These cells were split on Friday and coming in yesterday these clusters of dead cells were apparent throughout all the flasks that were split. I believe they are dead cells because some were attached and some were floating so yesterday I aspirated the media and washed the cells in PBS then replaced with pre-warmed DMEM & 10% FBS in the hope that I could get rid of these from the culture but unfortunately no. The medium does not contain antibiotics as the cells are used to produce rAAV.
Have also been having issues with helper-free triple transfection to produce rAAV with these cells and wondering if the reason is due to the health of the culture beforehand as I cannot find anything wrong with the seeding or transfection protocol itself.
It may be a contamination of your culture and the clumps are dead cells. Can you maintain your cells for a few passages with antibiotics in the medium to get rid of possible contamination or to ensure that there isn't any before then switching back to antibiotic-free medium?
Good luck!
They look to be contamination in your culture. I would recommend changing your media to one containing antibiotics for a few passages to eliminate the problem then go back to you antibiotic-free. Also ensure everything you are using is cleaned with 70% EtOH, gloves are worn in the hood and exposed skin is covered. You may also need to replace the filters in the hood if they are not filtering properly I would also recommend removing the tray in the disassemble and clean your hood in particular the area under the tray and ensure your UV is working properly.
Those are dead cells. I would recommend a few passages with antibiotics or thawing a new vial of cells after a good deep clean of everything. Including the hood.
Also avoid allowing the cells to grow to over-confluence which can lead to those clumps.
Thank you all for your replies and I will give everything a good deep clean and start over.
Have you resolved your issue with your cultures? If not, I have a different idea about what you are seeing. How do you split your cells ? With Trypsin or other method ?
Hi Maureen, it has reoccured this new year but the exact same pattern in my colleagues flasks. We split the cells using TrypLE first removing the existing media then adding TryplE for 5 minutes and monitor detachment/tap flask. Then add more DMEM+10%FBS and make a well mixed suspension before taking a volume of a given split ratio to a new sterile flask and then top up with media. I am curious to hear your theory too.
I was thinking this may be clumps of cells that were transferred after you trypsinizing. I have see this happen before. Do you see any clumps of cells detaching during your Trypsinizaton ?
You might try gently passing your cells through an 18-21 gauge needle a couple of times before replating them. Or you could try to do a quick (~1min) centrifugation to get the clumps to pellet and then transfer from the suspended cells. Or you could try incubating in Trypsin a bit longer (and at 37 degrees).
Best of luck !
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