## 2 Materials and Methods
### Biocompatibility Assessment  
The biocompatibility of the micro-porous membrane filter was evaluated by performing cytotoxicity tests, using L-929 (mouse fibroblast) and A-549 (human alveolar basal epithelial cell) cells, using two recommended methods: A direct contact method and an extract dilution method, which have been previously described [28; 29]. The two types of cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L D-glucose, 1.0 mM sodium pyruvate, 10% fetal bovine serum, and 1% antibiotic antimyotic solution, including 10,000 units of penicillin, 10 mg of streptomycin, and 25 g of amphotericin B per mL (Sigma-Aldrich Co., St. Louis, MO, USA).  
The morphology of the cells was visualized under the micro-porous membrane filter using the direct contact method by transfecting the L-929 and A-549 cells with green fluorescent protein (GFP), as previously described [30; 31]. Briefly, films of high-density polyethylene (HDPE, thickness 0.5 mm), a negative control, and polyurethane containing zinc diethylditicorabamate (PU-ZDEC, thickness 0.5 mm), a positive control, were supplied by the Hatano Research Institute's Food and Drug Safety Center, and the films (1 \(\times\) 1 cm\({}^{2}\)) were sterilized using ethylene oxide gas for use as the negative and positive controls. Pre-cultured GFP-transfected L-929 and A-549 cells were plated in 6-well plates (Costar Corp., Greenwich, CT, USA) at 1.5 \(\times\) 10\({}^{5}\) cells per well, and the plates were pre-incubated for 24 h at 37 \({}^{\circ}\)C in an incubator (5% CO\({}_{2}\) conditions). The samples, the Pd-Ni membrane filter, and the HDPE and PU-ZDEC films were overlaid onto the cells to form a confluent monolayer in the center of each well, and were further incubated for 24 h. To confirm the morphological changes by the Pd-Ni membrane filter, the GFP-transfected L-929 and A-549 cells in each well were imaged using an automated imaging reader (Cytation 3; BioTek Instrument, Inc., Winooski, VT, USA) with fluorescence optics (excitation at 465 nm and emission at 503 nm).  
Assessment of the biocompatibility of the Pd-Ni membrane filter using an extract dilution method as previously described [28] was achieved by placing the samples, Pd-Ni membrane filter, HDPE, and PU-ZDEC films in screw-capped tubes containing 2x DMEM and agitated at 100 rpm for 48 h in a 37 \({}^{\circ}\)C incubator. After incubation, the extracts were then serially diluted, two-fold, by adding fresh media containing FBS. The L-929 and A-549 cells were plated in the 24-well plate and incubated with the same density of cells and conditions as described above. After incubating for 24 h, the medium was replaced with diluted extract media and incubated under the same conditions for a further 24 h. The cells were incubated with a Cell Counting Kit-8 (CCK-8) solution (Dojindo Laboratories, Kumamoto, Japan) for 15 min after washing three times with DPBS. The cell viabilities in each sample were calculated by measuring the absorbance of each well at 450 nm on a multimodal micro-plate reader (Cytation 3). Cell viability was expressed as a percentage of surviving cells in relation to the number of control cells.