Heparin solution

For mammosphere culture, MDA MB 231 cells (ATCC, Manassas, VA, USA) with less than ten passages were grown to about 70%–80% confluence and then enzymatically detached with trypsin (Nacalai Tesque, Kyoto, Japan), gently pipetted and suspended into single cells and passed through a 40 μm cell strainer (Corning, Lowell, MA, USA). A quantity of 1 mL of MDA MB 231 cells suspension was cultured in 24 well ultra-low attachment plates (Corning, Lowell, MA, USA) at 20,000 cells/mL in Mammocult complete media (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 4 μg/mL of heparin solution (Stem Cell Technologies, Vancouver, BC, Canada), 0.48 μg/mL hydrocortisone stock solution (Stem Cell Technologies, Vancouver, BC, Canada), and 1% antibiotics (Nacalai Tesque, Kyoto, Japan). After 7 days of culture in the incubator in 5% CO₂ and at 37 °C, mammospheres were used for further experiment [29 (link)].

PB and BM were isolated and analyzed as previously described (61 (link), 71 (link), 72 (link)). Briefly, PB was collected in fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS), 2% fetal bovine serum, and 2 mM EDTA] supplemented with 0.0004% heparin solution (STEMCELL Technologies Inc.). RBCs were removed by incubation in 1% dextran solution in PBS at 37°C for 25 min. The cells in the supernatant were isolated and subjected to room-temperature RBC lysis solution (STEMCELL Technologies Inc.). PB cells were stained with conjugated antibodies targeting CD4, CD8, NK1.1, CD19, CD11b, Gr-1, CD45.1, and CD45.2. BM cells were isolated from tibias, femurs and hip bones that were crushed with a pestle and mortar. Lineage depletion (B220, CD4, CD8, CD11b, Gr-1, and Ter119) or c-kit enrichment was performed using MACS magnetic microbead kits (Miltenyi Biotec) according to the manufacturer’s instructions. BM cells were stained with conjugated antibodies against B220, CD4, CD8, CD11b, Gr-1, Ter119, cKit, Sca1, CD48, CD150, CD45.1, CD45.2, CD105, CD41, CD16/32, and MHC class II. Streptavidin-BV510 was used for biotin identification. Propidium Iodide (1 μg/ml; Molecular Probes) was used to distinguish viability. Flow cytometry analysis and assisted cell sorting were performed using Beckman Coulter CyAn ADP, Becton Dickinson (BD) LSRFortessa X-20, and BD Aria III.

In order to obtain human tumor spheroids with stemness feature, the MCF7 cells were detached and suspended as individual cells at 1 × 10⁵ cells/mL in a Mammo Cult Human Medium Kit (STEMCELL Technology, Cat# 05620) included with hydrocortisone (STEMCELL Technology, Cat# 07925) and heparin solution (STEMCELL Technology, Cat# 07980), and cultured for 14 days onto a 100 mm ultra-low attachment dish (corning). To use the appropriate size of spheroids in the microfluidic device, the generated spheroids were collected and filtered in two consecutive filtration steps: (a) 40-μm filtration, in order to exclude all the spheroids smaller than 40 μm; and (b) 100-μm filtration to exclude aggregates larger than 100 μm and centrifuged by 250× g for 5 min to separate them from the supernatant. Dried plat containing spheroids were suspended in collagen Type-I solution on ice for loading in microfluidic devices [28 (link)].

Cells (5 × 10²) were seeded in 96-well ultra-low attachment culture plates (Corning Life Sciences) in MammoCult™ Basal Medium (Stemcell) with 4 µg/ml Heparin Solution (Stemcell) and 0.48 µg/ml Hydrocortisone Stock Solution (Stemcell) for 10–14 days. Spheres were counted under an inverted microscope in triplicate wells.

mNSCs were maintained in ultra-low attachment flasks in culture medium with 1:1 ratio of DMEM/F-12 (Invitrogen, 11330-032) and Neurobasal A (Invitrogen, 10888-022) consisting of 1% each of HEPES Buffer Solution 1 M, sodium pyruvate solution 100 nM, non-essential amino acids solution 10 mM, Glutamax-I Supplement and Penicillin/Streptomycin solution. The media was supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech. Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980). Cells were dissociated using Accutase (StemCell Tech. Inc., 07922) and passaged every 3 or 4 days.