Long-term in vitro Culture of Cryptosporidium parvum

Nigel Yarlett; Mary Morada

doi:10.21769/BioProtoc.2947

Long-term in vitro Culture of Cryptosporidium parvum

Bio Protoc. 2018 Aug 5;8(15):e2947. doi: 10.21769/BioProtoc.2947.

Authors

Nigel Yarlett^{1

2}, Mary Morada¹

Affiliations

¹ Haskins Laboratories, Pace University, New York, NY, USA.
² Department of Chemistry and Physical Sciences, Pace University, New York, NY, USA.

Abstract

Continuous in vitro growth of Cryptosporidium parvum has proved difficult and conventional in vitro culture techniques result in short-term (2-5 days) growth of the parasite resulting in thin-walled oocysts that fail to propagate using in vitro cultures, and do not produce an active infection using immunosuppressed or immunodeficient mouse models (Arrowood, 2002). Here we describe the use of hollow fiber bioreactors (HFB) that simulate in vivo conditions by providing oxygen and nutrients to host intestinal cells from the basal surface and permit the establishment of a low redox, high nutrient environment on the apical surface. When inoculated with 10⁵ C. parvum (Iowa isolate) oocysts the bioreactor produced 10⁸ oocysts per ml (20 ml extra-capillary volume) after 14 days, and was 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 were indistinguishable from the parent Iowa isolate used to initiate the culture. HFB produced oocysts had similar percent excystation profiles to the parent Iowa isolate.

Keywords: Cell culture; Cryptosporidia; Cryptosporidium parvum; Hollow fiber bioreactor; Parasitology; Protozoans.