Bead Sprouting Assay for Early-Stage Angiogenesis Assessment: An In Vitro Bead-Based Model to Study Endothelial Sprouting in the Presence of Pericytes

Abstract

Source: Azam, S. H. et al. Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay. J. Vis. Exp. (2018).

This video demonstrates a modified microcarrier beads-based assay incorporating pericytes to study endothelial sprouting during the early stages of angiogenesis. The assay provides an in vitro experimental model to understand molecular mechanisms of angiogenesis and the effect of anti-angiogenetic therapeutics.

Protocol

1. Preparing Beads for Gel Implantation

1. Take a culture containing microcarrier beads coated with Human Umbilical Vein Endothelial Cells (HUVEC) and pericytes.
2. Examine the dishes containing the microcarrier-coated beads under the microscope using the 20X objective to ensure that all beads have been sufficiently coated by endothelial cells.
3. Vigorously pipette the plated solution of cells to detach them from the 6-cm plate and place the solution into a tube. Wash the plate 2X additional times with complete media and transfer to the tube to collect all residual beads that may be adhered to the plate.
   1. Avoid introducing air bubble....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sterile Pipette tips	VWR
Pipettors	Eppendorf
Complete EGM2 Media Bullet Kit	Lonza	CC-3162	HUVEC Media
MEM	Gibco	11095114	10T1/2 Media
DMEM	Gibco	11965118	NHLF Media
Tissue culture-grade PBS	Gibco	14190-144	Magnesium and calcium free
Accutase	Life Technologies	A1110501	For lifting HUVEC
Trypsin	Life Technologies	15050065	For lifting 10T 1/2 and NHLF
HUVEC	Lonza	C2517A
10T 1/2	ATCC
NHLF	ATCC
Cytodex 3 microcarrier beads	Sigma	C3275
Tissue culture-coated 6 and 10 cm plates	Corning
Fibrinogen from bovine plasma	Sigma	F8630
Thrombin	Sigma	t9549
Aprotinin	Sigma	a3428
Falcon Round-Bottom Tubes	Corning
Tissue culture incubator and hood
24-well glass bottom plates	MatTek	P24G1.513F	Glass-bottom plates are needed only if the sprouts are going to be imaged. If not, tissue culture plastic is also acceptable.