Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking. Currently, Cryptosporidium oocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains (Moredun, Iowa) produce sufficient infectious oocysts to infect subsequent cultures. Oocyst identity was confirmed by specific staining (Crypt-a-glo, Vicia Villosa lectin, Sporo-glo), PCR-based amplification of Cryptosporidium-specific genes, lipidomics fingerprinting, and atomic force microscopy (AFM). Antibody-stained oocysts produced unstained oocysts confirming production of novel oocysts. Infected cultures could be cryoconserved and continued to produce infectious oocysts after resuscitation. Transmission electron microscopy identified all key Cryptosporidium life cycle stages. Infected cultures produced thick-walled (primarily involved in Cryptosporidium transmission between organisms) and thin-walled oocysts (important for Cryptosporidium propagation within a host/tissue) as indicated by DAPI staining (only thin-walled oocysts are permeable to DAPI staining, thus allowing visualisation of sporozoites) and AFM. In conclusion, we present a novel, easy-to-handle cell culture system that enables the propagation, cryopreservation and detailed investigation of Cryptosporidium at a laboratory scale. Its availability will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/134270 doi: bioRxiv preprint first posted online May. 4, 2017; 3 Cryptosporidiosis causes a significant number of deaths in children and immunocompromised individuals [1][2][3][4] . It is caused by species of the genus Cryptosporidium, in humans typically by C. parvum and C. hominis. The Cryptosporidium species belong to the phylum Apicomplexa and it has recently been proposed for the species to be reclassified as a member of the subclass of gregarina 5,6 . They are waterborne pathogens, and cryptosporidiosis has commonly been associated with disease in developing countries [7][8][9] . However, more recent molecular epidemiology studies suggested that the disease is also an increasing health concern in developed countries and may have reached epidemic levels 1, 2, 10-14 . Only one moderately effective drug (nitazoxanide) is available for the treatment of cryptosporidiosis.More effective drugs are urgently needed 10,15,16 .Cryptosporidium is a parasite that invades host cells, within the boundaries of the host cell membrane, residing intracellularly yet extra-cytoplasmic sometimes referred to simply as epicellular 17 . Cryptosporidium typically infects e...