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Method for amplifying candidate stem cell

A technology of hematopoietic stem cells and nuclear cells, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of anti-leukemia and graft-versus-host effect changes, reduced proliferation ability, and failure to meet clinical use requirements, etc. , to reduce the differentiation of hematopoietic stem cells, promote the generation of stromal cells, and meet the requirements of clinical use

Inactive Publication Date: 2011-09-28
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

However, the above methods have the following problems: (1) the scale of culture is difficult to scale up, which cannot meet the requirements of clinical use; (2) the co-cultivation of heterologous stromal cells and stromal cells and hematopoietic stem cells may have the problem of immune rejection, while the same The preparation process of source stromal cells is cumbersome and takes a long time; (3) Cytokines alone cannot maintain long-term hematopoiesis in vitro, and the amount of cytokines is large and expensive, which greatly limits the large-scale expansion of hematopoietic stem cells
However, when hematopoietic stem cells are separated and purified from mononuclear cells, part of them will inevitably be lost, and the highly purified hematopoietic stem cells will completely lose other auxiliary cells, which not only leads to a decrease in proliferation ability, but also causes graft-versus-leukemia and graft-versus-host effects. will change

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  • Method for amplifying candidate stem cell
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Embodiment 1

[0033] A method of expanding hematopoietic stem cells,

[0034] 1) Separation of mononuclear cells:

[0035] Obtaining nucleated cells from human umbilical cord blood: add methylcellulose solution with a weight concentration of 0.125% to human umbilical cord blood at a volume ratio of 4:1, let stand at room temperature, and settle red blood cells; take the supernatant at 1500rpm / min Centrifuge, the sediment is nucleated cells.

[0036] Density gradient centrifugation, the Ficoll lymphocyte separation solution with a density of 1.077 / ml was added to the nucleated cells in a volume ratio of 1:1, 1500rpm / mim, 30min, and the mononuclear cells in the middle layer were aspirated; it contained CD34 + The number of cells is 0.9% of the total number of cells; after washing with PBS (1500rpm / min, 5min), adjust the cell density for later use.

[0037] 2) Preparation of microencapsulated mononuclear cells:

[0038] A. Prepare sodium alginate with B-L-guluronic acid weight content of 31.2% and α-D-...

Embodiment 2

[0051] A method of expanding hematopoietic stem cells,

[0052] 1) Separation of mononuclear cells:

[0053] Density gradient centrifugation, the Ficoll lymphocyte separation solution with a density of 1.077 / ml was added to the human bone marrow fluid at a volume ratio of 1.5:1, 1500rpm / mim, 30min, and the mononuclear cells in the middle layer were aspirated; it contained CD34 + Number of cells 1.9% of the total number of cells; After washing with PBS (1500rpm / min, 5min), adjust the cell density for later use;

[0054] 2) Preparation of microencapsulated mononuclear cells:

[0055] A. Prepare sodium alginate with a weight content of 37% β-L-guluronic acid and 63% α-D-mannuronic acid into 1.0% w / v sodium alginate solution I;

[0056] Sodium alginate containing 62% by weight of β-L-guluronic acid and 38% by weight of α-D-mannuronic acid is prepared into 2.5% w / v sodium alginate solution II;

[0057] Mix sodium alginate solution I and sodium alginate solution II in a volume ratio of 1:1 to ...

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Abstract

The invention relates to a method for amplification of hemopoietic stem cells. The method comprises the steps of separation of mononuclear cells, preparation and culture of microencapsulated mononuclear cells, and amplification of hemopoietic stem cells. The method can realize the amplification of hemopoietic stem cell as well as carries out culture on a large scale, and can play a significant role in blood biology and clinic application and research.

Description

Technical field [0001] The invention relates to a method for amplifying hematopoietic stem cells under three-dimensional conditions. Specifically, it is to prepare microencapsulated mononuclear cells, and use the physical and chemical properties of microcapsules and microencapsulated materials to promote the production of stromal cells, and realize the three-dimensional co-culture of hematopoietic stem cells and homologous stromal cells. The stromal cells secrete extracellular matrix and cells at the same time. Factors, which together form a hematopoietic microenvironment in the microcapsules, can inhibit / delay the differentiation of hematopoietic stem cells and achieve effective expansion of hematopoietic stem cells with only a small amount of cytokines. Background technique [0002] Hematopoietic stem cells have a wide range of clinical applications. They have a wide range of applications in supportive therapy, immunotherapy, replacement therapy, and gene therapy for blood syst...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N5/08C12N5/0786C12N5/0789
Inventor 马小军李双月王为于炜婷
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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