Protocol for generating airway organoids from 2D air liquid interface-differentiated nasal epithelia for use in a functional CFTR assay

We present a protocol to generate organoids from air-liquid-interface (ALI)-differentiated nasal epithelia. We detail their application as cystic fibrosis (CF) disease model in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent forskolin-induced swelling (FIS) assay. We describe steps for isolation, expansion and cryostorage of nasal brushing-derived basal progenitor cells, and their differentiation in ALI cultures. Furthermore, we detail the conversion of differentiated epithelial fragments into organoids of healthy controls and CF subjects for validating CFTR function and modulator responses.For complete details on the use and execution of this protocol, please refer to Amatngalim et al.1

Automated summary

We propose a protocol for generating organoids from ALI-differentiated nasal epithelia, which can be used as a cystic fibrosis disease model in the CFTR-dependent FIS assay. We provide step-by-step instructions for the isolation, expansion, and cryostorage of basal progenitor cells derived from nasal brushings, as well as their differentiation in ALI cultures. Additionally, we describe the conversion of differentiated epithelial fragments into organoids for CFTR function validation and modulator response testing. For complete details, please refer to Amatngalim et al.