Materials and Reagents

Collagenase I (Life Technologies, Invitrogen^TM, catalog number: 17100-017 )
Endothelial cell growth supplement (ECGS) (Sigma-Aldrich, catalog number: E2759 )
M199 medium
Fetal calf serum (FCS)
Phosphate buffered saline (PBS)
Pen/Strep
Sodium pyruvate
Antibiotic
Ethanol
Collagenase solution
Culture medium (see Recipes)

Equipment

Procedure

Wash all tools, glassware and tray in detergent and then put them in ethanol for 20 min under UV in the culture hood.
Prewarm PBS and collagenase I (100 ml of PBS for one cord) at 37 °C.
Dilute 1 ml of collagenase (20 mg/ml) into 20 ml of warm PBS, spin at 1,000 x g for 5 min.
Discard the pellet and filter the supernatant using syringe filter under sterile conditions.
Take the cord from the bottle, cut ~1 cm from each site (because the ends may be contaminated). Check if the cord is damaged (holes etc), if so discard it.
There are three blood vessels in the cord, find the biggest one.
Cut the very end of the needle, insert it in the biggest blood vessel hole, fix the needle with the clamp.
Screw on the syringe, wash the cord with warm PBS until no blood is leaking from the cord.
Put the second clamp at the other end of the cord.
Pump 20 ml of collagenase solution into cord, be careful not to blow up the cord.
Incubate at 37 °C for 10 min in a sterilized beaker, seal with parafilm.
Cut the lower end open. Put all collagenase I to a 50 ml tube, spin at 1,000 x g for 5 min.
Remove supernatnat, resuspend pellet in 3 ml complete M199 medium.
Plate on to a 3 cm tissue culture dish.

Recipes

References