Cell Culture Under Flow: Applications and Experimental Examples

This page summarizes common cell culture under flow applications and wall shear stress assay applications for cell models exposed to defined fluid flow under controlled in vitro conditions. It is intended for researchers planning endothelial, epithelial, vascular, immune cell interaction, barrier, mechanotransduction, and 3D disease model studies under flow.

The focus is on selecting the right application for your biological question. Depending on the model, cells can be cultured as 2D monolayers under flow, in matrix-based 3D models, or in membrane-based co-cultures using optically suitable porous membranes. Each application chapter connects the biological context with experimental examples and suitable ibidi solutions.

Staining of spheroids (blue, red) and HUVECs (green) after 3 days in co-culture under perfusion. Cell nuclei: DAPI (blue); actin filaments: Phalloidin-iFluor 647 (red); CD31: Alexa Fluor 488 labeled anti-CD31 antibody (green).

Find the Right Flow Application for Your Research

Cell culture under flow can be used for a wide range of applications, such as:

• Endothelial mechanobiology, shear stress response, and vascular flow conditioning
• Lymphatic endothelial flow biology
• Atherosclerosis, disturbed flow, and vascular dysfuncion
• Thrombosis and platelet adhesion under flow
• Immune cell recruitment, interaction, and transendothelial migration
• Barrier models, permeability, and blood–brain barrier research
• Kidney tubule epithelial models and nephrotoxicity
• Airway epithelial flow and mucociliary biology
• Gut epithelial barrier and host–microbiome interaction
• Tumor microenvironment, extravasation, and drug delivery
• Viral and bacterial infection under flow

Many additional cell culture under flow applications are possible depending on the cell type, flow profile, culture format, and readout. If your specific research question is not listed here, our application specialists can help you choose the most suitable ibidi flow setup. Please contact us for individual support.

The table below highlights selected high-priority application areas that can be addressed with ibidi flow products and are described in more detail in the following chapters.

Endothelial Mechanobiology and Vascular Flow Conditioning

Endothelial cells are highly sensitive to wall shear stress. Defined laminar flow can be used to condition endothelial monolayers and to analyze morphology, alignment, cytoskeletal organization, junction formation, inflammatory signaling, gene expression, and mechanotransduction.

Culturing Human Umbilical Vein Endothelial Cells (HUVECs) Under Flow

Immunofluorescence staining allows the comparison of static and flow-cultured endothelial cells and can reveal differences in cytoskeletal organization. In this example, flow-conditioned HUVECs were stained against VE-cadherin and compared with static cultures.

VE-cadherin staining highlights adherens junctions in both static and flow-conditioned HUVECs. Under static conditions, the cells are generally larger and show a less organized actin cytoskeleton. In contrast, flow-conditioned cells are oriented and display distinct F-actin stress fibers, indicating flow-dependent reorganization of the endothelial cell layer.

HUVECs were cultured for 5 days under static conditions (left, 0 dyn/cm²) or under flow at 10 dyn/cm² (right) in a µ-Slide I ^0.4 Luer ibiTreat, using the ibidi Pump System. The cells were stained for VE-cadherin (green), F-actin (red), and nuclei (blue). Imaging was performed using a Nikon Eclipse microscope at 60× magnification.

Culturing Pulmonary Endothelial Cells (HPMECs) Under Shear Stress

When cultured under flow, human pulmonary microvascular endothelial cells (HPMECs) show changes in morphology, cytoskeletal organization, and endothelial cell–cell contacts compared with static culture. This example demonstrates how phase contrast and fluorescence microscopy can be used to assess endothelial responses to defined shear stress conditions.

Fluorescence microscopy of HPMECs comparing static culture (left) and flow culture (right) after 72 h in the µ-Slide I ^0.6 Luer, coated with Collagen IV. A flow of 2.3 dyn/cm² was applied using the ibidi Pump System. Fluorescence labels: β-actin (green), VE-cadherin (red), and DAPI (blue). Scale bars 100 µm. Data by Daniel Bourquain, Robert Koch Institute, Berlin.

Atherosclerosis, Disturbed Flow, and Vascular Dysfuncion

Atherosclerosis-related mechanisms are strongly linked to local flow patterns. Non-uniform, oscillatory, or disturbed-flow-like conditions can be used to analyze endothelial activation, inflammatory signaling, monocyte adhesion, mechanosensitive membrane domains, and vascular dysfunction.

Disturbed Flow in a Custom In Vitro Arteriovenous Fistula Model

Disturbed flow plays an important role in endothelial dysfunction and vascular remodeling in arteriovenous fistulas. In this study by Xiao et al., the authors established a custom in vitro arteriovenous fistula (AVF) model that formed a 60° bifurcated flow system. Human umbilical vein endothelial cells were seeded on a transparent polycarbonate membrane (PCM), which was inserted into the model.

To better mimic AVF-like pulsatile hemodynamics, the authors connected the model to a modified ibidi Pump System. A side tube with a small-diameter needle was added upstream of the main tube, allowing residual low-flow perfusion during the low-flow phase instead of complete flow interruption. By adjusting the pump pressure, the setup generated alternating high- and low-flow phases. This allowed the authors to correlate computational fluid dynamics with endothelial morphology, focal adhesion junctions, glycocalyx organization, and flow-sensitive marker expression in defined regions of laminar, oscillatory, and disturbed flow.

Schematic representation of a custom in vitro arteriovenous fistula model for studying disturbed flow and endothelial cell function. A tailored, cell-seeded transparent polycarbonate membrane was inserted into a 60° bifurcated flow model and perfused using a modified ibidi Pump System to generate pulsatile AVF-like flow conditions. Adapted from: Xiao Z, et al. (2026), doi:10.1016/j.actbio.2026.01.044, licensed under CC BY 4.0.

Immune Cell Recruitment, Interaction, and Transendothelial Migration

Flow assays are widely used to study immune cell and leukocyte rolling, adhesion, and transmigration. In these assays, immune cells are perfused over an activated endothelial monolayer, a protein-coated surface, a membrane, or a matrix-based model. Readouts can include rolling velocity, adhesion count, transmigration events, and live cell tracking.

Rolling and Adhesion Assay Under Defined Shear Stress

Rolling and adhesion assays are commonly used to analyze leukocyte–endothelial interactions under flow. In this example, cerebrovascular endothelial cells (CVECs) were cultured as a 2D endothelial monolayer in the µ-Slide VI ^0.4 and stimulated with LPS to induce an inflammatory endothelial phenotype. Granulocytes were then perfused over the activated endothelial layer using the ibidi Pump System at 1 dyn/cm², enabling live microscopic analysis of granulocyte rolling, adhesion, and cell–cell interactions under defined shear stress.

Rolling and adhesion assay with granulocytes perfused over an LPS-stimulated cerebrovascular endothelial cell monolayer in the µ-Slide VI ^0.4. The assay enables visualization of leukocyte–endothelial interactions under defined shear stress. Image courtesy of Gediminas Cepinskas, University of Western Ontario, Canada.

Barrier Models and Permeability

Barrier models under flow are used to study endothelial or epithelial barrier integrity, permeability, compound transport, and immune cell interaction or transmigration. Depending on the model, cells can be cultured on a membrane, on a channel surface, or in combination with a 3D matrix.

Barrier Co-Culture Models Under Flow

Porous membrane co-culture assays under flow enable the cultivation of two cell layers in separate but interacting compartments. One cell monolayer can be exposed to flow while soluble factors, cell–cell communication, or barrier-related responses are analyzed across the membrane. This assay format is suitable for endothelial and epithelial barrier models, vascular co-culture systems, immune cell interaction studies, transmigration studies, and microscopy of living or fixed cell layers under defined shear stress conditions.

Transmigration of cells across a cell monolayer on the membrane into a 3D gel matrix with embedded cells using the µ-Slide ibiPore SiN.

A Dynamic Blood–Brain Barrier Model for Microscopy and Permeability Analysis

The blood–brain barrier is a highly selective vascular interface that restricts drug entry into the brain, making physiologically relevant in vitro models essential for permeability testing and CNS drug development. In this study by Choublier et al., the authors developed a custom two-compartment BBB device in which hCMEC/D3 brain endothelial cells were cultured on a semi-permeable membrane and exposed to uniform laminar shear stress using the ibidi Pump System. The setup enabled long-term endothelial culture under flow, direct microscopy-based analysis of barrier morphology and junction markers, and permeability measurements under static and dynamic conditions.

Schematic representation of a dynamic blood–brain barrier model for microscopy and permeability analysis. Brain endothelial cells were cultured on a semi-permeable membrane in a custom two-compartment device and perfused under uniform laminar shear stress using the ibidi Pump System. Adapted from: Choublier N, et al. (2021), doi:10.3390/app11125584, licensed under CC BY 4.0.

Tumor Microenvironment, Extravasation, and Drug Delivery

Perfused tumor microenvironment models can combine endothelial layers, immune cells, tumor spheroids, hydrogel matrices, and dynamic compound delivery. These models are suitable for studying extravasation, immune-tumor interaction, carrier penetration, drug accumulation, and viability under flow.

Monocyte-Mediated Drug Delivery in a 3D Tumor Model Under Flow

Immune cell trafficking offers a promising route to improve drug delivery into solid tumors, where vascular barriers and dense extracellular matrices often limit passive nanoparticle accumulation. In this study by Chang et al., the authors developed monocyte-adhesive peptidyl liposomes, referred to as monocyte-mediated drug carriers (MMDCs), designed to hitchhike on circulating monocytes. Using ibidi flow-based assays, they first analyzed the interaction between MMDCs and monocytes under defined shear stress. They then established a 3D tumor microenvironment model with collagen-embedded tumor spheroids and an endothelial barrier to investigate monocyte adhesion, transendothelial migration, and selective MMDC accumulation in cancer-containing matrices under dynamic flow conditions.

Schematic illustration of MMDC-assisted monocyte homing in a 3D tumor microenvironment model under flow. The setup enabled analysis of monocyte adhesion, transendothelial migration, and tumor-directed carrier accumulation. Graphical abstract created by ibidi, based on: Chang CY, et al. (2025), doi:10.1016/j.jconrel.2025.113672.

PD-L1-Targeted Immunoliposomes in a Cervical Cancer-on-a-Chip Model

Cancer-on-a-chip models provide a controlled approach to study how therapeutic nanocarriers behave in a 3D tumor-like environment under perfusion. In this study by Fobian et al., PD-L1-targeted immunoliposomes were investigated in a dynamic cervical cancer model combining tumor spheroids, an extracellular matrix, and defined flow. The setup allowed the authors to assess how flow influences immunoliposome transport, penetration into the 3D matrix, and delivery toward PD-L1-expressing tumor cells in a more physiologically relevant in vitro system.

Schematic representation of a dynamic cervical cancer-on-a-chip model for studying PD-L1-targeted immunoliposome delivery. Tumor spheroids were embedded in an extracellular matrix and exposed to controlled perfusion, enabling the analysis of nanocarrier transport, matrix penetration, and tumor-associated accumulation under flow. Reproduced from: Fobian SF, et al. (2025), doi:10.1016/j.jconrel.2025.01.014, licensed under CC BY 4.0.

Plan Your ibidi Flow Setup

Frequently Asked Questions About Wall Shear Stress Assay Applications