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The physiological shear stress is dependent on the type of tissue and vessel. According to literature “normal” shear stress values in a human body vary between 0 and ~60 dyn/cm². It is crucial to determine the relevant shear stress range that applies to the cell type in use. It would be optimal to compare to values found in literature and then test the impact of several shear stresses on the marker being investigated.
The ibidi Pump System is suitable for introducing suspension cells into the circulating medium. Keep in mind that the medium (and, therefore, the suspension cells) is recirculating over the cell layer. The unspecific activation of the suspension cells is very low compared to peristaltic pumps.
The suspension cells can be introduced to the system by adding them to the medium reservoirs or injecting them through an extra injection port.
In addition, the ibidi Pump System is optimal for imaging the cells during rolling and adhesion.
In rolling and adhesion assays, apply the same parameters as for a standard flow conditioning of an endothelial cell layer. There are two things to consider: Firstly, the endothelial cell layer should be flow-conditioned for several days before performing a rolling adhesion experiment. The shear stress for this period might be different from the shear stress applied during the rolling adhesion. Secondly, in adhesion assays, the speed of the perfused medium also plays a role. A variation of the shear stress might be needed. Also, it is necessary to test the optimal flow rate/shear stress.
The cells must be firmly attached to the bottom of the channel slide before starting the flow. Adherence time is dependent on the type of cell, additions made to the medium, and the cell culture surface. When using HUVEC on a collagen-coated surface, adherence time is very short. The cells attach to the surface within half an hour and can be exposed to shear stress after this time. In order to give the cells the opportunity to adapt to the new environmental conditions, a stepwise increase of the shear stress is recommended. Details for HUVEC are given in Application Note 13.
The adaptive response of cells to the mechanical shear stress takes up to several days. Depending on which marker you are investigating, it may be necessary to condition the cell layer for up to several days. Test your parameter in a long-term experiment to determine the correct time point of measurement.
The physiological shape of endothelial cells greatly varies between different types of blood vessels. Not all types of endothelial cells are elongated in their physiological environment.
Given our experience, we can offer some guidelines for HUVEC: HUVEC elongate in the first period of flow. In order to cover the whole surface, the cells increase their surface by elongating in the flow direction. After this flow period, the cell layer grows to an oriented cobblestone pattern, which no longer shows significant elongation.
Please find more details in our scientific poster.
The number of cells in a flow assay can easily be increased by serially connecting several slides to one Perfusion Set/Fluidic Unit. Application Note 25 gives a detailed protocol for the connection of several slides to one Fluidic Unit.
Additionally, the number of cells in a flow channel can be increased by seeding cells, in two separate steps, onto the bottom and ceiling of the channel.
The ibidi Pump System is designed for a continuous, unidirectional flow. However, with some slight modifications, and an additional Fluidic Unit, oscillatory flow (the fast switching of the flow direction), and even pulsatile flow (the oscillation between zero flow and desired flow) are possible.
No. The ibidi Pump needs to be controlled by the PumpControl software that is installed on a computer.
Only the Fluidic Unit with the mounted Perfusion Set and Slide is placed inside the incubator. The ibidi Pump stays outside and is connected to the Fluidic Unit with an air pressure tubing and a switching cable. This ensures that the operation of the pump does not heat up the incubator conditions.
The sterility of the ibidi Pump System is ensured once you have attached the Perfusion Set to the µ-Slide, and have closed the reservoirs with sterile filters. Afterwards, the system can be handled in a non-sterile environment.
Certain parts of the Perfusion Sets are autoclavable.
The Perfusion Sets are composed of syringes, filters, silicone tubing and PP-adapters that connect the tubing. Syringes and filters are not autoclavable, and we recommend replacing these parts with the Filter/Reservoir Sets. The silicone tubing and the adapters are autoclavable. Nevertheless, substances in the medium may adhere to the plastic surface and can be difficult to remove. When re-using the tubing, keep in mind that the number of cycles that the tubing can resist is limited. Observe the affected parts and then vary the position of the tubing slightly by pulling up and down in the valve slots.
You can find more information on sterility and cleaning in the instructions for the ibidi Pump System.
The ibidi Pump System is almost maintenance free. There are only few parts that need to be checked from time to time.
The silica beads can be regenerated by heating them up to at least 120°C for 8 hours. After drying the beads, place them in a vessel with a tight lock for storage.
The ibidi Pump and the Fluidic Unit can be wiped down with an alcohol-soaked paper towel. The inside of the pump is protected by sterile filters at the inlet and outlet. The Perfusion Sets can be cleaned with 70% alcohol as well, but adherent substances at the silicon tubing might not be fully removed. The slides are for single use only!
Using positive air pressure, the rear air inlet of the ibidi Pump must be connected to the incubator. For this option, we supply additional air pressure tubing and a drying bottle to prevent condensation inside the pump. For a detailed description see the ibidi Pump instructions.
Even though the ibidi Pump System is designed and optimized to work with ibidi µ-Slides, any flow channels can be used to study shear stress on cells. You will need to find a way to attach the flow channel to the Perfusion Set. We recommend using standard female Luer adapters on a custom-flow chamber. This helps to connect the ibidi Perfusion Sets to the flow chamber.
Also keep in mind that the pump only supplies an air pressure between -95 mbar and +95 mbar. This means that the flow resistance must not be too high. Furthermore, the PumpControl software will not be able to calculate the shear stress from the given air pressure, when you use any slides other than the ibidi µ-Slides, which are specifically created for this purpose.
There are only two reasons why you would need an upgrade of your ibidi Pump System:
The shear stress in the straight channel regions of the µ-Slide y-shaped is exactly double the shear stress found in the branched regions. This is only valid in the homogeneous regions of the channels, because there is an inhomogeneous shear stress in the 30° and 45° branching regions, and the two, middle-kink regions.
Find detailed information on shear stress/shear rates and flow rates in the µ-Slide y-shaped in Application Note 18.
Turbulent flow characteristics cannot be truly achieved in small channels when working with normal flow rates. The Reynolds number (Re), a dimensionless number, is used to characterize different flow regimes. Laminar flows occur at low Reynolds numbers whereas turbulent flows occur at high Reynolds numbers. The critical Reynolds number that indicates laminar flow in pipes and biological vessels is Re=2000. A Reynolds number of above approximately Re 4000 is most likely to represent a turbulent flow.
At a very high flow rate of 60 ml/min in a µ-Slide y-shaped, the Reynolds number is still very low at around Re 200.
Yes. With a special setup, the µ-Slide III 3in1 can be fully controlled with the ibidi Pump System. In this manner, adherent cells in the slide can be exposed to alternating gradients using laminar flow. For further information, please contact [email protected] .
The use of µ-Slide VI 0.1 is advisable when the cell number in an experiment is limited (e.g. primary mouse cells), or if immunofluorescence with a minimum of reagents should be performed.