HUVECs

Obtención de células endoteliales a partir de cordón umbilical humano (HUVECs)

Reactivos & equipamiento
• Vena Umbilical humana
• Colagenasa tipo IV
• Medio de cultivo para célulasendoteliales (M199)
• Jeringa 10-20 mL
• Bisturí (esteril)
• 2 Cánulas
• PBS 1X (esteril)
• Hilo quirúrgico
• Bandeja de disección
• Gabinete de Bioseguridad
•Incubador O2/CO2


Procedimiento

1. Clean umbilical cord with a clean gauze.
2. Place clean cords on a tray. Make sure they are no injured
3. Place one end on top of a petri dishinside the tissue culture hood.
4. Cut one end with a sterile surgical blade.
5. Screw up the needle into the stopcock, 3-way luer-lock.
6. Look for the vein (the widest vessel); see arrow.7. Place the needle into the vein.
8. Open the clamp.
9. Place the free end of the cord inside the clamp and screw it up with your fingers.
10. Screw it up tighter with a surgical forcep.11. Leave the clamped cord on a tray.
12. Open a sterile irrigation solution bottle.
13. Fill out 50 ml syringes with irrigation solution.
14. Fit the end of the syringe onto the top ofthe 3-way luer-lock. Apply a gentle flow through the vein.
15. Squeeze the cord gently to remove remaining irrigation solution.
16. Close the 3-way luer-lock and place the lid back on top.
17.Put the cord back on a tray.
18. Fill out 50 ml syringes with 0.1% collagenase solution.
19. Clamp the free end with a hemostatic forcep and apply collagenase solution through the other end.20. Apply this solution until the cord is moderately swollen and close the 3-way luer-lock.
21. Place them onto a tray and leave them into the incubator for 20 minutes.
22. After thisincubation time, remove the tray from the incubator.
23. They must look softened because of the collagenase treatment.
24. Open again the irrigation solution bottle inside the hood.
25. Fill again...