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Culture system and method for amplifying hemopoietic stem cells as well as application thereof

A technology of hematopoietic stem cells and medium, applied in the direction of cell culture active agent, blood/immune system cells, tissue culture, etc., to achieve the effect of large quantity, low expansion rate and low cost

Inactive Publication Date: 2019-04-09
NEWISH TECH (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MS275 has been reported to promote the generation of human induced pluripotent stem cells (iPSCs), while the effect of MS275 on the maintenance and expansion of hematopoietic stem cells has not been reported

Method used

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  • Culture system and method for amplifying hemopoietic stem cells as well as application thereof
  • Culture system and method for amplifying hemopoietic stem cells as well as application thereof
  • Culture system and method for amplifying hemopoietic stem cells as well as application thereof

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preparation example Construction

[0040] The preparation of the umbilical cord blood hematopoietic stem cells includes: diluting the umbilical cord blood 2 to 3 times with normal saline, adding lymphocyte separation medium, centrifuging at 1500 to 2000 rpm / min for 20 minutes to obtain mononuclear cell layer (PBMC), washing with normal saline and resuspending Obtain PBMC cell aggregates; then separate CD34+ cells by magnetic beads method.

[0041] StemSpanSFEM II is a serum-free medium, the manufacturer is StemCell Technologies, the product number is 09655;

[0042] Recombinant human stem cell factor rhSCF (recombined human stem cell factor), the manufacturer is Stemimmune LLC, the product number is HHM-SF-1000;

[0043] Recombinant human thrombopoietin rhTPO (recombined human thrombopoietin), the manufacturer is Stemimmune LLC, the product number is HHM-TP-0100;

[0044] Recombinant human FMS-like tyrosine kinase 3 ligand rhFLT3L (recombined human FMS-liketyrosinekinase 3ligand), the manufacturer is Stemimmun...

Embodiment 1

[0060] 1. Obtain umbilical cord blood mononuclear cells;

[0061] (1) Dilute the umbilical cord blood with normal saline for 2 to 3 times, mix well and add dropwise to 0.4 times the volume of lymphocyte separation solution, taking care not to damage the interface;

[0062] (2) Use 1500-2000rpm / min to centrifuge for 20min. Due to different densities, the centrifuge tube is divided into four layers from top to bottom: the first layer is the plasma layer, the second layer is the ring-shaped milky white mononuclear cell layer (PBMC), and the second layer is the plasma layer. The third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer;

[0063] (3) Use a straw to carefully draw the second layer of milky white mononuclear cell layer (PBMC) into another 50ml centrifuge tube, add physiological saline, and centrifuge again at 1500-2000rpm / min for 5-10min;

[0064] (4) Discard the supernatant and add physiological saline to resuspend, fi...

Embodiment 2

[0074] The contents of factors in the medium of each group are shown in Table 1:

[0075] Table 1 The content of factors in the medium of each group

[0076]

MS275

SCF

TPO

FLT3

group 1

1μM

80ng / ml

30ng / ml

90ng / ml

group 2

1μM

100ng / ml

50ng / ml

100ng / ml

group 3

1μM

120ng / ml

70ng / ml

110ng / ml

group 4

0

80ng / ml

30ng / ml

90ng / ml

[0077] Each substance was added to StemSpan SFEM II serum-free medium at the concentrations shown in Table 1.

[0078] The CD34+ umbilical cord blood hematopoietic stem cells prepared in Example 1 were suspended and inoculated in the cell culture medium of each group for culture. Cells were seeded at a density of 1 × 10 in a 24-well plate 4 cells / well, placed at 37°C, 5% CO 2 Incubator cultivation. According to the state of cell culture, 500 μL of fresh cell culture medium of each group was added every 2 days, and a large number of hem...

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Abstract

The invention relates to the technical field of cell culture, in particular to a culture system and method for amplifying hemopoietic stem cells as well as an application thereof. Research shows thatin an amplifying culture process of hemopoietic stem cells of umbilical cord blood, MS275 is added while cell factors are added, and the effects of increasing the number of the hemopoietic stem cellsand improving the CFU colony formation capacity of the hemopoietic stem cells are realized, so that the hemopoietic stem cells can be in a proliferation and non-differentiation state and meet the clinical transplantation demand. The culture system and method are convenient to operate and low in cost, more hemopoietic stem cells are obtained, and the defects that hemopoietic stem cells in the priorart are low in amplification rate, prone to differentiation and the like are overcome.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a culture system, method and application for expanding hematopoietic stem cells. Background technique [0002] Hematopoietic stem cell transplantation is a common and effective treatment for various blood diseases and immune system diseases such as leukemia, lymphoma, aplastic anemia, and thalassemia. Hematopoietic stem cells generally come from three sources: bone marrow, peripheral blood and umbilical cord blood. Compared with bone marrow and peripheral blood hematopoietic stem cells, umbilical cord blood hematopoietic stem cells are easy to obtain, rich in sources, and have no damage to donors and no side effects, so they have become an important source of hematopoietic stem cell transplantation donors. [0003] At present, the bottleneck of umbilical cord blood hematopoietic stem cell transplantation technology lies in its low cell content. The number of hematopoietic s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2501/125C12N2501/145C12N2501/16C12N2501/26
Inventor 陈立功刘德芳孙忠杰严小娥马骏凡
Owner NEWISH TECH (BEIJING) CO LTD
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