Adult human hepatogenic stem cell line HN, preparation method thereof and application

A technology of stem cell lines and liver stem cells, applied in cell dissociation methods, liver cells, artificial cell constructs, etc., can solve the problems of limited application of liver cell transplantation and low survival rate, and achieve the effect of stable characteristics and uniform components

Inactive Publication Date: 2017-12-29
洪丰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the problem of hepatocyte allogeneic rejection, the large-scale expansion culture technology of hepatocytes in vitro and the low survival rate of frozen hepatocytes after resuscitation have not been effectively resolved, the application of hepatocyte transplantation is greatly limited

Method used

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  • Adult human hepatogenic stem cell line HN, preparation method thereof and application
  • Adult human hepatogenic stem cell line HN, preparation method thereof and application
  • Adult human hepatogenic stem cell line HN, preparation method thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Isolation and primary culture of human liver stem cells

[0046]1. Experimental materials and reagents

[0047] 1. Tissue source: "Normal liver tissue" adjacent to the tumor excised from patients with hepatic hemangioma.

[0048] 2. Perfusion buffer: D-Hank's balanced salt solution containing 20mM HEPES.

[0049] 3. Circulating buffer: Hank's balanced salt solution containing 0.1% collagenase (Sigma-Adlrich).

[0050] 4. Enzyme buffer: PBS solution containing 0.05% collagenase (Sigma-Adlrich), 0.025% trypsin (Sigma-Adlrich), 0.02% EDTA, 0.004% DNase I (Sigma-Adlrich).

[0051] 5. DMEM liquid medium and fetal bovine serum are all products of Gibco Company.

[0052] 6. Percoll is a product of Pharmacia.

[0053] 7. Complete medium: containing 10% fetal bovine serum, 0.3U / mL insulin (Sigma-Adlrich), 0.3ng / mL epidermal growth factor (Epithelial growth factor, EGF, PeProTech), 0.2ng / mL stem cell growth factor ( Stem cell factor, SCF, PeProTech), 100 U / mL peni...

Embodiment 2

[0068] Example 2: Subculture and cryopreservation of adult human liver stem cells

[0069] 1. Reagents

[0070] 1. Trypsin solution: PBS containing 0.25% trypsin (Sigma-Adlrich), 0.02% EDTA.

[0071] 2. Freezing solution: DMEM medium containing 10% DMSO and 30% fetal bovine serum.

[0072] 2. Experimental method:

[0073] 1. When the cells grow to 90% confluence, discard the old medium, add 2mL PBS and gently swirl the culture flask to wash the cells.

[0074] 2. Discard the PBS, then add 2mL PBS to wash the cells once.

[0075] 3. Discard the PBS, add trypsin solution to just cover the cells, observe the digestion situation under an inverted phase-contrast microscope, and quickly discard the digestion solution when the cell protrusions retract.

[0076] 4. Add 2mL of complete medium to stop the action of trypsin, draw the medium in the bottle and gently blow the cells repeatedly.

[0077] 5. Collect the blown cells and place them in a 15mL centrifuge tube, mix well and t...

Embodiment 3

[0082] Example 3: Identification of Adult Human Liver Stem Cells

[0083] 1. Reagents

[0084] 1. QIAGEN RNeasy Mini Extraction Kit (Germany; Qiagen; 74104)

[0085] 2. ABI applied biosytenms RNA to cDNA Master Mix Reverse Transcription Kit (USA; ABI Company; 4472908)

[0086] 3. Primer sequences specific to each gene in the PCR amplification reaction (synthesized by Shanghai Bioengineering Co., Ltd.)

[0087]

[0088]

[0089] 4. dNTPmix: Contains 2mM each of dATP, dCTP, dGTP, and dTTP

[0090] 5. Taq DNA polymerase

[0091] 2. Experimental method:

[0092] 1. Adult human liver stem cells were cultured in a 6-well plate using stem cell medium. After growing to 90% confluence, the cells were collected by digestion with 0.25% trypsin-EDTA, added with 10 mL of sterile PBS, centrifuged at 1200 rpm for 8 min at room temperature.

[0093] 2. Cell pellets were extracted with QIAGEN RNeasy Mini Extraction Kit for total RNA.

[0094] 3. Then take 2ug of total RNA and use A...

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Abstract

The invention relates to an adult human hepatogenic stem cell line HN, a preparation method thereof and an application. The human hepatogenic stem cell line is collected at the China General Microbiological Culture Collection Center, the collection address is No. 3, first yard, Beichen West Road, Chaoyang District, Beijing, the collection date is May 15, 2017, and the collection number is CGMCC No. 13844. The human hepatogenic stem cell line is used for in vitro and vivo directional induction of liver cells and hepatic duct epithelial cells, preparation of biological artificial livers, resistance of acute hepatic failure, chronic hepatic failure and alcoholic fatty livers, inhibition of liver fibrosis and anti-tumor effects. The cells have the biological characteristics that the cells have typical epithelial cell morphology and strong multiplication capacity, grow to achieve contact inhibition and density inhibition, bidirectionally express liver cell specific marker albumin, cytokeratin 8 and biliary epithelial cell specific marker cytokeratin 7 and 9, have various functions of the liver cells, have multi-directional differentiation potential and avoid tumorigenicity.

Description

technical field [0001] The invention relates to an adult human liver-derived stem cell line, which belongs to the field of medical biotechnology. Background technique [0002] Liver disease is a huge threat to human health, especially hepatitis, liver cirrhosis, and liver cancer caused by hepatitis viruses are particularly serious in my country, and subsequent liver failure is the most common cause of death. Orthotopic liver transplantation is considered to be the only effective method for the treatment of end-stage liver disease, but its clinical application is greatly limited due to lack of liver source. [0003] Hepatocyte transplantation is a good alternative for the treatment of liver-related diseases, such as acute liver failure, genetic metabolic liver disease, and chronic liver failure caused by viruses. Due to the problem of hepatocyte allogeneic rejection, large-scale expansion and culture of hepatocytes in vitro, and the low survival rate of cryopreserved hepatoc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A61K35/407A61P1/16A61P35/00A61L27/38C12R1/91
CPCC12N5/0672A61K35/407A61L27/3804A61L27/3839A61L2430/28C12N5/067C12N2509/00
Inventor 洪丰洪叶毕研贞陈煜卢永科
Owner 洪丰
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