ibidi Application Guide

Cell Culture
Under Flow
Cell Culture Under Flow:
An Overview  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 2Why Cell Culture Under Flow?  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 2The Impact of Flow / Shear Stress on Cells .  .  .  .  .  . 3The Different Types of Flow .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 4Applications of Flow Assays .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 6

The Flow Assay Experiment: Planning,
Setup, and Analysis .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 8Experimental Workflow .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 8Sample Preparation .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 8Flow Conditioning of Adherent Cells .  .  .  .  .  .  .  .  .  .  . 9Staining and Image Acquisition .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 9Quantitative and Statistical Analysis .  .  .  .  .  .  .  .  .  .  . 9Questions to Ask Before Starting an Experiment  .  . 10
Choosing the Optimal Setup for Your Experiment  .  . 11

Cells Under Flow:Experimental Examples  .  .  .  .  .  .  .  .  .  .  . 14
Immunofluorescence Staining of FlowConditioned Endothelial Cells .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 14

Selected Publications

Impedance Measurements Under Flow
Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

T. G. Walsh et al. Stabilization of Brain Microvascular Endothelial
Barrier Function by Shear Stress Involves VE cadherin Signaling
Leading to Modulation of pTyr Occludin Levels. Journal of
Cellular Physiology, 2011, 10.1002/jcp.22655
T. Keeley, R. Siow, R. Jacob and G. Mann. A PP2A-mediatedfeedback mechanism controls Ca2+-dependentNOsynthesis
under physiological oxygen. The FASEB Journal, 2017, 10.1096/
fj.201700211R
A. Sabine et al. FOXC2 and fluid shear stress stabilize postnatal
lymphatic vasculature. The Journal of Clinical Investigation,
2015, 10.1172/JCI80454

www.

.com

Cell Culture Under Flow:
An Overview
Why Cell Culture Under Flow?
Liquids are a crucial component of every living
species. Many cell types are surrounded by moving
fluids. Examples are:• vascular endothelial cells that form the inner
layer of blood vessels,• lymphatic endothelial cells that form the inner
layer of lymphatic vessels,• epithelial cells of the kidney and the lung.
This liquid flow causes shear stress, a mechanicalforce that influences the cell morphology andbehavior in many ways.
In many standard in vitro experiments, cells arecultured without flow. Under these static conditions,shear stress-dependent cellular changes cannot betaken into account. In contrast, in vitro cell culture
under flow simulates this mechanical stimulus andinduces a more physiological, in vivo-like behavior.
Working under flow conditions can be especially
important when using cells that occur in biofluidic
systems, such as endothelial or epithelial cells.

Endothelial cells in blood vessels (top) are under continuous
flow. Using in vitro cell culture under flow (bottom), these
physiological conditions can be simulated.

Immunofluorescence of human umbilical vein endothelial cells (HUVEC) under static conditions and under flow. Cytoskeletal F-actin was
stained with phalloidin (red). VE-Cadherins (green) mark the adherence junctions. Nuclei are stained using DAPI (blue). Left: HUVEC,
static culture, 0 dyn/cm2, 5 days, μ-Dish 35 mm, high. Right: HUVEC, flow-conditioned, 10 dyn/cm², 2 days, µ-Slide I 0.4 Luer.

2

The Impact of Flow / Shear Stress on Cells
Shear stress is the mechanical force induced by thefriction of liquid against the apical cell membrane.
In vivo, several adherent cell types are exposed
to mechanical shear stress in biofluidic systems,
such as blood and lymphatic vessels or nephrons.
This mechanical stimulus has a great impact on
the physiological behavior and adhesion propertiesof cells. Cells react to shear stress by changes
in ion channel activation, gene expression, andreorganization of the whole cell layer.
The shear stress is measured in dyne/cm² (dyn/cm²).
Physiological shear stress values range from 0.5 to120 dyn/cm² and depend on the vessel type (e.g.,artery or vein), the tissue (e.g., brain, connective
tissue, or heart), and the size of the organism (e.g.,mouse, rat, or human).

No shear stress

StaticShear stress

Perfusion

HUVEC cultured under flow conditions (20 dyn/cm²) in a µ-Slide I 0.4 Luer over 9 days. For visualization of the cytoskeleton, the cells weretransduced with the adenoviral vector rAV CMV-LifeAct-TagRFP 24 hours prior to the experiment.

VesselShear stress(dyn/cm²)

ReferenceAorta~ 1–22

C.P. Cheng, R.J. Herfkens, C.A. Taylor. Comparison of abdominal aortic
hemodynamics between men and women at rest and during lower limb exercise.
J Vasc Surg, 2003, 10.1067/mva.2002.107
read abstract

Arteries~ 10–70

C.P. Cheng, R.J. Herfkens, C.A. Taylor. Abdominal aortic hemodynamic conditionsin healthy subjects aged 50-70 at rest and during lower limb exercise: in vivo
quantification using MRI. Atherosclerosis, 2003, 168(2):323–31
read abstract

Veins

~ 1–6

A.M. Malek, S.L. Alper, S. Izumo. Hemodynamic shear stress and its role inatherosclerosis. JAMA, 1999, 282(21):2035–42
read abstract

Capillaries~ 3–95

A.G. Koutsiaris, et al. Volume flow and wall shear stress quantification in thehuman conjunctival capillaries and post-capillary venules in vivo. Biorheology,
2007, 44(5–6):375–86
read abstract3

The Different Types of Flow
Several types of flow with defined characteristics occur in different tissues and settings. Basically, flow types
can be subdivided into laminar flow and turbulent flow:
Type of flow

Physiological occurrence

Laminar flow

Common, in many healthy vessels

Unidirectionallaminar flow

In most small healthy biologicalvessels

Pulsatile laminarflow

Flow rate

Flow direction

Generation withibidi Pump/Slides
Yes

Constant

Constant

Yes

In large arterial vessels due to
Periodicallyfluctuations caused by the heartbeat changing

Constant

Yes

Oscillatory laminarflow

Accepted as a means of turbulencesimulation using flow chambers

Constant

Periodicallychanging

Yes

Turbulent flow

Rare, during pathophysiologicalprocesses

Changing

Changing

No

Laminar Flow

Laminar Flow

Laminar flow is defined as the movement of liquids without
turbulences. The fluid flows in parallel layers with no disruption
between them. Laminar flow can be subdivided into thefollowing:• Unidirectional laminar flow
• Pulsatile laminar flow
• Oscillatory laminar flow

Laminar flow profile. Arrows represent the
distribution of velocities.

Unidirectional Laminar FlowExperimentally, unidirectional laminar flow is achieved by
perfusing medium through low-walled channels, and by keeping
both the flow direction and velocity constant over time.

Flow rate

Unidirectional laminar flow is encountered in most small healthy
biological vessels, such as small arteries and veins. In vivo,
certain cells, such as endothelial cells and kidney epithelial cells,
are constantly exposed to flow.

Unidirectional Laminar Flow
1.0
0.5
0.0
-0.5
-1.0

Time

ibidi Solutions:• The ibidi Pump System is ideally suited for creating a unidirectional laminar flow.
• The µ-Slide I Luer Family and the µ-Slide VI 0.4 Channel Slides are recommended for homogeneous
unidirectional laminar flow experiments. Using these slides, homogeneous laminar shear stress covers
the whole channel area, except for a thin band on either side of the channel walls and near the reservoirs.
The width of these bands is proportional to the channel’s height.

4

Non-Uniform Laminar Flow
Unidirectional laminar flow can occur in a non-uniform pattern. Here, the flow direction is constant whereas theflow rate spatially varies across the cell layer. In vivo, a non-uniform laminar flow occurs at vessel branching sites.
Experimentally, non-uniform laminar shear stress can be achieved by a special channel geometry, which
generates flow rate variations at specific sites within a slide.

ibidi Solutions:
The µ-Slide y-shaped has been designed for studies of non-uniform flow using the ibidi Pump System.
The shear stress level depends on the slide region. For details, please refer to Application Note 18 (PDF).

Pulsatile Laminar Flow
Flow rate

Pulsatile laminar flow is encountered in large arterial vessels
due to the fluctuations caused by the heartbeat. Experimentally,
this type of flow can be mimicked by employing a flow with aperiodically changing flow rate and constant flow direction.

Pulsatile Laminar Flow
1.0
0.5
0.0
-0.5
-1.0

Time

Oscillatory Laminar Flow

T. Hosoya, et al. Differential Responses of the Nrf2-Keap1 System to Laminarand Oscillatory Shear Stresses in Endothelial Cells. J Biol Chem, 2005,
10.1074/jbc.M502551200
read abstract

Flow rate

Oscillatory laminar flow is used as a means of simulating
turbulences. The flow direction is changed at regular intervals.
Besides during valve switching, the flow rate is constant.

Oscillatory Laminar Flow
1.0
0.5
0.0
-0.5
-1.0

Time

ibidi Solutions:• The ibidi Pump System is ideal for creating pulsatile or oscillatory laminar flow. Find details in theInstruction Manual for Pulsatile Flow (PDF) and Instruction Manual for the ibidi Pump System (PDF).
• The µ-Slide I Luer family and the µ-Slide VI 0.4 can be used for pulsatile or oscillatory laminar flow assays.

Turbulent Flow

Turbulent Flow

Turbulent flow is characterized by unpredictable changes in bothflow rate and direction over time. In vivo, turbulences are rare
and can only be found during pathophysiological processes.

Turbulent flow profile. Arrows represent the
distribution of velocities.

ibidi Solutions:Due to physical reasons, turbulent flow cannot be achieved in ibidi flow chambers using physiological flow
regimes. Oscillatory laminar flow, which is accepted as a means of simulating turbulences, can be createdusing the ibidi Pump System. Find the details in the Instruction Manual for the ibidi Pump System (PDF).

5

Applications of Flow Assays
Long-Term Cell Culture Under Flow
Principle
In vivo, endothelial cells develop and differentiate
under shear stress conditions. When starting cellbased assays with endothelial cells, you should
consider the possible influence of this mechanicalforce on cell morphology and physiology. To bringthe cells to a more physiological, in vivo-like state,
they are cultured under flow for hours up to several
weeks, generating more relevant results. In otherwords, flow conditioning is crucial for any kind ofinvestigations using cells that are physiologicallyunderlying flow conditions.

Flow characteristics

Continuous laminar flow,
Non-uniform flow,
Oscillatory flow (needed for
disturbed flow simulation),
Pulsatile flow

Experiment duration

Hours, up to several weeks

Application Examples• Investigating the influence of shear stress on endothelial cell physiology with various experimental
readouts, such as immunofluorescence, western
blot, qPCR, and FACS
• Preparing the cell layer for subsequent functional
assays, such as rolling and adhesion or transmigration assays

Recommended pumps ibidi Pump System
Recommended
μ-Slidesμ-Slide I Luer Family
μ-Slide VI 0.4
μ-Slide y-shaped

Flow characteristics

Continuous laminar flow

Experiment duration

Minutes to hours

Rolling and Adhesion Assays
Principle
In a rolling and adhesion assay, leukocytes and/or
platelets are perfused over a surface with a protein
coating or a cell layer. Their adhesion to and theirinteraction with the surface can be analyzed undervarious conditions (e.g., after gene knockdown ordrug treatment).

Application Examples• Investigating the adhesion of platelets and leukocytes on endothelial cells or matrix protein layers
Schulz C, et al. (2009) Novel Methods for Assessment ofPlatelet and Leukocyte Function Under Flow - Application of
Epifluorescence and Two-Photon Microscopy in a Small Volume
Flow Chamber Model. Open Biol J 2(1):130–136.
read abstract

6

Recommended pumps ibidi Pump System
Syringe pump
Peristaltic pump
Recommended
μ-Slidesμ-Slide I Luer Family
μ-Slide VI 0.4
μ-Slide y-shaped

Defined Liquid Exchange
Principle
A channel slide and a pump are used to supply
the cells with exactly defined amounts of culture
medium and/or any supplement of interest.

Application Examples• Defined exchange of the cell culture medium• Live Ca2+ -imaging• Live cell imaging of drug-stimulated adherent cells• Live cell imaging of cellular stainings

Flow characteristics

Short periods of laminar
flow

Experiment duration

Minutes to hours

Recommended pumps Manual liquid delivery
Syringe pump
Peristaltic pump
Recommended
μ-Slides3D Cell Culture

μ-Slide VI 0.4
μ-Slide I Luer Family

Cells in 3D culture

Principle
Cells are cultured in a 3D gel matrix. An interstitial
flow is applied directly to the gel to supply the cellswith nutrients, O2, and CO2 to maintain optimal
conditions.

Nutrients
O2, CO2

Application Examples• 3D culture of single cells, spheroids or organoids
in a gel matrix (e.g., with hepatocytes, fibroblasts,
muscle cells, kidney cells, and stem cells)

Gel matrix

Flow characteristics

Very low interstitial flow

Experiment duration

Hours, up to several weeks

Recommended pumps ibidi Pump System
Recommended
μ-Slidesμ-Slide VI 0.4
μ-Slide I Luer Family

ECIS Flow Assays
ECIS (Electric Cell-substrate Impedance Sensing)is a platform to measure morphological andphysiological changes in an adherent cell layer bymeans of impedance. Living cells can be measured
directly in the vessel without any disturbance, andwithout requiring any staining. The ECIS Flow Arrayenables the researcher to combine any type of flow
assay with parallel impedance measurement.
Find more details about ECIS flow assays here.

7

The Flow Assay Experiment:
Planning, Setup, and Analysis
Experimental Workflow
Amplification Plot

Fluorescence

3
2.4
1.8
1.2
0.6
0
0

Cell Seeding Into
Channel Slides

Flow Conditioningof Adherent Cells

Staining and Image
Acquisition

5

10

15

20

25

Cycle

30

35

40

Quantitative andStatistical Analysis

In a flow experiment, in vivo-like conditions are mimicked for cells that are physiologically exposed to shear
stress (e.g., endothelial cells and epithelial cells).
The ibidi Pump System and the ibidi Channel Slides offer diverse possibilities for conducting flow assays andallow for the adaptation of many experimental parameters to various cellular and environmental requirements.

Sample Preparation

Procedure:
The cell suspension is filled into the channel slide. After cellattachment, the flow experiment can be started. ibidi provides
channel slides with different channel heights and geometries,
which are suitable for various experimental conditions. For
details, please refer to the chapter “The Optimal Perfusion Set
and Slide for Your Application”.

ibidi Products for This Step8µ-Slide I Luerµ-Slide VI 0.1µ-Slide VI 0.4µ-Slide y-shaped

sticky-Slide I Luer

sticky-Slide VI 0.4

Flow Conditioning of Adherent Cells
Procedure
After connecting the channel slide to the ibidi Pump System,
the flow can be applied. Under flow, the cell culture medium iscontinuously pumped through the channel slide. This results inshear stress that influences the cell behavior and physiology.
The flow type, flow rate, and the assay duration depend on theexperimental setup. Find more details about how to plan the
optimal experimental setup in the chapter “Questions to Ask
Before Starting an Experiment”.

ibidi Products for This Step

ibidi Pump System

ibidi Heating System,
Universal Fit

ibidi Gas
Incubation System

Staining and Image Acquisition
Procedure
Using live cell microscopy, images or videos of the flow-conditioned
cell layer can be directly acquired within the channel slide. Further,immunofluorescence stainings and subsequent imaging can be
performed directly in the channel slide.

ibidi Products for This Step

ibidi Mounting
MediumQuantitative and Statistical Analysis
Amplification Plot

Procedure

Fluorescence

3
2.4
1.8
1.2
0.6
0
0

5

10

15

20

25

Cycle

30

35

40

After the flow experiment, the cells can be easily analyzed
using, e.g., Western blot, qPCR, FACS, and more downstream
methods.

9

Questions to Ask Before Starting an Experiment
These parameters have to be defined in order to set up your experiment correctly:Φ• Pump type

• Tubing / reservoir size

• Slide typeincluding channel geometry
and surface/coating

ml
min

s

=

2 ml · 60 mint[s]• Flow rate

(see shear stress tables
in Application Note 11(PDF), or use the software
calculation)

The following questions help you to define these important parameters:
Which cell type are you planning toinvestigate?

How long will the experiment last (hours,
days, weeks)?

Knowing your cell type of interest is essential for all
subsequent assay planning.

The duration of an experiment is important forchoosing the perfusion system that fits your needs
best. Please have a look at the different pumpsystems and their applications here.

What is the typical shear stress level of yourcell type?
The physiological shear stress level of your cell
type defines your choice of the pump, tubing, andchannel slides.

Which kind of shear stress do you want toinvestigate?
Knowing the type of shear stress that will be applied
to your cells is important for choosing the optimal
perfusion system for your assay. Please have a lookat the different types of shear stress and suitablepump systems here.

Which cell culture medium are you planning touse? Which viscosity does this medium have?
The viscosity of the medium is important for shear
stress calculation. Please find an exact descriptionof shear stress calculation in the Application Note
11: “Shear Stress and Shear Rates for ibidiµ-Slides” (PDF).

What is the available amount of medium, medium supplements, and number of cells?
The amount of medium/supplements and thenumber of cells that are available for the flow assay,
influence your choice of the reservoir and tubing
size. For more details, please have a look at the
Perfusion Set Selection Suide.

10

Should your assay be a one-way setup withdefined medium conditions, or a circular
setup, where the medium is slowly conditioned by the cells?
The type of setup determines the choice of asuitable pump. Please have a look at the different
pump systems and their applications here.

Which coating is required for your cells toadhere optimally?
The coating of the channel slide should enableoptimal adherence and growth of your cell type.

Are the reagents to be used expensive or inexpensive? Are they available in large volumes?
The availability of reagents and their costs are an
important factor for choosing the size of the tubing
and reservoir. For more details, please have a look at
the Perfusion Set Selection Guide.

What are the endpoints of the experiment (e.g.,qPCR, western blot, FACS, immunofluorescencestaining)?
The method of endpoint analysis is importantfor calculating the needed cell number, which
influences the choice of the used cell culture vessel.

Choosing the Optimal Setup for Your Experiment
The Optimal Pump System for Your Experiment
Several pump systems are available on the market. Their different properties make them suitable for specificexperimental requirements.
A syringe pump consists of a syringe, which is mounted onto a device that moves the plunger in a definedvelocity. The outlet of the syringe can be connected to a tubing and a slide to create a flow in the slide. This easyto-use device is mainly suitable for short-term experiments, where a relatively low medium volume and a slow tomoderate flow rate are needed. More complex devices can also generate a circulating medium flow over the cells.
A peristaltic pump consists of a rotor with several contact points to the inserted tubing. The coils squeeze the tubing
and thus move the medium forward. The peristaltic pump is suitable for long-term flow in parallel flow chambers.
The ibidi Pump System generates a flow by applying an air pressure onto the medium-filled reservoirs. By
recirculation of the medium using a special switching pattern , a constant unidirectional flow is generated. This
makes it an ideal setup for applying defined shear stress in long-term cell culture. Using the ibidi Pump System,
it is possible to simulate continuous and pulsatile laminar flow, as well as oscillatory flow.
To help you find the pump system that fits your needs, please see the properties of the different pump systems,
and whether they are applicable for your flow type of choice, in the tables below:

Possible Flow Types
Syringe pump

Peristaltic pumpibidi Pump System

Circulating flow

Yes (only with push-andpull pumps)

Yes

Yes

Long-term unidirectional
flow

Yes
(limited by volume)

Yes

Yes

Short-term flow

Yes

Yes
(with limitations)Yes(with limitations)Pulsatile flow for
simulating heartbeats

No

No

Yes

Oscillatory flow for
simulating turbulences

Yes

No

Yes

Syringe pump

Peristaltic pumpibidi Pump System

Almost none
(only initial pulse)

Yes (undefined pulsation
via drive shaft, dependingon rotor type)

Almost none
(only during valve
activation)

Mechanical stress during Almost none
pumping on non-attached (sedimentation can be acells held in a reservoir
problem)StrongVery low

Combinable withmicroscopy

Yes

Difficult due to pulsation

Yes

Parallel experiments
possible

Yes

Yes

Yes

Setup within an
incubator

Difficult

DifficultYes

Long-term experiments
possible with low
medium volume

No

Yes

Yes

Programmability

Yes

Yes

Yes

Pump System Characteristics

Pulsation

If you have further questions, please contact ibidi or your local distributor for a personal consultation.

11

The Optimal Shear Stress for Your Experiment
Depending on the vessel type and tissue, the shear stress in a human body varies between almost 0 and over
100 dyn/cm². Information about the physiological shear stress that applies for your cell type of interest can befound in the chapter “The Impact of Flow / Shear Stress on Cells” and in the literature.
It is crucial to perform preliminary experiments to determine at which shear stress level and after which time of
conditioning the investigated protein or pathway reacts on the shear stress stimulus. Since the restructuring of thecell layer takes at least several days, it might be necessary to prolong the conditioning to such a long time frame.
Find detailed information about the shear stress-regulated factors and the corresponding response times in this review:
P.F. Davies. Flow-Mediated Endothelial Mechanotransduction. Physiological Reviews, 1995, 10.1152/
physrev.1995.75.3.519
read abstract

The Optimal Perfusion Set and Slide for Your Application
ibidi provides several Channel Slides with different volumes and geometries. These can be combined with thedifferent ibidi Perfusion Sets, which are offered with varying inner diameters and tube lengths.
With each combination of a Perfusion Set and Channel Slide, a specific shear stress range can be achieved
using the ibidi Pump System. Depending on the shear stress your setup requires, choose a suitable combinationaccording to the Perfusion Set and µ-Slide Selection Guide.

General rules:• Higher shear stresses are created in Channel
Slides with lower channels.
• Lower shear stresses are created with smaller
inner diameters in the tubing.
Explore the detailed Perfusion Set and µ-Slide
Selection Guide.

Perfusion Set and µ-Slide Selection
Red
Yellow/GreenBlue(ID 1.6 mm) (ID 1.6 mm) (ID 0.8 mm)
Low shear stressHigh shear stressLong tubes for
microscopy (50 cm)

White(ID 0.8 mm)

Black(ID 0.5 mm)










Yellow(ID 0.5 mm)



Working volume (ml)

12

12

12

12

2.5

2.5

Standard reservoir* (ml)

10

10

10

10

2

2

* By changing the reservoir, different working volumes can be achieved. Reservoirs are available with a syringe volume of 2, 12, and 50 ml.

12

Suitability of Channel Slides for Different Shear Stress Ranges
The suitability of Channel Slides for different shear stress ranges depends on the channel height.
Please note: due to the very low channel height of 0.2–0.8 mm, the suitability of the Channel Slides for static
cell culture is limited. Demanding, fast proliferating cells might be starving already after several hours if no fresh
medium is added!

0.2 mmChannel height

0.4 mm

0.6 mm

0.8 mmµ-Slide I Luerµ-Slide III 3in1µ-Slide y-shapedµ-Slide VI 0.4
Static cultivation for long timeHigh shear stress

Low shear stress

Application Notes
AN 03: Growing Cells inµ-Channels (PDF)
Growing cells inside a µ-Slide VI0.4,
and a comparison between
channels and open wells

AN 13: HUVECs Under
Perfusion (PDF)
Setting up a flow experimentusing μ-Slide I and HUVECs

AN 11: Shear Stress andShear Rates (PDF)
Detailed information on shear
stress/shear rates and flow
rates in our channel slides

AN 25: Serial Connection of
Flow Chambers (PDF)
Setup protocol for connecting
several Luer-Slides to one
Fluidic Unit

AN 18: Shear Stress andShear Rates in µ-Slideµ-Shaped (PDF)
Detailed information on shear
stress / shear rates and flow
rates in our µ-Slide y-shaped

AN 31: Serial Connection ofµ-Slide VI 0.4 (PDF)
Protocol for connecting the six
channels of µ-Slide VI 0.4 to
one Fluidic Unit

AN 48: Shear Stress andShear Rates in ECIS Flow
Arrays (PDF)
Detailed information on shear
stress, shear rate, and flow rate
calculations in ECIS Flow Arrays

13

Cells Under Flow:Experimental Examples
Immunofluorescence Staining of Flow-Conditioned Endothelial Cells
After the flow experiment, immunofluorescence stainings can easily be done in the ibidi Channel Slides.
When comparing the cultivation of HUVEC under static and flow conditions, the differences in the cellularorganization and various cellular compartments (e.g., tight junctions) are clearly visible.
Find detailed information about immunofluorescence staining in ibidi µ-Slides and µ-Dishes here.

Adherence Junctions (VE-Cadherins)
Under static conditions, HUVEC are generally large with a chaotically structured actin skeleton. In contrast,
flow-conditioned cells are elongated and show distinct stress fibers. F-actin was stained with phalloidin (red).
VE-cadherins (green), which mark the adherence junctions, are present in both conditions. Nuclei are stainedusing DAPI (blue).

Static culture

Flow culture

HUVEC, static culture, 0 dyn/cm2, 5 days, µ-Dish 35 mm, ibiTreat.

HUVEC, flow-conditioned, 10 dyn/cm², 5 days, µ-Slide I 0.4 Luer, ibiTreat.

Tight Junctions (Claudin-5)
Claudin-5, a tight junction protein, can be found at the cell-cell contact zone of flow-conditioned cells after 5days (green). This shows that the impact of the mechanical shear stress is crucial for the differentiation of thecell layer. F-actin was stained with phalloidin (red). Nuclei are stained using DAPI (blue).

14

Static culture

Flow culture

HUVEC, static culture, 0 dyn/cm², 5 days, µ-Dish 35 mm, ibiTreat.

HUVEC, flow-conditioned 10 dyn/cm², 5 days, µ-Slide I 0.4 Luer, ibiTreat.

Golgi Apparatus

Von-Willebrand-Factor

The Golgi apparatus, stained with Anti-HumanGolgin-97 (red), is localized along the direction of
flow. F-actin was stained with phalloidin (green).
Nuclei are stained using DAPI (blue).

The von-Willebrand-Factor (vWF, stained withanti human von-Willebrand-Factor) is a typicalendothelial cell membrane marker. When the cells
are exposed to flow, the vWF-multimers elongate torods that are sticking to the cell membrane (green).
Nuclei are stained using DAPI (blue).

HUVEC, flow-conditioned, 10 dyn/cm², 4 days, µ-Slide I 0.4 Luer, ibiTreat.

HUVEC, flow-conditioned, 10 dyn/cm², 5 days, µ-Slide VI 0.4, ibiTreat.

Impedance Measurements Under Flow Stimulation
Mechanical perturbations have a profound effect on the characteristics of cell layers in vitro. In long-term experimentswith HUVEC, three different phenotypes can be observed: a round flat cell after seeding, elongated cells after 1–2 days,
and finally an oriented cobblestone appearance of a dense, compact cell layer. The morphological changes are accompanied by physiological changes of the endothelial cell monolayer, which can be measured by impedance monitoring.

Working Principle of Impedance
Measurements
Cells are cultivated in channels with electrodearrays. Depending on the state of development of
the endothelial cell monolayer, the gaps between
the cells (influenced by the cell-cell contacts) change
in size. These shifting gaps can be characterized
by their change in conductivity when applying AC
currents of different frequencies. The continuous
red lines in the scheme represent the ion currents
between the cells when applying the AC current.

Electrode

Cell layer

Capacitance (C)Resistance (R)For a detailed description, please refer to the applications website “Impedance-Based Cell Assays”.

Static culture

Flow culture

After seeding, cells grow to confluence in 2–3 days. During thistime, the conductivity is reduced, and the resistance rises to aplateau, which is then maintained under the subsequent static
culturing over several days.

Applying shear stress reduces the conductivity and increases the
impedance of the monolayer. Over a period of days the impedanceof the endothelial monolayer decreases. The physiological propertiesof the cell monolayer are altered when compared to the static conditions.

15

16

Manufacturer

North American Headquarters

ibidi GmbH
Lochhamer Schlag 1182166 Gräfelfing
Germany

ibidi USA, Inc.
2920 Marketplace Drive
Fitchburg, WI 53719
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