title: Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays.
abstract: OBJECTIVE
  The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host-microbial interactions).
  
  
  METHODS
  We created a large panel of human gastrointestinal epithelial cell lines (n=65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays.
  
  
  RESULTS
  We obtained epithelial lines from all accessible tissue sites within 2 weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3-1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarised monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli.
  
  
  CONCLUSIONS
  This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host-microbial interactions.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Kelli L
authors.initials: KL
authors.lastname: VanDussen
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Marinshaw
authors.affiliation: Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Nurmohammad
authors.initials: N
authors.lastname: Shaikh
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Miyoshi
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Clara
authors.initials: C
authors.lastname: Moon
authors.affiliation: Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Phillip I
authors.initials: PI
authors.lastname: Tarr
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Ciorba
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Thaddeus S
authors.initials: TS
authors.lastname: Stappenbeck
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001422:Bacterial Adhesion; D001681:Biological Assay / Q000379:methods*; D001706:Biopsy; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004847:Epithelial Cells / Q000166:cytology* / Q000382:microbiology; D005819:Genetic Markers; D006801:Humans; D007082:Ileum / Q000166:cytology; D007413:Intestinal Mucosa / Q000166:cytology* / Q000382:microbiology; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D012007:Rectum / Q000166:cytology; D015203:Reproducibility of Results; D018874:Spheroids, Cellular
chemical_list: D017077:Culture Media, Conditioned; D005819:Genetic Markers; D012333:RNA, Messenger
keywords: CELL BIOLOGY; DIFFERENTIATION; E. COLI; EPITHELIAL CELLS; INTESTINAL EPITHELIUM