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Medium and purpose thereof
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A culture medium and mercaptoethanol technology, applied in the direction of vertebrate cells, artificial cell constructs, animal cells, etc., can solve the problems of limited expansion, small proportion and number of endothelial cells, difficult clinical treatment, etc., and achieve clear composition , Avoid negative effects, Nutritious effect
Active Publication Date: 2015-02-25
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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Problems solved by technology
However, due to the small proportion and number of endothelial cells produced by these induction methods, and the limited expansion in vitro after sorting, it is difficult to be used for clinical treatment.
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Embodiment 1
[0045] refer to figure 1 , using the method for inducing embryonic stem cells to differentiate into endothelial cells in vitro of the present invention, culture embryonic stem cells according to the following steps:
[0047] Using the fourth medium, mouse fibroblasts were co-cultured with undifferentiated human embryonic stem cells (ie hESCs, H1 and H9) as trophoblast cells. Among them, the basic composition of the fourth medium is: 78% D-MEM / F-12 basic medium (GIBCO, No.12400-016), added 20% Knockout serum substitute (GIBCO, 10828), 1mM L- Glutamine (GIBCO, 35050), 0.1mM β-mercaptoethanol (Invitrogen, 21985-023), 1% non-essential amino acid (NEAA (English abbreviation), Invitrogen, 11140-050), and 5ng / ml basic fibroblastGrowth factor (rhbFGF (English abbreviation), Millipore, GF003). After the trophoblast cells are co-cultured, hESCs are observed. If passage is required, the hESCs to be passaged are digested into small cell ...
Embodiment 2
[0082] Flow cytometry and RT-PCR were performed on the cells obtained in Example 1 through the first induced differentiation culture, the cells subjected to the second induced differentiation culture, and the endothelial cells obtained through the third induced differentiation culture for 6 days , and observe the morphological changes of the cells in the three stages under a microscope.
[0088] (1) Discard the old medium and wash three times with 1×PBS.
[0089] (2) Add 0.25% trypsin to digest the cells to be tested, and observe the cells under a microscope. If the cell gap becomes larger and the cells are detached from the well plate, add new termination medium to terminate the digestion.
[0090] (3) Collect the ...
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Abstract
The present invention discloses a group of mediums and kits for in vitro inducing embryonic stem cells to differentiate into endothelial cells, purpose thereof for in vitro inducing embryonic stem cells to differentiate into endothelial cells, and endothelial cells or derivatives thereof. The group of mediums comprise: a first medium, which is an IMDM / F12 medium added with insulintransferrinselenium, BSA, non-essential amino acids, and rhBMP-4; a second medium, which is an IMDM / F12medium added with insulintransferrinselenium, BSA, non-essential amino acids, rhVEGF and rhbFGF; and a third medium, which is an EGM2 medium. The group of medium disclosed by the present invention can quickly and efficiently induce embryonic stem cells to differentiate to endothelial progenitor cells and endothelial cells.
Description
technical field [0001] The invention relates to the technical field of endothelial cell regeneration, in particular to a culture medium and its application. More specifically, the present invention relates to a group of culture media and kits for inducing embryonic stem cells to differentiate into endothelial cells in vitro, and their use in inducing embryonic stem cells to differentiate into endothelial cells in vitro, methods for inducing embryonic stem cells to differentiate into endothelial cells in vitro method, and endothelial cells or derivatives thereof. Background technique [0002] The cardiovascular system is the first system to emerge and function during embryonic development. The occurrence and development of blood vessels are not only important for the development and differentiation of organs in embryogenesis, but also for wound repair and reproductive function in adults. Vascular development also plays an important role in a range of pathological phenomena,...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 裴雪涛谢小燕岳文何丽娟李艳华南雪姚海雷房芳张博文
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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