I will be working with mesenchymal stem cells de-differentiated from adult adipose tissue. They will be grown in low serum conditions around 2% FBS as not to induce differentiation. To ensure a stable cell culture of viable cells in a 75 cm2 flask at a cell density of 5,000 cells per square cm, what would be the appropriate concentration of trypsin? Also should I use a special type of trypsin, gamma irradiated vs. EDTA? Should I dilute the trypsin with PBS before treating the cells?
Dear Maria,
We culture hUC-MSCs and We use 0.25% trypsin-EDTA (appx 1.5ml per T75 flask for 3 mins in the incubator) followed by inactivation with complete media.
Thanks.
We always used TrypLE select from Life technologies for MSCs (either WJ-MSCs, cardiac MSCs...) always preferred the gentle detatching versus standard trypsin. Obviously costs a bit more, but you may also have more flexibility in your incubation time, or adjust it according to special requirements, as it does not damage cells nor needs inactivation.
Dear Maria,
I agree with Carolyn, lower concentration of Trypsin (0.05%, LifeTechnologies) is better for adipose tissue derived MSC. Trypsin should not be warmed to 37oC, room temperature is just fine. Cells detach very quickly under these conditions (2-3 minutes).
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