I’d like some advice regarding sterilizing a two-vessel bioreactor, specifically the tubing. The first vessel will be a media bottle and the second will be a custom-fabricated housing for a tissue scaffold. The two vessels will be connected to each other via two tubes. Tubing will transfer media from vessel one to vessel two, and vice versa via peristaltic pump. Vessel one will have ports for aeration, sampling, and addition. Agitation will be done via a stir bar, with a magnetic stirrer under the vessel.
I will sterilize the scaffold prior to seeding it and placing it within the second vessel. I’m not sure how to best sterilize the reactor. The problem I have regards sterilization of everything else. Ideally I’ll have as much of the reactor hooked up as possible prior to sterilization, thereby minimizing the risk of contamination. I will need to access the second vessel however, in order to place the seeded scaffold within it.
One way to deal with sterilization is to hook tubing with foil-covered ends to the first vessel, which will be put in a sterilization bag. The second vessel will be disassembled and placed in a second sterilization bag. Both vessels will be autoclaved, and assembled under a hood. The problem with this method is I have serious concerns regarding the sterilization of the lumina of the tubing. No steam will reach it. A possible solution is to run the autoclave for a very long time, so as to increase the likelihood of sterilizing enclosed parts of the bioreactor.
Another method of sterilizing the reactor is to use dry heat on everything except the scaffold and the polypropylene cap of the media bottle. This presents its own problems, though. I’d need to connect quite a bit of tubing under a hood, hoping that I don’t introduce any contamination.
I’ve never heard of autoclaving being an acceptable practice for silicone tubing. Should I give it a try, though? If this is inadvisable, do you think that there’s a high likelihood of risking contamination as a result of assembling everything under a hood? There’s no avoiding having to assemble some of the bioreactor under the hood. Having to assemble a polypropylene cap, alone with a a barbed assembly, tubing, etc. seems like it might be problematic.
Can anyone offer any advice? Thanks.
I don't have much experience, but doing dry heat and then hooking tubing up in the hood seems fine to me!
You could autoclave everything except the tubing if steam reaching the lumen is the concern.
Update: At least for the tubing I'm working with, both autoclaving and EtO seem to be acceptable sterilization techniques.
https://www.thomassci.com/Laboratory-Supplies/Tubing/_/Tygon-S3-E-3603-Laboratory-Tubing
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