Mammocult proliferation supplement

After treatment of MHCC97H or SMMC‐7721 cells with agomiR‐378a‐3p versus agomiRcontrol or antagomiR‐378a‐3p versus antagomiRcontrol (200 nM) for 48 h, the generated MHCC97H‐agomiR‐378a‐3p versus MHCC97H‐agomiRcontrol as well as SMMC‐7721‐antagomiR‐378a‐3p versus SMMC‐7721‐antagomiRcontrol cells were redispensed into six‐well ultralow adherent plates (Corning, Corning, NY, USA) at a density of 4 × 10³ cells/2 mL complete MammoCult™ medium (MammoCult™ basal medium plus 10% MammoCult™ proliferation supplement) (STEMCELL Technologies, Vancouver, BC, Canada) per well. One week after incubation, the number of tumor microspheres was counted.

Single cells were individually seeded through cell sorting (BD FACS Jazz) in 96 well Ultra-low attachment plates containing MammoCult™ Basal medium (Human) (Stem Cell Tech) supplied with hydrocortisone 0.48 mg/mL, Heparin solution 0.2% and 10% MammoCult™ Proliferation Supplement (Human). After 21 days, primary tumorspheres formed were measured both in size and number using inverted microscope (Olympus CKX41).

Forty-eight hours post transfection, cells were harvested and used to form spheres using MammoCult™ media (STEM CELL Technologies, Vancouver, BC, Canada) supplemented with 10% MammoCult™ Proliferation Supplement (STEM CELL Technologies, Canada), 0.2% heparin, and 0.5% hydrocortisone according to the manufacturer’s instructions. Afterwards, 40,000 cells/well of MCF-7 per condition were seeded in 2 mL complete MammoCult media in low adherent 6-well culture plates and incubated for 7 days. On day 7, spheres (>60 µm) were counted and mammosphere-forming efficiency (% MFE) was calculated as follows: % MFE = (number of mammospheres per well/number of seeded cells per well) × 100 [46 (link)]. To passage the spheres, 500 μL of pre-warmed Trypsin-EDTA was added for 1 min at 37 °C and the spheres were broken. Then, cells were seeded according to the previously mentioned densities. Carl Zeiss ZEN image software was used for the acquisition of bright field images of the mammospheres.

MDA-MB-2312 cells overexpressing miR-22 and empty vector cells were seeded at a concentration of 4 × 10³ cells/cm² in Mammocult media (StemCell Technologies) containing the Mammocult Proliferation Supplement (StemCell Technologies), 4 µg/mL heparin (StemCell Technologies), 0.48 µg/mL hydrocortisone (StemCell Technologies), and 500 U/mL Penicillin-Streptomycin (Thermo Fisher). Mammospheres grew in this media for 16 days, and the medium was changed every 2 days. Mammospheres were given 5 µL/mL doses of anti-miR-22 LNA, SCR-LNA for a final concentration of 500 nM, and VHL every 2 days when the medium was changed.

For mammosphere formation, 1 × 10³ cells/well were plated in 24-well ultra-low attachment plates (Corning, NY, USA) with MammoCult medium enriched with MammoCult proliferation supplement (10% v/v), hydrocortisone (0.48 µg/ml), and heparin (4 µg/ml) (StemCell Technologies, Vancouver, BC, Canada) and cultured for 7 days. After 7 days of culture the size and number of formed mammospheres were quantified using a Cytation3 cell imager (BioTek).