Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Khalil, Hassan A.; Nie, Wenxian; Edwards, Robert A.; Yoo, James J.

doi:10.3791/50611

Cited by 32 publications

(32 citation statements)

References 14 publications

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“…To make the HI‐μTP, the spinner ﬂask bioreactor (250 ml, CELLSPIN; Integra Biosciences) was inoculated with a human subepithelial myoﬁbroblast (ISEMFs) extracted ileal biopsy (Khalil, Nie, Edwards, & Yoo, ; Lahar et al, ; Supporting Information S1) after informed consent at a cell density of 10 5 cells/ml and home‐made (Martorina, Casale, Urciuolo, Netti, & Imparato, ) gelatin microbead (Supporting Information S2) density of 2 mg/ml (corresponding to 5 × 10 3 beads/mg), to obtain an initial ratio of 10 cells per microbead. The culture suspension was stirred intermittently at 10 rpm (5 min stirring and 30 min static incubation) for the ﬁrst 6 hr postinoculation to allow cell adhesion, and then continuously at 30 rpm up to 10 days.…”

Section: Methodsmentioning

confidence: 99%

Intestine‐on‐chip device increases ECM remodeling inducing faster epithelial cell differentiation

Gregorio

Corrado

Sbrescia

et al. 2019

Biotech & Bioengineering

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An intestine‐on‐chip has been developed to study intestinal physiology and pathophysiology as well as intestinal transport absorption and toxicity studies in a controlled and human similar environment. Here, we report that dynamic culture of an intestine‐on‐chip enhances extracellular matrix (ECM) remodeling of the stroma, basement membrane production and speeds up epithelial differentiation. We developed a three‐dimensional human intestinal stromal equivalent composed of human intestinal subepithelial myofibroblasts embedded in their own ECM. Then, we cultured human colon carcinoma‐derived cells in both static and dynamic conditions in the opportunely designed microfluidic system until the formation of a well‐oriented epithelium. This low cost and handy microfluidic device allows to qualitatively and quantitatively detect epithelial polarization and mucus production as well as monitor barrier function and ECM remodeling after nutraceutical treatment.

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Section: Methodsmentioning

confidence: 99%

Intestine‐on‐chip device increases ECM remodeling inducing faster epithelial cell differentiation

Gregorio

Corrado

Sbrescia

et al. 2019

Biotech & Bioengineering

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“…The advantage of primary culture is that these cells are relatively unmanipulated and may better reflect the in vivo environment compared to immortalized cell lines 9 .…”

Section: Discussionmentioning

confidence: 99%

Isolation of Myofibroblasts from Mouse and Human Esophagus

Gargus

Niu

Shaker

2015

JoVE

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Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express α-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders. Video LinkThe video component of this article can be found at

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“…We isolated colonocytes and stromal cells following previously published methods with minor modifications (30, 31). Briefly, colons were removed and mucosa scrape-isolated and minced with razor blade into 2 mm pieces.…”

Section: Methodsmentioning

confidence: 99%

ADAM17 is a Tumor Promoter and Therapeutic Target in Western Diet–associated Colon Cancer

Mustafi

Dougherty

Mustafi

et al. 2017

Clinical Cancer Research

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Purpose Epidermal growth factor receptors (EGFR) are required for tumor promotion by Western diet (WD). The metalloprotease, ADAM17 activates EGFR by releasing pro-EGFR ligands. ADAM17 is regulated by G-protein coupled receptors, including CXCR4. Here we investigated CXCR4-ADAM17 crosstalk and examined the role of ADAM17 in tumorigenesis. Experimental Design We used CXCR4 inhibitor, AMD3100 and ADAM17 inhibitor, BMS566394 to assess CXCR4-ADAM17 crosstalk in colon cancer cells. We compared expression of CXCR4 ligand, CXCL2, and ADAM17 in mice fed WD versus standard diet. Separately, mice were treated with marimastat, a broad-spectrum ADAM17 inhibitor, or AMD3100 to assess EGFR activation by ADAM17 and CXCR4. Using Apc mutant Min mice, we investigated effects of ADAM17/10 inhibitor INCB3619 on tumorigenesis. To assess effects of colonocyte ADAM17, mice with ADAM17 conditional deletion were treated with azoxymethane (AOM). ADAM17 expression was also compared in colonocytes from primary human colon cancers and adjacent mucosa. Results CXCL12 treatment activated colon cancer cell EGFR signals, and CXCR4 or ADAM17 blockade reduced this activation. In vivo, WD increased CXCL12 in stromal cells and TGFα in colonocytes. Marimastat or AMD3100 caused >50% reduction in EGFR signals (p<0.05). In Min mice, INCB3619 reduced EGFR signals in adenomas and inhibited intestinal tumor multiplicity (p<0.05). In the AOM model, colonocyte ADAM17 deletion reduced EGFR signals and colonic tumor development (p<0.05). Finally, ADAM17 was up-regulated >2.5-fold in human malignant colonocytes. Conclusions ADAM17 is a WD-inducible enzyme activated by CXCL12-CXCR4 signaling, suggesting the pathway: Western diet->CXCL12->CXCR4->ADAM17->TGFα‐>EGFR. ADAM17 might serve as a druggable target in chemoprevention strategies.

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Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Cited by 32 publications

References 14 publications

Intestine‐on‐chip device increases ECM remodeling inducing faster epithelial cell differentiation

Intestine‐on‐chip device increases ECM remodeling inducing faster epithelial cell differentiation

Isolation of Myofibroblasts from Mouse and Human Esophagus

ADAM17 is a Tumor Promoter and Therapeutic Target in Western Diet–associated Colon Cancer

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