human colonic fibroblast ccd18co  (ATCC)

Journal: Advanced Science

Article Title: Direct Extracellular Matrix Modulation Attenuates Intestinal Fibrosis via a Fibronectin‐Targeted Approach

doi: 10.1002/advs.202519433

Figure Lengend Snippet: Fibronectin, as the ECM scaffold, accumulates in fibrotic intestines. (A, B) Matrixome analysis in clinical specimens (A colon and B ileum). In the pie chart, red refers to increased ECM in Crohn's intestine; blue refers to decreased ECM in Crohn's intestine. The bar plots show the relative abundance of fibronectin in the control and Crohn's group ( n = 8). (C) Gross specimens and H&E staining of native and decellularized intestinal tissues. (D) Schematic workflow of intestinal decellularization and proteomic sequencing. Mice colons were bisected longitudinally; one segment underwent PBS rinsing (control), and the other underwent decellularization. Both were subjected to proteomic sequencing and analysis. Groups: control ( n = 3), fibrosis ( n = 4). (E, F) Matrixome analysis in the intestinal fibrosis model. The gray region in the pie chart refers to fibronectin. The bar plots show the relative abundance of fibronectin in the control and fibrosis group under native or decellularized conditions. (G) Representative Masson's trichrome staining and fibronectin IHC of healthy (Control group, n = 10) and fibrotic (Fibrosis group, n = 10) human intestinal tissues. (H) Quantification of submucosal thickness and fibronectin abundance in human specimens. (I) Fibronectin transcription levels change in fibrotic cell models ( n = 6). (J–L) Relative abundance of soluble fibronectin and fibronectin fibrils in fibrotic cell models ( n = 3). (M–O) Differences in fibronectin abundance and morphological changes in cell models ( n = 5). (P, Q) Colocalization of fibronectin fibrils with type I/III collagen fibers in vitro and in vivo. Human intestinal fibroblasts CCD18Co were used for cell models. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001. Continuous data were analyzed using Student's t‐test. Nonparametric distributions were analyzed by Mann‐Whitney/Wilcoxon rank‐sum tests. IHC, immunohistochemistry; ECM, extracellular matrix; DSS, dextran sulfate sodium salt; TNBS, 2,4,6‐trinitrobenzenesulfonic acid. MFI, mean fluorescence intensity. Scale bar: 2.5 mm and 100 µm (G, Masson staining); 500 and 100 µm (G, Fibronectin); 20 µm (M); 100 µm (P and Q).

Article Snippet: Human colonic fibroblast CCD18Co (ATCC Cat# CRL‐1459, RRID:CVCL_2379, Lot Number: 70045368, contamination‐free confirmed by ATCC), derived from normal colon tissue of a 2.5‐month‐old female, was cultured in fibroblast medium containing 1% penicillin‐streptomycin, 2% fetal bovine serum, and 1% fibroblast growth factor [ ].

Techniques: Control, Staining, Sequencing, In Vitro, In Vivo, MANN-WHITNEY, Immunohistochemistry, Fluorescence

Journal: Advanced Science

Article Title: Direct Extracellular Matrix Modulation Attenuates Intestinal Fibrosis via a Fibronectin‐Targeted Approach

doi: 10.1002/advs.202519433

Figure Lengend Snippet: Blocking fibronectin assembly impedes fibrillogenesis and ameliorates intestinal fibrosis. (A) Schematic of pUR4 targeting fibronectin domains (detailed in ). (B) Proposed mechanism of pUR4 action on extracellular matrix. (C) Molecular docking of pUR4 with fibronectin. (D) The relative abundance of soluble fibronectin and fibronectin fibers with pUR4 treatment. (E) ELISA of fibronectin in cell culture supernatants. (F) Representative images (of three biological replicates) depicting pUR4 effect on fibronectin (white arrows) and collagen (yellow arrows) fibers; white boxes show magnified regions. (G) The relative abundance of soluble collagen and collagen fibers with pUR4 treatment. (H) Schematic of R1R2 targeting fibronectin domains. (I) Molecular docking of R1R2 with fibronectin. (J) Proposed mechanism of R1R2 action on ECM. (K) R1R2 effects on fibronectin/collagen fibers; conventions as in (F). (L) DSS model dosing protocol: Mice received daily i.p. pUR4 (25 mg/kg) or scrambled pUR4 (equimolar) from DSS initiation until endpoint (Control, n = 7; DSS+scrambled pUR4, n = 9; DSS+pUR4, n = 9). (M, N) Macroscopic colon images (M) and Masson's trichrome staining (N) in the DSS model. (O) Quantification of colon length, collagen volume fraction, and submucosal thickness. (P) TNBS model dosing: Protocol as in (L) with TNBS challenge (Control, n = 5; TNBS+scrambled pUR4, n = 7; TNBS+pUR4, n = 12). (Q, R) Macroscopic colon images (M) and Masson's trichrome staining (N) in the TNBS model. (S) Quantification of colon length, collagen volume fraction, and submucosal thickness. Human intestinal fibroblasts CCD18Co were used for in vitro studies. Data are presented as mean ±SD. * p < 0.05; ** p < 0.01; *** p < 0.001. Continuous data were analyzed using ANOVA for unpaired groups. Nonparametric distributions were analyzed by Kruskal‐Wallis tests, with Dunn's post hoc testing for multiple comparisons. IHC, immunohistochemistry; ECM, extracellular matrix; DSS, dextran sulfate sodium salt; TNBS, 2,4,6‐trinitrobenzenesulfonic acid. Scale bar: 20 µm (J. K) and 5 µm (Enlarged); 100 µm (N, R).

Article Snippet: Human colonic fibroblast CCD18Co (ATCC Cat# CRL‐1459, RRID:CVCL_2379, Lot Number: 70045368, contamination‐free confirmed by ATCC), derived from normal colon tissue of a 2.5‐month‐old female, was cultured in fibroblast medium containing 1% penicillin‐streptomycin, 2% fetal bovine serum, and 1% fibroblast growth factor [ ].

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Staining, In Vitro, Immunohistochemistry

Journal: Advanced Science

Article Title: Direct Extracellular Matrix Modulation Attenuates Intestinal Fibrosis via a Fibronectin‐Targeted Approach

doi: 10.1002/advs.202519433

Figure Lengend Snippet: Fibronectin in the extracellular matrix regulates cellular activation and fibrillogenesis. (A) Transcriptional impact of pUR4 and R1R2 on FN1 and COL1A1. (B) Schematic workflow for ECM coating experiments. (C) The gene expression changes of fibroblasts in different ECM coating matrices. low FN (5µg/mL), high FN (50µg/mL), and collagen (5µg/mL). (D) Collagen I/III morphology on FN‐coated vs. non‐coated matrices. Background FN fluorescence was high on FN‐coated matrices with low intracellular signal, but low in non‐coated systems with prominent intracellular FN accumulation (yellow circles: single cells; white boxes: magnified regions; white arrows: ECM fibers). (E) Decellularization model workflow. HIFs were cultured at 2.5 × 10^4 cells per well (12‐well plate) for 3 days before decellularized. Low fibronectin group: derived from CCD18co. High fibronectin group: derived from TGFβ‐treated CCD18co. (F) Validation of decellularization: Pre‐treatment showed intact cell structures, fibrous FN, and soluble collagen. Post‐decellularization preserved FN fibers while eliminating cells and collagen. (G) Transcriptional differences induced by varying FN levels in decellularized matrices. (H) Cell morphology modulation by FN density: Low FN promoted spindle shapes with diffuse α‐SMA; high FN induced flattened, polygonal cells with enlarged size and α‐SMA‐rich stress fibers. (I, J) Quantification of cell size and α‐SMA MFI (8 sights from 4 biological replicates). (K) Thick‐layer decellularization model workflow. HIFs were cultured at 1 × 10^5 cells per well (12‐well plate) for 5 days before decellularized. Low fibronectin group: derived from CCD18co. High fibronectin group: derived from TGFβ‐treated CCD18co. (L) Presentation of the remaining fibronectin ECM in the thick‐layer decellularization model. In the low fibronectin group, fibronectin is diffusely distributed across the dish bottom, appearing as punctate spots along with a small number of thin filaments. In the high fibronectin group, a greater amount of fibronectin is diffusely distributed across the entire dish bottom, accompanied by abundant thick, fibrous bundles. (M) Transcriptional differences in the thick‐layer decellularization model. Data are presented as mean ±SD. * p < 0.05; ** p < 0.01; *** p < 0.001. Continuous data were analyzed using Student's t‐test or ANOVA for unpaired groups. Nonparametric distributions were analyzed by Mann‐Whitney/Wilcoxon rank‐sum tests or Kruskal‐Wallis tests, with Dunn's post hoc testing for multiple comparisons. FN, fibronectin; ECM, extracellular matrix; MFI, mean fluorescence intensity. Scale bar: 50 and 10 µm (Enlarged).

Article Snippet: Human colonic fibroblast CCD18Co (ATCC Cat# CRL‐1459, RRID:CVCL_2379, Lot Number: 70045368, contamination‐free confirmed by ATCC), derived from normal colon tissue of a 2.5‐month‐old female, was cultured in fibroblast medium containing 1% penicillin‐streptomycin, 2% fetal bovine serum, and 1% fibroblast growth factor [ ].

Techniques: Activation Assay, Gene Expression, Fluorescence, Cell Culture, Derivative Assay, Biomarker Discovery, MANN-WHITNEY

Journal: Advanced Science

Article Title: Direct Extracellular Matrix Modulation Attenuates Intestinal Fibrosis via a Fibronectin‐Targeted Approach

doi: 10.1002/advs.202519433

Figure Lengend Snippet: Integrin α5β1 serves as the cellular receptor bridging ECM fibronectin to the cell. (A) STRING protein interaction map of major fibronectin integrin receptors. (B, C) Representative images and quantification of major integrin receptor expression in clinical specimens (Control, n = 6; Fibrosis, n = 6). (D) Representative images of major integrin receptors in animal models. (E) Integrin expression differences in fibroblasts under TGFβ activation ( n = 3). (F) Integrin expression differences in fibroblasts in the ECM coating model ( n = 3). (G) Integrin expression differences in fibroblasts in the decellularization model ( n = 3). (H–K) Distribution of integrins and fibronectin in fibroblasts and their pericellular ECM. (L) Co‐localization analysis of integrins and fibronectin. The selected parts for co‐localization analysis can be found in Figure . Human intestinal fibroblasts CCD18Co were used for cell models. Data are presented as mean ±SD. Continuous data were analyzed using Student's t‐test. Nonparametric distributions were analyzed by Mann‐Whitney. * p < 0.05; ** p < 0.01; *** p < 0.001. FN, fibronectin; ECM, extracellular matrix. Scale bar: 100 µm (B, D); 50 µm (H–K).

Article Snippet: Human colonic fibroblast CCD18Co (ATCC Cat# CRL‐1459, RRID:CVCL_2379, Lot Number: 70045368, contamination‐free confirmed by ATCC), derived from normal colon tissue of a 2.5‐month‐old female, was cultured in fibroblast medium containing 1% penicillin‐streptomycin, 2% fetal bovine serum, and 1% fibroblast growth factor [ ].

Techniques: Expressing, Control, Activation Assay, MANN-WHITNEY

Journal: Advanced Science

Article Title: Direct Extracellular Matrix Modulation Attenuates Intestinal Fibrosis via a Fibronectin‐Targeted Approach

doi: 10.1002/advs.202519433

Figure Lengend Snippet: Fibronectin regulates intestinal fibrosis via integrin‐mediated mechanotransduction. (A, B) Schematic representations of ATN161 acting on fibronectin domains. (C) ATN161 does not alter ECM fibronectin or collagen morphology. (D) Gene expression differences in integrin‐mediated mechanotransduction pathways (FAK/SRC and ROCK/RhoA) in the ECM coating model. (E) Gene expression differences in integrin‐mediated mechanotransduction pathways in the decellularization model. (F–H) Impact of ATN161 on fibroblast activation and mechanotransduction pathway expression. (I, J) Effect of ATN161 on fibroblast activation in fibronectin‐coated and decellularized matrices. (K) Dosing regimen for TNBS model: Mice received ATN161 (1 mg/kg every two days, i.p.) or equimolar ATN163 from TNBS initiation until endpoint (Control, n = 6; TNBS+ATN161, n = 6; TNBS+ATN163, n = 8). (L) Macroscopic colon appearance in the TNBS model. (M) Masson's trichrome staining in the TNBS model. (N) Quantification of colon length, collagen volume fraction, and submucosal thickness in the TNBS model. (O) Dosing regimen for DSS model (parameters as in K; Control, n = 8; DSS+ATN161, n = 8; DSS+ATN163, n = 8). (P) Macroscopic colon appearance in the DSS model. (Q) Masson's trichrome staining in the DSS model. (R) Quantification of colon length, collagen volume fraction, and submucosal thickness in the DSS model. Human intestinal fibroblasts CCD18Co were used for cell models. Data are presented as mean ±SD. Continuous data were analyzed using Student's t‐test or ANOVA for unpaired groups. Nonparametric distributions were analyzed by Mann‐Whitney/Wilcoxon rank‐sum tests or Kruskal‐Wallis tests, with Dunn's post hoc testing for multiple comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001. ECM, extracellular matrix; DSS, dextran sulfate sodium salt; TNBS, 2,4,6‐trinitrobenzenesulfonic acid. Scale bar: 20 µm (C); 100 µm (M, Q).

Article Snippet: Human colonic fibroblast CCD18Co (ATCC Cat# CRL‐1459, RRID:CVCL_2379, Lot Number: 70045368, contamination‐free confirmed by ATCC), derived from normal colon tissue of a 2.5‐month‐old female, was cultured in fibroblast medium containing 1% penicillin‐streptomycin, 2% fetal bovine serum, and 1% fibroblast growth factor [ ].

Techniques: Gene Expression, Activation Assay, Expressing, Control, Staining, MANN-WHITNEY

Journal: Animal Cells and Systems

Article Title: Bromelain effectively suppresses Kras-mutant colorectal cancer by stimulating ferroptosis

doi: 10.1080/19768354.2018.1512521

Figure Lengend Snippet: ACSL-4 induced ferroptosis. (a) Four different CRCs, CacO 2 , NCI-H508, HCT-116, and DLD-1, were cultured with different concentrations of erastin (left panel) or 5 μM erastin with or without bromelain (right panel) and cell death was analyzed. (b) Four different CRCs, CacO 2 , NCI-H508, HCT-116, and DLD-1, and normal colon cells, CCD18co, were treated with or without bromelain and the expression level of ACSL-4 was analyzed. (c) DLD-1 cells were treated with erastin, siACSL-4, or bromelain. Cell death was quantified by PI staining coupled with flow cytometry (upper panel) and lipid ROS was measured (lower panel). (d) Schematic diagram describing the action of bromelain in ferroptosis. The results are shown as the mean, with error bars representing the 95% confidence interval (lower/upper limit); * p < 0.05.

Article Snippet: Human normal colon fibroblasts CCD18co (CRL-1459, ATCC, Manassas, VA, USA) were grown in Eagle's Minimum Essential Medium (Gibco) supplemented with 10% fetal bovine serum, 1% antibiotic/anti-mycotic, 2 mM GlutaMAX, and 0.1 mM non-essential amino acids (Invitrogen).

Techniques: Cell Culture, Expressing, Staining, Flow Cytometry