AbstractCryptosporidiumparasites 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,Cryptosporidiumoocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two differentCryptosporidium parvumstrains (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 ofCryptosporidium-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 keyCryptosporidiumlife cycle stages. Infected cultures produced thick-walled (primarily involved inCryptosporidiumtransmission between organisms) and thin-walled oocysts (important forCryptosporidiumpropagation 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 ofCryptosporidiumat a laboratory scale. Its availability will accelerate research onCryptosporidiumand the development of anti-Cryptosporidiumdrugs.
Vapor-based micro/nano-partitioning of fluoro-functional group immobilization for long-term stable cell patterning
