Isolation and culture of human umbilical cord venous endothelial cells and proliferation experiments
Isolation and culture of human umbilical cord venous endothelial cells and proliferation experiments
Vascular endothelial cells are not only involved in the regulation of vascular permeability and coagulation processes, but also play an important role in many processes such as immune regulation, transplant rejection, tumor metastasis, and inflammation. Endothelial cell culture is an important means to study the function of endothelial cells in vitro. in the 70s Eric et al. established the method of endothelial cell isolation and in vitro culture. (Source: Basic Medical Immunology Laboratory Guide, edited by Jin Boquan and Li Enshan, 1st edition, Beijing: World Book Press, 1990).
Operation method
Isolation and culture of human umbilical cord venous endothelial cells and proliferation experiments
Principle
In vitro, endothelial cells can proliferate rapidly in the presence of endothelial cell growth factor and can be passed on, which can be used as a model for the study of endothelial cell proliferation, cytokine secretion, and phenotype.
Materials and Instruments
Collagenase, serum
Cord Buffor Streptomycin Sulfate Raw Buried Saline EDTA-Na Gelatin
Cannula Hemostat Clamp Syringe Gloves Filament Lunch Box Screeder Centrifuge Oscillator Culture Bottle CO Incubator Microscope Ultra-clean Bench Well Plate
Move
I. Isolation of umbilical cord endothelial cells
1. Umbilical cord collection
Clamp both ends of the umbilical cord with sterile hemostatic forceps, cut the umbilical cord on the outside of the forceps, and put it into a lunch box containing Cord Buffer at 4 ℃. The umbilical cord should not be left at 4 ℃ for more than 24 hours.
2. Umbilical cord endothelial cell isolation
(1) With sterile gloves, remove the umbilical cord and sterilize the inside of the hemostat with iodine and alcohol cotton balls before Cut off both ends of the umbilical cord with sterile scissors.
(2) Find a vein at one end of the cord, insert an umbilical vein cannula, and tie it off with 2 thick silk threads. The lumen of the vein was flushed with a 30 ml syringe of Cord Buffer until the umbilical blood was washed out.
(3) Drive out the residual fluid in the venous cavity, clamp the other end of the umbilical cord with hemostatic forceps, use a 10 ml syringe to connect the needle and inject 37 ℃ blood into the umbilical cord. Connect the needle, inject about 6-8 ml of 0.1% collagenase preheated at 37 ℃, and put it into the Cord Buffer at 37 ℃ to keep warm for 6-8 ml. Cord Buffer for 6-10 min.
(4) Aspirate out the cell suspension digested by collagenase from the venous lumen and add it to a centrifuge tube pre-filled with 20 ml Cord Buffer.
(5) Centrifuge at 1500 rpm for 10 min. Discard the supernatant, resuspend the cells with 5-7 ml of 20% FCS, RPMT 1640, transfer to culture flasks, and incubate in 5% CO Put the cells into the incubator with 5% CO
Preparation of crude extract of bovine hypothalamic endothelial cell growth factor
1. Take fresh bovine hypothalamus, add 1.5 times the volume of ice saline. Homogenize with tissue homogenizer, 0.5 min each time, 6 times in total.
2. mechanically shake with a shaker for about 1.5 h at 4 ℃.
3. Centrifuge at 10000 g for 40 min and collect the supernatant.
4. Add streptomycin sulfate at 0.5%, overnight at 4 ℃.
5. Centrifuge at 20,000 g for 40 min and collect the supernatant.
6. Measure 280 nm and 260 nm OD value by UV spectrophotometry and calculate the protein content according to the formula.
7. Filter, separate and store at -20 ℃.
Cultivation and characterization of endothelial cells
1. culture bottle coated with gelatin
Add 3 ml of 0.2% gelatin into the culture flask (25 cm) to cover the bottom of the flask, and store at 4 ℃. Prepare. Discard the gelatin solution before use.
2. Primary culture
Endothelial cells collected from umbilical cord vein were transferred into gelatin-coated culture flasks ( 25 cm), 5-7 ml of 20% calf serum 199 medium was added, and endothelial cell growth factor crude extract and heparin were added at a final concentration of 20 μg/ml and 100 μg/ Endothelial growth factor crude extract and heparin were added at final concentrations of 20 μg/ml and 100 μg/ml, respectively. The solution was changed once every 3-4 days.
3. Surrogate culture
When the endothelial cell growth spreads to the bottom of the bottle, it can be passed on. Aspirate off the medium and wash the flasks with serum-free RPMI1640 2 ml once. Wash the culture flask with RPMI1640 2 ml once. Add 0.1% trypsin, 0.02% EDTA-Na 3 ml, and incubate at 37 ℃. Wait for most of the cells were curled up and rounded under the microscope, 3 ml of serum-containing medium could be added to terminate the digestion of trypsin, and the cells were blown with the elbow burette to blow. Collect the cell suspension, add it to the centrifuge tube, centrifuge at 1500 rpm for 10 min, discard the supernatant, resuspend the cells in complete medium, transfer it to the incubator. medium to resuspend the cells and transfer to culture flasks for expansion.
4. Endothelial cell identification
Under the light microscope, the endothelial cells were polygonal or spindle-shaped, with one or two clear nuclei. The endothelial cells are polygonal or spindle-shaped under the light microscope. Under transmission electron microscopy, characteristic Weible-Palade vesicles may be present in some endothelial cells. Indirect immunofluorescence Indirect immunofluorescence staining, endothelial cells are positive for VIIIR:Ag.
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