Generation of Primary Mammospheres: A Technique to Determine Cell Potency

A single breast cancer stem cell gives rise to a cluster of cells called a primary mammosphere. To obtain them, first culture a breast cancer cell line until 70% to 80% confluent, to ensure the cells are in their growth phase. Add Tripsin-EDTA to detach cells from the flask, and add a medium with serum to stop the action of Tripsin.

Now, take the cells in a tube and centrifuge to obtain the cell pellet. Resuspend the pellet in the mammosphere medium without serum and pass the suspension through a cell strainer cap filter, to get a single cell suspension. This suspension consists of stem cells, progenitor cells, and differentiated cells showing different protein expressions.

CD44 and CD24 are cell surface proteins characteristic of breast cancer stem cells. They are positive for CD44 expression and negative for CD24. Isolate cells from the cell suspension using fluorescence or magnetic activated cell sorting. Next, take an aliquot and ad trypan blue stain to the cells, and calculate the number of viable cells per milliliter using a hemocytometer slide to find the seeding density.

Finally, seed the cells in a six well ultra low adherent plate, so that cells remain suspended. Incubate