We are been having trouble lately when using dispase for more than three minutes. Our feeder cells are coming out with our colonies in the wash step as a one layered mess. When we re-plate our cells to expand, there are this ugly strings of MEF cells all over the plate. We thought it was mycoplasm but our culture is negative for it. We also tried to dilute our dispase (StemCell @ 5U/ml) and change the incubation time from 5 minutes to 3 minutes but with no luck. Could it be the gelatin we use when plating the MEFs? We have looked at the MEF plates before using to plate our iPSCs and it look fine. Any thoughts?
Thanks Ying. We order our feeder cells already treated with MMC.
Hi Marylin,
We are using dispase on a regular basis for our hiPSC without problems. It is better than EDTA, at least from my experience. The protocol: Discard hES media, wash once the cells with DMEM/F12, add 320 ul of dispase to one well of 6-well, put in the incubator for 5-7 min (check using microscope that the cells are detaching), than suck the dispase (you won'y lose cells, don't worry), wash twice with DMEM/F12, add 1 ml of hES medium, triturate the cells, and split to a new gelatin-coated with mitomycin-C treated MEFs as feeder cells.
We use dispase of 1 mg/ml from StemCell technologies, cat # 07923. [PAY ATTENTION THAT YOU ARE USING 5MG/ML, which I assume is the problem].
Good Luck,
Dorit
We haven't use dispase for splitting our human iPSCs but have very good experience with EDTA (i.e. Versene) for splitting iPSCs grown on vitronectin-coated plates and cultured in E8 medium. This is a gentle, enzyme-free and time-saving procedure that allows both the stable passaging of iPSCs adapted to feeder-free conditions and their efficient cryopreservation. If you continue having problems with dispase you may consider switching to EDTA procedure because it will also reduce your costs associated with generation or purchase of MEFs, which itself may unnecessarily introduce variability in your iPSC cultures.
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