Biotase

Except otherwise indicated, Panc-1 or BxPC-3 cells grown to 80% confluency on 8-well culture slides or tissue culture flasks (BD Biosciences, Bedford MA, USA), and 3D spheroids (Panc-1, 79 single spheroids equivalent to 2 × 10⁶ cells) cultivated for 7 days in 3D culture spinner flasks were washed once with HBSS (Biochrom GmbH, Berlin, Germany) and 3 times with serum-free culture medium. The cells were supplemented with serum-free culture media containing 40 µg/mL bovine testes hyaluronidase I-S or hyaluronidase IV-S (Sigma Aldrich, Missouri USA) or 20 µg/mL to 40 µg/mL collagenase, (clostridopeptidase A, from Sigma Aldrich) and further cultured for 4 h at standard culture conditions. Thereafter, the medium was removed and complete medium supplemented with MNP at different concentrations ranging from 5 to 50 µg Fe/mL were added and the cells further cultured for 2–24 h. Cells aimed for subsequent magnetic hyperthermia treatment were grown in large tissue culture flasks, treated with enzymes and exposed to MNP (50 µgFe/mL) as described above. Thereafter, the cells were washed 3 times with HBSS to removed free MNPs, and dissociated with Biotase (Biochrom GmbH Berlin, Germany), and counted, and 1 × 10⁷ cells were pelleted and dispensed in 200 µL prewarmed complete culture medium for exposure in an alternating magnetic field.

As a model for neuronal cells, SHSY-5Y cells were used [33 (link)]. SHSY-5Y cells were cultivated in flasks with RPMI medium (Merck, Darmstadt, Germany), supplemented with penicillin/streptomycin (1%) and FCS (10%). For their dissemination, cells were washed with PBS and detached from the flasks with biotase (Biochrom). Cells were counted in a Trypan Blue exclusion assay in Neubauer’s cell chamber. For experiments with RPE supernatants, 300,000 cells per well were disseminated on collagen A–coated 24-well plates. For the experiments with organ culture supernatants 10,000 cells per well were seeded on uncoated 96-well plates. When cells reached a confluence of 85%, they were treated for 24 h at 37 °C with supernatants and controls, respectively.