FAQ: ibidi Pump System and Cell Culture under Flow

• Which shear stress should be applied to endothelial cells?
• Can rolling and adhesion assays be performed in the system?
• Which shear stress would be suitable for rolling and adhesion assays?
• How long should the cells attach before starting the flow?
• How long should the shear stress be applied to the cell layer?
• Why are my cells not elongated under flow conditions?
• How can I increase the number of cells in the flow assay?
• Which flow types are possible with the ibidi Pump System?
• Is the ibidi Pump System a stand-alone system?
• Which parts of the ibidi Pump System must be placed inside the incubator?
• How do I maintain sterility within the perfusion system?
• How often can the Perfusion Sets be reused?
• Which parts of the ibidi Pump System are exchangeable?
• How can I regenerate the silica beads?
• How can the parts of the ibidi Pump System be cleaned?
• How can I ensure that my cells are supplied with incubated air?
• Can I use any slides, other than the ibidi µ-Slides, with the ibidi Pump System?
• How can I upgrade my ibidi Pump System?
• What is the difference between the shear stress in the branched regions and the single channel of the µ-Slide y-shaped?
• Is the flow pattern in µ-Slide y-shaped turbulent?
• Can the µ-Slide III ³ⁱⁿ¹ be controlled using the ibidi Pump System?
• Which specific applications use the µ-Slide VI ^0.1?

Which shear stress should be applied to endothelial cells?

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.

Can rolling and adhesion assays be performed in the system?

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.

Which shear stress would be suitable for rolling and adhesion assays?

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.

How long should the cells attach before starting the flow?

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.

How long should the shear stress be applied to the cell layer?

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.

Why are my cells not elongated under flow conditions?

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.

How can I increase the number of cells in the flow assay?

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.

Which flow types are possible with the ibidi Pump System?

The ibidi Pump System is designed for a continuous, unidirectional flow. However, with some slight modifications, and an additional Fluidic Unit, oscillating flow (the fast switching of the flow direction), and even pulsatile flow (the oscillation between zero flow and desired flow) are possible.

Is the ibidi Pump System a stand-alone system?

No. The ibidi Pump needs to be controlled by the PumpControl software that is installed on a computer.

Which parts of the ibidi Pump System must be placed inside the incubator?

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.

How do I maintain sterility within the perfusion system?

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.

How often can the Perfusion Sets be re-used?

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.

Which parts of the ibidi Pump System are exchangeable?

The ibidi Pump System is almost maintenance free. There are only few parts that need to be checked from time to time.

1. Silica gel from the drying bottle
   The Silica beads are coated with an orange indicator, which turns white when saturated with moisture. The Silica gel can be used until all of the orange color is gone. For recovery, place the Silica beads in a glass Petri dish, then place the Petri dish into a drying oven at 120 °C for at least 8 hours. The beads will turn orange again, once the Silica has been recovered. After the beads cool down to room temperature, they can be placed back into the drying bottle for use.
2. Replacement filters for Perfusion Sets
   The filters on the Perfusion Sets can become clogged, if they come in contact with the medium. If the pores of the filter are blocked, the correct flow rate cannot be obtained. If this happens during an experiment, immediately replace the filter with a new one. Replacement parts are available from ibidi (Filter/Reservoir Sets, #10971, #10972, #10974).
3. Alternative filter replacement
   It is also possible to replace only the filters by removing the black tubing ring and the sealing gasket from the old filter and slipping both over the new filter. The filters are Teflon air filters with a pore size of 0.2 µm (Sartorius, Minisart{textregistered}SRP15 17558-------K).
4. Pinch Valves of the Fluidic Unit
   The pinch valves have a defined life time that is dependent on the number of switching events experienced. If the pinch valves are soiled or do not function properly, contact ibidi or your local distributor for replacement or repair.

How can I regenerate the silica beads?

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.

How can the parts of the ibidi Pump System be cleaned?

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!

How can I ensure that my cells are supplied with incubated air?

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.

Can I use any slides, other than the ibidi µ-Slides, with the ibidi Pump System?

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.

How can I upgrade my ibidi Pump System?

There are only two reasons why you would need an upgrade of your ibidi Pump System:

1. You have an ibidi Pump without the latest firmware (FW 1.10 or higher). In this case, contact ibidi or your local distributor for an upgrade.
2. You have a standard Fluidic Unit setup for the 10 ml reservoirs, but you want to work with a greater or smaller amount of medium.
   ibidi provides three different reservoir sizes: 2 ml, 10 ml and 50 ml. For each of them, a different Reservoir Holder is required. Please contact ibidi to get this supplementary equipment.

What is the difference between the shear stress in the branched regions and the single channel of the µ-Slide y-shaped?

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.

Is the flow pattern in µ-Slide y-shaped turbulent?

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.

Can the µ-Slide III ³ⁱⁿ¹ be controlled by using the ibidi Pump System?

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] .

Which specific applications use the µ-Slide VI ^0.1?

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.