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Watch our movie Long-Term Cell Culture Under Perfusion (MV 21) to get more information on applications and handling.

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Contact ibidi for a free demo of the ibidi Pump System.

 

ibidi WEBINAR

Listen to ibidi’s recorded webinar "Cell Cultivation under Flow".

PRACTICAL COURSE

Register for a 2 day Laboratory Course at ibidi Munich / Germany: Cell Cultivation under Perfusion and Live Cell Imaging.

ibidi FAQs

Find answers to frequently asked questions on cell culture under flow.

Setting Up a Flow Experiment

In order to set up an experiment correctly, you should first answer the following questions:

  1. Which kind of experiment is being planned: Adhesion assay or cell culture under flow conditions?
  2. What shear stress or shear rate will be applied?
  3. How long will the experiment last?
  4. Should it be a one-way setup or a circular setup?
  5. Are the reagents to be used expensive or inexpensive? Are they available in large volumes?
  6. Should the experiment be performed at 37 °C and with an atmosphere of 5% CO2?
  7. What other factors may be of importance in the experiment?

Suitability of Various Channel Heights for Flow or Longer Term Static Cultivation


Impedance Measurements Under Flow Stimulation

In vivo, endothelial cells develop and differentiate under shear stress conditions. When starting cell-based assays that use endothelial cells, you should consider the possible influence of this mechanical force on cell morphology and physiology.

There is growing evidence that under in vitro conditions, the mechanical perturbations have a profound effect on the characteristics of the cell layer. In long-term experiments with HUVEC, three different phenotypes can be observed: a round flat cell after seeding, elongated cells after 1-3 days, and finally a cobblestone appearance of a dense compact cell layer. The morphological changes are subsequently accompanied by physiological changes of the endothelial cell monolayer, which is measured by impedance monitoring.

Working Principle of Impedance Measurements

Cells are cultivated in channels with electrode arrays. Depending on the morphological appearance 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.

For a detailed description, please refer to the applications website "Impedance-Based Cell Assays".

Conductivity and Impedance Change of an Endothelial Cell Monolayer

Electrode with HUVEC under static conditions
Electrode with HUVEC under shear stress conditions
Static culture: After seeding, cells grow to confluence in 2 - 3 days. During this time, the conductivity is reduced, and the resistance rises to a plateau, which is then maintained under the subsequent static culturing over several days.
Flow culture: Applying shear stress reduces the conductivity and increases the impedance of the monolayer. Over a period of days the impedance of the endothelial monolayer decreases. The physiological properties of the cell monolayer are altered when compared to the static conditions.

Immunofluorescence Staining of Flow-Conditioned Endothelial Cells

Immunofluorescence stainings can easily be done in ibidi channel μ-Slides, subsequent to the performance of your specific experiment. When comparing the cultivation of HUVEC under static and flow conditions, the differences between static culture and flow conditioning are clearly visible.

Find detailed information about immunufluorescence staining in ibidi µ-Slides and µ-Dishes here.

Adherence Junctions (VE-Cadherins)

Flow-conditioned cells are elongated and show distinct stress fibers, whereas static culturing generates cells with a bigger surface and a chaotically structured actin skeleton. VE-Cadherins (adherence junction proteins of endothelial cells) are present in both conditions.

Red – F-Actin (Phalloidin-Alexa 633),
Green – VE-Cadherin (VE-Cadherin (D87F2) XP, Rabbit mAb),
Blue – Cell nuclei (DAPI)

HUVEC, flow-conditioned, 10 dyn/cm², 5 days,
µ-Slide I 0.4 Luer, ibiTreat
HUVEC, static culture, 0 dyn/cm2, 5 days,
µ-Dish 35 mm, ibiTreat

Tight Junctions (Claudin-5)

Claudin-5, a tight junction protein, can be found at the cell-cell contact zone when the cells are flow-conditioned for five days. This shows that the impact of the mechanical shear stress is crucial for the differentiation of the cell layer.

Red – F-Actin (Phalloidin-Alexa 633)
Green – VE-Cadherin (VE-Cadherin (D87F2) XP)
Blue – Cell nuclei (DAPI)

HUVEC, flow-conditioned 10 dyn/cm², 5 days,
µ-Slide I 0.4 Luer, ibiTreat
HUVEC, static culture, 0 dyn/cm², 5 days,
µ-Dish 35 mm, ibiTreat

Golgi Apparatus

The Golgi apparatus is localized along the direction of flow.

Red – Golgi apparatus (Anti-Human-Golgin-97, mAb, CDF4)
Green – F-Actin (Phalloidin-Alexa 488)
Blue – Cell nuclei (DAPI)

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

Von-Willebrand-Factor

The von-Willebrand-Factor (vWF) is a typical endothelial cell membrane marker. When the cells are exposed to flow, the vWF-multimers elongate to rods that are sticking to the cell membrane.

Green – von-Willebrand-Factor (Anti-Human-von-Willebrand-Factor IgG)
Blue – Cell nuclei (DAPI)

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