I have a single-cell clones from a CRISPR cell line that have become contaminated. I would like to not have to re-generate the line and instead recover what we have. They are cultured in DMEM with 10% FBS and 1% Pen/Strep. I have also added Normocin as well, but still have some contamination issues leading to poor cell health. I think I may have identified the source of the contamination, butI hate to loose the 6 months of time it took to get these cells. I also recognize that I might need to start over, but was hoping that there might be some tricks of the trade out there that we could try first.
Is the contamination intracellular?
If it isn't you could try serial washing steps with really gentle centrifugations to pellet the cells (or even leaving the cells to pellet on their own). If the contamination is bacteria they will pellet slower than your cells and therefore doing a high number of washing steps in PBS should highly reduce the amount of contamination.
For example if your cells are adherent aspirate the media and wash the cells in the plate with pbs 3-4 times before tripsinizing the cells. Then you could do 10 washing steps with 10 ml of PBS each, resuspending everytime the pellet of cells in a small volume (~500ml) vigorously (without killing your cells) so you improve the possibilities to detach any bacteria that could be in contact with your cells.
Then you could use a higher concentration of your antibiotic/antimicotic solution and change media frequently with hard washes of the plate/flask with PBS.
If the cell is really precious I would do that, but honestly depending how high is the contamination it is possible you wont be able to get ride of it. If you try this and don't discard the cells, culture them in a separate incubator and strongly clean up everything in the culture room, use more rounds of UV before and after... maintaining a contaminated culture is a risk for any other using the same culture room and even more the same incubator/hood
Hi Daniel Stanton
One method you can try is to split your cells equally into 6-well plates and apply different concentrations of antibiotic. Then identify the well that has the maximum concentration with little impact on cell morphology and death and keep these cells for at least 14 days whilst replenishing antibiotic every 2-3 days.
Hope this helps and good luck!
Dear Daniel Stanton
It is impossible to eliminate contamination entirely, but you can reduce the frequency of contamination by gaining a thorough understanding of their sources and following good aseptic techniques. I would prefer discarding the contaminated culture because such cultures would be less fit to work with for future assays, and it is mere waste of time and resources. Further, use of antibiotics in the process of eliminating contaminants may cross react with the cells and interfere with the cellular processes under investigation.
I do understand that you have spent 6 months of your time and these cells are precious, but honestly speaking you should not go ahead with these cells. Even if you succeed to get rid of contamination by various methods mentioned by David Rincón Fernández-Pacheco and Jack Hopkins , which you should try, you may not be working with healthy cells anymore. I am glad that you have identified the source of contamination. This will be helpful.
Regards.
Hi Daniel Stanton,
it is very hard to eliminate bacterial contamination from cell culture. However, since your cells are extremely precious, you can try a combination of multiple strategies. Whenever you sub-culture your cells or remove the spent media, give them gentle washing using sterile PBS multiple times. After trypsinizing your cells, when you centrifuge them, wash the cell pellet repeatedly with sterile media.
Further, you may add a combination of 3rd generation antibiotics like Amoxicillin and Ciprofloxacin at 20-50ug/ml and 10-20ug/ml. These will not kill your mammalian cells but definitely eliminate bacterial contamination since AMX is bacteriostatic and CIP is bactericidal in nature. You can passage your cells in media containing AMX and CIP (sterile) for 2-4 generations followed by passaging in ideal growth media (with PenStrep). Good luck!
Hi, Daniel. I am encountering a similar issue. I wanted to ask what worked for you? Best.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line