I am a student in my final few weeks of a summer research project. I am working on MIN6 cells which I am currently culturing, however as of the start of the week they appear to be struggling to grow, and their are lots of dead cells floating in the media, and I cant seem to find an explanation.
I had one flask of quite confluent cells (80-85%), which I chose to split into four flasks on Thursday last week. On Monday I checked the cells again and they were only about 50% confluent, with many dead cells floating. I have split one flask into 4 in the past and growth was previously much quicker. I changed the media and checked them again later in the day but there were still some dead cells. I also noticed that where before individual cells were usually quite large and spread out across the plastic surface before, now they are much smaller.
I have not changed my technique for splitting cells at all-after trypsinisation, I collect the cells into a tube and centrifuge them at 1000rpm for 5 mins, take off the old media, resuspend in the necessary media and use that in the new flasks. I have also made a bottle of fresh media (DMEM with 25mM glucose, 10%FBS, L-Glutamine and a small amount of medium beta-mercaptoethanol), but there still seem to be plenty of dead cells. I also can't see any signs of contamination down the microscope, and no media colour changes. The cells are quite old (p43) but I still don't think this is old enough to be a problem.
Can anyone suggest anything else I might be doing wrong, and how I could potentially remedy it? Would it just be that I should leave them to grow longer and I just split them to hard? Thanks for any help you can give.
*Unfortunately I cant provide any pictures as our microscope camera is currently not working
Those cells are quite old and this may underlie the problems you are having especially if you consider the possibility that they were older than passage one when they were first thawed. Other than this you may want to consider the possibility of heavy mycoplasma infection which you can check via a PCR of the DNA derived from the media, or via DAPI stain and confocal microscopy in which DNA staining will show up in areas other than the nucleus. If this is the problem then you can treat it quickly and pretty cheaply by using antibiotics. Finally your culture method looks fairly good, but from what I know many people will culture these cells in 15% FBS. You may also want to change the media more frequently and just cope with the slow growth until you finish your summer out.
I hope this helps
Andrew
Hi James,
There's a lot of reasons why your cells may not be growing fast. I believe MIN6 cells grow very slow in general, and like to be in close proximity to other cells in culture. Splitting the cells 1:4 doesn't sound that sparse, but you could try at a bit higher density to see if that helps.
Remember passage number is all relative. Does P43 mean 43 passages since someone in your lab froze them down, or since you bought them commercially? If you have the cells available, maybe it wouldn't hurt to thaw out another batch
Best of luck,
Jesse
Perhaps it's time to thaw another vial, all the above suggestions are great. Growth slows down with passage number, low density when split up, "old" medium, infection and contaminants. Cell culture work is tricky. Sometimes it's best to check if you have cells from an earlier passage and see how those are. Just to be on the safe side, when you thaw them prepare fresh medium. Good luck
cells tend to die if they are not seeded in proper number during expansion. the problem i see here is splitting a single 85% confluent flask into four flasks.
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