A Planar Culture Model of Human Absorptive Enterocytes Reveals Metformin Increases Fatty Acid Oxidation and Export

A Leaky Human Colon Model Reveals Uncoupled Apical/Basal Cytotoxicity in EarlyClostridioides difficileToxin Exposure

“…4D, E) . This finding is consistent with previous jejunal TEER studies even after 12 days of differentiation, 36 supporting the notion that hCE monolayers have a different barrier strength profile than hSIE or Caco-2 monolayers, which exhibit lower barrier strength profiles. Thus, differentiated Caco-2 cells, which have a baseline TEER that is much lower than that of differentiated hCE and is more consistent with ISCs and hSIE, may not accurately report C. difficile toxin sensitivities in the colon.…”

“…Therefore, we sought to understand whether these transcriptomic differences translated to functional differences that are associated with a tight epithelial barrier, which is a hallmark of a mature gut epithelium. We have previously established a method for culturing healthy human jejunal monolayers and tracking cell maturation using transepithelial electrical resistance (TEER), 36 a non-invasive quantitative technique that is widely accepted to measure epithelial integrity. 55 Here, we sought to apply similar principles to differentiate hCE (DC) monolayers from multiple human donors and characterize variation in barrier properties between donors as measured by TEER (Fig.…”

“…Surgical specimens of human small and large intestines were obtained from donors at HonorBridge (formerly Carolina Donor Services, Durham, NC). Crypts from desired regions (Jej, DC) of each donor were detached from the specimen as previously described 36, 37, 39 using a chelating buffer 78 composed of EDTA (2 mM), dithiothreitol (DTT, 0.5 mM, freshly added), Na 2 HPO 4 (5.6 mM), KH 2 PO 4 (8.0 mM), NaCl (96.2 mM), KCl (1.6 mM), sucrose (43.4 mM), D-sorbitol (54.9 mM), pH 7.4. Released crypts were expanded as a monolayer on a neutralized collagen hydrogel as described previously.…”

“…Released crypts were expanded as a monolayer on a neutralized collagen hydrogel as described previously. [36][37][38] Briefly, crypts were placed on the top of 1 mg/mL collagen hydrogels (1 ml into each well of 6-well plate) at a density of 10,000 crypts/well, overlaid with 3 mL of Expansion Media (EM) containing 10 mmol/L Y-27632 (S1049; SelleckChem, Houston, TX), and incubated at 37ºC in 5% CO2. See Table 1 for small intestine and colon EM formulations.…”

“…Here, we establish a physiologically relevant in vitro model of C. difficile TcdA/B toxicity using primary adult intestinal stem cell (ISC)-derived hCE monolayers. Building upon our previous methods of culturing primary ISCs from healthy human donor organs and differentiating these ISCs into a functional epithelial layer, [36][37][38] we develop a highly sensitive assay system for evaluating both early-stage intestinal barrier injury and late-stage cytotoxic events associated with TcdA/B. Our tissue engineering approach focuses on producing an epithelium that closely mimics in vivo colonic tissue, which we use to assess C. difficile-relevant transcriptional and phenotypic differences between hCE, human small intestinal epithelium (hSIE), and Caco-2 cells.…”