Hi, everyone. I am new in cell culturing. Recently I was culturing murine stem cells (E14) , but I had a problem during cell passage. I could hardly get single cells after trypsin digestion. I had to pipet cells up and down numerous times to separate the cells, which was said to damage the cells a lot.
My procedure for trypsin digestion is as follows:
1. For a 60 mm dish, add 1 ml 0.25% Trypsin-EDTA (Gibco) after aspirating the medium.
2. Incubate the dish in 37 oC, 5% CO2 for 1 minute.
3. Add 2 ml culture medium. Pipet up and down several times.
After these steps I still saw a lot of cells aggregated under a microscope. Continuing pipet could help, but that needed many times.
Hi! As mentioned above, FCS-containing cell culture medium contains trypsin inhibitors. It might help to rinse the cells with PBS after aspirating the cells, before adding trypsin. For highest trypsonation efficiency, warm the trypsin to 37 degrees Celsius before adding it to the cells.
I also agree with Kersti and Elisa..I always use the following steps.. 1. First aspirate the spent medium and wash the plates with sterile PBS (appx. 5ml, pre-warmed). 2 Add 1.5-2 ml of pre-warmed trypsin. 3. Incubate at 37 degree upto 5mins or less and then tap to see the cell detachment. 4. Add FBS containing washing buffer/medium (pre-warmed)...for safety use 10times the volume of tryspsin. 5. Pipette gently several times. That's it...For single cell morphology we routinely culture mES/iPS cells on E-cadherin based matrix (E-cadherin Fc). If your research focuses on single cell morphology you can try this matrix. You can find more information in the attached paper link. Thanks.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0000015
Thank you, everyone! Actually I did rinse the cells with DPBS after removing the medium, just forgot to add to my procedure in the initial ask.
I was taught to use the medium, trpsin and other solutions incubated in a refrigerator, without pre-warm in 37 oC. I also doubted on their efficiency under a lower temperature. Can anyone give me some suggestions? Thanks a lot!
The optimum temperature of Trypsin is 37.1...so I think it's better to use that temperature for pre-warming. To avoid autolysis of trypsin it should be stored in -20 or -80 though.
I work with mammary stem cells and I use Accutase to get a single cell suspension. It is much more gentle on the cells than Trypsin. If cells are still not dissociated, I then use Trypsin 0.05%.
Do you culture your cells in 2-D or 3-D?
I also use Accutase (less aggressive than trypsin) 5 min at 37°C, followed by mechanical disaggregation, for adult human stem cells and adult mouse stem cells.
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