Magnetic 3D Cell Aggregation and Patterning

To form three dimensional (3D) cell aggregates, cells, with or without miRNA/siRNA transfection, were incubated overnight in RPMI with 10% FBS, with a magnetic gold–polymer–iron oxide hydrogel (Nanoshuttle-PL, NS) (n3D Biosciences, Houston TX) to allow the attachment of NS to the cells [36 (link), 37 (link), 74 (link)]. 5 uM of CellTracker Green CMFDA Dye (Molecular Probes) was added to the cells and incubated for 30 minutes. The cells were washed with PBS, trypsinized, resuspended in RPMI with 10% FBS, and then magnetically levitated to the air-water interface for 4 hours, where they formed 3D structures with an ECM. Next, the 3D structures were mechanically disrupted by pipetting, and the resulting suspensions of aggregates were transferred to a 96-well plate, where they were formed or ‘printed’ into either a dot or a ring shape by placing a dot or ring magnet ‘drive’ underneath the plate for 15 minutes. Using either phase contrast or fluorescence, the 3D culture area was automatically imaged over time by an IncuCyte ZOOM live-cell imaging system (Essen BioSciences, Ann Arbor, Michigan), and the area of the dense 3D spheroidal regions was recorded for each well.