Quantification of Self-renewal in Murine Mammosphere Cultures

So mammosphere cultures provide an easily accessible motor system to study and dissect important aspects of normal in cancer breast stem cell behaviors, including their ability to self renew and to differentiate. Our quantitative approach aims at the evaluation of the grow rate of mammosphere cultures in an objective manner, allowing a direct comparison of different cell types and conditions. For the first trial, I would advise keeping a tight control over the incubation times to avoid over digestion and to ensure an accurate cell count before each plating.

Demonstrating the procedure with Charolaise Bull will be Errico D'Elia, a technician from my laboratory. After harvesting the four abdominal and lower thoracic mammory glands from 5 to 30, 8 to 10 week old female mice, place up to 20 glands per 100 millimeter petri dish in a minimal volume of DPBS. Mince the tissues into smaller pieces and add 10 milliliters of digestive medium to the fragments.

Use a 25 milliliters serological pipette to transfer the tissue pieces into a 50 milliliter conical tube and seal the tube with paraffin film. Then incubate the fragments on a rotator at 0.03 X G for 2 1/2 hours at 37 degrees and 5%carbon dioxide with humidity. At the end of the incubation, if all the fragments can be passed through the P1000 pipette tip, sediment the tissue slurry by centrifugation and carefully decant the supernatant.

Re suspend the pellet in 3 milliliters of PBS and filter the tube contents sequentially through one 100, 170, 140 micrometers cell strainer into a new conical tube, washing each strainer with 2 milliliters of fresh DPBS after each filter. Pellet the cells by centrifugation and decant the supernatant re suspending the cells in the remaining DPBS. Mix the cells with an equal volume of ammonium chloride potassium lysis buffer and lyse the red blood cells on ice for up to 5 minutes.

At the end of the incubation, arrest the lysis with 10 milliliters of DPBS and collect the cells by centrifugation. Confirm the presence of a white pellet and re suspend the pellet in 1 to 5 milliliters of mammosphere medium for counting. Then plate the cells onto non-tissue culture-treated ultra low adhesion six well plates at a 2 x 10 to the fifth viable cells per milliliter of mammosphere medium concentration for a 7 to 10 day incubation at 37 degrees Celsius and 5%carbon dioxide.

At the end of the 7 to 10 day culture, collect the primary mammospheres from each well of the cell culture plate and sediment the mammospheres by cetrifugation. After decanting the supernatant, use a P200 pipette with a filtered tip to pipette the mammospheres approximately 100 times. The generation of single cells patients is critical for the accurate quantification of the mammosphere cell for new lability.

Visually inspect extent of the segregation and use the cell strainer if necessary. Re suspend the disassociated mammospheres with 1 to 5 milliliters of fresh mammosphere medium for counting and plate 2 X 10 to the fourth viable cells per milliliter on polyhema coated ultra low adhesion six well plates for a 5 to 7 day culture. Then, after 5 to 7 days, count the number of spheres per well.

At the end of each passage, use a digital camera mounted on a stereoscope to scan the entire surface of all the wells to image the spheres. After saving the images, as tif files, import the stereoscope images as an image sequence into ImageJ and set the scale and the type of image to 8-bit. Duplicate the stack of images and select Subtract Background from the Process tab.

Check the Light background and Sliding paraboloid options and click OK to process all of the images of the stack. Select Adjust and Threshold from the Image tab and click apply. A pop up window will appear.

Select Default as the Method and Light as the Background. Check the Calculate threshold for each image box and click OK.To process all of the images in the stack, select Watershed and click Yes. Select Open and click Yes and select Erode and click Yes.

Next, select Analyze Particles from the Analyze menu and set the minimum size threshold at 10, 000 micrometers squared and the Circularity between 0.50 and 1.00. Select the option Ellipses from the Show drop down menu and check Summarize, Exclude on edges, and In situ Show. Then click OK to process all of the images of the stack.

To calculate the cumulative growth curve for each passage register the number of plated cells and the number of cells in spheres counted per well and calculate the sphere size at the end of each passage. Infer the number of plated spheres, by dividing the number of cells plated for each passage by the sphere size calculated at the end of the previous passage. Calculate the cumulative cell number for each well per passage and the cumulative sphere number for each well per passage and plot the data points on a semi-logarithmic scale.

Then an exponential trend line to the data points and calculate the coefficient of determination to measure the goodness of the fit. Then depict the equation of the trend line as a natural exponential function to infer the growth rate of the culture. In this experiment, the sphere and cell numbers of five consecutive passages, for three independent experiments were determined.

Here at the cumulative cell and sphere numbers per passage are shown. As observed Myc activation, leads to increased sphere and cell growth rates. Biological and relevant time points can be selected for cell collection or for performing gene expression analysis or other functional assets to evaluate cell differentiation, migration, and invasion.

This is a simple and cost-effective approach with multiple applications. For example, it can be used in direct discovery to measure the effects on Cancer stem cell proliferation and self-renewal.