Developing in vitro culturing techniques of the Apicomplexan parasite Cryptosporidium parvum

Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa, whose members are parasites of the GI tract and airways. Cryptosporidium is the second largest cause of childhood diarrhoea in children under two and is associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods, in particular those which can support the complete growth of the parasite long term, while producing a high yield of oocysts. While numerous cell lines have been reported as maintaining the parasite, there remain no options for maintaining the parasite for longer than a week. The current cell line of choice, HCT-8, can only maintain infection for three days. Recently, our lab has successfully cultivated C. parvum in the oesophageal cancer derived cell line COLO-680N, and can maintain infection for over a week. Given the success of this cell line, a panel of cancer derived cell lines were grown in the presence of C. parvum for one week, with development assessed with fluorescent microscopy and PCR. Four cell lines were used to establish cultures for three weeks. The lung adenocarcinoma cell line, HCC4006, gave the highest oocyst yield of 5.8 x 105 oocysts per mL, a nine fold return on the initial inoculum. In addition, to tackle the issue of long term oocyst production in vitro, a simple, low cost bioreactor system using the COLO-680N cell line was established which produced infectious oocysts for 13 weeks. This method of oocyst production will be used to establish further cell lines as long term culture platforms. Further work aims to characterise these cell lines to establish factors which promote parasite development, and to further improve yield with media supplementation.