Tryple express reagent

Parental Chinese hamster ovary cells (CHO-K1, ATCC: CRL-11268) were cultivated in F-12 Nut Mix (Ham) medium (Thermo Fisher Scientific Inc., ref. 21765-029) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Euroclone S.p.A., cat. no. ECS0180L) and 1% (v/v) penicillin/streptomycin solution (Lonza, cat. no. DE17-602E). Heat inactivation of FBS was performed by incubation in 56°C water bath for 30 min.
Human embryonic kidney cells (HEK-293T, ATCC: CRL-11268) and HeLa cells (HeLa, ATCC: CCL-2) were cultivated in Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific Inc., ref. 11960-044) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Euroclone S.p.A., cat. no. ECS0180L), 1% (v/v) L-Glutamine (Lonza, cat. no. 17-605E), and 1% (v/v) penicillin/streptomycin solution (Lonza, cat. no. DE17-602E).
All cell lines were cultivated in 75 mL cell culture flasks at 37°C in a humidified atmosphere containing 5% CO₂. Subculture was performed every 3-4 days, at about 80% confluence. After 2x wash using Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific Inc., ref. 14040-091), cells were detached from the surface of the culture flask by using a TrypLE Express reagent (Thermo Fisher Scientific Inc., ref. 12605-010). Viable cell numbers were determined using a TC20 Automated Cell Counter (Bio-Rad Laboratories, Inc.).

Mammosphere formation assay was performed by adaptation of a previously published protocol [41 ]. Briefly, to generate primary mammospheres, cells were harvested with trypsin, centrifuged and re-suspended in mammosphere medium (DMEM/F12 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin with the addition of 20 ng/ml recombinant human epidermal growth factor (EGF), 10 ng/ml recombinant human basic fibroblast growth factor (bFGF) and 1× B27 supplement (Life Technologies). Cells were then counted and passed through a 25G needle to obtain a single cell suspension. Then, 10³ cells/well were seeded in ultra-low attachment 24-well plates (Corning, Rome, Italy) in 800 μl of mammosphere medium. Plates were incubated at 37 °C for one week, and images were obtained at DM IL LED Inverted Microscope (Leica, Wetzlar, Germany). Mammospheres were counted and split by harvesting the mammospheres with PBS, centrifuging and re-suspending with TrypLE Express Reagent (ThermoFisher Scientific) as an alternative to trypsin. The single cell suspension was then again counted, and seeded at the same starting concentration in the mammosphere medium. After 1 week, mammospheres were counted and it was possible to calculate the Mammosphere Forming Efficiency (%) using the following equation:
MFE (%) = (# of mammospheres per well)/(# of cells seeded per well) × 100.

The performance of the straight microfluidic chip was assessed using HNC cell lines Fadu (ATCC^®HTB43^TM), 2A3 (ATCC^®CRL3212^TM) and CAL27 (CRL^®2095 ^TM) sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured under standard conditions in humidified incubators at 37 °C, 5% CO₂ in RPMI 1640-Glutamax (Life Technologies, Inc., Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS) and 1% Penicillin/Streptomycin. Cells were lifted using Tryple Express reagent (ThermoFisher Scientific, Waltham, MA, USA), resuspended in media and gently mixed on a shaker prior to experiments. Cell lines for spiking and recovery experiments were labelled with CellTracker™ (Life Technologies, Inc., Carlsbad, CA, USA) as per manufacturer’s instructions. Cell line authenticity was confirmed by short tandom repeat (STR) profiling with GenePrint^® 10 system (Promega, Madison, WI, USA) by the Genomic Research Centre (/IHBI/QUT). Unlabelled spike-in cells were stained with the CellSearch (Menarini Silicon Biosystems, Bologna, Italy) antibody cocktail targeting cytokeratin-8,18,19, CD45 and DAPI.