Abstract
Multiple sampling strategies to cover different or dynamic stages of malignant continuum with organoid cultures provide a valuable platform for epithelium homeostasis, transformation, and cancer progression. Here, we present a protocol to initiate, culture, passage, and characterize organoids from normal and cancer-prone human esophageal tissues. We describe steps for multiple sampling of malignant continuum and the initiation and maintenance of multi-stage organoids. We then detail procedures for the histological characterization of organoids and co-culture systems based on organoids and stromal cells. For complete details on the use and execution of this protocol, please refer to Chen et al.1.