I think that it is standard practice to operate in a darkroom when observing fluorescence or luminescence.
Recently, microscopes that can be used outside of a darkroom have been introduced by various companies, and fluorescence observation has been made possible.
When you are doing a fluorescence imaging or luminescence imaging, and for the long-term cell culture...
How do you replace the medium in the dark?
And do you encounter any problems while doing so?
In this article, I will suggest one answer to such a problem.
table of contents
The darkroom is really "dark"
Manual media exchange is a "gamble"
Media replacement is still necessary
The option of replacing the medium with a self-made pump
Single-handed usage of a cell perfusion device
Summary
The darkroom is really "dark"
To undertake this experiment, I visited an actual darkroom.
After our culture equipment was set up, the culture environment prepared, and the sample (cell) set and observed, the lighting was turned off.
The room was so dark that I couldn't see a thing. I was only just able to make out my surroundings thanks to the brightness of my PC monitor.
Manual media exchange is a "gamble"
Under such circumstances, performing the procedure is problematic: to open the lid of the culture device, then the lid of the dish, to suck the medium out so as to not touch the cells, and then to gently pour in the new medium (protecting the cells in the process) makes such a task almost impossible!
(If I’ve lost you, I'm sorry!)
If I were to do it, alone, I would probably do so while feeling quite anxious about whether or not the culture was being fed and would have to predict the required amount of food needed, in any shortfall. In a nutshell, it would be a gamble.
And if this was being done for an important sample operation, I am sure I would be even more nervous, no doubt with trembling hands and sweat dripping into my eyes.
Media replacement is still necessary
Despite such a situation, cells with an active metabolism, including ES cells and iPS cells and organoids, which have recently become a hot topic, die as soon as they do not exchange medium.
And I understand that there is roughly a medium exchange taking place every other day.
Observing active cells and organoids in real time, every time, would be the ideal. I think there are many researchers and professionals who would agree with me.
The option of replacing the medium with a self-made pump
"Then you can pump it from the outside."
If you’ve thought that, you are a perfusion maniac (like me)!
To replace the medium with an external pump, I’d propose the following steps should be taken:
Prepare a tube to access the dish.
Connect the pump to it.
To prevent overflow, the flow rate of the pump on the supply side and the waste liquid side should be controlled.
Determine the flow rate so that the cells do not peel off.
I think it would be a system that would work.
However, starting from the beginning, a lot of time-consuming verification would be required before such an experiment could take place.
Single-handed usage of a cell perfusion device: I was able to perform the task using a cell perfusion device held in one hand!
One solution to such a problem is the cell perfusion system.
A dedicated lid and appropriate pump setting reduce the overflow during the replacement of the medium.
Tubes are specially prepared, so no further preparation is required.
The operation is performed via the touch panel, so working in a pitch-black darkroom is safe.
Click here for the video of the actual medium exchange operation ↓
Advanced media replacement work is possible
In addition to solving the problem of medium replacement in the darkroom, performing each of the following functions is also now a possibility.
Temperature change during medium replacement can be suppressed
By supplying the medium through the tube heater, it is possible to prevent the temperature drop of the sample in the replacement medium.
Automatic medium exchange by program operation
By enabling programming to take place with a touch panel, it is possible to automatically replace the medium, such as "Replace the medium 0 times after ○○ time, repeat it 5 times".
Replacement mediums can now be supplied at a slower speed
By reducing the pump flow rate, it is possible to change the medium in a much milder fashion, at a rate which could not be achieved when performing the task manually.
Other easily programmable options can be provided for continuous perfusion, chemical administration, and mixing.
For more information, please review the product information page or contact us.
Summary
To summarize:
Conducting a medium exchange in a pitch black darkroom has been very difficult, if not almost impossible, until now.
It could be done with a self-made pump, but determining the appropriate conditions was problematic.
The cell perfusion system we have developed enables advanced media exchange to take place effectively, at a level of accuracy which could not have been performed in the past or manually.
But will it be?
Thank you for reading to the end.
Footnote:
I intend to deliver further content that can be understood by those who are exchanging mediums in the dark room.
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