Generation of Agarose-Based FFPE Cancer Organoids for Morphology Preservation

Formalin-fixed paraffin-embedded (FFPE) slides are essential for histological and immunohistochemical analyses of organoids. Conventional preparation of FFPE slides from organoids embedded in basement membrane extract (BME) presents several challenges. During the fixation step, dehydration often causes collapse of the BME, which normally supports the three-dimensional architecture of organoids. As a result, organoids may lose their original morphology, particularly in the case of cystic or structurally delicate types, leading to distortion and reduced reliability in downstream histological evaluation. Here, we introduce a straightforward protocol that improves the reliability of FFPE slide preparation for BME-based organoids by enhancing sample integrity and sectioning quality. By using 2% agarose as a mold during the embedding process, organoids grown in BME were effectively stabilized, enabling reliable preservation of their morphology throughout FFPE slide preparation. This method effectively addresses the difficulties in processing structurally delicate organoids and allows robust preparation of diverse cancer organoid morphologies-such as cystic, dense, and grape-like structures-while maintaining their native three-dimensional architecture. Our approach simplified the technical process while ensuring reliable histopathological analysis, making it a valuable tool for cancer research and personalized medicine. Key features • FFPE preparation applicable to diverse cancer organoid morphologies, including cystic, dense, and grape-like, while preserving three-dimensional architecture. • Agarose molding allows intact retrieval and fixation of BME domes, preventing collapse and maintaining organoid 3D architecture during FFPE preparation. • Compatible with diagnostic IHC/IF markers (pan-CK, CK19, p63, Ki-67, p53) across cancer organoids.