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Method for acquiring macrophages with phagocytic functions by means of pluripotent stem cell differentiation

A technology of grouping cells and cytokines, applied in the direction of non-embryonic pluripotent stem cells, artificially induced pluripotent cells, biochemical equipment and methods, etc., can solve the problem of immature iPS induction differentiation method, few sources of macrophages, and poor quality Guarantee and other issues to achieve the effect of low cost, good growth, and reduced pollution risk

Active Publication Date: 2018-12-25
赛元生物科技(杭州)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first object of the present invention is to provide a group of cell induction medium, the second object of the present invention is to provide a kind of cell induction culture method, the third object of the present invention is to provide the above-mentioned cell induction medium or cell induction culture method in the induction Induced differentiation of pluripotent stem cells or embryoid bodies into macrophages, the fourth object of the present invention is to provide a medium for inducing pluripotent stem cells or embryoid bodies to differentiate into macrophages comprising the above-mentioned cell induction medium A kit for phagocytes to alleviate the technical problems of the existing technology that there are few sources of macrophages, the number of cells is low or the quality cannot be guaranteed, and the iPS induction differentiation method is immature

Method used

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  • Method for acquiring macrophages with phagocytic functions by means of pluripotent stem cell differentiation
  • Method for acquiring macrophages with phagocytic functions by means of pluripotent stem cell differentiation
  • Method for acquiring macrophages with phagocytic functions by means of pluripotent stem cell differentiation

Examples

Experimental program
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Effect test

Embodiment 1

[0133] Example 1 Induction of hiPSCs to form embryoid bodies (EBs)

[0134] hiPSCs are obtained from human peripheral blood mononuclear cells (PBMC) through the existing method of inducing reprogramming: a certain amount of PBMCs are electroporated into episomes containing reprogramming genes, and after clones appear, they are picked on matrigel culture plates to continue culturing , to obtain reprogrammed iPS.

[0135] Prewarm (15-25°C) sufficient amounts of mTeSR1, DMEM / F12 and Versene for cell passage. The Rock kinase inhibitor Y-27632 was added to the medium so that the final concentration was 3 μM.

[0136] a) Wash the original well with 1ml DPBS;

[0137] b) Absorb DPBS, add 1ml Versene+Y27632;

[0138] c) Incubate at 37°C for 4 minutes;

[0139] d) Use a pipette with a 1000ul tip to pipette 1-2 times and remove the cells (usually the cells are still in larger clumps to form better EBs);

[0140] e) Immediately transfer the cells to a centrifuge tube containing DMEM...

Embodiment 2

[0142] Example 2 Induction of embryoid bodies (EBs) to differentiate into macrophages

[0143] Schematic flow chart of embryoid body (EB) induced differentiation into macrophages as shown in figure 1 shown.

[0144] Step a) remove the mTeSR1 medium of the embryoid body in f) of embodiment 1, and use the first medium (STEMdiff TM APEL TM 2. 10ng / ml BMP4, 5ng / ml bFGF) were incubated for 24 hours, and the embryoid bodies were differentiated into mesoderm cells;

[0145] Step b) remove the first culture medium in step a), start to use the second culture medium (STEMdiff TM APEL TM 2, 10ng / ml BMP4, 5ng / ml bFGF, 50ng / ml VEGF, 100ng / ml SCF) incubate and culture mesoderm cells to obtain hematopoietic cells, during which a new second medium is replaced every other day;

[0146] Step c) remove the second culture medium in step b), start to use the third culture medium (STEMdiff TM APEL TM 2, 10ng / ml bFGF, 50ng / ml VEGF, 50ng / ml SCF, 10ng / ml IGF1, 25ng / ml IL-3, 50ng / ml M-CSF, 50ng / ...

Embodiment 3

[0152] Example 3 Detection of Cell Differentiation Morphology

[0153] Observe the cell morphology under a microscope and take pictures, the results are as follows Figure 2A-Figure 2E shown.

[0154] Figure 2A For iPS cells on day 0, Figure 2B For day 2 mesoderm cells, Figure 2C For day 7 hematopoietic cells, Figure 2D For myeloid cells on day 17, Figure 2E Mature macrophages on day 28.

[0155] It can be seen from the figure that the iPS cells were well differentiated and a large number of mature macrophages were obtained.

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Abstract

The invention relates to the technical field of cell culture, and particularly provides a method for acquiring macrophages with phagocytic functions by means of pluripotent stem cell differentiation.Induced differentiation can be quickly and substantially carried out on cells of embryoid bodies by the aid of cell induced culture media to obtain the macrophages. First six culture media include serum-free culture media, basic nutrient substances which are required by growth, proliferation and differentiation of cells in various phases can be provided by the serum-free culture media, exogenous substances can be prevented, and accordingly pollution risks can be reduced. A seventh culture medium contains serum, FBS (fetal bovine serum) is additionally added into the seventh culture medium, andaccordingly growth of the macrophages can be effectively maintained. Each culture medium further contains diversified cell factors, and accordingly directional differentiation of the cells can be promoted. The method has the advantages that the method for carrying out induced culture on the cells is simple, is high in efficiency and low in cost and has important significance in clinical treatmentand research, and the high-quality and high-purity macrophages can be obtained in short periods by means of substantial amplification.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for obtaining macrophages with phagocytic function through differentiation of pluripotent stem cells. Background technique [0002] Macrophages are one of the important immune cells in the body. They are distributed in most tissues and organs. They play the role of antigen presentation and production of corresponding cytokines in non-specific immunity to clear bacteria, viruses, fungal pathogens and specific immune responses. role. And macrophages are a kind of cells with multiple differentiation sources, monocytes, CD34 + Hematopoietic stem cells and early T lymphocytes can all differentiate into macrophages under certain conditions. [0003] At present, there are two main sources of human macrophages used in in vitro experiments, one is tumor-derived cell lines, such as U937 and THP-1 cell lines; the other is derived from primary cells such as peripheral blood m...

Claims

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Application Information

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IPC IPC(8): C12N5/0786C12N5/074
CPCC12N5/0645C12N5/0696C12N2500/90C12N2501/105C12N2501/115C12N2501/125C12N2501/405C12N2501/22C12N2501/2303C12N2506/45C12N2501/155C12N2501/165
Inventor 张进张丽
Owner 赛元生物科技(杭州)有限公司
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