neural progenitor cell death in multi-well plate - (Oct/10/2005 )
Recently I was trying to do a proliferation study for neural progenitor cells in 48 well plates. After a day, some wells appeared to have no viable cells. I kept the plates for 5 days to whilst I tried to work out what had gone wrong.
In the “good” wells, cells attached and proliferated to confluence. In the “bad” wells, the cells seemed to have exploded on contact with the substrate. There was no obvious contamination in these wells after 5 days. The good wells and bad wells were randomly distributed in the 3 plates.
All plates had poly-ornithine + laminin coating and cells from one stock at the same seeding density (50 cells/mm2). The difference between plates was the basal media, and the incubators used.
Our cells are maintained in antibiotic-free media. Infections are rare, and when they do occur they are bacterial and clearly visible after 1-2 days. We have conditioned media screened for mycoplasma by PCR every 6 months, this has always come back negative.
I’m really stumped; does this situation ring any bells for any of you? 
I just wanted to bump my old question, since this problem is still popping up whenever cell experiments are done in multi-well plates.
What I can add to my original post is that we routinely culture without antibiotics in flasks, but use them for large experiments done in multi-well plates, since they will be exposed to so much handling.
In plates cultured by the same user, plates with antibiotics had random "dead wells" whereas those cultured without antibiotics were normal. It is really odd!
Any ideas???
are the cells that die in one incubator, and the cells that live in a different incubator?
have you tried growing the cells in the same type of flask you culture the cells in, with the antibiotics?
it seems as though the antibiotics are the problem. perhaps they're too concentrated, or they're bad and causing major toxicity, or the cells are sensitive to them....
Vetticus
have you tried growing the cells in the same type of flask you culture the cells in, with the antibiotics?
it seems as though the antibiotics are the problem. perhaps they're too concentrated, or they're bad and causing major toxicity, or the cells are sensitive to them....
Vetticus
Thanks for your reply Vetticus!
This has occurred in two different incubators. In the last case, plates with random dead wells were side by side with the live plates in one incubator. The only difference in that case was the antibiotic- but if the antibiotics were bad, or too concentrated, shouldn't the effect be similar in all the wells?
We haven't cultured with antibiotics in flasks.
Another weird thing is that this doesn't happen directly after plating. The cells can look great for up to 4 days before exploding overnight
I'm starting to wonder if the ethanol faerie is visiting certain wells...
Hi Oryx, a couple more questions.
What antibiotic(s) are you using and at what concentration?
Do you add the antibiotics to your medium or to the wells?
If you add them to the wells is the same volume of medium present in each well?
What is the volume of medium in each well?
The issue here appears to be the antibiotics, as you do not notice any contamination.
My suggestion would be that your cell type may be particularly sensitive to the antibiotics (as well as concentration) that you're using, but its not yet lethal to the cells, so that only the more susceptible ones (poor adherence maybe?) will gradually fall to the "attack" by the antibiotics over time. and when the effect is sufficient to take a toll on the cells, they collectively lyse.
Thanks karyotyper and chayedan for your replies!
The antibiotics are bought as a stock solution from invitrogen, and are diluted in media to a final conc of 100IU/ml pen and 100ug/ml strep. For 48 well plates we use 0.5ml media per well, and the media is changed every other day.
I think you're right about the sensitivity... the last experiment showed "surviving" wells with antibiotics to have about half the number of cells as in the antibiotic-free wells, but there weren't enough of them to do proper statistics.
The cells' adherence to the culture substrate normally changes with time. In growth media the cells will start to pile as they approach confluence and the piles can loosen as they get larger. I don't think that the piling affects the sensitivity as we see the effect in wells that are treated for differentiation, and not piling in the same way.
Hi again Oryx
Your antibiotics are added at standard concentrations but they would appear to be the culprit given your observations. I'd double check the calculations on the addition of the stock solution just to make sure you're not accidently out by a large factor.....unlikely, but stranger things have happened 
If you must have antibiotics you could halve the concentration and see if that helps. Alternatively seek another broad spectrum antibiotic with a different action (eg. gentamycin). I know Invitrogen recently (past couple of years) changed from pen/strep to gentamicin as a routine antibiotic in their specialised media for prenatal human cell culture. Can't recall their reason for changing though.
If contamination in your lab is relatively rare without antibiotics you could bite the bullet and try and proceed without them. Good luck!
i was reading up material on cell culture deaths without apparent contamination causes and i realised that there could be a possibility of viral contamination of your cultures.
To quote John Ryan (Corning, Incorporated):
Since cytopathic viruses usually destroy
the cultures they infect, they tend to be
self-limiting. Thus, when cultures selfdestruct
for no apparent reason and no
evidence of common biological contaminants
can be found, cryptic viruses are
often blamed. (See Figures 3a and 3b.)
They are perfect culprits, unseen and
undetectable; guilty without direct evidence.
This is unfortunate, since the real
cause of this culture destruction may be
something else, possibly mycoplasma or
a chemical contaminant, and as a result
will go undetected to become a more
serious problem.
if your lab is able to fund some assays to detect any viral contaminants, it might be worth a try if you find that your cultures are still not doing well and lysing at random.
Good luck!
karyotyper, that's an interesting point about invitrogen! I'll see what I can dig up.
As we do our routine culture without antibiotics, it would be no issue to go the same way for multi-well experiments. We just tried to protect ourself from contamination losses over large numbers of samples that have to be handled for 10 days. Unfortunately, for the remaining runs in this particular set we need to keep the conditions the same- especially since there could be a difference in the proliferation rate.
Chayedan, I think I've read that contamination guide too, when I was hunting a good reference for people to use in the lab, in case they saw something funky happening in their cultures