Method for separating and culturing endothelial clone formative cell
A technique of separating and culturing endothelium, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of inconvenient bone marrow sampling, difficult cord blood sampling and operation, and low number of EPCs
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Example Embodiment
[0062] Example 2 Umbilical cord-derived ECFCs primary cell culture and observation of the passage process
[0063] The primary cells of umbilical cord-derived ECFCs isolated by the method of Example 1 adhered more than 24-36 hours, and spindle-shaped and round adherent cells were seen. After culturing for 3-7 days, scattered cell clones were observed under an inverted microscope. It is fusiform, polygonal, round, and the cell body is large. After 7-10 days, the growth rate was fast, and many cell clones appeared. About 10-14 days, 80% of the cells were confluent and arranged like paving stones. After 10 consecutive passages, the cell morphology did not change significantly. ( figure 1 )
[0064] figure 1 Cell morphology of umbilical cord-derived ECFCs. (A), 36h of primary cell culture; (B), 5th day of primary cell culture; (C), 14th day of primary cell culture; (D), P1 generation cells; (E), P3 generation cells; (F), P10 passage cells.
Example Embodiment
[0065] Example 3 Determination of the growth curve of umbilical cord-derived ECFCs
[0066] The adherent cells obtained according to the method of Example 1 were 80% confluent and digested with 0.25% trypsin, and then the cells were digested by 2 × 10 4 Cells / well were seeded in a 24-well plate, 3 wells were digested every 24 h, cells were collected, and viable cells were counted with 0.4% trypan blue, observed for a total of 7 days, and the growth curve was drawn.
[0067] The growth curve of umbilical cord-derived ECFCs basically conformed to the law of cell growth curve. It suggested that the umbilical cord-derived ECFCs proliferated relatively stable in vitro, and the growth state was not abnormal. The cells proliferated rapidly, and in the logarithmic growth phase, the cell doubling time was about (43.53±5.38)h( figure 2 ).
Example Embodiment
[0068] Example 4 Cell cycle assay of umbilical cord-derived ECFCs
[0069] In the logarithmic phase of the growth of the umbilical cord-derived ECFCs obtained in Example 1, the cells were digested, fixed in 75% ethanol for 1 h at 4°C, incubated with 10 μg / mL RNase A at 37°C for 30 min, and 50 μg / mL propidium iodide was added at 4°C to protect from light. After incubation for 5 min, flow cytometer (FACA Calibur, USA) was used for detection, and ModiFIT software was used to analyze the results.
[0070] Flow cytometry analysis of the cell cycle showed that G 0 / G 1 period accounted for 75.58%, S+G 2 Cells in +M phase accounted for only 24.42% ( image 3 ).
[0071] The results showed that most cells of umbilical cord-derived ECFCs were in G0 / G1 phase, and a few were in S phase, which had typical stem cell proliferation characteristics.
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap