Application Note 14

Live Cell Imaging Setups Under Flow
Live cell imaging is one possible read-out of cell culture assays under flow conditions.
Depending on the duration and temperature requirements of the experiment, there areseveral possible setups with different advantages and disadvantages, which are shownand discussed in this Application Note.

Related Topics:
ibidi Pump System instruction manual
ibidi Heating System
ibidi Gas Incubation System
Application Guide Live Cell Imaging
Application Guide Cell Culture Under Flow

Keywords:
Live cell imaging, cell culture under flow, shear stress, perfusion assay, stage top
incubation, pump, microscopy

Table of contents
1.
2.
3.
4.
5.

Introduction .......................................................................................................... 2
Setup Without Incubation ..................................................................................... 4
Setup With Stage Top Incubation ........................................................................ 5
Setup With Stage Top Incubation and Bench Top Incubator ............................... 6
Setup With Cage plus Stage Top Incubator ......................................................... 7_____________________________________________________________________________________________________
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1. Introduction
When setting up a flow experiment on the microscope, you might encounter somebasic physical challenges to consider.
1.1. Stable Conditions:
Healthy cells need stable conditions such as temperature, pH, ion concentration, etc.
Depending on the specific cell type the requirements may vary. Stage top incubatorsand cage incubators help to ensure homogeneous, stable conditions. Perfusion ofmedia into these incubation setups has the potential to change environmental
conditions for the cell.
1.2. Air Bubbles:
Temperature gradients can create air bubbles over time. The physical principle
explaining this effect is the solubility of gas in liquids and plastic. At highertemperatures, water and plastic can absorb less gas than at lower temperatures. Everytime the medium temperature changes, gas is taken up or released by the medium.
This can also happen across the tubing wall, especially when silicone tubing is used.
If cold medium warms up along the circuit, it releases gas, which cannot escapebecause it is trapped in the tubing or channel (Figure 1).
Eventually, the tubing can become clogged by air bubbles leading to a disturbance offlow. This is especially true in adapters, where the cross section is widened. Here theflow velocity goes down and the gas bubbles can gather to form big bubbles, blocking
the whole channel cross section (Figure 2).
Favorable Parameters
Some parameters favor stability of the running experiment, and reduce the formationof gas bubbles. To evaluate for the presence of gas bubbles within the system, onemust setup and perform a test run of the entire live-cell/perfusion system. This is the
only way to verify gas bubble formation over time.
ibidi recommends checking that the following parameters are being met:
• Homogeneous temperature over the whole tubing system prevents gas
bubble formation.
• High humidity (>70%) over the whole tubing system prevents evaporation.
• Homogeneous flow velocity rather pushes gas bubbles out, then keeping andenlarging them in regions with lower flow velocity. Adapters are inevitable, andalways somehow change the flow velocity. That means in reality an optimal
homogeneous flow velocity is not attainable.
• Overpressure (positive pressure) creates a pressure drop between inside thetubing and outside, rather pushing the gas out of the tubing. On the other hand,
an underpressure (negative pressure) creates a pressure drop sucking in gas
from outside the tubing and thus favoring gas bubble formation.
• Short term experiments do not give gas bubbles the time to develop.
• Introduction of a bubble trap can help catching gas bubbles before they enter
the channel with the cells. Bubble traps have a certain capacity until they are
exhausted and save time for the experiment.
The principle of a bubble trap is shown in Figure 3. A simple bubble trap can bemade joining two syringe parts by melting the edges (Figure 4).
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Figure 1 Gas uptake and bubble formation along a temperature gradient caused by different
gas solubilities of the medium.

Figure 2 Formation of large bubbles at widening cross section of the tubing.

Figure 3 Principle of a bubble trap

Figure 4 Example of a simple DIY bubble trap.

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2. Setup Without IncubationMaterial:• ibidi Pump System placed next to the microscope• Inverted microscope

Pro:
••
•
•

Homogeneous medium temperature along the perfusion tubing.
Low probability of gas bubble development.
Very stable over longer time periods.
Very low costs and easy handling.

Con:• Cells are at room temperature.
• No CO2 supply to the cell culture medium (carbonate buffers are not functional).
Suited for:• Cells or organisms that can be cultivated at room temperature.
• Experiments without cells.
• Potentially for short term imaging, if cells tolerate the temperature drop.

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3. Setup With Stage Top Incubation
Material:• ibidi Pump System placed next to the microscope• ibidi Stage Top Incubation System (Gas Mixer and Temperature Controller)
controls temperature, CO2, and humidity in the stage top incubation chamber.
• Inverted microscope

Pro:
••

Cells are at warm temperature.
Low costs.

Con:• The temperature gradient along the perfusion tubing promotes the generationof air bubbles.
• Low CO2 input to the culture medium. The exposure time in the incubationchamber (supplied with CO2) is very short.
It helps to suck in the atmosphere from the stage top incubator to the pumpback port. Like this CO2 enriched gas is applied on top of the medium
reservoirs.
• Not stable over long time periods.
Suited for:• Short term experiments.

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4. Setup With Stage Top Incubation and Bench Top Incubator

Material:• ibidi Pump System• Bench top incubator• ibidi Stage Top Incubation System (Gas Mixer and Temperature Controller)
controls temperature, CO2, and humidity in the stage top incubation chamber.
• Inverted Microscope

Pro:
••
•

Cells can be kept at 37°C since cooling of the medium only occurs in thetubing leading from the incubator to the stage top incubation.
CO2 supply to the medium is provided to the medium reservoirs and channel
slide. For optimal CO2 supply, the pump back port must be connected to thesmall incubator’s atmosphere.
Stable over medium time periods.

Con:• The temperature gradient along the perfusion tubing favours the generation of
air bubbles during long time periods.
Suited for:• Medium time periods with cells that need to be kept warm.
• Longer time periods with an additional bubble trap.

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5. Setup With Cage Plus Stage Top Incubator
Material:• ibidi Pump System placed next to the microscope• ibidi Stage Top Incubation System (Gas Mixer and Temperature Controller)
controls temperature, CO2, and humidity in the stage top incubation chamber.
• Inverted Microscope equipped with heated cagePro:•••

Homogeneous temperature along the whole perfusion tubing and reservoirs
prevents air bubbles.
Optimal temperature at the cells.
Stable over long time periods.

Con:• Dry atmosphere inside the cage incubator could favor the generation of airbubbles.
• Low CO2 supply to the culture medium. The dwell time in the incubationchamber is very short.
It helps to suck in the atmosphere from the stage top incubator to the pumpback port. Like this, CO2 enriched gas is applied on top of the medium
reservoirs.
• High costs.
Suited for:• Long term experiments.

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