A novel <i>in</i> - <i>situ</i> fixation and embedding method improves lung cancer organoid histopathology

Patient-derived lung cancer organoid provides a valuable platform for various applications. Histopathological examination of organoids is crucial to verify their consistency with the original tissue. Traditional methods require collecting multiple Matrigel domes and dissociating organoids from the matrix before histopathology. In this study, we report a fast and convenient method for making a paraffin block using only one Matrigel droplet. Organoids cultured in a 24-well plate were fixed with formalin <i>in situ</i> for 72 hours, pre-embedded in 3% agarose to block the Matrigel dome, and processed through routine dehydration, embedding, sectioning, and staining. We compared this method with others, including traditional small specimen paper wrapping and collecting dissociated organoids in EP tubes before embedding. Using only a single droplet, this approach yields a large number of organoids per section. Our method eliminates the need for Matrigel dissociation, effectively reduces organoids loss during fixation and paraffin embedding processes. In lung cancer organoids, tumor markers were consistent with the corresponding original tumor tissues. This novel protocol allows <i>in-situ</i> processing of patient-derived lung cancer organoids from a single Matrigel dome. It significantly reduces culture costs and time and is particularly useful for the histological examination of early-stage organoids.