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Method for preparing vascular endothelial cells and kit specially used for method
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A technology of vascular endothelium and kit, applied in the field of biomedicine, which can solve the problems of high price, uncertain culture medium composition, limited amplification ability, etc.
Active Publication Date: 2018-05-18
TSINGHUA UNIV
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Problems solved by technology
At present, it is difficult to obtain a large number of vascular endothelial cells, and the expansion ability is limited, the price is expensive, the composition of the culture medium is uncertain, and it contains animal-derived components, which greatly limits the relevant clinical applications.
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Embodiment 1
[0075] Embodiment 1, the preparation of nutrient solution
[0077] Take recombinant human serum albumin, dissolve it with sterile PBS buffer, and then filter it with a filter membrane to obtain a stock solution of recombinant human serum albumin with a concentration of 50 mg / mL.
[0078] Take human transferrin, dissolve it with sterile PBS buffer, and then filter it with a filter membrane to obtain a human transferrin stock solution with a concentration of 1 mg / mL.
[0079] Take vitamin C, dissolve it with cell culture grade water, and then filter it with a filter membrane to obtain a vitamin C stock solution with a concentration of 50 mg / mL.
[0080] Take sodium selenite, dissolve it with cell culture grade water, and then filter it with a filter membrane to obtain a sodium selenite stock solution with a concentration of 100 μg / mL.
[0081] Take CHIR-99021 and dissolve it with DMSO to obtain a stock solution of CHIR-99021 with a con...
Embodiment 2
[0093] Example 2, the acquisition and detection of vascular endothelial cells
[0094] Human pluripotent stem cells are H1 cells, H7 cells, H9 cells or CD34-iPSC cells.
[0095] The culture solution II is the culture solution II-1, the culture solution II-2 or the culture solution II-3.
[0096] The culture solution I is the culture solution I-1, the culture solution I-2, the culture solution I-3, the culture solution I-4 or the culture solution I-5.
[0097] 1. Acquisition of vascular endothelial cells
[0098] 1. Take human pluripotent stem cells grown to a confluence of 70%-80%, discard the supernatant, and then add PBS buffer to wash twice (the purpose is to remove dead cells and residual cell metabolic waste).
[0099] The morphology of human pluripotent stem cells is shown in figure 1 (The upper left is H1 cells, the upper right is H7 cells, the lower left is H9 cells, and the lower right is CD34-iPSC cells).
[0100] 2. After completing step 1, first add an appropri...
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Abstract
The invention discloses a method for preparing vascular endothelial cells and a kit specially used for the method. The kit comprises a nutrient solution II and a nutrient solution I, wherein the nutrient solution II includes serum albumin, transferrin, vitamin C, sodium selenite, VEGF, bFGF, and a basic nutrient solution free of serum, and the nutrient solution I includes serum albumin, transferrin, vitamin C, sodium selenite, BMP4, a GSK3 inhibitor, and a basic nutrient solution free of serum. It is proved through experiments that vascular endothelial cells can be obtained via the preparationmethod, and the nutrient solutions used in the preparation method allow human pluripotent stem cells to be fast efficiently differentiated into vascular endothelial cells. The nutrient solutions aredefined in chemical components and free of animal protein or insulin. Vascular endothelial cells can be massively produced through the preparation method, and the vascular endothelial cells are stablein quality and high in safety. The method has significant application value.
Description
technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing vascular endothelial cells and a special kit thereof. Background technique [0002] Human pluripotent stem cells (including human induced pluripotent stem cells and human embryonic stem cells) can be differentiated into functional cells of various tissues and organs, used to study human development, make disease models, and replace damaged and diseased cells through celltransplantation , Promote the body's wound repair and treat diseases. Stem cells and regenerative medicine will change the traditional treatment methods for diseases such as necrosis and injury, and bring about revolutionary changes in the mechanism research and clinical treatment of diseases. [0003] Vascular endothelial cells are important constituent cells of human blood vessels, and have important application value in injury repair, vascular reconstruction, treatment of ischemic diseases, t...
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