is hepatic progenitor cell treatment, including a cell transplantation and a bioartificial liver support system, providing excellent human hepatocyte source for cytotoxicity test platform in the drug screening, virus infection and drug screening platform etc.

Description

technical field [0001] The invention relates to a method for expanding and preparing human liver progenitor cells in vitro. The culture method and the human liver progenitor cells prepared by the culture method can be used for liver cell therapy, including liver cell transplantation, and artificial liver support system in vitro, cytotoxicity test platform in drug screening, hepatitis virus infection and drug screening platform, etc. Background technique [0002] Orthotopic liver transplantation is currently the most effective therapy for saving the lives of patients with end-stage liver disease. However, the cost is high, the technique is complicated, and especially the source of donor livers is in severe shortage, and many patients die while waiting for the donor livers. Moreover, after the whole liver transplantation, the patient has to take expensive anti-immune rejection drugs for life, which seriously affects the quality of life of the patient. As a potential alternat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/0735C12N5/074
Inventor 芦银雪
Owner 芦银雪
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