Method for culturing placental blood hematopoietic stem cells

A technology of hematopoietic stem cells and culturing methods, which can be applied to cell culture active agents, blood/immune system cells, animal cells, etc., can solve the problems of poor activity and proliferation ability of hematopoietic stem cells, and achieve stable and effective cultivation methods, enhanced metabolism, and matching. reasonable effect

Pending Publication Date: 2019-04-05
金时代进出口贸易重庆有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a method for culturing placental blood hematopoietic

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  • Method for culturing placental blood hematopoietic stem cells

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Embodiment 1

[0037] A method for culturing placental blood hematopoietic stem cells, comprising the following steps:

[0038] (1) Collect placental blood, add an equal volume of diluent to dilute to obtain placental blood dilution, then add 4% sedimentation solution to the placental blood dilution to precipitate, then centrifuge, discard the supernatant, To obtain a precipitate, add the resuspension solution to the precipitation, mix well, then add the separation solution, centrifuge, take the cloud layer, add the resuspension solution to the cloud layer, mix well, and centrifuge to obtain mononuclear cells, and put them into the mononuclear cells Add the resuspension to obtain the cell resuspension, the density of mononuclear cells in the cell resuspension is 1-2×10 8 a / mL;

[0039] (2) Add CD34 to the cell suspension + Antibody, mix well, incubate at room temperature for 10min, then add CD34 + Immunomagnetic beads, mix well, incubate at room temperature for 10 minutes, wash with new w...

Embodiment 2

[0059] A method for culturing placental blood hematopoietic stem cells, comprising the following steps:

[0060] (1) Collect placental blood, add an equal volume of diluent to dilute to obtain placental blood dilution, then add 8% sedimentation solution to the placental blood dilution to precipitate, then centrifuge, discard the supernatant, To obtain a precipitate, add the resuspension solution to the precipitation, mix well, then add the separation solution, centrifuge, take the cloud layer, add the resuspension solution to the cloud layer, mix well, and centrifuge to obtain mononuclear cells, and put them into the mononuclear cells Add the resuspension to obtain the cell resuspension, the density of mononuclear cells in the cell resuspension is 1-2×10 8 a / mL;

[0061] (2) Add CD34 to the cell suspension + Antibody, mix well, incubate at room temperature for 20min, then add CD34 + Immunomagnetic beads, mix well, incubate at room temperature for 20 minutes, wash with new w...

Embodiment 3

[0081] A method for culturing placental blood hematopoietic stem cells, comprising the following steps:

[0082] (1) Collect placental blood, add an equal volume of diluent to dilute to obtain placental blood diluent, then add a sedimentation solution with a mass percentage of 6% to the placental blood diluent to precipitate, then centrifuge, discard the supernatant, To obtain a precipitate, add the resuspension solution to the precipitation, mix well, then add the separation solution, centrifuge, take the cloud layer, add the resuspension solution to the cloud layer, mix well, and centrifuge to obtain mononuclear cells, and put them into the mononuclear cells Add the resuspension to obtain the cell resuspension, the density of mononuclear cells in the cell resuspension is 1-2×10 8 a / mL;

[0083] (2) Add CD34 to the cell suspension + Antibody, mix well, incubate at room temperature for 15min, then add CD34 + Immunomagnetic beads, mix well, incubate at room temperature for 1...

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Abstract

The invention discloses a method for culturing placental blood hematopoietic stem cells. The method comprises the following steps that (1) placental blood is collected, diluted by 0.3-1.0 wt% of a sodium chloride solution, precipitated by 0.3-1.0 wt% of a methylcellulose solution, re-suspended by human AB plasma with concentration of 12-16% and separated and purified by a separation solution to obtain mononuclear cells; (2) a CD34<+> antibody is added to a mononuclear cell resuspension solution for incubation, then CD34<+> immunomagnetic beads are added for incubation, washing is performed with a new washing solution, and the mononuclear cell resuspension solution is placed in a magnetic field and subjected to standing to obtain CD34<+> cells; and (3) the CD34<+> cells are inoculated on aplacental blood hematopoietic stem cell culture medium for culture to obtain the placental blood hematopoietic stem cells. The culture method is stable and effective, the hematopoietic stem cells canproliferate for a long time without differentiation, the problem that cell apoptosis is promoted due to the fact that the hematopoietic stem cells secrete a large number of oxidation products in the process of proliferation is solved, and the amplification rate and cell activity of the hematopoietic stem cells are improved.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a culture method for placental blood hematopoietic stem cells. Background technique [0002] Hematopoietic stem cells are the adult stem cells in the blood system, which are a heterogeneous group with the ability of long-term self-renewal and the potential to differentiate into various mature blood cells. It is a type of adult stem cell with the longest and most in-depth research history, and it has important guiding significance for the study of various stem cells, including tumor stem cells. Because hematopoietic stem cells have the potential of multi-directional differentiation, transplanting hematopoietic stem cells into the body is an effective method for the treatment of leukemia, lymphoma and other malignant tumors, metabolic diseases, autoimmune diseases and congenital immune deficiencies that seriously endanger human health. alleviating the suffering of...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2501/998C12N2501/905C12N2501/90C12N2500/76C12N2500/34C12N2500/38C12N2500/30C12N2500/05
Inventor 赵翔宋彩霞曹玉昊叶劲涛王仁杰黄启程邱阳李国焱唐玲胡小东
Owner 金时代进出口贸易重庆有限公司
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