Ez bindshut 2

MIA PaCa-2 and PANC-1 cells were pre-treated with 200 or 400 μg/mL TiOxNPs for one hour followed by 2 or 5 Gy radiation treatment. Next, cells were trypsinized, counted, and plated in ultra-low attachment 96-wells plates (EZ-BindShut II, AGC Techno Glass, Shizuoka, Japan) at a density of 1000 cells/well. The cells were maintained in serum-free alpha MEM supplemented with B27 (Life Technologies), 10 ng/mL rhEGF (PeproTech), and 10 ng/mL rhbFGF (PeproTech), and then mixed with 1% methylcellulose. The number of spheres over 20 μM was evaluated after 10–12 days using BZ analysis software on a Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).
For the second passage, spheres were dissociated with 500 μL accutase for 5 to 10 days at 37 °C until a single cell suspension was obtained, followed by incubation with 200 or 400 μg/mL TiOxNPs for one hour followed by 2 or 5 Gy radiation treatment. Cells were then cultured and analyzed after 10–12 days.

SAS cells were incubated in SFM on a cell culture plate with a low-adhesion surface (EZ-BindShut^®II of AGC TECHNO GLASS, Japan) at 37°C in a 5% CO₂ incubator for several days (Figure 1A-i). SFM (~10 mL) contained 5 mL DMEM, 5 mL Ham's F-12K (Kaighn's) Medium, 200 ng epidermal growth factor, 200 ng basic fibroblast growth factor, and 0.2 mL B-27 (×50). A Spheroid Catch, which was obtained from Fukae Kasei Co. Ltd. (Kobe Japan), was set up in a collection tube (e.g., 50 mL centrifuge tube; T2318, Sigma-Aldrich). SAS cells were collected and transferred gently from the culture plate to the Spheroid Catch set up in a collection tube (Figure 1A-i~iii). The plate was rinsed with PBS to collect the spheroids that were tightly attached to the culture plate, and the spheroids were transferred from the culture plate to the Spheroid Catch. After gravity filtration, the selection of large spheroids was increased by removing small-sized spheroids by centrifuging the collection tube containing Spheroid Catch at 190 × g for 5 s at room temperature. (Figure 1A-iv). The mesh was detached by creating a small hole at the bottom of the Spheroid Catch with a needle or a tip of forceps (Figure 1A-v) and transferred (Figure 1A-vi) to a conventional culture plate (diameter, 3.5 cm) containing 1 mL accumax (Innovative Cell Technologies) and incubated at 37°C in a 5% CO₂ incubator for 7 min to disperse the aggregated cells (Figure 1A-vii). Then, the cells were mixed with 2 mL standard culture medium (DMEM containing 10% FBS) and the mixture was transferred to a 15-mL centrifuge tube (T1818, Sigma-Aldrich). The cells were collected by centrifugation (190 × g) for 5 min at room temperature (Figure 1A-viii). The supernatant was discarded and the solution including precipitated cells at the bottom (~1 mL) was transferred to a 1.5 mL microfuge tube (Figure 1A-iX). The cells were disaggregated by repeated suction and release using a 1 mL syringe equipped with a 25 G needle (Figure 1A-x), which completely disperses the spheroids into single cells. The number of single cells was counted. In selection step #2 (Figure 1A-xi), cells from selection step #1 were resuspended in SFM and plated at density of ~400/cm² in an EZ-BindShut^®II (e.g., 3,100 cells per a 10-cm diameter plate). This selection step may be repeated five times (Figure 1A-xii~xv). To establish a cell line, a single cell was selected by resuspending ~100 cells in 10 mL of SFM, transferring 0.1 mL of SFM into each well of a 96-well plate, and incubating for more than a week at 37°C in a 5% CO₂ incubator until spheroids were observed in each well.