I'm growing mammospheres from breast cancer stem cells and there are single cells grow in the media. Also I noticed that the mammospheres are not regular circles and the largest diameter does not exceed 50 micron. any Idea??
One of the hallmarks of the "Transformed State", a cancer cell, is the ability to grow in suspension culture, such as when cells are cultured in soft agar or in an untreated, polystyrene culture dish. This reflects adherence-independent ability of cancer cells to grow; cells aggregate to form mammosphere, revealing that cells adhere to one another, but they barely adhere to the polystyrene substratum. Under such culture conditions, normal cells (with the exception of bone marrow hematopoietic progenitors cells) do not grow because their growth is dependent on adhesion to the extracellular matrix proteins that are deposited on the surface of the culture dish.
Single cells are often observed in a sphere formation media. Some of them will form spheres in a longer culture (2-4 weeks).
If shperes are too small, try to increase FGFb and EGF concentrations.
Amorphous spheres can be formed in 3 various occasions:
-cell dencity in too high, "false spheres" are formed due to cell clamping;
-cells are not properly tripsinized prior plating- again "false spheres";
-patient individual features.
First two options won't give spheres when replated for 2-nd and 3-rd SFAs.
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