Kelli L VanDussen1, Jeffrey M Marinshaw2, Nurmohammad Shaikh3, Hiroyuki Miyoshi1, Clara Moon1, Phillip I Tarr4, Matthew A Ciorba2, Thaddeus S Stappenbeck1. 1. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA. 2. Division of Gastroenterology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA. 3. Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA. 4. 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.
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). DESIGN: 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. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
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). DESIGN: We created a large panel of humangastrointestinal 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. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
Entities:
Keywords:
CELL BIOLOGY; DIFFERENTIATION; E. COLI; EPITHELIAL CELLS; INTESTINAL EPITHELIUM
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