Home › Cell Biology › A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions
Cell Biology JoVE (Open Access) Citable · DOI

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

DOI: 10.3791/50959-v
What you'll learn
  • ✓ Assemble and prime a 3D flow chamber device for cell extravasation studies
  • ✓ Culture endothelial cells on microporous inserts under physiological conditions
  • ✓ Quantify chemokine-directed cell migration across endothelial barriers under shear stress
  • ✓ Evaluate the multi-step extravasation cascade in vitro
Protocol

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Difficulty
advanced
Total time
~3–5 days (including endothelial cell culture preparation and assay execution)
Model organism
Human endothelial cells (HUVEC or similar); circulating cell populations (e.g., leukocytes, cancer cells)
Biosafety
BSL-1

Steps

1
Precoat microporous inserts with collagen

Apply collagen solution to microporous membrane inserts to establish a basement membrane-like substrate. Allow coating to dry and prepare inserts for endothelial cell seeding.

▶ 02:14
2
Culture endothelial cells on upper inserts

Seed and culture endothelial cells on the collagen-coated microporous inserts until a confluent monolayer forms. This creates the barrier mimicking the endothelial lining of blood vessels.

▶ 03:44
3
Assemble and prime 3D flow chamber device

Mount the cell-seeded insert into the flow chamber apparatus and establish physiological flow conditions. Prime the system with appropriate medium to eliminate air bubbles and stabilize shear stress.

▶ 04:57
4
Perform circulating cell assay and collect samples

Introduce circulating cells (labeled with fluorescent markers) into the flow chamber under shear stress and chemokine gradient conditions. Collect samples at defined timepoints to quantify cell extravasation and migration.

▶ 07:59
5
Analyze extravasation results and outcomes

Quantify migrated cells using microscopy or flow cytometry. Evaluate step-wise progression through the extravasation cascade (rolling, adhesion, transmigration) under physiological flow.

▶ 09:19
💬 Comments coming soon