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 the plate for 5 to 10 days without disturbing the plate until spheres are 40 microns in diameter. In the following protocol, we will generate primary mammospheres from a breast cancer cell line.

- Working under a sterile culture hood, begin this procedure with MCF7 or MDA-MB-231 cells that are 70% to 80% confluent. Aspirate the medium from the flask. Wash the cells twice with PBS, then add Tripsin-EDTA and incubate for two to six minutes. Following detachment, quench by adding mammosphere medium containing 10% FBS.

Once the cells have detached, transfer them to a 15 milliliter conical centrifuge tube and spin it 200 times g at room temperature for five minutes. Following this centrifugation decant the supernatant, then resuspended the cells in one to five milliliters of mammosphere medium. Pipette up and down 10 times to break up the cell pellet.

Next, transfer the cell suspension to a 40 micron cell straining cap filter and collect the flow through in the attached tube to obtain a single cell suspension. Pipette a 20 microliter aliquot of the resuspended cells onto a hemocytometer and use a microscope to examine it. If cell clusters are observed as seen here, use a syringe to pass out the suspension in and out of a 25 gauge needle one or two times.

Once the cells have been dispersed into a single cell suspension as shown here, proceed to isolate CD44 positive, CD24 negative cellular subsets via fluorescence activated cell sorting or magnetic activated cell sorting using LS-column to positively select the CD44 positive cells. Since the desired cells attach to the walls of the LS-column, discard the flowthrough, then remove cells from the column, apply five milliliters of buffer and apply and depress the plunger supplied with the column to flush out the desired cells.

Next, use an LD-column to further purify the population by negative selection. Here, the CD24 positive cells attach to the LD-columns. Collect the flowthrough which contains the CD24 negative cells. Next, using flow cytometry confirm the phenotypes of all isolated cells. Using the trypan blue exclusion method, calculate the density of viable cells.

After appropriately diluting the cell suspension, seed each well of a six well ultralow attachment plate with 500 to 4,000 cells per centimeter square in 2 milliliters of complete mammosphere medium. Incubate the plates taking care not to disturb them for 5 to 10 days until spheres are observed. Continue culturing until the spheres were at least 40 microns in diameter but have not yet started to turn apoptotic.