Dear Linda Scavo,

I regularly use 0.25% trypsin-EDTA in PBS solution to lift off the MSCs (or be it any cells). I do as follows:

1. Remove the media.

2. Wash with PBS , three times.

3, Add 0.25% trypsin-EDTA (around 1mL for 100mm dish).

4. Incubate @37C for 2-3 minutes.

5. Check cells under microscope for detachment.

6. Add around 3-4 mL of serum supplemented media (to neutralize the trypsin-EDTA)

7. Mix thoroughly with pipette (to make a single cell suspension).

8. Collect in tubes.

9. Centrifuge.

10. Disperse well in the required solution for further use.

[sometimes 0.05% trypsin-EDTA solution also works, but takes little longer time]

Might be other researchers clarify about using the cell detachment solutions tat you have mentioned.

Good luck. 

Regards, 

Arun

Thanks a lot Arun ...but the trypsine will harm the Msc ... i read we can use accutase or accumax . ..it's more gentil ..... What do you think about them ?? 

Best regards 

Hi Linda,

I feel that the damage to MSC (or any type of cell) due to trypsin occurs due to long incubation time and addition of inadequate serum supplemented media to neutralize. 

I always make sure that cells are never exposed to trypsin more than 5 minutes.

Most of my seniors, other colleagues, we have been following this, and have further passaged MSCs, characterized them and have also differentiated them successfully.

If we properly wash the cells with PBS (to clear all the previous culture media), I could effectively remove the cells within 2-3 minutes even with 0.05% trypsin.  [sometimes people miss out or wash insufficiently,  which in turn will result in longer exposure of cells to trypsin, which could definitely have some negative effects].

Might be people with more expertise could suggest something. Lets wait for other to reply..

Best regards.