Isolation and Culture of Human Colon Epithelial Cells Using a Modified Explant Technique Employing a Noninjurious Approach

Mohammadpour, Hamid

doi:10.1385/1-59259-861-7:237

Cited by 4 publications

(4 citation statements)

References 8 publications

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“…Mucosal strips were transferred to a new container with 200ml of RPMI 1640 with 1mM EDTA, 10% Fetal Bovine Serum (FBS) and antibiotic/antimycotic solution (RPMI-EDTA-FBS) with a stir bar and stirred at room temperature at 60rpm for a minimum of 4 hours to release cells from basal lamina. These isolated colon cells were cultured as previously described [40], and propagated in Epithelial Growth Media consisting of RPMI-1640 medium supplemented with 5% fibroblast conditioned media, antibiotic/antimycotic solution (penicillin, streptomycin, amphotericin B), 2mM L-glutamine, 10% FBS, insulin (5µg/ml) and transferrin (5µg/ml).…”

Section: Methodsmentioning

confidence: 99%

Enhanced sensitivity of colon tumour cells to natural killer cell cytotoxicity after mild thermal stress is regulated through HSF1-mediated expression of MICA

Dayanç

Bansal

Gure

et al. 2013

International Journal of Hyperthermia

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Purpose Previously we showed that mild thermal stress increased Natural Killer (NK) cell - mediated tumor cytotoxicity and that this could be blocked by anti-NKG2D or anti-MICA antibodies. Here, we investigated the role of the transcription factor HSF1 in thermal regulation of MICA expression in tumor cells in vitro and in vivo. Materials and Methods Hyperthermia experiments were conducted in vitro and in mice using a target temperature of 39.5°C. Apoptotic cells and NK cells in situ were visualized by use of the TUNEL assay or expression of NKp46 respectively. Using Colo205 cells, HSF1 message was blocked utilizing siRNA while luciferase reporter assays were used to measure the activity of the MICA promoter in vitro. Cell surface MICA was measured by flow cytometry. Results Following WBH, tumor tissues showed an increase in NK cells and apoptosis. Mild thermal stress resulted in a transient increase in surface MICA and enhanced NK cytotoxicity of the Colo205 colon cancer cell line. Silencing (mRNA) HSF1 expression in Colo205 cells prevented the thermal enhancement of MICA message and surface protein levels, with partial loss of thermally enhanced NK cytotoxicity. Mutations of the HSF1 binding site on the MICA promoter implicated HSF1 in the thermal enhancement of MICA. Some, but not all, patient-derived colon tumor derived xenografts also exhibited an enhanced MICA message expression after WBH. Conclusions Upregulation of MICA expression in Colo205 cells and enhanced sensitivity to NK cell killing following mild thermal stress is dependent upon HSF1.

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Section: Methodsmentioning

confidence: 99%

Enhanced sensitivity of colon tumour cells to natural killer cell cytotoxicity after mild thermal stress is regulated through HSF1-mediated expression of MICA

Dayanç

Bansal

Gure

et al. 2013

International Journal of Hyperthermia

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“…During past decades, primary intestinal cell cultures have been obtained from the small intestine (Perreault and Beaulieu, 1998;Aldhous et al, 2001) or from the colon (Baten et al, 1992;Fonti et al, 1994;Deveney et al, 1996;Grossmann et al, 2003;Mohammadpour, 2005), but a rapid loss of the differentiated charac-teristics of cells was observed. As a consequence, these primary cell models could be used only for short-term toxicity studies.…”

Section: Introductionmentioning

confidence: 99%

Gene Expression Profiling of Systems Involved in the Metabolism and the Disposition of Xenobiotics: Comparison between Human Intestinal Biopsy Samples and Colon Cell Lines

Bourgine¹,

Billaut-Laden²,

Happillon³

et al. 2012

Drug Metab Dispos

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ABSTRACT:Intestinal cell lines are used as in vitro models for pharmacological and toxicological studies. However, a general report of the gene expression spectrum of proteins that are involved in the metabolism and the disposition of xenobiotics in these in vitro systems is not currently available. To fill this information gap, we systematically characterized the expression profile of 377 genes encoding xenobiotic-metabolizing enzymes, transporters, and nuclear receptors and transcription factors in intestinal mucosa (ileum, ascending colon, transverse colon, descending colon, and rectum) from five healthy subjects and in five commonly used intestinal cell lines (Caco-2, C2BBe1, HT29, T84, and FHC).

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“…Isolation of viable colonic epithelial cells is therefore an important approach for applicable proteomic studies. Methods used include a microbial metalloenzyme-based dispase-collagenase digestion method (72, 73), explant-based cultures (74), and more recently, an EDTA-perfusion method (75, 76). For example, using the EDTA-perfusion, in combination with MACS-based immuno-subtraction of immune cells with anti-CD3ε, -B220, -CD11b, -CD11c, and -NK1.1 mAbs, Mizoguchi et al obtained highly purified intact epithelial cells (76).…”

Section: Enrichment Of Specific Cell Typesmentioning

confidence: 99%

Applications of proteomics in the study of inflammatory bowel diseases

Alex¹,

Guček²,

Li³

2009

Inflammatory Bowel Diseases

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Inflammatory bowel diseases (IBD) are chronic, heterogeneous, and multi-factorial intestinal inflammatory disorders. Major challenges in IBD research include identification of major pathogenic alterations of genes/proteins as well as effective biomarkers for early diagnosis, prognosis, and prediction of therapeutic response. Since proteins govern cellular structure and biological function, a wide selection of proteomic approaches enables effective characterization of IBD pathogenesis by investigating the dynamic nature of protein expression, cellular and subcellular distribution, post-translational modifications, and interactions at both cellular and subcellular levels. The aims of this review are to 1) highlight the current status of proteomic studies of IBD and 2) introduce the available and emerging proteomic technologies that have potential applications in the study of IBD. These technologies include various mass spectrometry technologies, quantitative proteomics (2D-PAGE, ICAT, SILAC, iTRAQ), protein/antibody arrays, and multi-epitope-ligand cartographie. This review also presents information and methodologies, from sample-selection and enrichment to protein-identification, that are not only essential but also particularly relevant to IBD research. The potential future application of these technologies is expected to have a significant impact on the discovery of novel biomarkers and key pathogenic factors for IBD.

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Isolation and Culture of Human Colon Epithelial Cells Using a Modified Explant Technique Employing a Noninjurious Approach

Cited by 4 publications

References 8 publications

Enhanced sensitivity of colon tumour cells to natural killer cell cytotoxicity after mild thermal stress is regulated through HSF1-mediated expression of MICA

Enhanced sensitivity of colon tumour cells to natural killer cell cytotoxicity after mild thermal stress is regulated through HSF1-mediated expression of MICA

Gene Expression Profiling of Systems Involved in the Metabolism and the Disposition of Xenobiotics: Comparison between Human Intestinal Biopsy Samples and Colon Cell Lines

Applications of proteomics in the study of inflammatory bowel diseases

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