HUVEC isolation and cloning (JUMC)

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SOP: Isolation of human umbilical cord vein endothelial cells (HUVEC) (JUMC)

Reagents
• Medium EBM (Endothelial Basal Medium), Clonetics, cat. No. CC – 3121
• EGM Single Quots, cat. No. CC- 4133
Kit Content:
 CC- 4017 hEGF (human Epithelial Growth Factor) 0,5ml
 CC – 4035 Hydrocortisone 0,5ml
 CC – 4981 GA – 1000 0,5ml
 CC – 4092 BBE (Brain Bovine Extract) 2,0ml
 CC – 4101 FBS (Fetal Bovine Serum) 10ml

• Antibiotics (penicillin, streptomycin) Sigma
• Medium M-199 Sigma cat.No. M5017
• Collagenase type IA 0,1%, Sigma cat. No. C 2674,
• Tripsin Gibco cat. No. 25300
• PBS (without Ca and Mg) Biomed, Lublin
• EDTA Sigma cat. No. E5134

Required equipment
• catheters with needles 1,2 x 4,5 mm
• syringes 20ml
• plastic clips

Preparation of medium to HUVEC culture: Mix EBM medium with EGM Single Quots and antibiotics: penicilin (10 U/ml), streptomycin (10µg/ml)

Procedure:

1. Freshly umbilical cord is used. (up to two hours after delivery, stored on ice in refrigerator ( +4oC)
2. Place two catheters into the umbilical vein at both edges of cord using plastic clip clamps to close the vein lumen around the catheters.
3. Flush gently the lumen of vein using syringe with 50ml of M-199 medium with penicilin (10 U/ml), streptomycin (10µg/ml) to remove the content (rest of blood, cloths).
4. Fill up the vein lumen with M-199 medium containing 0,1 % collagenase (ca 15ml).
5. Incubate in 37oC for 25min.
6. Collect the vein lumen content (medium with collagenase and cells) using syringes and place it in the 50ml plastic tube (Falcon)
7. Wash the vein lumen twice with 50ml of M-199 medium collecting effluent to the same tube.
8. Fill the tube up to 50ml with M-199 medium and centrifuge at room temperature 100xg for 10 min
9. Discard supernatant, and fill again the tube with M-199 medium gradually up to 50 ml dispersing with the pipette the cellular pellet to wash cells.
10. Centrifuge at 100xg, room temperature for 10min.
11. Discard supernatant
12. Suspend the pellet in 10ml of EBM medium with EGM Single Quots Transfer the cells to the sterile 75 cm2 culture flask.

Cell culture is maintained at 37oC in humidified air containing 5 % CO2. Isolated cells population is tested by immunostaining for presence of alpha-actin and desmin (Becton Dickinson) (to exclude presence of SMC and fibroblasts) and factor VIII (Becton Dickinson) (to confirm presence of endothelial cells)

Cloning and maintenance of HUVEC
Medium EBM medium with EGM Single Quots changed twice per week. The incubation of 0,05% Tripsin with 0,01% EDTA dissolved in PBS for up to 5 min at room temperature is used for cell passage detachment. Passage is performed after obtaining the cultured cell confluency (5-9 days ). For HUVEC culture is recommended a 1:2 subculturing ratio. The cells up to 3-4 passages are only used for experiments.


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