title: Human primary intestinal epithelial cells as an improved in vitro model for Cryptosporidium parvum infection.
abstract: The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens.
authors.affiliation: Infectious Diseases Division, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA. alcastel@utmb.edu
authors.forename: Alejandro
authors.initials: A
authors.lastname: Castellanos-Gonzalez
authors.forename: Miguel M
authors.initials: MM
authors.lastname: Cabada
authors.forename: Joan
authors.initials: J
authors.lastname: Nichols
authors.forename: Guillermo
authors.initials: G
authors.lastname: Gomez
authors.forename: A Clinton
authors.initials: AC
authors.lastname: White
mesh_terms: D015415:Biomarkers; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D016785:Cryptosporidium parvum / Q000502:physiology*; D004847:Epithelial Cells / Q000469:parasitology* / Q000648:ultrastructure; D006790:Host-Parasite Interactions; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*
chemical_list: D015415:Biomarkers