Gravitational and acoustic micromanipulation platforms for high-content histological analysis of organoids and spheroids

Three-dimensional cell culture models (3D-CCMs), such as organoids and spheroids, are powerful tools for studying organ development and drug testing. Histology is a valuable imaging technique for its ability to reveal tissue architecture and biomarker localization at arbitrary depths within 3D-CCMs. However, conventional histological workflows are often low-throughput and labor-intensive, primarily due to the random embedding of 3D-CCMs within hydrogel blocks. To address these limitations, this thesis developed and validated two micromanipulation platforms based on gravitational and acoustic principles to enable precise spatial organization of 3D-CCMs during embedding. These platforms successfully meet the need for high-content histological analysis and are ready for implementation in laboratory, where they can support more efficient and standardized workflows. We first developed the HistoBrick platform, a structured hydrogel block containing microwells that passively aligned 3D-CCMs of homogeneous sizes on a single plane via sedimentation. The HistoBrick also supported the co-embedding of multiple experimental conditions, significantly increasing throughput of histological workflows and reducing reagent use. A major contribution of this work was the development and validation of a novel composite hydrogel composed of poly(ethylene glycol) diacrylate (PEGDA) and gelatine, specifically designed for cryosectioning. This PEGDA-gelatine hydrogel enabled seamless fabrication of the HistoBrick while preserving fragile tissue integrity, such as photoreceptors in retinal organoids. By improving sample preparation, the HistoBrick enabled long-term analysis of photoreceptors in retinal organoids, which was previously too time- and labor-intensive using conventional embedding methods. While the HistoBrick platform effectively aligned homogeneous 3D-CCMs, the reliance on passive sedimentation limits equatorial alignment in heterogeneous samples. To address this limitation, we developed a second platform that uses acoustic levitation to achieve precise equatorial alignment of 3D-CCMs for enhanced histological analysis. The platform levitated 3D-CCMs within compartmentalized wells, supporting the co-embedding of multiple experimental conditions within a single block. As a result, the platform enabled planar alignment of 3D-CCMs by their center of mass with higher precision than its sedimentation-based counterpart, significantly enhancing the information content of histological sections for size-heterogeneous 3D-CCMs beyond the capabilities of the HistoBrick. After enabling precise 3D-CCM positioning for histology, the scope of this work was extended to explore broader applications of acoustic micromanipulation. To this end, a third platform was developed using a novel transducer: piezoelectric micromachined ultrasonic transducer (PMUT) array. PMUT arrays generated spatially programmable bulk acoustic waves in fluid. We demonstrated, for the first time, determ