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

Published: April 30, 2023

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 Take a culture containing microcarrier beads coated with Human Umbilical Vein Endothelial Cells (HUVEC) and pericytes. 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. 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…

Declarações

The authors have nothing to disclose.

Materials

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.

Tags

Play Video

Citar este artigo
Bead Sprouting Assay for Early-Stage Angiogenesis Assessment: An In Vitro Bead-Based Model to Study Endothelial Sprouting in the Presence of Pericytes. J. Vis. Exp. (Pending Publication), e21168, doi: (2023).

View Video