Fig. 5. Geometric control in microchip hanging drop 3D culture.

(A) Process flow 3D shape formation. Chip with annular circle, square, and triangle channels is fabricated such that the depth of the channel is ~150 μm. Internal well surfaces are made hydrophilic through oxygen plasma, after which cells with media are loaded in a single step and centrifuged. Oil shear is performed for droplet formation, as the cavity between outer border of the channel and inner post causes the droplet to conform to the channel shape. The chip is inverted for hanging drop, and after 24-hour incubation of cells, dense 3D shapes are generated. (B) Fluorescence image of shape formation of different sizes seen 1 day after cell seeding. Outer diameter (circle) or side lengths (square and triangle) of shape channels are mentioned on top left corner of each image. (C) Volumetric mapping of the shapes in the green box seen in (B). (D) Bar graph of volumes of annular circle, square, and triangle shapes for each channel size for days 1 to 3 after cell seeding. (E) Bar graph of cell death (green volume normalized to red volume) in annular circle, square, and triangle shapes for each channel size for days 1 to 3 after cell seeding.