Method for disintegrating human pluripotent stem cells into macrophages
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A macrophage, volume percentage technology, applied in the biological field, can solve problems such as hindering clinical application, long differentiation cycle, and low efficiency
Active Publication Date: 2019-12-24
TSINGHUA UNIV
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Problems solved by technology
The main problems of this method are the long differentiation cycle, low efficiency, and animal-derived components, which hinder its potential clinical application.
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Embodiment 1
[0159] Example 1. Differentiation of human pluripotent stem cells into macrophages
[0160] Two types of human pluripotent stem cells were used in this example, namely H1 cells and CD34-iPSC cells.
[0161] 1. Inoculate human pluripotent stem cells in a 6-well plate (2.5×10 per well 5 cells) were cultured in TeSR-E8 medium at 37°C until the cell confluency was 70%-80%.
[0162] 2. After completing step 1, take the six-well plate, suck off the culture supernatant, and add PBS buffer preheated to 37°C to wash twice. At this point, the morphology of H1 cells and CD34-iPSC cells is shown in figure 1 (Scale bar 100 μm).
[0163] 3. After completing step 2, take the six-well plate, add 1ml of cell digestion solution Accutase to each well, let it stand at 37°C for 3-5min, then add an appropriate amount of RPMI 1640 basal culture medium to stop the digestion, and collect the cells by centrifugation.
[0164] 4. Inoculate the cells collected in step 3 on a petri dish (the petri dis...
Embodiment 2
[0174] Example 2, Detection of cells in the process of differentiation of pluripotent stem cells into macrophages
[0175] 1. Flow cytometric detection of cell A and cell B
[0176] 1. Get the cell A obtained in step 9 and the cell B obtained in step 12 in Example 1, and resuspend the cells in PBS buffer containing 5% (v / v) fetal bovine serum to obtain cell A suspension (containing 1 × 10 5 cells), cell B suspension (containing 1×10 5 cells).
[0177] 2. Add PE-labeled CD43 antibody (volume ratio 1:50) and APC-labeled CD34 antibody (volume ratio 1:50) to the A cell suspension in step 1, respectively, and add to the B cell suspension in step 1 PE-labeled CD43 antibody (volume ratio 1:50), APC-labeled CD34 antibody (volume ratio 1:50), FITC-labeled CD45 antibody (volume ratio 1:50), FITC-labeled CD14 antibody (volume ratio 1:50 ), APC-labeled CD11b antibody (volume ratio 1:50), PE-labeled CD163 antibody (volume ratio 1:50), incubate at room temperature for 20 minutes in the d...
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Abstract
The invention discloses a method for disintegrating human pluripotent stem cells into macrophages. The invention provides a reagent kit for disintegrating the pluripotent stem cells into the macrophages. The reagent kit comprises a complete set of culture fluid which can disintegrate the pluripotent stem cells into the macrophages. A monolayer cell culture method is adopted, firstly, hemutopoiesisstem cells are obtained through disintegrating, and then through further adding IL3 and M-CSF for stimulation, CD14 and CD163 positive macrophages are obtained. The method has the advantages that chemical components are definite, animal-source components do not exist, the safety of preparation cells is greatly increased, and the method has the characteristics of short in time consumption, high indisintegrating efficiency, low in cost and the like. Through the adoption of the preparation method provided by the invention, human macrophages can be produced in a large-scale manner, the quality is stable, the safety is high, and massive cell sources are provided for tissue project, medicine research and development and cell therapy.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for differentiating macrophages from human pluripotent stem cells. Background technique [0002] Human pluripotent stem cells include human embryonic stem cells and human induced pluripotent stem cells, which can differentiate into various types of cells in the human body. They can be used to make disease models, conduct drug toxicity tests, and replace damaged and diseased cells through cell transplantation to promote Body wound repair and disease treatment. [0003] Macrophages are important natural immune cells of the human body. They have important application value in resisting pathogenic microbial infection, removing necrotic cells and tissue debris, and studying the occurrence and development of atherosclerosis. At present, it is difficult to obtain a large number of human macrophages, and it is difficult to expand and genetically manipulate, which greatly limits rel...
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