A Microcarrier Bead-Based Three-Dimensional Angiogenesis Model Using Human Umbilical Vein Endothelial Cells

Summary

This protocol describes a microcarrier bead-based three-dimensional angiogenesis model using human umbilical vein endothelial cells. The assay allows direct visualization of endothelial sprouting and lumen-like structure formation and can be applied to studies of angiogenic mechanisms and therapeutic interventions.

Abstract

Angiogenesis is a multistep biological process involving endothelial cell activation, migration, elongation, lumen formation, branching, and anastomosis. Conventional two-dimensional culture systems are useful for the preliminary evaluation of drugs, genes, or culture conditions, but they do not adequately recapitulate the spatial organization of endothelial morphogenesis within a three-dimensional extracellular matrix. The present protocol describes a microcarrier bead-based three-dimensional angiogenesis assay using human umbilical vein endothelial cells (HUVECs). The HUVECs are first seeded onto microcarrier beads bearing chemically coupled acid-denatured porcine type I collagen and then embedded in a basement membrane matrix. Oral mucosal fibroblasts are subsequently seeded on top of the gel to provide paracrine support for endothelial sprouting and vascular-like structure formation. The culture medium is replaced every 2 days, and sprouting behavior and angiogenic phenotypes are monitored by inverted microscopy. Under appropriate culture conditions, endothelial sprouts emerge within several days, followed by the formation of lumen-like structures and inter-sprout connections. This method provides a convenient and reproducible platform for in vitro studies of endothelial morphogenesis and can be further applied to investigations of gene manipulation, pharmacological intervention, and disease modeling.

Introduction

Endothelial sprouting is a key event in angiogenesis and involves the emergence, migration, proliferation, and alignment of endothelial cells from pre-existing vascular structures, followed by lumen formation and the establishment of interconnected vascular-like networks^1,2. Although conventional two-dimensional culture systems and gel-based tube formation assays are technically straightforward, they are limited in their ability to recapitulate the spatial remodeling of endothelial cells within a three-dimensional microenvironment^3,4. The classical mic....

Protocol

Human umbilical vein endothelial cells used in this protocol were isolated and cultured from human umbilical cord tissues collected from the Department of Obstetrics, The First People’s Hospital of Yunnan Province. Written informed consent was obtained from all donors prior to sample collection. All procedures involving human-derived materials were approved by the Medical Ethics Committee of The First People’s Hospital of Yunnan Province (approval no. KHLL2023-KY198). Human oral mucosal fibroblasts (HOMFs) were kindly provided by the research group of Dr. Jiemei Zhai at the Affiliated Stomatological Hospital of Kunming Medical University. The reagents and ....

Results

Angiogenesis in the three-dimensional HUVEC model

In successfully established cultures, endothelial cells are uniformly distributed on the surface of the microcarrier beads, and no obvious bead aggregation is observed. Dynamic observation showed progressive endothelial sprouting and microvascular-like network formation on days 7, 9, and 11 (Figure 1A). Quantitative analysis further confirmed the progressive angiogenic response in this model. Using.......

Discussion

The present protocol describes a modified three-dimensional endothelial sprouting model based on microcarrier beads. Figure 3 provides a schematic overview of the experimental workflow. The protocol was adapted from the classical angiogenesis assay reported by Nakatsu and Hughes and optimized to meet the objectives of the present study^8,9. In the classical system, endothelial cells are coated onto collagen-coated microcar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tube	Beyotime	FTUB306	Clear, nuclease-free
15 mL centrifuge tube	Haier	311005	Conical-bottom, sterile, bag-packed
24-well cell culture plate	Haier	612003	Flat-bottom, tissue culture-treated, individually wrapped, for adherent cell culture
Automated cell counting slide	Countstar	12-0005-50	Disposable slide for automated cell counter, 50 slides/box
CO2 cell culture incubator	Thermo Fisher Scientific	3110	Used for cell culture at 37 °C with 5% CO2
Cytodex 3 microcarriers	Cytiva	17048503	Dry powder; collagen-coated dextran microcarrier beads
Dotted Line plugin	N/A	N/A	Region-of-interest visualization plugin used for displaying outlined vascularized areas
Dulbecco’s Modified Eagle Medium (DMEM), high glucose	EvaCell (EVA Life Sciences)	E2102	DMEM basic (1×), containing 4.5 g/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate, 500 mL
Endothelial Cell Medium (ECM)	ScienCell Research Laboratories	1001	Endothelial cell culture medium supplemented with additional FBS to a final concentration of 10% before use
Fetal bovine serum (FBS)	Gibco	10099141C	Qualified, single-use format, Australia origin, 500 mL
Human oral mucosal fibroblasts (HOMFs)	Jiemei Zhai research group, The Affiliated Stomatological Hospital of Kunming Medical University	N/A	Primary fibroblasts isolated from human oral mucosa; kindly provided by another laboratory
Human umbilical vein endothelial cells (HUVECs)	In-house	N/A	Primary endothelial cells isolated from human umbilical veins; no catalog number
ImageJ software	National Institutes of Health (NIH), USA	N/A	Open-source software; version 1.54p
Inverted microscope	Olympus, Japan	N/A	For bright-field, phase-contrast, and fluorescence imaging
Laser scanning confocal microscope	Nikon, Japan	N/A	For confocal imaging;model not specified
Matrigel basement membrane matrix	Corning	354262	High concentration (HC), phenol red-free, LDEV-free, 10 mL
Penicillin-Streptomycin (Pen-Strep)	Gibco	15140-122	Penicillin-streptomycin solution for cell culture, 100×, 100 mL
Phosphate-buffered saline (PBS)	LiJi Bio	AC08L011	1×, sterile, 500 mL
T25 cell culture flask	ABCBIO	ABC707008	T25, vented cap, tissue culture-treated, for adherent cell culture
Trypsin-EDTA (0.25%)	Gibco	25200-056	0.25% trypsin-EDTA solution, 100 mL