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Method for differentiating macrophages from hiPS

A technology of macrophages and cells, applied in the field of genetic engineering, can solve problems such as production limitations, achieve the effect of increasing the number, avoiding the chance of contamination, and simplifying the differentiation steps

Active Publication Date: 2021-12-24
SHANGHAI CHILDRENS MEDICAL CENT AFFILIATED TO SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, current differentiation protocols require co-cultivation with stromal cells, isolation of monocytes, or complex reaction equipment, and yields are limited

Method used

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  • Method for differentiating macrophages from hiPS
  • Method for differentiating macrophages from hiPS

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Embodiment 1

[0024] The solutions of the present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention will not be limited. Embodiment 1 (the scheme of p8000)

[0025] A method for human induced pluripotent stem cells to differentiate macrophages, comprising the steps of:

[0026] 1), culture of human induced pluripotent stem cells (hiPSCs): no feeder layer, no serum Medium (cellapy, Human Pluripotent Stem Cell Medium) to culture hiPSCs, and passage when the cells reach 70%-80% density. Discard the medium, add 0.5mM EDTA to cover the cells, digest and incubate at 37°C for 4-5 minutes. Suck off the EDTA liquid, add iPS culture medium, pass passage at a ratio of 1:6, and place the plate in an incubator with a temperature of 37°C and 5% carbon dioxide.

[0027] 2), we use the EB-centrifugation protocol for hematopoietic differentiation: when hiPSCs grow to about 70% density, discard the culture medium, add Tryp...

Embodiment 2

[0030] Embodiment 2 (the scheme of p3500)

[0031] 1), culture of human induced pluripotent stem cells (hiPSCs): no feeder layer, no serum Medium (cellapy, Human Pluripotent Stem Cell Medium) to culture hiPSCs, and passage when the cells reach 70%-80% density. Discard the medium, add 0.5mM EDTA to cover the cells, digest and incubate at 37°C for 4-5 minutes. Suck off the EDTA liquid, add iPS culture medium, pass passage at a ratio of 1:6, and place the plate in an incubator with a temperature of 37°C and 5% carbon dioxide.

[0032] 2), we use the EB-centrifugation protocol for hematopoietic differentiation: but hiPSCs grow to about 70% density, discard the culture medium, add TrypLE Express ((Gbico) digestion solution that can cover hiPSCs, digest at room temperature for 2 minutes. Then discard Remove the digestion solution, resuspend the cells with APEL (STEMCELL Technologies) supplemented with 10mMY27632 (STEMCELL Technologies), 10ng / ml BMP4 (R&D) and 10ng / ml recombinan...

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Abstract

The invention relates to a method for differentiating macrophages from hiPS. The method comprises the following steps of: 1) culturing human induced pluripotent stem cells (hiPSCs) into embryoid EB; and (2) differentiating the embryoid EB into macrophages in an induced differentiation way, wherein an initial cell planting mode is that 8000 cells are cultured in each hole of each EB. The invention solves the problems of long time consumption and low yield of differentiation of macrophages by iPS. Macrophage differentiation time is shortened, and the number of differentiated macrophages is increased. Moreover, the steps of trophoblast cells and mononuclear cell sorting are not needed, so that a differentiation step is simplified, unnecessary pollution opportunities are also avoided, and clinical-grade macrophage differentiation is established.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular, the invention relates to a method for differentiating macrophages from hiPS (human induced pluripotent stem cells). Background technique [0002] Macrophages are the most plastic cell type in the hematopoietic system and play an important role in growth and development, internal environment stability, tissue repair and immunity. In addition, macrophages are closely associated with tumors. As immune cells, macrophages have a dual role in the tumor microenvironment. Tumor-associated macrophages (TAMs) promote tumor growth, including supporting tumor-associated angiogenesis and promoting tumor cell invasion, migration, and intravascular invasion. In esophageal, breast, and pancreatic cancers, the researchers observed that more advanced tumors were often accompanied by a high density of TAMs. Therefore, macrophages are important targets for immunotherapy. [0003] In re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786C12N5/074A61K35/15A61P35/00
CPCC12N5/0645A61K35/15A61P35/00C12N2506/45C12N2501/115C12N2501/155C12N2501/165C12N2501/125C12N2501/22C12N2501/2303Y02A50/30
Inventor 李彦欣李姗姗冯海忠
Owner SHANGHAI CHILDRENS MEDICAL CENT AFFILIATED TO SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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