Hello everyone!
I am working with human keratinocytes, derived from skin punch biopsies (4 mm). Once I isolate the proliferative skin cells, I seed them in one well (in a 24 well plate). They grow nicely, you can appreciate they maintain a good morphology and proliferate at a normal speed. The problem is that, when these cells reach 70-80% confluence and I try to rise them by using Trypsin-EDTA, they don't fully detach from the well.
Normally, I use Trypsin-EDTA at a 0.25% concentration (in HBSS), at 37ºC during 5 minutes, but it does not seem enough for cells to detach. I have tried using it for a longer time, but cells suffer a lot from this process, so they don't grow very good in the subcultures. Do you have any experience, recommendation or any ideas on how can we improve this process?
We would like to reach at least a T25 or T75 in the third or fourth passage in order to be able to perform some experiments in these cells, but as I am not able to detach all of the cells, I am really struggling.
Hello Antonio Segovia Zafra
You may use the following protocol.
After removal of culture medium, wash the cells with PBS (without Ca2+ / Mg2+) to remove all traces of serum. Aspirate PBS and give a brief rinse with pre-warmed trypsin/EDTA (few secs). Then add pre-warmed trypsin/EDTA solution to completely cover the monolayer of cells and place in 37 °C incubator for ~3 minutes. Do not incubate cells with either too high a trypsin concentration or expose the cells for too long a period as this will damage cell membrane and kill the cells. Observe the cells under microscope.
The detached cells appear rounded and refractile under microscope. If less than 90% of cells are detached, incubate the plate for another 2 minutes and observe the cells under microscope for every 30 seconds. Once cells appear detached, add pre-warmed complete growth media to inactivate trypsin. Gently disperse the medium by pipetting over the cell layer surface several times to ensure the recovery of most of the cells.
There are several factors that could cause difficulty in detaching the cells from the substratum such as cell type, population density, serum concentration in the growth medium and potency of trypsin.
Hope this protocol helps!
Good Luck.
Malcolm Nobre's answer is the best answer，add pre-warmed complete growth media to inactivate trypsin，I usually resuspend the cells twice with PBS (without Ca2+ / Mg2+)，More keratinocytes adherence
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