Journal: Archives of Toxicology

Article Title: Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

doi: 10.1007/s00204-022-03228-y

Figure Lengend Snippet: EpiIntestinal™ tissue recapitulates unique 3D tissue architecture and barrier function of normal human small intestines. a SEM was used to visualize villi (left panel) and microvilli (right panel) structures on EpiIntestinal™ tissue. Scale bar = 10 μm. b Immunofluorescence staining identifies villin (green) and DNA stain (blue) in the 3D EpiIntestinal reconstructed microtissue. Scale bar = 10 μm. c F-actin (green) and DAPI (blue) immunohistochemical staining of cryosections (25 µm) of OCT embedded, EpiIntestinal™ tissue. Scale bar = 20 µm. d Alamar blue viability assay of 3D tissue. e TEER measurement of tissue barrier integrity and f FITC-dextran assay of tissue permeability. The results from three independent experiments are shown. Each experiment consisted of a single microtissue per test condition. *** p < 0.001

Article Snippet: Henceforth, we aimed to evaluate whether the MatTek 3D EpiIntestinal™ microtissue is a suitable technology platform to assess genotoxicity in the human gut.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Viability Assay, Permeability

Journal: Archives of Toxicology

Article Title: Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

doi: 10.1007/s00204-022-03228-y

Figure Lengend Snippet: Development of MN cytome assay in EpiIntestinal™ tissue. a Prevalence of binucleated (BN) and mononucleated (Mono) cells dissociated from EpiIntestinal™ tissue following 10 days’ cytochalasin B exposure. One thousand total cells per microtissue were scored to determine the percentage of mononucleated and binucleated cells. b Microscopic images of BN and Mono cells dyed with acridine orange. Scale bar = 10 µm. c Treatment regimen of 3D EpiIntestinal™ tissue over 10 days. d Various cell types and nuclear anomalies identified and evaluated in MN cytome assay. The Mitomycin C (2 μg/ml) treated EpiIntestinal™ tissue were dissociated, fixed and differentially stained with acridine orange (see “ ”). Scale bar = 10 μm. The results from three independent experiments are shown. Each experiment consisted of a single microtissue per test condition. *** p < 0.001

Article Snippet: Henceforth, we aimed to evaluate whether the MatTek 3D EpiIntestinal™ microtissue is a suitable technology platform to assess genotoxicity in the human gut.

Techniques: Staining

Journal: Archives of Toxicology

Article Title: Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

doi: 10.1007/s00204-022-03228-y

Figure Lengend Snippet: Assessment of cell proliferation in EpiIntestinal™ tissue. a Cell proliferation of EpiIntestinal™ tissue harvested at day 1, 7 and 11 post tissue arrival. PD = population doubling. b TK6 cell proliferation at 48 h and 72 h post cell seeding. The results from three independent experiments are shown. Each experiment consisted of a single microtissue per test condition. * p < 0.05 and ** p < 0.01

Article Snippet: Henceforth, we aimed to evaluate whether the MatTek 3D EpiIntestinal™ microtissue is a suitable technology platform to assess genotoxicity in the human gut.

Techniques:

Journal: Archives of Toxicology

Article Title: Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

doi: 10.1007/s00204-022-03228-y

Figure Lengend Snippet: Prevalence of background micronuclei (MN) in EpiIntestinal™ tissue. a Immunofluorescence staining of active caspase-3 (green) and DAPI (blue) in 0.1% DMSO (solvent) treated EpiIntestinal™ tissue, differentiate early apoptotic cells ( i – iii ) and genuine MN (red arrow, iv ) from DNA fragment(s) stemming from early apoptotic cells (white arrow, v ). Scale bar = 10 µm. b Prevalence of basal MN in EpiIntestinal™ tissue on day 1, 7 and 11. Dissociated single cells were fixed in Carnoy’s fixative and 4% formaldehyde (FA). 4% FA (total) represents total basal MN scored from the fixed cells, while 4% FA (normalized) represents the population of MN after exclusion of DNA fragment(s) stemming from early apoptotic cells in the same samples. One thousand total cells were scored per microtissue. The results from three independent experiments are shown. Each experiment consisted of a single microtissue per test condition

Article Snippet: Henceforth, we aimed to evaluate whether the MatTek 3D EpiIntestinal™ microtissue is a suitable technology platform to assess genotoxicity in the human gut.

Techniques: Immunofluorescence, Staining

Journal: Archives of Toxicology

Article Title: Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

doi: 10.1007/s00204-022-03228-y

Figure Lengend Snippet: Genotoxicity assessment of reference chemicals exposed-3D EpiIntestinal™ tissue. Tissues were exposed to test articles: a mitomycin C, b vinblastine sulphate, and c benzo(a)pyrene; for 10 days via apical treatment. Genotoxicity was assessed until cell viability was decreased by approximately 50%. Tissues were dissociated to single cells, fixed in Carnoy’s fixative and 4% formaldehyde (FA) and scored according to the criteria described in “ ”. 4% FA (total) represents total MN scored from the fixed cells, while 4% FA (normalized) represents the population of MN after exclusion of DNA fragment(s) stemming from early apoptotic cells from the MN scoring. One thousand total cells were scored per microtissue to determine the frequency of MN induction. The results from three independent experiments are shown. Each experiment consisted of a single microtissue per test dose. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: Henceforth, we aimed to evaluate whether the MatTek 3D EpiIntestinal™ microtissue is a suitable technology platform to assess genotoxicity in the human gut.

Techniques:

Journal: Archives of Toxicology

Article Title: Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

doi: 10.1007/s00204-022-03228-y

Figure Lengend Snippet: RICyt assay distinguishes genotoxins from non-genotoxin. a The genotoxin, mitomycin C, and non-genotoxic agent, phenformin HCl, were applied to the apical surfaces of 3D EpiIntestinal™ in vitro model tissues at various doses for 10 days. Tissues were dissociated into single cell suspensions, fixed in Carnoy’s fixative. One thousand total cells were scored per microtissue to determine the frequency of MN induction. Cell viability of treated 3D EpiIntestinal™ tissues are presented. The results from three independent experiments are shown. Each experiment consisted of a single microtissue per test dose. b A parallel study was conducted in which TK6 lymphocytic cells were exposed to mitomycin C and phenformin HCl at various doses for 24 h. Single cell suspensions were then fixed and scored. One thousand cells had been scored per treated sample for MN induction. Cell viability profile of treated TK6 cells are presented. The TK6 MN data were generated from independent duplicates. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: Henceforth, we aimed to evaluate whether the MatTek 3D EpiIntestinal™ microtissue is a suitable technology platform to assess genotoxicity in the human gut.

Techniques: In Vitro, Generated

Journal: Archives of toxicology

Article Title: Urolithin A attenuates arsenic-induced gut barrier dysfunction

doi: 10.1007/s00204-022-03232-2

Figure Lengend Snippet: (A) and (C) iAs+3 (0, 1, 5, 10, 20, 25, 50 μM) (B) and (D) iAs+3 (25 μM) with UroA (0, 5, 10, 25, 50 μM) were added to the apical compartment of EpiIntestinal 3D microtissues grown on Transwell inserts and incubated at 37 °C for 24 h and 48 h, respectively. FITC-dextran was added to these cells (apical compartment) and incubated for 2 h at 37°C and FITC-dextran levels in the bottom chamber well (basal compartment) was measured. *p < 0.05, **p < 0.01, ***p < 0.001, Statistics performed using one-way ANOVA in GraphPad Prism software. Error bar, mean ± SEM (n=4).

Article Snippet: Primary human small intestinal epithelial cultures EpiIntestinal 3D microtissues (SMI-100 and SMI-196) were obtained from MatTek Corporation (Ashland, MA, USA) and cultured according to the manufacturer’s instructions (24-well and 96-well format respectively) in a specially formulated culture medium (MatTek Corporation).

Techniques: Incubation, Software

Journal: Archives of toxicology

Article Title: Urolithin A attenuates arsenic-induced gut barrier dysfunction

doi: 10.1007/s00204-022-03232-2

Figure Lengend Snippet: For chronic exposure of iAs+3 in human intestinal organoids, iAs+3 (0, 1, 5 μM) with or without UroA (10 μM) were added to the apical compartment of EpiIntestinal 3D microtissues grown on Transwell inserts and incubated at 37 °C for 14 days. Fresh treatments were added every alternate days and supernatants were stored. (A) On 7th and 14th day, FITC-dextran was added to these cells (apical compartment) and incubated for 2 h at 37°C and FITC-dextran levels in the bottom chamber well (basal compartment) was measured. (B) EpiIntestinal 3D microtissues were treated with iAs3+ (1, 5 μM) in presence or absence of UroA (10μM) for 14 days. The supernatants were collected every 3 days for 14 days and pooled. LDH release in these cumulative supernatants at day 14 was measured. (C) IL-8 levels and (D) TNFα levels in supernatants were measured. *p < 0.05, **p < 0.01, ***p < 0.001, Statistics performed using one-way ANOVA in GraphPad Prism software. Error bar, mean ± SEM (n=4).

Article Snippet: Primary human small intestinal epithelial cultures EpiIntestinal 3D microtissues (SMI-100 and SMI-196) were obtained from MatTek Corporation (Ashland, MA, USA) and cultured according to the manufacturer’s instructions (24-well and 96-well format respectively) in a specially formulated culture medium (MatTek Corporation).

Techniques: Incubation, Software

Journal: RSC Chemical Biology

Article Title: Imaging therapeutic peptide transport across intestinal barriers

doi: 10.1039/d1cb00024a

Figure Lengend Snippet: Summary of in vitro and ex vivo intestinal barrier models for studying peptide transport. (A) Example of micro-engineered scaffolds to generate crypt-villus architecture of human small intestinal epithelium. Top left, an electron microscopy image of the PDMS stamp used to create, top right, a micromolded collagen scaffold in a modified Transwell insert; bottom, visualization of cellular differentiation and polarization using immunostaining. Left, absorptive enterocytes localized on the villi (ALP, red) and proliferative cells localized in the crypt (EdU, green). Right, terminally differentiated tissues of the intestine expressing human cytokeratin 20 localized on villi tips (KRT20, red) and stem cells localized to crypts and adjacent regions (Olfm4, green). Reproduced from with permission from Elsevier, copyright 2017. (B) Top, OrganoPlate by MIMETAS: schematic of the three lane system at the center of each channel network, consisting of a Caco-2 cell tubular lane, an extracellular matrix (ECM) gel lane, and a perfusion lane. Bottom, 3D reconstruction of a confocal z-stack showing the Caco-2 cell tubular morphology visualized by staining the tight junction protein ZO-1 (red), the brush border-protein Ezrin (green), and DNA (blue). White arrows indicate the apical (A) and basal sides (B). Reproduced from with permission from Springer Nature, copyright 2017. (C) EpiIntestinal™ model, an ex vivo model for studying drug absorption in the small intestine based on primary human cell-based organotypic small intestinal micro-tissues. Left, an illustration showing different types of cells and the microporous membrane underneath. Right, immmunostained cross-sections of the reconstructed microtissues showing cytokeratin-19 stained columnar epithelial cells (CK-19, red), villin stained apical surface of epithelium (green) and vimentin stained fibroblasts in the underlying ECM substrate (white). Reproduced from with permission from Springer Nature, copyright 2018. (D) Left, two types of organoid morphology, basal-out and apical-out, can be produced, with the latter potentially facilitating studies of peptide transport across the intestinal barrier from the apical to the basal side. Middle, organoids imaged using modulation contrast microscopy. Right, confocal microscopy images with nuclei in blue, ZO-1 (green) and β-catenin (red) illustrate how the orientation of the organoid organization is flipped when going from the basal-out to the apical-out system. Reproduced from with permission from Cell Press, copyright 2019.

Article Snippet: The EpiIntestinal model (MatTek, MA) is a high-throughput, human primary cell-based, 3D microtissue model that can be kept in culture for up to a month ( ).

Techniques: In Vitro, Ex Vivo, Electron Microscopy, Modification, Cell Differentiation, Immunostaining, Expressing, Staining, Membrane, Produced, Microscopy, Confocal Microscopy