Two- and Three-Dimensional Human Intestinal Tissue Models for Cryptosporidium Infection

Conventional cell cultures utilizing transformed or immortalized cell lines or primary human epithelial cells have played a fundamental role in furthering our understanding of Cryptosporidium infection. However, they remain inadequate with respect to their inability to emulate in vivo conditions and support long-term growth and completion of the parasite's life cycle. Previously, we developed a three-dimensional (3D) silk scaffold-based model using transformed human intestinal epithelial cells (IECs). This model supported C. parvum infection for up to 2 weeks and resulted in completion of the life cycle of the parasite. However, transformed IECs are not representative of primary human IECs.Human intestinal organoids (hIOs) or human intestinal enteroids (hIEs) are 3D cultures derived from Lgr5⁺ stem cells isolated from the crypts of human intestinal biopsies or surgical intestinal tissues. These multicellular cultures can be induced to differentiate into enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and tuft cells. hIEs better represent human intestinal structure and function than immortalized, IEC lines. Recently, significant progress has been made in the development of technologies to culture hIEs in vitro. When grown in a 3D matrix, hIEs provide a spatial organization resembling the native human intestinal epithelium. Additionally, they can be dissociated and grown as monolayers in tissue culture plates, permeable supports, or silk scaffolds that enable mechanistic studies of pathogen infections. They can also be co-cultured with other human cells such as macrophages, myofibroblasts, and immune cells. The hIEs grown in these novel culture systems recapitulate the physiology, the 3D architecture, and functional diversity of native intestinal epithelium and provide a powerful and promising new tool to study Cryptosporidium-host cell interactions and screen for interventions ex vivo. In this chapter, we describe a 3D silk scaffold-based model using transformed IECs co-cultured with human intestinal myofibroblasts as well as 2D and 3D hIE-derived models of C. parvum infection.