24 well ultra low cluster plates

Three human non-small cell lung cancer (NSCLC) cell lines, A-549, PC-9, NCI-H358, were maintained in RPMI 1640 medium (Cellgro, Corning Inc.) supplemented with 10% fetal bovine serum (Cellgro, Corning Inc.) and 1% penicillin-streptomycin-glutamine (Cellgro, Corning Inc.) in standard culture conditions. All cells were grown to 80% confluence before harvesting. Cells were seeded onto 24-well ultra-low cluster plates (Costar, Corning Inc.) at 1×10⁵ cells per well, and shaken for ~10 minutes to promote aggregation. Cells were incubated for 5 days and then exposed to either free drug (cisplatin or paclitaxel) or nanoparticles (two- or three-layer) with one of these drugs. Drug concentrations ranged from 0 to 1024 (μM cisplatin or nM paclitaxel) in 4X increments (0, 0.0625, 0.25, 1, etc.) for 48 h. Spheroids were exposed to the same concentration of nanoparticle-loaded drug as free drug, with the dose calculated by considering the loading efficiency from the HPLC data showing the drug concentration in the nanoparticles and the percent drug released at 48 h. Negative controls (without drug and without nanoparticles) were seeded and incubated under the same conditions. At the end point spheroids were disaggregated using trypsin (0.05%), and cell viability was assessed via trypan blue exclusion counts.