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Long-term in-vitro culture and directional differentiation system and method for liver stem cell

An in vitro culture system and directional differentiation technology, applied in the direction of cell culture active agents, biochemical equipment and methods, artificial cell constructs, etc. System maintenance, etc.

Active Publication Date: 2015-03-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

) At present, it is still not possible to stably maintain the differentiation system at a specific intermediate developmental stage, such as the tissue stem cell stage
At the same time, there is currently no effective method to induce the differentiation of human pluripotent stem cells into hepatic stem cells that can be stably expanded.
[0004] Therefore, there is still a lack of effective means to expand hepatic stem cells in this field, especially the inability to achieve long-term in vitro culture and directed differentiation of hepatic stem cells under chemically defined culture conditions.

Method used

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  • Long-term in-vitro culture and directional differentiation system and method for liver stem cell
  • Long-term in-vitro culture and directional differentiation system and method for liver stem cell
  • Long-term in-vitro culture and directional differentiation system and method for liver stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Expansion of mouse embryonic liver stem cells

[0054] Take mouse embryos at 12.5 days pregnant, take out the embryonic liver, digest with Accutase (Invitrogen), and inoculate the single cell suspension in 0.5% Matrigel (BD Bioscience) or 20 μg / ml Laminin (Invitrogen) 6-well culture plate, the medium is DMEM / F12 (Invitrogen Company), containing insulin-transferrin-sodium selenite mixture (Invitrogen Company), 3 μM CHIR99021 (Tocris Company), 2 μM E-616452 (Sigma- Aldrich), 5 μM lysophosphatidic acid (Sigma-Aldrich), 0.5 μM sphingosine-1-phosphate (Sigma-Aldrich), 20 ng / mL epidermal growth factor (R&D Systems) and 50 μg / mL human Recombinant albumin (R&D Systems). Cells were subcultured with Accutase enzyme when grown to 70% confluency.

[0055] This culture condition can selectively expand embryonic liver stem cells, such as figure 1 As shown, the presence of vascular endothelial cells and fibroblasts could no longer be detected after five generations of ce...

Embodiment 2

[0059] Example 2: Expansion of liver stem cells obtained by differentiation of human pluripotent stem cells

[0060] Human pluripotent stem cells H1 and Hues9 cells (Wicell Company) were cultured in 6-well culture plates coated with 0.5% Matrigel, and the medium was DMEM / F12, containing 1% N2 (Invitrogen Company), 1% B27 (Invitrogen Company) and 20ng / ml bFGF (Invitrogen). When the cells grow to 50% confluence, replace the medium with DMEM / F12, B27 (insulin-free), and 100ng / ml Activin A (R&D Systems) for four days to induce cell differentiation into endoderm cells; then replace the medium with DMEM / F12 , containing 1% N2, 1% B27, 5 μM SB431542 (Sigma-Aldrich Company) and 10ng / ml BMP4 (Invitrogen Company) were cultured for 2 days to induce cells to further differentiate into the ventral foregut; then replace the medium with DMEM / F12, Containing 1% N2, 1% B27, 10ng / ml bFGF and 10ng / ml BMP4 cultured for 5 days to induce cells to differentiate into hepatoblasts. The medium was re...

Embodiment 3

[0063] Example 3: Inducing hepatic stem cells to differentiate into mature hepatocytes

[0064] Inoculate mouse or human hepatic stem cells cultured in vitro into 6-well culture plates coated with 0.5% Matrigel (10000 cells / well). The medium is DMEM / F12, containing insulin-transferrin-sodium selenite mixture, 20ng / mL hepatocyte growth factor, 20ng / mL Oncostatin M, 100nM dexamethasone, 1μM γ-secretase inhibitor Compound E and 2 μM of transforming growth factor beta receptor inhibitor E-616452.

[0065] After two weeks of in vitro culture, the expression of mature hepatocyte marker molecules was significantly up-regulated; under this induction condition, hepatic stem cells can differentiate into mature hepatocytes expressing and secreting ALB in a high proportion; the differentiated cells have a low density of uptake of acetylation The ability of lipoproteins, while taking up fluorescein-labeled bile acid and secreting bile acid into the intercellular bile duct; differentiated ...

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Abstract

The invention relates to the technical field of biomedical engineering and particularly relates to a long-term in-vitro culture and directional differentiation system and method for a liver stem cell. The long-term in-vitro culture and directional differentiation system comprises an amplification culture medium with specific chemical components, and a differential medium with specific chemical components, wherein the amplification culture medium is used for carrying out in-vitro culture of a mouse or human liver stem cell, and the differential medium is used for carrying out induced differentiation on the mouse or human liver stem cell to form a matured liver cell. By using the long-term in-vitro culture and directional differentiation system and method, a selectively-amplified liver stem cell in mother cells of the liver can be obtained from a mouse embryonic liver tissue or through human multipotential stem cell differentiation, the liver stem cell can be cultured for more than 20 generations under such a condition, and the stable molecular phenotype of the liver stem cell is maintained. By using the long-term in-vitro culture and directional differentiation system and method, the cultured mouse or human liver stem cell can be further subjected to induced differentiation to form the matured liver cell with functions of secreting albumin, metabolizing urea and the like.

Description

technical field [0001] The present invention relates to the technical field of biomedical engineering, in particular to a system and method for long-term in vitro culture and directional differentiation of hepatic stem cells, including an expansion medium with defined chemical composition, which is used for in vitro culture of mouse or human hepatic stem cells; and a chemically defined differentiation medium for inducing the differentiation of mouse or human hepatic stem cells into mature hepatocytes. Background technique [0002] In my country, more than 300,000 patients die from liver disease every year, but the annual liver transplantation can only meet the needs of 5% of patients with liver disease. The scarcity of transplantable livers severely limits the clinical application of this treatment. Existing preclinical and clinical studies have shown that mature hepatocytes or hepatic stem cells can replant and rebuild the liver after transplantation, but both mature hepat...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/071C12Q1/02A61K35/407A61P1/16A61L27/38
CPCC12N5/067C12N2501/12C12N2501/15C12N2501/237C12N2501/39C12N2501/734
Inventor 李文林吕林洁张木子孙平新
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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