Hi,
I am trying to isolate MSCs from peripheral blood for the generation of iPSCs, wich will be used for neural differentiation. I would like to ask if anyone has succeed in isolating MSCs from peripheral blood without any stimulating factor and what protocol they used for the isolation and the culture of the cells. I am currently trying to isolate MSCs from PBMCs wich i take using the ficol method and try to culture them in 24 plate(half embeded with matrigel and half of the plate without) using DMEM-F12 with 20% FBS and 1%PenStrep, with medium change every third day.
Ahmed Lotfy Thank you for your answer. Have you tried Ficoll for isolating MSCs from peripheral blood in your lab? Can you give me some details regarding the plates you used, the medium change timeline and when did you see your MSCs expancion?
Hi Georgios Dafnopoulos
First at all, it's a very interesting work you're facing.
Regarding the isolation of PB-MSCs, I recommend the article by Lin et. al (Article Characterisation of multipotent stem cells from human periph...
),in which they describe a protocol for isolation of PB-MSCs without any stimulation factor. In addition, the growth medium they use is composed of A-MEM, 17% FBS and 1% antibiotics for the first passage, once confluent to 60% they use 10% FBS. The initial density is 0.5x10^6 cells/cm2. Finally, they suggest hypoxia as optimal for the culture of PB-MSCs. Although the reality is that hypoxia equipment is very expensive and not all groups can afford it.You may also find useful this review in which they cite several articles in which they have worked with PB-MSCs and analyze each protocol used (Article Application of Mesenchymal Stem Cells in Inflammatory and Fi...
).Best regards.
Peripheral blood contains very few mscs, so it must be very hard to culture them. To increase the stem cells, bone marrow stem cells need to be mobilze to enter the circulation..and the donor need to be given drugs to mobiluze thr stem cells
Dear Georgios Dafnopoulos
As we know the MSCs are rare in PBMC and it is definitely hard to isolate them from PBMC without any specific isolation techniques.
The isolated PBMC with Ficol definitely contains MSCs but the MSCs numbers are very low to make cell-cell interactions for expansion and growth.
I recommend you enrich the MSCs after PBMC isolation via MACS/FACS/Easy-sep/ etc (Isolation techniques) then expand them.
Best regards,
Fatemeh.
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