Aspirate the medium and wash the cells with 1x PBS

Add 300 µl accutase to a T-25 flask and incubate at 37 °C, 5% CO₂ for 3-5 minutes

Resuspend the cells in 10 ml PBS and transfer the cell suspension to a 15-ml falcon tube

Centrifuge at 500 g for 5 minutes at 20 °C

Aspirate the supernatant and dissolve the pellet in 10 ml M-199 medium (serum-free)

Count the cells using a Luna Cell Counter

Prepare cell suspension for hanging drops

5000 cells in 25 µl drop

For 40 spheroids: Kultur HUVEC hAMSC Medium um

10% HUVECs + 90% hAMSCs 104 1.8* ⁴104 1.8⁵105 1000 µl

For 10 ml medium: Reagent Stock solution End concentration Volume for 10 ml l

M-199 8 ml

FBS 100% 20% 2 ml

ECGS 10 mg/ml 10 µg/ml 10 µl

Heparin 5000 I.E./ml 15 I.E./ml 30 µl

Pipette 25 µl drops on the inner side of the lid of a pertridish (hydrophobic)

Carefully flip the lid

Put the lid on the bottom of the petridish filled with 20 ml PBS

Incubate for 48 hours at 37 °C, 5% CO₂

Prepare gel mix (500 µl/µ-dish)

Gel mix for 2 gels

Reagent Volume for 1000 µl

HPL 200 µl

Aprotinin 58 µl

M-199 741 µl

ECGS 1 µl

Aprotinin is added to prevent fibrinolysis.

Mix 500 µl Gel mix with 100 µl thrombin

Carefully pipette the gel mix on the window of the µ-dish (avoid bubbles)

Polymerize the gels at 37 °C for at least 20 minutes

Pick a spheroid with a 200 µl pipette tip (avoid transferring too much medium) and place the spheroid on the gel (positioning according to the picture)

Repeat step 16 until 5 spheroids are transferred to the gel

Put the µ-dish in a bigger pertridish with PBS (to prevent dehydration of the gels) and incubate at 37 °C, 5% CO₂overnight

After 24 hours sprouts are visible

Add medium to the gel (500 µl/gel), pipette carefully at the border of the dish

For 2 ml medium:

Reagent Stock solution End concentration Volume for 2 ml

M-199 1.6 ml

HPL 100% 20% 400 µl

ECGS 10 mg/ml 10 µg/ml 2 µl

Incubate at 37 °C, 5% CO₂