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This chapter presents a stepwise optimization of a Matrigel®-based in vitro angiogenesis assay using human umbilical vein endothelial cells (HUVECs) to achieve reproducible and quantifiable tube formation. Critical parameters—including culture surface, matrix composition and thickness, coating temperature and incubation time, growth factor–defined serum-free medium, and cell seeding density—were systematically evaluated. Optimal conditions were identified as 3 mg/mL Matrigel (100 μL/well), polymerized at 22 °C for 90 min, with HUVECs seeded at 20,000 cells/cm2 and cultured in a defined growth factor cocktail. Standardized imaging and ImageJ-based analysis enabled robust quantification of network features. This protocol improves consistency, stability, and comparability across angiogenesis studies.

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Stepwise Optimization of a Matrigel-Based In Vitro Angiogenesis Assay for Reproducible and Quantifiable 2D-Tube Formation Using HUVECs

  • 实验方案

  • 发表日期：

    2026年

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  • Bilge Alptekin,
  • İbrahim Alptekin,
  • Ezel Erkan,
  • Ferda Topal Celikkan,
  • Alp Can

摘要

This chapter presents a stepwise optimization of a Matrigel®-based in vitro angiogenesis assay using human umbilical vein endothelial cells (HUVECs) to achieve reproducible and quantifiable tube formation. Critical parameters—including culture surface, matrix composition and thickness, coating temperature and incubation time, growth factor–defined serum-free medium, and cell seeding density—were systematically evaluated. Optimal conditions were identified as 3 mg/mL Matrigel (100 μL/well), polymerized at 22 °C for 90 min, with HUVECs seeded at 20,000 cells/cm2 and cultured in a defined growth factor cocktail. Standardized imaging and ImageJ-based analysis enabled robust quantification of network features. This protocol improves consistency, stability, and comparability across angiogenesis studies.