Discussion

Long term in vitro growth of Cryptosporidium parvum has proved difficult with the majority of vitro studies being performed in 2-3 days, after which time they fail to propagate in in vitro or in vivo. Using a hollow fiber bioreactor (HFB) to grow human intestinal epithelial cells we can simulate in vivo conditions by providing oxygen and nutrients to host intestinal cells from the basal surface, while permitting the establishment of a low redox, high nutrient environment on the apical surface. When inoculated with 105 C. parvum (Iowa isolate) oocysts the bioreactor produces 108 oocysts per ml (20 ml extra-capillary volume) and has been maintained for over 2 years. In vivo infectivity studies using a TCR-α-immune deficient mouse model showed that oocysts produced from the bioreactor at 6, 12 and 18 months are indistinguishable from the parent Iowa isolate used to initiate the culture. In addition, the HFB produced oocysts have similar percent excystation profiles to the parent Iowa isolate. The technique provides for the first time the opportunity to perform long term in vitro studies with Cryptosporidium parvum.