Generation of Primary Mammospheres: A Technique to Determine Cell Potency

Protocol

1. Generation of Primary Mammospheres from Human Breast Cancer Cell Lines

NOTE: Perform the following steps under a sterile culture hood.

1. Prepare Mammosphere Media containing DMEM/F12 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin. Prepare complete media immediately before use by adding 20 ng/ml recombinant human epidermal growth factor (EGF; Sigma), 10 ng/ml recombinant human basic fibroblast growth factor (bFGF; R&D Systems) and 1x B27 supplement.
2. Aspirate media from flask containing adherent MCF-7 or MDA-MB-231 cells (or a breast cancer cell line of your choice; 70-80% confluence), wash twice in 1x PBS, and trypsinize cells.
3. Centrifuge cells at 200 x g at room temperature for 5 min. Decant supernatant and resuspend cells in 1-5 ml of mammosphere media. Pipette up and down and if necessary use a 40 µm cell strainer cap filter to obtain single-cell suspension. Use a hemocytometer to ensure a single cell suspension has formed (if not, use a 25 G needle to syringe the cell suspension up to 3 times).
4. If necessary isolate CD44+CD24- cellular subset via FACS using anti-CD24-phycoerythrin (PE) and anti-CD44-fluorescein isothiocyanate (FITC) monoclonal antibodies⁹. Alternatively, isolate such population using a magnetic-activated cell sorting (MACS) system with anti-CD44 and anti-CD24-biotin combined microbeads as per manufacturer’s instructions. Perform positive selection using LS columns, and negative selection using LD columns and confirm the phenotypes of all isolated cells by flow cytometry.
5. Calculate the number of viable cells per ml using trypan blue.
   NOTE: Cell viability is calculated as the number of viable cells divided by the total number of cells within the grids on the hemocytometer. Cells that take up trypan blue are considered non-viable. The following procedure is used to accurately determine proportion of viable cells.
   1. Prepare a 0.4% solution of trypan blue in PBS. Add 0.1 ml of trypan blue stock solution to 1 ml of cells. Load a hemocytometer and examine immediately under a microscope at low magnification. Count the number of total cells and the number of blue staining cells. Viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100.
   2. To calculate the number of viable cells per ml of culture, use the formula below. Remember to correct for the dilution factor.
      Number of viable cells × 10⁴ × 1.1 = cells/ml culture
6. Resuspend a pre-determined amount of cells in 2 ml of complete mammosphere media in each well of a 6-well ultra-low adherent plate. The seeding density is normally 500-4,000 cells/cm² cell per well. Optionally use low attachment 24-well plates while seeding cells at the same density in 0.5 ml of media.
   NOTE: We recommend optimization of seeding density and time of culture for each cell lines of interest.
7. Incubate ultra low-attachment 6-well plates at 37 °C and 5% CO₂ for 5-10 days (depending on the cell line and the size of mammospheres) without disturbing the plates, particularly during the growth phase of the first 5 days.

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Materials

Name	Company	Catalog Number	Comments
DMEM/F12	Lonza	CC-3151
2 mM L-glutamine	Sigma Aldrich	G8540
100 U/ml Penicillin & Streptomycin	Sigma Aldrich	P4083
20 ng/ml Recombinant human epidermal growth factor (EGF)	Sigma Aldrich	E9644
20 ng/ml Recombinant human basic fibroblast growth factor (bFGF)	R&D systems	233-FB-025
1x B27 supplement	Invitrogen	17504-044
Phosphate buffered saline (PBS)	Thermo Scientific	12399902
0.5% trypsin/0.2% EDTA	Sigma Aldrich	59418C
Fetal calf serum	First Link UK	02-00-850
Trypan blue	Sigma Aldrich	93595
Low attachment 6-well plates	Corning	CLS3814