Patterning of concave wells using monodisperse bubbles for the creation of spheroids.

Multicellular organisms consist of three-dimensional organized environments. 3D cell culturing shows better in vivo cellular behavior than 2D culture, offering better cellular communication and signaling pathways. Concave wells provide high production of spheroids with reproducible sizes by culturing inside uniform wells. This work is an innovative way to create concave wells by using mono-dispersed bubbles as a patterning force. First, we designed a 3D-printed millifluidic coflow device for the production of homogeneous bubbles. This allows the fabrication of the concave wells to be cost-effective and widely available. Agarose gel was formed inside a 35-mm dish, and bubbles were deposited on top of the agarose. When hardened, concave wells were left behind. By controlling the flow rate, we changed the bubble size and thus the size of the wells.  

We forged 1.7 mm diameter concave wells in a 35-mm dish, creating ~ 50 concave wells per dish. In each, we cultured 0.5x10^6 human embryonic kidney cells (HEK293). After 24 hours, we had a spherical construct with an average diameter of 0.008mm, and after two weeks, spheroids had grown to 1.4 mm diameter. In conclusion, this is a cost-effective and high-throughput method to create homogeneous wells that can be used for 3D cell culture.