Abstract
Oxygen (O
2
) transport is a major limitation in high-density cell culture, particularly as three-dimensional (3D) spheroids are increasingly used to better mimic in vivo tissue environments. Here, we developed a poly(4-methyl-1-pentene) (PMP)-based microwell platform, leveraging its lower molecular absorption and greater mechanical stability than the commonly used O
2
-permeable polydimethylsiloxane (PDMS), while retaining high O
2
permeability. We measured O
2
permeability in 50-μm-thick PMP membranes and found approximately 200-fold greater O
2
permeability than the conventional 1-mm polystyrene culture-plate material. PMP membrane-based microwell plates with 350-μm-diameter wells maintained spatial separation of pre-formed hepatocyte spheroids while supplying O
2
from the plate bottom, synergistically enhancing spheroid oxygenation. Compared with non-O
2
-permeable plates, the platform reduced localized hypoxia and hypoxia-associated cell injury while improving spheroid viability and growth. Collectively, PMP-based O
2
-permeable microwell plates provide a promising, scalable, high-throughput platform for spheroid culture with multiple applications in tissue engineering, drug screening and beyond.