Currently I am working on isolation of mesenchymal stem cells from human umbilical cord. Since MSCs have the unique properties of self renewal and differentiation into various lineages, my protocol demands to keep them undifferentiated. Nanog and LIF have been mentioned as factors responsible for undifferentitation of MSCs. However their roles are not clear. Kindly help.
Hello,
I worked on isolation and culture of WJ-MSCs and our main culture protocol is mainly adherent to those published by others. Low-glucose DMEM, plus 10% FBS, NEAAs, antibiotics, and cells can be maintained up to passage 15th, maintaining morphology and markers expression.
In these conditions, if you maintain the standard passaging ratio, the medium alone will not be sufficient to promote any differentiation process. Never tried one of the mentioned factors: in our hands cells do not need them.
Hope to have been of help.
Giampiero
Hi,
You can culture the cells in DMF10 medium ( %10FBS, %1 Pen/Strep, %1 L-glu, %60 DMEM-LG, %40 MCDB201), when your cells become adherent change the medium in every 3 or 4 days and when you reach the %70-90 confluency, you have to passage the cells, so you maintained the cells as you want, if you don't passage the cells, just you would have older and apoptotic cells, you can't promote any differantiation in this medium.
Good Luck!
Hello
I agree withe reserchers also , i add this styp .
1. Inject 5mL DMEM-low glucose with 10% FBS.
2. Harvest cells by massaging the cord tissue.
3. Centrifuge at 300xg for 10 minutes at room temperatures.
4. Remove supernatant, add culture medium and plate.
5. Incubate at 37°C, 5% CO2 incubator overnight and change the medium in every 3 or 4 days and when you reach the %70-90 confluency
Good Luck!
Hi
I totally agree with previous comments, be careful to passage them before they reach critical confluence or they will differentiate/ undergo apoptosis.
That's one of the main critical point.
Good luck
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