mesenchymal stem cells - fixation for staining (Apr/18/2002 )
I have currently started working with human mesenchymal stem cells (hMSC). I am having trouble with the cells lifting in culture (some) and when I try to fix them (most) before using a von Kossa stain. Any ideas how I could a) make a stronger matrix or b) use a different, stronger fixative that agrees with silver nitrate staining?
I suggest U
Rocky Tuan and Rob Mauck at NIH have cultured hMSCs from multiple donors in a synthetic ECM biomaterial called PuraMatrix peptide hydrogel. They had a poster at Orthopedic Research Society ORS showing better differentiation to mature chondrocytes with great expression of proteoglycans, GAGs, aggrecan and collagen. Alan Grodzinsky from MIT and esteemed cartilage regeneration researcher is an inventor of the material and it's clinically compatible.
I've cultured chondrocytes and it works great for tissue engineering.
Where did you obtain human mesencyhymal stem cell? Maybe you can find answer from cambrex.com
quote name='s4hees' date='Apr 18 2002, 01:36 PM' post='2726']
I have currently started working with human mesenchymal stem cells (hMSC). I am having trouble with the cells lifting in culture (some) and when I try to fix them (most) before using a von Kossa stain. Any ideas how I could a) make a stronger matrix or use a different, stronger fixative that agrees with silver nitrate staining?
I too have this problem with my hMSC cultures. Apparently it is pretty common, and is not due to a 'weak' matrix but due to the production of lots of collagen causing the culture to contract upon itself.
What fixative are you currently using?
I fix primarily in ethanol, and prior to calcium/mineralization staining, fix a second time with 4% formalin-this seems to give the culture a bit more mechanical stability. However, I have never used Von Kossa so am not sure about compatibility of formalin with that.
Also, if you do more than one assay on the same plate make sure you wash the plate very gently between assays, and try not to shake it, and if pipetting stain etc into the well aim your pipette at the sides of the well, not directly onto the cell monolayer to minimise mechanical disturbance to the cells
Hope this helps!
I got problem bout mesenchymal stem cell derived human placenta..the cell cant survive in complete medium consisting DMEM(high glucose),FBS,Pen-sterp..the sample is amniotic stem cell..i need ur help to solve this problem..i don know which of growth factor suitable for amniotic stem cell.
hello,you how much FBS are you supplementing these cells with?My cells grew pretty well with 15% FBS.Other than that you can also try bFGF.IMEM with 10% FBS also shows good growth.