Cell strainer

Human surgical specimens [10 PNET, eight PDAC, three intraductal papillary mucinous neoplasm (IPMN), one metastasis of renal cancer, one gallbladder cancer, one duodenal cancer, six pancreas specimens, and one liver specimen] were obtained in accordance with the Declaration of Helsinki Principles and the guidelines of the Ethics Committee of the National Cancer Center (Tokyo, Japan). To examine the infectivity of each virus, for a flow cytometric analysis, pancreatic tumor tissues were mechanically processed into single cells with a sterile scalpel or scissors, and filtered with 100 μm nylon mesh (Cell Strainer; Thermo Fisher Scientific, Waltham, MA). For the preparation of single cells, fibroblasts were not removed, since major population of cells prior to the analysis was tumor cells, not stromal cells. For immunohistochemical staining, tissues were sliced into small pieces (1–4 mm in diameter) with a sterile scalpel. The prepared cells and tissue slices were cultured in RPMI‐1640 medium with 10% FBS.

In one series of experiment, eight to 10 newborn (P1–P3) mice were anesthetized on ice and the brains were quickly removed to place in ice-cold Hank’s balanced salt solution (Hank’s solution). The brain was cut coronally at 300 µm thickness with tissue chopper and the SCN region was dissected. Collected SCN tissues were dissociated using papain (13~20 units/ml; Worthington Biochem. Corp.) and DNase I (1 mg/ml) at 37 °C for 20 min. The dissociated cells were washed with 50% horse serum and collected by spin-down at 1,500 rpm for 5 min. The cells were dispersed using pipette in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cell suspension was filtrated with cell strainer (ø 40 µm; Falcon, Thermo Fisher Scientific). The 200 µl of filtrated cell suspension was placed onto the center hole (ø 1.2 mm) of culture dish at a density of 3.5–11.0 × 10⁴ cells/ml (5.8 ± 3.2 × 10⁴ cells/ml). One day before seeding, the dispersed cortical glial cells were pre-cultured in the culture dish. One hour after seeding, the dish was washed with culture medium to remove excessive cells suspended in the dish. Afterward, the culture medium was exchanged twice a week until imaging experiment.

T84 epithelial cells were seeded into 6-well plates (1×10⁶/well) cultured for 48 h and following the experimental treatment, cells were stained with tetramethyl rhodamine-ethyl ester (TMRE: 400 nM, 40 min, 37°C; ThermoFisher) dye for mitochondrial membrane potential. FCCP (10 μM) was used as a positive control for loss of mitochondrial membrane potential. Cells were then washed with PBS, dissociated with accutase (Sigma), and 1 mL PBS (37°C) was added to each well followed by gentle pipetting to increase dissociation. Cells were pelleted and re-suspended in 700 μL phenol red-free culture medium, passed through a 100 µm cell strainer (#22363549; ThermoFisher) and transferred to polystyrene 5 mL flow tubes (Falcon), and stained with DAPI(5 min). prior to assessment. A Becton-Dickson FACS CANTO cytometer supported by the BD FACS Diva software (BD Biosciences, Franklin Lakes, NJ) was used to measure fluorescence signal intensity from the dyes. The TMRE signal was detected by a PE laser (561-red) and the DAPI signal was detected by a BV421 laser (405-violet). The DAPI signal was used to exclude dead cells and mitochondrial membrane potential was measured as percent of change in mean fluorescence intensity (MFI) of TMRE compared to control cells, where lower MFI indicates increased mitochondrial membrane depolarization. Data were analyzed using Flow-Jo analysis software.^{36 (link)}

H1(WA01), H9 (WA09) and DF19 (iPS DF19-9-7T) were all purchased from the National Stem Cell Bank at www.wicell.org. H1 and H9 were initially expanded in hESCs media composed of 80% DMEM/F12 (cat#11330057, Life Technologies), 20% knockout serum replacer (cat#10828028, Life Technologies), 1% NEAA (cat# 11140050, Life Technologies), 4 ng/ml bFGF (cat# 100-18B, PeproTech), 1 uM L-Glutamine (G8540, Sigma) and 0.07 µM beta-mercaptoethanol (ES-007-E, Millipore) on MEF feeder cells (GSC-6001M, mitomycin inactivated CF-1 cells, GlobalStem), whereas DF19 was expanded in TeSR1 media (5850, Stem Cell Technologies) on matrigel (354234, BD). For culturing on inactivated hMSCs, all cell lines were grown in hESCs media supplemented with different concentrations of bFGF. Cells were long-term passaged using 1 mg/ml collagenase (cat# 17104-019, Life Technologies) and colonies were detached by physical scraping using the tip of a glass pipet after 7 minutes of collagenase incubation. Single cell dissociation was carried out using accutase (cat# A1110501, Life Technologies) by following the provided manufacturer's protocol. Cells were passed through a sterile cell strainer (cat# 08-771-01, Thermofisher Scientific) to achieve single cell homogeneity and counted using the Countess automated cell counter (cat# C10227, Life Technologies), average of 3 chambers/sample.

Wildtype mice were mated and at e15 the females were euthanized and the embryos were removed. The females were not further used. The embryos were decapitated and cortex tissue was dissected and used for primary cultures. Culture set-up is previously described in Perland et al. (2016) (link).Briefly, the cortex samples were pooled, washes in PBS-Glucose and dissociated using papain (Thermo Fisher Scientific) and DNAse (Thermo Fisher Scientific) for 30 min. Thereafter, mechanical dissociation was performed by pipetting before filtering the cell solution through a cell strainer (Thermo Fisher Scientific). The cells were diluted in plating media consisting of DMEM:F12 (Gibco, Invitrogen) supplemented with 2 mM GlutaMax, 1 mM Na-Pyruvate, 10% FBS and 1% Pen strep, all supplied from Invitrogen, and plated on Poly-L-Lysine (Sigma) coated coverslips (12 mm, #1.5) (Menzel-Gläser, Thermo Fischer Scientific) in 24-well plates (Nonclone delta, Thermo Fischer Scientific) or on Poly-L-Lysine coated 6-well plates (Nunclone delta, Thermo Fischer Scientific). The cell was incubated at 37°C, 5% CO₂ for 3 h before media change to NeurobasalA media (Gibco, Invitrogen) supplemented with 2 mM Glutamax, 1 mM Na-Pyruvate, 1 % Pen-strep and 1x B27 Supplement. 75% of the media was changed every third day and the cells allowed to grow for 9 days.

Mouse breast cancer 4T1 cells (CRL-2539™, ATCC), EMT6 (CRL-2755™, ATCC), and normal mouse mammary HC11 cells (CRL-3062™, ATCC) were routinely cultured in RPMI 1640 (10-040-CV, Corning) with 10% FBS (10082-147, Gibco) and 1% penicillin plus streptomycin supplement (SV30010, Citiva) at 37 °C in a high humidity, 5% CO2 and 95% air incubator. Three-dimensional cancer stem cell (CSC) spheroid culture was performed as previously described [34 (link), 35 (link)]. Briefly, 4T1 cells were cultured using ultralow attachment plates (3471, Corning) in serum free RPMI1640 supplemented with 2% SM1 supplement (5711, StemCell Technologies), 20 ng/mL EGF (78,006, StemCell Technologies), 20 ng/ml FGF (78,003, StemCell Technologies) and 4 µg/ml heparin (7980, StemCell Technologies) for 7 days. Under these conditions, tumor cells with stemness continue to proliferate and grow into spheres, while other cells in the culture are either unable to proliferate or die. At day 7, CSC spheroids, greater than 40 μm, were enriched and collected using a 40 μm cell strainer (50-196-0596, ThermoFisher). CSC spheroids smaller than 40 μm and non-proliferating non-CSCs pass through the strainer and are discarded.