Modular microsystem for epithelial cell culture and electrical characterisation

We have realised a microsystem for the culture and electrical characterisation of epithelial cell layers for cell-based diagnostic applications. The main goal of this work is to achieve both cell culture and impedimetric and potentiometric characterisation on a single device. The miniaturised cell culture system enables the uses of scarce epithelial cells, as obtained from transgenic mice or from human biopsies. The device is completely modular and offers high flexibility: a polycarbonate membrane used as cell substrate is glued in between two moulded Polydimethylsiloxane (PDMS) layers to form a sandwich, which is placed between two stacks, containing the microfluidic channels and integrated measurement electrodes. The polycarbonate membrane sandwich can be removed, replaced or analysed at any time. We have characterised the impedimetric properties of our microsystem, demonstrated epithelial cell layer growth within it, and have done the initial electrical characterisation of epithelial cell layers.     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.     

Stochastic model of autocrine and paracrine signals in cell culture assays

Autocrine signaling systems are commonly studied under cell culture conditions. In a typical cell culture assay, a layer of liquid medium covers a random two-dimensional dispersion of cells, which secrete ligands. In a growing number of experiments, it is important to characterize the spatial range of autocrine and paracrine cell communication. Currently, the spatial distribution of diffusing signals can be analyzed only indirectly, from their effects on the intracellular signaling or physiological responses of autocrine cells. To directly characterize the spatial range of secreted ligands, we propose a stochastic model for autocrine cell cultures and analyze it using a combination of analytical and computational tools. The two main results derived within the framework of this model are 1), an expression for the fraction of autocrine trajectories, i.e., the probability for a ligand to be trapped by the same cell from which it has been secreted; and 2), an expression for the spatial distribution of trapping points of paracrine trajectories. We test these analytical results by stochastic simulations with efficient Brownian dynamics code and apply our model to analyze the spatial operation of autocrine epidermal growth factor receptor systems.     

A culture system for the development of the preimplantation rat embryo

We describe a system for the culture of 1-cell rat embryos through to the blastocyst stage, using co-culture on specific feeder cell layers and particular defined media. We show that the use of rat uterine epithelial cells as a feeder layer, together with either M16M, CZB or HECM-1 media at 38.5 degrees C can improve the in vitro development of cultured rat embryos. There was considerable variation in the culture conditions, which were optimal for each strain of rat tested. We show that the 4 to 8-cell embryos are viable after reimplantation and that the second 4 to 8-cell block in the rat can be overcome using HECM-1 in a co-culture system, thus enabling the in vitro culture of rat embryos up to the blastocyst stage.     

Regulation of cell proliferation in a stratified culture system of epithelial cells from prostate tissue

Mechanisms controlling epithelial proliferation and differentiation in the prostate have been primarily investigated in mouse models. The regulation of proliferation and differentiation is poorly understood in human prostate epithelial cells. In vivo, the glandular prostate epithelium consists of a p63-positive proliferating basal cell layer and a post-mitotic p27-positive secretory cell layer. We have established an organized stratified culture system of human primary prostate epithelial cells to gain insight into mechanisms regulating proliferation and differentiation. In this system, expression of p63 is observed in the bottom layer. In addition, BrdU incorporation persists even though cells are confluent. In contrast, in the upper layer, p63 expression is greatly diminished, p27 is expressed, and the cells are growth arrested. Overexpression of cyclin D1 or knockdown of p27 does not increase proliferation. After inactivation of the nuclear phosphoprotein Rb, the cell layers remain organized and cell proliferation increases only in the bottom layer. Furthermore, the expression of p63 remains confined to the bottom layer after Rb inactivation. Altogether, this in vitro model recapitulates certain aspects of in vivo growth regulation and differentiation and suggests that the loss of Rb family proteins in human cells trigger hyperplasia but is not sufficient for transformation.This work was supported by the Departments of Pathology and Urology at Weill Medial College, by grants DAMD-17-02-1-0159, MEDC-GR-355, and P30 CA015704-30, and by grant RO1CA84069 to B.E.C.     

Glycosylation and annexin II cell surface translocation mediate airway epithelial wound repair

Glycosylation of cell surface proteins can regulate multiple cellular functions. We hypothesized that glycosylation and expression of glycoproteins after epithelial injury is important in mediating repair. We report the use of an in vitro culture model of human airway epithelial cells (1HAEo(-)) to identify mediators of epithelial repair. We characterized carbohydrate moieties associated with repair by their interaction with the lectin from Cicer arietinum, chickpea agglutinin (CPA). Using CPA, we identified changes in cell surface glycosylation during wound repair. Following mechanical wounding of confluent monolayers of 1HAEo(-) cells, CPA staining increases on the cell surface of groups of cells in proximity to the wound edge. Blocking the CPA carbohydrate ligand inhibited wound repair highlighting the role of the CPA carbohydrate ligand in epithelial repair. Annexin II (AII), a calcium-dependent, membrane-associated protein, was identified as a protein associated with the CPA ligand. By membrane protein biotinylation and immunodetection, we have shown that following mechanical wounding, the presentation of AII on the cell surface increases coordinate with repair. Cell surface AII accumulates in proximity to the wound. Furthermore, translocation of AII to the cell surface is N-glycosylation dependent. We are the first to demonstrate that following injury, N-glycosylation events and AII presentation on the cell surface of airway epithelial cells are important mediators in repair.     

Fibroblast growth factor signalling controls successive cell behaviours during mesoderm layer formation in Drosophila

Fibroblast growth factor (FGF)-dependent epithelial-mesenchymal transitions and cell migration contribute to the establishment of germ layers in vertebrates and other animals, but a comprehensive demonstration of the cellular activities that FGF controls to mediate these events has not been provided for any system. The establishment of the Drosophila mesoderm layer from an epithelial primordium involves a transition to a mesenchymal state and the dispersal of cells away from the site of internalisation in a FGF-dependent fashion. We show here that FGF plays multiple roles at successive stages of mesoderm morphogenesis in Drosophila. It is first required for the mesoderm primordium to lose its epithelial polarity. An intimate, FGF-dependent contact is established and maintained between the germ layers through mesoderm cell protrusions. These protrusions extend deep into the underlying ectoderm epithelium and are associated with high levels of E-cadherin at the germ layer interface. Finally, FGF directs distinct hitherto unrecognised and partially redundant protrusive behaviours during later mesoderm spreading. Cells first move radially towards the ectoderm, and then switch to a dorsally directed movement across its surface. We show that both movements are important for layer formation and present evidence suggesting that they are controlled by genetically distinct mechanisms.     

Discrete models of autocrine cell communication in epithelial layers

Pattern formation in epithelial layers heavily relies on cell communication by secreted ligands. Whereas the experimentally observed signaling patterns can be visualized at single-cell resolution, a biophysical framework for their interpretation is currently lacking. To this end, we develop a family of discrete models of cell communication in epithelial layers. The models are based on the introduction of cell-to-cell coupling coefficients that characterize the spatial range of intercellular signaling by diffusing ligands. We derive the coupling coefficients as functions of geometric, cellular, and molecular parameters of the ligand transport problem. Using these coupling coefficients, we analyze a nonlinear model of positive feedback between ligand release and binding. In particular, we study criteria of existence of the patterns consisting of clusters of a few signaling cells, as well as the onset of signal propagation. We use our model to interpret recent experimental studies of the EGFR/Rhomboid/Spitz module in Drosophila development.     

Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium

We have previously shown that a basic 64-kilodalton (no. 3 in the catalog of Moll et al.) and an acidic 55-kilodalton (no. 12) keratin are characteristic of suprabasal cell layers in cultured rabbit corneal epithelial colonies, and therefore may be regarded as markers for an advanced stage of corneal epithelial differentiation. Moreover, using an AE5 mouse monoclonal antibody, we showed that the 64-kilodalton keratin marker is expressed suprabasally in limbal epithelium but uniformly (basal layer included) in central corneal epithelium, suggesting that corneal basal cells are in a more differentiated state than limbal basal cells. In conjunction with previous data implicating the centripetal migration of corneal epithelial cells, our data support a model of corneal epithelial maturation in which corneal epithelial stem cells are located in the limbus, the transitional zone between the cornea and conjunctiva. In the present study, we analyzed the expression of the 64-kilodalton keratin in developing human corneal epithelium by immunohistochemical staining. At 8 weeks of gestation, the presumptive corneal epithelium is composed of a single layer of cuboidal cells with an overlying periderm; neither of these cell layers is AE5 positive. At 12-13 weeks of gestation, some superficial cells of the three- to four-layered epithelium become AE5 positive, providing the earliest sign of overt corneal epithelial differentiation. At 36 weeks, although the epithelium is morphologically mature (four to six layers), AE5 produces a suprabasal staining pattern, this being in contrast to the adult epithelium which exhibits uniform staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity

Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two- to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 µg/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair. c-met protooncogene; transepithelial resistance; Madin-Darby canine kidney cell     

Mucociliary interactions and mucus dynamics in ciliated human bronchial epithelial cell cultures

The airway epithelial surface liquid is generally considered to be composed of two layers, a periciliary layer and a continuous thick mucus layer moving in bulk. This view may not be appropriate for all areas of the lung. Our hypothesis, that mucus may form a discontinuous layer with dynamic attachments to the surface, is investigated using a culture system. We used live-cell confocal microscopy to investigate thin mucus layers and fluorescent beads and exogenous MUC5B to visualize mucus dynamics on ciliated human bronchial cultures. A continuous mucus layer was not observed. In sparsely ciliated cultures, mucus attached to ciliated cells; however, in highly ciliated cultures, mucus formed strands several hundred micrometers long. As with increases in ciliation, increases in bead concentration caused the appearance of mucus strands. We confirmed the involvement of mucins in the binding of mucus to cilia by adding labeled purified MUC5B to the cultures. These data suggest that mucins may have an intrinsic ability to form attachments to cilia. The significance of these findings is that aberrant modulation of such an intrinsic property may explain the initiation of highly adherent mucus in cystic fibrosis lung disease.     

An adherent mucus layer attenuates the genotoxic effect of colibactin

The human gastrointestinal tract is a complex ecosystem in which epithelial cells and microorganisms of the intestinal microbiota live in symbiosis. Certain members of the microbiota, in particular Escherichia coli strains of the B2 phylotype, carry the polyketide synthase‐island encoding the genotoxin colibactin. Colibactin is a nonribosomal peptide or polyketide‐nonribosomal peptide hybrid of still unsolved structure, which induces DNA double strand breaks (DSBs) in eukaryotic cells. However, direct contact between live bacteria and host cell is required in order to elicit these genotoxic effects. In this study, we used a variety of cell culture models, among them, a 3D cell culture approach based on decellularised small intestinal submucosa, to investigate whether the intestinal mucus layer has the potential to interfere with colibactin activity. We demonstrate that the expression of mucins and the formation of an adherent mucus layer significantly increased with increasing complexity of cell culture. Moreover, we show that the presence of an adherent mucus layer on epithelial cells attenuates the genotoxic activity of colibactin, by preventing the induction of DNA‐DSBs. Removal of the adherent mucus layer restored the occurrence of DNA‐DSBs.     

Induction of cAMP receptors by disaggregation of the multicellular complexes of Dictyostelium discoideum

Subcultivated rat lingual epithelial cells when grown on collagen gels at a liquid-gas interface achieve a highly ordered state that closely resembles the parent tissue. Three distinct cell layers are present; basal, spinous, and keratinized. Basal cells are cuboidal in shape and form a complex interface with the underlying collagen fibrils. Spinous cells form a layer 5–10 cells thick and, with the exception of keratohyalin granules, possess an organellar complement identical with native cells, including membrane-coating granules. The keratinized cell layer increases in thickness as a function of time spent in culture. Forty or more plies of terminally differentiated cells are observed following a 30-day culture period. Terminally differentiated cells while retaining pycnotic nuclei and some other organellar debris are principally envelope-enclosed squames filled with tonofilaments. Keratinization is a continuing process which occurs simultaneously across the full expanse of the culture surface. The high degree of tissue organization observed appears to be the result of feeding the cultures from the undersurface.     

A comparison of primary oesophageal squamous epithelial cells with HET‐1A in organotypic culture

Background Information. Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5‐year survival of less than 15%. Recent evidence suggests that stromal—epithelial interactions are fundamental in carcinogenesis. The advent of co‐culture techniques permits the investigation of cross‐talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET‐1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro. Results. When grown in an organotypic culture with normal fibroblasts, the oesophageal carcinoma cell lines OE21 (squamous) and OE19 (adenocarcinoma) morphologically resembled the tumour of origin with evidence of stromal invasion and mucus production, respectively. However, HET‐1A cells, which were derived from normal squamous oesophageal cells, appeared dysplastic and failed to display evidence of squamous differentiation. By comparison with primary oesophageal epithelial cells, the HET‐1A cells were highly proliferative and did not express the epithelial markers E‐cadherin or CK5/6 (casein kinase 5/6), or the stratified epithelial marker ΔNp63, but did express the mesenchymal markers vimentin and N‐cadherin. Conclusion. Studies of epithelial carcinogenesis will benefit from culture systems which allow manipulation of the stromal and epithelial layers independently. We have developed an organotypic culture using primary oesophageal squamous cells and fibroblasts in which a stratified epithelium with a proliferative basal layer that stains strongly for ΔNp63 develops. This model will be suitable for the study of the molecular events in the development of Barrett's oesophagus. The most commonly used normal oesophageal squamous cell line, HET‐1A, does not have the characteristics of normal oesophageal squamous cells and should not be used in models of the normal oesophageal epithelium. Until more representative cell lines are available, future studies in oesophageal cancer will be reliant on the availability and manipulation of primary tissue.     

Cell proliferation in bovine mammary epithelium: identification of the primary proliferative cell population

Histologic analyses indicate that a lightly staining cell population present in mammary parenchyma may function as mammary stem cells. We performed an analysis of mammary epithelial cell proliferation in prepubertal bromodeoxyuridine (BrdU)-injected Holstein heifers to investigate this hypothesis. We observed light, dark, and intermediate staining cells in histologic sections stained with basic fuschin and azure II. Light cells comprised 10% of the total parenchymal cell population but accounted for 50% of the cell proliferation. Intermediate cells comprised 60% of the cell population and 43% of proliferating cells. Dark cells comprised 30% of the parenchymal cell population but only 7% of proliferating cells. The distribution of BrdU+ cells across basal, embedded, and lumenal parenchymal cell layers was correlated with the fraction of total parenchymal cells present in each layer (r=0.99). However, the proportion of mitotic cells observed in the basal cell layer was only half of what would be predicted by the BrdU-labeling data. This observation suggests that some basal cells either arrest in G(2) or migrate into the suprabasal epithelial layers before undergoing mitosis. These results strongly support the concept that lightly staining mammary parenchymal cells are the primary proliferative cell population.     

A Two‐Resonance Tapping Cavity for an Optimal Light Trapping in Thin‐Film Solar Cells

An optimal photon absorption in thin film photovoltaic technologies can only be reached by effectively trapping the light in the absorber layer provided a considerable portion of the photons is rejected or scattered out of such layer. Here, a new optical cavity is proposed that can be made to have a resonant character at two different nonharmonic frequencies when adjusting the materials or geometry configurations of the partially transmitting cavity layers. Specific configurations are found where a reminiscence of such two fundamental resonances coexists leading to a broadband light trapping. When a PTB7‐Th:PC₇₁BM organic cell is integrated within such cavity, a power conversion efficiency of 11.1% is measured. This study also demonstrates that when materials alternative to organics are used in the photoactive cell layer, a similar cavity can be implemented to also obtain the largest light absorption possible. Indeed, when it is applied to perovskite cells, an external quantum efficiency is predicted that closely matches its corresponding internal one for a broad wavelength range.     

Metalloprotease disintegrin-mediated ectodomain shedding of EGFR ligands promotes intestinal epithelial restitution

EGF receptor (EGFR) promotes intestinal epithelial restitution, an important early process in the reepithelialization of ulcers. During epithelial restitution, the mechanism of EGFR activation is not known. We evaluated the role of TNF-converting enzyme (TACE), a metalloprotease disintegrin that proteolytically processes plasma membrane-anchored EGFR ligand precursors into their mature active forms, in wound-induced EGFR activation and epithelial restitution. With the use of scrape-wounded rat intestinal epithelial-1 (RIE-1) cell monolayers to model epithelial ulceration and restitution, we observed the rapid wound-dependent release of EGFR ligands into culture medium. RIE-1 cells express TACE, and treatment with phorbol ester, an established TACE stimulus, triggered the extracellular release of an EGFR ligand, transforming growth factor-alpha. Blockade of TACE using TNF processing inhibitor (TAPI-1), a specific hydroxamate inhibitor of metalloprotease disintegrins, prevented release of EGFR ligands from wounded RIE-1 cell monolayers. The restitution of wounded RIE-1 cell monolayers was also dose-dependently inhibited by TAPI-1, establishing the role of metalloprotease disintegrins in this process. These results have established a mechanism of EGFR activation in wounded intestinal epithelium and show an important functional role for metalloprotease disintegrin-mediated ectodomain shedding during intestinal epithelial restitution. Therefore, activation of the TACE-EGFR system might promote the healing of intestinal tract ulcers in patients.     

Influence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o-: implications for the use of this cell line as an in vitro model for bronchial drug absorption studies

Air-interfaced culture (AIC) versus liquid-covered culture (LCC) conditions are known to have different effects on the differentiated phenotype of several cell types, including lung epithelial cells. We report the influence of culture conditions such as apical medium volume on the development of intercellular junctions in the human epithelial cell line 16HBE14o-. Immunofluorescence staining of the tight-junctional protein, ZO-1, has revealed its presence in cells grown in both AIC and LCC. However, only LCC-grown cells exhibit protein ZO-1 localized as a zonula-occludens-like regular belt connecting neighboring cells. The presence of typical tight junctions has been confirmed by electron microscopy. Immunostaining for occludin, claudin-1, connexin43, and E-cadherin has demonstrated intercellular junction structures only in the cells in LCC. These morphological findings have been paralleled by higher transepithelial electrical resistance values and similar fluxes of the hydrophilic permeability marker, fluorescein-Na, under LCC compared with AIC conditions. We conclude that the formation of functional 16HBE14o- cell layers requires the presence of an apical fluid volume, in contrast to other culture conditions for airway epithelial cells.     

Correlated motion and oscillation of neighboring cells in vitro

It has long been realized that fibroblastic and epithelial cells establish recognizable patterns in tissue culture. This behavior implies that neighboring cells interact with one another to produce organized populations. Interaction between cells that are separated by many intervening cells is also possible and is demonstrated here using a special configuration of a biosensor referred to as electric cell-substrate impedance sensing (ECIS). Normally the electrical impedance of a single electrode covered with a confluent cell layer is measured, and the morphological changes of the cells are reflected in the impedance. In this case the cells are cultured on two closely spaced electrodes whose impedances are measured independently as a function of time, and communication between the cell populations is revealed as a correlation between these two time series. We also report for the first time another striking manifestation of dynamic cell interaction, where confluent layers of Madin-Darby canine kidney cells (MDCK) on a single electrode are observed to oscillate in synchrony with a period of approximately 2.5 h.