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Method for amplifying candidate stem cell in vitro

A technology for in vitro expansion of hematopoietic stem cells, applied in the field of in vitro expansion of hematopoietic stem cells, can solve the problems of reduced proliferative ability, difficulty in expanding the scale of culture, and inability to maintain it, so as to promote the generation of stromal cells, enhance biological effects, and avoid the preparation process Effect

Inactive Publication Date: 2009-03-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

However, the above-mentioned approaches have the following problems: (1) the scale of culture is difficult to enlarge and cannot meet the requirements of clinical use; (2) 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 in vitro
  • Method for amplifying candidate stem cell in vitro
  • Method for amplifying candidate stem cell in vitro

Examples

Experimental program
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Embodiment 1

[0035] A method for expanding hematopoietic stem cells in vitro,

[0036] 1) Isolation of mononuclear cells:

[0037] Acquisition of nucleated cells in human umbilical cord blood: add the methylcellulose solution with a weight concentration of 0.125% into human umbilical cord blood at a volume ratio of 4:1, leave it at room temperature, and settle red blood cells; take the supernatant at 1500rpm / min Centrifuge, and the sediment is nucleated cells.

[0038] 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).

[0039] 2) Preparation of microencapsulated mononuclear cells:

[0040] A. Sodium alginate with a weight content of 31.2% of β-L-guluronic acid and a weigh...

Embodiment 2

[0054] A method for expanding hematopoietic stem cells in vitro,

[0055] 1) Isolation of mononuclear cells:

[0056] 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);

[0057] 2) Preparation of microencapsulated mononuclear cells:

[0058] 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;

[0059] 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;

[0060] Sodium alginate solution I and sodium alginate solution II wer...

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Abstract

The invention relates to a method for in vitro 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 effective amplification of hemopoietic stem cell as well as carries out culture on a large scale, thereby stimulating the development of blood biology and clinic application and research.

Description

technical field [0001] The invention relates to a technical method for amplifying hematopoietic stem cells under three-dimensional conditions, specifically preparing microencapsulated mononuclear cells, using the physical and chemical properties of microcapsules and microencapsulated materials to promote the generation of stromal cells, and realizing the homologous hematopoietic stem cells. Three-dimensional co-culture of stromal cells, stromal cells secrete extracellular matrix and cytokines at the same time, and together constitute a hematopoietic microenvironment in the microcapsules, inhibit / delay the differentiation of hematopoietic stem cells and promote hematopoietic stem cells with only a small amount of cytokines effective amplification. 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 and gene therapy for blood sy...

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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