Human Umbilical Vein Endothelial Cell (HUVEC) Culture

Summary

Human umbilical vein endothelial cells (HUVEC) can be used (1) as a model cell for in vitro experiments and (2) to study the biological properties of vascular endothelial cells and their relationship with various diseases.

Operation method

Human Umbilical Vein Endothelial Cell (HUVEC) Culture

Principle

In this experiment, fresh umbilical cords of healthy maternal newborns were used, and the umbilical vein endothelial progenitor cells were obtained by collagenase type I with digestion and isolation. Collagenase is the most ideal vascular endothelial cell separating enzyme, with strong digestive effect on the intercellular matrix, which can make the epithelial cells and collagenous tissues successfully separated without damaging the endothelial cells, and the isolated vascular endothelial cells are more in number, with less contamination of heterogeneous cells and strong cell apposition ability.

Materials and Instruments

Isolated Human Umbilical Cord Venous Endothelial Cells HUVEC
PBS Collagenase M199 medium Trypsin EDTA DMEM medium
Needles Surgical forceps Centrifuge tubes Cryo-centrifuge Elbow Gelatin Constant temperature incubator Microscope

Move

1. Store 15-20 cm of neonatal cord in sterile PBS solution. (Note: Store at 4°C for a maximum of 24 hours and at room temperature for no more than 6 hours or discard)

2. Insert a blunt needle into the umbilical cord vein tube and rinse 3-5 times with sterile PBS solution until the blood is clean.

3. Clamp the lower end of the umbilical cord with surgical forceps and add 15 ml of collagenase (1 mg/ml) to digest the cord for 15-20 minutes at room temperature, shaking the cord up and down from time to time. 4.

4. After digestion, the lower end of the surgical forceps is loosened and the digestive fluid flows into a 50 ml sterile centrifuge tube, and the cord is rinsed 2-3 times with sterile PBS. 5.

5. Centrifuge the collected fluid (2000 rpm) for 3 minutes. 6.

6. Pour off the supernatant, add 10ml of M199 medium (with 10U/ml of bFGF), blow the cells with a bent tube, transfer all the liquid into a gelatin-coated culture bottle and incubate at 37℃. (Note: Add 3-4ml of sterile 1% gelatin solution into each culture bottle, shake well to make the gelatin solution completely cover the bottom of the bottle, and incubate at 37℃ for at least 2 hours, and pour out the gelatin solution before use, as the gelatin coating is good for cell attachment. The gelatin coating will help the cells to adhere to the wall.)

7. After 24 hours of incubation, pour off the medium and wash with sterile PBS solution for 2-3 times to wash away the red cells and dead cells, and add 10 ml of fresh M199 medium. 8.

8. Change the medium every 2 days (2/3 of the medium each time). 9.

9. Generally speaking, after 5-7 days of incubation, the cells can grow to 80-90% monolayer, and then can be passed on. 10.

10. Pour off the medium, wash with sterile PBS solution 2-3 times, add 2-3ml digestion solution (0.25% trypsin + 0.1% EDTA) to digest the cells, observe under the microscope, once the cells become rounded, add 2-3 times of serum DMEM medium to terminate the reaction.

11. Blow down the cells with a bent tube and transfer the digested cells to a 50 ml sterile centrifuge tube and centrifuge at 2000 rpm for 3 minutes. 12. Pour off the supernatant and add the supernatant to the DMEM medium.

12. Pour off the supernatant and add 10 ml of fresh medium. Generally, one bottle of cells can be passed on for 3-4 bottles, and the culture should be passed on accordingly. 13.

13. Generally, 2-3 generations of cells (cultured for about 20 days) can be used to do various experiments with the best results.

Caveat

1. Strict sterilization. Use three sets of instruments to take materials.

2. Strictly carry out aseptic operation to prevent bacterial, fungal and mycoplasma contamination and avoid chemical substance contamination.

3. Flame sterilize the mouth of the bottle and the pipette before aspirating the liquid; always use Hank'S liquid to cool the pipette after being flame sterilized. Prevent scalding and scalded cells. When aspirating liquids, avoid bottle mouth and pipette contact collision.

4. Before the centrifuge tube is put into the table, the mouth and wall of the tube should be sterilized.

Common Problems

From the patent "A method for isolation and culture of human umbilical vein endothelial cells HUVEC and their transmission

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