Plastic pillar inserts for three-dimensional (3D) cell cultures in 96-well plates

We have developed a plastic pillar insert to facilitate miniaturized three-dimensional (3D) cell cultures in 96-well plates by forming 3D hydrogel droplets containing cells (about 1 μL) on the tip of the pillar insert. Hemispherical 3D droplets containing cells were formed simply by immersing the tip of the pillar insert into a mixture of poly-l-lysine (PLL) and BaCl2 in a cell-encapsulation apparatus, followed by dipping it into cell suspension in alginate. Compared to traditional 3D cell culture platforms such as polymer scaffolds in 96 wells and 3D hanging drop plates, it allows us to easily change cell growth media or expose 3D cells to reagents by immersing the tip of the pillar inserts in different 96 wells filled with growth media or reagents. As a proof of concept, A549 and PC9 cell lines from human non-small cell lung cancer (NSCLC) were grown on the pillar insert and tested for cytotoxicity with Erlotinib. Both cells on the tip of the pillar insert grew over time, forming 3D structures unlike traditional 2D cell monolayer cultures and mimicking in vivo-like cellular microenvironment. The number of cells in alginate droplets was linearly proportional to the cell seeding density. The doubling time of A549 and PC9 cells were 15.9 h and 16.1 h, respectively, which were similar to those obtained from traditional 2D cell cultures. IC50 values from A549 and PC9 cells exposed to Erlotinib for 3 days were 15.2 ± 7.0 μM and 1.7 ± 0.4 nM, respectively, indicating that PC9 cells with EGFR mutation are highly sensitive to Erlotinib. Interestingly, the IC50 value of 3D PC9 cells grown on the pillar insert was 6 times lower than those obtained from 2D PC9 cells grown on the surface of 96-wells, whereas the IC50 value of 3D A549 cells were 5 times higher than those from 2D A549 cells. The result may represent that A549 and PC9 cells grown on the pillar insert are better mimicking what happens in humans.