Summary
This protocol describes a 3D endothelial tube formation assay using HUVECs co-cultured with pericytes or mesenchymal stromal cells in LunaX ECM. The model supports mechanistic angiogenesis research, preclinical screening and evaluation of pro- or anti-angiogenic treatments.
What you’ll do
- Prepare LunaX MultiMatrix Low Stiffness ECM and fresh photoinitiator.
- Combine HUVECs with pericytes or MSCs at a 10:1 ratio.
- Encapsulate the co-culture in 30 µL LunaX ECM domes.
- Crosslink the matrix to approximately 1.5 kPa and maintain it in supplemented endothelial medium.
- Monitor capillary-like networks by brightfield or fluorescence microscopy.
Protocol at a glance
- Cell model: HUVECs below passage 8 with pericytes or MSCs.
- Cell ratio: 6 × 10⁶ HUVECs and 6 × 10⁵ support cells per mL of final ECM.
- Crosslinking: 50 seconds at 10 mW/cm² to achieve approximately 1.5 kPa.
- Medium: EGM-2 supplemented with 50 ng/mL each of SDF-1, FGF-2 and VEGF.
- Expected timing: Initial networks within 2–3 days, with continued maturation during culture.
Expected outcome
Capillary-like structures and interconnected endothelial networks should appear within 2–3 days and mature into lumen-containing tubules with continued culture. Structures can be assessed by brightfield microscopy or stained for CD31, F-actin and nuclear markers.
Critical parameters
- Use healthy HUVECs below passage 8 and harvest both cell populations at 70–80% confluence.
- Completely quench and remove trypsin or other proteases before encapsulation.
- Use gentle centrifugation and pipetting to preserve endothelial and support-cell function.
- Prepare photoinitiator fresh, avoid bubbles and prevent dome disturbance during crosslinking.
- Replace 70% of the supplemented medium every 2 days and include relevant matrix, monoculture and pharmacological controls.
