Introduction

Here we describe potential solutions to problems that may be experienced during a cell culture experiment with vitro.alive (2D-chamber). It is recommended to read through the Troubleshooting-section before setting up the first experiment.

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Troubleshooting

Problem	Potential Solution
The slide still looks dirty after disinfecting it.'	If the slide came with a surface residue, wash it with hot soap water before disinfecting it.
The lab does not have a suitable medium container for the experiment.	It is possible to use a standard glass flask. Use the provided cap. See separate instructions for how to set up the medium container.
There is smoke or smell from the device.	Unplug immediately. Remember to never heat the medium without ongoing flow. Contact mailto:[email protected].
The lab does not have a way to gas the medium.	Place the medium container in an incubator or use a pH buffer. Use a filter to cover the air opening. See separate instructions for how to set up the medium container.
There are bubbles in the chamber after 24 hours.	Bubbles can occur if there is liquid from the cleaning procedure on top of the bubble trap. Also check that the medium container is located at least 20 cm above vitro.alive. The pump must push the liquid. Make sure that the silicone tubes and chamber (including the O-ring) are cleaned well before use.
The cells detached from the glass slide during the perfusion.	Re-consider the levels of shear stress, increase the incubation time and/or try coating the glass slide. It is also possible to run the experiment with a plastic slide.
The locking pin is stuck after the experiment.	Medium residues can solidify and cause the locking pin to get stuck. Use the “pin pusher” to push out the locking pin. It can help to press on the chamber. If it is still stuck, try dissolving the medium by wetting the locking pin with double distilled water.
The locking pin is stuck in the chamber after storage.	Use the “pin pusher” to push out the locking pin. If it is still stuck, wet the chamber lock with water or soap water to dissolve any medium residues, press on the chamber and push out the locking pin. After running the experiment, clean the chamber lock before storage. The chamber should be stored opened and disconnected from vitro.alive.
Bubbles when closing the chamber after adding the sample slide.	Consider if there is enough medium added to the glass slide before closing the chamber. Open the chamber. Remove the bubbles on the medium surface with a pipette. Close the chamber slowly. If there are any small bubbles left in the chamber, seal the connectors with disinfected fingertips (use gloves!) and gently tilt and tap on the chamber. Lead the bubbles away from the cells to a corner, and then to the outlet.
The chamber is stuck.	Wiggle the chamber back and forth and alternate which side that is being pulled out. If it is still stuck, gently push a spatula in between the chamber and the device. It is enough to create a small gap for the connectors to release the chamber.
There was contamination during the perfusion.	Never skip the pre- and post-experiment cleaning protocol. If the cleaning protocol has been following, contamination most likely comes from the stage where the slide was placed into the chamber or an unsterile medium container. Make sure to always handle the sample slide with a sterile tool. The medium container and silicone tubes must be autoclaved before the experiment. All openings to air in the medium container must be completely sealed or covered with a filter.
It is difficult to connect the silicone tubes to the connectors.	Make sure that the silicone tubes have the correct diameter.
The pump is not pulling liquid.	Check that the pump is pulling liquid in the right direction. Check that the silicone tubes are compatible with the pump. Also, check that the tubes are not squeezed anywhere.
Water is dripping out of the device after storage.	Make sure to dry the device for 3 hours with the supplied air-pump before storing it in a dry place. This is important to increase the longevity of vitro.alive.
vitro.alive does not turn on.	Check that the power supply is plugged in correctly. If it Is still not turning on, contact Vitroscope at mailto:[email protected].
The display is unresponsive or acting strangely.	Try rebooting the device. The device might also need a software update. Contact Vitroscope at mailto:[email protected].
The Internet does not connect.	Try rebooting the device. Contact Vitroscope at mailto:[email protected].

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Additional information

https://www.vitroscope.no/

https://www.frontiersin.org/articles/10.3389/fbioe.2019.00091/full