These cells must have been very adhered time as the mesenchymal cells from peripheral blood have a slow growth for confluence and end up staying longer to follow the following passage, hence the difficulty. trypsin for 3 minutes usually loose them as associated with light taps on the bottle. Good luck.

Thank you! You recommend what concentration of trypsin? I've used 0.25% trypsin without successful... 

I recommend the 0,25% Trypsin/ EDTA during 3 to 5 minutes in 37°C.