Hi Farah Aqel

My recommendation for any cell line that experiences low viability with trypsin is to switch to TrypLE which is a much more gentle dissociation medium. Add 2ml of prewarmed media to 75 cm2 flask and observe cell detachment every 5 minutes to check that there is no damage. Once the majority of cells have detached, tap the flask a few times and pipette up and down gently (expel cells at 1 ml per second), then continue with the rest of your tissue culture procedure.

TrypLE™ Express Enzyme (1X) is from Thermofisher.

Hope this helps!

Switching to TrypLE (milder detach agent) and also incubate it with 37 celsius will help.

Like others, I highly recommend switching to TrypLE Express Enzyme. It is a game-changer for subculturing epithelial cells: detachment guaranteed in 5 to 10 min at 37 degrees without harsh physical tapping, a significant increase in cell viability, no need to add serum to stop the enzyme (just dilute it at 1:5 [since my media is expensive, I even dilute with DMEM (no serum) and then spin them and resuspend them in their own appropriate media].

Other possibilities

In order to ensure that serum and ca mg are removed/reduced ,and thus to ensure the activity of the Trypsin is optimal, pre wash the cells with PBS or saline without Ca & Mg prior to treatment.

Also incubated the cells with the trypsin at 37c to enhance things

The primary cell line may become more easily removed with adaption on several passages, but then it will of course become more like the Perminant cell line.

Use of others types of plastic ware or special treatments may be possible to help