A new murine esophageal organoid culture method and organoid-based model of esophageal squamous cell neoplasia

Abstract

Summary Organoids mimic the physiologic and pathologic events of organs. However, no consensus on esophageal organoid (EO) culture methods has been reached. Moreover, organoid models reproducing esophageal squamous cell carcinoma (ESCC) initiation have been unavailable. Herein, we sought to develop an esophageal minimum essential organoid culture medium (E-MEOM) for culturing murine EOs and establishing an early ESCC model. We formulated E-MEOM to grow EOs from a single cell with clonal expansion, maintenance, and passage. We found that EOs cultured in E-MEOM were equivalent to the esophageal epithelium by histological analysis and transcriptomic study. Trp53 knockout and Kras G12D expression in EOs induced the development of esophageal squamous neoplasia, an early lesion of ESCC. Here we propose the new formula for EO culture with minimum components and the organoid model recapitulating ESCC initiation, laying the foundation for ESCC research and drug discovery.

Establishing transgenic murine esophageal organoids

“…Similar to our study ( Zheng et al., 2021 ), adenoviral vector transduction into mouse intestinal organoids is feasible ( Wang et al., 2014 ), and this method was successful in our previous study ( Zheng et al., 2021 ). According to our previous report, the viral infection efficiency was more than 80% when we measured the GFP-positive cells in the microscope.…”

“…We usually perform the passaging 8–10 days after seeding 1,000 cells on the 48-well plate. We have reported that the average size of the EOs is around 25,000 μm 2 at Day 10 in the microscopic images ( Zheng et al., 2021 ). If different scales of culture are used, the morphology of EOs and the culture medium should be carefully monitored.…”

“…According to our previous report, the viral infection efficiency was more than 80% when we measured the GFP-positive cells in the microscope. Since we knocked out Trp53 with Cre-LoxP recombination in our previous study ( Zheng et al., 2021 ), we used Nutlin to select Trp53 KO organoids. The selection of reagents and methods can vary based on the experimental design and the genes of interest.…”

“…We also describe how to establish transgenic EOs using viral transduction. For complete details on the use and execution of this protocol, please refer to Zheng et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Zheng et al. (2021) .…”