Spontaneous formation of stably-attached and 3D-organized hepatocyte aggregates on oxygen-permeable polydimethylsiloxane membranes having 3D microstructures

In order to enhance the viability and the differentiated functions of primary hepatocytes in cultures, it appears important to have them organized within a three-dimensional (3D) structure which promotes extensive cell-cell contacts, but also to be adequately supplied with oxygen. Here, we report a simple methodology satisfying these two fundamental but sometimes conflicting issues: primary rat hepatocytes were cultured on polydimethylsiloxane (PDMS) membranes with 3D-pillared microstructures with various dimensions, so that the cells could organize themselves around the pillars into various kinds of 3D multicellular aggregates, while being continuously supplied with oxygen by diffusion through the PDMS membrane. As expected, under such conditions, hepatocyte cultures exhibited higher albumin secretion and urea synthesis rates. It appeared then that as the spacing decreased between the pillars, the cells were more stably organized into smaller spherical aggregates and displayed the highest albumin secretion rates. Such a simple design is likely to be included in a new drug/chemical screening system in a practical microplate format, but also appears applicable to microfluidic devices.