Method for amplifying candidate stem cell

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

Inactive Publication Date: 2009-03-11
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
  • Method for amplifying candidate stem cell
  • Method for amplifying candidate stem cell

Examples

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Embodiment 1

[0033] A method of expanding hematopoietic stem cells,

[0034] 1) Isolation of mononuclear cells:

[0035]Acquisition of 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, and the sediment is nucleated cells.

[0036] Density gradient centrifugation, add Ficoll lymphocyte separation medium with a density of 1.077 / ml to the nucleated cells at a volume ratio of 1:1, 1500rpm / mim, 30min, absorb the mononuclear cells in the middle layer; it contains CD34 + The number of cells is 0.9% of the total number of cells; the cell density is adjusted after washing with PBS (1500 rpm / min, 5 min).

[0037] 2) Preparation of microencapsulated mononuclear cells:

[0038] A. Sodium alginate with a weight content of 31.2% of β-L-guluronic acid and a weight content of 6...

Embodiment 2

[0051] A method of expanding hematopoietic stem cells,

[0052] 1) Isolation of mononuclear cells:

[0053] Density gradient centrifugation, add Ficoll lymphocyte separation medium with a density of 1.077 / ml to the human bone marrow fluid at a volume ratio of 1.5:1, 1500rpm / mim, 30min, absorb the mononuclear cells in the middle layer; it contains CD34 + The number of cells is 1.9% of the total number of cells; the cell density is adjusted after washing with PBS (1500rpm / min, 5min);

[0054] 2) Preparation of microencapsulated mononuclear cells:

[0055] A. Sodium alginate with a weight content of 37% of β-L-guluronic acid and a weight content of 63% of α-D-mannuronic acid was prepared 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 was formulated into 2.5% w / v sodium alginate solution II;

[0057] Mix sodium alginate solution I and sodium alginate solution II at a volu...

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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 expanding hematopoietic stem cells under three-dimensional conditions. Specifically, microencapsulated mononuclear cells are prepared, and the physical and chemical properties of microcapsules and microencapsulated materials are used to promote the formation of stromal cells, so as to realize the three-dimensional co-cultivation of hematopoietic stem cells and homologous stromal cells, and the stromal cells secrete extracellular matrix and cells simultaneously. Factors together constitute a hematopoietic microenvironment in the microcapsules, which can inhibit / delay the differentiation of hematopoietic stem cells and realize the 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 application prospects, and they have a wide range of uses in supportive treatment, immunotherapy, replacement therapy a...

Claims

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Application Information

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Patent Type & Authority Applications(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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