Dear All,
I am working with thermo fisher H9 cell line, human induced pluripotent stem cell.
I made a mutation on them with crispr cas-9 I obtain single colonies and I propagate my clones. I have 4 stocks for eah colonies, (1:3) of a fully confluent six well plate frozen for each stock.
After a while now I a thaw 6 of the clones in to 6 well plate. and seed them with 50 ug gentamicin containing mTeasr1 medium. When 2 of them got 80% confluent other 4 was at 60%. So I double fed these 2 colonies to passage them all one day later.
My confluent plates had a major cell death next day, I take the death cells and seed them in a coated 24 well plate and they didn't attached. But the rest in the plate the very small colonies were growth again.
Meanwhile, when other 4 well reached %80 confluent, they also died as the first 2 well. Now again changed medium, I added Rock inhibitor for 24h and I hope them to grow again.
My question, is that Why do they started to die after 80%. Before while I was making stocks, I was even waited them 1 day after they reached 100% confluence. They never died before.
I am coating with geltrex, the same lot with everyone, so coating cannot be a problem.
I am using same medium, it also cannot be the problem.
There is no visible contamination, mycoplasm contamination cannot be occur due to 50ug/ml gentamicin.
The Only change in my cells compared the stocking period is I started to add gentamicin to my medium. Can it be a problem?
I have solved the problem. Probably none will face with it but just in case. If you combine pen/strep+gentamicin; It will be resulted in cell death. I removed pen/strep now there is no problem.
G is not a problem. Gibco sells amphotericin/gentamicin sol-n (A/G) https://www.thermofisher.com/order/catalog/product/R01510#/R01510 and its ok with hESCs. You can grow hESCs with P/S and A/G and colonies are fine. I dont recommend buying hESCs from thermofisher, for original early passage stocks go to wiCell https://www.wicell.org/. You need to explain more how you culture hESCs. If you dissociate them with versene (EDTA sol-n, or cell-dissociation buffer based on EDTA) then colonies change and do not produce compact pancake-like nice colonies we all like, and indeed may start "falling apart" when touching each other (high density). I see potential problem with passaging and the way you culture hESCs. It might be that off-site targeting generated a point mutation but 99% chance you are consistently passaging hESCs with EDTa-based methods. I cannot see how P/S may impact hESC growth
Hey igor, thank you for your answer.
Yes Gentamicin is not a problem, P/S alone also is not a problem. My problem was due to mixing them. Because my cells were growing perfectly till I start to treat them combine antibiotics. And immediately after I start to treat them with only Gentamicin the problem is solved. As I said previously, the problem was combining P/S+Gentamicin.
Just to answer your question; I use gentle cell disassociation reagent to passage them. but since I was always using the same reagent for passaging, it was not the problem. Also my cell line is characterized by exome sequencing, it has no off-target mutation. The more, since I also have untreated control line that had same problem, also CRISPR cannot be the reason of cell death.
When I eliminate every option, and since my cells turned into normal when I changed P/S+G combination into G; I can only say that the cell death was because of high antibiotics toxicity. Before my problems, my culture was antibiotic free. So I only faced with this problem when I started to use P/S+G combination.
Thanks for your valuable answer.
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