Method for improving multiplication capacity and performance of umbilical blood endothelial cells
A technology of endothelial cell proliferation and endothelial cells, which is applied in the field of improving the proliferation ability and performance of umbilical cord blood endothelial cells, so as to improve the therapeutic effect, enhance the angiogenesis ability of cells, and reduce the amount of cells
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Embodiment 1
[0033]This embodiment provides a method for improving the proliferation ability and performance of cord blood endothelial cells, and the specific steps are as follows:
[0034]Umbilical cord blood and umbilical cord specimens were obtained from placenta delivered by healthy mothers through aseptic operations. The tests for hepatitis B, hepatitis C, syphilis, AIDS, cytomegalovirus, TORCH, mycoplasma, chlamydia, G-6PD, and thalassaemia were all negative. After the specimens are collected, they are transported back to the blood bank to maintain 4~8℃ transportation conditions. Follow the methods below:
[0035]Step 1. Preparation of umbilical cord mesenchymal stem cells
[0036]Mesenchymal stem cells are extracted from the umbilical cord tissue of healthy people, and the obtained mesenchymal stem cells are cultured and expanded in vitro. After the primary cells are collected, they are subcultured in a ratio of 1:3. When the cells are about 80% of the density at the bottom of the bottle, trypsin ...
Embodiment 2
[0056]Example 2 Identification of Mesenchymal Stem Cells and Endothelial Cells
[0057]1. Identify mesenchymal stem cells and endothelial cells
[0058]1) Mesenchymal stem cells are identified by the following methods
[0059]a) Morphological analysis and cell counting of mesenchymal stem cells: Observe the cell status on an inverted microscope and take pictures of the cells cultured to the second generation.
[0060]b) Phenotype determination by flow cytometry: Take the second generation cells, collect them by 0.25% trypsinization, and wash them twice with normal saline by centrifugation. Add mouse IgG-FITC and IgG-PE (BD Company, USA) to the isotype control tube, and add mouse anti-human CD105-PE, CD90-FITC, CD73-PE, CD45-FITC, and CD34-PE to the detection tube (BD Company, USA) ) 5μL each. Incubate at room temperature for 30 min in the dark, and detect by flow cytometry (Fascalibur, BD Company, USA).
[0061]2) Identification of endothelial cells by the following methods
[0062]a) Morphological ...
Embodiment 3
[0068]Example 3 Effect verification
[0069]To verify the proliferation of cells co-cultured with endothelial cells and mesenchymal cells and their ability to form blood vessels, the details are as follows:
[0070]Grouping design: mixed group (group A, cells co-cultured with endothelial cells and mesenchymal cells), endothelial cell group (OEC group or B group) and mesenchymal stem cell group (MSC group or C group).
[0071]Refer to Example Step 3: OEC group took 2 mL of cell suspension A and added 8 mL of endothelial cell culture medium. In the MSC group, 2 mL of cell suspension B was added to 8 mL of endothelial cell culture medium. In the mixed group A, 2 mL of cell suspension A and cell suspension B were added to 6 mL of endothelial cell culture medium. The three groups were added to T75 culture flasks pre-plated with collagen and cultured. This experiment was repeated 3 times, and the results of each experiment were analyzed according to Example 3, Example 4, and Example 5.
[0072]1. Ve...
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