I am a master student works with biomaterial, since I want to get some in vitro studying results for my new type of nanofiber scaffold, I has tried 3 times to culture HUVECs cells. But at first of two times, I cannot see any cells from the scaffold through coverslips, the third time when I start to culture the cells, then the day before seeding, I saw some fiber-like tissue form in the cell culture dish and all cells dead.
I have checked the all medium and all solution, to sure that was new.
So my question likes, besides my skill problems, how to check contamination happens at the beginning and how to prevent from it? Can someone gives me some actual solution about it. Thank you.
Hello Xu,
The "fibrotic" tissue sounds like fungal contamination to me. Are you the only one having problems in the incubator/hood? If not, maybe is a sterility issue of the facility.
If the problem happens only to you, I suggest to detect where the problem comes from: You can try to first culture: your medium alone, your medium with your scaffolds and your scaffolds with PBS. Let them without cells and wait 48 hours at 37C to see if something grows, so you can detect the problem.
I suspect that, since the problem only happens in the seeding, your sterilisation technique of the scaffold may not be enough.
I recommend: 30-45 min in 70% ethanol, followed by three washes with sterile PBS (5 min each) and 2 hours under UV. Of course, when handling the material use autoclaved materials (forceps). Also, when fabricating your scaffolds, try to maintain them clean and, if possible, in sterile conditions.
Hope it helps
Hello Xu,
The "fibrotic" tissue sounds like fungal contamination to me. Are you the only one having problems in the incubator/hood? If not, maybe is a sterility issue of the facility.
If the problem happens only to you, I suggest to detect where the problem comes from: You can try to first culture: your medium alone, your medium with your scaffolds and your scaffolds with PBS. Let them without cells and wait 48 hours at 37C to see if something grows, so you can detect the problem.
I suspect that, since the problem only happens in the seeding, your sterilisation technique of the scaffold may not be enough.
I recommend: 30-45 min in 70% ethanol, followed by three washes with sterile PBS (5 min each) and 2 hours under UV. Of course, when handling the material use autoclaved materials (forceps). Also, when fabricating your scaffolds, try to maintain them clean and, if possible, in sterile conditions.
Hope it helps
Hi Xu,
considering that you are the only one experiencing the problem, and that it is not related to the media and media components, it has to rely in the sterilization process. As already suggested combine the ethanol 70% and the UV. After sterilization put one or 2 samples in cell culture media without cells, If after 24-48 hours nothing grows proceed with the actual experiment with cells.
Best of luck!
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