title: Detection of epithelial-cell injury, and quantification of infection, in the HCT-8 organoid model of cryptosporidiosis.
abstract: BACKGROUND
  Intestinal cells grown in microgravity produce a three-dimensional tissue assembly, or "organoid," similar to the human intestinal mucosa, making it an ideal model for enteric infections such as cryptosporidiosis.
  
  
  METHODS
  HCT-8 cells were grown in a reduced-gravity, low-shear, rotating-wall vessel (RWV) and were infected with Cryptosporidium parvum oocysts. Routine and electron microscopy (EM), immunolabeling with fluorescein-labeled Vicia villosa lectin and phycoerythrin-labeled monoclonal antibody to a 15-kD surface-membrane protein, and quantitative polymerase chain reaction (qPCR) using probes for 18s rRNA of C. parvum and HCT-8 cells were performed.
  
  
  RESULTS
  The RWV allowed development of columnar epithelium-like structures. Higher magnification revealed well-developed brush borders at the apical side of the tissue. Incubation with C. parvum resulted in patchy disruption of the epithelium and, at the surface of several epithelial cells, in localized infection with the organism. EM revealed irregular stunting of microvilli, foci of indistinct tight junctions, and areas of loose paracellular spaces. qPCR showed a 1.85-log (i.e., 70-fold) progression of infection from 6 h to 48 h of incubation.
  
  
  CONCLUSIONS
  The HCT-8 organoid displayed morphologic changes indicative of successful and quantifiable infection with C. parvum. The HCT-8 organoid-culture system may have application in interventional in vitro studies of cryptosporidiosis.
authors.affiliation: Center for Global Health, University of Virginia, Charlottesville,VA 22901, USA. ca6t@virginia.edu
authors.forename: Cirle
authors.initials: C
authors.lastname: Alcantara Warren
authors.forename: Raul V
authors.initials: RV
authors.lastname: Destura
authors.forename: Jesus Emmanuel A D
authors.initials: JE
authors.lastname: Sevilleja
authors.forename: Luis F
authors.initials: LF
authors.lastname: Barroso
authors.forename: Humberto
authors.initials: H
authors.lastname: Carvalho
authors.forename: Leah J
authors.initials: LJ
authors.lastname: Barrett
authors.forename: Alison D
authors.initials: AD
authors.lastname: O'Brien
authors.forename: Richard L
authors.initials: RL
authors.lastname: Guerrant
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D003457:Cryptosporidiosis / Q000469:parasitology / Q000473:pathology*; D016785:Cryptosporidium parvum / Q000502:physiology*; D004847:Epithelial Cells / Q000469:parasitology* / Q000473:pathology; D006801:Humans; D009940:Organoids / Q000166:cytology* / Q000469:parasitology*