Ocular cell monolayers cultured on biodegradable substrates.

The aim of this study was to culture retinal pigment epithelial (RPE) and corneal endothelial cells on biodegradable substrates for future use in monolayer transplantation in the eye. The biodegradable polymers, poly-l-lactic (PLLA) and poly-dl-lactic-co-glycolic acid (85:15) (PLGA) (both of molecular weight 105 kd) were the biomaterials used. All materials were seeded with either pig/human retinal pigment epithelial cells or rabbit corneal endothelial cells and were maintained in tissue culture conditions. Upon confluency, the cell density was calculated and cell viability determined. All monolayers were stained with phalloidin-rhodamine for F-actin and antibodies to the tight junction (zonula occludens) protein, ZO1, to demonstrate the presence of tight junctions. The final cell density of human RPE monolayers on PLLA films was 2950 cells/mm(2) (+/-185). The final cell density of pig RPE on PLLA and PLGA film was 2350 cells/mm(2) (+/-152 and 178, respectively). Rabbit corneal endothelial cells had a final cell density of 2650 cells/mm(2) (+/-164). F-actin staining revealed a circumferential ring of actin filaments in all of the cells grown on substrates. ZO(1) immunohistochemistry demonstrated staining along the lateral cell borders of all cell types. The successful culture of retinal pigment epithelial and corneal endothelial monolayers on these substrates may have potential for transplanting cell monolayers in the eye to improve vision.     

白蛋白对内皮细胞单层屏障功能的增强作用

我们建立了体外灌流内皮细胞单层研究血管能透性的方法，可以测定内皮单层的滤过系数（Ｋｆ）和渗透压筢射系数（σ），研究了白蛋白对内皮单层通透屏障功能的影响。不同浓度白蛋白溶液（１，５，１０，２０ｍｇ／ｍｌ）灌流单纯的滤膜时，其流量变化不大，灌流膜上致密生长的内皮细胞单层时，流量和Ｋｆ值随白蛋白浓度的增加而降低。同时，蛋白清除率也不断减小，σ增大，说明白蛋白通过与内皮细胞的作用使内皮单层对水和大分子物质     

Penetration of endothelial cell monolayers by Borrelia burgdorferi.

The ability of Borrelia burgdorferi, the agent of Lyme disease, to penetrate cultured human umbilical vein endothelial cell monolayers was investigated. After 4 h of coincubation, approximately 7.7% of added bacteria passed through the host cell monolayers. Electron microscopy revealed that the borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing through them.     

Microcavity substrates casted from self-assembled microsphere monolayers for spheroid cell culture

Multicellular spheroids are an important 3-dimensional cell culture model that reflects many key aspects of in vivo microenvironments. This paper presents a scalable, self-assembly based approach for fabricating microcavity substrates for multicellular spheroid cell culture. Hydrophobic glass microbeads were self-assembled into a tightly packed monolayer through the combined actions of surface tension, gravity, and lateral capillary forces at the water-air interface of a polymer solution. The packed bead monolayer was subsequently embedded in the dried polymer layer. The surface was used as a template for replicating microcavity substrates with perfect spherical shapes. We demonstrated the use of the substrate in monitoring the formation process of tumor spheroids, a proof-of-concept scale-up fabrication procedure into standard microplate formats, and its application in testing cancer drug responses in the context of bone marrow stromal cells. The presented technique offers a simple and effective way of forming high-density uniformly-sized spheroids without microfabrication equipment for biological and drug screening applications.     

High precision measurement of electrical resistance across endothelial cell monolayers

Effects of vasoactive agonists on endothelial permeability was assessed by measurement of transendothelial electrical resistance (TEER) of human umbilical vein endothelial cells (HUVECs) grown on porous polycarbonate supports. Because of the low values of TEER obtained in this preparation (< 5 cm²) a design of an Ussing type recording chamber was chosen that provided for a homogeneous electric Held across the monolayer and for proper correction of series resistances. Precision current pulses and appropriate rates of sampling and averaging of the voltage signal allowed for measurement of < 0.1 resistance changes of the endothelium on top of a 21 series resistance of the support and bathing fluid layers. Histamine (10 M) and thrombin (10 U/ml) induced an abrupt and substantial decrease of TEER, bradykinin (1 M) was less effective, PAF (380 nM) and LTC₄ (1 M) had no effect TEER was also reduced by the calcium ionophore A-23187 (10 M). The technique allows for measurements of TEER in low resistance monolayer cultures with high precision and time resolution. The results obtained extend previous observations in providing quantitative data on the increase of permeability of HUVECs in response to vasoactive agonists.Supported by grant P-10435-M from the Austrian Science Foundation to BRB.     

Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies

Cell shapes induced by cell-substratum interactions are linked with proliferation, differentiation or apoptosis of cells. To clarify the relevance of specific surface characteristics, we applied self-assembled monolayers (SAM) of alkyl silanes exhibiting a variety of terminating functional groups. We first characterised the SAMs on glass or silicon wafers by measuring wettability, layer thickness and roughness. Water contact angle data revealed that methyl (CH(3)), bromine (Br), and vinyl (CH=CH(2)) groups lead to hydrophobic surfaces, while amine (NH(2)) and carboxyl (COOH) functions lead to moderately wettable surfaces, and polyethylene glycol (PEG) and hydroxyl (OH) groups created wettable substrata. The surfaces were found to be molecular smooth except for one type of NH(2) surface. The SDS-PAGE analysis of proteins adsorbed from bovine serum to the SAMs showed less protein adsorption to PEG and OH than to CH(3), NH(2) and COOH. Immunoblotting revealed that a key component of adsorbed proteins is vitronectin while fibronectin was not detectable. The interaction of human fibroblasts with CH(3), PEG and OH terminated SAMs was similarly weak while strong attachment, spreading, fibronectin matrix formation and growth were observed on COOH and NH(2). The strong interaction of fibroblasts with the latter SAMs was linked to an enhanced activity of integrins as observed after antibody-tagging of living cells.     

Correlating fibronectin adsorption with endothelial cell adhesion and signaling on polymer substrates

Fibronectin (Fn) adsorption was studied on different commercial polymer surface chemistries, including tissue culture polystyrene (TCPS), bacteriologic polystyrene (BPS), fluoropolymer Teflon AF, and poly-L-lactide (PLLA). Antibody probes detected the availability of Fn's cell binding domain on adsorbed Fn in the competitive presence and absence of bovine serum albumin (BSA). Domain availability was highest for Fn adsorbed on TCPS, especially in the presence of either serum albumin or dilute serum. Attachment and growth efficiencies for human umbilical venous endothelial cells (HUVECs) cultured on surfaces preadsorbed with Fn in serum and serum-free media correlated with antibody cell-binding domain availability: TCPS > BPS, Teflon AF > PLLA. Intracellular signaling from the GTPase, RhoA, was highest (RhoA:RhoGDI inhibitor ratio) in cells cultured on the Teflon AF surfaces, indicating that despite lower attached cell numbers on Teflon AF compared to TCPS, cell signaling remained activated after 24 h of growth. Up-regulated cellular Fn mRNA messages, assessed using RT-PCR techniques, supported HUVECs' producing the endogenous extracellular matrix (ECM) protein Fn in order to attach and survive on the suboptimal Teflon AF culture surfaces.     

Mylar and Teflon-AF as cell culture substrates for studying endothelial cell adhesion

The textured and opaque nature of Dacron and ePTFE has prevented the use of these fabrics in conventional cell culture techniques normally employed to optimize cell attachment and retention. This lack of optimization has led, in part, to the poor performance of endothelialization strategies for improving vascular graft patency. Here we show that thin, transparent films of Mylar and Teflon-AF are viable in vitro cell culture mimics of Dacron and ePTFE vascular graft materials, particularly for the study of protein mediated endothelial cell (EC) attachment, spreading and adhesion. Glass substrates were used as controls. X-ray photoelectron spectroscopy (XPS) and contact angle analysis showed that Mylar and Teflon-AF have surface chemistries that closely match Dacron and ePTFE. (125)I radiolabeling was used to quantify fibronectin (FN) adsorption, and FN and biotinylated-BSA "dual ligand" co-adsorption onto glass, Mylar and Teflon-AF substrates. Native human umbilical vein endothelial cells (HUVEC) and streptavidin-incubated biotinylated-HUVEC (SA-b-HUVEC) spreading was measured using phase contrast microscopy. Cell retention and adhesion was determined using phase contrast microscopy under laminar flow. All surfaces lacking protein pre-treatment, regardless of surface type, showed the lowest degree of cell spreading and retention. Dual ligand treated Mylar films showed significantly greater SA-b-HUVEC spreading up to 2 h, but were similar to HUVEC on FN treated Mylar at longer times; whereas SA-b-HUVEC spreading on dual ligand treated Teflon-AF was never significantly different from HUVEC on FN treated Teflon-AF at any time point. SA-b-HUVEC retention was significantly greater on dual ligand treated Mylar compared to HUVEC on FN treated Mylar over the entire range of shear stresses tested (3.54-28.3 dynes/cm(2)); whereas SA-b-HUVEC retention to dual ligand and HUVEC retention to FN treated Teflon-AF gave similar results at each shear stress, with only the mid-range of stresses showing significant difference in cell retention to Teflon-AF.     

Polypyrrole-heparin composites as stimulus-responsive substrates for endothelial cell growth.

Heparin is a potent anticoagulant which can be immobilized on biomaterial surfaces to increase their hemocompatability. In the present work, we have electrochemically synthesized composites comprising heparin and the electrically conducting polymer polypyrrole. The incorporation and exposure of heparin were controlled by varying key conditions of polymer synthesis (i.e., applied current and synthesis time). The resulting composite polymers were electroactive after synthesis and the amount of heparin exposed in the polymer could be increased (up to threefold) by switching the polymers from their oxidized to reduced states. Polymer reduction was achieved by either application of negative potentials (-0.4 to -0.7 V for 90 s) or exposure to aqueous reductant (0.1M sodium dithionite for 30 min). Heparin-polypyrrole composites remained stable after autoclaving, displaying no significant loss of electroactivity, and had a shelf life of at least 2 years postautoclaving. Finally, the composites were found to be excellent substrates for the growth of human endothelial cells.     

Modification of endothelial cell functions by Hantaan virus infection: prolonged hyper-permeability induced by TNF-alpha of hantaan virus-infected endothelial cell monolayers

Summary. Serious vascular leakage is central to the pathogenesis of hantavirus infections. However, there is no evidence suggesting the hantavirus infection of endothelial cells directly causes obvious cell damage or morphological alteration either in vivo or in vitro. In this study, we examined whether Hantaan virus (HTNV) infection modifies the barrier function of endothelial cell monolayers upon the exposure to pro-inflammatory cytokines. Low levels (1ng/ml) of tumor necrosis factor-alpha initially increased the permeability in both HTNV-infected and uninfected monolayers similarly. Thereafter, however, these monolayers showed significant difference. The HTNV-infected monolayers remained irreversibly hyper-permeable during the experimental period up to 4 days, while the uninfected monolayers completely recovered the barrier function. The prolonged hyper-permeability of HTNV-infected monolayers was not associated with cell death or gap formation in the monolayers, and was independent from their nitric oxide or prostaglandin production. These results are the first evidence that hantavirus infection modifies barrier function of endothelial cell monolayers and suggest that HTNV-infection of endothelial cells may contribute to the increased vascular leakage through the prolonged response to cytokines.     

Dietary fish oil diminishes lymphocyte adhesion to macrophage and endothelial cell monolayers.

To further investigate the immunomodulatory effects of dietary lipids, rats were fed on a low-fat diet or on high-fat diets that contained hydrogenated coconut, olive, safflower, evening primrose or fish oil as the principal fat source. The fish oil diet decreased the level of expression of CD2, CD11a, CD18 and CD44 on the surface of freshly prepared lymphocytes and of CD2, CD11a, CD18, CD54 (intercellular adhesion molecule-1; ICAM-1) and CD62L (L-selectin) on the surface of concanavalin A (Con A)-stimulated lymphocytes. The olive oil diet also resulted in decreased expression of some adhesion molecules. The fish or olive oil diets, and to a lesser extent the safflower or evening primrose oil diets, decreased the adhesion of both freshly prepared and Con A-stimulated lymphocytes to macrophage monolayers. The fish oil diet, and to a lesser extent the olive or evening primrose oil diets, reduced the ability of Con A-stimulated lymphocytes to adhere to untreated endothelial cells. Furthermore, the fish oil diet resulted in a 50% reduction in Con A-stimulated lymphocyte adhesion to tumour necrosis factor-alpha (TNF-alpha)-stimulated endothelial cells. This study demonstrates that dietary lipids affect the expression of functionally important adhesion molecules on the surface of lymphocytes. Furthermore, this study suggests that such diet-induced effects on adhesion molecule expression might alter the ability of lymphocytes to bind to macrophages and to endothelial cells. Of the diets studied fish oil causes the most significant effects. The results of this study suggest that a reduction in cellular infiltration may partly explain the protective effect of a fish-oil-rich diet against the development of inflammatory and cardiovascular diseases.     

Cocaine-induced increase in the permeability function of human vascular endothelial cell monolayers.

The effects of cocaine on endothelial cell macromolecular transport, electrical resistance, and morphology were assessed. In confluent endothelial monolayers grown on microporus filters, cocaine (0.01 to 1 mmol/L) induced a rapid concentration-dependent increase in permeability to peroxidase and low density lipoprotein. Along with increased transport, the cocaine effect was paralleled by a decrease in transendothelial electrical resistance. Alterations in membrane resistance were fully reversible following washout of the drug, providing evidence that cocaine does not cause permanent injury to the integrity of the monolayer. Cocaines major metabolites, benzoylecgonine and ecgonine methyl ester, had minimal effect on electrical resistance properties, whereas monolayer impedance was markedly depressed by the novel cocaine/alcohol metabolite, cocaine ethyl ester (cocaethylene). Morphologic studies of cocaine-treated endothelial cells revealed a marked disruption of F-actin and the formation of intercellular gaps; no evidence of cell lysis and/or detachment was noted. Forskolin, a potent activator of adenylate cyclase known to promote the endothelial cell barrier function, impaired cocaine-induced changes in electrical resistance and morphology. Cocaine, however, had no effect on resting levels of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) in confluent endothelial monolayers. In summary, the results indicate that cocaine directly induces structural defects in the endothelial cell barrier which enhance the transport of macromolecular tracers, the mechanism does not appear to involve intracellular cAMP.     

Adherence of human saphenous vein endothelial cell monolayers to tissue-engineered biomatrix vascular conduits

This study investigates the adherence and retention under in vitro flow conditions of endothelium grown on the luminal surface of 4-mm-internal-diameter, biomatrix vascular conduits. The biomatrix vascular conduits are produced in living animals and consist of a naturally formed extracellular matrix wall incorporating a polyester mesh. We propose that the microarchitecture of the luminal surface may be conducive to endothelial cell (EC) seeding and to the formation of a firmly adherent endothelium without prior treatment of the surface with cell adhesives. ECs were isolated from segments of human saphenous vein and grown to confluence in a culture. Cultured cells were characterized by morphology and immunocytochemistry with anti-CD31, Von Willebrand factor, smooth muscle actin, cytokeratin, and the lectin Ulex Europaeus agglutinin. After culture, ECs were seeded (1 x 10(6) cell/mL) by rotation onto the luminal surface of 20-cm-long, biomatrix vascular conduits (n = 3). The seeded conduits were incubated for 72 h, and at set time points postseeding (1, 24, 48, and 72 h), the morphology, percentage luminal surface cover, and cell density (cell/cm(2)) were determined from en face preparations and histological cross sections. After 72 h in culture, the seeded conduits were subjected to a nonpulsatile flow for 1 h with culture media. A flow rate of 480 mL/min generated physiological-range shear stresses of 12 dyn/cm(2) on the endothelialized surface. After the flow, the conduits were fixed for histology, and the EC morphology and percentage luminal surface cover were determined. Quantification of the extent of luminal surface endothelialization, preflow and postflow, and cell densities at confluence was performed with digital imaging light microscopy and image analysis software. An analysis of the results demonstrated that confluent EC monolayers may be established on the luminal surface of biomatrix vascular conduits within 48 h. The formed endothelium was firmly adherent and was retained under physiological-range flow.     

Lymphocyte migration through cultured endothelial cell monolayers derived from the blood-retinal barrier.

Lymphocyte migration across endothelial monolayers, derived from the rat blood-retinal barrier, was recorded in vitro using time-lapse video microscopy. Syngeneic lymphocytes were plated out onto endothelial cell monolayers for 4 hr and their surface motility and transmonolayer migration recorded and quantified. Under resting conditions lymphocytes, obtained from peripheral lymph nodes (PLN), were small, rounded and static with less than 5% migrating across the monolayer. Activation of the lymphocytes with concanavalin A (Con A) increased their size and surface motility on both interferon-gamma (IFN-gamma)-treated and resting endothelia, but did not alter the number migrating across the monolayer. Similar results were also found for phytohaemagglutinin (PHA)-activated lymphocytes. Interleukin-2 (IL-2)-dependent CD4+ T-cell lines specifically recognizing either retinal soluble antigen (S-Ag) or bovine serum albumin (BSA) exhibited significantly greater surface motility over the endothelial monolayers than the mitogen-activated PLN lymphocytes. By 4 hr, in excess of 50% of the T-cell line lymphocytes had migrated across the endothelial monolayer. Treatment of the endothelial cells with IFN-gamma caused a small, but not significant, increase in the level of T-cell line lymphocyte migration. These results suggest that the migration of lymphocytes across central nervous system-derived endothelia is primarily dependent upon the state and mode of lymphocyte activation.     

The promotion of neuronal maturation on soft substrates

Microenvironmental mechanical properties of stem cell niches vary across tissues and developmental stages. Accumulating evidence suggests that matching substrate elasticity with in vivo tissue elasticity facilitates stem cell differentiation. However, it has not been established whether substrate elasticity can control the maturation stage of cells generated by stem cell differentiation. Here we show that soft substrates with elasticities commensurable to the elasticity of the brain promote the maturation of neural stem cell-derived neurons. In the absence of added growth factors, neurons differentiated on soft substrates displayed long neurites and presynaptic terminals, contrasting with the bipolar immature morphology of neurons differentiated on stiff substrates. Further, soft substrates supported an increase in astrocytic differentiation. However, stiffness cues could not override the dependency of astrocytic differentiation on Notch signaling. These results demonstrate that substrate elasticity per se can drive neuronal maturation thus defining a crucial parameter in neuronal differentiation of stem cells.     

Ingestion of Staphylococcus aureus by bovine endothelial cells results in time- and inoculum-dependent damage to endothelial cell monolayers.

Cultured endothelial cells phagocytize Staphylococcus aureus, but the resultant effects are unknown. Monolayers of cultured bovine endothelial cells with or without [3H]adenine label were exposed to 100, 10, or 1 S. aureus organism per endothelial cell for 3.5 h. Lysostaphin was then applied to all cultures to destroy extracellular but not phagocytized S. aureus. In cultures treated for only 20 min with lysostaphin, S. aureus multiplied exponentially after a 9- to 12-h lag period. In cultures treated continuously with lysostaphin, numbers of S. aureus remained constant or decreased. These results indicate that S. aureus became extracellular and multiplied but did not multiply intracellularly. In parallel experiments, the release of 3H-adenine from prelabeled endothelial cell monolayers was assayed to indicate cytotoxicity. Results indicated that the loss of 3H-adenine from endothelial cell monolayers depended on the following: (i) the size of the S. aureus inoculum, (ii) the strain of S. aureus, and (iii) the length of time after exposure to S. aureus. S. aureus endocarditis and persistent septicemia could arise, at least in part, from ingestion of S. aureus by host endothelium. The intracellular location would afford S. aureus protection from host defenses and antibiotics. Eventual damage to endothelial cells could expose collagen, thus resulting in platelet adherence and vegetation formation. Intracellular S. aureus would be continuously released into the circulation, possibly accounting for the persistent bacteremia that is found in S. aureus endovascular infections.     

Panning T cells on vascular endothelial cell monolayers: a rapid method for enriching naive T cells

A key functional/phenotypic difference between naive and memory T cells is the ability of memory and activated T cells to home to sites of inflammation by adhering to vascular endothelial cells. To determine if this trait could be used to separate naive T cells from memory T cells, CD4+ T cells were incubated with monolayers of IFN-gamma-primed vascular endothelial cells after which the phenotypic and functional characteristics of the nonadherent population were assayed. The nonadherent population 1) contained a five-fold decrease in the frequency of cells displaying the CD44(high)/CD45RB(low) "memory" phenotype and 2) responded well to allostimulation but displayed a reduced ability to respond to immobilized anti-CD3 antibody and, when isolated from ovalbumin-immunized mice, displayed a reduced recall response to ovalbumin in vitro. These studies demonstrate that rwo brief incubations of T cells with monolayers of IFN-gamma-primed endothelial cells can significantly enrich for naive T cells as determined by both phenotypic and functional analyses.     

Fibronectin fragments released from phorbol ester-stimulated pulmonary artery endothelial cell monolayers promote neutrophil chemotaxis.

We have recently shown that monolayer cultures of calf pulmonary artery endothelial (CPAE) cells pretreated with phorbol myristate acetate (PMA) generate a conditioned medium that is chemotactic for human polymorphonuclear leucocytes (PMNL). Fibronectin (Fn) is a multidomain protein found in the plasma and subendothelial extracellular matrix that induces attachment and migration of a variety of cell types. The present study was designed to evaluate the role of Fn or fragments of Fn present in conditioned medium from phorbol ester-stimulated endothelial cells as potential chemotactic factors for human PMNL. A large number of Fn fragments were revealed by Western immunoblotting of serum-free conditioned medium 4 hr after treatment of CPAE monolayers with PMA. Gelatin-Sepharose affinity chromatography of 4-hr conditioned medium demonstrated chemotactic activity for PMNL in both gelatin-binding and non-gelatin-binding fractions. The addition of bovine Fn antiserum to the conditioned medium inhibited PMNL chemotaxis in a dose-dependent manner while having no effect on PMNL chemotaxis generated by zymosan-activated serum. One site on the Fn molecule known to interact with phagocytic cells is the cell-binding domain containing the Arg-Gly-Asp (RGD) sequence. Pretreatment of PMNL with a RGD-containing peptide (1 mM GRGDSPK) for 10 min completely inhibited the expression of chemotactic activity present in conditioned medium and in the gelatin-binding and non-gelatin-binding fractions. PMNL chemotaxis was not stimulated by either intact Fn or the RGD-containing septapeptide tested over a wide concentration range. However, incubation of PMNL with a purified 120,000-MW fragment of Fn containing the cell-binding domain stimulated chemotaxis in a dose-dependent manner. In contrast, a purified 45,000 MW fragment of Fn containing the gelatin-binding domain was not chemotactic for PMNL. When a monoclonal antibody directed against the cell-binding domain of Fn was incubated with conditioned medium, a significant reduction in PMNL chemotaxis was observed. These results demonstrate that phorbol ester-stimulated pulmonary artery endothelial cells release Fn fragments and suggest an important role for Fn fragments containing the cell-binding domain in stimulating the migration of PMNL.     

Fibronectin anchorage to polymer substrates controls the initial phase of endothelial cell adhesion

Early stages of the adhesion of human endothelial cells onto a set of smooth polymer films were analyzed to reveal the modulation of cell-matrix interactions by the physicochemical constraints of predeposited fibronectin (FN). Hydrophobic and hydrophilic polymer substrates, consisting of poly(octadecene-alt-maleic anhydride) and poly(propene-alt-maleic anhydride) films, were coated with similar amounts of FN at conditions of either covalent or noncovalent immobilization. The well-defined substrates permit variation of the anchorage of FN at invariant topography, pliability, and molecular composition. Although all of the compared FN coatings were effective in stimulating attachment of endothelial cells, the initial formation of cell-matrix adhesions was found to be controlled by the type of interaction between predeposited FN and the underlying substrate. Covalent linkage and hydrophobic interactions of the predeposited FN with the polymer films interfered with the rapid generation of focal and fibrillar adhesions. It was demonstrated that this was caused by the fact that only weakly bound FN could become readily reorganized by the adherent cells. Upon prolonged culture periods at standard cell culture conditions, secretion and deposition of organized extracellular matrix by the attached cells was found to balance out the differences of the substrates.