摘要Biliary cryptosporidiosis caused by Cryptosporidium is life-threatening in immunocompromised individuals,particularly those with acquired immune deficiency syndrome(AIDS),resulting in AIDS cholangiopathy.However,studies of biliary cryptosporidiosis have lagged due to the lack of in vitro models that allow complete pathogen development.Here we describe procedures for the generation of mouse cholangiocyte organoids(Chol-orgs).The Chol-orgs,which ex-pressed stem and biliary cell markers,could be clonally expanded for three months and stored in liquid nitrogen for more than one year.Combined with cell differentiation using the air-liquid interface(ALI)approach,we established a culture system for C.parvum.ALI cultures using Chol-orgs have supported～50-fold amplification of the pathogen and generated viable oocysts in vitro.In addition,we analyzed the transcriptome of Chol-ALI cultures infected with the lid subtype of C.parvum to characterize host cell responses.RNA-seq analysis revealed that C.parvum upregulated immune and inflammatory responses and downregulated metabolic and cell proliferation signaling pathways in Chol-orgs.A similar system using bovine cholangiocytes also supported the complete development of C.parvum in vitro.These in vitro models provide convenient methods to study biliary cryptosporidiosis and other hepatic infections and to develop effective therapies for AIDS cholangiopathy.