A method for obtaining macrophages with phagocytic function through differentiation of pluripotent stem cells

A cell and cell induction technology, applied in the field of cell culture, can solve the problems of immature iPS induction and differentiation method, few sources of macrophages, and unguaranteed quality, and achieve the effects of low cost, good growth, and reduced pollution risk.

Active Publication Date: 2022-06-07
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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  • A method for obtaining macrophages with phagocytic function through differentiation of pluripotent stem cells
  • A method for obtaining macrophages with phagocytic function through differentiation of pluripotent stem cells
  • A method for obtaining macrophages with phagocytic function through differentiation of pluripotent stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0133] Example 1 Induction of hiPSC to form embryoid body (EB)

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

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

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

[0137] b) Aspirate 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 cells still in larger clumps form better EBs);

[0140] e) Immediately transfer the cells to a centrifuge tube containing DMEM / F12 to dilute Versene at a ratio...

Embodiment 2

[0142] Example 2 Embryoid bodies (EB) induce differentiation into macrophages

[0143] Schematic diagram of the flow chart of embryoid body (EB) induction into macrophages figure 1 shown.

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

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

[0146] Step c) remove the second medium in step b), and start using the third medium (STEMdiff TM APEL TM 2. Hematopoietic cells were incubated with 10ng / ml bFGF, 50ng / ml VEGF, 50ng / ml SCF, 10ng / ml IGF1, 25ng / ml IL-3, 50ng / ml M-CSF, 50ng / ml GM-...

Embodiment 3

[0152] Example 3 Detection of cell differentiation and morphology

[0153] The cell morphology was observed under the microscope and photographed, and the results were as follows Figures 2A-2E shown.

[0154] Figure 2A for day 0 iPS cells, Figure 2B for day 2 mesoderm cells, Figure 2C for the hematopoietic cells on day 7, 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 specifically provides a method for obtaining macrophages with phagocytic function through differentiation of pluripotent stem cells. The cell induction medium can realize rapid and large-scale induction of embryoid somatic cells to differentiate into macrophages. Among them, the first six media include serum-free media, which can provide basic nutrients for cell growth, proliferation and differentiation at various stages, and avoid the introduction of exogenous substances to reduce the risk of contamination. The seventh medium contains serum and additionally added FBS, which can well maintain the growth of macrophages. Each medium contains various cytokines which can promote the directional differentiation of cells. The cell induction culture method provided by the present invention is simple, efficient, low in cost, and can be expanded in a large amount in a short period to obtain high-quality and high-purity macrophages, which is of great significance for clinical treatment and research.

Description

technical field [0001] The present 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 of the body and are distributed in most tissues and organs. They perform antigen presentation and production of corresponding cytokines in the clearance of bacteria, viruses, and fungal pathogens in non-specific immunity and in specific immune responses. effect. And macrophages are a kind of multi-differentiated cells, monocytes, CD34 + Hematopoietic stem cells and early T lymphocytes can 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 primary cells such as peripheral blood mononuclear cells...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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