Culture of Intestinal Epithelial Cell Monolayers and Their Use in Multiplex Macromolecular Permeability Assays for In Vitro Analysis of Tight Junction Size Selectivity.

LIGHT Signals Directly to Intestinal Epithelia to Cause Barrier Dysfunction via Cytoskeletal and Endocytic Mechanisms

Intestinal barrier function is reduced in inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) and interleukin (IL)-13, which are up-regulated in IBD, induce barrier defects that are associated with myosin light chain kinase (MLCK) activation and increased claudin-2 expression, respectively, in cultured intestinal epithelial monolayers. Here we report that these independent signaling pathways have distinct effects on tight junction barrier properties and interact in vivo. MLCK activation alters size selectivity to enhance paracellular flux of uncharged macromolecules without affecting charge selectivity and can be rapidly reversed by MLCK inhibition. In contrast, IL-13-dependent claudin-2 expression increases paracellular cation flux in vitro and in vivo without altering tight junction size selectivity but is unaffected by MLCK inhibition in vitro. In vivo, MLCK activation increases paracellular flux of uncharged macromolecules and also triggers IL-13 expression, claudin-2 synthesis, and increased paracellular cation flux. We conclude that reversible, MLCK-dependent permeability increases cause mucosal immune activation that, in turn, feeds back on the tight junction to establish long-lasting barrier defects. Interactions between these otherwise distinct tight junction regulatory pathways may contribute to IBD pathogenesis.

Sealing the ducts

The broad spectrum of interepithelial junctions in skin and lung

Intestinal barrier function relies primarily on the assembly and integrity of tight junctions, which forms a size-selective barrier. This barrier restricts paracellular movement of solutes in various types of epithelia. Of note, extracellular Ca2+ concentration affects tight junction assembly. Therefore, the removal and re-addition of Ca2+ into cell culture medium of cultured intestinal epithelial cells causes destabilization and reassembly of tight junction to membrane periphery near apical surface, respectively. Based on this principle, the Ca2+-switch assay was established to investigate tight junction assembly in fully differentiated intestinal epithelial cells. This chapter provides a stepwise protocol for culture of intestinal epithelial cell monolayers using T84 cell line as an in vitro model and the Ca2+-switch assay for evaluating tight junction assembly.

We have previously shown that growth of the oral squamous cell carcinoma cell line SAS, is resistant to cetuximab in monolayer culture conditions, even though epidermal growth factor receptor (EGFR) was phosphorylated, but the growth of SAS aggregates was sensitive to cetuximab. In the present study, we demonstrate differences in the EGFR signaling pathways utilized by SAS cells in monolayer and suspension cultures at the molecular level. Cetuximab treatment of SAS cells in monolayer cultures inhibits the phosphorylation of EGFR and ERK, and reduces the cell migratory potency, but not cell proliferation. AG1478 treatment reduces the phosphorylation of EGFR, ERK and AKT, and affects cell growth in monolayer cultures. The phosphorylation levels of EGFR and AKT are significantly higher in SAS cell aggregates compared to monolayer cultures. Treatment with cetuximab and AG1478 reduces the growth of SAS aggregates and eliminates the phosphorylation of EGFR and AKT. Furthermore, proliferation of SAS aggregates is also inhibited by LY294002 and MK2206, which are inhibitors of PI3K and AKT, respectively. In addition, treatment with the lipid raft disruptor filipin III reduced the phosphorylation levels of EGFR and Akt in SAS aggregates, but not in SAS monolayer culture. These results suggest the possibility that ligands in the serum stimulate the phosphorylation of EGFR localized in lipid rafts leading to PI3K-AKT activation, which results in the growth of SAS aggregates, therefore resulting in the sensitivity of SAS aggregates to cetuximab.

We have previously shown that growth of the oral squamous cell carcinoma cell line SAS is resistant to cetuximab in monolayer culture conditions even though epidermal growth factor receptor (EGFR) was phosphorylated, whereas growth of SAS aggregates was sensitive to cetuximab. In the present study, we demonstrate differences in the EGFR signaling pathways utilized by SAS cells in monolayer and suspension cultures at the molecular level. Cetuximab treatment of SAS cells in monolayer cultures inhibits the phosphorylation of EGFR and ERK and reduces the cell migratory potency, but not cell proliferation. AG1478 treatment reduces the phosphorylation of EGFR, ERK, and AKT, and affects cell growth in monolayer cultures. The phosphorylation levels of EGFR and AKT are significantly higher in SAS cell aggregates than in monolayer cultures. Treatment with cetuximab and AG1478 reduces the growth of SAS aggregates and eliminates the phosphorylation of EGFR and AKT. Furthermore, proliferation of SAS aggregates is also inhibited by LY294002 and MK2206, which are inhibitors of PI3K and AKT, respectively. In addition, treatment with the lipid raft disruptor filipin III reduced the phosphorylation levels of EGFR and Akt in SAS aggregates, but not in SAS monolayer cultures. These results suggest that ligands in the serum stimulate the phosphorylation of EGFR localized in lipid rafts, leading to PI3K-AKT activation, which results in the growth of SAS aggregates, potentially increasing the sensitivity of SAS aggregates to cetuximab.

During intramembranous bone formation, preosteoblasts condense, differentiate into osteoblasts and deposit bone matrix. We compared the differentiation process of rat calvarial osteoblast-like cells inoculated as micromasses, which mimic the in vivo condensation process, with cells inoculated as monolayers. The cells were analysed morphologically at 1,2 and 3 weeks by light microscopy (alkaline phosphatase activity, mineralization), by transmission electron microscopy, and biochemically (collagen typing, alkaline phosphatase activity, protein and DNA content). The cells inoculated as monolayers formed alkaline phosphatase positive and mineralized nodules during the culture period. The cells inoculated as a micromass formed a large mineralized area consisting of smaller nodules. The ultrastructure of the cells in both culture systems showed the typical features of osteoblasts and osteocytes. The main difference between monolayer and micromass cultures was found after 1 week in culture. The cells inoculated as a micromass formed a multilayer of cells. The cytoplasm contained rER, mitochondria, vesicles and ribosomes. There were abundant collagen fibrils in membrane folds and in the extracellular matrix. This was in contrast to the cells in monolayer culture which showed hardly any collagen fibrils in the extracellular matrix. The promotion of the differentiation was also confirmed by biochemical data showing that the DNA content was lower in the micromass than in the monolayer cultures during the culture period. These results show that micromass, as compared to monolayer, culture promotes the differentiation of rat osteoblast-like cells in vitro.

Fibrin microbeads (FMB) as a 3D platform for kidney gene and cell therapy

Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5-7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm2) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

Cigarette smoke inhibits osteogenic differentiation and proliferation of human osteoprogenitor cells in monolayer and three-dimensional collagen gel culture

The effects of creatine (Cr) supplementation on primary rat osteoblast-like cells cultured as monolayer and micromass were investigated. Cr was added to the medium at concentrations of either 10 or 20 mM. At various time points, the cell cultures were analyzed morphologically, metabolically and biochemically. The degree of differentiation of primary osteoblast-like cell cultures was higher in micromass cultures compared to monolayer cultures, as judged by alkaline phosphatase (ALP) activity and extent of mineralization. In both culture systems, Cr supplementation showed positive effects, which were dependent on the organizational level of the osteoblast-like cells in such a way that the cells in monolayer culture showed significantly increased metabolic activity, ALP activity and mineralization in the presence of Cr than without the supplement. In micromass cultures, Cr also significantly enhanced ALP activity and mineralization, without affecting metabolic activity. The effect of Cr on ALP activity was more pronounced at higher concentrations of Cr, but 20 mM Cr already showed some adverse effects on cell viability. In conclusion, chemically pure Cr added to low serum cell culture medium has a stimulatory effect on metabolic activity, differentiation and mineralization of osteoblast-like cells indicating that Cr supplementation could also be used as a potential clinical intervention to stimulate cell growth, differentiation and mineralization during bone repair in vivo.

In cell culture, both endothelial and epithelial cell monolayers have been found to generate structurally similar tight junctional complexes, as assessed by thin complexes of the two cell types are, at least in part, responsible for the very different permeability characteristics of native endothelial and epithelial cell monolayers. The purpose of this work was to compare cultured endothelial and epithelial cells with respect to the function of their tight junctional complexes in regulating the movement of macromolecules and ions across the cell monolayers, and define functional parameters to characterize the tight junctional complexes. Bovine aorta endothelial cells and T84 colonic carcinoma epithelial cells were cultured on a microporous membrane support. The permeability coefficients of inulin, albumin, and insulin were determined with the cell monolayers and compared with the permeability coefficients obtained with 3T3-C2 fibroblasts, a cell line that does not generate tight junctions. Electrical resistance measurements across the monolayer-filter systems were also compared. The permeability coefficient of albumin across the endothelial cell monolayer compared favorably with other reported values. Likewise, the electrical resistance across the T84 cell monolayer was in good agreement with published values. Utilizing permeability coefficients for macromolecules as an index of tight junction function, we found that a distinction between a lack of tight junctions (fibroblasts), the presence of endothelial tight junctions, and the presence of epithelial tight junctions was readily made. However, when utilizing electrical resistance as an index of tight junction function, identical measurements were obtained with fibroblasts and endothelial cells. This indicates that more than one index of tight junction function is necessary to characterize the junctional complexes. Although structurally similar, epithelial cell and endothelial cell tight junctions perform very different functions, and, from our data, we conclude that the demonstration of tight junctional structures by electron microscopy is not relevant to the functional nature of the junction: structure does not imply function. A minimal assessment of tight junction function should rely on both the determination of the electrical resistance across the cell monolayer, and the determination of the permeability coefficients of selected macromolecules.

Epithelial tight junctions define the paracellular permeability of the intestinal barrier. Molecules can cross the tight junctions via two distinct size-selective and charge-selective paracellular pathways: the pore pathway and the leak pathway. These can be distinguished by their selectivities and differential regulation by immune cells. However, permeability increases measured in most studies are secondary to epithelial damage, which allows non-selective flux via the unrestricted pathway. Restoration of increased unrestricted pathway permeability requires mucosal healing. By contrast, tight junction barrier loss can be reversed by targeted interventions. Specific approaches are needed to restore pore pathway or leak pathway permeability increases. Recent studies have used preclinical disease models to demonstrate the potential of pore pathway or leak pathway barrier restoration in disease. In this Review, we focus on the two paracellular flux pathways that are dependent on the tight junction. We discuss the latest evidence that highlights tight junction components, structures and regulatory mechanisms, their impact on gut health and disease, and opportunities for therapeutic intervention.