• Home
  • Search Results
  • Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays.

Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays.

Gut (2014-07-11)
Kelli L VanDussen, Jeffrey M Marinshaw, Nurmohammad Shaikh, Hiroyuki Miyoshi, Clara Moon, Phillip I Tarr, Matthew A Ciorba, Thaddeus S Stappenbeck
ABSTRACT

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). 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. 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. This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host-microbial interactions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Methanol, anhydrous, 99.8%
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%, poly-coated bottles
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, 99.93%
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
Thiazolyl Blue Tetrazolium Bromide, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥97.5% (HPLC)
Sigma-Aldrich
Thiazolyl Blue Tetrazolium Bromide, 98%
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With 15 mM HEPES and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
L-Glutamine, meets USP testing specifications, suitable for cell culture, 99.0-101.0%, from non-animal source
Sigma-Aldrich
HEPES buffer solution, 1 M in H2O
Sigma-Aldrich
L-Glutamine, ReagentPlus®, ≥99% (HPLC)
Sigma-Aldrich
DAPT, ≥98% (HPLC), solid
SAFC
HEPES
SAFC
L-Glutamine
Sigma-Aldrich
HEPES, BioUltra, for molecular biology, ≥99.5% (T)
SAFC
HEPES
Sigma-Aldrich
HEPES, BioXtra, pH 5.0-6.5 (1 M in H2O), ≥99.5% (titration)
Sigma-Aldrich
L-Glutamine, BioUltra, ≥99.5% (NT)
Sigma-Aldrich
L-Glutamine, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
HEPES, BioXtra, suitable for mouse embryo cell culture, ≥99.5% (titration)
Sigma-Aldrich
HEPES, anhydrous, free-flowing, Redi-Dri, ≥99.5%
Supelco
Methanol solution, contains 0.10 % (v/v) formic acid, UHPLC, suitable for mass spectrometry (MS), ≥99.5%
Supelco
Methanol solution, NMR reference standard, 4% in methanol-d4 (99.8 atom % D), NMR tube size 5 mm × 8 in.
Sigma-Aldrich
L-Glutamine
Sigma-Aldrich
Methanol, NMR reference standard
Sigma-Aldrich
Methanol solution, NMR reference standard, 4% in methanol-d4 (99.8 atom % D), NMR tube size 3 mm × 8 in.
Supelco
Methanol solution, contains 0.1 % (v/v) trifluoroacetic acid, 5 % (v/v) water, suitable for HPLC
Sigma-Aldrich
Methanol solution, (Methanol:Dimethyl sulfoxide 1:1 (v/v))
Sigma-Aldrich
Methanol-12C, 99.95 atom % 12C