Abstract
A major bottleneck in the progress of Cryptosporidium research is the lack of accessible cryopreservation of Cryptosporidium oocysts. Here, we present a protocol for the cryopreservation of Cryptosporidium isolates using enteroids. We describe the steps for the establishment of enteroid cultures and cryopreservation of C. parvum-infected HCT-8 cultures. We then detail procedures for the recovery and propagation of frozen parasites using enteroids. For complete details on the use and execution of this protocol, please refer to Deng et al.1.