Development of an Organ-on-a-Chip for Correlative Microscopy: Visualizing Early Osteogenesis in 3D with High Resolution

Organs-on-a-chip are of increasing interest for studying tissue development in health and disease. In the past years, platforms have been developed to analyze developmental biological processes by light microscopy and biochemical assays. While these show great potential for pharmaceutical applications, retrieving ultrastructural information about microtissues remains challenging. In this study, an organ-on-a-chip is presented that is suitable for analysis by correlative light and electron microscopy. Using osteogenic cultures in which the cells and deposited collagen matrix align between pillars, the applicability of this platform for high-resolution, longitudinal monitoring of the microtissue development during multiple weeks with 3D live fluorescence microscopy is shown. The analysis of complete microtissues showed the development of two distinct actin patterns during differentiation. Furthermore, intact microtissues were extracted and cryo-fixed by high-pressure freezing, to enable processing for volume electron microscopy. The region of interest for electron microscopy was selected based on the fluorescence images and the resulting FIB/SEM image stack shows ultrastructural information about the cellular organization, collagen alignment, and mineralization in the cultures. The platform developed in this study shows great potential for increasing our understanding of processes underlying structural organization of tissues in health and disease, by allowing ultrastructural analysis of tissues during development.