Journal: Journal of visualized experiments : JoVE

Article Title: Generation of Multicellular Human Primary Endometrial Organoids

doi: 10.3791/60384

Figure Lengend Snippet: Table of Materials

Article Snippet: The presence of stromal cells not only provides the support for epithelial cells but also provides the necessary paracrine actions that have been established to be important for endometrial hormone response 4 , 8 , 9 . table ft1 table-wrap mode="anchored" t5 caption a7 Name of Material/Equipment Company Catalog Number Comments/Description Collagenase, Type II, powder Thermo Fisher Scientific 17101015 DNase I Sigma-Aldrich D4513 Dispase Corning 354235 Hank's Balanced Salt Solution (HBSS) Corning 21-022-CV 1x without calcium, magnesium, and phenol red MammoCult Human Medium Kit STEMCELL Technologies 05620 supplemented with 2uL/ml heparin and 5 uL/ml hydrocortisone Heparin solution STEMCELL Technologies 07980 added to MammoCult media Hydrocortisone stock solution STEMCELL Technologies 07925 added to MammoCult media Penicillin-Streptomycin Thermo Fisher Scientific 15140122 MicroTissues ® 3D Petri Dish ® Sigma-Aldrich Z764043 Agarose HS, molecular biology grade Denville Scientific CA3510-6 Ammonium chloride (NH4Cl) Amresco 0621 Sodium bicarbonate (NaHCO3) Sigma-Aldrich S6014 EDTA Fisher Scientific BP120-1 β-Estradiol Sigma-Aldrich E2257 Testosterone Sigma-Aldrich 86500 Phosphate buffered saline, pH 7.4 Sigma-Aldrich P3813 Paraformaldehyde, 16% solution Electron Microscopy Sciences 15710 Direct-zolTM RNA MicroPrep Zymo Research R2060 Hematoxylin Stain Solution Thermo Fisher Scientific 3530-32 Modified Harris formulation, mercury free Eosin Stain VWR 95057-848 Purified mouse anti-E-cadherin antibody BD Biociences 610181 Clone 36 Recombinant anti-vimentin antibody [EPR3776] Abcam ab92547 Trichrome Stain Abcam ab150686 Estrogen Receptor (SP1), rabbit monoclonal antibody Thermo Fisher Scientific RM-9101-S Progesterone Receptor, PgR 1294, unconjugated, culture supernatant Agilent Technologies M356801-2 Fluoroshield with DAPI, histology mounting medium Sigma-Aldrich F6057 Open in a separate window The new multicellular endometrial organoid offers a model system of the endometrium that is simple to generate and that incorporates both epithelial and stromal cells.

Techniques: Saline, Electron Microscopy, Staining, Modification, Formulation, Purification, Recombinant

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: Biology

Article Title: Burn-Related Glycocalyx Derangement and the Emerging Role of MMP8 in Syndecan Shedding

doi: 10.3390/biology14030269

Figure Lengend Snippet: Effects of MMP8 treatment on pulmonary glycocalyx in vitro. ( A ) UMAP representation of scRNA-seq analysis from healthy human lung tissue, identifying 14 distinct cell clusters. ( B ) Feature Plot displaying the glycocalyx module score (GMS) and ( C ) degrading enzyme module score (DEMS), calculated based on the gene sets outlined in . ( D ) Dot plot showing syndecan gene expression, with dot size indicating the percentage of expressing cells and color intensity reflecting average expression levels. ( E ) Immunofluorescence imaging of untreated and rhMMP8-treated EpiAlveolar™ 3D lung models, incorporating alveolar epithelial cells (EpiCs), fibroblasts (FBs), and endothelial cells (ECs). The 20× magnification images display DAPI-stained nuclei (blue) and SDC1 fluorescence (red). ( F ) ELISA-based quantitative analysis of SDC1, SDC4, and HA in EpiAlveolar™ model supernatants, with significant differences ( p < 0.05) marked in red. ( G ) SAECs treated with APMA, rhMMP8 alone, and activated rhMMP8, with SDC1, SDC4, and HA levels in supernatants analyzed via ELISA. Statistical significance was assessed using two-tailed unpaired t -tests and the Kruskal–Wallis test followed by Dunn’s multiple comparisons, with significant results (adj. p < 0.05) highlighted in red.

Article Snippet: EpiAlveolar TM 3D Small Airway Human MicroTissues were purchased from MatTek (MatTek Corporation, Ashland, MA, USA, cat#: ALV-100-MM).

Techniques: In Vitro, Gene Expression, Expressing, Immunofluorescence, Imaging, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test

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