3D Culture Platform for Enabling Large-Scale Imaging and Control of Cell Distribution into Complex Shapes by...

While organoid differentiation protocols have been widely developed, local control of initial cell seeding position and imaging of large-scale organoid samples with high resolution remain challenging. 3D bioprinting is an effective method to achieve control of cell positioning, but existing methods mainly rely on the use of synthetic hydrogels that could compromise the native morphogenesis of organoids. To address this problem, we developed a 3D culture platform that combines 3D printing with a cube device to enable an unrestricted range of designs to be formed in biological hydrogels. We demonstrated the formation of channels in collagen hydrogel in the cube device via a molding process using a 3D-printed water-soluble mold. The mold is first placed in uncured hydrogel solution, then easily removed by immersion in water after the gel around it has cured, thus creating a mold-shaped gap in the hydrogel. At the same time, the difficulty in obtaining high-resolution imaging on a large scale can also be solved as the cube device allows us to scan the tissue sample from multiple directions, so that the imaging quality can be enhanced without having to rely on higher-end microscopes. Using this developed technology, we demonstrated (1) mimicking vascular structure by seeding HUVEC on the inner walls of helix-shaped channels in collagen gels, and (2) multi-directional imaging of the vascular structure in the cube device. Thus, this paper describes a concerted method that simultaneously allows for the precise control of cell positioning in hydrogels for organoid morphogenesis, and the imaging of large-sized organoid samples. It is expected that the platform developed here can lead to advancements in organoid technology to generate organoids with more sophisticated structures.