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 microcarrier bead-based three-dimensional sprouting assay reproduces several stages of angiogenesis in vitro, including endothelial sprouting, elongation, lumen formation, and network assembly, and has therefore become a widely used method for angiogenesis research.

In the present protocol, a three-dimensional microcarrier bead-based sprouting model using normal human umbilical vein endothelial cells is established. Based on previously reported endothelial microcarrier bead sprouting assays^5,6,7, this method preserves the basic framework of endothelial cell coating onto microcarrier beads, embedding in a three-dimensional matrix, and fibroblast overlay culture, while substituting a basement membrane matrix for the conventional fibrin gel to simplify the procedure and facilitate routine imaging. The basement membrane matrix was selected in this modified protocol for several practical and biological reasons. Experimentally, it provides a ready-to-use three-dimensional matrix that allows rapid gel formation, simple bead embedding, and convenient routine microscopic observation without the additional polymerization steps required for fibrin gel preparation. Biologically, the basement membrane matrix contains extracellular matrix components that support endothelial cell adhesion, migration, sprout extension, and lumen-like structure formation. Therefore, this modification is suitable for studies requiring direct visualization of endothelial sprouting behavior, comparison of angiogenic phenotypes, and evaluation of pharmacological or genetic interventions in a three-dimensional culture environment. However, because the basement membrane matrix differs from fibrin gel in matrix composition, mechanical properties, degradability, and bioactive cues, the present assay should be interpreted as a modified microcarrier bead-based sprouting model rather than a direct replacement of the classical fibrin-based assay. These matrix-related differences may influence sprouting kinetics, lumen stability, and network morphology, and should be considered when comparing results across different three-dimensional angiogenesis systems. This model is suitable for observing endothelial sprouting behavior in three dimensions and can be further applied to studies of pharmacological intervention, gene manipulation, and angiogenic mechanisms.

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 the equipment used are listed in the Table of Materials.

1. Cell preparation

1. Culture human umbilical vein endothelial cells (HUVECs) in endothelial cell growth medium supplemented with additional fetal bovine serum (FBS) to a final concentration of 10% at 37 °C in a humidified incubator with 5% CO₂. Cells used for the assay should be free of contamination, exhibit stable growth, and be in the logarithmic growth phase. Use cells at passage 4–6.
2. Culture human oral mucosal fibroblasts (HOMFs) in high-glucose basal medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO₂. Use cells at passage 4–6.
3. Before microcarrier bead coating, confirm that both cell types are at an appropriate confluence. Avoid overconfluent cultures or excessive enzymatic digestion, as these may adversely affect subsequent sprouting.

2. Preparation of microcarrier beads

1. Hydrate 0.5 g of dry  microcarrier beads in 50 mL of PBS for at least 3 h. After washing, resuspend the beads in 50  mL of PBS to obtain a 10 mg/mL microcarrier bead suspension, corresponding to approximately 3 × 10⁴ beads/mL.
2. Transfer the  suspension to a siliconized glass bottle, sterilize it by autoclaving at 115 °C for 15 min, and store it at 4 °C until use.
3. Before endothelial cell coating, remove the storage solution and wash the beads with sterile PBS. Equilibrate the beads in endothelial cell growth medium at 37 °C for at least 30 min.
4. Gently resuspend the equilibrated beads immediately before use to avoid sedimentation and bead aggregation.
5. Before cell coating, examine a small aliquot of the bead suspension under an inverted microscope. Proceed only when the beads are fully hydrated, evenly suspended, and free of visible aggregates or fragmented particles.

3. Coating endothelial cells onto microcarrier beads

1. Detach HUVECs using trypsin and resuspend the cells in endothelial cell growth medium.
2. Coat the microcarrier beads at a density of 1,500 cells per bead. Mix 3 × 10⁶ HUVECs with 2,000 microcarrier beads in 1.5 mL of endothelial cell growth medium.
3. Transfer the cell-bead mixture into a microcentrifuge tube and incubate for 12 h in a cell culture incubator. Throughout the full 12 h incubation period, gently invert the tube once every 20 min to maintain adequate contact between the cells and beads and to promote uniform endothelial cell attachment.
4. After 12 h, transfer the coated beads into a 25 cm² cell culture flask containing 4 mL of endothelial cell growth medium and continue incubation overnight to allow firm attachment of endothelial cells to the microcarrier bead surface.
5. Before embedding, examine representative coated beads under an inverted microscope. Proceed to matrix embedding only when more than 80% of the examined beads show a continuous and relatively uniform endothelial cell layer on the bead surface, with minimal free-floating cells, no obvious cell clumps, and no severe bead aggregation. Beads with uneven coating, large cell clusters, or extensive aggregation should not be used for embedding.
6. If the beads adhere to the bottom of the flask, gently shake the flask to release them. Avoid vigorous pipetting, which may cause bead aggregation or cell detachment.

4. Embedding the coated beads in a three-dimensional matrix

1. Transfer the endothelial cell-coated microcarrier beads into a 15 mL centrifuge tube and allow the beads to settle by gravity. Remove the excess medium and add an appropriate volume of fresh medium.
2. Mix the microcarrier bead suspension with prechilled basement membrane matrix at a 1:1 (v/v) ratio to obtain a final bead concentration of 500 beads/mL.
3. Add 100 µL of the matrix-bead suspension to the center of each well of a 24-well plate, allowing the mixture to form a single dome-shaped gel droplet without contacting the wall of the well. Gently mix the suspension immediately before dispensing and avoid introducing air bubbles.
4. Leave the 24-well plate at room temperature for 5 min to allow initial gelation and stabilization of the gel droplet. Then incubate the plate upright at 37 °C in 5% CO₂ for 15 min, followed by an additional 15 min incubation in the inverted position to ensure complete gelation and proper gel formation.
5. After the gel has fully solidified, proceed immediately to fibroblast overlay culture.

5. Fibroblast overlay culture

1. Resuspend 3 × 10⁴ human oral mucosal fibroblasts (HOMFs) in 1 mL of endothelial cell growth medium and gently add the cell suspension along the wall of each well, allowing the cells to distribute over the gel surface without directly disrupting the gel dome.
2. Continue incubation in endothelial cell growth medium at 37 °C in a humidified incubator with 5% CO₂. Human oral mucosal fibroblasts are used as supporting cells because they provide stromal-derived paracrine signals that promote endothelial sprout extension, stabilization, and vascular-like remodeling in the three-dimensional culture system.

6. Culture maintenance and microscopic observation

1. Replace the culture medium every 2 days. During medium changes, slowly aspirate the spent medium and add fresh culture medium along the wall of the well to avoid disturbing the gel.
2. Observe the cultures daily or every other day under an inverted microscope using bright-field or phase-contrast mode. For longitudinal quantitative analysis, select multiple fields of view in each well on day 7 and photograph the same predefined fields on days 9 and 11.
3. Under optimal culture conditions, obvious endothelial sprouting is typically observed on days 4–6, lumen-like structures appear on days 5–7, and interconnected microvascular-like networks gradually form after day 7.
4. Define successful sprouting as the presence of multiple endothelial extensions growing radially from the bead surface into the surrounding three-dimensional matrix. A sprout should be counted only when it clearly extends beyond the bead surface and remains connected to the bead-associated endothelial cell layer.
5. Define lumen-like structure formation as the appearance of elongated endothelial structures with a visible central clear space or tube-like morphology. Inter-sprout connections are defined as direct contacts or continuous structures formed between adjacent sprouts.
6. Cultures with extensive cell death, severe bead aggregation, disrupted gel structure, abundant bubbles, matrix collapse, or only short, irregular cell protrusions should be considered suboptimal and excluded from quantitative analysis.

7. Image acquisition and quantitative analysis

1. Select multiple fields of view in each well on day 7 and acquire images from the same predefined fields on days 9 and 11. Images should be collected using identical magnification, exposure settings, and imaging conditions at all time points.
2. Quantify the number of sprouts per microcarrier bead and the vascularized area.
   1. For vascularized area measurement, use open-source image analysis software. Open the acquired image and manually delineate the boundary of the vascularized region of interest (ROI) using the polygon or freehand selection tool. Add the selected ROI to the ROI manager.
   2. For visualization, use a region-of-interest visualization plugin to display the outlined ROI. Enable area measurement in the measurement settings and measure the selected ROI to obtain the vascularized area. Apply the same analysis settings to all images.
   3. For angiogenic rate analysis, use the vascularized area on day 7 as the baseline, and calculate the relative change in vascularized area on days 9 and 11.
3. Count the number of individual sprouts independently by three observers, and use the average value for analysis.

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