Organoid-based in vitro systems to model Cryptosporidium parvum infection in 2D and 3D.

Typical cancer cell-based culture systems cannot support the full life cycle of Cryptosporidium parvum , despite its monoxenous life cycle which is completed in the small intestine of a single host. There is a block to fertilization and zygote formation in vitro . In this paper, we adapted a 2D organoid derived monolayer system and a 3D inverted enteroid system for use in C. parvum culture. 3D inverted enteroids were successfully infected by C. parvum without the need for microinjection and supported subculture of C. parvum . Using the 2D organoid derived monolayer (ODM) system, the infection can be maintained for at least 3 weeks with new oocyst production throughout. Fertilization was confirmed based on successful mating of two strains of C. parvum. We demonstrated that the apparent block to fertilization in typical cell culture is overcome using ODMs.