In Vitro Culture of Cryptosporidium spp. Using Stem Cell-Derived Intestinal Epithelial Monolayers.

Cryptosporidium parvum has a complex life cycle consisting of asexual and sexual phases that culminate in oocyst formation in vivo. The most widely used cell culture platforms to study C. parvum only support a few days of growth and do not allow the parasite to proceed past the sexual stages to complete oocyst formation. Additionally, these cell culture platforms are mostly adenocarcinoma cell lines, which do not adequately model the parasite's natural environment in the small intestine. We present a method for generating mouse primary intestinal epithelial cell monolayers that support long-term Cryptosporidium parvum growth, as well as human primary intestinal epithelial cell monolayers that facilitate long-term growth of C. parvum and C. hominis. Stem cells are grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an "air-liquid interface" that enhanced host cell differentiation and supported Cryptosporidium growth including all stages of the life cycle. The use of primary intestinal cells to grow Cryptosporidium in vitro will be a valuable tool for studying host-parasite interactions using a convenient in vitro model that more closely resembles the natural niche in the intestine.