Enhanced oxygen permeability in membrane-bottomed concave microwells for the formation of pancreatic islet spheroids

Oxygen availability is a critical factor in regulating cell viability that ultimately contributes to the normal morphogenesis and functionality of human tissues. Among various cell culture platforms, construction of 3D multicellular spheroids based on microwell arrays has been extensively applied to reconstitute in vitro human tissue models due to its precise control of tissue culture conditions as well as simple fabrication processes. However, an adequate supply of oxygen into the spheroidal cellular aggregation still remains one of the main challenges to producing healthy in vitro spheroidal tissue models. Here, we present a novel design for controlling the oxygen distribution in concave microwell arrays. We show that oxygen permeability into the microwell is tightly regulated by varying the poly-dimethylsiloxane (PDMS) bottom thickness of the concave microwells. Moreover, we validate the enhanced performance of the engineered microwell arrays by culturing non-proliferated primary rat pancreatic islet spheroids on varying bottom thickness from 10 lm to 1050 lm. Morphological and functional analyses performed on the pancreatic islet spheroids grown for 14 days prove the long-term stability, enhanced viability, and increased hormone secretion under the sufficient oxygen delivery conditions. We expect our results could provide knowledge on oxygen distribution in 3-dimensional spheroidal cell structures and critical design concept for tissue engineering applications.