We are trying to isolate MSC by explant cultures of tissue pieces. In a 10 cm2 petridish (polystyrene) there is a good number of cells growing out of the explants.
After trypsination (warmed up trypsin, 3-4 minutes at 37°, soft tapping to remove all cells) and transfer to the chamberslides (8-Well, Permanox, Nunc) almost all cells are lost.
What might be the reason?
Dear Mathias, if the chamberslides are made out of glass, try coating them, e.g. with 1% gelatin, so that the cells can adhere better to the surface. Adherence to the glass surface might also be affected by the medium supplements that you use for culture. In case you grow your cells with human platelet lysate or AB-serum (e.g. 5%PRP or 5% ABS in DMEM) they might have a harder time adhering to the glass surface than with medium containing fetal calf serum (e.g. 10% FCS in DMEM). Good Luck, Guido
Another thing to note about MSC isolation is that you will typically get the most multipotent cells of the mixed culture to come off first, so I wouldn't go much longer than 4 minutes when trypsinizing if you are trying to achieve a pure population. Consider using HBSS instead of PBS to wash 0.05% Trypsin/EDTA to be more gentle on the cells and prevent senescence at earlier passages.
You can try fibronectin coating(could be a morphology change if cultured for longer but could serve your purpose).Also try using plastic chamber slides without coating,the MSCs should attach and survive for further staining.
Trypsin is too harsh; use collagenase instead to obtain clumps of tissues for culture. MSCs grow much better in fibronectin (BD) -precoated non-TC well plates under hypoxia (3-5%). And you'd better use quality MSC medium, such as the kit from ABM (TM022).
Hiii
we are trying to isolate msc from umbilical cord through explant culture. we have followed standard protocol however after removal of explants from tissue culture flask we have found some layers are suspending on media. the flask is quite confluent but the attached cells are not looking like msc (neither round nor elongated). should we go for trypsinization?
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