Collective cell migration without proliferation: density determines cell velocity and wave velocity

NASA Astrophysics Data System (ADS)

Tlili, Sham; Gauquelin, Estelle; Li, Brigitte; Cardoso, Olivier; Ladoux, Benoît; Delanoë-Ayari, Hélène; Graner, François

2018-05-01

Collective cell migration contributes to embryogenesis, wound healing and tumour metastasis. Cell monolayer migration experiments help in understanding what determines the movement of cells far from the leading edge. Inhibiting cell proliferation limits cell density increase and prevents jamming; we observe long-duration migration and quantify space-time characteristics of the velocity profile over large length scales and time scales. Velocity waves propagate backwards and their frequency depends only on cell density at the moving front. Both cell average velocity and wave velocity increase linearly with the cell effective radius regardless of the distance to the front. Inhibiting lamellipodia decreases cell velocity while waves either disappear or have a lower frequency. Our model combines conservation laws, monolayer mechanical properties and a phenomenological coupling between strain and polarity: advancing cells pull on their followers, which then become polarized. With reasonable values of parameters, this model agrees with several of our experimental observations. Together, our experiments and model disantangle the respective contributions of active velocity and of proliferation in monolayer migration, explain how cells maintain their polarity far from the moving front, and highlight the importance of strain-polarity coupling and density in long-range information propagation.

Opinion: Building epithelial architecture: insights from three-dimensional culture models.

PubMed

O'Brien, Lucy Erin; Zegers, Mirjam M P; Mostov, Keith E

2002-07-01

How do individual cells organize into multicellular tissues? Here, we propose that the morphogenetic behaviour of epithelial cells is guided by two distinct elements: an intrinsic differentiation programme that drives formation of a lumen-enclosing monolayer, and a growth factor-induced, transient de-differentiation that allows this monolayer to be remodelled.

In Vitro Model of Tumor Cell Extravasation

PubMed Central

Jeon, Jessie S.; Zervantonakis, Ioannis K.; Chung, Seok; Kamm, Roger D.; Charest, Joseph L.

2013-01-01

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium. PMID:23437268

Surgery-derived reactive oxygen species produced by polymorphonuclear leukocytes promote tumor recurrence: studies in an in vitro model.

PubMed

van Grevenstein, Wilhelmina M U; Aalbers, Arend G J; Ten Raa, Sander; Sluiter, Wim; Hofland, Leo J; Jeekel, Hans; van Eijck, Casper H J

2007-06-01

Tissue injury induces the acute phase response, aimed at minimizing damage and starting the healing process. Polymorphonuclear leukocytes (PMNs) respond to the presence of specific chemoattractants and begin to appear in large numbers. The aim of this study was to investigate the influence of reactive oxygen species (ROS) produced by PMNs on the interaction between colon carcinoma cells and mesothelial cells. An experimental human in vitro model was designed using Caco-2 colon carcinoma cells and primary cultures of mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the mesothelium with stimulated PMNs and unstimulated PMNs. Mesothelial cells were also incubated with xanthine/xanthine oxidase (X/XO) complex producing ROS after which adhesion of Caco-2 cells was investigated and the expression of adhesion molecules (ICAM-1, VCAM-1, and CD44) by means of enzyme immunoassay. In the control situation the average adhesion of Caco-2 cells to the mesothelial monolayers was 23%. Mesothelial monolayers incubated with unstimulated PMNs showed a 25% increase of tumor cell adhesion (P < 0.05). The adhesion of tumor to the monolayers incubated with the N-formyl-methionyl-leucyl-phenylalanine-stimulated PMNs increased with 40% (P < 0.01). Incubation of the mesothelium with X/XO resulted in an enhancement of adhesion of Caco-2 cells of 70% and an up-regulation of expression of ICAM-1, VCAM-1, and CD44. This study reveals an increase of tumor cell adhesion to the mesothelium induced by incubating the mesothelial monolayers with PMNs. PMNs are producing a number of products, like proteolytic enzymes, cytokines, and ROS. These factors up-regulate the expression of adhesion molecules and in that way stimulate the adhesion of tumor to the mesothelium.

Effects of structure on the interactions between five natural antimicrobial compounds and phospholipids of bacterial cell membrane on model monolayers

USDA-ARS?s Scientific Manuscript database

Monolayers composed of bacterial phospholipids were used as model membranes to study interactions of naturally occurring phenolic compounds 2,5-dihydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and the plant essential oil compounds carvacrol, cinnamaldehyde, and geraniol, previously found to be...

Characterisation of FAP-1 expression and CD95 mediated apoptosis in the A818-6 pancreatic adenocarcinoma differentiation system.

PubMed

Winterhoff, Boris J N; Arlt, Alexander; Duttmann, Angelika; Ungefroren, Hendrik; Schäfer, Heiner; Kalthoff, Holger; Kruse, Marie-Luise

2012-03-01

The present study investigated the expression and localisation of FAP-1 (Fas associated phosphatase-1) and CD95 in a 3D differentiation model in comparison to 2D monolayers of the pancreatic adenocarcinoma cell line A818-6. Under non-adherent growth conditions, A818-6 cells differentiate into 3D highly organised polarised epithelial hollow spheres, resembling duct-like structures. A818-6 cells showed a differentiation-dependent FAP-1 localisation. Cells grown as 2D monolayers revealed FAP-1 staining in a juxtanuclear cisternal position, as well as localisation in the nucleus. After differentiation into hollow spheres, FAP-1 was relocated towards the actin cytoskeleton beneath the outer plasma membrane of polarised cells and no further nuclear localisation was observed. CD95 surface staining was found only in a subset of A818-6 monolayer cells, while differentiated hollow spheres appeared to express CD95 in all cells of a given sphere. We rarely observed co-localisation of CD95 and FAP-1 in A818-6 monolayer cells, but strong co-localisation beneath the outer plasma membrane in polarised cells. Analysis of surface expression by flow cytometry revealed that only a subset (36%) of monolayer cells showed CD95 surface expression, and after induction of hollow spheres, CD95 presentation at the outer plasma membrane was reduced to 13% of hollow spheres. Induction of apoptosis by stimulation with agonistic anti-CD95 antibodies, resulted in increased caspase activity in both, monolayer cells and hollow spheres. Knock down of FAP-1 mRNA in A818-6 monolayer cells did not alter resposiveness to CD95 agonistic antibodies. These data suggested that CD95 signal transduction was not affected by FAP-1 expression in A818-6 monolayer cells. In differentiated 3D hollow spheres, we found a polarisation-induced co-localisation of CD95 and FAP-1. A tight control of receptor surface representation and signalling induced apoptosis ensures controlled removal of individual cells instead of a "snowball effect" of apoptotic events. Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Characterisation of the membrane affinity of an isoniazide peptide conjugate by tensiometry, atomic force microscopy and sum-frequency vibrational spectroscopy, using a phospholipid Langmuir monolayer model.

PubMed

Hill, Katalin; Pénzes, Csanád Botond; Schnöller, Donát; Horváti, Kata; Bosze, Szilvia; Hudecz, Ferenc; Keszthelyi, Tamás; Kiss, Eva

2010-10-07

Tensiometry, sum-frequency vibrational spectroscopy, and atomic force microscopy were employed to assess the cell penetration ability of a peptide conjugate of the antituberculotic agent isoniazide. Isoniazide was conjugated to peptide (91)SEFAYGSFVRTVSLPV(106), a functional T-cell epitope of the immunodominant 16 kDa protein of Mycobacterium tuberculosis. As a simple but versatile model of the cell membrane a phospholipid Langmuir monolayer at the liquid/air interface was used. Changes induced in the structure of the phospholipid monolayer by injection of the peptide conjugate into the subphase were followed by tensiometry and sum-frequency vibrational spectroscopy. The drug penetrated lipid films were transferred to a solid support by the Langmuir-Blodgett technique, and their structures were characterized by atomic force microscopy. Peptide conjugation was found to strongly enhance the cell penetration ability of isoniazide.

3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems.

PubMed

Howes, Amy L; Richardson, Robyn D; Finlay, Darren; Vuori, Kristiina

2014-01-01

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

3-Dimensional Culture Systems for Anti-Cancer Compound Profiling and High-Throughput Screening Reveal Increases in EGFR Inhibitor-Mediated Cytotoxicity Compared to Monolayer Culture Systems

PubMed Central

Howes, Amy L.; Richardson, Robyn D.; Finlay, Darren; Vuori, Kristiina

2014-01-01

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery. PMID:25247711

Effects of dexamethasone on C6 astrocytoma radiosensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lordo, C.D.; Stroude, E.C.; Del Maestro, R.F.

1989-05-01

Brain-tumor patients often undergo radiation therapy while receiving corticosteroids for the treatment of cerebral edema. Studies have demonstrated that dexamethasone is radioprotective in a number of cell lines. The C6 astrocytoma cell line is well established in vitro and is modulated by dexamethasone treatment. It has therefore been hypothesized that dexamethasone-treated C6 astrocytoma cells would be more resistant to radiation-induced damage. The present study was carried out to assess this hypothesis using both the in vitro C6 astrocytoma monolayer and three-dimensional multicellular spheroid models. Dexamethasone was inhibitory to the C6 astrocytoma cells in the monolayer preparation, increasing their doubling timemore » by 13%. In the spheroid cultures, dexamethasone treatment decreased the number of cells per spheroid by 46%. Dexamethasone did not affect the plating efficiency of either the cells from the monolayer experiment or those dissociated from spheroids, however, suggesting that the inhibitory effect was not tumoricidal. At a clinical concentration (1.94 x 10(-5) M), dexamethasone did not significantly influence plating efficiency of irradiated C6 astrocytoma cells in monolayer or three-dimensional spheroid cultures.« less

Transport of ginkgolides with different lipophilicities based on an hCMEC/D3 cell monolayer as a blood-brain barrier cell model.

PubMed

Ma, Shuwei; Liu, Xingyan; Xu, Qingrun; Zhang, Xiantao

2014-10-02

In this report, the transport of ginkgolides with different lipophilicities was investigated using an hCMEC/D3 cell monolayer as a blood-brain barrier (BBB) cell model in vitro in an attempt to explain ginkgolide transport path mediated by lipophilicity. The log P values of ginkgolides were determined by measuring the distribution of the molecule between oil and water. Additionally, the cytotoxicity of ginkgolides on hCMEC/D3 cells was assayed with the MTT method. Ginkgolide contents were determined with an ultra performance liquid chromatograph equipped with an evaporative light scattering detector (ULPC-ELSD) method. Apparent permeability coefficients (Papp) and efflux ratios (PappBL→AP/PappAP→BL) were then calculated to describe the transport characteristics of ginkgolide. The transport of ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide J across the hCMEC/D3 cell monolayer was non-directional. Additionally, ginkgolide C transport on the cell monolayer was time- and concentration-dependent in the paracellular pathway controlled by cytochalasin D (a tight junction modulator). The transport of ginkgolide N, ginkgolide L, and ginkgolide K across the cell monolayer displayed clear directionality at low ginkgolide concentrations. This behavior indicated that the transport of ginkgolide N, ginkgolide L, and ginkgolide K was influenced by the transcellular pathway containing an efflux protein accompanied by the paracellular pathway for passive diffusion. Additionally, the transport of ginkgolide K was increased significantly by co-culturing with a P-gp inhibitor. These findings provide important information for elucidating ginkgolide transport pathways and may be beneficial for the design of ginkgolide molecules with high neuroprotective effects. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Colin D.A.; Sayer, Rachel; Windass, Amy S.

2008-12-15

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysismore » of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.« less

Molecular Monolayers for Electrical Passivation and Functionalization of Silicon-Based Solar Energy Devices.

PubMed

Veerbeek, Janneke; Firet, Nienke J; Vijselaar, Wouter; Elbersen, Rick; Gardeniers, Han; Huskens, Jurriaan

2017-01-11

Silicon-based solar fuel devices require passivation for optimal performance yet at the same time need functionalization with (photo)catalysts for efficient solar fuel production. Here, we use molecular monolayers to enable electrical passivation and simultaneous functionalization of silicon-based solar cells. Organic monolayers were coupled to silicon surfaces by hydrosilylation in order to avoid an insulating silicon oxide layer at the surface. Monolayers of 1-tetradecyne were shown to passivate silicon micropillar-based solar cells with radial junctions, by which the efficiency increased from 8.7% to 9.9% for n + /p junctions and from 7.8% to 8.8% for p + /n junctions. This electrical passivation of the surface, most likely by removal of dangling bonds, is reflected in a higher shunt resistance in the J-V measurements. Monolayers of 1,8-nonadiyne were still reactive for click chemistry with a model catalyst, thus enabling simultaneous passivation and future catalyst coupling.

Modeling an Excitable Biosynthetic Tissue with Inherent Variability for Paired Computational-Experimental Studies.

PubMed

Gokhale, Tanmay A; Kim, Jong M; Kirkton, Robert D; Bursac, Nenad; Henriquez, Craig S

2017-01-01

To understand how excitable tissues give rise to arrhythmias, it is crucially necessary to understand the electrical dynamics of cells in the context of their environment. Multicellular monolayer cultures have proven useful for investigating arrhythmias and other conduction anomalies, and because of their relatively simple structure, these constructs lend themselves to paired computational studies that often help elucidate mechanisms of the observed behavior. However, tissue cultures of cardiomyocyte monolayers currently require the use of neonatal cells with ionic properties that change rapidly during development and have thus been poorly characterized and modeled to date. Recently, Kirkton and Bursac demonstrated the ability to create biosynthetic excitable tissues from genetically engineered and immortalized HEK293 cells with well-characterized electrical properties and the ability to propagate action potentials. In this study, we developed and validated a computational model of these excitable HEK293 cells (called "Ex293" cells) using existing electrophysiological data and a genetic search algorithm. In order to reproduce not only the mean but also the variability of experimental observations, we examined what sources of variation were required in the computational model. Random cell-to-cell and inter-monolayer variation in both ionic conductances and tissue conductivity was necessary to explain the experimentally observed variability in action potential shape and macroscopic conduction, and the spatial organization of cell-to-cell conductance variation was found to not impact macroscopic behavior; the resulting model accurately reproduces both normal and drug-modified conduction behavior. The development of a computational Ex293 cell and tissue model provides a novel framework to perform paired computational-experimental studies to study normal and abnormal conduction in multidimensional excitable tissue, and the methodology of modeling variation can be applied to models of any excitable cell.

Comparative proteome analysis of monolayer and spheroid culture of canine osteosarcoma cells.

PubMed

Gebhard, Christiane; Miller, Ingrid; Hummel, Karin; Neschi Née Ondrovics, Martina; Schlosser, Sarah; Walter, Ingrid

2018-04-15

Osteosarcoma is an aggressive bone tumor with high metastasis rate in the lungs and affects both humans and dogs in a similar way. Three-dimensional tumor cell cultures mimic the in vivo situation of micro-tumors and metastases and are therefore better experimental in vitro models than the often applied two-dimensional monolayer cultures. The aim of the present study was to perform comparative proteomics of standard monolayer cultures of canine osteosarcoma cells (D17) and three-dimensional spheroid cultures, to better characterize the 3D model before starting with experiments like migration assays. Using DIGE in combination with MALDI-TOF/TOF we found 27 unique canine proteins differently represented between these two culture systems, most of them being part of a functional network including mainly chaperones, structural proteins, stress-related proteins, proteins of the glycolysis/gluconeogenesis pathway and oxidoreductases. In monolayer cells, a noticeable shift to more acidic pI values was noticed for several proteins of medium to high abundance; two proteins (protein disulfide isomerase A3, stress-induced-phosphoprotein 1) showed an increase of phosphorylated protein species. Protein distribution within the cells, as detected by immunohistochemistry, displayed a switch of stress-induced-phosphoprotein 1 from the cytoplasm (in monolayer cultures) to the nucleus (in spheroid cultures). Additionally, Western blot testing revealed upregulated concentrations of metastasin (S100A4), triosephosphate isomerase 1 and septin 2 in spheroid cultures, in contrast to decreased concentrations of CCT2, a subunit of the T-complex. Results indicate regulation of stress proteins in the process of three-dimensional organization characterized by a hypoxic and nutrient-deficient environment comparable to tumor micro-metastases. Osteosarcoma is an aggressive bone tumor that early spreads to the lungs. Three-dimensional tumor cell cultures represent the avascular stage of micro-tumors and metastases, and should therefore represent a better experimental in vitro model compared to two-dimensional monolayer cultures. Significant differences have been reported in response to drug and radiation treatment between these two culture systems. A gel-based proteomic investigation was performed to compare protein patterns of a canine osteosarcoma cell line cultivated under those two conditions, to learn more about altered cell composition and its impact on cell behaviour. Due to the fact that the canine osteosarcoma is an accepted model for the human disease, results will be relevant for the human species as well. Copyright © 2018 Elsevier B.V. All rights reserved.

Monolayer to MTS: using SEM, HIM, TEM and SERS to compare morphology, nanosensor uptake and redox potential in MCF7 cells

NASA Astrophysics Data System (ADS)

Jamieson, L. E.; Bell, A. P.; Harrison, D. J.; Campbell, C. J.

2015-06-01

Cellular redox potential is important for the control and regulation of a vast number of processes occurring in cells. When the fine redox potential balance within cells is disturbed it can have serious consequences such as the initiation or progression of disease. It is thought that a redox gradient develops in cancer tumours where the peripheral regions are well oxygenated and internal regions, further from vascular blood supply, become starved of oxygen and hypoxic. This makes treatment of these areas more challenging as, for example, radiotherapy relies on the presence of oxygen. Currently techniques for quantitative analysis of redox gradients are limited. Surface enhanced Raman scattering (SERS) nanosensors (NS) have been used to detect redox potential in a quantitative manner in monolayer cultured cells with many advantages over other techniques. This technique has considerable potential for use in multicellular tumour spheroids (MTS) - a three dimensional (3D) cell model which better mimics the tumour environment and gradients that develop. MTS are a more realistic model of the in vivo cellular morphology and environment and are becoming an increasingly popular in vitro model, replacing traditional monolayer culture. Imaging techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM) and helium ion microscopy (HIM) were used to investigate differences in morphology and NS uptake in monolayer culture compared to MTS. After confirming NS uptake, the first SERS measurements revealing quantitative information on redox potential in MTS were performed.

Variable Effects of Autophagy Induction by Trehalose on Herpesviruses Depending on Conditions of Infection

PubMed Central

Meier, Jeffery L.; Grose, Charles

2017-01-01

Trehalose is a non-reducing sugar formed from two glucose units. Trehalose induces abundant autophagy in cultured cells and also reduces the rate of aggregation of the huntingtin protein in the animal model of Huntington disease, a chronic neurological disease in humans. The mechanism of this effect on autophagy is now known to be caused by starvation secondary to inhibition of a family of glucose transporters known as the solute carrier 2 or the glucose transporter family. Variable effects of trehalose treatment have been observed during infections with two herpesviruses—human cytomegalovirus and varicella-zoster virus. The reasons for differing results have now been delineated. These differences are caused by two variables in conditions of infection: timing of addition of trehalose and type of inoculum (cell-free virus vs. infected cells). When monolayers pretreated with trehalose were inoculated with cell-free virus, there was a decline in virus spread by as much as 93 percent when compared with untreated monolayers. However, when monolayers were inoculated with infected cells rather than cell-free virus, there was no decline in virus spread. These results demonstrated that the effect of trehalose was limited to monolayers that were starved when inoculated with cell-free virus. In contrast, sufficient virus was already present in infected cell inocula so as to minimize any inhibitory effect of a starved monolayer. These results also showed that trehalose did not specifically inhibit a herpesvirus; rather, addition of trehalose to cell culture media altered the intracellular environment. PMID:28356891

Variable Effects of Autophagy Induction by Trehalose on Herpesviruses Depending on Conditions of Infection.

PubMed

Meier, Jeffery L; Grose, Charles

2017-03-01

Trehalose is a non-reducing sugar formed from two glucose units. Trehalose induces abundant autophagy in cultured cells and also reduces the rate of aggregation of the huntingtin protein in the animal model of Huntington disease, a chronic neurological disease in humans. The mechanism of this effect on autophagy is now known to be caused by starvation secondary to inhibition of a family of glucose transporters known as the solute carrier 2 or the glucose transporter family. Variable effects of trehalose treatment have been observed during infections with two herpesviruses-human cytomegalovirus and varicella-zoster virus. The reasons for differing results have now been delineated. These differences are caused by two variables in conditions of infection: timing of addition of trehalose and type of inoculum (cell-free virus vs. infected cells). When monolayers pretreated with trehalose were inoculated with cell-free virus, there was a decline in virus spread by as much as 93 percent when compared with untreated monolayers. However, when monolayers were inoculated with infected cells rather than cell-free virus, there was no decline in virus spread. These results demonstrated that the effect of trehalose was limited to monolayers that were starved when inoculated with cell-free virus. In contrast, sufficient virus was already present in infected cell inocula so as to minimize any inhibitory effect of a starved monolayer. These results also showed that trehalose did not specifically inhibit a herpesvirus; rather, addition of trehalose to cell culture media altered the intracellular environment.

Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model.

PubMed

Schmutzer, Michael; Aszodi, Attila

2017-04-01

The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in ex vivo systems than in in vitro cell cultures. I have therefore established an ex vivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p = 0.0083) and P2 (p < 0.05). Suspension-cultured cells did not promote cartilage repair when compared with implanted monolayer-cultured chondrocytes and pellets: 24.0 ± 0.66% for suspension cells, 46.4 ± 2.9% for monolayer cells, and 127.64 ± 0.90% for pellets (p < 0.0001) of the original defect volume (percentage of defect). Additional cultivation with chondrogenesis-promoting growth factors TGF-β1 and BMP-2 revealed an enhancing effect on cartilage repair in all settings. The advantage and innovation of this system over in vitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such ex vivo explants might serve as a better model system to mimic clinical situations. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chitcholtan, Kenny, E-mail: kenny.chitcholtan@otago.ac.nz; Asselin, Eric, E-mail: Eric.Asselin@uqtr.ca; Parent, Sophie, E-mail: Sophie.Parent@uqtr.ca

2013-01-01

Three-dimensional (3D) in vitro models have an invaluable role in understanding the behaviour of tumour cells in a well defined microenvironment. This is because some aspects of tumour characteristics cannot be fully recapitulated in a cell monolayer (2D). In the present study, we compared growth patterns, expression of signalling molecules, and metabolism-associated proteins of endometrial cancer cell lines in 3D and 2D cell cultures. Cancer cells formed spherical structures in 3D reconstituted basement membrane (3D rBM), and the morphological appearance was cell line dependent. Cell differentiation was observed after 8 days in the 3D rBM. There was reduced proliferation, detectedmore » by less expression of PCNA in 3D rBM than in 2D cell monolayers. The addition of exogenous epidermal growth factor (EGF) to cancer cells induced phosphorylation of EGFR and Akt in both cell culture conditions. The uptake of glucose was selectively altered in the 3D rBM, but there was a lack of association with Glut-1 expression. The secretion of vascular endothelial growth factor (VEGF) and prostaglandin E{sub 2} (PGE{sub 2}) was selectively altered in 3D rBM, and it was cell line dependent. Our data demonstrated that 3D rBM as an in vitro model can influence proliferation and metabolism of endometrial cancer cell behaviour compared to 2D cell monolayer. Changes are specific to individual cell types. The use of 3D rBM is, therefore, important in the in vitro study of targeted anticancer therapies.« less

Increasing the throughput and productivity of Caco-2 cell permeability assays using liquid chromatography-mass spectrometry: application to resveratrol absorption and metabolism.

PubMed

Li, Yongmei; Shin, Young Geun; Yu, Chongwoo; Kosmeder, Jerome W; Hirschelman, Wendy H; Pezzuto, John M; van Breemen, Richard B

2003-12-01

The Caco-2 cell monolayer permeability assay has become a standard model of human intestinal absorption and transport. This paper reviews recent progress in increasing the throughput of Caco-2 cell monolayer assays and in expanding the scope of this assay to include modeling intestinal drug metabolism. The state-of-the-art in Caco-2 cell monolayer permeability assays combines multi-well plates fitted with semi-permeable inserts on which Caco-2 cells have been cultured with liquid chromatography-mass spectrometry (LC-MS) or LC-tandem mass spectrometry (LC-MS-MS) for the quantitative analysis of test compounds and the identification of their intestinal metabolites. After reviewing the progress in increasing the throughput of Caco-2 cell monolayer assays for both modeling human intestinal permeability or transport and the metabolism of xenobiotic compounds, we demonstrate the application of LC-MS and LC-MS-MS to the measurement of resveratrol permeability and metabolism in the Caco-2 model. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is a polyphenolic compound occurring in grapes, peanuts and other food sources, that is under investigation as a cancer chemoprevention agent. The apparent permeability coefficient for apical (AP) to basolateral (BL) movement of resveratrol was 2.0 x 10(-5)cm/sec. Resveratrol was not a substrate for P-glycoprotein or the multi-drug resistance associated proteins (MRP). No phase I metabolites were observed, but the phase II conjugates resveratrol-3-glucuronide and resveratrol-3-sulfate was identified based on LC-MS and LC-MS-MS analysis and comparison with synthetic standards. Although these data indicate that resveratrol diffuses rapidly across the intestinal epithelium, extensive phase II metabolism during absorption might reduce resveratrol bioavailability.

Analysis of cell flux in the parallel plate flow chamber: implications for cell capture studies.

PubMed Central

Munn, L L; Melder, R J; Jain, R K

1994-01-01

The parallel plate flow chamber provides a controlled environment for determinations of the shear stress at which cells in suspension can bind to endothelial cell monolayers. By decreasing the flow rate of cell-containing media over the monolayer and assessing the number of cells bound at each wall shear stress, the relationship between shear force and binding efficiency can be determined. The rate of binding should depend on the delivery of cells to the surface as well as the intrinsic cell-surface interactions; thus, only if the cell flux to the surface is known can the resulting binding curves be interpreted correctly. We present the development and validation of a mathematical model based on the sedimentation rate and velocity profile in the chamber for the delivery of cells from a flowing suspension to the chamber surface. Our results show that the flux depends on the bulk cell concentration, the distance from the entrance point, and the flow rate of the cell-containing medium. The model was then used in a normalization procedure for experiments in which T cells attach to TNF-alpha-stimulated HUVEC monolayers, showing that a threshold for adhesion occurs at a shear stress of about 3 dyn/cm2. Images FIGURE 1 FIGURE 2 PMID:7948702

Human RPE Stem Cells Grown into Polarized RPE Monolayers on a Polyester Matrix Are Maintained after Grafting into Rabbit Subretinal Space

PubMed Central

Stanzel, Boris V.; Liu, Zengping; Somboonthanakij, Sudawadee; Wongsawad, Warapat; Brinken, Ralf; Eter, Nicole; Corneo, Barbara; Holz, Frank G.; Temple, Sally; Stern, Jeffrey H.; Blenkinsop, Timothy A.

2014-01-01

Summary Transplantation of the retinal pigment epithelium (RPE) is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC) as an alternative RPE source. Polarized monolayers of adult hRPESC-derived RPE grown on polyester (PET) membranes had near-native characteristics. Trephined pieces of RPE monolayers on PET were transplanted subretinally in the rabbit, a large-eyed animal model. After 4 days, retinal edema was observed above the implant, detected by spectral domain optical coherence tomography (SD-OCT) and fundoscopy. At 1 week, retinal atrophy overlying the fetal or adult transplant was observed, remaining stable thereafter. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. Taken together, the xeno-RPE survived with retained characteristics in the subretinal space. These experiments support that adult hRPESC-derived RPE are a potential source for transplantation therapies. PMID:24511471

Transport of decursin and decursinol angelate across Caco-2 and MDR-MDCK cell monolayers: in vitro models for intestinal and blood-brain barrier permeability.

PubMed

Madgula, Vamsi L; Avula, Bharathi; Reddy V L, Niranjan; Khan, Ikhlas A; Khan, Shabana I

2007-04-01

Decursin (DE) and decursinol angelate (DA) were isolated from the roots of Angelica gigas (Apiaceae) and purified by HPLC. DE and DA have been reported to exhibit significant neuropharmacological activities, but their intestinal transport and permeability in terms of CNS penetration across the blood-brain barrier (BBB) are unknown. This study was undertaken to evaluate the IN VITRO intestinal and BBB transport of DE and DA using Caco-2 and MDR-MDCK cell monolayer models, respectively. The bidirectional transport of DE and DA across Caco-2 and MDR-MDCK monolayers was examined for 2 hours. Integrity of the monolayer was determined by TEER value and by monitoring the transport of Lucifer yellow (Ly) across the monolayers. Quantitation of DE and DA was performed by HPLC. DE and DA exhibited bidirectional transport with a Papp value in the range of 9.0-12.0x10(-6) cm/sec and 7.2-11.7x10(-6) cm/sec in Caco-2 and MDR-MDCK monolayers, respectively. The TEER values were in the range of 410-440 and 1170-1230 ohm cm2 for Caco-2 and MDR-MDCK monolayers, respectively. Ly measurement, the fluorescent marker of passive paracellular diffusion, resulted in Papp values of 2.5-5.0x10(-6) in Caco-2 and 6.0-8.0x10(-6) cm/sec in MDR-MDCK monolayers, confirming that the monolayer integrity was intact at the end of the experiment. Caco-2:human colonic adenocarcinoma DA:decursinol angelate DE:decursin Ly:Lucifer yellow MDCK:Madin-Darby canine kidney MDR:multidrug resistant Papp:apparent permeability TEER:transepithelial electrical resistance.

Characterizing the mechanics of cultured cell monolayers

PubMed Central

Peter, Loic; Bellis, Julien; Baum, Buzz; Kabla, Alexandre J.; Charras, Guillaume T.

2012-01-01

One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics. PMID:22991459

Quantification of Malignant Breast Cancer Cell MDA-MB-231 Transmigration across Brain and Lung Microvascular Endothelium

PubMed Central

Fan, Jie; Fu, Bingmei M.

2015-01-01

Tumor cell extravasation through the endothelial barrier forming the microvessel wall is a crucial step during tumor metastasis. However, where, how and how fast tumor cells transmigrate through endothelial barriers remain unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through brain and lung microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in the structural components (integrity) of endothelial monolayers were quantified by confocal microscopy. Endothelial monolayer permeability to albumin Palbumin was also quantified under the same conditions. We found that about 98% of transmigration occurred at the joints of endothelial cells instead of cell bodies; tumor cell adhesion and transmigration degraded endothelial surface glycocalyx and disrupted endothelial junction proteins, consequently increased Palbumin; more tumor cells adhered to and transmigrated through the endothelial monolayer with higher Palbumin; Palbumin and tumor transmigration were increased by vascular endothelial growth factor (VEGF), a representative of cytokines, and lipopolysaccharides (LPS), a typical systemic inflammatory factor, but reduced by adenosine 3′, 5′-cyclic monophosphate (cAMP). These results suggest that reinforcing endothelial structural integrity is an effective approach for inhibiting tumor extravasation. PMID:26603751

Endocytic reawakening of motility in jammed epithelia

NASA Astrophysics Data System (ADS)

Malinverno, Chiara; Corallino, Salvatore; Giavazzi, Fabio; Bergert, Martin; Li, Qingsen; Leoni, Marco; Disanza, Andrea; Frittoli, Emanuela; Oldani, Amanda; Martini, Emanuele; Lendenmann, Tobias; Deflorian, Gianluca; Beznoussenko, Galina V.; Poulikakos, Dimos; Ong, Kok Haur; Uroz, Marina; Trepat, Xavier; Parazzoli, Dario; Maiuri, Paolo; Yu, Weimiao; Ferrari, Aldo; Cerbino, Roberto; Scita, Giorgio

2017-05-01

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.

Comparative study of expression and activity of glucose transporters between stem cell-derived brain microvascular endothelial cells and hCMEC/D3 cells.

PubMed

Al-Ahmad, Abraham J

2017-10-01

Glucose constitutes a major source of energy of mammalian brains. Glucose uptake at the blood-brain barrier (BBB) occurs through a facilitated glucose transport, through glucose transporter 1 (GLUT1), although other isoforms have been described at the BBB. Mutations in GLUT1 are associated with the GLUT1 deficiency syndrome, yet none of the current in vitro models of the human BBB maybe suited for modeling such a disorder. In this study, we investigated the expression of glucose transporters and glucose diffusion across brain microvascular endothelial cells (BMECs) derived from healthy patient-derived induced pluripotent stem cells (iPSCs). We investigated the expression of different glucose transporters at the BBB using immunocytochemistry and flow cytometry and measured glucose uptake and diffusion across BMEC monolayers obtained from two iPSC lines and from hCMEC/D3 cells. BMEC monolayers showed expression of several glucose transporters, in particular GLUT1, GLUT3, and GLUT4. Diffusion of glucose across the monolayers was mediated via a saturable transcellular mechanism and partially inhibited by pharmacological inhibitors. Taken together, our study suggests the presence of several glucose transporters isoforms at the human BBB and demonstrates the feasibility of modeling glucose across the BBB using patient-derived stem cells. Copyright © 2017 the American Physiological Society.

Mechanical Model of Geometric Cell and Topological Algorithm for Cell Dynamics from Single-Cell to Formation of Monolayered Tissues with Pattern

PubMed Central

Kachalo, Sëma; Naveed, Hammad; Cao, Youfang; Zhao, Jieling; Liang, Jie

2015-01-01

Geometric and mechanical properties of individual cells and interactions among neighboring cells are the basis of formation of tissue patterns. Understanding the complex interplay of cells is essential for gaining insight into embryogenesis, tissue development, and other emerging behavior. Here we describe a cell model and an efficient geometric algorithm for studying the dynamic process of tissue formation in 2D (e.g. epithelial tissues). Our approach improves upon previous methods by incorporating properties of individual cells as well as detailed description of the dynamic growth process, with all topological changes accounted for. Cell size, shape, and division plane orientation are modeled realistically. In addition, cell birth, cell growth, cell shrinkage, cell death, cell division, cell collision, and cell rearrangements are now fully accounted for. Different models of cell-cell interactions, such as lateral inhibition during the process of growth, can be studied in detail. Cellular pattern formation for monolayered tissues from arbitrary initial conditions, including that of a single cell, can also be studied in detail. Computational efficiency is achieved through the employment of a special data structure that ensures access to neighboring cells in constant time, without additional space requirement. We have successfully generated tissues consisting of more than 20,000 cells starting from 2 cells within 1 hour. We show that our model can be used to study embryogenesis, tissue fusion, and cell apoptosis. We give detailed study of the classical developmental process of bristle formation on the epidermis of D. melanogaster and the fundamental problem of homeostatic size control in epithelial tissues. Simulation results reveal significant roles of solubility of secreted factors in both the bristle formation and the homeostatic control of tissue size. Our method can be used to study broad problems in monolayered tissue formation. Our software is publicly available. PMID:25974182

Intrinsic Cell Stress is Independent of Organization in Engineered Cell Sheets.

PubMed

van Loosdregt, Inge A E W; Dekker, Sylvia; Alford, Patrick W; Oomens, Cees W J; Loerakker, Sandra; Bouten, Carlijn V C

2018-06-01

Understanding cell contractility is of fundamental importance for cardiovascular tissue engineering, due to its major impact on the tissue's mechanical properties as well as the development of permanent dimensional changes, e.g., by contraction or dilatation of the tissue. Previous attempts to quantify contractile cellular stresses mostly used strongly aligned monolayers of cells, which might not represent the actual organization in engineered cardiovascular tissues such as heart valves. In the present study, therefore, we investigated whether differences in organization affect the magnitude of intrinsic stress generated by individual myofibroblasts, a frequently used cell source for in vitro engineered heart valves. Four different monolayer organizations were created via micro-contact printing of fibronectin lines on thin PDMS films, ranging from strongly anisotropic to isotropic. Thin film curvature, cell density, and actin stress fiber distribution were quantified, and subsequently, intrinsic stress and contractility of the monolayers were determined by incorporating these data into sample-specific finite element models. Our data indicate that the intrinsic stress exerted by the monolayers in each group correlates with cell density. Additionally, after normalizing for cell density and accounting for differences in alignment, no consistent differences in intrinsic contractility were found between the different monolayer organizations, suggesting that the intrinsic stress exerted by individual myofibroblasts is independent of the organization. Consequently, this study emphasizes the importance of choosing proper architectural properties for scaffolds in cardiovascular tissue engineering, as these directly affect the stresses in the tissue, which play a crucial role in both the functionality and remodeling of (engineered) cardiovascular tissues.

Calcium oscillations in wounded fibroblast monolayers are spatially regulated through substrate mechanics

NASA Astrophysics Data System (ADS)

Lembong, Josephine; Sabass, Benedikt; Stone, Howard A.

2017-08-01

The maintenance of tissue integrity is essential for the life of multicellular organisms. Healing of a skin wound is a paradigm for how various cell types localize and repair tissue perturbations in an orchestrated fashion. To investigate biophysical mechanisms associated with wound localization, we focus on a model system consisting of a fibroblast monolayer on an elastic substrate. We find that the creation of an edge in the monolayer causes cytosolic calcium oscillations throughout the monolayer. The oscillation frequency increases with cell density, which shows that wound-induced calcium oscillations occur collectively. Inhibition of myosin II reduces the number of oscillating cells, demonstrating a coupling between actomyosin activity and calcium response. The spatial distribution of oscillating cells depends on the stiffness of the substrate. For soft substrates with a Young’s modulus E ~ 360 Pa, oscillations occur on average within 0.2 mm distance from the wound edge. Increasing substrate stiffness leads to an average localization of oscillations away from the edge (up to ~0.6 mm). In addition, we use traction force microscopy to determine stresses between cells and substrate. We find that an increase of substrate rigidity leads to a higher traction magnitude. For E  <  ~2 kPa, the traction magnitude is strongly concentrated at the monolayer edge, while for E  >  ~8 kPa, traction magnitude is on average almost uniform beneath the monolayer. Thus, the spatial occurrence of calcium oscillations correlates with the cell-substrate traction. Overall, the experiments with fibroblasts demonstrate a collective, chemomechanical localization mechanism at the edge of a wound with a potential physiological role.

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

PubMed

Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

2017-08-01

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effect of shear stress on water and LDL transport through cultured endothelial cell monolayers.

PubMed

Kang, Hongyan; Cancel, Limary M; Tarbell, John M

2014-04-01

Previous animal experiments have shown that the transport of LDL into arterial walls is shear stress dependent. However, little work has probed shear effects on LDL transport in vitro where conditions are well defined and mechanisms are more easily explored. Therefore, we measured shear induced water and LDL fluxes across cultured bovine aortic endothelial (BAEC) monolayers in vitro and developed a three-pore model to describe the transport dynamics. Cell apoptosis was quantified by TdT-mediated dUTP nick end labeling (TUNEL) assay. We also examined the role of nitric oxide (NO) in shear induced water and LDL fluxes by incubating BAEC monolayers with an NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA). Our results show that direct exposure of endothelial monolayers to 12 dyn/cm2 shear stress for 3 h elicited a 2.37-fold increase in water flux (Jv), a 3.00-fold increase in LDL permeability (Pe), a 1.32-fold increase in LDL uptake, and a 1.68-fold increase in apoptotic rate. L-NMMA treatment of BAEC monolayers blocked shear induced Jv response, but had no significant effect on shear responses of Pe and cell apoptosis. A long time shear exposure (12 h) of endothelial monolayers reduced Pe and apoptotic rate close to the baseline. These results suggest that an acute change in shear stress from a static baseline state induces increases in water flux that are mediated by an NO dependent mechanism. On the other hand, the permeability of endothelial monolayers to LDL is enhanced by a short term-shear application and reduced nearly to the baseline level by a longer time shear exposure, positively correlated to the leaky junctions forming around apoptotic cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Effect of shear stress on water and LDL transport through cultured endothelial cell monolayers

PubMed Central

Kang, Hongyan; Cancel, Limary M.; Tarbell, John M.

2014-01-01

Previous animal experiments have shown that the transport of LDL into arterial walls is shear stress dependent. However, little work has probed shear effects on LDL transport in vitro where conditions are well defined and mechanisms are more easily explored. Therefore, we measured shear induced water and LDL fluxes across cultured bovine aortic endothelial (BAEC) monolayers in vitro and developed a three-pore model to describe the transport dynamics. Cell apoptosis was quantified by TdT-mediated dUTP nick end labeling (TUNEL) assay. We also examined the role of nitric oxide (NO) in shear induced water and LDL fluxes by incubating BAEC monolayers with a NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA). Our results show that direct exposure of endothelial monolayers to 12 dyn/cm2 shear stress for 3 hours elicited a 2.37-fold increase in water flux (Jv), a 3.00-fold increase in LDL permeability (Pe), a 1.32-fold increase in LDL uptake, and a 1.68-fold increase in apoptotic rate. L-NMMA treatment of BAEC monolayers blocked shear induced Jv response, but had no significant effect on shear responses of Pe and cell apoptosis. A long time shear exposure (12 h) of endothelial monolayers reduced Pe and apoptotic rate close to the baseline. These results suggest that an acute change in shear stress from a static baseline state induces increases in water flux that are mediated by a NO dependent mechanism. On the other hand, the permeability of endothelial monolayers to LDL is enhanced by a short term-shear application and reduced nearly to the baseline level by a longer time shear exposure, positively correlated to the leaky junctions forming around apoptotic cells. PMID:24583416

Puerarin transport across a Calu-3 cell monolayer - an in vitro model of nasal mucosa permeability and the influence of paeoniflorin and menthol.

PubMed

Zhang, Lin; Du, Shou-Ying; Lu, Yang; Liu, Chang; Tian, Zhi-Hao; Yang, Chang; Wu, Hui-Chao; Wang, Zhen

2016-01-01

Nasal administration is a high-potential delivery system, particularly because it can provide a pathway from the nose to the brain. The objective of this research is to characterize puerarin transport across a Calu-3 cell monolayer used as a model of the nasal mucosa and to evaluate the influence of puerarin in combination with paeoniflorin and menthol to explore the enhanced mechanism of the permeability at the cell level. The apparent permeability coefficients (P app) of puerarin bidirectional transport were both <1.5×10(-6) cm/s, and the efflux ratio was <1.5, indicating that puerarin alone exhibited poor absorption and that its transport primarily occurred by passive diffusion through the cell monolayer. When puerarin was coad ministered with paeoniflorin, the P app was not changed (P>0.05). However, the addition of menthol significantly (P<0.05) improved the P app of puerarin in both directions. Moreover, based on immunofluorescence experiments and transepithelial electrical resistance measurements, the data indicated that the drug compatibility opened tight junctions and weakened the barrier capabilities of epithelial cells, thereby promoting the permeability of puerarin.

A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

PubMed

Koban, Robert; Neumann, Markus; Daugs, Aila; Bloch, Oliver; Nitsche, Andreas; Langhammer, Stefan; Ellerbrok, Heinz

2018-02-01

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction.

PubMed

Miyahara, Yoshinori; Nagaya, Noritoshi; Kataoka, Masaharu; Yanagawa, Bobby; Tanaka, Koichi; Hao, Hiroyuki; Ishino, Kozo; Ishida, Hideyuki; Shimizu, Tatsuya; Kangawa, Kenji; Sano, Shunji; Okano, Teruo; Kitamura, Soichiro; Mori, Hidezo

2006-04-01

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.

Interaction of 3‧,4‧,6‧-trimyristoyl-uridine derivative as potential anticancer drug with phospholipids of tumorigenic and non-tumorigenic cells

NASA Astrophysics Data System (ADS)

Salis, Luiz Fernando Grosso; Jaroque, Guilherme Nuñez; Escobar, Jhon Fernando Berrío; Giordani, Cristiano; Martinez, Alejandro Martinez; Fernández, Diana Margarita Márquez; Castelli, Francesco; Sarpietro, Maria Grazia; Caseli, Luciano

2017-12-01

Investigating the mechanism of action of drugs whose pharmaceutical activity is associated with cell membranes is fundamental to comprehending the biochemical and biophysical processes that occur on membrane surfaces. In this work, we investigated the interaction of an ester-type derivative of uridine, 3‧,4‧,6‧-trimyristoyl uridine, with models for cell membranes formed by lipid monolayers at the air-water interface. For that, selected lipids have been chosen in order to mimic tumorigenic and non-tumorigenic cells. For mixed monolayers with 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dihexadecanoyl-sn-glycero-3-phospho-L-serine (DPPS), the surface pressure-area isotherms exhibited a noticeable shift to lower areas in relation to the areas predicted for ideal mixtures, indicating a condensation of the monolayer structure. Changes in the viscoelastic properties of the interfacial film could be inferred by analyzing the compressibility modulus of the monolayer. Structural and morphological changes were also evidenced by using vibrational spectroscopy and Brewster angle microscopy, respectively, with distinctive effects on DPPC and DPPS. As conclusion we can state that the lipid composition of the monolayer modulates the interaction with this lipophilic drug, which may have important implications in understanding how this drug acts on specific sites of the cellular membrane.

Physics of active jamming during collective cellular motion in a monolayer.

PubMed

Garcia, Simon; Hannezo, Edouard; Elgeti, Jens; Joanny, Jean-François; Silberzan, Pascal; Gov, Nir S

2015-12-15

Although collective cell motion plays an important role, for example during wound healing, embryogenesis, or cancer progression, the fundamental rules governing this motion are still not well understood, in particular at high cell density. We study here the motion of human bronchial epithelial cells within a monolayer, over long times. We observe that, as the monolayer ages, the cells slow down monotonously, while the velocity correlation length first increases as the cells slow down but eventually decreases at the slowest motions. By comparing experiments, analytic model, and detailed particle-based simulations, we shed light on this biological amorphous solidification process, demonstrating that the observed dynamics can be explained as a consequence of the combined maturation and strengthening of cell-cell and cell-substrate adhesions. Surprisingly, the increase of cell surface density due to proliferation is only secondary in this process. This analysis is confirmed with two other cell types. The very general relations between the mean cell velocity and velocity correlation lengths, which apply for aggregates of self-propelled particles, as well as motile cells, can possibly be used to discriminate between various parameter changes in vivo, from noninvasive microscopy data.

Mechanical characterization of disordered and anisotropic cellular monolayers

NASA Astrophysics Data System (ADS)

Nestor-Bergmann, Alexander; Johns, Emma; Woolner, Sarah; Jensen, Oliver E.

2018-05-01

We consider a cellular monolayer, described using a vertex-based model, for which cells form a spatially disordered array of convex polygons that tile the plane. Equilibrium cell configurations are assumed to minimize a global energy defined in terms of cell areas and perimeters; energy is dissipated via dynamic area and length changes, as well as cell neighbor exchanges. The model captures our observations of an epithelium from a Xenopus embryo showing that uniaxial stretching induces spatial ordering, with cells under net tension (compression) tending to align with (against) the direction of stretch, but with the stress remaining heterogeneous at the single-cell level. We use the vertex model to derive the linearized relation between tissue-level stress, strain, and strain rate about a deformed base state, which can be used to characterize the tissue's anisotropic mechanical properties; expressions for viscoelastic tissue moduli are given as direct sums over cells. When the base state is isotropic, the model predicts that tissue properties can be tuned to a regime with high elastic shear resistance but low resistance to area changes, or vice versa.

An oscillating dynamic model of collective cells in a monolayer

NASA Astrophysics Data System (ADS)

Lin, Shao-Zhen; Xue, Shi-Lei; Li, Bo; Feng, Xi-Qiao

2018-03-01

Periodic oscillations of collective cells occur in the morphogenesis and organogenesis of various tissues and organs. In this paper, an oscillating cytodynamic model is presented by integrating the chemomechanical interplay between the RhoA effector signaling pathway and cell deformation. We show that both an isolated cell and a cell aggregate can undergo spontaneous oscillations as a result of Hopf bifurcation, upon which the system evolves into a limit cycle of chemomechanical oscillations. The dynamic characteristics are tailored by the mechanical properties of cells (e.g., elasticity, contractility, and intercellular tension) and the chemical reactions involved in the RhoA effector signaling pathway. External forces are found to modulate the oscillation intensity of collective cells in the monolayer and to polarize their oscillations along the direction of external tension. The proposed cytodynamic model can recapitulate the prominent features of cell oscillations observed in a variety of experiments, including both isolated cells (e.g., spreading mouse embryonic fibroblasts, migrating amoeboid cells, and suspending 3T3 fibroblasts) and multicellular systems (e.g., Drosophila embryogenesis and oogenesis).

Escherichia coli K1 invasion increases human brain microvascular endothelial cell monolayer permeability by disassembling vascular-endothelial cadherins at tight junctions.

PubMed

Sukumaran, Sunil K; Prasadarao, Nemani V

2003-11-01

We investigated the permeability changes that occur in the human brain microvascular endothelial cell (HBMEC) monolayer, an in vitro model of the blood-brain barrier, during Escherichia coli K1 infection. An increase in permeability of HBMECs and a decrease in transendothelial electrical resistance were observed. These permeability changes occurred only when HBMECs were infected with E. coli expressing outer membrane protein A (OmpA) and preceded the traversal of bacteria across the monolayer. Activated protein kinase C (PKC)-alpha interacts with vascular-endothelial cadherins (VECs) at the tight junctions of HBMECs, resulting in the dissociation of beta-catenins from VECs and leading to the increased permeability of the HBMEC monolayer. Overexpression of a dominant negative form of PKC-alpha in HBMECs blocked the E. coli-induced increase in permeability of HBMECs. Anti-OmpA and anti-OmpA receptor antibodies exerted inhibition of E. coli-induced permeability of HBMEC monolayers. This inhibition was the result of the absence of PKC-alpha activation in HBMECs treated with the antibodies.

Mathematical model for cell competition: Predator-prey interactions at the interface between two groups of cells in monolayer tissue.

PubMed

Nishikawa, Seiya; Takamatsu, Atsuko; Ohsawa, Shizue; Igaki, Tatsushi

2016-09-07

The phenomenon of 'cell competition' has been implicated in the normal development and maintenance of organs, such as in the regulation of organ size and suppression of neoplastic development. In cell competition, one group of cells competes with another group through an interaction at their interface. Which cell group "wins" is governed by a certain relative fitness within the cells. However, this idea of cellular fitness has not been clearly defined. We construct two types of mathematical models to describe this phenomenon of cell competition by considering the interaction at the interface as a predator-prey type interaction in a monolayer tissue such as epithelium. Both of these models can reproduce several typical experimental observations involving systems of mutant cells (losers) and normal cells (winners). By analyzing one of the model and defining an index for the degree of fitness in groups of cells, we show that the fate of each group mainly depends on the relative carrying capacities of certain resources and the strength of the predator-prey interaction at the interface. This contradicts the classical hypothesis in which the relative proliferation rate determines the winner. Copyright © 2016 Elsevier Ltd. All rights reserved.

Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

NASA Astrophysics Data System (ADS)

Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.

1988-05-01

The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.

Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions.

PubMed

Álvarez, Romina S; Sacerdoti, Flavia; Jancic, Carolina; Paton, Adrienne W; Paton, James C; Ibarra, Cristina; Amaral, María M

2016-01-01

Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20-30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.

Changes in barrier function of a model intestinal epithelium by intraepithelial lymphocytes require new protein synthesis by epithelial cells.

PubMed Central

Taylor, C T; Murphy, A; Kelleher, D; Baird, A W

1997-01-01

BACKGROUND: Elements of the mucosal immune system may play an important part in regulating epithelial barrier function in the intestinal tract. Intraepithelial lymphocytes (IELs) represent a subtype of immunocyte which is strategically placed to regulate epithelial function at most mucosal sites. AIMS AND METHODS: An IEL derived cell line (SC1) was used to examine its effects on the model epithelium T84--a tumour derived cell line which retains the phenotype of colonic crypt cells. Transepithelial electrical resistance (TER) was used as a marker of epithelial integrity. RESULTS: Coculture of T84 cells with SC1 produced a significant fall in TER as did exposure of T84 monolayers to IEL derived supernatant. Recombinant interferon-gamma (rIFN gamma) also reduced TER in T84 monolayers. Cycloheximide prevented the effects of IEL supernatant and of rIFN gamma on TER. The fall in TER in response to rIFN gamma was attenuated by blocking antibodies, which did not alter the fall in resistance induced by IEL supernatant. Fractions of IEL supernatant, separated on the basis of size, evoked temporally distinct changes in TER. Ultrastructural studies support the hypothesis that the slow onset but severe fall in TER indicates catastrophic effects on the monolayer. The more rapid onset fall in TER was not associated with gross changes in monolayer morphology. Reduction of TER by IEL supernatant was not influenced by inhibitors of tyrosine phosphatase or of protein kinase C. Although herbimycin did reduce the rapid onset change in TER, the tyrosine kinase inhibitor genistein did not alter responses to IEL supernatant. CONCLUSIONS: Mucosal T cells may influence barrier function by a process involving new protein synthesis by epithelial cells. This model may have relevance in some inflammatory conditions of the gastrointestinal tract. Images PMID:9203943

Mathematical modelling of the transport of hydroxypropyl-β-cyclodextrin inclusion complexes of ranitidine hydrochloride and furosemide loaded chitosan nanoparticles across a Caco-2 cell monolayer.

PubMed

Sadighi, Armin; Ostad, S N; Rezayat, S M; Foroutan, M; Faramarzi, M A; Dorkoosh, F A

2012-01-17

Chitosan nanoparticles (CS-NPs) have been used to enhance the permeability of furosemide and ranitidine hydrochloride (ranitidine HCl) which were selected as candidates for two different biopharmaceutical drug classes having low permeability across Caco-2 cell monolayers. Drugs loaded CS-NPs were prepared by ionic gelation of CS and pentasodium tripolyphosphate (TPP) which added to the drugs inclusion complexes with hydroxypropyl-β-cyclodextrin (HP-βCD). The stability constants for furosemide/HP-βCD and ranitidine HCl/HP-βCD were calculated as 335 M(-1) and 410 M(-1), whereas the association efficiencies (AE%) of the drugs/HP-βCD inclusion complexes with CS-NPs were determined to be 23.0 and 19.5%, respectively. Zetasizer and scanning electron microscopy (SEM) were used to characterise drugs/HP-βCD-NPs size and morphology. Transport of both nano and non-nano formulations of drugs/HP-βCD complexes across a Caco-2 cell monolayer was assessed and fitted to mathematical models. Furosemide/HP-βCD-NPs demonstrated transport kinetics best suited for the Higuchi model, whereas other drug formulations demonstrated power law transportation behaviour. Permeability experiments revealed that furosemide/HP-βCD and ranitidine HCl/HP-βCD nano formulations greatly induce the opening of tight junctions and enhance drug transition through Caco-2 monolayers. Copyright © 2011 Elsevier B.V. All rights reserved.

Respiratory syncytial virus infection disrupts monolayer integrity and function in cystic fibrosis airway cells.

PubMed

Kong, Michele; Maeng, Patrick; Hong, Jeong; Szczesniak, Rhonda; Sorscher, Eric; Sullender, Wayne; Clancy, John Paul

2013-09-19

Respiratory Syncytial Virus (RSV) infection is a common contributor to pulmonary symptoms in children with cystic fibrosis (CF). Here we examined RSV infection in immortalized bronchial epithelial cells (CFBE41o-) expressing wild-type (wt) or F508del cystic fibrosis transmembrane conductance regulator (CFTR), for monolayer integrity and RSV replication. CFBE41o- monolayers expressing wt or F508del CFTR were grown on permeable supports and inoculated with RSV A2 strain. Control experiments utilized UV-inactivated RSV and heat-killed RSV. Monolayer resistance and RSV production was monitored for up to six days post-infection. Within 24 h, a progressive decrease in monolayer resistance was observed in RSV infected F508del CFBE41o- cells, while the monolayer integrity of RSV infected wt CFTR CFBE41o- cells remained stable. RSV replication was necessary to disrupt F508del CFBE41o- monolayers as UV-irradiated and heat killed RSV had no effect on monolayer integrity, with an earlier and much more pronounced peak in RSV titer noted in F508del relative to wt CFTR-expressing cells. RSV infection of wt CFBE41o- monolayers also resulted in blunting of CFTR response. These findings identify an enhanced sensitivity of CFBE41o- cells expressing F508del CFTR to RSV infection, replication and monolayer disruption independent of the cellular immune response, and provide a novel mechanism by which cystic fibrosis airway epithelia are susceptible to RSV-dependent injury.

Cellular dosimetry of (111)In using monte carlo N-particle computer code: comparison with analytic methods and correlation with in vitro cytotoxicity.

PubMed

Cai, Zhongli; Pignol, Jean-Philippe; Chan, Conrad; Reilly, Raymond M

2010-03-01

Our objective was to compare Monte Carlo N-particle (MCNP) self- and cross-doses from (111)In to the nucleus of breast cancer cells with doses calculated by reported analytic methods (Goddu et al. and Farragi et al.). A further objective was to determine whether the MCNP-predicted surviving fraction (SF) of breast cancer cells exposed in vitro to (111)In-labeled diethylenetriaminepentaacetic acid human epidermal growth factor ((111)In-DTPA-hEGF) could accurately predict the experimentally determined values. MCNP was used to simulate the transport of electrons emitted by (111)In from the cell surface, cytoplasm, or nucleus. The doses to the nucleus per decay (S values) were calculated for single cells, closely packed monolayer cells, or cell clusters. The cell and nucleus dimensions of 6 breast cancer cell lines were measured, and cell line-specific S values were calculated. For self-doses, MCNP S values of nucleus to nucleus agreed very well with those of Goddu et al. (ratio of S values using analytic methods vs. MCNP = 0.962-0.995) and Faraggi et al. (ratio = 1.011-1.024). MCNP S values of cytoplasm and cell surface to nucleus compared fairly well with the reported values (ratio = 0.662-1.534 for Goddu et al.; 0.944-1.129 for Faraggi et al.). For cross doses, the S values to the nucleus were independent of (111)In subcellular distribution but increased with cluster size. S values for monolayer cells were significantly different from those of single cells and cell clusters. The MCNP-predicted SF for monolayer MDA-MB-468, MDA-MB-231, and MCF-7 cells agreed with the experimental data (relative error of 3.1%, -1.0%, and 1.7%). The single-cell and cell cluster models were less accurate in predicting the SF. For MDA-MB-468 cells, relative error was 8.1% using the single-cell model and -54% to -67% using the cell cluster model. Individual cell-line dimensions had large effects on S values and were needed to estimate doses and SF accurately. MCNP simulation compared well with the reported analytic methods in the calculation of subcellular S values for single cells and cell clusters. Application of a monolayer model was most accurate in predicting the SF of breast cancer cells exposed in vitro to (111)In-DTPA-hEGF.

Involvement of PI3K and ROCK signaling pathways in migration of bone marrow-derived mesenchymal stem cells through human brain microvascular endothelial cell monolayers.

PubMed

Lin, Mei-Na; Shang, De-Shu; Sun, Wei; Li, Bo; Xu, Xin; Fang, Wen-Gang; Zhao, Wei-Dong; Cao, Liu; Chen, Yu-Hua

2013-06-04

Bone marrow-derived mesenchymal stem cells (MSC) represent an important and easily available source of stem cells for potential therapeutic use in neurological diseases. The entry of circulating cells into the central nervous system by intravenous administration requires, firstly, the passage of the cells across the blood-brain barrier (BBB). However, little is known of the details of MSC transmigration across the BBB. In the present study, we employed an in vitro BBB model constructed using a human brain microvascular endothelial cell monolayer to study the mechanism underlying MSC transendothelial migration. Transmigration assays, transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) flux assays showed that MSC could transmigrate through human brain microvascular endothelial cell monolayers by a paracellular pathway. Cell fractionation and immunofluorescence assays confirmed the disruption of tight junctions. Inhibition assays showed that a Rho-kinase (ROCK) inhibitor (Y27632) effectively promoted MSC transendothelial migration; conversely, a PI3K inhibitor (LY294002) blocked MSC transendothelial migration. Interestingly, adenovirus-mediated interference with ROCK in MSC significantly increased MSC transendothelial migration, and overexpression of a PI3K dominant negative mutant in MSC cells could block transendothelial migration. Our findings provide clear evidence that the PI3K and ROCK pathways are involved in MSC migration through human brain microvascular endothelial cell monolayers. The information yielded by this study may be helpful in constructing gene-modified mesenchymal stem cells that are able to penetrate the BBB effectively for cell therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Physics of active jamming during collective cellular motion in a monolayer

PubMed Central

Garcia, Simon; Hannezo, Edouard; Elgeti, Jens; Joanny, Jean-François; Silberzan, Pascal; Gov, Nir S.

2015-01-01

Although collective cell motion plays an important role, for example during wound healing, embryogenesis, or cancer progression, the fundamental rules governing this motion are still not well understood, in particular at high cell density. We study here the motion of human bronchial epithelial cells within a monolayer, over long times. We observe that, as the monolayer ages, the cells slow down monotonously, while the velocity correlation length first increases as the cells slow down but eventually decreases at the slowest motions. By comparing experiments, analytic model, and detailed particle-based simulations, we shed light on this biological amorphous solidification process, demonstrating that the observed dynamics can be explained as a consequence of the combined maturation and strengthening of cell−cell and cell−substrate adhesions. Surprisingly, the increase of cell surface density due to proliferation is only secondary in this process. This analysis is confirmed with two other cell types. The very general relations between the mean cell velocity and velocity correlation lengths, which apply for aggregates of self-propelled particles, as well as motile cells, can possibly be used to discriminate between various parameter changes in vivo, from noninvasive microscopy data. PMID:26627719

Quantification of stromal vascular cell mechanics with a linear cell monolayer rheometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elkins, Claire M., E-mail: cma9@stanford.edu; Fuller, Gerald G.; Shen, Wen-Jun

2015-01-15

Over the past few decades researchers have developed a variety of methods for measuring the mechanical properties of whole cells, including traction force microscopy, atomic force microscopy (AFM), and single-cell tensile testing. Though each of these techniques provides insight into cell mechanics, most also involve some nonideal conditions for acquiring live cell data, such as probing only one portion of a cell at a time, or placing the cell in a nonrepresentative geometry during testing. In the present work, we describe the development of a linear cell monolayer rheometer (LCMR) and its application to measure the mechanics of a live,more » confluent monolayer of stromal vascular cells. In the LCMR, a monolayer of cells is contacted on both top and bottom by two collagen-coated plates and allowed to adhere. The top plate then shears the monolayer by stepping forward to induce a predetermined step strain, while a force transducer attached to the top plate collects stress information. The stress and strain data are then used to determine the maximum relaxation modulus recorded after step-strain, G{sub r}{sup 0}, referred to as the zero-time relaxation modulus of the cell monolayer. The present study validates the ability of the LCMR to quantify cell mechanics by measuring the change in G{sub r}{sup 0} of a confluent cell monolayer upon the selective inhibition of three major cytoskeletal components (actin microfilaments, vimentin intermediate filaments, and microtubules). The LCMR results indicate that both actin- and vimentin-deficient cells had ∼50% lower G{sub r}{sup 0} values than wild-type, whereas tubulin deficiency resulted in ∼100% higher G{sub r}{sup 0} values. These findings constitute the first use of a cell monolayer rheometer to quantitatively distinguish the roles of different cytoskeletal elements in maintaining cell stiffness and structure. Significantly, they are consistent with results obtained using single-cell mechanical testing methods, suggesting that the rheology-based LCMR technique may be a useful tool for rapid analysis of cell mechanics by shearing an entire cell monolayer.« less

Artificial sweetener saccharin disrupts intestinal epithelial cells' barrier function in vitro.

PubMed

Santos, P S; Caria, C R P; Gotardo, E M F; Ribeiro, M L; Pedrazzoli, J; Gambero, A

2018-06-25

Consumption of non-nutritive sweeteners (NNS) is a dietary practice used by those who wish to lose weight or by patients on a sugar-restricted diet such as those with DM2. Although these substances are safe, possible biological interactions with the digestive tract, particularly in relation to intestinal permeability, have not been studied. Thus, the current work sought to investigate the action of different NNS on intestinal permeability using an in vitro Caco-2 cell model. Caco-2 cells were incubated with acesulfame K, aspartame, saccharin, or sucralose at equimolar concentrations. Acesulfame K, aspartame, and sucralose did not disrupt monolayer integrity in the cells. However, saccharin increased paracellular permeability and decreased transepithelial electrical resistance (TEER) via a non-cytotoxic mechanism. The levels of the tight junction protein claudin-1 were reduced in Caco-2 cells that had previously been exposed to saccharin. The inhibition of nuclear factor-κB (NF-κB) was able to prevent the reduction in TEER induced by saccharin treatment. Thalidomide, as an inhibitor of ubiquitin ligase, was able to prevent the decrease in claudin-1 protein expression and the TEER reduction in Caco-2 cells. Saccharin disrupts monolayer integrity and alters paracellular permeability in a Caco-2 cell monolayer model, via a mechanism involving NF-κB activation, resulting in the ubiquitination of the tight junction protein claudin-1. Saccharin consumption may potentially alter the intestinal integrity in humans.

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

PubMed Central

Salomon, Julie; Gaston, Cécile; Magescas, Jérémy; Duvauchelle, Boris; Canioni, Danielle; Sengmanivong, Lucie; Mayeux, Adeline; Michaux, Grégoire; Campeotto, Florence; Lemale, Julie; Viala, Jérôme; Poirier, Françoise; Minc, Nicolas; Schmitz, Jacques; Brousse, Nicole; Ladoux, Benoit; Goulet, Olivier; Delacour, Delphine

2017-01-01

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture. PMID:28084299

Cellular interactions of a lipid-based nanocarrier model with human keratinocytes: Unravelling transport mechanisms.

PubMed

Silva, Elisabete; Barreiros, Luísa; Segundo, Marcela A; Costa Lima, Sofia A; Reis, Salette

2017-04-15

Knowledge of delivery system transport through epidermal cell monolayer is vital to improve skin permeation and bioavailability. Recently, nanostructured lipid carriers (NLCs) have gained great attention for transdermal delivery due to their biocompatibility, high drug payload, occlusive properties and skin hydration effect. However, the nanocarriers transport related mechanisms in epidermal epithelial cells are not yet understood. In this research, the internalization and transport pathways of the NLCs across the epidermal epithelial cell monolayer (HaCaT cells) were investigated. The 250nm sized witepsol/miglyol NLCs, prepared by hot homogenization had reduced cytotoxicity and no effect on the integrity of cell membrane in human HaCaT keratinocytes. The internalization was time-, concentration- and energy-dependent, and the uptake of NLCs was a vesicle-mediated process by macropinocytosis and clathrin-mediated pathways. 3% of NLCs were found at the apical membrane side of the HaCaT monolayer through exocytosis mechanism. Additionally, the endoplasmic reticulum, Golgi apparatus and microtubules played crucial roles in the transport of NLCs out of HaCaT cells. NLCs were transported intact across the human keratinocytes monolayer, without disturbing the tight junction's structure. From the transcytosis data only approximately 12% of the internalized NLCs were passed from the apical to the basolateral side. The transcytosis of NLCs throughout the HaCaT cell monolayer towards the basolateral membrane side requires the involvement of the endoplasmic reticulum, Golgi apparatus and microtubules. Our findings may contribute to a systematic understanding of NLCs transport across epidermal epithelial cell monolayers and their optimization for clinical transdermal application. Transdermal drug delivery is a challenging and growing area of clinical application. Lipid nanoparticles such as nanostructured lipid carriers (NLCs) have gained wide interest for transdermal drug delivery. However these nanocarriers' interactions with epidermal epithelial barrier are yet unknown. Unveiling the mechanisms involved in NLCs transport across the epidermal epithelial monolayers will contribute with valuable information to achieve enhanced skin permeability, superior bioavailability and consequently improved therapeutic effect. With our present work we could certainly provide researchers and clinicians guidance for the design of optimized transdermal delivery systems, based on the nanomaterials and biological interactions. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Automatic Cell Segmentation in Fluorescence Images of Confluent Cell Monolayers Using Multi-object Geometric Deformable Model.

PubMed

Yang, Zhen; Bogovic, John A; Carass, Aaron; Ye, Mao; Searson, Peter C; Prince, Jerry L

2013-03-13

With the rapid development of microscopy for cell imaging, there is a strong and growing demand for image analysis software to quantitatively study cell morphology. Automatic cell segmentation is an important step in image analysis. Despite substantial progress, there is still a need to improve the accuracy, efficiency, and adaptability to different cell morphologies. In this paper, we propose a fully automatic method for segmenting cells in fluorescence images of confluent cell monolayers. This method addresses several challenges through a combination of ideas. 1) It realizes a fully automatic segmentation process by first detecting the cell nuclei as initial seeds and then using a multi-object geometric deformable model (MGDM) for final segmentation. 2) To deal with different defects in the fluorescence images, the cell junctions are enhanced by applying an order-statistic filter and principal curvature based image operator. 3) The final segmentation using MGDM promotes robust and accurate segmentation results, and guarantees no overlaps and gaps between neighboring cells. The automatic segmentation results are compared with manually delineated cells, and the average Dice coefficient over all distinguishable cells is 0.88.

Alternative functional in vitro models of human intestinal epithelia

PubMed Central

Kauffman, Amanda L.; Gyurdieva, Alexandra V.; Mabus, John R.; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J.

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport. PMID:23847534

Alternative functional in vitro models of human intestinal epithelia.

PubMed

Kauffman, Amanda L; Gyurdieva, Alexandra V; Mabus, John R; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

PubMed

Fernando, Elizabeth H; Dicay, Michael; Stahl, Martin; Gordon, Marilyn H; Vegso, Andrew; Baggio, Cristiane; Alston, Laurie; Lopes, Fernando; Baker, Kristi; Hirota, Simon; McKay, Derek M; Vallance, Bruce; MacNaughton, Wallace K

2017-11-01

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner. Copyright © 2017 the American Physiological Society.

An insight of in vitro transport of PEGylated non-ionic surfactant vesicles (NSVs) across the intestinal polarized enterocyte monolayers.

PubMed

Primavera, Rosita; Palumbo, Paola; Celia, Christian; Cinque, Benedetta; Carata, Elisabetta; Carafa, Maria; Paolino, Donatella; Cifone, Maria Grazia; Di Marzio, Luisa

2018-06-01

PEGylated non-ionic surfactant-based vesicles (NSVs) are promising drug delivery systems for the local, oral and systemic administrations of therapeutics. The aim of this study was to test the cellular biocompatibility and transport of Nile Red-loaded NSVs (NR-NSVs) across the Caco-2-cell monolayers, which represent an in vitro model of human intestinal epithelium. The NR-NSVs assumed a spherical shape with a mean size of 140 nm, and a narrow size distribution. The NR-NSVs did not modify Caco-2 cell viability, which remained unaltered in vitro up to a concentration of 1 mM. The transport studies demonstrated that the NR-NSVs moved across the Caco-2 monolayers without affecting the transepithelial electrical resistance. These results were supported by flow cytometry analysis, which demonstrated that NR-NSVs were internalized inside the Caco-2 cells. Nanoparticle tracking and Transmission Electron Microscopy (TEM) analysis showed the presence of NR-NSVs in the basolateral side of the Caco-2 monolayers. TEM images also showed that NSVs were transported intact across the Caco-2 monolayers, thus demonstrating a predominant transcytosis mechanism of transport through endocytosis. The NSVs did not affect the integrity of the membrane barrier in vitro, and can potentially be used in clinics to increase the oral bioavailability and delivery of therapeutics. Copyright © 2018 Elsevier B.V. All rights reserved.

3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

PubMed

Matsusaki, Michiya; Hikimoto, Daichi; Nishiguchi, Akihiro; Kadowaki, Koji; Ohura, Kayoko; Imai, Teruko; Akashi, Mitsuru

2015-02-13

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Transport and metabolism of delta sleep-inducing peptide in cultured human intestinal epithelial cell monolayers.

PubMed

Augustijns, P F; Borchardt, R T

1995-12-01

A cultured human intestinal epithelial (Caco-2) cell monolayer was used to study the transport and metabolism of delta sleep-inducing peptide [DSIP (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu)]. DSIP is of interest because it has been reported to be capable of permeating biological barriers (e.g. blood-brain barrier), and this property has been related to its solution conformation. When applied to the apical (AP) side of Caco-2 cell monolayers, DSIP was rapidly metabolized (8.2 +/- 1.1% remaining after a 2-hr incubation), affording Trp as the major metabolite and Trp-Ala as a minor metabolite. When DSIP was added to the basolateral (BL) side of the monolayer, the same metabolites were detected, but the peptide was more stable (70.6 +/- 3.0% remaining after a 2-hr incubation). Inclusion of bestatin, an inhibitor of aminopeptidases, at concentrations up to 0.29 mM with DSIP on the AP side of the Caco-2 cell monolayer increased the stability of the peptide only slightly but dramatically altered the distribution of the metabolites (Trp-Ala became the major metabolite, and Trp became the minor metabolite). Inclusion of other aminopeptidase inhibitors (e.g. amastatin, puromycin) alone, dipeptidylpeptidase IV inhibitors (e.g. diprotin A, Gly-Pro) alone, inhibitors of proteases that require heavy metals for proper activity (e.g. EDTA, 1,10-phenanthroline) alone, or cysteine protease inhibitors (e.g. leupeptin) alone did not lead to significant stabilization of the peptide. However, inclusion of a combination of 0.29 mM bestatin and 1 mM diprotin A with DSIP on the AP side of the monolayers resulted in a substantial increase in the stability of the peptide (83.2 +/- 3.7% remaining after a 2-hr incubation). However, under these conditions, a new metabolite (Trp-Ala-Gly-Gly-Asp-Ala-Ser) was observed with a formation that could be inhibited by inclusion of 1 mM captopril, an inhibitor of peptidyl dipeptidase A. Therefore, the stability of DSIP could be further increased (95.1 +/- 1.6% remaining after a 2-hr incubation) by incubating the peptide with 0.29 mM bestatin, 1 mM diprotin A, and 1 mM captopril. However, even when the major metabolic pathways were inhibited on the AP side of the cell monolayer, no DSIP was detected on the BL side of a Caco-2 cell monolayer. These results suggest that a yet unidentified metabolic pathway is preventing the AP-to-BL flux of DSIP or that DSIP has lower "intrinsic" ability to permeate across cultured intestinal epithelial cells than across cultured brain endothelial cells, a cell culture model of the blood-brain barrier.

A facile in vitro model to study rapid mineralization in bone tissues.

PubMed

Deegan, Anthony J; Aydin, Halil M; Hu, Bin; Konduru, Sandeep; Kuiper, Jan Herman; Yang, Ying

2014-09-16

Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required. We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT). Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture. This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate size determines the mineralization rate. Mineral content depended on aggregate size and culture duration. Thus, our culture system may provide a good model to study regulation factors at different development phases of the osteoblastic lineage.

Three-dimensional Model of Tissue and Heavy Ions Effects

NASA Technical Reports Server (NTRS)

Ponomarev, Artem L.; Sundaresan, Alamelu; Huff, Janice L.; Cucinotta, Francis A.

2007-01-01

A three-dimensional tissue model was incorporated into a new Monte Carlo algorithm that simulates passage of heavy ions in a tissue box . The tissue box was given as a realistic model of tissue based on confocal microscopy images. The action of heavy ions on the cellular matrix for 2- or 3-dimensional cases was simulated. Cells were modeled as a cell culture monolayer in one example, where the data were taken directly from microscopy (2-d cell matrix), and as a multi-layer obtained from confocal microscopy (3-d case). Image segmentation was used to identify cells with precise areas/volumes in an irradiated cell culture monolayer, and slices of tissue with many cell layers. The cells were then inserted into the model box of the simulated physical space pixel by pixel. In the case of modeled tissues (3-d), the tissue box had periodic boundary conditions imposed, which extrapolates the technique to macroscopic volumes of tissue. For the real tissue (3-d), specific spatial patterns for cell apoptosis and necrosis are expected. The cell patterns were modeled based on action cross sections for apoptosis and necrosis estimated from current experimental data. A spatial correlation function indicating a higher spatial concentration of damaged cells from heavy ions relative to the low-LET radiation cell damage pattern is presented. The spatial correlation effects among necrotic cells can help studying microlesions in organs, and probable effects of directionality of heavy ion radiation on epithelium and endothelium.

Mycoplasma pulmonis Inhibits Electrogenic Ion Transport across Murine Tracheal Epithelial Cell Monolayers

PubMed Central

Lambert, Linda C.; Trummell, Hoa Q.; Singh, Ashvani; Cassell, Gail H.; Bridges, Robert J.

1998-01-01

Murine chronic respiratory disease is characterized by persistent colonization of tracheal and bronchial epithelial cell surfaces by Mycoplasma pulmonis, submucosal and intraluminal immune and inflammatory cells, and altered airway activity. To determine the direct effect of M. pulmonis upon transepithelial ion transport in the absence of immune and inflammatory cell responses, primary mouse tracheal epithelial cell monolayers (MTEs) were apically infected and assayed in Ussing chambers. M. pulmonis-infected MTEs, but not those infected with a nonmurine mycoplasma, demonstrated reductions in amiloride-sensitive Na+ absorption, cyclic AMP, and cholinergic-stimulated Cl− secretion and transepithelial resistance. These effects were shown to require interaction of viable organisms with the apical surface of the monolayer and to be dependent upon organism number and duration of infection. Altered transport due to M. pulmonis was not merely a result of epithelial cell death as evidenced by the following: (i) active transport of Na+ and Cl−, albeit at reduced rates; (ii) normal cell morphology, including intact tight junctions, as demonstrated by electron microscopy; (iii) maintenance of a mean transepithelial resistance of 440 Ω/cm2; and (iv) lack of leakage of fluid from the basolateral to the apical surface of the monolayer. Alteration in epithelial ion transport in vitro is consistent with impaired pulmonary clearance and altered airway function in M. pulmonis-infected animals. Furthermore, the ability of M. pulmonis to alter transport without killing the host cell may explain its successful parasitism and long-term persistence in the host. Further study of the MTE-M. pulmonis model should elucidate the molecular mechanisms which mediate this reduction in transepithelial ion transport. PMID:9423868

[Study on transport mechanism of baicalin in Scutellariae radix extracts and effect of Angelica dahurica extracts on transport of baicalin by Caco-2 cell monolayer model].

PubMed

Liang, Xin-Li; Zhu, Meng-Liang; Zhao, Li-Jun; Zhao, Guo-Wei; Liao, Zheng-Gen; Cao, Yun-Chao; Yang, Ming

2013-07-01

To study the transport mechanism of baicalin of Scutellariae Radix extracts and the effect of Angelica dahurica extracts on the intestinal absorption of baicalin by using Caco-2 cell monolayer model, in order to analyze the effect mechanism of Angelica dahurica extracts on the intestinal absorption of baicalin. The Caco-2 cell monolayer model was established with human colonic adenocarcinoma cells, and used to study the effect of pH, time, drug concentration and temperature on the transport of baicalin in Scutellariae Radix extracts, the effect of P-gp and MRP protein-dedicated inhibitors on the bidirectional transport of baicalin in Caco-2 cell model, and the effect of angelica root extracts on baicalin absorption and transport. Baicalin was absorbed well at 37 degrees C and under pH 7.4 condition and concentration dependent. Its proteins became inactive at 4 degrees C, with a low transport. The bi-drectional transfer PDR was 0. 54. After P-gp inhibitor verapamil and MRP inhibitor probenecid were added, the value of PappBL-AP of baicalin decreased, but without any difference in PDR. The transport of baicalin was improved by 2.34, 3.31 and 3.13 times, after A. dahurica extract coumarin, volatile oil, and mixture of coumarin and volatile oil. The transport mechanism of baicalin is mainly passive transfer and supplemented with efflux proteins involved. A. dahurica extracts can enhance the absorption of baicalin, which may be related to the passive transfer merchanism of baicalin. A. dahurica extracts' effect in opening the close junction among cells may be related to its expression or function in inhibiting efflux proteins.

Vertical uniformity of cells and nuclei in epithelial monolayers.

PubMed

Neelam, Srujana; Hayes, Peter Robert; Zhang, Qiao; Dickinson, Richard B; Lele, Tanmay P

2016-01-22

Morphological variability in cytoskeletal organization, organelle position and cell boundaries is a common feature of cultured cells. Remarkable uniformity and reproducibility in structure can be accomplished by providing cells with defined geometric cues. Cells in tissues can also self-organize in the absence of directing extracellular cues; however the mechanical principles for such self-organization are not understood. We report that unlike horizontal shapes, the vertical shapes of the cell and nucleus in the z-dimension are uniform in cells in cultured monolayers compared to isolated cells. Apical surfaces of cells and their nuclei in monolayers were flat and heights were uniform. In contrast, isolated cells, or cells with disrupted cell-cell adhesions had nuclei with curved apical surfaces and variable heights. Isolated cells cultured within micron-sized square wells displayed flat cell and nuclear shapes similar to cells in monolayers. Local disruption of nuclear-cytoskeletal linkages resulted in spatial variation in vertical uniformity. These results suggest that competition between cell-cell pulling forces that expand and shorten the vertical cell cross-section, thereby widening and flattening the nucleus, and the resistance of the nucleus to further flattening results in uniform cell and nuclear cross-sections. Our results reveal the mechanical principles of self-organized vertical uniformity in cell monolayers.

Effect of functional end groups of silane self assembled monolayer surfaces on apatite formation, fibronectin adsorption and osteoblast cell function

PubMed Central

Toworfe, G.K.; Bhattacharyya, S.; Composto, R.J.; Adams, C.S.; Shapiro, I.M.; Ducheyne, P.

2008-01-01

Bioactive glass (BG) can directly bond to living bone without fibrous tissue encapsulation. Key mechanistic steps of BG’s activity are attributed to calcium phosphate formation, surface hydroxylation and fibronectin (FN) adsorption. In the present study, self-assembled monolayers (SAMs) of alkanesilanes with different surface chemistry (OH, NH2, and COOH) were used as a model system to mimic BG’s surface activity. Calcium phosphate (Ca-P) was formed on SAMs by immersion in a solution which simulates the electrolyte content of physiological fluids. FN adsorption kinetics and monolayer coverage was determined on SAMs with or without Ca-P coating. The surface roughness was also examined on these substrates before and after FN adsorption. The effects of FN-adsorbed, Ca-P coated SAMs on the function of MC3T3-E1 were evaluated by cell growth, expression of alkaline phosphatase activity, and actin cytoskeleton formation. We demonstrate that, although the FN monolayer coverage and the rms roughness are similar on −OH and −COOH terminated SAMs with or without Ca-P coating, higher levels of ALP activity, more actin cytoskeleton formation and more cell growth are obtained on −OH and −COOH terminated SAMs with Ca-P coating. In addition, although the FN monolayer coverage is higher on Ca-P coated −NH2 terminated SAMs and SiOx surfaces, higher levels of ALP activity and more cell growth are obtained on Ca-P coated −OH and −COOH terminated SAMs. Thus with same Ca-P coatings, different surface functional groups have different effects on the function of osteoblastic cells. These findings represent new insights into the mechanism of bioactivity of BG and, thereby, may lead to designing superior constructs for bone grafting. PMID:19012271

Expression and functional activity of P-glycoprotein in passaged primary human nasal epithelial cell monolayers cultured by the air-liquid interface method for nasal drug transport study.

PubMed

Cho, Hyun-Jong; Choi, Min-Koo; Lin, Hongxia; Kim, Jung Sun; Chung, Suk-Jae; Shim, Chang-Koo; Kim, Dae-Duk

2011-03-01

P-glycoprotein (P-gp) is an efflux transporter encoded by the multidrug resistance gene (MDR1), which is also known as the human ABCB1 gene (ATP-binding cassette, subfamily-B). The objectives of this study were to investigate the expression of P-gp in passaged primary human nasal epithelial (HNE) cell monolayer, cultured by the air-liquid interface (ALI) method, and to evaluate its feasibility as an in-vitro model for cellular uptake and transport studies of P-gp substrates. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify the expression of the MDR1 gene. Transport and cellular uptake studies with P-gp substrate (rhodamine123) and P-gp inhibitors (verapamil and cyclosporin A) were conducted to assess the functional activity of P-gp in HNE cell monolayers cultured by the ALI method. MDR1 gene expression in primary HNE cell monolayers cultured by ALI method was confirmed by RT-PCR. The apparent permeability coefficient (P(app) ) of the P-gp substrate (rhodamine123) in the basolateral to apical (B to A) direction was 6.9 times higher than that in the apical to basolateral (A to B) direction. B to A transport was saturated at high rhodamine123 concentration, and the treatment of P-gp inhibitors increased cellular uptake of rhodamine123 in a time- and concentration-dependent manner. These results support the MDR1 gene expression and the functional activity of P-gp in primary HNE cell monolayers cultured by the ALI method. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Incorporation of in vitro digestive enzymes in an intestinal epithelial cell line model for protein hazard identification.

PubMed

Markell, Lauren K; Wezalis, Stephanie M; Roper, Jason M; Zimmermann, Cindi; Delaney, Bryan

2017-10-01

Relatively few proteins in nature produce adverse effects following oral exposure. Of those that do, effects are often observed in the gut, particularly on intestinal epithelial cells (IEC). Previous studies reported that addition of protein toxins to IEC lines disrupted monolayer integrity but innocuous dietary proteins did not. Studies presented here investigated the effects of innocuous (bovine serum albumin, β-lactoglobulin, RuBisCO, fibronectin) or hazardous (phytohaemagglutinin-E, concanavalin A, wheat germ agglutinin, melittin) proteins that either were untreated or exposed to digestive enzymes prior to addition to Caco-2 human IEC line monolayers. At high concentrations intact fibronectin caused an increase in monolayer permeability but other innocuous proteins did not whether exposed to digestive enzymes or not. In contrast, all untreated hazardous proteins and those that were resistant to digestion (ex. wheat germ agglutinin) disrupted monolayer integrity. However, proteins sensitive to degradation by digestive enzymes (ex. melittin) did not adversely affect monolayers when exposed to these enzymes prior to addition to IEC line monolayers. These results indicate that in vitro exposure of proteins to digestive enzymes can assist in differentiating between innocuous and hazardous proteins as another component to consider in the overall weight of evidence approach in protein hazard assessment. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Sum Frequency Generation of Interfacial Lipid Monolayers Shows Polarization Dependence on Experimental Geometries.

PubMed

Li, Bolin; Li, Xu; Ma, Yong-Hao; Han, Xiaofeng; Wu, Fu-Gen; Guo, Zhirui; Chen, Zhan; Lu, Xiaolin

2016-07-19

Sum frequency generation (SFG) vibrational spectroscopy has been widely employed to investigate molecular structures of biological surfaces and interfaces including model cell membranes. A variety of lipid monolayers or bilayers serving as model cell membranes and their interactions with many different molecules have been extensively studied using SFG. Here, we conducted an in-depth investigation on polarization-dependent SFG signals collected from interfacial lipid monolayers using different experimental geometries, i.e., the prism geometry (total internal reflection) and the window geometry (external reflection). The different SFG spectral features of interfacial lipid monolayers detected using different experimental geometries are due to the interplay between the varied Fresnel coefficients and second-order nonlinear susceptibility tensor terms of different vibrational modes (i.e., ss and as modes of methyl groups), which were analyzed in detail in this study. Therefore, understanding the interplay between the interfacial Fresnel coefficients and χ((2)) tensors is a prerequisite for correctly understanding the SFG spectral features with respect to different experimental geometries. More importantly, the derived information in this paper should not be limited to the methyl groups with a C3v symmetry; valid extension to interfacial functional groups with different molecular symmetries and even chiral interfaces could be expected.

PR01 Molecular Pathogenesis of Rickettsioses and Development of Anti-Rickettsial Treatment by Combinatorial Peptide-Based Libraries

DTIC Science & Technology

2006-02-01

likely reflecting similar cell death rates in all monolayers at late time points. By the end of the experiment at 120 hours, all monolayers showed a...50-55% increase in permeability when compared to the controls. 2. Cell death rates in rickettsiae-infected SV-HCEC monolayers In order to...necrotic cell death. Quantification of cell death was performed by determining the percent of total cells staining positive for PI. Cell death rates did

Interaction of capsaicinoids with cell membrane models does not correlate with pungency of peppers

NASA Astrophysics Data System (ADS)

Geraldo, Vananélia P. N.; Ziglio, Analine C.; Gonçalves, Débora; Oliveira, Osvaldo N.

2017-04-01

Mixed monolayers were prepared using phospholipids in order to mimic cell membranes and fractions of capsaicinoids (extracted from Malagueta, Caps-M, and Bhut Jolokia, Caps-B, peppers). According to their surface-pressure isotherms and polarization-modulated infrared reflection absorption spectra (PM-IRRAS), weak molecular-level interactions were observed between Caps and phospholipids. Both Caps-M and Caps-B penetrated into the alkyl tail region of the monolayer, interacted with the phosphate group of the phospholipids and affected hydration of their Cdbnd O groups. Since the physiological activity of Caps is not governed solely by interaction with cell membranes, it should require participation of a neuronal membrane receptor, e.g. vanilloid receptor (TRPV1).

Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump.

PubMed

Mita, Sachiko; Suzuki, Hiroshi; Akita, Hidetaka; Stieger, Bruno; Meier, Peter J; Hofmann, Alan F; Sugiyama, Yuichi

2005-01-01

Bile salts are predominantly taken up by hepatocytes via the basolateral Na(+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains, whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that coexpressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep-expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep-coexpressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent K(m) value of 13.9 +/- 4.7 microM for cells expressing Ntcp alone, which is comparable with 22.2 +/- 4.5 microM for cells expressing both Ntcp and Bsep and V(max) values of 15.8 +/- 4.2 and 60.8 +/- 9.0 pmol.min(-1).mg protein(-1) for Ntcp alone and Ntcp and Bsep-coexpressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate was also observed across the double transfectant. This double-expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.

A dynamic cellular vertex model of growing epithelial tissues

NASA Astrophysics Data System (ADS)

Lin, Shao-Zhen; Li, Bo; Feng, Xi-Qiao

2017-04-01

Intercellular interactions play a significant role in a wide range of biological functions and processes at both the cellular and tissue scales, for example, embryogenesis, organogenesis, and cancer invasion. In this paper, a dynamic cellular vertex model is presented to study the morphomechanics of a growing epithelial monolayer. The regulating role of stresses in soft tissue growth is revealed. It is found that the cells originating from the same parent cell in the monolayer can orchestrate into clustering patterns as the tissue grows. Collective cell migration exhibits a feature of spatial correlation across multiple cells. Dynamic intercellular interactions can engender a variety of distinct tissue behaviors in a social context. Uniform cell proliferation may render high and heterogeneous residual compressive stresses, while stress-regulated proliferation can effectively release the stresses, reducing the stress heterogeneity in the tissue. The results highlight the critical role of mechanical factors in the growth and morphogenesis of epithelial tissues and help understand the development and invasion of epithelial tumors.

Subretinal Implantation of Retinal Pigment Epithelial Cells Derived From Human Embryonic Stem Cells: Improved Survival When Implanted as a Monolayer

PubMed Central

Diniz, Bruno; Thomas, Padmaja; Thomas, Biju; Ribeiro, Ramiro; Hu, Yuntao; Brant, Rodrigo; Ahuja, Ashish; Zhu, Danhong; Liu, Laura; Koss, Michael; Maia, Mauricio; Chader, Gerald; Hinton, David R.; Humayun, Mark S.

2013-01-01

Purpose. To evaluate cell survival and tumorigenicity of human embryonic stem cell–derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM). Methods. Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker). Results. The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group. Conclusions. hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects. PMID:23833067

Growth of cultured corneal endothelial cells onto a vitreous carbon matrix.

PubMed

Wickham, M G; Cleveland, P H; Binder, P S; Akers, P H

1983-01-01

Fourth passage cells of a rabbit corneal endothelial line were grown for 1 week in flasks containing pieces of a reticulated vitreous carbon matrix. The rate of cell growth in flasks containing the matrix was consistent with that in control flasks. Small fragments of the vitreous carbon material lying on the flask floor were covered by the monolayers as the cells grew to confluency. Vertical growth of cells onto larger pieces of the matrix proceeded in a staged fashion with maximum cell density on pieces of the matrix closest to the floor of the flask. As defined by scanning electron microscopy, cell growth occurred to a level at least 600 microns above the floor of the flask and the confluent monolayer. This novel culture procedure should be a model situation for study of many different aspects of the in vitro capabilities of corneal endothelial cells.

CdSe magic-sized quantum dots incorporated in biomembrane models at the air-water interface composed of components of tumorigenic and non-tumorigenic cells.

PubMed

Goto, Thiago E; Lopes, Carla C; Nader, Helena B; Silva, Anielle C A; Dantas, Noelio O; Siqueira, José R; Caseli, Luciano

2016-07-01

Cadmium selenide (CdSe) magic-sized quantum dots (MSQDs) are semiconductor nanocrystals with stable luminescence that are feasible for biomedical applications, especially for in vivo and in vitro imaging of tumor cells. In this work, we investigated the specific interaction of CdSe MSQDs with tumorigenic and non-tumorigenic cells using Langmuir monolayers and Langmuir-Blodgett (LB) films of lipids as membrane models for diagnosis of cancerous cells. Surface pressure-area isotherms and polarization modulation reflection-absorption spectroscopy (PM-IRRAS) showed an intrinsic interaction between the quantum dots, inserted in the aqueous subphase, and Langmuir monolayers constituted either of selected lipids or of tumorigenic and non-tumorigenic cell extracts. The films were transferred to solid supports to obtain microscopic images, providing information on their morphology. Similarity between films with different compositions representing cell membranes, with or without the quantum dots, was evaluated by atomic force microscopy (AFM) and confocal microscopy. This study demonstrates that the affinity of quantum dots for models representing cancer cells permits the use of these systems as devices for cancer diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

PubMed Central

Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

2006-01-01

Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

Intestinal transport of sophocarpine across the Caco-2 cell monolayer model and quantification by LC/MS.

PubMed

Sun, Sen; Zhang, Hai; Sun, Fengfeng; Zhao, Liang; Zhong, Yanqiang; Chai, Yifeng; Zhang, Guoqing

2014-06-01

Sophocarpine is a biologically active component obtained from the foxtail-like sophora herb and seed that is often orally administered for the treatment of cancer and chronic bronchial asthma. The aim of this study was to develop a rapid and specific LC/MS method for the determination of sophocarpine and to explore its transcellular transport mechanism across the Caco-2 (the human colon adenocarcia cell lines) monolayer cell transwell model. Caco-2 cells were seeded on permeable polycarbonate membranes and incubated for 21 days. Before the experiment, the trans-epithelial electric resistance, integrity and alkaline phosphatase activity of the Caco-2 monolayers were verified and used in subsequent experiments. In the Caco-2 model constructed, many influencing factors were investigated, including time, concentration, pH and different protein inhibitors. The results suggested that sophocarpine was transported mainly by passive diffusion. The flux of sophocarpine was time- and concentration-dependent, and the pH also had an effect on its transportation. The PappBA was higher than PappAB , indicating that a polarized transport might exist for sophocarpine. MK-571 and reserpine, inhibitors of the multidrug resistance associated protein 2 and the breast cancer resistance protein, decreased the efflux of sophocarpine, while verapamil had no effect on its transport. These results revealed that sophocarpine is absorbed mainly by passive diffusion, and that a carrier-mediated mechanism is also involved in the transport of sophocarpine. Copyright © 2014 John Wiley & Sons, Ltd.

New insights into the effects of biomaterial chemistry and topography on the morphology of kidney epithelial cells.

PubMed

Hulshof, Frits; Schophuizen, Carolien; Mihajlovic, Milos; van Blitterswijk, Clemens; Masereeuw, Rosalinde; de Boer, Jan; Stamatialis, Dimitrios

2018-02-01

Increasing incidence of renal pathology in the western world calls for innovative research for the development of cell-based therapies such as a bioartificial kidney (BAK) device. To fulfil the multitude of kidney functions, the core component of the BAK is a living membrane consisting of a tight kidney cell monolayer with preserved functional organic ion transporters cultured on a polymeric membrane surface. This membrane, on one side, is in contact with blood and therefore should have excellent blood compatibility, whereas the other side should facilitate functional monolayer formation. This work investigated the effect of membrane chemistry and surface topography on kidney epithelial cells to improve the formation of a functional monolayer. To achieve this, microtopographies were fabricated with high resolution and reproducibility on polystyrene films and on polyethersulfone-polyvinyl pyrrolidone (PES-PVP) porous membranes. A conditionally immortalized proximal tubule epithelial cell line (ciPTEC) was cultured on both, and subsequently, the cell morphology and monolayer formation were assessed. Our results showed that L-dopamine coating of the PES-PVP was sufficient to support ciPTEC monolayer formation. The polystyrene topographies with large features were able to align the cells in various patterns without significantly disruption of monolayer formation; however, the PES-PVP topographies with large features disrupted the monolayer. In contrast, the PES-PVP membranes with small features and with large spacing supported well the ciPTEC monolayer formation. In addition, the topographical PES-PVP membranes were compatible as a substrate membrane to measure organic cation transporter activity in Transwell® systems. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Pseudoislet formation enhances gene expression, insulin secretion and cytoprotective mechanisms of clonal human insulin-secreting 1.1B4 cells.

PubMed

Green, Alastair D; Vasu, Srividya; McClenaghan, Neville H; Flatt, Peter R

2015-10-01

We have studied the effects of cell communication on human beta cell function and resistance to cytotoxicity using the novel human insulin-secreting cell line 1.1B4 configured as monolayers and pseudoislets. Incubation with the incretin gut hormones GLP-1 and GIP caused dose-dependent stimulation of insulin secretion from 1.1B4 cell monolayers and pseudoislets. The secretory responses were 1.5-2.7-fold greater than monolayers. Cell viability (MTT), DNA damage (comet assay) and apoptosis (acridine orange/ethidium bromide staining) were investigated following 2-h exposure of 1.1B4 monolayers and pseudoislets to ninhydrin, H2O2, streptozotocin, glucose, palmitate or cocktails of proinflammatory cytokines. All agents tested decreased viability and increased DNA damage and apoptosis in both 1.1B4 monolayers and pseudoislets. However, pseudoislets exhibited significantly greater resistance to cytotoxicity (1.5-2.7-fold increases in LD50) and lower levels of DNA damage (1.3-3.4-fold differences in percentage tail DNA and olive tail moment) and apoptosis (1.3-1.5-fold difference) compared to monolayers. Measurement of gene expression by reverse-transcription, real-time PCR showed that genes involved with insulin secretion (INS, PDX1, PCSK1, PCSK2, GLP1R and GIPR), cell-cell communication (GJD2, GJA1 and CDH1) and antioxidant defence (SOD1, SOD2, GPX1 and CAT) were significantly upregulated in pseudoislets compared to monolayers, whilst the expression of proapoptotic genes (NOS2, MAPK8, MAPK10 and NFKB1) showed no significant differences. In summary, these data indicate cell-communication associated with three-dimensional islet architecture is important both for effective insulin secretion and for protection of human beta cells against cytotoxicity.

Transport of surface engineered polyamidoamine (PAMAM) dendrimers across IPEC-J2 cell monolayers.

PubMed

Pisal, Dipak S; Yellepeddi, Venkata K; Kumar, Ajay; Palakurthi, Srinath

2008-11-01

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAM(G4) dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P(app)) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.

Investigation of transport mechanism of exendin-4 across Madin Darby canine kidney cell monolayers.

PubMed

Wang, Mengshu; Sun, Bingxue; Feng, Jiao; Zhang, Haihong; Liu, Bin; Li, Chun; Chen, Yan; Zhang, Yong; Kong, Wei

2012-01-01

The purpose of this study was to investigate the transport mechanism of exendin-4 using Madin Darby canine kidney (MDCK) cell monolayer as an in vitro model of the human intestinal barrier. The roles of active and passive mechanisms of exendin-4 in the cell models were well studied and the corresponding contributions of the transcelluar and paracellular pathway to exendin-4 transport were also evaluated. Moreover, the apparent permeability coefficient (P(app)) values of exendin-4 were determined in the presence of chitosan, sodium decanoate and ethylenediaminetetraacetic acid (EDTA) to further confirm the relative transport mechanism and to evaluate their potential utility in future formulation design. The results revealed the low transport capacity of exendin-4 (P(app), 0.10±0.06×10(-6) cm/s). And exendin-4 transport across the cell models was time and concentration-dependence, direction and energy-independence, and similar to the passive transport marker. Drug efflux and active transport were not observed. In the presence of absorption enhancers, the P(app) value significantly increased up to 2.2-11.9 folds without apparent cytotoxicity, which is comparable to that of the paracellular transport marker. And the order of enhancement was to the effect of chitosan>EDTA>sodium decanoate, and the order of safety was sodium decanoate≈chitosan>EDTA. These findings demonstrated that exendin-4 transport across MDCK cell monolayer mainly by passive paracellular pathway, which agrees with the result of confocal laser scanning microscopy. And these absorption enhancers can be used as potential safe ingredients to improve oral efficacy of exendin-4.

Miscibility and interactions of animal and plant sterols with choline plasmalogen in binary and multicomponent model systems.

PubMed

Hąc-Wydro, Katarzyna; Luty, Katarzyna

2014-04-01

In this work miscibility and interactions of sterols with choline plasmalogen (PC-plasm) in Langmuir monolayers were studied. Moreover, the properties of cholesterol/phosphatidylcholine/plasmalogen mixtures of different PC-plasm concentration were investigated. The foregoing systems were treated as a model of cancer cell membranes, which are of higher plasmalogen level than normal cells. Finally, the influence of β-sitosterol and stigmasterol (phytosterols differing in anticancer potency) on these mixtures was verified. The properties of monolayers were analyzed based on the parameters derived from the surface pressure-area isotherms and images taken with Brewster Angle Microscope. It was found that at 30% of sterol in sterol/plasmalogen monolayer the lipids are immiscible and 3D crystallites are formed within the film. Cholesterol molecules mix favorably with PC-plasm at Xchol ≥ 0.5, while the investigated phytosterols only at their prevailing proportion in binary system. The increase of choline plasmalogen in cholesterol/phosphatidylcholine monolayer causes destabilization of the system. Moreover, the incorporation of phytosterols into cholesterol/phosphatidylcholine+PC-plasm mixtures disturbed membrane morphology and this effect was stronger for β-sitosterol as compared to stigmasterol. It was concluded that the presence of vinyl ether bond at sn-1 position in PC-plasm molecule strongly affects miscibility of choline plasmalogen with sterols. The comparison of the collected data with those reported in literature allowed one to conclude that miscibility and interactions of sterols with PC-plasm are less favorable than those with phosphatidylcholine. It was also suggested that overexpression of plasmalogens in cancer cell membranes may be a factor differentiating sensitivity of cells to anticancer effect of phytosterols. Copyright © 2014 Elsevier B.V. All rights reserved.

Human adipose-derived stem cell spheroid treated with photobiomodulation irradiation accelerates tissue regeneration in mouse model of skin flap ischemia.

PubMed

Park, In-Su; Chung, Phil-Sang; Ahn, Jin Chul; Leproux, Anais

2017-11-01

Skin flap grafting is a form of transplantation widely used in plastic surgery. However, ischemia/reperfusion injury is the main factor which reduces the survival rate of flaps following grafting. We investigated whether photobiomodulation (PBM) precondition prior to human adipose-derived stromal cell (hASC) spheroid (PBM-spheroid) transplantation improved skin tissue functional recovery by the stimulation of angiogenesis and tissue regeneration in skin flap of mice. The LED had an emission wavelength peaked at 660 ± 20 nm (6 J/cm 2 , 10 mW/cm 2 ). The expression of angiogenic growth factors in PBM-spheroid hASCs was much greater than that of not-PBM-treated spheroid or monolayer-cultured hASCs. From immunochemical staining analysis, the hASCs of PBM-spheroid were CD31 + , KDR + , and CD34 + , whereas monolayer-cultured hASCs were negative for these markers. To evaluate the therapeutic effect of hASC PBM-spheroid in vivo, PBS, monolayer-cultured hASCs, and not-PBM-spheroid were transplanted into a skin flap model. The animals were observed for 14 days. The PBM-spheroid hASCs transplanted into the skin flap ischemia differentiated into endothelial cells and remained differentiated. Transplantation of PBM-spheroid hASCs into the skin flap ischemia significantly elevated the density of vascular formations through angiogenic factors released by the skin flap ischemia and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of PBM-spheroid hASCs significantly improved functional recovery compared with PBS, monolayer-cultured hASCs, and not-PBM-spheroid treatment. These findings suggest that transplantation of PBM-spheroid hASCs may be an effective stem cell therapy for the treatment of skin flap ischemia.

The cell line NCl-H441 is a useful in vitro model for transport studies of human distal lung epithelial barrier.

PubMed

Salomon, Johanna J; Muchitsch, Viktoria E; Gausterer, Julia C; Schwagerus, Elena; Huwer, Hanno; Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

2014-03-03

The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP(+) uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm(2), after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors. In transport studies, Rh123 exhibited net secretion, which again was inhibitable by bona fide P-gp modulators. The uptake of ASP(+) was time- and temperature-dependent with Km = 881.2 ± 195.3 μM and Vmax = 2.07 ± 0.26 nmol/min/mg protein. TEA, amantadine, quinidine, and verapamil significantly inhibited ASP(+) uptake into NCl-H441 cells, whereas the effect of d- and l-carnitine and ergothioneine, two OCTN substrates, was less pronounced. NCl-H441 cells are the first cell line of human distal lung epithelial origin with the ability to form monolayers with appreciable barrier properties. Moreover, drug transporter expression and activity in NCl-H441 cells was consistent with what has been reported for human alveolar epithelial cells in primary culture.

The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pajęcka, Kamilla, E-mail: kpaj@novonordisk.com; Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus; Nielsen, Malik Nygaard

Background and aims: Nephropathy involves pathophysiological changes to the glomerulus. The primary glomerular endothelial cells (GEnCs) have emerged as an important tool for studying glomerulosclerotic mechanisms and in the screening process for drug-candidates. The success of the studies is dependent on the quality of the cell model. Therefore, we set out to establish an easy, reproducible model of the quiescent endothelial monolayer with the use of commercially available extracellular matrices (ECMs). Methods: Primary hGEnCs were seeded on various ECMs. Cell adhesion was monitored by an impedance sensing system. The localization of junctional proteins was assessed by immunofluorescence and the barriermore » function by passage of fluorescent dextrans and magnitude of VEGF response. Results: All ECM matrices except recombinant human laminin 111 (rhLN111) supported comparable cell proliferation. Culturing hGEnCs on rhLN521, rhLN511 or fibronectin resulted in a physiologically relevant barrier to 70 kDa dextrans which was 82% tighter than that formed on collagen type IV. Furthermore, only hGEnCs cultured on rhLN521 or rhLN511 showed plasma-membrane localized zonula occludens-1 and vascular endothelial cadherin indicative of proper tight and adherens junctions (AJ). Conclusion: We recommend culturing hGEnCs on the mature glomerular basement membrane laminin - rhLN521 – which, as the only commercially available ECM, promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined AJs and physiological perm-selectivity. - Highlights: • rhLN521, rhLN511 and hFN assure physiologically relevant permeability. • rhLN521 and rhLN511 ensure best cell morphology and adherens junction formation. • Collagen IV and I based coating results in disorganized hGEnC monolayer. • Physiologically relevant ECM may lead to down-regulation of self-produced matrices.« less

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

PubMed Central

Phelps, Edward A.; Cianciaruso, Chiara; Santo-Domingo, Jaime; Pasquier, Miriella; Galliverti, Gabriele; Piemonti, Lorenzo; Berishvili, Ekaterine; Burri, Olivier; Wiederkehr, Andreas; Hubbell, Jeffrey A.; Baekkeskov, Steinunn

2017-01-01

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells. PMID:28401888

Neutral amino acid transport across brain microvessel endothelial cell monolayers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Audus, K.L.; Borchardt, R.T.

1986-03-01

Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with (/sup 3/H)-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional,more » and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, ..cap alpha..-methyldopa, L-DOPA, ..cap alpha..-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB.« less

Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

DOE PAGES

Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; ...

2016-05-27

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed betweenmore » the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.« less

Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

PubMed Central

Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

2016-01-01

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

A hybrid computational model to explore the topological characteristics of epithelial tissues.

PubMed

González-Valverde, Ismael; García-Aznar, José Manuel

2017-11-01

Epithelial tissues show a particular topology where cells resemble a polygon-like shape, but some biological processes can alter this tissue topology. During cell proliferation, mitotic cell dilation deforms the tissue and modifies the tissue topology. Additionally, cells are reorganized in the epithelial layer and these rearrangements also alter the polygon distribution. We present here a computer-based hybrid framework focused on the simulation of epithelial layer dynamics that combines discrete and continuum numerical models. In this framework, we consider topological and mechanical aspects of the epithelial tissue. Individual cells in the tissue are simulated by an off-lattice agent-based model, which keeps the information of each cell. In addition, we model the cell-cell interaction forces and the cell cycle. Otherwise, we simulate the passive mechanical behaviour of the cell monolayer using a material that approximates the mechanical properties of the cell. This continuum approach is solved by the finite element method, which uses a dynamic mesh generated by the triangulation of cell polygons. Forces generated by cell-cell interaction in the agent-based model are also applied on the finite element mesh. Cell movement in the agent-based model is driven by the displacements obtained from the deformed finite element mesh of the continuum mechanical approach. We successfully compare the results of our simulations with some experiments about the topology of proliferating epithelial tissues in Drosophila. Our framework is able to model the emergent behaviour of the cell monolayer that is due to local cell-cell interactions, which have a direct influence on the dynamics of the epithelial tissue. Copyright © 2017 John Wiley & Sons, Ltd.

Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

PubMed

Gao, Bin; Wang, Lin; Han, Shuang; Pingguan-Murphy, Belinda; Zhang, Xiaohui; Xu, Feng

2016-08-01

Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.

New mouse tumor model system (RIF-1) for comparison of end-point studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Twentyman, P.R.; Brown, J.M.; Gray, J.W.

1980-03-01

A new tumor model system (RIF-1) was developed that is very suitable for studies in which clonogenic survival is compared with growth delay and control probability following various forms of treatment. The tumor was a radiation-induced sarcoma in the inbred female C3H/Km mouse. It had a low median tumor dose, had a satisfactory plating efficiency direct from in vivo to in vitro, was nonimmunogenic or minimally immunogenic, and metastasized only at a relatively advanced stage of growth. The cell line grew either as a monolayer on plastic dishes, as tumor spheroids in spinner culture, as lung nodules following injection ofmore » a single-cell suspension into the tall veins of syngeneic mice, or as a solid tumor. Both diploid and tetraploid clonogenic cells were found in monolayer cultures of the RIF-1 line.« less

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases1

PubMed Central

Zozulya, Alla L.; Reinke, Emily; Baiu, Dana C.; Karman, Jozsef; Sandor, Matyas; Fabry, Zsuzsanna

2007-01-01

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1α increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1α -induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS. PMID:17182592

Impedance spectroscopy for the detection and identification of unknown toxins

NASA Astrophysics Data System (ADS)

Riggs, B. C.; Plopper, G. E.; Paluh, J. L.; Phamduy, T. B.; Corr, D. T.; Chrisey, D. B.

2012-06-01

Advancements in biological and chemical warfare has allowed for the creation of novel toxins necessitating a universal, real-time sensor. We have used a function-based biosensor employing impedance spectroscopy using a low current density AC signal over a range of frequencies (62.5 Hz-64 kHz) to measure the electrical impedance of a confluent epithelial cell monolayer at 120 sec intervals. Madin Darby canine kidney (MDCK) epithelial cells were grown to confluence on thin film interdigitated gold electrodes. A stable impedance measurement of 2200 Ω was found after 24 hrs of growth. After exposure to cytotoxins anthrax lethal toxin and etoposide, the impedance decreased in a linear fashion resulting in a 50% drop in impedance over 50hrs showing significant difference from the control sample (~20% decrease). Immunofluorescent imaging showed that apoptosis was induced through the addition of toxins. Similarities of the impedance signal shows that the mechanism of cellular death was the same between ALT and etoposide. A revised equivalent circuit model was employed in order to quantify morphological changes in the cell monolayer such as tight junction integrity and cell surface area coverage. This model showed a faster response to cytotoxin (2 hrs) compared to raw measurements (20 hrs). We demonstrate that herein that impedance spectroscopy of epithelial monolayers serves as a real-time non-destructive sensor for unknown pathogens.

Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing.

PubMed

Yu, Linfen; Chen, Michael C W; Cheung, Karen C

2010-09-21

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Moreover, continuous dynamic perfusion allows the establishment of long term cell culture and subsequent multicellular spheroid formation. A droplet-based microfluidic system was used to form alginate beads with entrapped breast tumor cells. After gelation, the alginate beads were trapped in microsieve structures for cell culture in a continuous perfusion system. The alginate environment permitted cell proliferation and the formation of multicellular spheroids was observed. The dose-dependent response of the tumor spheroids to doxorubicin, and anticancer drug, showed multicellular resistance compared to conventional monolayer culture. The microsieve structures maintain constant location of each bead in the same position throughout the device seeding process, cell proliferation and spheroid formation, treatment with drug, and imaging, permitting temporal and spatial tracking.

Traction force dynamics predict gap formation in activated endothelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valent, Erik T.; Nieuw Amerongen, Geerten P. van; Hinsbergh, Victor W.M. van

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneousmore » distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps. - Highlights: • Endothelial monolayers exert dynamic- and heterogeneous traction forces. • High traction forces correlate with junctional areas and the F-actin cytoskeleton. • Newly formed inter-endothelial gaps are characterized by opposing traction forces. • Force stability is a key feature controlling endothelial permeability.« less

Maximising the use of freshly isolated human hepatocytes.

PubMed

Evans, Peter J

2016-01-01

Freshly isolated human hepatocytes are the best model for predicting adverse drug reactions. However, their preparation and use present the investigator with many variables that are beyond their control. These include operation continuity and timing, size and number of cut surfaces on liver tissue and the prior history of the patient. To exploit the potential of freshly isolated human hepatocytes a method is required to preserve the cells in their initial in vivo like state. This experimental pausing allows experiments to be prioritised at convenient times of the day. A novel approach for selecting viable human hepatocytes by functional attachment to a gelatin gel is described rather than relying on their physical characteristics. The cells are preserved as a monolayer on the semi-solid support at 10°C as single spherical entities. The hepatocytes can be released into suspension, when required, by a temperature transition to 37°C for 20min. The cells can be used in suspension or as a monolayer. The length of preservation depends upon the source tissue. Hepatocytes from normal liver can be maintained for at least 4days and demonstrated to have the same level of CYP3A4 and the enzymes involved in glucuronidation and sulphation as freshly isolated cells. Cells from fatty liver, attached to gelatin, vary in their preservation time but it is at least 24h and so confluent monolayers, that survive at 37°C can be generated the following day. The technique enables freshly isolated human hepatocytes to be used more effectively. They can be preserved in times of plenty so more experimentation is possible. Alternatively, with poorer fatty cells the initial attachment on gelatin enables confluent monolayers of lipid rich cells to be studied. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of the intestinal permeability of rosemary (Rosmarinus officinalis L.) extract polyphenols and terpenoids in Caco-2 cell monolayers.

PubMed

Pérez-Sánchez, Almudena; Borrás-Linares, Isabel; Barrajón-Catalán, Enrique; Arráez-Román, David; González-Álvarez, Isabel; Ibáñez, Elena; Segura-Carretero, Antonio; Bermejo, Marival; Micol, Vicente

2017-01-01

Rosemary (Rosmarinus officinalis) is grown throughout the world and is widely used as a medicinal herb and to season and preserve food. Rosemary polyphenols and terpenoids have attracted great interest due to their potential health benefits. However, complete information regarding their absorption and bioavailability in Caco-2 cell model is scarce. The permeation properties of the bioactive compounds (flavonoids, diterpenes, triterpenes and phenylpropanoids) of a rosemary extract (RE), obtained by supercritical fluid extraction, was studied in Caco-2 cell monolayer model, both in a free form or liposomed. Compounds were identified and quantitated by liquid chromatography coupled to quadrupole time-of-flight with electrospray ionization mass spectrometry analysis (HPLC-ESI-QTOF-MS), and the apparent permeability values (Papp) were determined, for the first time in the extract, for 24 compounds in both directions across cell monolayer. For some compounds, such as triterpenoids and some flavonoids, Papp values found were reported for the first time in Caco-2 cells.Our results indicate that most compounds are scarcely absorbed, and passive diffusion is suggested to be the primary mechanism of absorption. The use of liposomes to vehiculize the extract resulted in reduced permeability for most compounds. Finally, the biopharmaceutical classification (BCS) of all the compounds was achieved according to their permeability and solubility data for bioequivalence purposes. BCS study reveal that most of the RE compounds could be classified as classes III and IV (low permeability); therefore, RE itself should also be classified into this category.

Three dimensional culture of the murine osteoblastic cell line OCT-1 on collagen coated microcarriers

NASA Astrophysics Data System (ADS)

Lau, P.; Hellweg, C. E.; Kirchner, S.; Baumstark-Khan, C.

2005-08-01

During long-term space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. Bone loss during space flight is about 1-2% per month. Bone is continually being remodelled under the influence of three types of highly specialized cells. Osteoblasts, the bone forming cells, osteoclasts, the bone resorbing cells and finally osteocytes preserve the homeostasis of bone formation and resorption. In vitro 3- dimensional cell culture of osteoblastic cell lines on microcarrier beads might be a better model to evaluate changes in bone cell morphology, function and differentiation under influence of spaceflight related factors than the conventional 2-D monolayer culture technique. Furthermore, it allows production of a greater amount of cells compared to the monolayer culture. Aim of this study is to examine the effects of culturing the immortalized murine osteoblastic cell line OCT-1 in a 3- dimensional environment on cell morphology and proliferation rate.

Analysis and comparison of oxygen consumption of HepG2 cells in a monolayer and three-dimensional high density cell culture by use of a matrigrid®.

PubMed

Weise, Frank; Fernekorn, Uta; Hampl, Jörg; Klett, Maren; Schober, Andreas

2013-09-01

By the use of a MatriGrid® we have established a three-dimensional high density cell culture. The MatriGrid® is a culture medium permeable, polymeric scaffold with 187 microcavities. In these cavities (300 μm diameter and 207 μm deep) the cells can growth three-dimensionally. For these experiments we measured the oxygen consumption of HepG2 cell cultures in order to optimize cultivation conditions. We measured and compared the oxygen consumption, growth rate and vitality under three different cultivation conditions: monolayer, three-dimensional static and three-dimensional actively perfused. The results show that the cells in a three-dimensional cell culture consume less oxygen as in a monolayer cell culture and that the actively perfused three-dimensional cell culture in the MatriGrid® has a similar growth rate and vitality as the monolayer culture. Copyright © 2013 Wiley Periodicals, Inc.

Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

PubMed

Tan, Kah Yong; Teo, Kim Leng; Lim, Jessica F Y; Chen, Allen K L; Choolani, Mahesh; Reuveny, Shaul; Chan, Jerry; Oh, Steve Kw

2015-08-01

Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Effects of osmolarity on human epithelial conjunctival cells using an electrical technique.

PubMed

Bellotti, Mariela; Bast, Walter; Berra, Alejandro; Bonetto, Fabian J

2011-12-01

The purpose of this study is to report the effect of different media osmolarity on a cell line monolayer of normal human conjunctival epithelia (IOBA-NHC) using Electric Cell-substrate Impedance Sensing (ECIS). We built our own ECIS system. We fabricated biocompatible microelectrodes. We used a monolayer of IOBA-NHC cells with media at different osmolarities (315, 360, 446, and 617 mOsm/l). When there is an increase in hyperosmolarity, there is a slight decrease in the measured resistance of the naked microelectrode (without cells), whereas its capacitance remained practically unchanged. The evaluation of resistance and capacitance of a microelectrode covered by a monolayer of IOBA-NHC in relation to a naked microelectrode showed no difference in the standard media (315 mOsm/l), a small difference with 360 mOsm/l, and significant differences with hyperosmolarities of 446 mOsm/l and 610 mOsm/l. The resistance with a confluent cell monolayer is up to three times greater compared to the value of the resistance of the naked electrode with standard media. Both resistance and capacitance measurements for the cell monolayer were sensitive to changes in osmolarity.

Topological defects in epithelia govern cell death and extrusion

NASA Astrophysics Data System (ADS)

Saw, Thuan Beng; Doostmohammadi, Amin; Nier, Vincent; Kocgozlu, Leyla; Thampi, Sumesh; Toyama, Yusuke; Marcq, Philippe; Lim, Chwee Teck; Yeomans, Julia M.; Ladoux, Benoit

2017-04-01

Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling, oncogenic transformation and overcrowding of cells. Despite the important linkage of cell extrusion to developmental, homeostatic and pathological processes such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity, caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell-cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo, with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.

The effect of neighboring cells on the stiffness of cancerous and non-cancerous human mammary epithelial cells

NASA Astrophysics Data System (ADS)

Guo, Xinyi; Bonin, Keith; Scarpinato, Karin; Guthold, Martin

2014-10-01

Using an Atomic Force Microscope (AFM) with a 5.3 μm diameter spherical probe, we determined mechanical properties of individual human mammary epithelial cells. The cells were derived from a pair of cell lines that mimic cell progression through four phases of neoplastic transformation: normal (non-transformed), immortal, tumorigenic, and metastatic. Measurements on cells in all four phases were taken over both the cytoplasmic and nuclear regions. Moreover, the measurements were made for cells in different microenvironments as related to cell-cell contacts: isolated cells; cells residing on the periphery of a contiguous cell monolayer; and cells on the inside of a contiguous cell monolayer. By fitting the AFM force versus indentation curves to a Hertz model, we determined the pseudo-elastic Young’s modulus, E. Combining all data for the cellular subregions (over nucleus and cytoplasm) and the different cell microenvironments, we obtained stiffness values for normal, immortal, tumorigenic, and metastatic cells of 870 Pa, 870 Pa, 490 Pa, and 580 Pa, respectively. That is, cells become softer as they advance to the tumorigenic phase and then stiffen somewhat in the final step to metastatic cells. We also found a distinct contrast in the influence of a cell’s microenvironment on cell stiffness. Normal mammary epithelial cells inside a monolayer are stiffer than peripheral cells, which are stiffer than isolated cells. However, the microenvironment had a slight, opposite effect on tumorigenic and little effect on immortal and metastatic cell stiffness. Thus, the stiffness of cancer cells is less sensitive to the microenvironment than normal cells. Our results show that the mechanical properties of a cell can depend on cancer progression and microenvironment (cell-cell interactions).

Tumour necrosis factor alpha changes porcine intestinal ion transport through a paracrine mechanism involving prostaglandins.

PubMed Central

Kandil, H M; Berschneider, H M; Argenzio, R A

1994-01-01

Prostaglandins stimulate electrogenic anion secretion and inhibit sodium chloride absorption in cryptosporidium induced pig diarrhoea. Because tumour necrosis factor alpha (TNF alpha) is an early mediator of inflammation and stimulates prostaglandin secretion, we investigated its effect on intestinal ion transport. Cryptosporidium infected pig ileum showed higher macrophage infiltration and tissue TNF alpha-like activity than uninfected tissues (p < 0.05, n = 4 and p < 0.05, n = 12, respectively). TNF alpha treatment of control porcine ileal mucosa increased the short circuit current (Isc), a measurement of net anion secretion in this model (p < 0.001, n = 23). This effect was blocked by 10(-6) M indomethacin and Cl- replacement. Neither acute treatment nor preincubation of colonic intestinal epithelial cell monolayers (T84) with TNF alpha stimulated the Isc. However, co-mounting of TNF alpha preincubated pig jejunal fibroblasts (P2JF) monolayers back to back with untreated T84 monolayers dose-dependently induced an indomethacin sensitive increase in Isc compared with values in untreated co-mounted monolayers (p < 0.001, n = 11). These data suggest that in infectious diarrhoea, TNF alpha may induce Cl- secretion through a paracrine mechanism involving prostaglandin release from subepithelial cells, for example fibroblasts. PMID:8063221

Can microcarrier-expanded chondrocytes synthesize cartilaginous tissue in vitro?

PubMed

Surrao, Denver C; Khan, Aasma A; McGregor, Aaron J; Amsden, Brian G; Waldman, Stephen D

2011-08-01

Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on Millicell™ filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue.

The Elementary Mass Action Rate Constants of P-gp Transport for a Confluent Monolayer of MDCKII-hMDR1 Cells

PubMed Central

Tran, Thuy Thanh; Mittal, Aditya; Aldinger, Tanya; Polli, Joseph W.; Ayrton, Andrew; Ellens, Harma; Bentz, Joe

2005-01-01

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized. PMID:15501934

In Vitro Effect of Laser-Induced Hydrodynamics on Cancer Cells.

PubMed

Elagin, V V; Pavlikov, A I; Yusupov, V I; Shirmanova, M V; Zagaynova, E V; Bagratashvili, V N

2015-11-01

We studied the effect of laser-induced hydrodynamic on viability of Colo-26 murine colon carcinoma cells in vitro. Laser-induced hydrodynamics was generated by a laser (λ=1.56 μ, power 3 W, 5 min exposure); to this end, the fiber end was submersed into a buffer above the cell monolayer. It was found that laser-induced hydrodynamics destructed the monolayer at standoff distances of between the working end of the laser fiber to cell monolayer of 1 and 5 mm and triggers apoptotic and necrotic death in remaining cells at a distance of 4 mm from the emitter.

Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

PubMed

Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

2014-10-01

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

Normal flora: living vehicles for non-invasive protein drug delivery.

PubMed

Shao, Jun; Kaushal, Gagan

2004-11-22

Feasibility to use probiotic bacteria as a living protein delivery system through oral route was assessed in vitro. Lactococcus lactis transformed with a plasmid to express and secret beta-lactamase was used to deliver beta-lactamase through Caco-2 monolayer, an intestine epithelium. Transport of beta-lactamase through Caco-2 monolayer was carried out in the transwells. The viability and integrity of the cell monolayers co-cultured with L. lactis was examined by trypan blue exclusion method and by measuring the transport of mannitol and propranolol as well as the transepithelial electrical resistance (TEER). Results show that it is feasible to use cell culture technique to evaluate the drug delivery by normal flora. The transport rate of beta-lactamase when delivered by L. lactis was 2.0 +/- 0.1 x 10(-2)h(-1) (n = 9) and through free solution form was 1.0 +/- 0.1 x 10(-2)h-1. When co-cultured with L. lactis, Caco-2 cell viability decreased to 98, 96, and 94% at 6, 8, and 10h, respectively. Transport of mannitol through Caco-2 cell monolayer was significantly increased and the transport of propranolol through Caco-2 cell monolayer was significantly decreased in the presence of L. lactis. Increase in the amount of protein delivered is probably due to the concentrate of the protein by L. lactis on the monolayer (absorption surface) and the opening of the tight junction of Caco-2 monolayer by L. lactis. copyright 2004 Elsevier B.V.

Overcrowding drives the unjamming transition of gap-free monolayers

NASA Astrophysics Data System (ADS)

Lan, Ganhui; Su, Tao

Collective cell motility plays central roles in various biological phenomena such as wound healing, cancer metastasis and embryogenesis. These are demonstrations of the unjamming transition in biology. However, contradictory to the typical density-driven jamming in particulate assemblies, cellular systems often get unjammed in highly packed, sometimes overcrowding environments. Here, we investigate monolayers' collective behaviors when cell number changes under the gap-free constraint. We report that overcrowding can unjam gap-free monolayers through increasing isotropic compression. We show that the transition boundary is determined by the isotropic compression and the cell-cell adhesion. Furthermore, we construct the free energy landscape for the T1 topological transition during monolayer rearrangement, and discover that the landscape evolves from single-barrier W shape to double-barrier M shape during the unjamming process. We also discover a distributed-to-disordered morphological transition of cells' geometry, coinciding with the unjamming transition. Our analyses reveal that the overcrowding and adhesion induced unjamming reflects the mechanical yielding of the highly deformable monolayer, suggesting an alternative mechanism that cells may robustly gain collective mobility through proliferation in confined environments, which differs from those caused by loosing up a packed particulate assembly. This work is supported by the GWU College Facilitating Funds.

STANDARDIZATION OF A FLUORESCENT-BASED QUANTITATIVE ADHESION ASSAY TO STUDY ATTACHMENT OF Taenia solium ONCOSPHERE TO EPITHELIAL CELLS In Vitro

PubMed Central

Chile, Nancy; Evangelista, Julio; Gilman, Robert H.; Arana, Yanina; Palma, Sandra; Sterling, Charles R; Garcia, Hector H.; Gonzalez, Armando; Verastegui, Manuela

2012-01-01

To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment. PMID:22178422

Test of Antifibrotic Drugs in a Cellular Model of Fibrosis Based on Muscle-Derived Fibroblasts from Duchenne Muscular Dystrophy Patients.

PubMed

Zanotti, Simona; Mora, Marina

2018-01-01

An in vitro model of muscle fibrosis, based on the use of primary human fibroblasts isolated from muscle biopsies of patients affected by Duchenne muscular dystrophies (DMD) and cultivated in monolayer and 3D conditions, is used to test the potential antifibrotic activity of pirfenidone (PFD). This in vitro model may be usefully also to evaluate the toxicity and efficacy of other candidate molecules for the treatment of fibrosis. The drug toxicity is evaluated using a colorimetric assay based on the conversion of tetrazolium salt (MTT) to insoluble formazan, while the effect of the drug on cell proliferation is measured with the bromodeoxyuridine incorporation assay. The efficacy of the drug is evaluated in fibroblast monolayers by quantitating synthesis and deposition of intracellular collagen with a spectrophotometric picrosirius red-based assay, and by quantitating cell migration using a "scratch" assay. The efficacy of PFD as antifibrotic drug is also evaluated in a 3D fibroblast model by measuring diameters and number of nodules.

A Human Pluripotent Stem Cell Model of Facioscapulohumeral Muscular Dystrophy-Affected Skeletal Muscles.

PubMed

Caron, Leslie; Kher, Devaki; Lee, Kian Leong; McKernan, Robert; Dumevska, Biljana; Hidalgo, Alejandro; Li, Jia; Yang, Henry; Main, Heather; Ferri, Giulia; Petek, Lisa M; Poellinger, Lorenz; Miller, Daniel G; Gabellini, Davide; Schmidt, Uli

2016-09-01

: Facioscapulohumeral muscular dystrophy (FSHD) represents a major unmet clinical need arising from the progressive weakness and atrophy of skeletal muscles. The dearth of adequate experimental models has severely hampered our understanding of the disease. To date, no treatment is available for FSHD. Human embryonic stem cells (hESCs) potentially represent a renewable source of skeletal muscle cells (SkMCs) and provide an alternative to invasive patient biopsies. We developed a scalable monolayer system to differentiate hESCs into mature SkMCs within 26 days, without cell sorting or genetic manipulation. Here we show that SkMCs derived from FSHD1-affected hESC lines exclusively express the FSHD pathogenic marker double homeobox 4 and exhibit some of the defects reported in FSHD. FSHD1 myotubes are thinner when compared with unaffected and Becker muscular dystrophy myotubes, and differentially regulate genes involved in cell cycle control, oxidative stress response, and cell adhesion. This cellular model will be a powerful tool for studying FSHD and will ultimately assist in the development of effective treatments for muscular dystrophies. This work describes an efficient and highly scalable monolayer system to differentiate human pluripotent stem cells (hPSCs) into skeletal muscle cells (SkMCs) and demonstrates disease-specific phenotypes in SkMCs derived from both embryonic and induced hPSCs affected with facioscapulohumeral muscular dystrophy. This study represents the first human stem cell-based cellular model for a muscular dystrophy that is suitable for high-throughput screening and drug development. ©AlphaMed Press.

Arsenite induces endothelial cell permeability increase through a reactive oxygen species-vascular endothelial growth factor pathway.

PubMed

Bao, Lingzhi; Shi, Honglian

2010-11-15

As a potent environmental oxidative stressor, arsenic exposure has been reported to exacerbate cardiovascular diseases and increase vascular endothelial cell monolayer permeability. However, the underlying mechanism of this effect is not well understood. In this paper, we test our hypothesis that reactive oxygen species (ROS)-induced vascular endothelial growth factor (VEGF) expression may play an important role in an arsenic-caused increase of endothelial cell monolayer permeability. The mouse brain vascular endothelial cell bEnd3 monolayer was exposed to arsenite for 1, 3, and 6 days. The monolayer permeability, VEGF protein release, and ROS generation were determined. In addition, VE-cadherin and zonula occludens-1 (ZO-1), two membrane structure proteins, were immunostained to elucidate the effects of arsenite on the cell-cell junction. The roles of ROS and VEGF in arsenite-induced permeability was determined by inhibiting ROS with antioxidants and immuno-depleting VEGF with a VEGF antibody. We observed that arsenite increased bEnd3 monolayer permeability, elevated the production of cellular ROS, and increased VEGF release. VE-cadherin and ZO-1 disruptions were also found in cells treated with arsenite. Furthermore, both antioxidant (N-acetyl cysteine and tempol) and the VEGF antibody treatments significantly lowered the arsenite-induced permeability of the bEnd3 monolayer as well as VEGF expression. VE-cadherin and ZO-1 disruptions were also diminished by N-acetyl cysteine and the VEGF antibody. Our data suggest that the increase in VEGF expression caused by ROS may play an important role in the arsenite-induced increase in endothelial cell permeability.

Evaluation of permeability alteration and epithelial-mesenchymal transition induced by transforming growth factor-β1 in A549, NCI-H441, and Calu-3 cells: Development of an in vitro model of respiratory epithelial cells in idiopathic pulmonary fibrosis.

PubMed

Togami, Kohei; Yamaguchi, Kotaro; Chono, Sumio; Tada, Hitoshi

2017-07-01

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease, which is accompanied by changes in lung structure. With regard to treatment, aerosolized drugs administered intrapulmonarily are rapidly distributed into the plasma and do not remain in the lungs due to damage to the alveolar epithelium that occurs from pulmonary fibrosis. In this study, we sought to develop an in vitro model of respiratory epithelial cells in IPF for the evaluation of the intrapulmonary distribution of aerosolized drugs. We investigated transforming growth factor (TGF)-β 1 -induced epithelial-mesenchymal transition (EMT) and permeability alteration in A549, NCI-H441, and Calu-3 cell monolayers. After TGF-β 1 treatment of A549, NCI-H441, and Calu-3 cells, EMT markers including E-cadherin and vimentin and tight junction proteins including claudins-1, -3, and -5 were stained using immunofluorescence methods and detected using immunoblotting methods. Transport experiments were performed using TGF-β 1 -treated cell monolayers and fluorescein isothiocyanate dextrans (FD; 4.4, 10, and 70kDa). In addition, TGF-β 1 -induced apoptosis and necrosis were evaluated by flow cytometry using Annexin V and ethidium homodimer III, respectively. In NCI-H441 cells, incomplete EMT, destruction of claudins-1 and -3, and enhancement of FD permeability were caused by TGF-β 1 treatment. In A549 cells, complete EMT occurred but was not adequate for transport experiments because of low transepithelial electrical resistance. Whereas in Calu-3 cells, no changes were observed. TGF-β 1 -induced apoptosis and necrosis were not observed in any of the cell lines. Incomplete EMT and permeability enhancement were observed in the alveolar epithelium of IPF. Therefore, our results indicate that TGF-β 1 -treated NCI-H441 cell monolayers may serve as a useful in vitro model of respiratory epithelial cells for IPF. Copyright © 2017 Elsevier Inc. All rights reserved.

Impact of jamming on collective cell migration

NASA Astrophysics Data System (ADS)

Nnetu, Kenechukwu David; Knorr, Melanie; Pawlizak, Steve; Fuhs, Thomas; Zink, Mareike; KäS, Josef A.

2012-02-01

Multi-cellular migration plays an important role in physiological processes such as embryogenesis, cancer metastasis and tissue repair. During migration, single cells undergo cycles of extension, adhesion and retraction resulting in morphological changes. In a confluent monolayer, there are inter-cellular interactions and crowding, however, the impact of these interactions on the dynamics and elasticity of the monolayer at the multi-cellular and single cell level is not well understood. Here we study the dynamics of a confluent epithelial monolayer by simultaneously measuring cell motion at the multi-cellular and single cell level for various cell densities and tensile elasticity. At the multi-cellular level, the system exhibited spatial kinetic transitions from isotropic to anisotropic migration on long times and the velocity of the monolayer decreased with increasing cell density. Moreover, the dynamics was spatially and temporally heterogeneous. Interestingly, the dynamics was also heterogeneous in wound-healing assays and the correlation length was fitted by compressed exponential. On the single cell scale, we observed transient caging effects with increasing cage rearrangement times as the system age due to an increase in density. Also, the density dependent elastic modulus of the monolayer scaled as a weak power law. Together, these findings suggest that caging effects at the single cell level initiates a slow and heterogeneous dynamics at the multi-cellular level which is similar to the glassy dynamics of deformable colloidal systems.

Evaluation of the intestinal permeability of rosemary (Rosmarinus officinalis L.) extract polyphenols and terpenoids in Caco-2 cell monolayers

PubMed Central

Arráez-Román, David; González-Álvarez, Isabel; Ibáñez, Elena; Segura-Carretero, Antonio; Bermejo, Marival; Micol, Vicente

2017-01-01

Rosemary (Rosmarinus officinalis) is grown throughout the world and is widely used as a medicinal herb and to season and preserve food. Rosemary polyphenols and terpenoids have attracted great interest due to their potential health benefits. However, complete information regarding their absorption and bioavailability in Caco-2 cell model is scarce. The permeation properties of the bioactive compounds (flavonoids, diterpenes, triterpenes and phenylpropanoids) of a rosemary extract (RE), obtained by supercritical fluid extraction, was studied in Caco-2 cell monolayer model, both in a free form or liposomed. Compounds were identified and quantitated by liquid chromatography coupled to quadrupole time-of-flight with electrospray ionization mass spectrometry analysis (HPLC-ESI-QTOF-MS), and the apparent permeability values (Papp) were determined, for the first time in the extract, for 24 compounds in both directions across cell monolayer. For some compounds, such as triterpenoids and some flavonoids, Papp values found were reported for the first time in Caco-2 cells.Our results indicate that most compounds are scarcely absorbed, and passive diffusion is suggested to be the primary mechanism of absorption. The use of liposomes to vehiculize the extract resulted in reduced permeability for most compounds. Finally, the biopharmaceutical classification (BCS) of all the compounds was achieved according to their permeability and solubility data for bioequivalence purposes. BCS study reveal that most of the RE compounds could be classified as classes III and IV (low permeability); therefore, RE itself should also be classified into this category. PMID:28234919

Modes of Cell Death Induced by Photodynamic Therapy Using Zinc Phthalocyanine in Lung Cancer Cells Grown as a Monolayer and Three-Dimensional Multicellular Spheroids.

PubMed

Manoto, Sello L; Houreld, Nicolette; Hodgkinson, Natasha; Abrahamse, Heidi

2017-05-16

Photodynamic therapy (PDT) involves interaction of a photosensitizer, light, and molecular oxygen which produces singlet oxygen and subsequent tumour eradication. The development of second generation photosensitizers, such as phthalocyanines, has improved this technology. Customary monolayer cell culture techniques are, unfortunately, too simple to replicate treatment effects in vivo. Multicellular tumour spheroids may provide a better alternative since they mimic aspects of the human tumour environment. This study aimed to profile 84 genes involved in apoptosis following treatment with PDT on lung cancer cells (A549) grown in a monolayer versus three-dimensional multicellular tumour spheroids (250 and 500 μm). Gene expression profiling was performed 24 h post irradiation (680 nm; 5 J/cm²) with zinc sulfophthalocyanine (ZnPcS mix ) to determine the genes involved in apoptotic cell death. In the monolayer cells, eight pro-apoptotic genes were upregulated, and two were downregulated. In the multicellular tumour spheroids (250 µm) there was upregulation of only 1 gene while there was downregulation of 56 genes. Apoptosis in the monolayer cultured cells was induced via both the intrinsic and extrinsic apoptotic pathways. However, in the multicellular tumour spheroids (250 and 500 µm) the apoptotic pathway that was followed was not conclusive.

Renal Epithelial Cyst Formation and Enlargement in vitro: Dependence on cAMP

NASA Astrophysics Data System (ADS)

Mangoo-Karim, Roberto; Uchic, Marie; Lechene, Claude; Grantham, Jared J.

1989-08-01

Cysts, a common abnormality of kidneys, are collections of urine-like fluid enclosed by a continuous layer of epithelial cells. Renal cysts derive from nephrons and collecting ducts and progressively enlarge as a consequence of epithelial proliferation and transepithelial fluid secretion. The initiation of cyst formation and the factors that control cyst enlargement are unknown. We used an in vitro model of renal cysts to explore the role of the cAMP signal transduction system in the formation and expansion of cysts. MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, arginine vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. All agonists were potentiated by 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor. The cell proliferation component of cyst enlargement was accelerated by cAMP agonists, as shown by the increased growth of MDCK cells in subconfluent monolayers. The fluid secretion component, reflected by the transepithelial movement of fluid across polarized monolayers of MDCK cells grown on permeable supports, was stimulated by cAMP agonists in the basolateral medium. Chloride levels were higher in the cyst fluid and the secreted fluid than in the bathing medium. We conclude that the development of MDCK cysts is dependent on cAMP. This signal transduction system may be an important modulator of epithelial cell proliferation and transepithelial fluid secretion in the kidney.

There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox globator

PubMed Central

2011-01-01

Background Epithelial folding is a common morphogenetic process during the development of multicellular organisms. In metazoans, the biological and biomechanical processes that underlie such three-dimensional (3D) developmental events are usually complex and difficult to investigate. Spheroidal green algae of the genus Volvox are uniquely suited as model systems for studying the basic principles of epithelial folding. Volvox embryos begin life inside out and then must turn their spherical cell monolayer outside in to achieve their adult configuration; this process is called 'inversion.' There are two fundamentally different sequences of inversion processes in Volvocaceae: type A and type B. Type A inversion is well studied, but not much is known about type B inversion. How does the embryo of a typical type B inverter, V. globator, turn itself inside out? Results In this study, we investigated the type B inversion of V. globator embryos and focused on the major movement patterns of the cellular monolayer, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. Isolated intact, sectioned and fragmented embryos were analyzed throughout the inversion process using light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques. We generated 3D models of the identified cell shapes, including the localizations of CBs. We show how concerted cell-shape changes and concerted changes in the position of cells relative to the CB system cause cell layer movements and turn the spherical cell monolayer inside out. The type B inversion of V. globator is compared to the type A inversion in V. carteri. Conclusions Concerted, spatially and temporally coordinated changes in cellular shapes in conjunction with concerted migration of cells relative to the CB system are the causes of type B inversion in V. globator. Despite significant similarities between type A and type B inverters, differences exist in almost all details of the inversion process, suggesting analogous inversion processes that arose through parallel evolution. Based on our results and due to the cellular biomechanical implications of the involved tensile and compressive forces, we developed a global mechanistic scenario that predicts epithelial folding during embryonic inversion in V. globator. PMID:22206406

Remodeling of the abdominal epithelial monolayer during the larva-pupa-adult transformation of Manduca.

PubMed

Nardi, James B; Bee, Charles Mark; Wallace, Catherine Lee

2018-06-01

During metamorphosis of insect epithelial monolayers, cells die, divide, and rearrange. In Drosophila undifferentiated diploid cells destined to form the adult cuticle of each abdominal segment segregate early in development from the surrounding polyploid larval epithelial cells of that segment as eight groups of diploid histoblast cells. The larval polyploid cells are programmed to die and be replaced by divisions and rearrangements of histoblast cells. By contrast, abdominal epithelial cells of Manduca larvae form a monolayer of cells representing different ploidy levels with no definitive segregation of diploid cells destined to form adult structures. These epithelial cells of mixed ploidy levels produce a thick smooth larval cuticle with sparsely distributed sensory bristles. Adult descendants of this larval monolayer produce a thinner cuticle with densely packed scale cells. The transition between these differentiated states of Manduca involves divisions of cells, changes in ploidy levels, and sorting of certain polyploid cells into circular rosette patches to minimize contacts of these polyploid cells with surrounding cells of equal or smaller size. Cells within the rosettes and some surrounding cells are destined to die and be replaced by remaining epithelial cells of uniform size and ploidy at pupa-adult apolysis. Copyright © 2018 Elsevier Inc. All rights reserved.

Modular control of endothelial sheet migration

PubMed Central

Vitorino, Philip; Meyer, Tobias

2008-01-01

Growth factor-induced migration of endothelial cell monolayers enables embryonic development, wound healing, and angiogenesis. Although collective migration is widespread and therapeutically relevant, the underlying mechanism by which cell monolayers respond to growth factor, sense directional signals, induce motility, and coordinate individual cell movements is only partially understood. Here we used RNAi to identify 100 regulatory proteins that enhance or suppress endothelial sheet migration into cell-free space. We measured multiple live-cell migration parameters for all siRNA perturbations and found that each targeted protein primarily regulates one of four functional outputs: cell motility, directed migration, cell–cell coordination, or cell density. We demonstrate that cell motility regulators drive random, growth factor-independent motility in the presence or absence of open space. In contrast, directed migration regulators selectively transduce growth factor signals to direct cells along the monolayer boundary toward open space. Lastly, we found that regulators of cell–cell coordination are growth factor-independent and reorient randomly migrating cells inside the sheet when boundary cells begin to migrate. Thus, cells transition from random to collective migration through a modular control system, whereby growth factor signals convert boundary cells into pioneers, while cells inside the monolayer reorient and follow pioneers through growth factor-independent migration and cell–cell coordination. PMID:19056882

Dual-Function Au@Y2O3:Eu3+ Smart Film for Enhanced Power Conversion Efficiency and Long-Term Stability of Perovskite Solar Cells.

PubMed

Kim, Chang Woo; Eom, Tae Young; Yang, In Seok; Kim, Byung Su; Lee, Wan In; Kang, Yong Soo; Kang, Young Soo

2017-07-28

In the present study, a dual-functional smart film combining the effects of wavelength conversion and amplification of the converted wave by the localized surface plasmon resonance has been investigated for a perovskite solar cell. This dual-functional film, composed of Au nanoparticles coated on the surface of Y 2 O 3 :Eu 3+ phosphor (Au@Y 2 O 3 :Eu 3+ ) nanoparticle monolayer, enhances the solar energy conversion efficiency to electrical energy and long-term stability of photovoltaic cells. Coupling between the Y 2 O 3 :Eu 3+ phosphor monolayer and ultraviolet solar light induces the latter to be converted into visible light with a quantum yield above 80%. Concurrently, the Au nanoparticle monolayer on the phosphor nanoparticle monolayer amplifies the converted visible light by up to 170%. This synergy leads to an increased solar light energy conversion efficiency of perovskite solar cells. Simultaneously, the dual-function film suppresses the photodegradation of perovskite by UV light, resulting in long-term stability. Introducing the hybrid smart Au@Y 2 O 3 :Eu 3+ film in perovskite solar cells increases their overall solar-to-electrical energy conversion efficiency to 16.1% and enhances long-term stability, as compared to the value of 15.2% for standard perovskite solar cells. The synergism between the wavelength conversion effect of the phosphor nanoparticle monolayer and the wave amplification by the localized surface plasmon resonance of the Au nanoparticle monolayer in a perovskite solar cell is comparatively investigated, providing a viable strategy of broadening the solar spectrum utilization.

Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate.

PubMed

Stewart-Hutchinson, Phillip J; Szasz, Taylor P; Jaeger, Emily R; Onken, Michael D; Cooper, John A; Morley, Sharon Celeste

2017-09-01

Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL -/- ) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL -/- B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration. © Society for Leukocyte Biology.

Ovarian cancer stem cells.

PubMed

Zeimet, A G; Reimer, D; Sopper, S; Boesch, M; Martowicz, A; Roessler, J; Wiedemair, A M; Rumpold, H; Untergasser, G; Concin, N; Hofstetter, G; Muller-Holzner, E; Fiegl, H; Marth, C; Wolf, D; Pesta, M; Hatina, J

2012-01-01

Because of its semi-solid character in dissemination and growth, advanced ovarian cancer with its hundreds of peritoneal tumor nodules and plaques appears to be an excellent in vivo model for studying the cancer stem cell hypothesis. The most important obstacle, however, is to adequately define and isolate these tumor-initiating cells endowed with the properties of anoikis-resistance and unlimited self-renewal. Until now, no universal single marker or marker constellation has been found to faithfully isolate (ovarian) cancer stem cells. As these multipotent cells are known to possess highly elaborated efflux systems for cytotoxic agents, these pump systems have been exploited to outline putative stem cells as a side-population (SP) via dye exclusion analysis. Furthermore, the cells in question have been isolated via flow cytometry on the basis of cell surface markers thought to be characteristic for stem cells.In the Vienna variant of the ovarian cancer cell line A2780 a proof-of-principle model with both a stable SP and a stable ALDH1A1+ cell population was established. Double staining clearly revealed that both cell fractions were not identical. Of note, A2780V cells were negative for expression of surface markers CD44 and CD117 (c-kit). When cultured on monolayers of healthy human mesothelial cells, green-fluorescence-protein (GFP)-transfected SP of A2780V exhibited spheroid-formation, whereas non-side-population (NSP) developed a spare monolayer growing over the healthy mesothelium. Furthermore, A2780V SP was found to be partially resistant to platinum. However, this resistance could not be explained by over-expression of the "excision repair cross-complementation group 1" (ERCC1) gene, which is essentially involved in the repair of platinated DNA damage. ERCC1 was, nonetheless, over-expressed in A2780V cells grown as spheres under stem cell-selective conditions as compared to adherent monolayers cultured under differentiating conditions. The same was true for the primary ovarian cancer cells B-57.In summary our investigations indicate that even in multi-passaged cancer cell lines hierarchic government of growth and differentiation is conserved and that the key cancer stem cell population may be composed of small overlapping cell fractions defined by various arbitrary markers.

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

PubMed Central

Correia, Cláudia; Koshkin, Alexey; Carido, Madalena; Espinha, Nuno; Šarić, Tomo; Lima, Pedro A.; Alves, Paula M.

2016-01-01

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. Significance The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs. PMID:27025693

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing.

PubMed

Correia, Cláudia; Koshkin, Alexey; Carido, Madalena; Espinha, Nuno; Šarić, Tomo; Lima, Pedro A; Serra, Margarida; Alves, Paula M

2016-05-01

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs. ©AlphaMed Press.

Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum.

PubMed

Berginc, Katja; Zakelj, Simon; Levstik, Lea; Ursic, Darko; Kristl, Albin

2007-05-01

Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M-S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M-S P(app) values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and alpha-CHC (alpha-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium. One is very likely an MCT (monocarboxylic acid cotransporter) isoform, inhibited by specific MCT inhibitor alpha-CHC, while the involvement of the second one with overlapping substrate/inhibitor specificities (most probably a member of the organic anion-transporting polypeptide family, inhibited at least partially by DIDS) could not be excluded.

Cytogenetic landscape of paired neurospheres and traditional monolayer cultures in pediatric malignant brain tumors.

PubMed

Zhao, Xiumei; Zhao, Yi-Jue; Lin, Qi; Yu, Litian; Liu, Zhigang; Lindsay, Holly; Kogiso, Mari; Rao, Pulivarthi; Li, Xiao-Nan; Lu, Xinyan

2015-07-01

New therapeutic targets are needed to eliminate cancer stem cells (CSCs). We hypothesize that direct comparison of paired CSCs and nonstem tumor cells (NSTCs) will facilitate identification of primary "driver" chromosomal aberrations that can serve as diagnostic markers and/or therapeutic targets. We applied spectral karyotyping and G-banding to matched pairs of neurospheres (CSC-enriched cultures) and fetal bovine serum-based monolayer cultures (enriched with NSTCs) from 16 patient-derived orthotopic xenograft mouse models, including 9 medulloblastomas (MBs) and 7 high-grade gliomas (HGGs), followed by direct comparison of their numerical and structural abnormalities. Chromosomal aberrations were detected in neurospheres of all 16 models, and 82.0% numerical and 82.4% structural abnormalities were maintained in their matching monolayer cultures. Among the shared abnormalities, recurrent clonal changes were identified including gain of chromosomes 18 and 7 and loss of chromosome 10/10q (5/16 models), isochromosome 17q in 2 MBs, and a new breakpoint of 13q14 in 3 HGGs. Chromothripsis-like evidence was also observed in 3 HGG pairs. Additionally, we noted 20 numerical and 15 structural aberrations that were lost from the neurospheres and found 26 numerical and 23 structural aberrations that were only present in the NSTCs. Compared with MBs, the neurosphere karyotypes of HGG were more complex, with fewer chromosomal aberrations preserved in their matching NSTCs. Self-renewing CSCs in MBs and pediatric HGGs harbor recurrent numerical and structural aberrations that were maintained in the matching monolayer cultures. These primary chromosomal changes may represent new markers for anti-CSC therapies. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Absorption mechanism of whey-protein-delivered curcumin using Caco-2 cell monolayers.

PubMed

Li, Ming; Cui, Jie; Ngadi, Michael O; Ma, Ying

2015-08-01

Curcumin (CCM) is a bioactive polyphenolic compound that suffers a low bioavailability because of its low water solubility. In this work β-lactoglobulin (β-Lg) and nanoemulsion were used as carriers to deliver curcumin. The pH stability of β-Lg-CCM was investigated. The digestion of β-Lg-CCM and the nanoemulsion was studied using an in vitro gastrointestinal model. The effect of different carriers on the permeability of curcumin was assessed using the Caco-2 cell monolayer model. The results revealed that the water solubility and the pH stability of curcumin significantly increased by binding with β-Lg. In SDS-PAGE experiments the β-Lg-CCM complex and nanoemulsion were found to be resistant to pepsin digestion but sensitive to trypsin. In the permeability experiment it was shown that the digested nanoemulsion and β-Lg-CCM improved significantly the permeation rate of curcumin. Copyright © 2015 Elsevier Ltd. All rights reserved.

The interaction of an antiparasitic peptide active against African sleeping sickness with cell membrane models.

PubMed

Pascholati, Cauê P; Lopera, Esteban Parra; Pavinatto, Felippe J; Caseli, Luciano; Nobre, Thatyane M; Zaniquelli, Maria E D; Viitala, Tapani; D'Silva, Claudius; Oliveira, Osvaldo N

2009-12-01

Zwitterionic peptides with trypanocidal activity are promising lead compounds for the treatment of African Sleeping Sickness, and have motivated research into the design of compounds capable of disrupting the protozoan membrane. In this study, we use the Langmuir monolayer technique to investigate the surface properties of an antiparasitic peptide, namely S-(2,4-dinitrophenyl)glutathione di-2-propyl ester, and its interaction with a model membrane comprising a phospholipid monolayer. The drug formed stable Langmuir monolayers, whose main feature was a phase transition accompanied by a negative surface elasticity. This was attributed to aggregation upon compression due to intermolecular bond associations of the molecules, inferred from surface pressure and surface potential isotherms, Brewster angle microscopy (BAM) images, infrared spectroscopy and dynamic elasticity measurements. When co-spread with dipalmitoyl phosphatidyl choline (DPPC), the drug affected both the surface pressure and the monolayer morphology, even at high surface pressures and with low amounts of the drug. The results were interpreted by assuming a repulsive, cooperative interaction between the drug and DPPC molecules. Such repulsive interaction and the large changes in fluidity arising from drug aggregation may be related to the disruption of the membrane, which is key for the parasite killing property.

Structural studies of the HIV-1 accessory protein Vpu in langmuir monolayers: synchrotron X-ray reflectivity.

PubMed Central

Zheng, S; Strzalka, J; Ma, C; Opella, S J; Ocko, B M; Blasie, J K

2001-01-01

Vpu is an 81 amino acid integral membrane protein encoded by the HIV-1 genome with a N-terminal hydrophobic domain and a C-terminal hydrophilic domain. It enhances the release of virus from the infected cell and triggers degradation of the virus receptor CD4. Langmuir monolayers of mixtures of Vpu and the phospholipid 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (DLgPC) at the water-air interface were studied by synchrotron radiation-based x-ray reflectivity over a range of mole ratios at constant surface pressure and for several surface pressures at a maximal mole ratio of Vpu/DLgPC. Analysis of the x-ray reflectivity data by both slab model-refinement and model-independent box-refinement methods firmly establish the monolayer electron density profiles. The electron density profiles as a function of increasing Vpu/DLgPC mole ratio at a constant, relatively high surface pressure indicated that the amphipathic helices of the cytoplasmic domain lie on the surface of the phospholipid headgroups and the hydrophobic transmembrane helix is oriented approximately normal to the plane of monolayer within the phospholipid hydrocarbon chain layer. At maximal Vpu/DLgPC mole ratio, the tilt of the transmembrane helix with respect to the monolayer normal decreases with increasing surface pressure and the conformation of the cytoplasmic domain varies substantially with surface pressure. PMID:11259297

Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.

1987-05-01

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of /sup 51/Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cellsmore » (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of /sup 51/Cr release from radiolabeled monolayers.« less

Study of iridium silicide monolayers using density functional theory

NASA Astrophysics Data System (ADS)

Popis, Minh D.; Popis, Sylvester V.; Oncel, Nuri; Hoffmann, Mark R.; ćakır, Deniz

2018-02-01

In this study, we investigated physical and electronic properties of possible two-dimensional structures formed by Si (silicon) and Ir (iridium). To this end, different plausible structures were modeled by using density functional theory and the cohesive energies calculated for the geometry of optimized structures, with the lowest equilibrium lattice constants. Among several candidate structures, we identified three mechanically (via elastic constants and Young's modulus), dynamically (via phonon calculations), and thermodynamically stable iridium silicide monolayer structures. The lowest energy structure has a chemical formula of Ir2Si4 (called r-IrSi2), with a rectangular lattice (Pmmn space group). Its cohesive energy was calculated to be -0.248 eV (per IrSi2 unit) with respect to bulk Ir and bulk Si. The band structure indicates that the Ir2Si4 monolayer exhibits metallic properties. Other stable structures have hexagonal (P-3m1) and tetragonal (P4/nmm) cell structures with 0.12 and 0.20 eV/f.u. higher cohesive energies, respectively. Our calculations showed that Ir-Si monolayers are reactive. Although O2 molecules exothermically dissociate on the surface of the free-standing iridium silicide monolayers with large binding energies, H2O molecules bind to the monolayers with a rather weak interaction.

Intercellular ice propagation: experimental evidence for ice growth through membrane pores.

PubMed Central

Acker, J P; Elliott, J A; McGann, L E

2001-01-01

Propagation of intracellular ice between cells significantly increases the prevalence of intracellular ice in confluent monolayers and tissues. It has been proposed that gap junctions facilitate ice propagation between cells. This study develops an equation for capillary freezing-point depression to determine the effect of temperature on the equilibrium radius of an ice crystal sufficiently small to grow through gap junctions. Convection cryomicroscopy and video image analysis were used to examine the incidence and pattern of intracellular ice formation (IIF) in the confluent monolayers of cell lines that do (MDCK) and do not (V-79W) form gap junctions. The effect of gap junctions on intracellular ice propagation was strongly temperature-dependent. For cells with gap junctions, IIF occurred in a directed wave-like pattern in 100% of the cells below -3 degrees C. At temperatures above -3 degrees C, there was a marked drop in the incidence of IIF, with isolated individual cells initially freezing randomly throughout the sample. This random pattern of IIF was also observed in the V-79W monolayers and in MDCK monolayers treated to prevent gap junction formation. The significant change in the low temperature behavior of confluent MDCK monolayers at -3 degrees C is likely the result of the inhibition of gap junction-facilitated ice propagation, and supports the theory that gap junctions facilitate ice nucleation between cells. PMID:11509353

Cytotoxicity of zinc oxide (ZnO) nanoparticles is influenced by cell density and culture format.

PubMed

Heng, Boon Chin; Zhao, Xinxin; Xiong, Sijing; Ng, Kee Woei; Boey, Freddy Yin-Chiang; Loo, Joachim Say-Chye

2011-06-01

A parameter that has often been overlooked in cytotoxicity assays is the density and confluency of mammalian cell monolayers utilized for toxicology screening. Hence, this study investigated how different cell seeding densities influenced their response to cytotoxic challenge with ZnO nanoparticles. Utilizing the same volume (1 ml per well) and concentration range (5-40 μg/ml) of ZnO nanoparticles, contradictory results were observed with higher-density cell monolayers (BEAS-2B cells) obtained either by increasing the number of seeded cells per well (50,000 vs. 200,000 cells per well of 12-well plate) or by seeding the same numbers of cells (50,000) within a smaller surface area (12-well vs. 48-well plate, 4.8 vs. 1.2 cm(2), respectively). Further experiments demonstrated that the data may be skewed by inconsistency in the mass/number of nanoparticles per unit area of culture surface, as well as by inconsistent nanoparticle to cell ratio. To keep these parameters constant, the same number of cells (50,000 per well) were seeded on 12-well plates, but with the cells being seeded at the edge of the well for the experimental group (by tilting the plate) to form a dense confluent monolayer, as opposed to a sparse monolayer for the control group seeded in the conventional manner. Utilizing such an experimental set-up for the comparative evaluation of four different cell lines (BEAS-2B, L-929, CRL-2922 and C2C12), it was observed that the high cell density monolayer was consistently more resistant to the cytotoxic effects of ZnO nanoparticles compared to the sparse monolayer for all four different cell types, with the greatest differences being observed above a ZnO concentration of 10 μg/ml. Hence, the results of this study demonstrate the need for the standardization of cell culture protocols utilized for toxicology screening of nanoparticles, with respect to cell density and mass/number of nanoparticles per unit area of culture surface.

Electronic Devices with Barium Barrier Film and Process for Making Same

DTIC Science & Technology

1998-08-20

structure of the barrier film on an atomic level 15 where the barrier .film is comprised of a plurality of contiguous monolayers, while FIG. 7B...yet another embodiment where the barrier film is comprised of a plurality of 20 contiguous monolayers in which different monolayers thereof are...barrier precursor compound effusion cell, for example a barium fluoride, strontium fluoride or the like effusion cell, is provided at 32, and has a

Electronic Devices with Barrier Film and Process for Making Same

DTIC Science & Technology

1998-08-20

the barrier film on an atomic level where the barrier film is comprised of a plurality of contiguous monolayers, while FIG. 7B shows another...embodiment where the barrier film is comprised of a plurality of contiguous monolayers in which different monolayers thereof are formed of different...compound effusion cell, for example a barium fluoride, strontium fluoride or the like effusion cell, is provided at 32, and has a shutter 33. A

Platinum Monolayer Electrocatalysts for Anodic Oxidation of Alcohols.

PubMed

Li, Meng; Liu, Ping; Adzic, Radoslav R

2012-12-06

The slow, incomplete oxidation of methanol and ethanol on platinum-based anodes as well as the high price and limited reserves of Pt has hampered the practical application of direct alcohol fuel cells. We describe the electrocatalysts consisting of one Pt monolayer (one atom thick layer) placed on extended or nanoparticle surfaces having the activity and selectivity for the oxidation of alcohol molecules that can be controlled with platinum-support interaction. The suitably expanded Pt monolayer (i.e., Pt/Au(111)) exhibits a factor of 7 activity increase in catalyzing methanol electrooxidation relative to Pt(111). Sizable enhancement is also observed for ethanol electrooxidation. Furthermore, a correlation between substrate-induced lateral strain in a Pt monolayer and its activity/selectivity is established and rationalized by experimental and theoretical studies. The knowledge we gained with single-crystal model catalysts was successfully applied in designing real nanocatalysts. These findings for alcohols are likely to be applicable for the oxidation of other classes of organic molecules.

Study of pharmaceutical industrial problems

NASA Technical Reports Server (NTRS)

Pincus, J. H.

1979-01-01

The growth of a human colon carcinoma cell line (SK-CO-1) and its production of carcinoembryonic antigen (CEA) in monolayer culture and on single layers of glass beads in unit gravity were evaluated. The limitations of using a microsphere-cell growth system in unit gravity were identified and how these may be overcome in space was considered. The project had the following tasks: (1) growth of cultured human colon carcinoma cells on a monolayer and CEA production; (2) evaluation of CEA production and release by SK-CO-1 cells grown on glass beads; (3) evaluation of other microcarriers for growing SK-CO-1 cells and determination of the minimum amount of culture medium needed for cell growth; and (4) growth of SK-CO-1 cells on collagen monolayers and CEA production.

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

PubMed

Min, Kyoung Ah; Rosania, Gus R; Kim, Chong-Kook; Shin, Meong Cheol

2016-03-01

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers

PubMed Central

Min, Kyoung Ah; Rosania, Gus R.; Kim, Chong-Kook; Shin, Meong Cheol

2016-01-01

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies. PMID:26746641

The Gene tia, Harbored by the Subtilase-Encoding Pathogenicity Island, Is Involved in the Ability of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli Strains To Invade Monolayers of Epithelial Cells

PubMed Central

Chiani, Paola; Michelacci, Valeria; Minelli, Fabio; Caprioli, Alfredo; Morabito, Stefano

2017-01-01

ABSTRACT Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia, previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia, present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli, including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains. PMID:28893912

The Gene tia, Harbored by the Subtilase-Encoding Pathogenicity Island, Is Involved in the Ability of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli Strains To Invade Monolayers of Epithelial Cells.

PubMed

Bondì, Roslen; Chiani, Paola; Michelacci, Valeria; Minelli, Fabio; Caprioli, Alfredo; Morabito, Stefano

2017-12-01

Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia , previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia , present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli , including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains. Copyright © 2017 American Society for Microbiology.

Probiotics promote endocytic allergen degradation in gut epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Chun-Hua; Liu, Zhi-Qiang; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON

Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barriermore » function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.« less

Reprogramming mediated radio-resistance of 3D-grown cancer cells.

PubMed

Xue, Gang; Ren, Zhenxin; Grabham, Peter W; Chen, Yaxiong; Zhu, Jiayun; Du, Yarong; Pan, Dong; Li, Xiaoman; Hu, Burong

2015-07-01

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy.

PubMed

Indovina, Paola; Collini, Maddalena; Chirico, Giuseppe; Santini, Maria Teresa

2007-02-20

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1alpha (hypoxia-inducible factor-1alpha) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (<100 microm) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.

Effects of method of detachment on electrophoretic mobility of mammalian cells grown in monolayer culture

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

A variety of proteolytic and micolytic enzumes, mechanical procedures, and changes in the ionic environment, especially Ca chelation, are used for dispersal of monolayer grown cells. If either chelating agents or mechanical dispersion are used alone, the cell yield is often low and suspensions of single cells are difficult to obtain. Confluent monolayers treated with EDTA tend to be released from their surfaces in sheets, and clumps of cells remain even after further incubation in EDTA. Crude trypsin is the most popular dispersal agent and is known to contain a variety of contaminating enzymes which contribute to the dispersal of cells. A variety of cell injuries resulting from the activity of proteolytic enzymes are reported. It is shown that crystalline trypsin is least harmful to cell integrity as judged by trypan blue uptake.

Culture on 3D Chitosan-Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells.

PubMed

Wang, Kui; Kievit, Forrest M; Erickson, Ariane E; Silber, John R; Ellenbogen, Richard G; Zhang, Miqin

2016-12-01

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

PubMed Central

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C.D.; Shaw, Lucy; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J.; Braker, Paul; Rahman, Ruman; Jones, George D.D.; Watson, Susan; Lowe, James; Kerr, Ian D.; Grundy, Richard G.; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3–4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents. PMID:20978004

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.

PubMed

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C D; Shaw, Lucy; Othman, Ramadhan; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J; Braker, Paul; Rahman, Ruman; Jones, George D D; Watson, Susan; Lowe, James; Kerr, Ian D; Grundy, Richard G; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.

In vitro effects of preserved and unpreserved anti-allergic drugs on human corneal epithelial cells.

PubMed

Guzman-Aranguez, Ana; Calvo, Patricia; Ropero, Inés; Pintor, Jesús

2014-11-01

Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures.

Electronic Devices with Cesium Barrier Film and Process for Making Same

DTIC Science & Technology

1998-08-20

interfacial structure of the barrier film on an atomic level where the barrier film is comprised of a plurality of contiguous monolayers, while FIG. 7B shows...another 20 embodiment where the barrier film is comprised of a plurality of contiguous monolayers in which different monolayers thereof are formed...compound effusion cell, for example a barium fluoride, strontium fluoride or the like effusion cell, is provided at 32, and has a shutter 33. A

Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds.

PubMed

Lobo, Anderson O; Antunes, Erica F; Palma, Mariana Bs; Pacheco-Soares, Cristina; Trava-Airoldi, Vladimir J; Corat, Evaldo J

2010-03-12

Monolayer formation of SaOS-2 (human osteoblast-like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non-toxicity and the flat spreading with monolayer formation of the SaOs-2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.

Uptake of 2S albumin allergens, Ber e 1 and Ses i 1, across human intestinal epithelial Caco-2 cell monolayers.

PubMed

Moreno, F Javier; Rubio, Luis A; Olano, Agustín; Clemente, Alfonso

2006-11-01

We have investigated the absorption rates of two purified major allergen 2S albumins, Ber e 1 from Brazil nuts (Bertholletia excelsa Humb. & Bonpl.) and Ses i 1 from white sesame seeds (Sesamum indicum L.), across human intestinal epithelial Caco-2 cell monolayers following gastrointestinal digestion in vitro. The transport from apical to basolateral side in cell monolayers was evaluated by RP-HPLC-UV and indirect competitive ELISA methods, being confirmed by western-blotting analysis. Significant amounts (approximately 15-25 nmol micromol(-1) initial amount/h) of intact Ber e 1 and Ses i 1 were found in the basolateral side. The absorption rates of both plant allergens through the cell monolayer were shown to be constant during the whole incubation period (4 h at 37 degrees C), verifying that the permeability of the membrane was not altered by the allergen digests. Our findings revealed that both purified 2S albumin allergens may be able to survive in immunologically reactive forms to the simulated harsh conditions of the gastrointestinal tract to be transported across the Caco-2 cell monolayers, so that they would be able to sensitize the mucosal immune system and/or elicit an allergic response.

The Volume of Three-Dimensional Cultures of Cancer Cells InVitro Influences Transcriptional Profile Differences and Similarities with Monolayer Cultures and Xenografted Tumors.

PubMed

Boghaert, Erwin R; Lu, Xin; Hessler, Paul E; McGonigal, Thomas P; Oleksijew, Anatol; Mitten, Michael J; Foster-Duke, Kelly; Hickson, Jonathan A; Santo, Vitor E; Brito, Catarina; Uziel, Tamar; Vaidya, Kedar S

2017-09-01

Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro. Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of applying these models for drug development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The apoptotic effects of silibinin on MDA-MB-231 and MCF-7 human breast carcinoma cells.

PubMed

Bayram, D; Çetin, E S; Kara, M; Özgöçmen, M; Candan, I A

2017-06-01

Silibinin is a bioactive flavonolignan extracted from milk thistle, known as Silybum marianum. Silibinin exerts strong antiproliferative, proapoptotic, and anti-inflammatory effects. Many studies have shown that silibinin inhibits experimentally induced malignancies of the liver, prostate, skin, and colon as well as promotes inhibition of the proliferation of cancer cell lines in vitro. This study aimed to investigate the effects of silibinin on the human breast carcinoma cell lines MDA-MB-231 and MCF-7 in monolayer and spheroid cultures. The MDA-MB-231 and MCF-7 cell lines were cultured in both monolayer and spheroid cultures. Cells were treated with silibinin at 24, 48, and 72 h of incubation. The 5-bromo-2'-deoxyuridine labeling index was used to determine the cells of the synthesis phase. Poly-ADP-ribose-polimerase immunohistochemical staining and the terminal deoxynucleotidyl transferase dUTP nick and labeling assay were used to determine the death of cells in both the monolayer and spheroid cultures. An half maximal inhibitory concentration dose of silibinin in MDA-MB-231 and MCF-7 cells was 100 µM/mL at 24, 48, and 72 h of incubation. Terminal deoxynucleotidyl transferase dUTP nick and labeling positive cells and active poly-ADP-ribose-polimerase were detected after treatment with silibinin in both the monolayer and spheroid cultures. The dead cell count was higher in the MDA-MB-231 and MCF-7 cell lines with silibinin applied than in the controls. Our study demonstrated that silibinin applications enhanced terminal deoxynucleotidyl transferase dUTP nick and labeling positive cells and active poly-ADP-ribose-polimerase in comparison to the control in both the monolayer and spheroid cultures.

Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

PubMed

Herron, Todd J; Rocha, Andre Monteiro Da; Campbell, Katherine F; Ponce-Balbuena, Daniela; Willis, B Cicero; Guerrero-Serna, Guadalupe; Liu, Qinghua; Klos, Matt; Musa, Hassan; Zarzoso, Manuel; Bizy, Alexandra; Furness, Jamie; Anumonwo, Justus; Mironov, Sergey; Jalife, José

2016-04-01

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers. © 2016 American Heart Association, Inc.

Active properties of living tissues lead to size-dependent dewetting

NASA Astrophysics Data System (ADS)

Perez-Gonzalez, Carlos; Alert, Ricard; Blanch-Mercader, Carles; Gomez-Gonzalez, Manuel; Casademunt, Jaume; Trepat, Xavier

Key biological processes such as cancer and development are characterized by drastic transitions from 2D to a 3D geometry. These rearrangements have been classically studied as a wetting problem. According to this theory, wettability of a substrate by an epithelium is determined by the competition between cell-cell and cell-substrate adhesion energies. In contrast, we found that, far from a passive process, tissue dewetting is an active process driven by tissue internal forces. Experimentally, we reproduced epithelial dewetting by promoting a progressive formation of intercellular junctions in a monolayer of epithelial cells. Interestingly, the formation of intercellular junctions produces an increase in cell contractility, with the subsequent increase in traction and intercellular stress. At a certain time, tissue tension overcomes cell-substrate maximum adhesion and the monolayer spontaneously dewets the substrate. We developed an active polar fluid model, finding both theoretically and experimentally that critical contractility to promote wetting-dewetting transition depends on cell-substrate adhesion and, unexpectedly, on tissue size. As a whole, this work generalizes wetting theory to living tissues, unveiling unprecedented properties due to their unique active nature.

Integrated quantitative fractal polarimetric analysis of monolayer lung cancer cells

NASA Astrophysics Data System (ADS)

Shrestha, Suman; Zhang, Lin; Quang, Tri; Farrahi, Tannaz; Narayan, Chaya; Deshpande, Aditi; Na, Ying; Blinzler, Adam; Ma, Junyu; Liu, Bo; Giakos, George C.

2014-05-01

Digital diagnostic pathology has become one of the most valuable and convenient advancements in technology over the past years. It allows us to acquire, store and analyze pathological information from the images of histological and immunohistochemical glass slides which are scanned to create digital slides. In this study, efficient fractal, wavelet-based polarimetric techniques for histological analysis of monolayer lung cancer cells will be introduced and different monolayer cancer lines will be studied. The outcome of this study indicates that application of fractal, wavelet polarimetric principles towards the analysis of squamous carcinoma and adenocarcinoma cancer cell lines may be proved extremely useful in discriminating among healthy and lung cancer cells as well as differentiating among different lung cancer cells.

In situ flat embedding of monolayers and cell relocation in the acrylic resin LR white for comparative light and electron microscopy studies.

PubMed

Steiner, M; Schöfer, C; Mosgoeller, W

1994-12-01

A simple and reliable method has been developed for the in situ LR White embedding of cell monolayers grown on glass cover-slips. Combined with cytochemical or immunological procedures, this technique allows light and/or electron microscopy investigations of a large number of cells in the same horizontal plane within a relatively short period of time. It can be applied to cells grown on microgrid finder cover-slips which allows a distinct site of even an individual cell of a monolayer to be studied at first at the light microscope level and subsequently at the electron microscope level. Hence, it is also suitable for controlling manipulation of single cells, followed by their serial sectioning after relocation in the electron microscope.

Transport of curcumin derivatives in Caco-2 cell monolayers.

PubMed

Zeng, Zhen; Shen, Zhe L; Zhai, Shuo; Xu, Jia L; Liang, Hui; Shen, Qin; Li, Qing Y

2017-08-01

Curcumin (Cur) is a strong natural antioxidant, who can prevent multiple diseases such as anti-cancer, anti-inflammatory, have a resistance to alzheimer's disease and various malignant diseases. But it has poor oral bioavailability due to its poor aqueous solubility, as well as instability. While its novel derivatives (CB and FE), showed better anti-tumor activity, better anti-oxidant activity and better stability than the original drug (Cur). The aim of this study was to study the intestinal transport of Cur, CB and FE using an in vitro Caco-2 cell monolayer model. The results showed that Cur had a lower permeability coefficient (1.13×10 -6 ±0.11×10 -6 cm/s) for apical-to-basolated (AP-BL) transport at 25μM, while the transport rate for AP to BL flux of CB (3.18×10 -6 ±0.31×10 -6 cm/s) and FE (5.28×10 -6 ±0.83×10 -6 cm/s) were significantly greater than that of Cur. The efflux ratio (ER) value at the concentration of 25μM was 1.31 for Cur, 1.26 for CB and 1.33 for FE, suggesting there was no active efflux involved in the translocation across the Caco-2 cell monolayers for the three compounds. Furthermore, the transport flux of CB and FE was in a concentration dependent manner, suggesting the intestinal transport mechanism in them was passive transport. In summary, the results demonstrated that both the intestinal permeability of CB and FE across Caco-2 cell monolayers was significantly improved compare to Cur. Thus they might show a higher oral bioavailability in vivo, and show the potential application in clinic or nutraceutical. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative and Mechanistic Assessment of Model Lipophilic Drugs in Micellar Solutions in the Transport Kinetics Across MDR1-MDCK Cell Monolayers.

PubMed

Ho, Norman F H; Nielsen, James; Peterson, Michelle; Burton, Philip S

2016-02-01

An approach to characterizing P-glycoprotein (Pgp) interaction potential for sparingly water-soluble compounds was developed using bidirectional transport kinetics in MDR1-MDCK cell monolayers. Paclitaxel, solubilized in a dilute polysorbate 80 (PS80) micellar solution, was used as a practical example. Although the passage of paclitaxel across the cell monolayer was initially governed by the thermodynamic activity of the micelle-solubilized drug solution, Pgp inhibition was sustained by the thermodynamic activity (i.e., critical micelle concentration) of the PS80 micellar solution bathing the apical (ap) membrane. The mechanistic understanding of the experimental strategies and treatment of data was supported by a biophysical model expressed in the form of transport events occurring at the ap and basolateral (bl) membranes in series whereas the vectorial directions of the transcellular kinetics were accommodated. The derived equations permitted the stepwise quantitative delineation of the Pgp efflux activity (inhibited and uninhibited by PS80) and the passive permeability coefficient of the ap membrane, the passive permeability at the bl membrane and, finally, the distinct coupling of these with efflux pump activity to identify the rate-determining steps and mechanisms. The Jmax/KM(∗) for paclitaxel was in the order of 10(-4) cm/s and the ap- and bl-membrane passive permeability coefficients were asymmetric, with bl-membrane permeability significantly greater than ap. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Paramyxovirus Infection Mimics In Vivo Cellular Dynamics in Three-Demensional Human Bronchio-Epithelial Tissue-Like Assemblies

NASA Technical Reports Server (NTRS)

Deatly, Anne M.; Lin, Yen-Huei; McCarthy, Maureen; Chen, Wei; Miller, Lynn Z.; Quiroz, Jorge; Nowak, Becky M.; Lerch, Robert A.; Udem, Stephen A.; Goodwin, Thomas J.

2012-01-01

Respiratory syncytial virus and parainfluenza virus cause severe respiratory disease, especially in infants, children and the elderly. An in vitro model that accurately mimics infection of the human respiratory epithelium (HRE) would facilitate vaccine development greatly. Monolayer cultures traditionally used to study these viruses do not accurately and precisely differentiate the replication efficiencies of wild type and attenuated viruses. Therefore, we engineered novel three-dimensional (3D) tissue-like assemblies (TLAs) of human broncho-epithelial (HBE) cells to produce a more physiologically relevant in vitro model of the HRE. TLAs resemble HRE structurally and by expression of differentiated epithelial cell markers. Most significantly, wild type viruses exhibited a clear growth advantage over attenuated strains in TLAs unlike monolayer cultures. In addition, the TLAs responded to virus infection by secreting pro-inflammatory mediators similar to the respiratory epithelia of infected children. These characteristics make the TLA model a valuable platform technology to develop and evaluate live, attenuated respiratory virus vaccine candidates for human use. Respiratory virus diseases, the most frequent and least preventable of all infectious diseases, range in severity from the common cold to severe bronchiolitis and pneumonia . Two paramyxoviruses, respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3), are responsible for a majority of the most severe respiratory diseases of infants and young children. RSV causes 70% of all bronchiolitis cases and is a major cause of morbidity and mortality worldwide, especially in infants. PIV3 causes 10-15% of bronchiolitis and pneumonia during infancy, second only to RSV, and 40% of croup in infants To date, licensed vaccines are not available to prevent these respiratory diseases. At present, traditional monkey kidney (Vero and LLC-MK2) and human (HEp-2) tissue culture cells and small animal models (mouse, cotton rat, guinea pig, ferret, and hamster) fail to accurately imitate viral replication and human disease states (8). Lacking an authentic model has impeded the development and evaluation of live, attenuated vaccine candidates. Development of a physiologically relevant in vitro tissue culture model that reproduces characteristics of the HRE, the primary target of RSV and PIV3, would aid in predicting clinical attenuation and safety of vaccine candidates. Successful tissue engineering of a 3D human intestinal model using novel NASA technology inspired the development of a tri-culture 3D model for the HRE. Sequential layering of primary mesenchymal cells (comprised of normal human fibroblasts and endothelial cells) followed by BEAS-2B epithelial cells derived from human bronchi and tracheae were recapitulated on Cultisphere and/or cytodex3 microcarriers in cylindrical vessels that rotate horizontally creating an organized epithelial structure. Horizontal rotation randomizes the gravity vector modeling aspects of microgravity. Mesenchymal and epithelial cells grown under these conditions reproduce the structural organization, multi-cellular complexity, and differentiation state of the HRE. The opportunity to study respiratory viruses in a nasal epithelium model is invaluable because the most promising respiratory virus vaccine candidates are live attenuated viruses for intranasal administration. Here we characterize the interactions of respiratory viruses and epithelial cells grown under modeled microgravity in comparison to gravity-ladened monolayers. 3D HBE TLAs and traditional monolayers (2D) are infected at 35 C, the upper temperature of the upper HRE, to simulate in vivo infection conditions. Growth kinetics of wild type (wt) RSV and PIV3 viruses were compared in 2D and 3D cells to that of strains attenuated in humans or rhesus macaques. This novel 3D HBE model also offers an opportunity to study whether the epithelial cell function, especially in host defenses recapitulated by mimicking the structural organization of the HRE. In vivo, airway epithelial cells play a significant and dynamic role in host defense by blocking paracellular permeability and modulating airway function through cellular interactions or tight junctions. As regulators of the innate immune response, epithelial cells constitutively express cytokines, chemokines, and colony stimulating factors including RANTES, IL-8, IL-6, GM-CSF, and G-CSF for proactive host defense. In response to viral infection, epithelial cells induce potent immuno-modulatory and pro-inflammatory cytokines that recruit phagocytic and inflammatory cells to clear the virus and enhance protection. Although disease pathogenesis is classically attributed to the cytopathic effects of the pathogen, severe disease states associated with RSV and PIV3 are attributed to the inflammatory response, especially in infants. RSV is a potent inducer of cytokines and pro-inflammatory mediators in epithelial cells in vivo. A differentiated human epithelial model independent of the complete functional immune system will help elucidate the role of epithelial cells in respiratory disease. We reported here, virus and host cell interactions in 3D HBE TLAs are similar to that in vivo. Because the epithelial cell organization of the TLAs impacts not only the expression of airway epithelial characteristics, but also cellular communication, the TLAs represent a more physiologically relevant model of the HRE than BEAS-2B or other non-tumour monolayer models of respiratory disease. As a result, wild type respiratory viruses have a clear growth advantage over attenuated viruses in TLAs unlike traditional monolayers. In addition, the TLAs respond to wild type virus infection by secreting pro-inflammatory mediators characteristic of infected HRE. TLAs expressing microbial defense mechanisms provide an excellent model to study the interactions of respiratory pathogens with their host and to identify the innate immunity mediators. Therefore, 3D HBE TLAs offer advantages for the study of respiratory viruses and the development of viral vaccine candidates.

Three-dimensional cell culture models for investigating human viruses.

PubMed

He, Bing; Chen, Guomin; Zeng, Yi

2016-10-01

Three-dimensional (3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover, these models bridge the gap between traditional two-dimensional (2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition, 3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.

Demonstration of increased anti-mycobacterial activity in peripheral blood monocytes after BCG vaccination in British school children.

PubMed Central

Cheng, S H; Walker, L; Poole, J; Aber, V R; Walker, K B; Mitchison, D A; Lowrie, D B

1988-01-01

A blood sample was taken from children aged 13-15 years immediately before BCG vaccination and 8 weeks after. The children were tuberculin skin-test negative to PPD-S before vaccination and positive after. Mononuclear cells were separated from the blood, infected with Mycobacterium microti at a low bacterium/monocyte ratio and allowed to form monolayers in microtitre wells. The infected monolayers were rinsed daily and the change in number of live bacteria in monolayers and supernatants was monitored by colony counts on agar. The cells were bacteriostatic during the first day, thereafter growth accelerated in pre-vaccination monolayers. When monolayers received pulsed exposures to autologous lymphocytes that had been incubated with whole dead tubercle bacilli the growth rates of M. microti were increased. However, growth rates in lymphocyte-pulsed monolayers were significantly lower after vaccination than before. It is proposed that this difference reflects the protective effect of vaccination. PMID:3219800

Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure

PubMed Central

Badie, Nima; Bursac, Nenad

2009-01-01

Systematic studies of cardiac structure-function relationships to date have been hindered by the intrinsic complexity and variability of in vivo and ex vivo model systems. Thus, we set out to develop a reproducible cell culture system that can accurately replicate the realistic microstructure of native cardiac tissues. Using cell micropatterning techniques, we aligned cultured cardiomyocytes at micro- and macroscopic spatial scales to follow local directions of cardiac fibers in murine ventricular cross sections, as measured by high-resolution diffusion tensor magnetic resonance imaging. To elucidate the roles of ventricular tissue microstructure in macroscopic impulse conduction, we optically mapped membrane potentials in micropatterned cardiac cultures with realistic tissue boundaries and natural cell orientation, cardiac cultures with realistic tissue boundaries but random cell orientation, and standard isotropic monolayers. At 2 Hz pacing, both microscopic changes in cell orientation and ventricular tissue boundaries independently and synergistically increased the spatial dispersion of conduction velocity, but not the action potential duration. The realistic variations in intramural microstructure created unique spatial signatures in micro- and macroscopic impulse propagation within ventricular cross-section cultures. This novel in vitro model system is expected to help bridge the existing gap between experimental structure-function studies in standard cardiac monolayers and intact heart tissues. PMID:19413993

Full membrane spanning self-assembled monolayers as model systems for UHV-based studies of cell-penetrating peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Franz, Johannes; Graham, Daniel J.; Schmüser, Lars

2015-03-01

Biophysical studies of the interaction of peptides with model membranes provide a simple yet effective approach to understand the transport of peptides and peptide based drug carriers across the cell membrane. Therein, the authors discuss the use of self-assembled monolayers fabricated from the full membrane-spanning thiol (FMST) 3-((14-((4'-((5-methyl-1-phenyl-35-(phytanyl)oxy-6,9,12,15,18,21,24,27,30,33,37-undecaoxa-2,3-dithiahenpentacontan-51-yl)oxy)-[1,1'-biphenyl]-4-yl)oxy)tetradecyl)oxy)-2-(phytanyl)oxy glycerol for ultrahigh vacuum (UHV) based experiments. UHV-based methods such as electron spectroscopy and mass spectrometry can provide important information about how peptides bind and interact with membranes, especially with the hydrophobic core of a lipid bilayer. Moreover, near-edge x-ray absorption fine structure spectra and x-ray photoelectron spectroscopy (XPS) data showed thatmore » FMST forms UHV-stable and ordered films on gold. XPS and time of flight secondary ion mass spectrometry depth profiles indicated that a proline-rich amphipathic cell-penetrating peptide, known as sweet arrow peptide is located at the outer perimeter of the model membrane.« less

Establishment and characterization of immortalized gingival epithelial and fibroblastic cell lines for the development of organotypic cultures.

PubMed

Bao, Kai; Akguel, Baki; Bostanci, Nagihan

2014-01-01

In vitro studies using 3D co-cultures of gingival cells can resemble their in vivo counterparts much better than 2D models that typically only utilize monolayer cultures with short-living primary cells. However, the use of 3D gingival models is still limited through lack of appropriate cell lines. We aimed to establish immortalized cell line models of primary human gingival epithelium keratinocytes (HGEK) and gingival fibroblasts (GFB). Immortalized cell lines (HGEK-16 and GFB-16) were induced by E6 and E7 oncoproteins of human papillomavirus. In addition, 3D multilayered organotypic cultures were formed by embedding GFB-16 cells within a collagen (Col) matrix and seeding of HGEK-16 cells on the upper surfaces. Cell growth was analyzed in both immortalized cell lines and their parental primary cells. The expression levels of cell type-specific markers, i.e. cytokeratin (CK) 10, CK13, CK16, CK18, CK19 for HGEK-16 and Col I and Col II for GFB-16, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Expansion of the primary cultures was impeded at early passages, while the transformed immortalized cell lines could be expanded for more than 30 passages. In 3D cultures, immortalized HGEK formed a multilayer of epithelial cells. qRT-PCR showed that cell-specific marker expression in the 3D cultures was qualitatively and quantitatively closer to that in human gingival tissue than to monolayer cultures. These results indicate that immortalized gingival fibroblastic and epithelial cell lines can successfully form organotypic multilayered cultures and, therefore, may be useful tools for studying gingival tissue in vitro. © 2014 S. Karger AG, Basel.

Arrhenius parameter determination as a function of heating method and cellular microenvironment based on spatial cell viability analysis.

PubMed

Whitney, Jon; Carswell, William; Rylander, Nichole

2013-06-01

Predictions of injury in response to photothermal therapy in vivo are frequently made using Arrhenius parameters obtained from cell monolayers exposed to laser or water bath heating. However, the impact of different heating methods and cellular microenvironments on Arrhenius predictions has not been thoroughly investigated. This study determined the influence of heating method (water bath and laser irradiation) and cellular microenvironment (cell monolayers and tissue phantoms) on Arrhenius parameters and spatial viability. MDA-MB-231 cells seeded in monolayers and sodium alginate phantoms were heated with a water bath for 3-20 min at 46, 50, and 54 °C or laser irradiated (wavelength of 1064 nm and fluences of 40 W/cm(2) or 3.8 W/cm(2) for 0-4 min) in combination with photoabsorptive carbon nanohorns. Spatial viability was measured using digital image analysis of cells stained with calcein AM and propidium iodide and used to determine Arrhenius parameters. The influence of microenvironment and heating method on Arrhenius parameters and capability of parameters derived from more simplistic experimental conditions (e.g. water bath heating of monolayers) to predict more physiologically relevant systems (e.g. laser heating of phantoms) were assessed. Arrhenius predictions of the treated area (<1% viable) under-predicted the measured areas in photothermally treated phantoms by 23 mm(2) using water bath treated cell monolayer parameters, 26 mm(2) using water bath treated phantom parameters, 27 mm(2) using photothermally treated monolayer parameters, and 0.7 mm(2) using photothermally treated phantom parameters. Heating method and cellular microenvironment influenced Arrhenius parameters, with heating method having the greater impact.

Process for Making a Semiconductor Device with Barrier Film Formation Using a Metal Halide and Products Thereof

DTIC Science & Technology

1998-08-20

structure of the barrier film on an atomic level where the barrier film is comprised of a plurality of contiguous monolayers, while FIG. 7B shows...another embodiment where the barrier film is comprised of a plurality of i contiguous monolayers in which different monolayers thereof are formed... effusion cell, for example a barium fluoride, strontium fluoride or the like effusion cell, is provided at 32, and has a shutter 33. A 15 shutter 35

Monte Carlo N-Particle (MCNP) Modeling of the Cellular Dosimetry of 64Cu: Comparison with MIRDcell S Values and Implications for Studies of Its Cytotoxic Effects.

PubMed

Cai, Zhongli; Kwon, Yongkyu Luke; Reilly, Raymond M

2017-02-01

64 Cu emits positrons as well as β - particles and Auger and internal conversion electrons useful for radiotherapy. Our objective was to model the cellular dosimetry of 64 Cu under different geometries commonly used to study the cytotoxic effects of 64 Cu. Monte Carlo N-Particle (MCNP) was used to simulate the transport of all particles emitted by 64 Cu from the cell surface (CS), cytoplasm (Cy), or nucleus (N) of a single cell; monolayer in a well (radius = 0.32-1.74 cm); or a sphere (radius = 50-6,000 μm) of cells to calculate S values. The radius of the cell and N ranged from 5 to 12 μm and 2 to 11 μm, respectively. S values were obtained by MIRDcell for comparison. MCF7/HER2-18 cells were exposed in vitro to 64 Cu-labeled trastuzumab. The subcellular distribution of 64 Cu was measured by cell fractionation. The surviving fraction was determined in a clonogenic assay. The relative differences of MCNP versus MIRDcell self-dose S values (S self ) for 64 Cu ranged from -0.2% to 3.6% for N to N (S N←N ), 2.3% to 8.6% for Cy to N (S N←Cy ), and -12.0% to 7.3% for CS to N (S N←CS ). The relative differences of MCNP versus MIRDcell cross-dose S values were 25.8%-30.6% for a monolayer and 30%-34% for a sphere, respectively. The ratios of S N←N versus S N←Cy and S N←Cy versus S N←CS decreased with increasing ratio of the N of the cell versus radius of the cell and the size of the monolayer or sphere. The surviving fraction of MCF7 /: HER2-18 cells treated with 64 Cu-labeled trastuzumab (0.016-0.368 MBq/μg, 67 nM) for 18 h versus the absorbed dose followed a linear survival curve with α = 0.51 ± 0.05 Gy -1 and R 2 = 0.8838. This is significantly different from the linear quadratic survival curve of MCF7 /: HER2-18 cells exposed to γ-rays. MCNP- and MIRDcell-calculated S values agreed well. 64 Cu in the N increases the dose to the N in isolated single cells but has less effect in a cell monolayer or small cluster of cells simulating a micrometastasis, and little effect in a sphere analogous to a tumor xenograft compared with 64 Cu in the Cy or on the CS. The dose deposited by 64 Cu is less effective for cell killing than γ-rays. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Increased Na+/H+ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease

PubMed Central

Olteanu, Dragos; Liu, Xiaofen; Liu, Wen; Roper, Venus C.; Sharma, Neeraj; Yoder, Bradley K.; Satlin, Lisa M.; Schwiebert, Erik M.

2012-01-01

Pathophysiological anomalies in autosomal dominant and recessive forms of polycystic kidney disease (PKD) may derive from impaired function/formation of the apical central monocilium of ductal epithelia such as that seen in the Oak Ridge polycystic kidney or orpk (Ift88Tg737Rpw) mouse and its immortalized cell models for the renal collecting duct. According to a previous study, Na/H exchanger (NHE) activity may contribute to hyperabsorptive Na+ movement in cilium-deficient (“mutant”) cortical collecting duct principal cell monolayers derived from the orpk mice compared with cilium-competent (“rescued”) monolayers. To examine NHE activity, we measured intracellular pH (pHi) by fluorescence imaging with the pH-sensitive dye BCECF, and used a custom-designed perfusion chamber to control the apical and basolateral solutions independently. Both mutant and rescued monolayers exhibited basolateral Na+-dependent acid-base transporter activity in the nominal absence of CO2/HCO3−. However, only the mutant cells displayed appreciable apical Na+-induced pHi recoveries from NH4+ prepulse-induced acid loads. Similar results were obtained with isolated, perfused collecting ducts from orpk vs. wild-type mice. The pHi dependence of basolateral cariporide/HOE-694-sensitive NHE activity under our experimental conditions was similar in both mutant and rescued cells, and 3.5- to 4.5-fold greater than apical HOE-sensitive NHE activity in the mutant cells (pHi 6.23–6.68). Increased apical NHE activity correlated with increased apical NHE1 expression in the mutant cells, and increased apical localization in collecting ducts of kidney sections from orpk vs. control mice. A kidney-specific conditional cilium-knockout mouse produced a more acidic urine compared with wild-type littermates and became alkalotic by 28 days of age. This study provides the first description of altered NHE activity, and an associated acid-base anomaly in any form of PKD. PMID:22301060

In vitro cell and tissue models for studying host-microbe interactions: a review.

PubMed

Bermudez-Brito, Miriam; Plaza-Díaz, Julio; Fontana, Luis; Muñoz-Quezada, Sergio; Gil, Angel

2013-01-01

Ideally, cell models should resemble the in vivo conditions; however, in most in vitro experimental models, epithelial cells are cultivated as monolayers, in which the establishment of functional epithelial features is not achieved. To overcome this problem, co-culture experiments with probiotics, dendritic cells and intestinal epithelial cells and three-dimensional models attempt to reconcile the complex and dynamic interactions that exist in vivo between the intestinal epithelium and bacteria on the luminal side and between the epithelium and the underlying immune system on the basolateral side. Additional models include tissue explants, bioreactors and organoids. The present review details the in vitro models used to study host-microbe interactions and explores the new tools that may help in understanding the molecular mechanisms of these interactions.

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

PubMed Central

Deppert, W; Hanke, K; Henning, R

1980-01-01

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. Images PMID:6255189

A mechanism enhancing macromolecule transport through paracellular spaces induced by Poly-L-Arginine: Poly-L-Arginine induces the internalization of tight junction proteins via clathrin-mediated endocytosis.

PubMed

Yamaki, Tsutomu; Kamiya, Yusuke; Ohtake, Kazuo; Uchida, Masaki; Seki, Toshinobu; Ueda, Hideo; Kobayashi, Jun; Morimoto, Yasunori; Natsume, Hideshi

2014-09-01

Poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules by disappearance of tight junction (TJ) proteins from cell-cell junctions. However, the mechanism of the disappearance of TJ proteins in response to PLA has been unclear. In this study, we investigated the mechanism of disappearance of TJ proteins from cell-cell junctions after the application of PLA to Caco-2 cell monolayers. The membrane conductance (Gt), FITC-dextran (FD-4) permeability, and localization of TJ proteins were examined after the treatment of Caco-2 cell monolayers with PLA in the presence of various endocytosis inhibitors. In addition, the localization of endosome marker proteins was also observed. Clathrin-mediated endocytosis inhibitors suppressed the increase in Gt and Papp of FD-4 induced by PLA, and also significantly suppressed the disappearance of TJ proteins induced by PLA. Furthermore, occludin, one of the TJ proteins, colocalized with early endosome and recycling endosomes after the internalization of occludin induced by PLA, and then was recycled to the cell-cell junctions. PLA induced the transient internalization of TJ proteins in cell-cell junctions via clathrin-mediated endocytosis, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs).

PubMed

DeStefano, Jackson G; Xu, Zinnia S; Williams, Ashley J; Yimam, Nahom; Searson, Peter C

2017-08-04

The endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood-brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells. To assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm -2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood-brain barrier were performed. Human brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood-brain barrier markers. The characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood-brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation of tight junctions decreases cell motility and prevents any morphological transitions, (3) flow serves to increase the contact area between cells, resulting in very low cell displacement in the monolayer, (4) since tight junctions are already formed under static conditions, increasing the contact area between cells does not cause upregulation in protein and gene expression of BBB markers, and (5) the increase in contact area induced by flow makes barrier function more robust.

A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes.

PubMed

Mueller, A J; Tew, S R; Vasieva, O; Clegg, P D; Canty-Laird, E G

2016-09-27

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

Effects of dexamethasone on angiotensin II-induced changes of monolayer permeability and F-actin distribution in glomerular endothelial cells.

PubMed

Fang, Junyan; Wang, Miao; Zhang, Wei; Wang, Yingdeng

2013-11-01

The aim of this study was to investigate the changes in monolayer permeability and F-actin distribution caused by angiotensin II (Ang II)-induced injury in glomerular endothelial cells (GENCs) and the effects of dexamethasone on these changes. GENCs isolated and cultured from Wistar rats were used to examine the changes in monolayer permeability and F-actin distribution induced by Ang II. GENC permeability was evaluated by measuring the diffusion of biotin-conjugated bovine serum albumin (biotin-BSA) across a cell monolayer. The expression levels and distribution of F-actin were assessed by flow cytometry. The biotin-BSA concentrations were measured by capture enzyme-linked immunosorbent assay. Ang II at a concentration of 10 mg/l increased the permeability of the GENC monolayer at 6 h and 12 h (P<0.05 and P<0.01, respectively) and caused F-actin depolymerisation at 6 h and 12 h (P<0.01). The two effects attributed to Ang II were significantly inhibited by dexamethasone treatment (P<0.01). The increased permeability of the GENC monolayer induced by Ang II was significantly correlated with the depolymerisation of F-actin. Dexamethasone abrogated the Ang II-mediated damage to GENCs indicating that it may play an important role in protecting GENCs from injury.

Interaction of cholesterol with sphingomyelins and acyl-chain-matched phosphatidylcholines: a comparative study of the effect of the chain length.

PubMed Central

Ramstedt, B; Slotte, J P

1999-01-01

In this study we have synthesized sphingomyelins (SM) and phosphatidylcholines (PC) with amide-linked or sn-2 linked acyl chains with lengths from 14 to 24 carbons. The purpose was to examine how the chain length and degree of unsaturation affected the interaction of cholesterol with these phospholipids in model membrane systems. Monolayers of saturated SMs and PCs with acyl chain lengths above 14 carbons were condensed and displayed a high collapse pressure ( approximately 70 mN/m). Monolayers of N-14:0-SM and 1(16:0)-2(14:0)-PC had a much lower collapse pressure (58-60 mN/m) and monounsaturated SMs collapsed at approximately 50 mN/m. The relative interaction of cholesterol with these phospholipids was determined at 22 degreesC by measuring the rate of cholesterol desorption from mixed monolayers (50 mol % cholesterol; 20 mN/m) to beta-cyclodextrin in the subphase (1.7 mM). The rate of cholesterol desorption was lower from saturated SM monolayers than from chain-matched PC monolayers. In SM monolayers, the rate of cholesterol desorption was very slow for all N-linked chains, whereas for PC monolayers we could observe higher desorption rates from monolayers of longer PCs. These results show that cholesterol interacts favorably with SMs (low rate of desorption), whereas its interaction (or miscibility) with long chain PCs is weaker. Introduction of a single cis-unsaturation in the N-linked acyl chain of SMs led to faster rates of cholesterol desorption as compared with saturated SMs. The exception was monolayers of N-22:1-SM and N-24:1-SM from which cholesterol desorbed almost as slowly as from the corresponding saturated SM monolayers. The results of this study suggest that cholesterol is most likely capable of interacting with all physiologically relevant (including long-chain) SMs present in the plasma membrane of cells. PMID:9929492

Protection against free radicals (UVB irradiation) of a water-soluble enzymatic extract from rice bran. Study using human keratinocyte monolayer and reconstructed human epidermis.

PubMed

Santa-María, C; Revilla, E; Miramontes, E; Bautista, J; García-Martínez, A; Romero, E; Carballo, M; Parrado, J

2010-01-01

The antioxidant capacity of a water-soluble enzymatic extract from rice bran (EERB) has been tested in two cell models: keratinocyte monolayers and human reconstructed epidermis. Cells were incubated in culture medium in presence of different amounts of EERB and were UVB irradiated. Cell population assessment (MTT assay) and MDA (malonaldehyde) production were evaluated. The EERB did not induce cytotoxic effect for concentrations inferior or equal to 100 microg/mL. Human keratinocyte monolayers were protected of irradiation preventing 33% the lipid peroxidation process at concentration of 10 microg/ml of EEBR. In reconstructed human epidermis, 100 microg/mL decreased lipid peroxidation process by 44% (p<0.01) with regards to irradiated negative control. This effect was comparable to that of vitamin E at 600 microg/mL. Our data indicate that EERB is potentially able to efficiently counteract UVB-induced response. The EERB, diluted at 10% with water has very good skin compatibility. This product showed a sun protection factor of 4.8+/-0.3. Thus we can propose EERB as a useful natural standardized extract in skin photoprotection with promising applications in the field of dermatology. Copyright 2009 Elsevier Ltd. All rights reserved.

Transport properties of puerarin and effect of Radix Angelicae Dahuricae extract on the transport of puerarin in Caco-2 cell model.

PubMed

Liang, Xin-Li; Zhao, Li-Jun; Liao, Zheng-Gen; Zhao, Guo-Wei; Zhang, Jing; Chao, Yun-Chao; Yang, Ming; Yin, Rong-Li

2012-12-18

Angelicae Dahurica (Hoffm.)Benth.& Hook.f.ex Franch.&Sav combined with Pueraria labota (Willd.)Ohwi has been widely used as herb-pairs in traditional Chinese medicine (TCM) for utilization of antipyretic analgesic and anti-inflammatory drugs, and modern pharmacological studies have shown that application compatibility of the two drugs has the effects of cardiovascular disease treatment. The previous study has proved that Radix Angelicae Dahuricae extract could enhance the intestinal absorption of puerarin in Pueraria. But the underlying compatibility mechanism of the two herbs remains unknown. In this study we tried to further evaluate the improvement of Radix Angelicae Dahuricae extract on the puerarin using the Caco-2 cell model and explore the transport properties of puerarin through the above research to discuss the possible effect mechanism of Radix Angelicae Dahuricae extract on the transport of puerarin and the underlying compatibility mechanism of the two herbs. The aim of this work was to study the transport properties of puerarin in Radix Pueraria across Caco-2 cell membrane and to explore how the Radix Angelicae Dahuricae extract affected the transport of puerarin using the well-characterized, human-based intestinal Caco-2 cell model as a platform. The bidirectional transport, and the effects of time, drug concentration, pH, P-gp inhibitors (Verapamil, Cyclosporin A), MRP inhibitor (MK-571) and EDTA-Na(2) (tight junction modulator) on the absorption of puerarin were observed. Then the influence of extract of Radix Angelicae Dahuricae on the transport of puerarin was studied. Drug concentration was measured by HPLC and the apparent permeability coefficients (Papp) and apparent permeability ratio (PDR) were calculated. The results showed that the transport (Papp) of puerarin in Caco-2 cell monolayer model had time and concentration dependence, and the transport showed saturation characteristics with the time and concentration of puerarin to a certain degree. The Papp of puerarin transported on Caco-2 cell monolayer model was significantly changed when the specified inhibitors of P-gp were added to the model and the PDR decreased from 1.74 to 0.43. The absorption of puerarin was improved when combined with Radix Angelicae Dahuricae. The intestinal absorption of puerarin is by passive diffusion as the dominating process and active transportation was mediated by P-gp and MRP transporter in Caco-2 cell monolayer model, and Radix Angelicae Dahuricae could enhance the intestinal absorption of puerarin. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The Plaque-Antiserum Method: an Assay of Virus Infectivity and an Experimental Model of Virus Infection

PubMed Central

De Flora, Silvio

1974-01-01

Areas of cytopathic effect can be circumscribed in cell monolayers by adding antiserum to the liquid nutrient medium after adsorption of virus. This procedure represents a simple and reliable tool for the titration of virus infectivity and provides an experimental model for studying some aspects of virus infection. Images PMID:4364462

A novel form of epithelial wound healing of the embryonic epidermis.

PubMed

Armstrong, Margaret T; Turlo, Kirsten; Elges, Chris J; Dayton, Sarah M; Lee, Janet; Armstrong, Peter B

2006-08-01

The embryonic epidermis of amniotes is a two-cell layer sheet with a periderm positioned superficial to the basal cell layer which, itself, attaches apically to the basal surface of the periderm and basally to the basal lamina. The presence of the periderm is essential to maintain the basal layer as a two-dimensional monolayer. Wounds to the epidermis that remove selectively just the periderm are healed by a stacking of the basal layer cells that constitute the wound bed. Basal cell stacking involves the desertion of the basal lamina by many of the cells so as to increase their contact area with other basal layer cells. This suggests that a preferential adhesion to the planar basal lamina is not important for the monolayered organization of the basal layer but, instead, association with inner surface of the planar periderm is the principal process that maintains the basal layer as a monolayer. The conversion of the basal layer from monolayer to multilayer during wound healing diminishes its planar area, resulting in movement of the wound borders toward the center of the wound. This is a novel scenario for wound healing.

Microscale temperature and SAR measurements in cell monolayer models exposed to millimeter waves.

PubMed

Zhadobov, Maxim; Alekseev, Stanislav I; Sauleau, Ronan; Le Page, Yann; Le Dréan, Yves; Fesenko, Evgeny E

2017-01-01

Due to shallow penetration of millimeter waves (MMW) and convection in liquid medium surrounding cells, the problem of accurate assessment of local MMW heating in in vitro experiments remains unsolved. Conventional dosimetric MMW techniques, such as infrared imaging or fiber optic (FO) sensors, face several inherent limits. Here we propose a methodology for accurate local temperature measurement and subsequent specific absorption rate (SAR) retrieval using microscale thermocouples (TC). SAR was retrieved by fitting the measured initial temperature rise to the numerical solution of an equivalent thermal model. It was found that the accuracy of temperature measurement depends on thermosensor size, that is, the smaller TC, the more accurate the temperature measurement. SAR determined using TC with lead diameters of 25 and 75 μm demonstrated 98.5% and 80.4% match with computed SAR, respectively. However, both TC provided the same temperature rises in long run (> 10 min). FO probe failed to measure adequately local heating both for short and long exposures due to the relatively large size of the probe sensor (400 μm) and time constant (0.6 s). Calculated SAR in the cell monolayer was almost two times lower than that in the surrounding liquid. It was shown that the impact of the cell monolayer on heating due to its small thickness (5 to 10 μm) can be considered as negligible. Moreover, we demonstrated the possibility of accurate measurement of MMW-induced thermal pulses (up to 10 °C) using 25 μm TC. Bioelectromagnetics. 38:11-21, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability

PubMed Central

Khan, Niamat; Lenz, Christof; Binder, Lutz; Pantakani, Dasaradha Venkata Krishna; Asif, Abdul R.

2016-01-01

Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea. Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability. Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability. Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the MPA-mediated increase in TJ permeability and leak flux diarrhea in organ transplant patients. PMID:27104530

Establishment of a cell-based wound healing assay for bio-relevant testing of wound therapeutics.

PubMed

Planz, Viktoria; Wang, Jing; Windbergs, Maike

Predictive in vitro testing of novel wound therapeutics requires adequate cell-based bio-assays. Such assays represent an integral part during preclinical development as pre-step before entering in vivo studies. Simple "scratch tests" based on defected skin cell monolayers exist, however these can solely be used for testing liquids, as cell monolayer destruction and excessive hydration limit their applicability for (semi-)solid systems like wound dressings. In this context, a cell-based wound healing assay is introduced for rapid and predictive testing of wound therapeutics independent of their physical state in a bio-relevant environment. A novel wound healing assay was established for bio-relevant and predictive testing of (semi-) solid wound therapeutics. The assay allows for physiologically relevant hydration of the tested wound therapeutics at the air-liquid interface and their removal without cell monolayer disruption. In a proof-of-concept study, the applicability and discriminative power could be demonstrated by examining unloaded and drug-loaded wound dressings with two different established wound healing actives (dexpanthenol and metyrapone) and their effect on skin cell behavior. The influence of the released drug on the cells´ healing behavior could successfully be monitored over time. Wound size assessment after 96h resulted in an eight fold smaller wound area for drug treated models compared to the ones treated with unloaded fibers and non-treated wounds. This assay provides valuable first insights towards the establishment of a valid screening and evaluation tool for preclinical wound therapeutic development from liquid to (semi-)solid systems to improve predictability in a simple, yet standardized way. Copyright © 2017 Elsevier Inc. All rights reserved.

In Vitro Effects of Preserved and Unpreserved Anti-Allergic Drugs on Human Corneal Epithelial Cells

PubMed Central

Calvo, Patricia; Ropero, Inés; Pintor, Jesús

2014-01-01

Abstract Purpose: Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Methods: Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. Results: The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. Conclusions: The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures. PMID:25100331

Human endothelial cells hollow fiber membrane bioreactor as a model of the blood vessel for in vitro studies.

PubMed

Ciechanowska, Anna; Ladyzynski, Piotr; Hoser, Grazyna; Sabalinska, Stanislawa; Kawiak, Jerzy; Foltynski, Piotr; Wojciechowski, Cezary; Chwojnowski, Andrzej

2016-09-01

Human endothelial cells are used in experimental models for studying in vitro pathophysiological mechanisms of different diseases. We developed an original bioreactor, which can simulate human blood vessel, with capillary polysulfone membranes covered with the human umbilical vein endothelial cells (HUVECs) and we characterized its properties. The elaborated cell seeding and culturing procedures ensured formation of a confluent cell monolayer on the inside surface of capillaries within 24 h of culturing under the shear stress of 6.6 dyn/cm(2). The optimal density of cells to be seeded was 60,000 cells/cm(2). Labeling HUVECs with carboxyfluorescein succinimidyl ester (CFSE) did not influence cells' metabolism. Flow cytometry-based analysis of HUVECs stained with CFSE demonstrated that in a presence of the shear stress cells' proliferation was much inhibited (after 72 h proliferation index was equal to 1.9 and 6.2 for cultures with and without shear stress, respectively) and the monolayer was formed mainly due to migration and spreading of cells that were physiologically elongated in a direction of the flow. Monitoring of cells' metabolism showed that HUVECs cultured in a presence of the shear stress preferred anaerobic metabolism and they consumed 1.5 times more glucose and produced 2.3 times more lactate than the cells cultured under static conditions. Daily von Willebrand factor production by HUVECs was near 2 times higher in a presence of the shear stress. The developed model can be used for at least 3 days in target studies under conditions mimicking the in vivo state more closely than the static HUVEC cultures.

Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro.

PubMed

Mießler, Katharina S; Markov, Alexander G; Amasheh, Salah

2018-01-01

During lactation, accumulation of milk in mammary glands (MG) causes hydrostatic pressure (HP) and concentration of bioactive compounds. Previously, a changed expression of tight junction (TJ) proteins was observed in mice MGs by accumulation of milk, in vivo. The TJ primarily determines the integrity of the MG epithelium. The present study questioned whether HP alone can affect the TJ in a mammary epithelial cell model, in vitro. Therefore, monolayers of HC11, a mammary epithelial cell line, were mounted into modified Ussing chambers and incubated with 10 kPa bilateral HP for 4 h. Short circuit current and transepithelial resistance were recorded and compared to controls, and TJ proteins were analyzed by Western blotting and immunofluorescent staining. In our first approach HC11 cells could withstand the pressure incubation and a downregulation of occludin was observed. In a second approach, using prolactin- and dexamethasone-induced cells, a decrease of short circuit current was observed, beginning after 2 h of incubation. With the addition of 1 mM barium chloride to the bathing solution the decrease could be blocked temporarily. On molecular level an upregulation of ZO-1 could be observed in hormone-induced cells, which was downregulated after the incubation with barium chloride. In conclusion, bilateral HP incubation affects mammary epithelial monolayers, in vitro. Both, the reduction of short circuit current and the change in TJ proteins may be interpreted as physiological requirements for lactation. Copyright © 2017 Elsevier Inc. All rights reserved.

A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

PubMed

Sabra, Georges; Vermette, Patrick

2013-02-01

The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered. Copyright © 2012 Wiley Periodicals, Inc.

In situ XAS of Pt monolayer model fuel cell catalysts: balance of na-nostructure and bimetallic interactions

NASA Astrophysics Data System (ADS)

Friebel, Daniel; Viswanathan, Venkat; Larsen, Ask; Miller, Daniel J.; Ogasawara, Hirohito; Anniyev, Toyli; O'Grady, Christopher P.; Nørskov, Jens; Nilsson, Anders

2012-02-01

The mechanism of the electrochemical oxygen reduction reaction (ORR) has been well understood based on DFT calculations, but there has been a lack of supporting experimental data, due to the difficulties of probing the electrocatalyst surface in situ. Our new approach using Pt monolayer model catalysts provides true surface sensitivity for - originally bulk sensitive - x-ray absorption spectroscopy (XAS) and, owing to the high resolution of the Bragg analyzer at SSRL beamline 6-2, allows for in situ detection of chemisorbed O and OH, whose stability can be used as a descriptor in predicting the activity of new ORR catalyst materials. Our ability to control the growth mode in the Pt/Rh(111) model system allows us to generate Pt nanostructures with highly different O affinities from identical starting materials.

Low-level stretching accelerates cell migration into a gap.

PubMed

Toume, Samer; Gefen, Amit; Weihs, Daphne

2017-08-01

We observed that radially stretching cell monolayers at a low level (3%) increases the rate at which they migrate to close a gap formed by in vitro injury. Wound healing has been shown to accelerate in vivo when deformations are topically applied, for example, by negative pressure wound therapy. However, the direct effect of deformations on cell migration during gap closure is still unknown. Thus, we have evaluated the effect of radially applied, sustained (static) tensile strain on the kinematics of en mass cell migration. Monolayers of murine fibroblasts were cultured on stretchable, linear-elastic substrates that were subjected to different tensile strains, using a custom-designed three-dimensionally printed stretching apparatus. Immediately following stretching, the monolayer was 'wounded' at its centre, and cell migration during gap closure was monitored and quantitatively evaluated. We observed a significant increase in normalised migration rates and a reduction of gap closure time with 3% stretching, relative to unstretched controls or 6% stretch. Interestingly, the initial gap area was linearly correlated with the maximum migration rate, especially when stretching was applied. Therefore, small deformations applied to cell monolayers during gap closure enhance en mass cell migration associated with wound healing and can be used to fine-tune treatment protocols. © 2016 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Effect of Foods and β-Cyclodextrin on the Bioaccessibility and the Uptake by Caco-2 Cells of Hydroxytyrosol from Either a Pure Standard or Alperujo.

PubMed

Malapert, Aurélia; Tomao, Valérie; Dangles, Olivier; Reboul, Emmanuelle

2018-05-09

Hydroxytyrosol bioaccessibility and absorption by the intestinal cells were studied using an in vitro digestion model and Caco-2 TC7 monolayers cells in culture in the presence and absence of β-cyclodextrin and foods. Hydroxytyrosol was either provided as a pure standard or in an alperujo powder. The presence of foods significantly decreased hydroxytyrosol bioaccessibility and absorption (-20 and -10%, respectively), while β-cyclodextrin had no effect. Moreover, the presence of other compounds from alperujo in the intestinal compartment reduced hydroxytyrosol absorption by Caco-2 cells compared to pure standard (-60%). The final bioavailability of hydroxytyrosol, defined as its quantity at the basolateral side of cultured cell monolayers compared to the initial amount in the test meal, was 6.9 ± 0.4, 31.1 ± 1.1, and 40.9 ± 1.5% when hydroxytyrosol was from alperujo or a standard administered with or without food, respectively. Our results show that conversely to foods, β-cyclodextrin does not alter hydroxytyrosol bioavailability.

Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

NASA Technical Reports Server (NTRS)

Marquette, Michele L.; Sognier, Marguerite A.

2013-01-01

An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

Neuron-Glia Adhesion is Inhibited by Antibodies to Neural Determinants

NASA Astrophysics Data System (ADS)

Grumet, M.; Rutishauser, U.; Edelman, G. M.

1983-10-01

Suspensions of embryonic chick neuronal cells adhered to monolayers of glial cells, but few neurons bound to control monolayers of fibroblastic cells from meninges or skin. Neuronal cell-glial cell adhesion was inhibited by prior incubation of the neurons with Fab' fragments of antibodies to neuronal membranes. In contrast, antibodies to the neural cell adhesion molecule (N-CAM) did not inhibit the binding. These results suggest that a specific adhesive mechanism between neurons and glial cells exists and that it is mediated by CAM's that differ from those so far identified.

Neutrophil-endothelial cell interactions on endothelial monolayers grown on micropore filters.

PubMed Central

Taylor, R F; Price, T H; Schwartz, S M; Dale, D C

1981-01-01

We have developed a technique for growing endothelial monolayers on micropore filters. These monolayers demonstrate confluence by phase and electron microscopy and provide a functional barrier to passage of radiolabeled albumin. Neutrophils readily penetrate the monolayer in response to chemotaxin, whereas there is little movement in the absence of chemotaxin. This system offers unique advantages over available chemotaxis assays and may have wider applications in the study of endothelial function. Images PMID:7007441

Proliferation of pulmonary endothelial cells: time-lapse cinematography of growth to confluence and restitution of monolayer after wounding.

PubMed

Ryan, U S; Absher, M; Olazabal, B M; Brown, L M; Ryan, J W

1982-01-01

A fundamental characteristic of vascular endothelium is that it exists as a monolayer, a condition that must be met in both vascular growth and repair. Maintenance of the monolayer is important both for the exchange of nutrients and for interactions between blood solutes and endothelial enzymes and transport systems. We have used time-lapse cinematography to compare proliferative behavior of bovine pulmonary endothelial cells in (1) establishment of a monolayer from a low-density seed (7.5 X 10(4) cells in a 60 mm dish) and (2) restitution of a confluent monolayer (approx. 2.9 x 10(6) cells in a 60 mm dish) following a mechanical wound (removal of cells from an area 5 x 15 mm by scraping). Culture 2 was not refed after wounding. In culture 2, approx. 30% of the cells accounted for repopulation (confluence in 40 hr). In culture 1, all cells entered into division. Participating cells of culture 2 began division immediately (69 divisions/filmed area in 10 hr, vs. four divisions in culture 1). Interdivision times (IDT) were longer and relatively constant in culture 1 until near confluence; none were less than 10 h, whereas in 2, 24% of the IDT's were less than or equal to 10 hr. Remarkably, IDTs of culture 2 decreased steadily until confluence was re-established. Cell migration in culture 1 was multidirectional while direction of migration in culture 2 was always into the wound area. Mean migration rate (MIG) in culture 2 was related to the site of origin of the cells, those dividing farthest from the unwounded area had fastest MIGs. Neither culture formed more than a single layer of cells. Although the cell kinetics of cultures 1 and 2 differed, the same goal, confluence, was achieved in either case.

Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

NASA Technical Reports Server (NTRS)

Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

2017-01-01

Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

Serratia marcescens is injurious to intestinal epithelial cells

PubMed Central

Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

2014-01-01

Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens. PMID:25426769

Serratia marcescens is injurious to intestinal epithelial cells.

PubMed

Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

2014-01-01

Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

Effect of Microcystin-LR on Cultured Rat Endothelial Cells

DTIC Science & Technology

1990-02-26

protection, silymarin , and dithioerythritol 19. ABSTRACT (Continue on reverse if necessary and identify by block number) Primary cultureLs of adult rat...8217 22.g-Ldenine nucleotides, and a small reduction of cell density in endothelial cell monolayers._ Silymarin at 0.2 mM but not(dithioerythritol;at 2.5...monolayers. Silymarin at 0.2 mM but not dithioerythritol at 2.5 mM, partially protected changes in endothelial cells produced by supernatants derived

Clonal differences in generation times of GPK epithelial cells in monolayer culture.

PubMed

Riley, P A; Hola, M

1980-01-01

Pedigrees of cells in eight clones of guinea pig keratocyte (GPK) cells in monolayer culture were analyzed from a time-lapse film. The generation times and the position in the field of observation were recorded up to the sixth generation when the cultures were still subconfluent. Statistical analysis of the results indicates that the position in the culture has less significance than the clonal origin of the cell in determining the interval between successive mitoses.

Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures.

PubMed

Gebhard, C; Gabriel, C; Walter, I

2016-06-01

Spheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold-free three-dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19-day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14- and 19-day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki-67 immunoreactivity showed an even distribution in two-dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis-associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold-free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell-cell and cell-matrix interactions. © 2015 The Authors. Anatomia, Histologia, Embryologia Published by Blackwell Verlag GmbH.

Permeability of rhynchophylline across human intestinal cell in vitro

PubMed Central

Ma, Bo; Wang, Jing; Sun, Jing; Li, Ming; Xu, Huibo; Sun, Guibo; Sun, Xiaobo

2014-01-01

Rhynchophylline (Rhy) is the major component of Uncaria species, which is used in Chinese traditional medicine for the treatment of central nervous system disorders. However, its oral bioavailability has not been known. This study aims to investigate the intestinal permeability and related mechanisms of Rhy using cultured human epithelial Caco-2 cells. The cytotoxicity of Rhy on Caco-2 cells was evaluated with MTT assay. The effect of Rhy on the integrity of Caco-2 cell monolayer was assayed with transepithelial electrical resistance. The permeability of Rhy across cell monolayer was assayed by measuring Rhy quantity in received side with HPLC. The effect of Rhy on the expression of P-glycoprotein and MDR1 was detected with Western blot and flow cytometry, respectively. In the concentration of Rhy, which did not produce toxicity on cell viability and integrity of Caco-2 cell monolayer, Rhy crossed the monolayer with velocity 2.76~5.57×10^-6 cm/sec and 10.68~15.66×10^-6 cm/sec from apical to basolateral side and from basolateral to apical side, respectively. The permeability of Rhy was increased by verapamil, a P-glycoprotein inhibitor, or rhodamine123, a P-glycoprotein substrate. Rhy revealed an induction effect on P-glycoprotein expression in Caco-2 cells. These results demonstrate the low permeability of Rhy in intro, and suggest that P-glycoprotein may underlie the mechanism. PMID:24966905

From single cells to tissue architecture-a bottom-up approach to modelling the spatio-temporal organisation of complex multi-cellular systems.

PubMed

Galle, J; Hoffmann, M; Aust, G

2009-01-01

Collective phenomena in multi-cellular assemblies can be approached on different levels of complexity. Here, we discuss a number of mathematical models which consider the dynamics of each individual cell, so-called agent-based or individual-based models (IBMs). As a special feature, these models allow to account for intracellular decision processes which are triggered by biomechanical cell-cell or cell-matrix interactions. We discuss their impact on the growth and homeostasis of multi-cellular systems as simulated by lattice-free models. Our results demonstrate that cell polarisation subsequent to cell-cell contact formation can be a source of stability in epithelial monolayers. Stroma contact-dependent regulation of tumour cell proliferation and migration is shown to result in invasion dynamics in accordance with the migrating cancer stem cell hypothesis. However, we demonstrate that different regulation mechanisms can equally well comply with present experimental results. Thus, we suggest a panel of experimental studies for the in-depth validation of the model assumptions.

Proteomic approach toward molecular backgrounds of drug resistance of osteosarcoma cells in spheroid culture system.

PubMed

Arai, Kazuya; Sakamoto, Ruriko; Kubota, Daisuke; Kondo, Tadashi

2013-08-01

Chemoresistance is one of the most critical prognostic factors in osteosarcoma, and elucidation of the molecular backgrounds of chemoresistance may lead to better clinical outcomes. Spheroid cells resemble in vivo cells and are considered an in vitro model for the drug discovery. We found that spheroid cells displayed more chemoresistance than conventional monolayer cells across 11 osteosarcoma cell lines. To investigate the molecular mechanisms underlying the resistance to chemotherapy, we examined the proteomic differences between the monolayer and spheroid cells by 2D-DIGE. Of the 4762 protein species observed, we further investigated 435 species with annotated mass spectra in the public proteome database, Genome Medicine Database of Japan Proteomics. Among the 435 protein species, we found that 17 species exhibited expression level differences when the cells formed spheroids in more than five cell lines and four species out of these 17 were associated with spheroid-formation associated resistance to doxorubicin. We confirmed the upregulation of cathepsin D in spheroid cells by western blotting. Cathepsin D has been implicated in chemoresistance of various malignancies but has not previously been implemented in osteosarcoma. Our study suggested that the spheroid system may be a useful tool to reveal the molecular backgrounds of chemoresistance in osteosarcoma. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Water filtration rate and infiltration/accumulation of low density lipoproteins in 3 different modes of endothelial/smooth muscle cell co-cultures.

PubMed

Ding, ZuFeng; Fan, YuBo; Deng, XiaoYan

2009-11-01

Using different endothelial/smooth muscle cell co-culture modes to simulate the intimal structure of blood vessels, the water filtration rate and the infiltration/accumulation of LDL of the cultured cell layers were studied. The three cell culture modes of the study were: (i) The endothelial cell monolayer (EC/Phi); (ii) endothelial cells directly co-cultured on the smooth muscle cell monolayer (EC-SMC); (iii) endothelial cells and smooth muscle cells cultured on different sides of a Millicell-CM membrane (EC/SMC). It was found that under the same condition, the water filtration rate was the lowest for the EC/SMC mode and the highest for the EC/Phi mode, while the infiltration/accumulation of DiI-LDLs was the lowest in the EC/Phi mode and the highest in the EC-SMC mode. It was also found that DiI-LDL infiltration/accumulation in the cultured cell layers increased with the increasing water filtration rate. The results from the in vitro model study therefore suggest that the infiltration/accumulation of the lipids within the arterial wall is positively correlated with concentration polarization of atherogenic lipids, and the integrity of the endothelium plays an important role in the penetration and accumulation of atherogenic lipids in blood vessel walls.

Isolation of High-Molecular-Weight DNA from Monolayer Cultures of Mammalian Cells Using Proteinase K and Phenol.

PubMed

Green, Michael R; Sambrook, Joseph

2017-07-05

This procedure is the method of choice for purification of mammalian genomic DNA from monolayer cultures when large amounts of DNA are required, for example, for Southern blotting. Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 × 10 7 cultured aneuploid cells (e.g., HeLa cells). © 2017 Cold Spring Harbor Laboratory Press.

Intra-hydrogel culture prevents transformation of mesenchymal stem cells induced by monolayer expansion.

PubMed

Jiang, Tongmeng; Liu, Junting; Ouyang, Yiqiang; Wu, Huayu; Zheng, Li; Zhao, Jinmin; Zhang, Xingdong

2018-05-01

In this study, we report that the intra-hydrogel culture system mitigates the transformation of mesenchymal stem cells (MSCs) induced by two-dimensional (2D) expansion. MSCs expanded in monolayer culture prior to encapsulation in collagen hydrogels (group eMSCs-CH) featured impaired stemness in chondrogenesis, comparing with the freshly isolated bone marrow mononuclear cells seeded directly in collagen hydrogels (group fMSCs-CH). The molecular mechanism of the in vitro expansion-triggered damage to MSCs was detected through genome-wide microarray analysis. Results indicated that pathways such as proteoglycans in cancer and pathways in cancer expansion were highly enriched in eMSCs-CH. And multiple up-regulated oncoma-associated genes were verified in eMSCs-CH compared with fMSCs-CH, indicating that expansion in vitro triggered cellular transformation was associated with signaling pathways related to tumorigenicity. Besides, focal adhesion (FA) and mitogen-activated protein kinase (MAPK) signaling pathways were also involved in in vitro expansion, indicating restructuring of the cell architecture. Thus, monolayer expansion in vitro may contribute to vulnerability of MSCs through the regulation of FA and MAPK. This study indicates that intra-hydrogel culture can mitigate the monolayer expansion induced transformation of MSCs and maintain the uniformity of the stem cells, which is a viable in vitro culture system for stem cell therapy.

The bovine placenta in vivo and in vitro.

PubMed

Haeger, J-D; Hambruch, N; Pfarrer, C

2016-07-01

The gross anatomic features (cotyledonary type) and histologic classification (synepitheliochorial) of the bovine placenta have been known for many years. Thorough ultrastructural analysis as well as a variety of descriptive studies dealing with the localization of cytoskeletal filaments, extracellular matrix, growth factor systems, steroid hormone receptors, and major histocompatibility complex have contributed further significant knowledge. However, this knowledge was not sufficient to solve clinical placenta-based problems, such as retained fetal membranes. Owing to the complexity of the fetomaternal interface in vitro, culture systems have been developed. As trophoblast giant cells (TGC) are thought to be key players in the cattle placenta, most cell culture models attempt to overcome the pitfall of losing the entire TGC population in vitro. Nevertheless, distinct cell line-based in vitro systems such as cell monolayers or 3-dimensional (co-culture) spheroids were generated for the fetal (trophoblast) and maternal (uterine epithelium) placental compartments. Monolayers have been used to study for example, growth factor or hormonal signaling and TGC formation, whereas spheroids served as models for, for example, trophoblast attachment, uterine epithelium depolarization, and also TGC formation. In the future, the use of more improved culture models might lead to better treatments of retained fetal membranes and increased prevention of embryonic loss. In addition, the in vitro models could shed more light on the mechanisms of the differentiation of uninucleate trophoblast into TGC. Copyright © 2016 Elsevier Inc. All rights reserved.

Inverse tissue mechanics of cell monolayer expansion.

PubMed

Kondo, Yohei; Aoki, Kazuhiro; Ishii, Shin

2018-03-01

Living tissues undergo deformation during morphogenesis. In this process, cells generate mechanical forces that drive the coordinated cell motion and shape changes. Recent advances in experimental and theoretical techniques have enabled in situ measurement of the mechanical forces, but the characterization of mechanical properties that determine how these forces quantitatively affect tissue deformation remains challenging, and this represents a major obstacle for the complete understanding of morphogenesis. Here, we proposed a non-invasive reverse-engineering approach for the estimation of the mechanical properties, by combining tissue mechanics modeling and statistical machine learning. Our strategy is to model the tissue as a continuum mechanical system and to use passive observations of spontaneous tissue deformation and force fields to statistically estimate the model parameters. This method was applied to the analysis of the collective migration of Madin-Darby canine kidney cells, and the tissue flow and force were simultaneously observed by the phase contrast imaging and traction force microscopy. We found that our monolayer elastic model, whose elastic moduli were reverse-engineered, enabled a long-term forecast of the traction force fields when given the tissue flow fields, indicating that the elasticity contributes to the evolution of the tissue stress. Furthermore, we investigated the tissues in which myosin was inhibited by blebbistatin treatment, and observed a several-fold reduction in the elastic moduli. The obtained results validate our framework, which paves the way to the estimation of mechanical properties of living tissues during morphogenesis.

Acanthamoeba culbertsoni isolated from a clinical case with intraocular dissemination: Structure and in vitro analysis of the interaction with hamster cornea and MDCK epithelial cell monolayers.

PubMed

González-Robles, Arturo; Omaña-Molina, Maritza; Salazar-Villatoro, Lizbeth; Flores-Maldonado, Catalina; Lorenzo-Morales, Jacob; Reyes-Batlle, María; Arnalich-Montiel, Francisco; Martínez-Palomo, Adolfo

2017-12-01

Acanthamoeba culbertsoni trophozoites, previously isolated from a human keratitis case with severe intraocular damage, were maintained in axenic culture. Co-incubation of amoebae with MDCK cell monolayers demonstrated an apparent preference of the amoebae to introduce themselves between the cells. The trophozoites appeared to cross the cell monolayer through the tight junctions, which resulted in decreased trans-epithelial resistance (TER) measurements. Unexpectedly, after co-incubation of amoebae with hamster corneas, we observed that the trophozoites were able to cross the different cell layers and reach the corneal stroma after only 12 h of interaction, in contrast to other Acanthamoeba species. These observations suggest that this A. culbertsoni isolate is particularly pathogenic. Further research with diverse methodologies needs to be performed to explain the unique behavior of this Acanthamoeba strain. Copyright © 2017 Elsevier Inc. All rights reserved.

Gene expression analysis of a porcine hepatocyte/bile duct in vitro differentiaion model

USDA-ARS?s Scientific Manuscript database

A serum-free, feeder-cell-dependent, inductive differentiation culture system of porcine hepatocytes and bile ductules was analyzed for differential gene expression on a porcine genome microarray. Primary cultures of baby pig hepatocytes (BPH) were matured in culture as a monolayer of hepatocytes w...

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

PubMed

Cerino, Giulia; Gaudiello, Emanuele; Grussenmeyer, Thomas; Melly, Ludovic; Massai, Diana; Banfi, Andrea; Martin, Ivan; Eckstein, Friedrich; Grapow, Martin; Marsano, Anna

2016-01-01

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment. © 2015 Wiley Periodicals, Inc.

Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation.

PubMed

Shen, Francis H; Werner, Brian C; Liang, Haixiang; Shang, Hulan; Yang, Ning; Li, Xudong; Shimer, Adam L; Balian, Gary; Katz, Adam J

2013-01-01

Healthy mammalian cells in normal tissues are organized in complex three-dimensional (3D) networks that display nutrient and signaling gradients. Conventional techniques that grow cells in a 2D monolayer fail to reproduce the environment that is observed in vivo. In recent years, 3D culture systems have been used to mimic tumor microenvironments in cancer research and to emulate embryogenesis in stem cell cultures. However, there have been no studies exploring the ability for adipose-derived stromal (ADS) cells in a 3D culture system to undergo osteogenic differentiation. To characterize and investigate the in vitro and in vivo potential for human ADS cells in a novel 3D culture system to undergo osteogenic differentiation. Basic science and laboratory study. Human ADS cells were isolated and prepared as either a 2D monolayer or 3D multicellular aggregates (MAs). Multicellular aggregates were formed using the hanging droplet technique. Cells were treated in osteogenic medium in vitro, and cellular differentiation was investigated using gene expression, histology, and microCT at 1-, 2-, and 4-week time points. In vivo investigation involved creating a muscle pouch by developing the avascular muscular interval in the vastus lateralis of male athymic rats. Specimens were then pretreated with osteogenic medium and surgically implanted as (1) carrier (Matrigel) alone (control), (2) carrier with human ADS cells in monolayer, or (3) human ADS cells as MAs. In vivo evidence of osteogenic differentiation was evaluated with micro computed tomography and histologic sectioning at a 2-week time point. Human ADS cells cultured by the hanging droplet technique successfully formed MAs at the air-fluid interface. Adipose-derived stromal cells cultured in monolayer or as 3D MAs retain their ability to self-replicate and undergo multilineage differentiation as confirmed by increased runx2/Cbfa2, ALP, and OCN and increased matrix mineralization on histologic sectioning. Multicellular aggregate cells expressed increased differentiation potential and extracellular matrix production over the same human ADS cells cultured in monolayer. Furthermore, MAs reseeded onto monolayer retained their stem cell capabilities. When implanted in vivo, significantly greater bone volume and extracellular matrix were present in the implanted specimens of MAs confirmed on both microCT and histological sectioning. This is the first study to investigate the capability of human ADS cells in a 3D culture system to undergo osteogenic differentiation. The results confirm that MAs maintain their stem cell characteristics. Compared with analogous cells in monolayer culture, the human ADS cells as MAs exhibit elevated levels of osteogenic differentiation and increased matrix mineralization. Furthermore, the creation of uniform spheroids allows for improved handling and manipulation during transplantation. These findings strongly support the concept that 3D culture systems remain not only a viable option for stem cell culture but also possibly a more attractive alternative to traditional culture techniques to improve the osteogenic potential of human adipose stem cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Advanced Cell Culture Techniques for Cancer Drug Discovery

PubMed Central

Lovitt, Carrie J.; Shelper, Todd B.; Avery, Vicky M.

2014-01-01

Human cancer cell lines are an integral part of drug discovery practices. However, modeling the complexity of cancer utilizing these cell lines on standard plastic substrata, does not accurately represent the tumor microenvironment. Research into developing advanced tumor cell culture models in a three-dimensional (3D) architecture that more prescisely characterizes the disease state have been undertaken by a number of laboratories around the world. These 3D cell culture models are particularly beneficial for investigating mechanistic processes and drug resistance in tumor cells. In addition, a range of molecular mechanisms deconstructed by studying cancer cells in 3D models suggest that tumor cells cultured in two-dimensional monolayer conditions do not respond to cancer therapeutics/compounds in a similar manner. Recent studies have demonstrated the potential of utilizing 3D cell culture models in drug discovery programs; however, it is evident that further research is required for the development of more complex models that incorporate the majority of the cellular and physical properties of a tumor. PMID:24887773

Advanced cell culture techniques for cancer drug discovery.

PubMed

Lovitt, Carrie J; Shelper, Todd B; Avery, Vicky M

2014-05-30

Human cancer cell lines are an integral part of drug discovery practices. However, modeling the complexity of cancer utilizing these cell lines on standard plastic substrata, does not accurately represent the tumor microenvironment. Research into developing advanced tumor cell culture models in a three-dimensional (3D) architecture that more prescisely characterizes the disease state have been undertaken by a number of laboratories around the world. These 3D cell culture models are particularly beneficial for investigating mechanistic processes and drug resistance in tumor cells. In addition, a range of molecular mechanisms deconstructed by studying cancer cells in 3D models suggest that tumor cells cultured in two-dimensional monolayer conditions do not respond to cancer therapeutics/compounds in a similar manner. Recent studies have demonstrated the potential of utilizing 3D cell culture models in drug discovery programs; however, it is evident that further research is required for the development of more complex models that incorporate the majority of the cellular and physical properties of a tumor.

Human brain microvascular endothelial cells resist elongation due to shear stress.

PubMed

Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

2015-05-01

Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier. Copyright © 2015 Elsevier Inc. All rights reserved.

[The relationship of the saturation density of multilayer cell cultures to their mass exchange with the medium].

PubMed

Akatov, V S; Lavrovskaia, V P

1991-01-01

Chinese hamster fibroblasts (CHF) and NIH 3T3 cells were cultured on a glass substrate at different distances from the porous membrane separating the cells from the perfusing medium. It is shown that with perfusion of medium above the membrane there is no movement of the medium near the cells. In both the types of culture, the cells grow in multilayers, however the multilayer character of growth in CHF is more pronounced than in NIH 3T3 cells. The saturation density of the cultures depends on the cell-membrane separation, and at separations of no more than 0.2 mm exceeds the saturation density in the monolayer by 8-10 fold. The dependences of the saturation density on separation are different for CHE and NIH 3T3 cells, indicating qualitative differences in the inhibition of cell growth in monolayers between these cultures. The results obtained indicate that the inhibition of cell growth in monolayer is due to mass exchange limitations, rather than to intercellular contact interactions.

Phosphorylation of the Tight Junction Protein Occludin Regulates Epithelial Monolayer Proliferation and Maturation

NASA Astrophysics Data System (ADS)

Bolinger, Mark Thomas

Barriers against the external environment are crucial for sustaining life in multicellular organisms, and form following convergent growth and development of cell-cell junctions. At least four types of epithelial cell-cell junctions exist, the most apical of which is known as the tight junction (TJ). A specific transmembrane protein known as occludin is highly phosphorylated on its C-terminal coiled-coil, and certain sites have been found to regulate specific aspects of TJ function, including the response to certain cytokines. Previously, our lab discovered a novel phosphosite at serine 471 that is located at a contact site with an important central organizer of the TJ, zonula occludens-1. Phosphoinhibitory, serine to alanine (S471A) occludin point mutant MDCK cell lines demonstrate that S471A monolayers are poorly organized compared to WT occludin (WT Occ) or phosphomimetic, serine to aspartic acid (S471D) lines. Additionally, S471A monolayers are composed of fewer, larger cells than controls, and exhibit proliferative arrest almost immediately following confluency, in contrast to control lines, which go through at least one additional round of proliferation. This phenotype can be recapitulated with a cell cycle inhibitor, demonstrating that confluent proliferation or cell packing is necessary for barrier maturation. G-protein coupled receptor kinase (GRK) was confirmed to be an S471 kinase by inhibitor experiments from a bioinformatically compiled candidate kinase list, and GRK inhibitors were able to recapitulate the phenotype of S471A lines. Finally, S471A expression perturbed purified coiled-coil stability as determined by NMR. Modeling of inter-coil interactions identified several possible hydrogen bonds that differ between the phosphorylated and non-phosphorylated forms. Expression of S471N (asparagine) transgenic occludin in vitro demonstrated highly organized border organization despite the lack of a negative charge at the S471 position. This result suggests that the border organization of p-S471 is not due to the negative charge at S471, and may be the result of differential intra-coil hydrogen bonding. In conclusion, cell packing is necessary for barrier maturation, and is regulated by the novel phosphosite, occludin S471. S471 is an important contributor to confluent proliferation, monolayer maturation, and barrier resistance, and plays a role in the barrier regulatory function of occludin.

Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK₁.

PubMed

Matsumoto, Takuya; Ishizaki, Yui; Mochizuki, Keika; Aoyagi, Mitsuru; Mitoma, Yoshiharu; Ishizaki, Shoichiro; Nagashima, Yuji

2017-07-17

This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK₁. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p -aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs).

Caveolae and caveolin-1 mediate endocytosis and transcytosis of oxidized low density lipoprotein in endothelial cells

PubMed Central

Sun, Shao-wei; Zu, Xu-yu; Tuo, Qin-hui; Chen, Lin-xi; Lei, Xiao-yong; Li, Kai; Tang, Chao-ke; Liao, Duan-fang

2010-01-01

Aim: To explore the mechanisms involved in ox-LDL transcytosis across endothelial cells and the role of caveolae in this process. Methods: An in vitro model was established to investigate the passage of oxidized low density lipoprotein (ox-LDL) through a tight monolayer of human umbilical vein endothelial cells (HUVEC) cultured on a collagen-coated filter. Passage of DiI-labeled ox-LDL through the monolayer was measured using a fluorescence spectrophotometer. The uptake and efflux of ox-LDL by HUVEC were determined using fluorescence microscopy and HPLC. Results: Caveolae inhibitors – carrageenan (250 μg/mL), filipin (5 μg/mL), and nocodazole (33 μmol/L)–decreased the transport of ox-LDL across the monolayer by 48.9%, 72.4%, and 79.8% as compared to the control group. In addition, they effectively decreased ox-LDL uptake and inhibited the efflux of ox-LDL. Caveolin-1 and LOX-1 were up-regulated by ox-LDL in a time-dependent manner and decreased gradually after depletion of ox-LDL (P<0.05). After treatment HUVEC with ox-LDL and silencing caveolin-1, NF-κB translocation to the nucleus was blocked and LOX-1 expression decreased (P<0.05). Conclusion: Caveolae can be a carrier for ox-LDL and may be involved in the uptake and transcytosis of ox-LDL by HUVEC. PMID:20835266

Kinin effects on ion transport in monolayers of HCA-7 cells, a line from a human colonic adenocarcinoma.

PubMed

Cuthbert, A W; Kirkland, S C; MacVinish, L J

1985-09-01

Using epithelial monolayers of HCA-7 cells, derived from a primary human colonic adenocarcinoma and grown on pervious supports, it is shown that responses to lysylbradykinin can be elicited from either side. It is proposed that kinin receptors are inserted into both apical and basolateral membrane domains.

The effect of polyethylene glycol-modified lipids on the interaction of HIV-1 derived peptide-dendrimer complexes with lipid membranes.

PubMed

Melikishvili, Sophie; Poturnayova, Alexandra; Ionov, Maksim; Bryszewska, Maria; Vary, Tomáš; Cirak, Julius; Muñoz-Fernández, María Ángeles; Gomez-Ramirez, Rafael; de la Mata, Francisco Javier; Hianik, Tibor

2016-12-01

In this study, dendrimers have been purposed as an alternative approach for delivery of HIV peptides to dendritic cells. We have investigated the interaction of dendriplexes formed from polyanionic HIV peptide Nef and cationic carbosilane dendrimer (CBD) with model lipid membranes - large unilamellar vesicles (LUVs), Langmuir monolayers and supported lipid membranes (sBLMs) containing various molar ratio of zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG 2000 ). In our experiments, the lipid membranes represented the model of the plasma membrane of the cell. PEGylated lipids were used in order to model glycocalyx which constitutes the outer leaflet of cellular membranes. The presence of PEGylated lipids resulted in an increase of the phase transition temperature of the lipid bilayer of LUVs, in a decrease of specific volume and adiabatic compressibility. Fluorescence anisotropy study suggests that PEGylated LUVs possessed higher lipid order and decreased fluidity when compared to zwitterionic DMPC vesicles. The interaction of dendriplexes with monolayers was accompanied by the formation of the aggregates as revealed by BAM experiments. This conclusion has been confirmed also by AFM imaging of sBLMs. We have demonstrated that dendriplexes interact with lipid membranes for all types of lipid composition. Moreover, the stronger interaction of cationic dendrimer/peptide complexes with lipid monolayers, vesicles and sBLMs was observed for membranes composed of zwitterionic lipids than for PEGylated lipid membranes. Increased concentration of PEGylated lipids made this interaction weaker. Copyright © 2016 Elsevier B.V. All rights reserved.

Recent Advances in Developing Platinum Monolayer Electrocatalysts for the O2 Reduction Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vukmirovic,M.B.; Sasaki, K.; Zhou, W.-P.

2008-09-15

For Pt, the best single-element catalyst for many reactions, the question of content and loading is exceedingly important because of its price and availability. Using platinum as a fuel-cell catalyst in automotive applications will cause an unquantifiable increase in the demand for this metal. This big obstacle for using fuel cells in electric cars must be solved by decreasing the content of Pt, which is a great challenge of electrocatalysis Over the last several years we inaugurated a new class of electrocatalysts for the oxygen reduction reaction (ORR) based on a monolayer of Pt deposited on metal or alloy carbon-supportedmore » nanoparticles. The possibility of decreasing the Pt content in the ORR catalysts down to a monolayer level has a considerable importance because this reaction requires high loadings due to its slow kinetics. The Pt-monolayer approach has several unique features and some of them are: high Pt utilization, enhanced (or decreased) activity, enhanced stability, and direct activity correlations. The synthesis of Pt monolayer (ML) electrocatalysts was facilitated by our new synthesis method which allowed us to deposit a monolayer of Pt on various metals, or alloy nanoparticles [1, 2] for the cathode electrocatalyst. In this synthesis approach Pt is laid down by the galvanically displacing a Cu monolayer, which was deposited at underpotentials in a monolayer-limited reaction on appropriate metal substrate, with Pt after immersing the electrode in a K{sub 2}PtCl{sub 4} solution.« less

Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells.

PubMed

Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M; Rice, Kenner C; Gannon, Brenda M; Fantegrossi, William E; Gonzalez, Carmen; Paule, Merle G; Ali, Syed F

2016-08-26

Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as "bath salts." These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM-2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic. Published by Elsevier Ireland Ltd.

Biodiversity and probiotic potential of yeasts isolated from Fura, a West African spontaneously fermented cereal.

PubMed

Pedersen, Line Lindegaard; Owusu-Kwarteng, James; Thorsen, Line; Jespersen, Lene

2012-10-01

Fura is a spontaneously fermented pearl millet product consumed in West Africa. The yeast species involved in the fermentation were identified by pheno- and genotypic methods to be Candida krusei, Kluyveromyces marxianus, Candida tropicalis, Candida rugosa, Candida fabianii, Candida norvegensis and Trichosporon asahii. C. krusei and K. marxianus were found to be the dominant species. Survival in pH 2.5 or in the presence of bile salts (0.3% (w/v) oxgall) and growth at 37°C were independently determined as indicators of the survival potential of the isolates during passage through the human gastrointestinal tract. Selected yeast species isolates were assessed for their probiotic potential. All of the examined yeast isolates survived and grew at human gastrointestinal conditions in pH 2.5, 0.3% (w/v) oxgall at 37°C. The effect on the transepithelial electrical resistance (TEER) across polarized monolayers of intestinal epithelial cells of human (Caco-2) and porcine (IPEC-J2) origin, were determined. The Caco-2 cells and IPEC-J2 cells displayed clearly different relative TEER results. The strains of C. krusei, K. marxianus, C. rugosa and T. asahii were able to increase the relative TEER of Caco-2 monolayers after 48h. In comparison, the relative TEER of IPEC-J2 monolayers decreased when exposed to the same yeasts, even though T. asahii did not differ significantly from Saccharomyces cerevisiae var. boulardii which is used as a human probiotic. C. tropicalis resulted in the largest relative TEER decrease for both the human and the porcine cell model assays. Hyphal growth was observed for C. albicans and C. tropicalis after 48h of incubation with polarized Caco-2 monolayers, whereas this was not the case for the remaining yeast species. In the present study new yeast strains with potential probiotic properties have been isolated to be used potentially as starter cultures for fura production. Copyright © 2012 Elsevier B.V. All rights reserved.

Carbon-1 versus Carbon-3 Linkage of d-Galactose to Porphyrins: Synthesis, Uptake, and Photodynamic Efficiency.

PubMed

Pereira, Patrícia M R; Rizvi, Waqar; Bhupathiraju, N V S Dinesh K; Berisha, Naxhije; Fernandes, Rosa; Tomé, João P C; Drain, Charles Michael

2018-02-21

The use of glycosylated compounds is actively pursued as a therapeutic strategy for cancer due to the overexpression of various types of sugar receptors and transporters on most cancer cells. Conjugation of saccharides to photosensitizers such as porphyrins provides a promising strategy to improve the selectivity and cell uptake of the photosensitizers, enhancing the overall photosensitizing efficacy. Most porphyrin-carbohydrate conjugates are linked via the carbon-1 position of the carbohydrate because this is the most synthetically accessible approach. Previous studies suggest that carbon-1 galactose derivatives show diminished binding since the hydroxyl group in the carbon-1 position of the sugar is a hydrogen bond acceptor in the galectin-1 sugar binding site. We therefore synthesized two isomeric porphyrin-galactose conjugates using click chemistry: one linked via the carbon-1 of the galactose and one linked via carbon-3. Free base and zinc analogs of both conjugates were synthesized. We assessed the uptake and photodynamic therapeutic (PDT) activity of the two conjugates in both monolayer and spheroidal cell cultures of four different cell lines. For both the monolayer and spheroid models, we observe that the uptake of both conjugates is proportional to the protein levels of galectin-1 and the uptake is suppressed after preincubation with an excess of thiogalactose, as measured by fluorescence spectroscopy. Compared to that of the carbon-1 conjugate, the uptake of the carbon-3 conjugate was greater in cell lines containing high expression levels of galectin-1. After photodynamic activation, MTT and lactate dehydrogenase assays demonstrated that the conjugates induce phototoxicity in both monolayers and spheroids of cancer cells.

Assessment of ABCG2-mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halwachs, Sandra

In humans, the ATP-binding cassette efflux transporter ABCG2 contributes to the fetoprotective barrier function of the placenta, potentially limiting the toxicity of transporter substrates to the fetus. During testing of chemicals including pesticides, developmental toxicity studies are performed in rabbit. Despite its toxicological relevance, ABCG2-mediated transport of pesticides in rabbit placenta has not been yet elucidated. We therefore generated polarized MDCK II cells expressing the ABCG2 transporter from rabbit placenta (rbABCG2) and evaluated interaction of the efflux transporter with selected insecticides, fungicides, and herbicides. The Hoechst H33342 accumulation assay indicated that 13 widely used pesticidal active substances including azoxystrobin, carbendazim,more » chlorpyrifos, chlormequat, diflufenican, dimethoate, dimethomorph, dithianon, ioxynil, methiocarb, propamocarb, rimsulfuron and toclofos-methyl may be rbABCG2 inhibitors and/or substrates. No such evidence was obtained for chlorpyrifos-methyl, epoxiconazole, glyphosate, imazalil and thiacloprid. Moreover, chlorpyrifos (CPF), dimethomorph, tolclofos-methyl and rimsulfuron showed concentration-dependent inhibition of H33342 excretion in rbABCG2-transduced MDCKII cells. To further evaluate the role of rbABCG2 in pesticide transport across the placenta barrier, we generated polarized MDCKII-rbABCG2 monolayers. Confocal microscopy confirmed correct localization of rbABCG2 protein in the apical plasma membrane. In transepithelial flux studies, we showed the time-dependent preferential basolateral to apical (B > A) directed transport of [{sup 14}C] CPF across polarized MDCKII-rbABCG2 monolayers which was significantly inhibited by the ABCG2 inhibitor fumitremorgin C (FTC). Using this novel in vitro cell culture model, we altogether showed functional secretory activity of the ABCG2 transporter from rabbit placenta and identified several pesticides like the insecticide CPF as potential rbABCG2 substrates. - Highlights: • Generation of MDCKII-rbABCG2 monolayers with epithelial barrier function • Detection of rbABCG2 in the apical plasma membrane of polarized MDCKII cells • Several pesticides interact with the ABCG2 transporter from rabbit placenta. • rbABCG2 mediates transport of the insecticide chlorpyrifos. • MDCKII-rbABCG2 cells are a suitable model to study transport in rabbit placenta.« less

Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures

PubMed Central

Mrozowska, Paulina S.

2016-01-01

MDCK II cells, a widely used model of polarized epithelia, develop into different structures depending on culture conditions: two-dimensional (2D) monolayers when grown on synthetic supports or three-dimensional (3D) cysts when surrounded by an extracellular matrix. The establishment of epithelial polarity is accompanied by transcytosis of the apical marker podocalyxin from the outer plasma membrane to the newly formed apical domain, but its exact route and regulation remain poorly understood. Here, through comprehensive colocalization and knockdown screenings, we identified the Rab GTPases mediating podocalyxin transcytosis and showed that different sets of Rabs coordinate its transport during cell polarization in 2D and 3D structures. Moreover, we demonstrated that different Rab35 effectors regulate podocalyxin trafficking in 2D and 3D environments; trafficking is mediated by OCRL in 2D monolayers and ACAP2 in 3D cysts. Our results give substantial insight into regulation of the transcytosis of this apical marker and highlight differences between trafficking mechanisms in 2D and 3D cell cultures. PMID:27138252

[Study of the effect of surface-active agents on living cells, used as component parts in microemulsions, based on their chemical structures and critical micelle-formative concentration (CMC)].

PubMed

Ujhelyi, Zoltán; Vecsernyés, Miklós; Bácskay, Ildikó

2013-01-01

The aim of this study was to examine the cellular effects of two nonionic amphiphilic tenside groups and their mixtures on human Caco-2 cell monolayers as dependent upon their chemical structures and physicochemical properties. The first group of polyethylene glycol esters is represented by Polysorbates and Labrasol alone and in blends, while the members of the second group:Capryol 90, Capryol PGMC, Lauroglycol 90 and Lauroglycol FCC were used as propylene glycol esters. They are increasingly used in SMEDDS as recent tensides or co-tensides to increase the solubility of hydrophobic drug. Critical micelle concentration was measured by determination of surface tension. CMC refers to the ability of solubilization of surfactants. Cytotoxicity tests were performed on Caco-2 cell monolayers by MTT and LDH methods. Caco-2 cell monolayers are convenient and reliable in vitro models of the gastrointestinal tract. Paracellular permeability was examined with Lucifer yellow assays. The integrity of cell monolayers was observed by TransEpithelial Electrical Resistance (TEER) measurements. Tight junction alterations effected by the surfactants were also characterized as evidence for paracellular pathway. Changes in sub cellular localization of the tight junction proteins: ZO-1, Claudin-land beta-cathenin, were examined by confocal laser scanning microscopy.The results of cytotoxicity assays were in agreement and showed significant differences among the cytotoxic properties of surfactants in a concentration-dependent manner. Polysorbates 20, 60, 80 are the most toxic compounds. In the case of Labrasol, the degree of esterification and lack of sorbit component decreased cytotoxicity. If the hydrophyl head was changed from polyethylene glycol to propylene glycol, the main determined factor of cytotoxicity was the monoester content and the length of carbon chain. In our CMC experiments, we found that only Labrasol showed expressed cytotoxicity above the CMC. It refers to good ability of micelle solubilization of Labrasol. In our paracellular transport experiments each of polyethylene glycol surfactants (Polysorbates and Labrasol) altered TEER values but propylene glycol esters did not modify the monolayer integrity. Polyethylene glycol esters alone and in blends (0.05% Labrasol--0.001% Polysorbates 20, 60, 80) were able to increase Lucifer yellow permeability significantly below the IC50 concentration. On the other hand Labrasol and Polysorbates 20 have expressed effect on tight junctions of Caco-2 monolayer. It could be concluded that polyethylene glycol ester-type tensides were able to enhance the paracellular permeability by the redistribution of junctional proteins. Our results might ensure useful data for selection of suitable tensides, co-tensides and tenside mixtures for SMEDDS formulations.

Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

PubMed

Thomas, Biju B; Zhu, Danhong; Zhang, Li; Thomas, Padmaja B; Hu, Yuntao; Nazari, Hossein; Stefanini, Francisco; Falabella, Paulo; Clegg, Dennis O; Hinton, David R; Humayun, Mark S

2016-05-01

To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

Cellular internalization, transcellular transport, and cellular effects of silver nanoparticles in polarized Caco-2 cells following apical or basolateral exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imai, Shunji; Morishita, Yuki; Hata, Tomoyuki

When considering the safety of ingested nanomaterials, it is important to quantitate their transfer across intestinal cells; however, little information exists about the effects of nanomaterial size or exposure side (apical versus basolateral epithelial surface) on nanomaterial transfer. Here, we examined cellular internalization and transcellular transport, and the effects of nanomaterials on Caco-2 monolayers after apical or basolateral exposure to Ag or Au nanoparticles with various sizes. After apical treatment, both internalization and transfer to the basolateral side of the monolayers were greater for smaller Ag nanoparticles than for larger Ag nanoparticles. In contrast, after basolateral treatment, larger Ag nanoparticlesmore » were more internalized than smaller Ag nanoparticles, but the transfer to the apical side was greater for smaller Ag nanoparticles. Au nanoparticles showed different rules of internalization and transcellular transport compared with Ag nanoparticles. Furthermore, the paracellular permeability of the Caco-2 monolayers was temporarily increased by Ag nanoparticles (5 μg/mL; diameters, ≤10 nm) following basolateral but not apical exposure. We conclude that the internalization, transfer, and effects of nanomaterials in epithelial cell monolayers depend on the size and composition of nanomaterials, and the exposure side. - Highlights: • Ag and Au nanoparticles can transfer across Caco-2 monolayers. • Cellular uptake of nanoparticles change between apical and basolateral exposure. • Basolateral Ag nanoparticle exposure increases the permeability of Caco-2 monolayers.« less

Optical properties of two-dimensional GaS and GaSe monolayers

NASA Astrophysics Data System (ADS)

Jappor, Hamad Rahman; Habeeb, Majeed Ali

2018-07-01

Optical properties of GaS and GaSe monolayers are investigated using first-principles calculations. The optical properties are studied up to 35 eV. Precisely, our results demonstrated that the optical properties appearance of GaS monolayer is comparative with GaSe monolayer with few informations contrasts. Moreover, the absorption begins in the visible region, although the peaks in the ultraviolet (UV) region. The refractive index values are 1.644 (GaS monolayer) and 2.01 (GaSe monolayer) at zero photon energy limit and increase to 2.092 and 2.698 respectively and both located in the visible region. Furthermore, we notice that the optical properties of both monolayers are obtained in the ultraviolet range and the results are significant. Accordingly, it can be used as a highly promising material in the solar cell, ultraviolet optical nanodevices, nanoelectronics, optoelectronic, and photocatalytic applications.

A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines.

PubMed

Adam, Aziza A A; van der Mark, Vincent A; Donkers, Joanne M; Wildenberg, Manon E; Oude Elferink, Ronald P J; Chamuleau, Robert A F M; Hoekstra, Ruurdtje

2018-01-01

Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBPα was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the in-vitro maintenance of primary hepatocytes and increase hepatic differentiation of stem cells.

Effective cancer laser-therapy design through the integration of nanotechnology and computational treatment planning models

NASA Astrophysics Data System (ADS)

Fisher, Jessica W.; Rylander, Marissa Nichole

2008-02-01

Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, the effectiveness of these therapies is limited due to nonspecific heating of target tissue which often leads to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to heat shock protein (HSP) induction in tumor regions where non-lethal temperature elevation occurs, thereby imparting enhanced tumor cell viability and resistance to subsequent chemotherapy and radiation treatments. Introducing multi-walled nanotubes (MWNT) into target tissue prior to laser irradiation increases heating selectivity permitting more precise thermal energy delivery to the tumor region and enhances thermal deposition thereby increasing tumor injury and reducing HSP expression induction. This study investigated the impact of MWNT inclusion in untreated and laser irradiated monolayer cell culture and cell phantom model. Cell viability remained high for all samples with MWNT inclusion and cells integrated into alginate phantoms, demonstrating the non-toxic nature of both MWNTs and alginate phantom models. Following, laser irradiation samples with MWNT inclusion exhibited dramatic temperature elevations and decreased cell viability compared to samples without MWNT. In the cell monolayer studies, laser irradiation of samples with MWNT inclusion experienced up-regulated HSP27, 70 and 90 expression as compared to laser only or untreated samples due to greater temperature increases albeit below the threshold for cell death. Further tuning of laser parameters will permit effective cell killing and down-regulation of HSP. Due to optimal tuning of laser parameters and inclusion of MWNT in phantom models, extensive temperature elevations and cell death occurred, demonstrating MWNT-mediated laser therapy as a viable therapy option when parameters are optimized. In conclusion, MWNT-mediated laser therapies show great promise for effective tumor destruction, but require determination of appropriate MWNT characteristics and laser parameters for maximum tumor destruction.

A physically based compact I-V model for monolayer TMDC channel MOSFET and DMFET biosensor.

PubMed

Rahman, Ehsanur; Shadman, Abir; Ahmed, Imtiaz; Khan, Saeed Uz Zaman; Khosru, Quazi D M

2018-06-08

In this work, a compact transport model has been developed for monolayer transition metal dichalcogenide (TMDC) channel MOSFET. The analytical model solves the Poisson's equation for the inversion charge density to get the electrostatic potential in the channel. Current is then calculated by solving the drift-diffusion equation. The model makes gradual channel approximation to simplify the solution procedure. The appropriate density of states obtained from the first principle density functional theory simulation has been considered to keep the model physically accurate for monolayer TMDC channel FET. The outcome of the model has been benchmarked against both experimental and numerical quantum simulation results with the help of a few fitting parameters. Using the compact model, detailed output and transfer characteristics of monolayer WSe 2 FET have been studied, and various performance parameters have been determined. The study confirms excellent ON and OFF state performances of monolayer WSe 2 FET which could be viable for the next generation high-speed, low power applications. Also, the proposed model has been extended to study the operation of a biosensor. A monolayer MoS 2 channel based dielectric modulated FET is investigated using the compact model for detection of a biomolecule in a dry environment.

A physically based compact I–V model for monolayer TMDC channel MOSFET and DMFET biosensor

NASA Astrophysics Data System (ADS)

Rahman, Ehsanur; Shadman, Abir; Ahmed, Imtiaz; Zaman Khan, Saeed Uz; Khosru, Quazi D. M.

2018-06-01

In this work, a compact transport model has been developed for monolayer transition metal dichalcogenide (TMDC) channel MOSFET. The analytical model solves the Poisson’s equation for the inversion charge density to get the electrostatic potential in the channel. Current is then calculated by solving the drift–diffusion equation. The model makes gradual channel approximation to simplify the solution procedure. The appropriate density of states obtained from the first principle density functional theory simulation has been considered to keep the model physically accurate for monolayer TMDC channel FET. The outcome of the model has been benchmarked against both experimental and numerical quantum simulation results with the help of a few fitting parameters. Using the compact model, detailed output and transfer characteristics of monolayer WSe2 FET have been studied, and various performance parameters have been determined. The study confirms excellent ON and OFF state performances of monolayer WSe2 FET which could be viable for the next generation high-speed, low power applications. Also, the proposed model has been extended to study the operation of a biosensor. A monolayer MoS2 channel based dielectric modulated FET is investigated using the compact model for detection of a biomolecule in a dry environment.

Growth of MCF-7 breast cancer cells and efficacy of anti-angiogenic agents in a hydroxyethyl chitosan/glycidyl methacrylate hydrogel.

PubMed

Wang, Hejing; Qian, Junmin; Zhang, Yaping; Xu, Weijun; Xiao, Juxiang; Suo, Aili

2017-01-01

Breast cancer negatively affects women's health worldwide. The tumour microenvironment plays a critical role in tumour initiation, proliferation, and metastasis. Cancer cells are traditionally grown in two-dimensional (2D) cultures as monolayers on a flat solid surface lacking cell-cell and cell-matrix interactions. These experimental conditions deviate from the clinical situation. Improved experimental systems that can mimic the in vivo situation are required to discover new therapies, particularly for anti-angiogenic agents that mainly target intercellular factors and play an essential role in treating some cancers. Chitosan can be modified to construct three-dimensional (3D) tumour models. Here, we report an in vitro 3D tumour model using a hydroxyethyl chitosan/glycidyl methacrylate (HECS-GMA) hydrogel produced by a series of chitosan modifications. Parameters relating to cell morphology, viability, proliferation, and migration were analysed using breast cancer MCF-7 cells. In a xenograft model, secretion of angiogenesis-related growth factors and the anti-angiogenic efficacy of Endostar and Bevacizumab in cells grown in HECS-GMA hydrogels were assessed by immunohistochemistry. Hydroxyethyl chitosan/glycidyl methacrylate hydrogels had a highly porous microstructure, mechanical properties, swelling ratio, and morphology consistent with a 3D tumour model. Compared with a 2D monolayer culture, breast cancer MCF-7 cells residing in the HECS-GMA hydrogels grew as tumour-like clusters in a 3D formation. In a xenograft model, MCF-7 cells cultured in the HECS-GMA hydrogels had increased secretion of angiogenesis-related growth factors. Recombinant human endostatin (Endostar), but not Bevacizumab (Avastin), was an effective anti-angiogenic agent in HECS-GMA hydrogels. The HECS-GMA hydrogel provided a 3D tumour model that mimicked the in vivo cancer microenvironment and supported the growth of MCF7 cells better than traditional tissue culture plates. The HECS-GMA hydrogel may offer an improved platform to minimize the gap between traditional tissue culture plates and clinical applicability. In addition, the anti-angiogenic efficacy of drugs such as Endostar and Bevacizumab can be more comprehensively studied and assessed in HECS-GMA hydrogels.

Nanoparticle transport across in vitro olfactory cell monolayers.

PubMed

Gartziandia, Oihane; Egusquiaguirre, Susana Patricia; Bianco, John; Pedraz, José Luis; Igartua, Manoli; Hernandez, Rosa Maria; Préat, Véronique; Beloqui, Ana

2016-02-29

Drug access to the CNS is hindered by the presence of the blood-brain barrier (BBB), and the intranasal route has risen as a non-invasive route to transport drugs directly from nose-to-brain avoiding the BBB. In addition, nanoparticles (NPs) have been described as efficient shuttles for direct nose-to-brain delivery of drugs. Nevertheless, there are few studies describing NP nose-to-brain transport. Thus, the aim of this work was (i) to develop, characterize and validate in vitro olfactory cell monolayers and (ii) to study the transport of polymeric- and lipid-based NPs across these monolayers in order to estimate NP access into the brain using cell penetrating peptide (CPPs) moieties: Tat and Penetratin (Pen). All tested poly(d,l-lactide-co-glycolide) (PLGA) and nanostructured lipid carrier (NLC) formulations were stable in transport buffer and biocompatible with the olfactory mucosa cells. Nevertheless, 0.7% of PLGA NPs was able to cross the olfactory cell monolayers, whereas 8% and 22% of NLC and chitosan-coated NLC (CS-NLC) were transported across them, respectively. Moreover, the incorporation of CPPs to NLC surface significantly increased their transport, reaching 46% of transported NPs. We conclude that CPP-CS-NLC represent a promising brain shuttle via nose-to-brain for drug delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

Transport of lipid nano-droplets through MDCK epithelial cell monolayer.

PubMed

Khatri, Pulkit; Shao, Jun

2017-05-01

This study aims to investigate the transport of lipid nano-droplets through MDCK epithelial cell monolayer. Nanoemulsions of self-nano-emulsifying drug delivery systems (SNEDDS) labeled with radioactive C18 triglyceride were developed. The effect of droplet size and lipid composition on the transport was investigated. The results showed that the lipid nano-droplet transport through MDCK cell monolayer was as high as 2.5%. The transport of lipid nano-droplets was higher for nanoemulsions of medium chain glycerides than the long chain glycerides. The transport was reduced by more than half when the average lipid nano-droplet size increased from 38nm to 261nm. The droplet size measurement verified the existence of lipid nano-droplets in the receiver chamber only when the nanoemulsions were added to the donor chamber but not when the surfactant or saline solution was added. Cryo-TEM images confirmed the presence of lipid nano-droplets in both donor and receiver chamber at the end of transport study. In conclusion, lipid nano-droplets can be transported through the cell monolayer. This finding may help to further explore the oral and other non-invasive delivery of macromolecules loaded inside SNEDDS. Copyright © 2017 Elsevier B.V. All rights reserved.

Photo-switchable Donor-Acceptor (D-A) Dyad Interfacial Self-Assembled Monolayers for Organic Photovoltaic Cells

DTIC Science & Technology

2015-11-05

AFRL-AFOSR-VA-TR-2015-0396 (HBCU) Photo-switchable Donor-Acceptor for Organic Photovoltaic Cells Luis Echegoyen UNIVERSITY OF TEXAS AT EL PASO Final...Acceptor (D-A) Dyad Interfacial Self-Assembled Monolayers for Organic Photovoltaic Cells 5a.  CONTRACT NUMBER 5b.  GRANT NUMBER FA9550-12-1-0053 5c...demonstrated using impedance spectroscopy for several triphenylamine-fullerene dyads, but their performance in photovoltaic devices was not remarkable, likely

RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

PubMed

Morabito, Caterina; Steimberg, Nathalie; Mazzoleni, Giovanna; Guarnieri, Simone; Fanò-Illic, Giorgio; Mariggiò, Maria A

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

RCCS Bioreactor-Based Modelled Microgravity Induces Significant Changes on In Vitro 3D Neuroglial Cell Cultures

PubMed Central

Mazzoleni, Giovanna; Fanò-Illic, Giorgio; Mariggiò, Maria A.

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions. PMID:25654124

Interaction of Soybean 7S Globulin Peptide with Cell Membrane Model via Isothermal Titration Calorimetry, Quartz Crystal Microbalance with Dissipation, and Langmuir Monolayer Study.

PubMed

Zou, Yuan; Pan, Runting; Ruan, Qijun; Wan, Zhili; Guo, Jian; Yang, Xiaoquan

2018-05-16

To understand the underlying molecular mechanism of the cholesterol-lowering effect of soybean 7S globulins, the interactions of their pepsin-released peptides (7S-peptides) with cell membrane models consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL) were systematically studied. The results showed that 7S-peptides were bound to DPPC/DOPC/CHOL liposomes mainly through van der Waals forces and hydrogen bonds, and the presence of higher CHOL concentrations enhanced the binding affinity (e.g., DPPC/DOPC/CHOL = 1:1:0, binding ratio = 0.114; DPPC/DOPC/CHOL = 1:1:1, binding ratio = 2.02). Compression isotherms indicated that the incorporation of 7S-peptides increased the DPPC/DOPC/CHOL monolayer fluidity and the lipid raft size. The presence of CHOL accelerated the 7S-peptide accumulation on lipid rafts, which could serve as platforms for peptides to develop into β-sheet rich structures. These results allow us to hypothesize that 7S-peptides may indirectly influence membrane protein functions via altering the membrane organization in the enterocytes.

Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation.

PubMed

Yu, L; Grist, S M; Nasseri, S S; Cheng, E; Hwang, Y-C E; Ni, C; Cheung, K C

2015-03-01

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.

Transepithelial transport of alpha-lipoic acid across human intestinal Caco-2 cell monolayers.

PubMed

Takaishi, Naoki; Yoshida, Kazutaka; Satsu, Hideo; Shimizu, Makoto

2007-06-27

Alpha-lipoic acid (LA) is used in dietary supplements or food with antioxidative functions. The mechanism for the intestinal absorption of alpha-lipoic acid was investigated in this study by using human intestinal Caco-2 cell monolayers. LA was rapidly transported across the Caco-2 cell monolayers, this transport being energy-dependent, suggesting transporter-mediated transport to be the mechanism involved. The LA transport was strongly dependent on the pH value, being accelerated in the acidic pH range. Furthermore, such monocarboxylic acids as benzoic acid and medium-chain fatty acids significantly inhibited LA transport, suggesting that a proton-linked monocarboxylic acid transporter (MCT) was involved in the intestinal transport of LA. The conversion of LA to the more antioxidative dihydrolipoic acid was also apparent during the transport process.

Interaction of antimicrobial arginine-based cationic surfactants with liposomes and lipid monolayers.

PubMed

Castillo, José A; Pinazo, Aurora; Carilla, Josep; Infante, M Rosa; Alsina, M Asunción; Haro, Isabel; Clapés, Pere

2004-04-13

The present work examines the relationship between the antimicrobial activity of novel arginine-based cationic surfactants and the physicochemical process involved in the perturbation of the cell membrane. To this end, the interaction of these surfactants with two biomembrane models, namely, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar lipid vesicles (MLVs) and monolayers of DPPC, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG), and Escherichia coli total lipid extract, was investigated. For the sake of comparison, this study included two commercial antimicrobial agents, hexadecyltrimethylammonium bromide and chlorhexidine dihydrochloride. Changes in the thermotropic phase transition parameters of DPPC MLVs in the presence of the compounds were studied by differential scanning calorimetry analysis. The results show that variations in both the transition temperature (Tm) and the transition width at half-height of the heat absorption peak (deltaT1/2) were consistent with the antimicrobial activity of the compounds. Penetration kinetics and compression isotherm studies performed with DPPC, DPPG, and E. coli total lipid extract monolayers indicated that both steric hindrance effects and electrostatic forces explained the antimicrobial agent-lipid interaction. Overall, in DPPC monolayers single-chain surfactants had the highest penetration capacity, whereas gemini surfactants were the most active in DPPG systems. The compression isotherms showed an expansion of the monolayers compared with that of pure lipids, indicating an insertion of the compounds into the lipid molecules. Owing to their cationic character, they are incorporated better into the negatively charged DPPG than into zwitterionic DPPC lipid monolayers.

The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression.

PubMed

Christopher, R A; Judge, S R; Vincent, P A; Higgins, P J; McKeown-Longo, P J

1999-10-01

Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin's amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin's amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.

Correlation of [RuCl3(dppb)(VPy)] cytotoxicity with its effects on the cell membranes: an investigation using Langmuir monolayers as membrane models.

PubMed

Sandrino, B; Tominaga, T T; Nobre, T M; Scorsin, L; Wrobel, E C; Fiorin, B C; de Araujo, M P; Caseli, L; Oliveira, O N; Wohnrath, K

2014-09-11

One of the major challenges in drug design is to identify compounds with potential toxicity toward target cells, preferably with molecular-level understanding of their mode of action. In this study, the antitumor property of a ruthenium complex, mer-[RuCl3(dppb)(VPy)] (dppb = 1,4-bis(diphenylphosphine)butane and VPy = 4-vinylpyridine) (RuVPy), was analyzed. Results showed that this compound led to a mortality rate of 50% of HEp-2 cell with 120 ± 10 μmol L(-1), indicating its high toxicity. Then, to prove if its mode of action is associated with its interaction with cell membranes, Langmuir monolayers were used as a membrane model. RuVPy had a strong effect on the surface pressure isotherms, especially on the elastic properties of both the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the negatively charged dipalmitoylphosphatidylglycerol (DPPG) phospholipids. These data were confirmed by polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). In addition, interactions between the positive group from RuVPy and the phosphate group from the phospholipids were corroborated by density functional theory (DFT) calculations, allowing the determination of the Ru complex orientation at the air-water interface. Although possible contributions from receptors or other cell components cannot be discarded, the results reported here represent evidence for significant effects on the cell membranes which are probably associated with the high toxicity of RuVPy.

Myosin-X functions in polarized epithelial cells

PubMed Central

Liu, Katy C.; Jacobs, Damon T.; Dunn, Brian D.; Fanning, Alan S.; Cheney, Richard E.

2012-01-01

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein–Myo10 localizes to lateral membrane cell–cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis. PMID:22419816

Enhanced detection of infectious hematopoietic necrosis virus by pretreatment of cell monolayers with polyethylene glycol

USGS Publications Warehouse

Batts, W.N.; Winton, J.R.

1989-01-01

To improve quantification of very low levels of infectious hematopoietic necrosis virus (IHNV) in samples of tissue, ovarian fluid, or natural water supplies, we tested the ability of polyethylene glycol (PEG) to enhance the sensitivity and speed of the plaque assay system. We compared 4, 7, and 10% solutions of PEG of molecular weight 6,000, 8,000, or 20,000 applied at selected volumes and for various durations. When cell monolayers of epithelioma papulosum cyprini (EPC), fathead minnow (FHM), chinook salmon embryo (CHSE-214), and bluegill fry (BF2) were pretreated with 7% PEG-20,000, they produced 4-17-fold increases in plaque assay titers of IHNV. The plaque assay titers of viral hemorrhagic septicemia virus, chum salmon reovirus, and chinook salmon paramyxovirus were also enhanced by exposure of CHSE-214 cells to PEG, but the titers of infectious pancreatic necrosis virus and Oncorhynchus masou virus were not substantially changed. Plaques formed by IHNV on PEG-treated EPC cells incubated at 15°C had a larger mean diameter at 6 d than those on control cells at 8 d; this suggests the assay could be shortened by use of PEG. Pretreatment of EPC cell monolayers with PEG enabled detection of IHNV in some samples that appeared negative with untreated cells. For example, when ovarian fluid samples from chinook salmon Oncorhynchus tshawytscha were inoculated onto untreated monolayers of EPC cells, IHNV was detected in only 11 of 51 samples; 17 of the samples were positive when PEG-treated EPC cells were used.PDF

Micro pore arrays in free standing cyclic olefin copolymer membranes: fabrication and surface functionalization strategies for in-vitro barrier tissue models

NASA Astrophysics Data System (ADS)

Gel, M.; Kandasamy, S.; Cartledge, K.; Be, C. L.; Haylock, D.

2013-12-01

In recent years there has been growing interest in micro engineered in-vitro models of tissues and organs. These models are designed to mimic the in-vivo like physiological conditions with a goal to study human physiology in an organ-specific context or to develop in-vitro disease models. One of the challenges in the development of these models is the formation of barrier tissues in which the permeability is controlled locally by the tissues cultured at the interface. In-vitro models of barrier tissues are typically created by generating a monolayer of cells grown on thin porous membranes. This paper reports a robust preparation method for free standing porous cyclic olefin copolymer (COC) membranes. We also demonstrate that gelatin coated membranes facilitate formation of highly confluent monolayer of HUVECs. Membranes with thickness in the range of 2-3 um incorporating micro pores with diameter approximately 20 um were fabricated and integrated with microfluidic channels. The performance of the device was demonstrated with a model system mimicking the endothelial barrier in bone marrow sinusoids.

Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study

PubMed Central

Palmela, Inês; Correia, Leonor; Silva, Rui F. M.; Sasaki, Hiroyuki; Kim, Kwang S.; Brites, Dora; Brito, Maria A.

2015-01-01

Ursodeoxycholic acid and its main conjugate glycoursodeoxycholic acid are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 activation and cell death, as well as interleukin-6 release and a loss of blood-brain barrier integrity. Here, we tested the preventive and restorative effects of these bile acids regarding the disruption of blood-brain barrier properties by UCB in in vitro conditions mimicking severe neonatal hyperbilirubinemia and using the same experimental blood-brain barrier model. Both bile acids reduced the apoptotic cell death induced by UCB, but only glycoursodeoxycholic acid significantly counteracted caspase-3 activation. Bile acids also prevented the upregulation of interleukin-6 mRNA, whereas only ursodeoxycholic acid abrogated cytokine release. Regarding barrier integrity, only ursodeoxycholic acid abrogated UCB-induced barrier permeability. Better protective effects were obtained by bile acid pre-treatment, but a strong efficacy was still observed by their addition after UCB treatment. Finally, both bile acids showed ability to cross confluent monolayers of human brain microvascular endothelial cells in a time-dependent manner. Collectively, data disclose a therapeutic time-window for preventive and restorative effects of ursodeoxycholic acid and glycoursodeoxycholic acid against UCB-induced blood-brain barrier disruption and damage to human brain microvascular endothelial cells. PMID:25821432

Oxygen consumption rate and mitochondrial density in human melanoma monolayer cultures and multicellular spheroids.

PubMed

Hystad, M E; Rofstad, E K

1994-05-15

Rate of oxygen consumption per cell has been shown in previous studies to decrease with increasing depth in the viable rim of multicellular spheroids initiated from rodent cells, human colon-carcinoma cells, and human glioma cells, due to progressive accumulation of quiescent cells during spheroid growth. The purpose of our work was to determine oxygen-consumption profiles in human melanoma spheroids. Monolayer cultures of 4 lines (BEX-c, COX-c, SAX-c, and WIX-c) and spheroid cultures of 2 lines (BEX-c and WIX-c) were subjected to investigation. Spheroids were initiated from monolayer cell cultures and grown in spinner flasks. Rate of oxygen consumption was measured with a Clarke-type electrode. Mitochondrial density was determined by stereological analysis of transmission electron micrographs. Thickness of viable rim and cell packing density were assessed by light microscopy of central spheroid sections. Cell-cycle distribution was determined by analysis of DNA histograms measured by flow cytometry. Cell volume was measured by an electronic particle counter. Rate of oxygen consumption per cell differed by a factor of approximately 1.8 between the 4 cell lines and was positively correlated to total volume of mitochondria per cell. Rate of oxygen consumption per cell and total volume of mitochondria per cell were equal for monolayer cell cultures, 600-microns spheroids and 1,200-microns spheroids of the same line. Mitochondrial density and location in the cell did not differ between cells at the spheroid surface, in the middle of the viable rim and adjacent to the central necrosis. Cell-cycle distribution, cell volume, and cell-packing density in the outer and inner halves of the viable rim were not significantly different. Consequently, the rate of oxygen consumption per cell in inner regions of the viable rim was probably equal to that at the spheroid surface, suggesting that oxygen diffusion distances may be shorter in some melanomas than in many other tumor types.

A novel cell culture technique for electron microscopy.

PubMed

Wang, F; Ledford, L B; Head, J F; Elliott, R L

1993-12-15

A simplified technique for the monolayer growth of cultured cells and their in situ embedment on the inner surface of the pyramidal portion of the Beem capsule for electron microscopy has been developed. The results demonstrated that the cell monolayers grew well on the surface of the Beem capsule and could be embedded in situ. Electron micrographs showed cells in their natural state of contact with one another. The plasma membrane and intracellular organelles were well preserved. This method minimizes many difficult steps and eliminates the disruption of cells by scraping, pelleting, or enzymatic reaction to remove them.

In vitro transport characteristics of EFdA, a novel nucleoside reverse transcriptase inhibitor using Caco-2 and MDCKII cell monolayers.

PubMed

Zhang, Wei; Parniak, Michael A; Sarafianos, Stefan G; Empey, Philip E; Rohan, Lisa C

2014-06-05

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a novel nucleoside reverse transcriptase inhibitor with a unique mechanism of action and highly potent activity against both wild-type and clinically relevant drug resistant HIV-1 variants. Furthermore, in vivo efficacy and safety evaluations have shown EFdA to be a promising therapeutic candidate for use in the treatment of HIV infection. However, little is known about the pharmacokinetic and biopharmaceutical properties of EFdA. In this study, we evaluated cellular EFdA transport using Caco-2 and Madin-Darby Canine Kidney II (MDCKII) in vitro cell models. Studies using Caco-2 cell monolayers showed that EFdA efflux ratios were >2.0, suggesting that active drug transport mechanisms may play a role in EFdA flux. ABCB1 transporter (PGP1) inhibition was assessed using the acetomethoxy derivate of calcein (calcein-AM) as a fluorescent probe in both wild-type MDCKII and PGP1 overexpressing MDCKII cells. Nonetheless, our data showed that EFdA is not a substrate of PGP1. Additionally, comparative bidirectional flux of EFdA and Lucifer yellow (LY, a well-known paracellular marker) was studied over a range of EFdA concentrations. In MDCKII monolayers, EFdA had an apparent permeability coefficient (Papp) (a-b) of <1×10(-6)cm/s. The Papp values significantly increased in the presence of the paracellular permeability enhancer, indicating that EFdA primarily permeates via the paracellular route. Copyright © 2014 Elsevier B.V. All rights reserved.

Precise engineering of dapivirine-loaded nanoparticles for the development of anti-HIV vaginal microbicides.

PubMed

das Neves, José; Sarmento, Bruno

2015-05-01

Polymeric nanoparticles (NPs) have the potential to provide effective and safe delivery of antiretroviral drugs in the context of prophylactic anti-HIV vaginal microbicides. Dapivirine-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) NPs were produced by an emulsion-solvent evaporation method, optimized for colloidal properties using a 3-factor, 3-level Box-Behnken experimental design, and characterized for drug loading, production yield, morphology, thermal behavior, drug release, in vitro cellular uptake, cytotoxicity and pro-inflammatory potential. Also, drug permeability/membrane retention in well-established HEC-1-A and CaSki cell monolayer models as mediated by NPs was assessed in the absence or presence of mucin. Box-Behnken design allowed optimizing monodisperse 170nm drug-loaded NPs. Drug release experiments showed an initial burst effect up to 4h, followed by sustained 24h release at pH 4.2 and 7.4. NPs were readily taken up by different genital and macrophage cell lines as assessed by fluorescence microscopy. Drug-loaded NPs presented lower or at least similar cytotoxicity as compared to the free drug, with up to around one-log increase in half-maximal cytotoxic concentration values. In all cases, no relevant changes in cell pro-inflammatory cytokine/chemokine production were observed. Dapivirine transport across cell monolayers was significantly decreased when mucin was present at the donor side with either NPs or the free drug, thus evidencing the influence of this natural glycoprotein in membrane permeability. Moreover, drug retention in cell monolayers was significantly higher for NPs in comparison with the free drug. Overall, obtained dapivirine-loaded PLGA NPs possess interesting technological and biological features that may contribute to their use as novel safe and effective vaginal microbicides. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

PubMed

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

Mechanical Characterization of Microengineered Epithelial Cysts by Using Atomic Force Microscopy.

PubMed

Shen, Yusheng; Guan, Dongshi; Serien, Daniela; Takeuchi, Shoji; Tong, Penger; Yobas, Levent; Huang, Pingbo

2017-01-24

Most organs contain interconnected tubular tissues that are one-cell-thick, polarized epithelial monolayers enclosing a fluid-filled lumen. Such tissue organization plays crucial roles in developmental and normal physiology, and the proper functioning of these tissues depends on their regulation by complex biochemical perturbations and equally important, but poorly understood, mechanical perturbations. In this study, by combining micropatterning techniques and atomic force microscopy, we developed a simple in vitro experimental platform for characterizing the mechanical properties of the MDCK II cyst, the simplest model of lumen-enclosing epithelial monolayers. By using this platform, we estimated the elasticity of the cyst monolayer and showed that the presence of a luminal space influences cyst mechanics substantially, which could be attributed to polarization and tissue-level coordination. More interestingly, the results from force-relaxation experiments showed that the cysts also displayed tissue-level poroelastic characteristics that differed slightly from those of single cells. Our study provides the first quantitative findings, to our knowledge, on the tissue-level mechanics of well-polarized epithelial cysts and offers new insights into the interplay between cyst mechanics and cyst physiology. Moreover, our simple platform is a potentially useful tool for enhancing the current understanding of cyst mechanics in health and disease. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

NASA Astrophysics Data System (ADS)

Bhattacharjee, Sourav; van Opstal, Edward J.; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

2013-06-01

The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size 45 nm) and polystyrene nanoparticles (PSNPs/size 50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

Human astrocytes/astrocyte conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells

PubMed Central

Siddharthan, Venkatraman; V. Kim, Yuri; Liu, Suyi; Kim, Kwang Sik

2009-01-01

The blood-brain barrier (BBB) is a structural and functional barrier that regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. We have previously developed the in vitro BBB model with human brain microvascular endothelial cells (HBMEC). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. In an attempt to develop the BBB model to mimic the in vivo condition we constructed the flow-based in vitro BBB model using HBMEC and human fetal astrocytes (HFA). We also examined the effect of astrocyte conditioned medium (ACM) in lieu of HFA to study the role of secreted factor(s) on the BBB properties. The tightness of HBMEC monolayer was assessed by the permeability of dextran and propidium iodide as well as by measuring the transendothelial electrical resistance (TEER). We showed that the HBMEC permeability was reduced and TEER was increased by non-contact, co-cultivation with HFA and ACM. The exposure of HBMEC to shear stress also exhibited decreased permeability. Moreover, HFA/ACM and shear flow exhibited additive effect of decreasing the permeability of HBMEC monolayer. In addition, we showed that the HBMEC expression of ZO-1 (tight junction protein) was increased by co-cultivation with ACM and in response to shear stress. These findings suggest that the non-contact co-cultivation with HFA helps maintain the barrier properties of HBMEC by secreting factor(s) into the medium. Our in vitro flow model system with the cells of human origin should be useful for studying the interactions between endothelial cells, glial cells, and secreted factor(s) as well as the role of shear stress in the barrier property of HBMEC. PMID:17368578

Effects of Fe particle irradiation on human endothelial barrier structure and function

NASA Astrophysics Data System (ADS)

Sharma, Preety; Guida, Peter; Grabham, Peter

2014-07-01

Space travel involves exposure to biologically effective heavy ion radiation and there is consequently a concern for possible degenerative disorders in humans. A significant target for radiation effects is the microvascular system, which is crucial to healthy functioning of the tissues. Its pathology is linked to disrupted endothelial barrier function and is not only a primary event in a range of degenerative diseases but also an important influencing factor in many others. Thus, an assessment of the effects of heavy ion radiation on endothelial barrier function would be useful for estimating the risks of space travel. This study was aimed at understanding the effects of high LET Fe particles (1 GeV/n) and is the first investigation of the effects of charged particles on the function of the human endothelial barrier. We used a set of established and novel endpoints to assess barrier function after exposure. These include, trans-endothelial electrical resistance (TEER), morphological effects, localization of adhesion and cell junction proteins (in 2D monolayers and in 3D tissue models), and permeability of molecules through the endothelial barrier. A dose of 0.50 Gy was sufficient to cause a progressive reduction in TEER measurements that were significant 48 hours after exposure. Concurrently, there were morphological changes and a 14% loss of cells from monolayers. Gaps also appeared in the normally continuous cell-border localization of the tight junction protein - ZO-1 but not the Platelet endothelial cell adhesion molecule (PECAM-1) in both monolayers and in 3D vessel models. Disruption of barrier function was confirmed by increased permeability to 3 kDa and 10 kDa dextran molecules. A dose of 0.25 Gy caused no detectible change in cell number, morphology, or TEER, but did cause barrier disruption since there were gaps in the cell border localization of ZO-1 and an increased permeability to 3 kDa dextran. These results indicate that Fe particles potently have impact on human endothelial barrier function and represent a risk for degenerative diseases in the space environment.

In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model.

PubMed

Tamai, Miho; Aoki, Mami; Nishimura, Akihito; Morishita, Koji; Tagawa, Yoh-ichi

2013-12-01

Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVL(mES), which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVL(mES) culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. L-Ornithine is an intermediate of the urea cycle, but supplemental L-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVL(mES), supplemental L-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVL(mES) displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVL(mES) will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.

Biomimetics in thin film design: Niche-like wrinkles designed for i-cell progenitor cell differentiation.

PubMed

Major, Roman; Lackner, Juergen M; Sanak, Marek; Major, Boguslaw

2017-11-01

The future and development of science are in interdisciplinary areas, such as biomedical engineering. Self-assembled structures, similar to stem cell niches, inhibit rapid cellular division processes and enable the capture of stem cells from blood flow. By modifying the surface topography and stiffness properties, progenitor cells were differentiated towards the formation of endothelial cell monolayers to effectively inhibit the coagulation cascade. Wrinkled material layers in the form of thin polymeric coatings were prepared. An optimized surface topography led to proper cell differentiation and influenced the appropriate formation of endothelial cell monolayers. Blood activation was decelerated by the formed endothelium. Copyright © 2017 Elsevier B.V. All rights reserved.

NaK-ATPase pump sites in cultured bovine corneal endothelium of varying cell density at confluence.

PubMed

Crawford, K M; Ernst, S A; Meyer, R F; MacCallum, D K

1995-06-01

The driving force for ion and water flow necessary for efficient deturgesence of the corneal stroma resides in the ouabain-sensitive sodium (Na) pump of corneal endothelial cells. Using a cell culture model of corneal endothelial cell hypertrophy, the authors examined the expression of Na pumps at the cell surface to see how this central element of the endothelial pump changed as corneal endothelial cell density decreased to a level associated with corneal decompensation in vivo. 3H-ouabain binding to NaK-ATPase at saturating conditions was used to quantitate the number of Na pump sites on cultured bovine corneal endothelial cells as the confluent density decreased from approximately 2750 cells/mm2 to approximately 275 cells/mm2. The mean number of Na pump sites per cell at confluence (1.92 +/- 0.07 x 10(6)) did not change as the cell density decreased 2.7-fold from 2763 cells/mm2 to 1000 cells/mm2. However, pump site expression doubled to approximately 4 x 10(6) sites/cell as the cell density decreased from 1000 cells/mm2 to 275 cells/mm2. Despite the incremental increase in Na pump site expression that occurred as the cells hypertrophied below a density of 1000/mm2 to achieve confluence, this increase was insufficient to prevent a decrease in Na pump site density of the intact monolayer, expressed as pump sites/mm2. The confluent cell density of cultured bovine corneal endothelial cells can be varied from that found in the normal native cornea to that associated with corneal decompensation. In confluent cultures with cell densities ranging from 2750 cells/mm2 to 1000 cells/mm2, the number of pump sites per cell remains relatively unchanged. Below cell densities of 1000 cells/mm2, the number of pump sites per cell progressively increases. The increased Na pump site abundance in markedly hypertrophied endothelial cells cannot adequately compensate for the progressive reduction in the number of transporting cells per unit area within the intact monolayer. Even when considered with the decrease in the size of the paracellular ion conductive pathway that is a consequence of progressive endothelial hypertrophy, the overall pumping capacity of the intact endothelial monolayer declines.

Engineering spheroids potentiating cell-cell and cell-ECM interactions by self-assembly of stem cell microlayer.

PubMed

Lee, Yu Bin; Kim, Eun Mi; Byun, Hayeon; Chang, Hyung-Kwan; Jeong, Kwanghee; Aman, Zachary M; Choi, Yu Suk; Park, Jungyul; Shin, Heungsoo

2018-05-01

Numerous methods have been reported for the fabrication of 3D multi-cellular spheroids and their use in stem cell culture. Current methods typically relying on the self-assembly of trypsinized, suspended stem cells, however, show limitations with respect to cell viability, throughput, and accurate recapitulation of the natural microenvironment. In this study, we developed a new system for engineering cell spheroids by self-assembly of micro-scale monolayer of stem cells. We prepared synthetic hydrogels with the surface of chemically formed micropatterns (squares/circles with width/diameter of 200 μm) on which mesenchymal stem cells isolated from human nasal turbinate tissue (hTMSCs) were selectively attached and formed a monolayer. The hydrogel is capable of thermally controlled expansion. As the temperature was decreased from 37 to 4 °C, the cell layer detached rapidly (<10 min) and assembled to form spheroids with consistent size (∼100 μm) and high viability (>90%). Spheroidization was significantly delayed and occurred with reduced efficiency on circle patterns compared to square patterns. Multi-physics mapping supported that delamination of the micro-scale monolayer may be affected by stress concentrated at the corners of the square pattern. In contrast, stress was distributed symmetrically along the boundary of the circle pattern. In addition, treatment of the micro-scale monolayer with a ROCK inhibitor significantly retarded spheroidization, highlighting the importance of contraction mediated by actin stress fibers for the stable generation of spheroidal stem cell structures. Spheroids prepared from the assembly of monolayers showed higher expression, both on the mRNA and protein levels, of ECM proteins (fibronectin and laminin) and stemness markers (Oct4, Sox2, and Nanog) compared to spheroids prepared from low-attachment plates, in which trypsinized single cells are assembled. The hTMSC spheroids also presented enhanced expression levels of markers related to tri-lineage (osteogenic, chondrogenic and adipogenic) differentiation. The changes in microcellular environments and functionalities were double-confirmed by using adipose derived mesenchymal stem cells (ADSCs). This spheroid engineering technique may have versatile applications in regenerative medicine for functionally improved 3D culture and therapeutic cell delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of a living membrane comprising a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane.

PubMed

Schophuizen, Carolien M S; De Napoli, Ilaria E; Jansen, Jitske; Teixeira, Sandra; Wilmer, Martijn J; Hoenderop, Joost G J; Van den Heuvel, Lambert P W; Masereeuw, Rosalinde; Stamatialis, Dimitrios

2015-03-01

The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2 mg ml(-1) 3,4-dihydroxy-l-phenylalanine (4 min coating, 1h dissolution) and 25 μg ml(-1) Coll IV (4 min coating). The successful transport of (14)C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models.

PubMed

Zambrano, Pablo; Suwalsky, Mario; Villena, Fernando; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

2017-01-29

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes. According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40-50 μM memantine was required to interact with isolated phosphatidylcholine bilayers. Copyright © 2016 Elsevier Inc. All rights reserved.

Menadione metabolism to thiodione in hepatoblastoma by scanning electrochemical microscopy

PubMed Central

Mauzeroll, Janine; Bard, Allen J.; Owhadian, Omeed; Monks, Terrence J.

2004-01-01

The cytotoxicity of menadione on hepatocytes was studied by using the substrate generation/tip collection mode of scanning electrochemical microscopy by exposing the cells to menadione and detecting the menadione-S-glutathione conjugate (thiodione) that is formed during the cellular detoxication process and is exported from the cell by an ATP-dependent pump. This efflux was electrochemically detected and allowed scanning electrochemical microscopy monitoring and imaging of single cells and groups of highly confluent live cells. Based on a constant flux model, ≈6 × 106 molecules of thiodione per cell per second are exported from monolayer cultures of Hep G2 cells. PMID:15601769

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

PubMed Central

Ivanov, I B; Hadjiiski, A; Denkov, N D; Gurkov, T D; Kralchevsky, P A; Koyasu, S

1998-01-01

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level. PMID:9649417

Solar cells based on particulate structure of active layer: Investigation of light absorption by an ordered system of spherical submicron silicon particles

NASA Astrophysics Data System (ADS)

Miskevich, Alexander A.; Loiko, Valery A.

2015-12-01

Enhancement of the performance of photovoltaic cells through increasing light absorption due to optimization of an active layer is considered. The optimization consists in creation of particulate structure of active layer. The ordered monolayers and multilayers of submicron crystalline silicon (c-Si) spherical particles are examined. The quasicrystalline approximation (QCA) and the transfer matrix method (TMM) are used to calculate light absorption in the wavelength range from 0.28 μm to 1.12 μm. The integrated over the terrestial solar spectral irradiance "Global tilt" ASTM G173-03 absorption coefficient is calculated. In the wavelength range of small absorption index of c-Si (0.8-1.12 μm) the integral absorption coefficient of monolayer can be more than 20 times higher than the one of the plane-parallel plate of the equivalent volume of material. In the overall considered range (0.28-1.12 μm) the enhancement factor up to ~1.45 for individual monolayer is observed. Maximum value of the spectral absorption coefficient approaches unity for multilayers consisting of large amount of sparse monolayers of small particles. Multilayers with variable concentration and size of particles in the monolayer sequences are considered. Absorption increasing by such gradient multilayers as compared to the non-gradient ones is illustrated. The considered structures are promising for creation of high efficiency thin-film solar cells.

Binding, uptake, and transport of hypericin by Caco-2 cell monolayers.

PubMed

Sattler, S; Schaefer, U; Schneider, W; Hoelzl, J; Lehr, C M

1997-10-01

The biological evaluation of hypericin in various test models is hampered by its very poor water solubility. In the present study cyclodextrin formulations and liposomal preparations were investigated for improved delivery and solubility of hypericin in aqueous buffer systems. Caco-2 cells, grown to tight monolayers on 96-well tissue culture plates as well as on Transwell polycarbonate filters, were used to study the membrane binding and the epithelial transport of hypericin. Cumulative transport of hypericin, which could not be measured without the use of cyclodextrins, in apical-to-basolateral direction from cyclodextrin-hypericin buffer solutions was 3-5% at 37 degrees C and approximately 0.12% at 4 degrees C after 5 h. After an incubation time of 1 h at 37 and 4 degrees C, 12.7% +/- 2.6% and 6.5% +/- 0.8%, respectively, of hypericin were found to be bound to or taken up by Caco-2 cells. Liposomal formulations markedly increased the solubility of hypericin in Krebs-Ringer buffer, but there was no effect observed on the binding and transport of hypericin delivered by liposomes in the Caco-2 cell model. Due to the fluorescence properties of hypericin, its interaction with the cells could be visualized by confocal laser scanning microscopy. The results indicate that a significant accumulation of the drug in the cell membrane and the cell nucleus membrane takes place. We conclude that hypericin is absorbed through the intestinal epithelium by passive transcellular diffusion and that increasing its solubility by cyclodextrin appears as a promising approach to increase its oral bioavailability for pharmaceutical formulations.

Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium.

PubMed

Quesnell, Rebecca R; Erickson, Jamie; Schultz, Bruce D

2007-01-01

In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. Bovine mammary epithelial (BME-UV cell line) cells were grown to confluence on permeable supports with a standard basolateral medium and either high-electrolyte (H-elec) or low-electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec medium at predetermined times prior to assay. Transepithelial electrical resistance (R(te)) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R(te) began within 24 h of H-elec medium exposure. Change from H-elec medium to L-elec medium time-dependently increased R(te). Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident, concomitant with the changes in R(te), although total occludin was unchanged. Neither substitution of Na(+) with N-methyl-d-glucosamine (NMDG(+)) nor pharmacological inhibition of transcellular Na(+) transport pathways abrogated the effects of apical H-elec medium on R(te). Tumor necrosis factor alpha, but not interleukin-1beta nor interleukin-6, in the apical compartment caused a significant decrease in R(te) within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance.

Absorption and Transport of Sea Cucumber Saponins from Apostichopus japonicus.

PubMed

Li, Shuai; Wang, Yuanhong; Jiang, Tingfu; Wang, Han; Yang, Shuang; Lv, Zhihua

2016-06-17

The present study is focused on the intestinal absorption of sea cucumber saponins. We determined the pharmacokinetic characteristics and bioavailability of Echinoside A and Holotoxin A₁; the findings indicated that the bioavailability of Holotoxin A₁ was lower than Echinoside A. We inferred that the differences in chemical structure between compounds was a factor that explained their different characteristics of transport across the intestine. In order to confirm the absorption characteristics of Echinoside A and Holotoxin A₁, we examined their transport across Caco-2 cell monolayer and effective permeability by single-pass intestinal perfusion. The results of Caco-2 cell model indicate that Echinoside A is transported by passive diffusion, and not influenced by the exocytosis of P-glycoprotein (P-gp, expressed in the apical side of Caco-2 monolayers as the classic inhibitor). The intestinal perfusion also demonstrated well the absorption of Echinoside A and poor absorption of Holotoxin A₁, which matched up with the result of the Caco-2 cell model. The results demonstrated our conjecture and provides fundamental information on the relationship between the chemical structure of these sea cucumber saponins and their absorption characteristics, and we believe that our findings build a foundation for the further metabolism study of sea cucumber saponins and contribute to the further clinical research of saponins.

Electronic Devices with Rubidium Barrier Film and Process for Making Same

DTIC Science & Technology

1998-08-20

barrier film is comprised of a plurality of contiguous monolayers, while FIG. 7B shows another embodiment of the 20 invention where the barrier film is... plurality of contiguous monolayers in which different monolayers thereof are formed of different types of metal atoms. -10- FIG. 8 is a schematic...system directed toward the substrate 26. A diffusion barrier precursor compound effusion cell, for example a barium fluoride, strontium fluoride or the

Intestinal and Blood-Brain Barrier Permeability of Ginkgolides and Bilobalide: In Vitro and In Vivo Approaches

USDA-ARS?s Scientific Manuscript database

In this study intestinal and blood brain barrier (BBB) transport of ginkgolides A, B, C, J and bilobalide, isolated from Ginkgo biloba (Family-Ginkgoaceae), was evaluated in Caco-2 and MDR1-MDCK cell monolayer models. Transepithelial transport was examined for 2 hours in both absorptive and secretor...

Generation of Cardiomyocytes from Pluripotent Stem Cells.

PubMed

Nakahama, Hiroko; Di Pasquale, Elisa

2016-01-01

The advent of pluripotent stem cells (PSCs) enabled a multitude of studies for modeling the development of diseases and testing pharmaceutical therapeutic potential in vitro. These PSCs have been differentiated to multiple cell types to demonstrate its pluripotent potential, including cardiomyocytes (CMs). However, the efficiency and efficacy of differentiation vary greatly between different cell lines and methods. Here, we describe two different methods for acquiring CMs from human pluripotent lines. One method involves the generation of embryoid bodies, which emulates the natural developmental process, while the other method chemically activates the canonical Wnt signaling pathway to induce a monolayer of cardiac differentiation.

Culture Models for Studying Thyroid Biology and Disorders

PubMed Central

Toda, Shuji; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Matsunobu, Aki; Yamamoto, Mihoko; Ootani, Akifumi; Yamasaki, Fumio; Koike, Eisuke; Sugihara, Hajime

2011-01-01

The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types. PMID:22363871

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritvos, O.; Ranta, T.; Jalkanen, J.

1988-05-01

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purifiedmore » 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.« less

Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

PubMed

Tong, Zhixiang; Martyn, Keir; Yang, Andy; Yin, Xiaolei; Mead, Benjamin E; Joshi, Nitin; Sherman, Nicholas E; Langer, Robert S; Karp, Jeffrey M

2018-02-01

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Surface chemistry from wettability and charge for the control of mesenchymal stem cell fate through self-assembled monolayers.

PubMed

Hao, Lijing; Fu, Xiaoling; Li, Tianjie; Zhao, Naru; Shi, Xuetao; Cui, Fuzhai; Du, Chang; Wang, Yingjun

2016-12-01

Self-assembled monolayers (SAMs) of alkanethiols on gold are highly controllable model substrates and have been employed to mimic the extracellular matrix for cell-related studies. This study aims to systematically explore how surface chemistry influences the adhesion, morphology, proliferation and osteogenic differentiation of mouse mesenchymal stem cells (mMSCs) using various functional groups (-OEG, -CH 3 , -PO 3 H 2 , -OH, -NH 2 and -COOH). Surface analysis demonstrated that these functional groups produced a wide range of wettability and charge: -OEG (hydrophilic and moderate iso-electric point (IEP)), -CH 3 (strongly hydrophobic and low IEP), -PO 3 H 2 (moderate wettability and low IEP), -OH (hydrophilic and moderate IEP), -NH 2 (moderate wettability and high IEP) and -COOH (hydrophilic and low IEP). In terms of cell responses, the effect of wettability may be more influential than charge for these groups. Moreover, compared to -OEG and -CH 3 groups, -PO 3 H 2 , -OH, -NH 2 and -COOH functionalities tended to promote not only cell adhesion, proliferation and osteogenic differentiation but also the expression of α v and β 1 integrins. This finding indicates that the surface chemistry may guide mMSC activities through α v and β 1 integrin signaling pathways. Model surfaces with controllable chemistry may provide insight into biological responses to substrate surfaces that would be useful for the design of biomaterial surfaces. Copyright © 2016 Elsevier B.V. All rights reserved.

Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

NASA Technical Reports Server (NTRS)

Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

2011-01-01

Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would otherwise die or be rendered reproductively inactive in 2-D culture.

Phases and phase transition in insoluble and adsorbed monolayers of amide amphiphiles: Specific characteristics of the condensed phases.

PubMed

Vollhardt, D

2015-08-01

For understanding the role of amide containing amphiphiles in inherently complex biological processes, monolayers at the air-water interface are used as simple biomimetic model systems. The specific characteristics of the condensed phases and phase transition in insoluble and adsorbed monolayers of amide amphiphiles are surveyed to highlight the effect of the chemical structure of the amide amphiphiles on the interfacial interactions in model monolayers. The mesoscopic topography and/or two-dimensional lattice structures of selected amino acid amphiphiles, amphiphilic N-alkylaldonamide, amide amphiphiles with specific tailored headgroups, such as amide amphiphiles based on derivatized ethanolamine, e.g. acylethanolamines (NAEs) and N-,O-diacylethanolamines (DAEs) are presented. Special attention is devoted the dominance of N,O-diacylated ethanolamine in mixed amphiphilic acid amide monolayers. The evidence that a first order phase transition can occur in adsorption layers and that condensed phase domains of mesoscopic scale can be formed in adsorption layers was first obtained on the basis of the experimental characteristics of a tailored amide amphiphile. New thermodynamic and kinetic concepts for the theoretical description of the characteristics of amide amphiphile's monolayers were developed. In particular, the equation of state for Langmuir monolayers generalized for the case that one, two or more phase transitions occur, and the new theory for phase transition in adsorbed monolayers are experimentally confirmed at first by amide amphiphile monolayers. Despite the significant progress made towards the understanding the model systems, these model studies are still limited to transfer the gained knowledge to biological systems where the fundamental physical principles are operative in the same way. The study of biomimetic systems, as described in this review, is only a first step in this direction. Copyright © 2014 Elsevier B.V. All rights reserved.

Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system

PubMed Central

Zhu, Huanqi; Scharnhorst, Kelsey S.; Stieg, Adam Z.; Gimzewski, James K.; Minami, Itsunari; Nakatsuji, Norio; Nakano, Haruko; Nakano, Atsushi

2017-01-01

Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals. PMID:28266620

E-cadherin Is Critical for Collective Sheet Migration and Is Regulated by the Chemokine CXCL12 Protein During Restitution*

PubMed Central

Hwang, Soonyean; Zimmerman, Noah P.; Agle, Kimberle A.; Turner, Jerrold R.; Kumar, Suresh N.; Dwinell, Michael B.

2012-01-01

Chemokines and other immune mediators enhance epithelial barrier repair. The intestinal barrier is established by highly regulated cell-cell contacts between epithelial cells. The goal of these studies was to define the role for the chemokine CXCL12 in regulating E-cadherin during collective sheet migration during epithelial restitution. Mechanisms regulating E-cadherin were investigated using Caco2BBE and IEC-6 model epithelia. Genetic knockdown confirmed a critical role for E-cadherin in in vitro restitution and in vivo wound repair. During restitution, both CXCL12 and TGF-β1 tightened the monolayer by decreasing the paracellular space between migrating epithelial cells. However, CXCL12 differed from TGF-β1 by stimulating the significant increase in E-cadherin membrane localization during restitution. Chemokine-stimulated relocalization of E-cadherin was paralleled by an increase in barrier integrity of polarized epithelium during restitution. CXCL12 activation of its cognate receptor CXCR4 stimulated E-cadherin localization and monolayer tightening through Rho-associated protein kinase activation and F-actin reorganization. These data demonstrate a key role for E-cadherin in intestinal epithelial restitution. PMID:22549778

Impact of in Vitro Gastrointestinal Digestion and Transepithelial Transport on Antioxidant and ACE-Inhibitory Activities of Brewer's Spent Yeast Autolysate.

PubMed

Vieira, Elsa F; das Neves, José; Vitorino, Rui; Dias da Silva, Diana; Carmo, Helena; Ferreira, Isabel M P L V O

2016-10-05

Brewer's spent yeast (BSY) autolysates may have potential applications as food ingredients or nutraceuticals due to their antioxidant and ACE-inhibitory activities. The impact of simulated gastrointestinal (GI) digestion, the interaction with intracellular sources of oxidative stress, the intestinal cell permeability of BSY peptides, and the antioxidant and ACE-inhibitory activities of BSY permeates were assayed. Gastrointestinal digestion of BSY autolysates enhanced antioxidant and ACE-inhibitory activities as measured in vitro. No cytotoxic effects were observed on Caco-2 cells after exposure to the digested BSY autolysates within a concentration range of 0.5 to 3.0 mg of peptides/mL. A protective role to induced oxidative stress was observed. The transepithelial transport assays indicate high apparent permeability coefficient (P app ) values for BSY peptides across Caco-2/HT29-MTX cell monolayer (14.5-26.1 × 10 -6 cm/s) and for Caco-2 cell monolayer model (12.4-20.8 × 10 -6 cm/s), while the antioxidant and ACE-inhibitory activities found in flux material from the basolateral side suggest transepithelial absorption of bioactive compounds.

Ultrasensitive Detection of Cu2+ Using a Microcantilever Sensor Modified with L-Cysteine Self-Assembled Monolayer.

PubMed

Xu, Xiaohe; Zhang, Na; Brown, Gilbert M; Thundat, Thomas G; Ji, Hai-Feng

2017-10-01

A microcantilever was modified with a self-assembled monolayer (SAM) of L-cysteine for the sensitively and selectively response to Cu(II) ions in aqueous solution. The microcantilever undergoes bending due to sorption of Cu(II) ions. The interaction of Cu(II) ions with the L-cysteine on the cantilever is diffusion controlled and does not follow a simple Langmuir adsorption model. A concentration of 10 -10  M Cu(II) was detected in a fluid cell using this technology. Other cations, such as Ni 2+ , Zn 2+ , Pb 2+ , Cd 2+ , Ca 2+ , K + , and Na + , did not respond with a significant deflection, indicating that this L-cysteine-modified cantilever responded selectively and sensitively to Cu(II).

Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide.

PubMed

Qiu, Zhifang; Mishra, Anuja; Li, Miao; Farnsworth, Steven L; Guerra, Bernadette; Lanford, Robert E; Hornsby, Peter J

2015-07-01

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy. Copyright © 2015. Published by Elsevier B.V.

Adsorption of GST-PI3Kgamma at the air-buffer interface and at substrate and nonsubstrate phospholipid monolayers.

PubMed

Hermelink, Antje; Kirsch, Cornelia; Klinger, Reinhard; Reiter, Gerald; Brezesinski, Gerald

2009-02-01

The recruitment of phosphoinositide 3-kinase gamma (PI3Kgamma) to the cell membrane is a crucial requirement for the initiation of inflammation cascades by second-messenger production. In addition to identifying other regulation pathways, it has been found that PI3Kgamma is able to bind phospholipids directly. In this study, the adsorption behavior of glutathione S-transferase (GST)-PI3Kgamma to nonsubstrate model phospholipids, as well as to commercially available substrate inositol phospholipids (phosphoinositides), was investigated by use of infrared reflection-absorption spectroscopy (IRRAS). The nonsubstrate phospholipid monolayers also yielded important information about structural requirements for protein adsorption. The enzyme did not interact with condensed zwitterionic or anionic monolayers; however, it could penetrate into uncompressed fluid monolayers. Compression to values above its equilibrium pressure led to a squeezing out and desorption of the protein. Protein affinity for the monolayer surface increased considerably when the lipid had an anionic headgroup and contained an arachidonoyl fatty acyl chain in sn-2 position. Similar results on a much higher level were observed with substrate phosphoinositides. No structural response of GST-PI3Kgamma to lipid interaction was detected by IRRAS. On the other hand, protein adsorption caused a condensing effect in phosphoinositide monolayers. In addition, the protein reduced the charge density at the interface probably by shifting the pK values of the phosphate groups attached to the inositol headgroups. Because of their strongly polar headgroups, an interaction of the inositides with the water molecules of the subphase can be expected. This interaction is disturbed by protein adsorption, causing the ionization state of the phosphates to change.

Adsorption of GST-PI3Kγ at the Air-Buffer Interface and at Substrate and Nonsubstrate Phospholipid Monolayers

PubMed Central

Hermelink, Antje; Kirsch, Cornelia; Klinger, Reinhard; Reiter, Gerald; Brezesinski, Gerald

2009-01-01

The recruitment of phosphoinositide 3-kinase γ (PI3Kγ) to the cell membrane is a crucial requirement for the initiation of inflammation cascades by second-messenger production. In addition to identifying other regulation pathways, it has been found that PI3Kγ is able to bind phospholipids directly. In this study, the adsorption behavior of glutathione S-transferase (GST)-PI3Kγ to nonsubstrate model phospholipids, as well as to commercially available substrate inositol phospholipids (phosphoinositides), was investigated by use of infrared reflection-absorption spectroscopy (IRRAS). The nonsubstrate phospholipid monolayers also yielded important information about structural requirements for protein adsorption. The enzyme did not interact with condensed zwitterionic or anionic monolayers; however, it could penetrate into uncompressed fluid monolayers. Compression to values above its equilibrium pressure led to a squeezing out and desorption of the protein. Protein affinity for the monolayer surface increased considerably when the lipid had an anionic headgroup and contained an arachidonoyl fatty acyl chain in sn-2 position. Similar results on a much higher level were observed with substrate phosphoinositides. No structural response of GST-PI3Kγ to lipid interaction was detected by IRRAS. On the other hand, protein adsorption caused a condensing effect in phosphoinositide monolayers. In addition, the protein reduced the charge density at the interface probably by shifting the pK values of the phosphate groups attached to the inositol headgroups. Because of their strongly polar headgroups, an interaction of the inositides with the water molecules of the subphase can be expected. This interaction is disturbed by protein adsorption, causing the ionization state of the phosphates to change. PMID:19186139

Rheology and microrheology of materials at the air-water interface

NASA Astrophysics Data System (ADS)

Walder, Robert Benjamin

2008-10-01

The study of materials at the air-water interface is an important area of research in soft condensed matter physics. Films at the air-water interface have been a system of interest to physics, chemistry and biology for the last 20 years. The unique properties of these surface films provide ideal models for 2-d films, surface chemistry and provide a platform for creating 2 dimensional analogue materials to cellular membranes. Measurements of the surface rheology of cross-linked F-actin networks associated with a lipid monolayer at the air-water interface of a Langmuir monolayer have been performed. The rheological measurements are made using a Couette cell. These data demonstrate that the network has a finite elastic modulus that grows as a function of the cross-linking concentration. We also note that under steady-state flow the system behaves as a power law fluid in which the effective viscosity decreases with imposed shear. A Langmuir monolayer trough that is equipped for simultaneous microrheology and standard rheology measurements has been constructed. The central elements are the trough itself with a full range of optical tools accessing the air-water interface from below the trough and a portable knife-edge torsion pendulum that can access the interface from above. The ability to simultaneously measure the mechanical response of Langmuir monolayers on very different length scales is an important step for our understanding of the mechanical response of two-dimensional viscoelastic networks. The optical tweezer microrheometer is used to study the micromechanical properties of Langmuir monolayers. Microrheology measurements are made a variety of surface pressures that correspond to different ordered phases of the monolayer. The complex shear modulus shows an order of magnitude increase for the liquid condensed phase of DPPC compared to the liquid expanded phase.

Crystallography and Molecular Arrangement of Polymorphic Monolayer J-Aggregates of a Cyanine Dye: Multiangle Polarized Light Fluorescence Optical Microscopy Study.

PubMed

Prokhorov, Valery V; Pozin, Sergey I; Perelygina, Olga M; Mal'tsev, Eugene I

2018-04-24

The molecular orientation in monolayer J-aggregates of 3,3-di(γ-sulfopropyl)-5,5-dichlorotiamonomethinecyanine dye has been precisely estimated using improved linear polarization measurements in the fluorescence microscope in which a multiangle set of polarization data is obtained using sample rotation. The estimated molecular orientation supplemented with the previously established crystallographic constraints based on the analysis of the well-developed two-dimensional J-aggregate shapes unambiguously indicate the staircase type of molecular arrangement for striplike J-aggregates with the staircases oriented along strips. The molecular transition dipoles are inclined at an angle of ∼25° to the strip direction, whereas the characteristic strip vertex angle ∼45° is formed by the [100] and [1-10] directions of the monoclinic unit cell. Measurements of the geometry of partially unwound tubes and their polarization properties support the model of tube formation by close-packed helical winding of flexible monolayer strips. In the tubes, the long molecular axes are oriented at a small angle in the range of 5-15° to the normal to the tube axis providing low bending energy. At a nanoscale, high-resolution atomic force microscopy imaging of J-aggregate monolayers reveals a complex quasi-one-dimensional organization.

An open-access microfluidic model for lung-specific functional studies at an air-liquid interface.

PubMed

Nalayanda, Divya D; Puleo, Christopher; Fulton, William B; Sharpe, Leilani M; Wang, Tza-Huei; Abdullah, Fizan

2009-10-01

In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays.

Tracing the 4000 year history of organic thin films: From monolayers on liquids to multilayers on solidsa)

NASA Astrophysics Data System (ADS)

Greene, J. E.

2015-03-01

The recorded history of organic monolayer and multilayer thin films spans approximately 4000 years. Fatty-acid-based monolayers were deposited on water by the ancients for applications ranging from fortune telling in King Hammurabi's time (˜1800 BC, Mesopotamia) to stilling choppy waters for sailors and divers as reported by the Roman philosopher Pliny the Elder in ˜78 AD, and then much later (1774) by the peripatetic American statesman and natural philosopher Benjamin Franklin, to Japanese "floating-ink" art (suminagashi) developed ˜1000 years ago. The modern science of organic monolayers began in the late-1800s/early-1900s with experiments by Lord Rayleigh and the important development by Agnes Pockels, followed two decades later by Irving Langmuir, of the tools and technology to measure the surface tension of liquids, the surface pressure of organic monolayers deposited on water, interfacial properties, molecular conformation of the organic layers, and phase transitions which occur upon compressing the monolayers. In 1935, Katherine Blodgett published a landmark paper showing that multilayers can be synthesized on solid substrates, with controlled thickness and composition, using an apparatus now known as the Langmuir-Blodgett (L-B) trough. A disadvantage of LB films for some applications is that they form weak physisorbed bonds to the substrate. In 1946, Bigelow, Pickett, and Zisman demonstrated, in another seminal paper, the growth of organic self-assembled monolayers (SAMs) via spontaneous adsorption from solution, rather than from the water/air interface, onto SiO2 and metal substrates. SAMs are close-packed two-dimensional organic crystals which exhibit strong covalent bonding to the substrate. The first multicomponent adsorbed monolayers and multilayer SAMs were produced in the early 1980s. Langmuir monolayers, L-B multilayers, and self-assembled mono- and multilayers have found an extraordinarily broad range of applications including controlled wetting, adhesion, electrochemistry, biocompatibility, molecular recognition, biosensing, cell biology, non-linear optics, molecular electronics, solar cells, read/write/erase memory, and magnetism.

Protective effects of Lactobacillus plantarum against epithelial barrier dysfunction of human colon cell line NCM460

PubMed Central

Liu, Zhi-Hua; Shen, Tong-Yi; Zhang, Peng; Ma, Yan-Lei; Moyer, Mary Pat; Qin, Huan-Long

2010-01-01

AIM: To investigate the effects of Lactobacillus plantarum (L. plantarum) in the intestinal permeability and expression of tight junction (TJ) using the normal human colon cell line NCM460. METHODS: Paracellular permeability of NCM460 monolayers was determined by transepithelial electrical resistance and dextran permeability. Expression of TJ proteins in NCM460 cell monolayers was detected by Western blotting and quantitative real-time polymerase chain reaction. RESULTS: L. plantarum played an important role in increasing transepithelial electrical resistance and decreasing the permeability to macromolecules of NCM460 monolayers against the disruption caused by enteropathogenic Escherichia coli (E. coli) or enteroinvasive E. coli. L. plantarum also prevented the decrease in the expression of TJ proteins and F-actin in NCM460 cells. CONCLUSION: L. plantarum can protect against dysfunction of NCM460 intestinal epithelial barrier caused by enteropathogenic E. coli or enteroinvasive E. coli, and thus can be a potential candidate of therapeutic agents for the treatment of intestinal diseases. PMID:21128328

Brucella dissociation is essential for macrophage egress and bacterial dissemination.

PubMed

Pei, Jianwu; Kahl-McDonagh, Melissa; Ficht, Thomas A

2014-01-01

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4-5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination.

Brucella dissociation is essential for macrophage egress and bacterial dissemination

PubMed Central

Pei, Jianwu; Kahl-McDonagh, Melissa; Ficht, Thomas A.

2014-01-01

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4–5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination. PMID:24634889

Conditioned medium of adipose-derived stromal cell culture in three-dimensional bioreactors for enhanced wound healing.

PubMed

Kwon, Sun Hyun; Bhang, Suk Ho; Jang, Hyeon-Ki; Rhim, Taiyoun; Kim, Byung-Soo

2015-03-01

It was previously shown that human adipose-derived stromal cell (hADSC)-conditioned medium (CM) promotes wound healing. An essential part of the wound healing process is neovascularization in the wound bed. We hypothesized that CM prepared from hADSCs cultured as spheroids in three-dimensional suspension bioreactors (spheroid CM) would contain much higher concentrations of angiogenic growth factors secreted by hADSCs, induce a higher extent of neovascularization in the wound bed, and improve wound healing as compared with CM prepared by conventional monolayer culture (monolayer CM). The concentrations of angiogenic growth factors (i.e., vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor) in spheroid CM were 20- to 145-fold higher than those in monolayer CM. Either fresh medium, monolayer CM, or spheroid CM was administered to full-thickness wounds created on the dorsal aspects of athymic mice. The monolayer CM promoted wound healing as compared with fresh medium or no treatment. Importantly, wound closure was faster, and dermal and epidermal regeneration was improved in the spheroid CM-treated mice compared with that in the monolayer CM-treated mice. The improved wound healing by spheroid CM may be attributed, at least in part, to enhanced neovascularization in the wound beds. The spheroid-based CM approach showed potential as a therapy for skin wound repair. Copyright © 2015 Elsevier Inc. All rights reserved.

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

PubMed

Gordon, Sheldon R; Wood, Meredith

2009-03-01

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence

PubMed Central

Lewis, Natasha S; Lewis, Emily EL; Mullin, Margaret; Wheadon, Helen; Dalby, Matthew J; Berry, Catherine C

2017-01-01

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies. PMID:28616152

Isolation and Characterization of Orientia tsutsugamushi from Rodents Captured following a Scrub Typhus Outbreak at a Military Training Base, Bothong District, Chonburi Province, Central Thailand

DTIC Science & Technology

2011-01-01

pended in M199 medium (Grand Island Biological Company [GIBCO), Grand Island, NY), and subsequently exposed to 3,000-rad irradiation in 60Co gamma... irradiator (Atomic Energy of Canada, Ltd., Ottawa, Canada). Irradiated L-929 cells were dispensed into three 50-cm2 flasks to develop a monolayer...monolayers of irradiated L-929 cells. Inoculated cells were incubated at room temperature for 1 hour on a shaker and then the BHI supernatant was dis

Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

PubMed

Qiu, Xiao-Xu; Liu, Yang; Zhang, Yi-Fan; Guan, Ya-Na; Jia, Qian-Qian; Wang, Chen; Liang, He; Li, Yong-Qin; Yang, Huang-Tian; Qin, Yong-Wen; Huang, Shuang; Zhao, Xian-Xian; Jing, Qing

2017-10-02

Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanyan; Hu, Yahui; Feng, Yidong

2014-01-15

Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity andmore » multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein inhibitors. • The accumulation of HZ08 increased via gene interference targeting P-glycoprotein. • HZ08 competitively bound to P-glycoprotein under the presence of verapamil.« less

Observations on the antibody-dependent cytotoxic cell by scanning electron microscopy.

PubMed Central

Inglis, J R; Penhale, W J; Farmer, A; Irvine, W J; Williams, A E

1975-01-01

The cytotoxic effect of human peripheral blood leucocytes on antibody-coated sheep erythrocyte monolayers has been investigated using scanning electron microscopy. Only a small proportion of leucocytes were found to adhere to the monolayers. A progressive destruction was observed beginning as small plaque-like areas of erythrocyte clearing which later became confluent. Three distinct cell types were found to be associated with the areas of lysis. No destruction was observed in control monolayers incubated for a similar period in the absence of either antibody of leucocytes. Surface changes in the erthrocytes adjacent to the leucocytes suggest that mechanical factors may be involved in erythrocyte lysis in this system. It is concluded that more than one leucocyte type may damage antibody-coated erythrocytes, possibly by a mechanism involving attachment to and mechanical disruption of the red cell membrane. Images FIG. 5 FIG. 2 FIG. 3 FIG. 1 FIG. 2 FIG. 4 PMID:1191386

Monoclonal antibodies directed against surface molecules of multicell spheroids

NASA Technical Reports Server (NTRS)

Martinez, Andrew O.

1993-01-01

The objective of this project is to generate a library of monoclonal antibodies (MAbs) to surface molecules of mammalian tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, three dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture; therefore, MCS make better in vitro model systems to study the interactions of mammalian cells. Additionally, they provide a functional assay for surface adhesion molecules.

In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

PubMed Central

Helms, Hans C; Abbott, N Joan; Burek, Malgorzata; Cecchelli, Romeo; Couraud, Pierre-Olivier; Deli, Maria A; Förster, Carola; Galla, Hans J; Romero, Ignacio A; Shusta, Eric V; Stebbins, Matthew J; Vandenhaute, Elodie; Weksler, Babette

2016-01-01

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This “blood-brain barrier” function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood–brain barrier models with a focus on their validation regarding a set of well-established blood–brain barrier characteristics. As an ideal cell culture model of the blood–brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described. PMID:26868179

Origin of the monolayer Raman signature in hexagonal boron nitride: a first-principles analysis.

PubMed

Ontaneda, Jorge; Singh, Anjali; Waghmare, Umesh V; Grau-Crespo, Ricardo

2018-05-10

Monolayers of hexagonal boron nitride (h-BN) can in principle be identified by a Raman signature, consisting of an upshift in the frequency of the E 2g vibrational mode with respect to the bulk value, but the origin of this shift (intrinsic or support-induced) is still debated. Herein we use density functional theory calculations to investigate whether there is an intrinsic Raman shift in the h-BN monolayer in comparison with the bulk. There is universal agreement among all tested functionals in predicting the magnitude of the frequency shift upon a variation in the in-plane cell parameter. It is clear that a small in-plane contraction can explain the Raman peak upshift from bulk to monolayer. However, we show that the larger in-plane parameter in the bulk (compared to the monolayer) results from non-local correlation effects, which cannot be accounted for by local functionals or those with empirical dispersion corrections. Using a non-local-correlation functional, we then investigate the effect of finite temperatures on the Raman signature. We demonstrate that bulk h-BN thermally expands in the direction perpendicular to the layers, while the intralayer distances slightly contract, in agreement with observed experimental behavior. Interestingly, the difference in in-plane cell parameter between bulk and monolayer decreases with temperature, and becomes very small at room temperature. We conclude that the different thermal expansion of bulk and monolayer partially 'erases' the intrinsic Raman signature, accounting for its small magnitude in recent experiments on suspended samples.

Origin of the monolayer Raman signature in hexagonal boron nitride: a first-principles analysis

NASA Astrophysics Data System (ADS)

Ontaneda, Jorge; Singh, Anjali; Waghmare, Umesh V.; Grau-Crespo, Ricardo

2018-05-01

Monolayers of hexagonal boron nitride (h-BN) can in principle be identified by a Raman signature, consisting of an upshift in the frequency of the E2g vibrational mode with respect to the bulk value, but the origin of this shift (intrinsic or support-induced) is still debated. Herein we use density functional theory calculations to investigate whether there is an intrinsic Raman shift in the h-BN monolayer in comparison with the bulk. There is universal agreement among all tested functionals in predicting the magnitude of the frequency shift upon a variation in the in-plane cell parameter. It is clear that a small in-plane contraction can explain the Raman peak upshift from bulk to monolayer. However, we show that the larger in-plane parameter in the bulk (compared to the monolayer) results from non-local correlation effects, which cannot be accounted for by local functionals or those with empirical dispersion corrections. Using a non-local-correlation functional, we then investigate the effect of finite temperatures on the Raman signature. We demonstrate that bulk h-BN thermally expands in the direction perpendicular to the layers, while the intralayer distances slightly contract, in agreement with observed experimental behavior. Interestingly, the difference in in-plane cell parameter between bulk and monolayer decreases with temperature, and becomes very small at room temperature. We conclude that the different thermal expansion of bulk and monolayer partially ‘erases’ the intrinsic Raman signature, accounting for its small magnitude in recent experiments on suspended samples.

Giant fluctuations and structural effects in a flocking epithelium

NASA Astrophysics Data System (ADS)

Giavazzi, Fabio; Malinverno, Chiara; Corallino, Salvatore; Ginelli, Francesco; Scita, Giorgio; Cerbino, Roberto

2017-09-01

Epithelial cells cultured in a monolayer are very motile in isolation but reach a near-jammed state when mitotic division increases their number above a critical threshold. We have recently shown that a monolayer can be reawakened by over-expression of a single protein, RAB5A, a master regulator of endocytosis. This reawakening of motility was explained in terms of a flocking transition that promotes the emergence of a large-scale collective migratory pattern. Here we focus on the impact of this reawakening on the structural properties of the monolayer. We find that the unjammed monolayer is characterised by a fluidisation at the single cell level, and by enhanced non-equilibrium large-scale number fluctuations at a larger length scale. Also, with the help of numerical simulations, we trace back the origin of these fluctuations to the self-propelled active nature of the constituents, and to the existence of a local alignment mechanism, leading to the spontaneous breaking of the orientational symmetry.

Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

PubMed

Huang, S K; Cheng, M; Hui, S W

1990-11-01

Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values.

The insertion and transport of anandamide in synthetic lipid membranes are both cholesterol-dependent.

PubMed

Di Pasquale, Eric; Chahinian, Henri; Sanchez, Patrick; Fantini, Jacques

2009-01-01

Anandamide is a lipid neurotransmitter which belongs to a class of molecules termed the endocannabinoids involved in multiple physiological functions. Anandamide is readily taken up into cells, but there is considerable controversy as to the nature of this transport process (passive diffusion through the lipid bilayer vs. involvement of putative proteic transporters). This issue is of major importance since anandamide transport through the plasma membrane is crucial for its biological activity and intracellular degradation. The aim of the present study was to evaluate the involvement of cholesterol in membrane uptake and transport of anandamide. Molecular modeling simulations suggested that anandamide can adopt a shape that is remarkably complementary to cholesterol. Physicochemical studies showed that in the nanomolar concentration range, anandamide strongly interacted with cholesterol monolayers at the air-water interface. The specificity of this interaction was assessed by: i) the lack of activity of structurally related unsaturated fatty acids (oleic acid and arachidonic acid at 50 nM) on cholesterol monolayers, and ii) the weak insertion of anandamide into phosphatidylcholine or sphingomyelin monolayers. In agreement with these data, the presence of cholesterol in reconstituted planar lipid bilayers triggered the stable insertion of anandamide detected as an increase in bilayer capacitance. Kinetics transport studies showed that pure phosphatidylcholine bilayers were weakly permeable to anandamide. The incorporation of cholesterol in phosphatidylcholine bilayers dose-dependently stimulated the translocation of anandamide. Our results demonstrate that cholesterol stimulates both the insertion of anandamide into synthetic lipid monolayers and bilayers, and its transport across bilayer membranes. In this respect, we suggest that besides putative anandamide protein-transporters, cholesterol could be an important component of the anandamide transport machinery. Finally, this study provides a mechanistic explanation for the key regulatory activity played by membrane cholesterol in the responsiveness of cells to anandamide.

Three-dimensional printing of Hela cells for cervical tumor model in vitro.

PubMed

Zhao, Yu; Yao, Rui; Ouyang, Liliang; Ding, Hongxu; Zhang, Ting; Zhang, Kaitai; Cheng, Shujun; Sun, Wei

2014-09-01

Advances in three-dimensional (3D) printing have enabled the direct assembly of cells and extracellular matrix materials to form in vitro cellular models for 3D biology, the study of disease pathogenesis and new drug discovery. In this study, we report a method of 3D printing for Hela cells and gelatin/alginate/fibrinogen hydrogels to construct in vitro cervical tumor models. Cell proliferation, matrix metalloproteinase (MMP) protein expression and chemoresistance were measured in the printed 3D cervical tumor models and compared with conventional 2D planar culture models. Over 90% cell viability was observed using the defined printing process. Comparisons of 3D and 2D results revealed that Hela cells showed a higher proliferation rate in the printed 3D environment and tended to form cellular spheroids, but formed monolayer cell sheets in 2D culture. Hela cells in 3D printed models also showed higher MMP protein expression and higher chemoresistance than those in 2D culture. These new biological characteristics from the printed 3D tumor models in vitro as well as the novel 3D cell printing technology may help the evolution of 3D cancer study.

No evidence for a direct role of HLA-B27 in pathological bone formation in axial SpA

PubMed Central

Neerinckx, Barbara; Kollnberger, Simon; Shaw, Jacqueline; Lories, Rik

2017-01-01

Objective The strong genetic association between HLA-B27 and ankylosing spondylitis has been known for over 40 years. HLA-B27 positivity is possibly associated with severity of ankylosis. We studied the in vitro and in vivo impact of HLA-B27 in models of chondrogenesis and osteogenesis. Methods Different in vitro differentiation systems were used to mimic endochondral and direct bone formation. ATDC5 cells and primary human periosteum-derived cells (hPDCs) were transduced with lentiviral vectors expressing HLA-B27 or HLA-B7. These cells and limb bud cells (from HLA-B27 transgenic and wild-type (WT) mice) were cultured in micromasses. To study direct osteogenesis in hPDCs, cells were cultured as monolayers and stimulated with osteogenic media. Chondrogenesis (COL2, ACAN, COL10) and osteogenesis (OSC, ALP, RUNX2) marker expression was studied by quantitative RT-PCR. Colorimetric tests were performed to measure proteoglycans, mineralization and collagens. Collagen antibody-induced arthritis (CAIA) was induced in HLA-B27 transgenic and WT mice. Clinical scoring and µCTs were performed. Statistical analyses were performed by two-way ANOVA. Results There was no difference in chondrogenesis markers or in colorimetric tests between HLA-B27+ and HLA-B7+ micromasses. Expression of osteogenesis markers and Alizarin red staining was comparable in the HLA-B27+ and the HLA-B7+ hPDCs in monolayers. HLA-B27 transgenic mice showed more severe arthritis compared with WT mice in the CAIA model. µCT analysis showed no increased bone formation in HLA-B27 transgenic mice. Conclusion HLA-B27 seems to enhance joint inflammation in the CAIA model. We could not document a direct effect of HLA-B27 on chondrogenesis or osteogenesis. PMID:28879048

Mercury-arc photolysis: a method for examining second messenger regulation of endothelial cell monolayer integrity.

PubMed

Patton, W F; Alexander, J S; Dodge, A B; Patton, R J; Hechtman, H B; Shepro, D

1991-07-01

Cell-cell apposition in bovine pulmonary endothelial cell monolayers was modulated by inducing transient increases in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) and 1,4,5-inositol triphosphate (IP3). This was accomplished by mercury-arc flash photolysis of o-nitrobenzyl derivatives of the second messengers (caged compounds). Second messenger release by the mercury-arc lamp was determined by radioimmunoassay of cAMP to have a t1/2 of approximately 8 min. Each second messenger induced the phosphorylation of a distinct subset of cytoskeletal proteins; however, both IP3 and cAMP increased vimentin phosphorylation. Actin isoform patterns were not altered by the second messengers. Intracellular pulses of IP3 in pulmonary endothelial cells caused disruption of endothelial monolayer integrity as determined by phase-contrast microscopy and by visualization of actin stress fibers with rhodamine-phalloidin. Intracellular pulses of cAMP increased cell-cell contact, cell surface area, and apposition. IP3 appeared to have its greatest effect on the actin peripheral band. In silicone rubber contractility assays this agent caused contraction of pulmonary microvascular endothelial cells as visualized by an increase in wrinkles beneath the cells. On the other hand, cAMP appeared to effect both the peripheral band and centralized actin domains. Caged cAMP caused relaxation of endothelial cells as visualized by a disappearance of wrinkles beneath the cells.

Size-tunable Lateral Confinement in Monolayer Semiconductors

DOE PAGES

Wei, Guohua; Czaplewski, David A.; Lenferink, Erik J.; ...

2017-06-12

Three-dimensional confinement allows semiconductor quantum dots to exhibit size-tunable electronic and optical properties that enable a wide range of opto-electronic applications from displays, solar cells and bio-medical imaging to single-electron devices. Additional modalities such as spin and valley properties in monolayer transition metal dichalcogenides provide further degrees of freedom requisite for information processing and spintronics. In nanostructures, however, spatial confinement can cause hybridization that inhibits the robustness of these emergent properties. Here in this paper, we show that laterally-confined excitons in monolayer MoS 2 nanodots can be created through top-down nanopatterning with controlled size tunability. Unlike chemically-exfoliated monolayer nanoparticles, themore » lithographically patterned monolayer semiconductor nanodots down to a radius of 15 nm exhibit the same valley polarization as in a continuous monolayer sheet. The inherited bulk spin and valley properties, the size dependence of excitonic energies, and the ability to fabricate MoS 2 nanostructures using semiconductor-compatible processing suggest that monolayer semiconductor nanodots have potential to be multimodal building blocks of integrated optoelectronics and spintronics systems« less

Modulation of human cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) in Caco-2 cell monolayers by selected commercial-source milk thistle and goldenseal products.

PubMed

Budzinski, Jason W; Trudeau, Vance L; Drouin, Cathy E; Panahi, Mitra; Arnason, J Thor; Foster, Brian C

2007-09-01

In this study, we used an in vitro Caco-2 cell monolayer model to evaluate aqueous extracts of commercial-source goldenseal (Hydrastis canadensis) and milk thistle (Silybum marianum) capsule formulations, their marker phytochemicals (berberine and silibinin, respectively), as well as dillapiol, vinblastine, and the HIV protease inhibitor saquinavir for their ability to modulate CYP3A4 and ABCB1 expression after short-term exposure (48 h). Both upregulation and downregulation of CYP3A4 expression was observed with extracts of varying concentrations of the two natural health products (NHPs). CYP3A4 was highly responsive in our system, showing a strong dose-dependent modulation by the CYP3A4 inhibitor dillapiol (upregulation) and the milk thistle flavonolignan silibinin (downregulation). ABCB1 was largely unresponsive in this cellular model and appears to be of little value as a biomarker under our experimental conditions. Therefore, the modulation of CYP3A4 gene expression can serve as an important marker for the in vitro assessment of NHP-drug interactions.

Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

PubMed Central

Di Giovanni, George D.; Rochelle, Paul A.

2012-01-01

This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. PMID:22038611

Regional Morphology and Transport of PAMAM Dendrimers Across Isolated Rat Intestinal Tissue.

PubMed

Hubbard, Dallin; Bond, Tanner; Ghandehari, Hamidreza

2015-12-01

Intestinal permeability of PAMAM dendrimers has been observed, giving rationale for their use in oral drug delivery as potential carriers of associated molecules. This study assessed the apparent permeability coefficients (Papp) of dendrimers across isolated rat intestinal regional mucosae, along with estimation of the maximum non-toxic concentration. Caco-2 monolayers were also used to assess the comparative Papp values between isolated mucosae and cell culture models. Concentrations from 0.1 to 10 mM of anionic and cationic dendrimers were tested in mucosae to assess their Papp, membrane TEER, [(14)C]-mannitol Papp, and histology. 0.1 mM concentrations of dendrimers were assessed over 120 min in Caco-2 cell monolayers as concentrations above that were cytotoxic. Jejunal transport of dendrimers was higher than transport in colonic epithelium. Monolayer Papp values of dendrimers were comparable to those of jejunal mucosae. Mucosae exposed to dendrimer concentrations of 10 mM for 120 min caused significant reduction in TEER and changes in tissue morphology; however, G3.5 was the only analogue that caused significant TEER reduction and morphological changes at 1 mM concentrations. Transport in jejunal mucosae appears to be the greatest indicating that the small intestinal will be the most likely region to target for oral drug delivery using PAMAM dendrimers. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Carboxymethyl starch mucoadhesive microspheres as gastroretentive dosage form.

PubMed

Lemieux, Marc; Gosselin, Patrick; Mateescu, Mircea Alexandru

2015-12-30

Carboxymethyl starch microspheres (CMS-MS) were produced from carboxymethyl starch powder (CMS-P) with a degree of substitution (DS) from 0.1 to 1.5 in order to investigate the influence of DS on physicochemical, drug release and mucoadhesion properties as well as interactions with gastrointestinal tract (GIT) epithelial barrier models. Placebo and furosemide loaded CMS-MS were obtained by emulsion-crosslinking with sodium trimetaphosphate (STMP). DS had an impact on increasing equilibrium water uptake and modulating drug release properties of the CMS-MS according to the surrounding pH. The transepithelial electrical resistance (TEER) of NCI-N87 gastric cell monolayers was not influenced in presence of CMS-MS, whereas that of Caco-2 intestinal cell monolayers decreased with increasing DS but recovered initial values at about 15h post-treatment. CMS-MS with increasing DS also enhanced furosemide permeability across both NCI-N87 and Caco-2 monolayers at pH gradients from 3.0 to 7.4. Mucoadhesion of CMS-MS on gastric mucosa (acidic condition) increased with the DS up to 55% for a DS of 1.0 but decreased on neutral intestinal mucosa to less than 10% with DS of 0.1. The drug release, permeability enhancement and mucoadhesive properties of the CMS-MS suggest CMS-MS with DS between 0.6 and 1.0 as suitable excipient for gastroretentive oral delivery dosage forms. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of Vibrational Culture Model Mimicking Vocal Fold Tissues.

PubMed

Kim, Dongjoo; Lim, Jae-Yol; Kwon, Soonjo

2016-10-01

The vocal folds (VFs) are connective tissues with complex matrix structures that provide the required mechanical properties for voice generation. VF injury leads to changes in tissue structure and properties, resulting in reduced voice quality. However, injury-induced biochemical changes and repair in scarred VF tissues have not been well characterized to date. To treat scarred VFs, it is essential to understand how physiological characteristics of VFs tissue change in response to external perturbation. In this study, we designed a simple vibrational culture model to mimic vibratory microenvironments observed in vivo. This model consists of a flexible culture plate, three linear actuators, a stereo splitter, and a function generator. Human vocal fold fibroblast (hVFF) monolayers were established on the flexible membrane, to which normal phonatory vibrations were delivered from linear actuators and a function generator. The hVFF monolayers were exposed to the vibrational stresses at a frequency of 205 Hz for 2, 6, and 10 h with maximum displacement of 47.1 μm, followed by a 6 h rest. We then observed the changes in cell morphology, cell viability, and gene expression related to extracellular matrix components. In our dynamic culture device mimicking normal phonatory frequencies, cell proliferation increased and expression of hyaluronic acid synthase 2 was downregulated in response to vibrational stresses. The results presented herein will be useful for evaluating cellular responses following VF injuries in the presence or absence of vibrational stresses.

Materials for Neural Differentiation, Trans-Differentiation, and Modeling of Neurological Disease.

PubMed

Gong, Lulu; Cao, Lining; Shen, Zhenmin; Shao, Li; Gao, Shaorong; Zhang, Chao; Lu, Jianfeng; Li, Weida

2018-04-01

Neuron regeneration from pluripotent stem cells (PSCs) differentiation or somatic cells trans-differentiation is a promising approach for cell replacement in neurodegenerative diseases and provides a powerful tool for investigating neural development, modeling neurological diseases, and uncovering the mechanisms that underlie diseases. Advancing the materials that are applied in neural differentiation and trans-differentiation promotes the safety, efficiency, and efficacy of neuron regeneration. In the neural differentiation process, matrix materials, either natural or synthetic, not only provide a structural and biochemical support for the monolayer or three-dimensional (3D) cultured cells but also assist in cell adhesion and cell-to-cell communication. They play important roles in directing the differentiation of PSCs into neural cells and modeling neurological diseases. For the trans-differentiation of neural cells, several materials have been used to make the conversion feasible for future therapy. Here, the most current applications of materials for neural differentiation for PSCs, neuronal trans-differentiation, and neurological disease modeling is summarized and discussed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Physical Model of the Dynamic Instability in an Expanding Cell Culture

PubMed Central

Mark, Shirley; Shlomovitz, Roie; Gov, Nir S.; Poujade, Mathieu; Grasland-Mongrain, Erwan; Silberzan, Pascal

2010-01-01

Abstract Collective cell migration is of great significance in many biological processes. The goal of this work is to give a physical model for the dynamics of cell migration during the wound healing response. Experiments demonstrate that an initially uniform cell-culture monolayer expands in a nonuniform manner, developing fingerlike shapes. These fingerlike shapes of the cell culture front are composed of columns of cells that move collectively. We propose a physical model to explain this phenomenon, based on the notion of dynamic instability. In this model, we treat the first layers of cells at the front of the moving cell culture as a continuous one-dimensional membrane (contour), with the usual elasticity of a membrane: curvature and surface-tension. This membrane is active, due to the forces of cellular motility of the cells, and we propose that this motility is related to the local curvature of the culture interface; larger convex curvature correlates with a stronger cellular motility force. This shape-force relation gives rise to a dynamic instability, which we then compare to the patterns observed in the wound healing experiments. PMID:20141748

Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

PubMed

Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

2016-01-01

As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

PubMed Central

Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

2014-01-01

Abstract. Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures. PMID:24390370

X-Ray Synchrotron and Neutron Reflectivity Studies of = Polymer-Modified Lipid Monolayers on Water

NASA Astrophysics Data System (ADS)

Smith, G. S.; Majewski, J.; Kuhl, T.; Israelachvili, J.; Kjaer, K.; Gerstenberg, M. C.; Als-Nielsen, J.

1997-03-01

We studied monolayers (at air-water interface) composed of mixtures of distearoyl phosphatidyl ethanolamine (DSPE) mixed with 1.3, 4.5 and 9% of the same lipid but modified by polyethylene glycol chains (PEG) covalently linked to its head group. The GID data yielded three reflections leading to a hexagonal unit cell a_H=4.7Åarea per lipid molecule = 38.3Åindependent of PEG concentration. The in-plane coherence lengths decreased from 360Åfor the pure lipid to 230Åfor 9.0% DSPE-PEG. The FWHM(q_z) of each of the Bragg rods increased with PEG-lipid concentration suggesting decreasing of the lengths of the coherently diffracting part of the hydrocarbon chains. Reflectivities show that both the density and the extension of the polymer segments increase with DSPE-PEG concentration and can be well modeled with a parabolic density profile. Our data indicates that the bulky hydrophilic polymer disrupts the lipid monolayer. This is attributed to an increase in lipid protrusions and a relaxation of the lateral force between PEG portions by staggering of the lipid headgroups.

Monolayer optical memory cells based on artificial trap-mediated charge storage and release

NASA Astrophysics Data System (ADS)

Lee, Juwon; Pak, Sangyeon; Lee, Young-Woo; Cho, Yuljae; Hong, John; Giraud, Paul; Shin, Hyeon Suk; Morris, Stephen M.; Sohn, Jung Inn; Cha, Seungnam; Kim, Jong Min

2017-03-01

Monolayer transition metal dichalcogenides are considered to be promising candidates for flexible and transparent optoelectronics applications due to their direct bandgap and strong light-matter interactions. Although several monolayer-based photodetectors have been demonstrated, single-layered optical memory devices suitable for high-quality image sensing have received little attention. Here we report a concept for monolayer MoS2 optoelectronic memory devices using artificially-structured charge trap layers through the functionalization of the monolayer/dielectric interfaces, leading to localized electronic states that serve as a basis for electrically-induced charge trapping and optically-mediated charge release. Our devices exhibit excellent photo-responsive memory characteristics with a large linear dynamic range of ~4,700 (73.4 dB) coupled with a low OFF-state current (<4 pA), and a long storage lifetime of over 104 s. In addition, the multi-level detection of up to 8 optical states is successfully demonstrated. These results represent a significant step toward the development of future monolayer optoelectronic memory devices.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Guohua; Czaplewski, David A.; Lenferink, Erik J.

Three-dimensional confinement allows semiconductor quantum dots to exhibit size-tunable electronic and optical properties that enable a wide range of opto-electronic applications from displays, solar cells and bio-medical imaging to single-electron devices. Additional modalities such as spin and valley properties in monolayer transition metal dichalcogenides provide further degrees of freedom requisite for information processing and spintronics. In nanostructures, however, spatial confinement can cause hybridization that inhibits the robustness of these emergent properties. Here in this paper, we show that laterally-confined excitons in monolayer MoS 2 nanodots can be created through top-down nanopatterning with controlled size tunability. Unlike chemically-exfoliated monolayer nanoparticles, themore » lithographically patterned monolayer semiconductor nanodots down to a radius of 15 nm exhibit the same valley polarization as in a continuous monolayer sheet. The inherited bulk spin and valley properties, the size dependence of excitonic energies, and the ability to fabricate MoS 2 nanostructures using semiconductor-compatible processing suggest that monolayer semiconductor nanodots have potential to be multimodal building blocks of integrated optoelectronics and spintronics systems« less

Na/Ca exchange in the basolateral membrane of the A6 cell monolayer: role in Cai homeostasis.

PubMed

Brochiero, E; Raschi, C; Ehrenfeld, J

1995-05-01

The presence of a Na/Ca exchanger in A6 cells was investigated by measuring intracellular calcium (Cai) fluctuations and the 45Ca fluxes through the basolateral membranes (blm) of the cell monolayer. Removal of Na+ from the medium produced a transient increase in Cai followed by a regulatory phase returning Cai to control levels in 3-4 min, this phase being greatly accelerated (< 60 s) by NaCl addition (apparent Km of approximately 5 mM Na+). The Cai increase was only found with the Na(+)-free medium on the basolateral side of the cell monolayer. A twofold increase in the 45Ca influx was observed under these conditions. In Ca(2+)- depleted cells, the initial Cai increase after Ca2+ addition to the medium was greater when the putative Na/Ca exchanger was not functioning (i.e. in a Na(+)-free medium). 45Ca effluxes through the blm of the monolayer were greatly and transiently increased by a Na(+)-free medium on the serosal side and blocked by orthovanadate (1 mM). The Cai increased induced by a hypo-osmotic shock was greater in cells bathed in a Na(+)-medium, conditions expected to block the activity of the Na/Ca exchanger. These findings support the hypothesis that a Na/Ca exchanger is present on the blm of A6 cells and affirm its role in Cai homeostasis in steady-state conditions and following osmotic shock. In addition, a Ca2+ pump also located on the blm and Ca2+ stores sensitive to inositol 1,4,5-trisphosphate were found to be implicated in Cai homeostasis.

Ki-67 as a molecular target for therapy in an in vitro three-dimensional model for ovarian cancer.

PubMed

Rahmanzadeh, Ramtin; Rai, Prakash; Celli, Jonathan P; Rizvi, Imran; Baron-Lühr, Bettina; Gerdes, Johannes; Hasan, Tayyaba

2010-11-15

Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67-directed antibody and subsequent light-triggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti-pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67-positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation. Copyright © 2010 AACR.

Ki-67 as a molecular target for therapy in an in vitro 3D model for ovarian cancer

PubMed Central

Rahmanzadeh, Ramtin; Rai, Prakash; Celli, Jonathan P.; Rizvi, Imran; Baron-Lühr, Bettina; Gerdes, Johannes; Hasan, Tayyaba

2010-01-01

Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully demonstrated. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we demonstrate for the first time, the specific delivery of a Ki-67 directed antibody and subsequent light-triggered death in a human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate encapsulating liposomes (PICELs) were constructed from anti-pKi-67 antibodies conjugated to fluorescein isothiocyanate, as a photoactivatable agent followed by encapsulation in non-cationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67 positive cancer cells both in monolayer and in 3D cultures of OVCAR-5 cells with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of: - 1. the exploitation of Ki-67 as a molecular target for therapy and - 2. specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation. PMID:21045152

Absorption and metabolism of the food contaminant 3-chloro-1,2-propanediol (3-MCPD) and its fatty acid esters by human intestinal Caco-2 cells.

PubMed

Buhrke, Thorsten; Weisshaar, Rüdiger; Lampen, Alfonso

2011-10-01

3-Chloro-1,2-propanediol (3-MCPD) fatty acid esters are formed upon thermal processing of fat-containing foods in the presence of chloride ions. Upon hydrolytic cleavage, these substances could release free 3-MCPD. This compound is toxicologically well characterised and displayed cancerogenic potential in rodent models. Recently, serious contaminations of different food products with 3-MCPD fatty acid esters have been reported. In regard to a risk assessment, the key question is to which degree these 3-MCPD fatty acid esters are hydrolysed in the human gut. Therefore, the aim of the present project was to examine the hydrolysis of 3-MCPD fatty acid esters and the resulting release of free 3-MCPD by using differentiated Caco-2 cells, a cellular in vitro model for the human intestinal barrier. Here, we show that 3-MCPD fatty acid esters at a concentration of 100 μM were neither absorbed by the cells nor the esters were transported via a Caco-2 monolayer. 3-MCPD-1-monoesters were hydrolysed in the presence of Caco-2 cells. In contrast, a 3-MCPD-1,2-diester used in this study was obviously absorbed and metabolised by the cells. Free 3-MCPD was not absorbed by the cells, but the substance migrated through a Caco-2 monolayer by paracellular diffusion. From these in vitro studies, we conclude that 3-MCPD-1-monoesters are likely to be hydrolysed in the human intestine, thereby increasing the burden with free 3-MCPD. In contrast, intestinal cells seem to have the capacity to metabolise 3-MCPD diesters, thereby detoxifying the 3-MCPD moiety.

Genetic changes in mammalian cells transformed by helium ions

NASA Astrophysics Data System (ADS)

Durante, M.; Grossi, G.; Yang, T. C.; Roots, R.

Midterm Syrian Hamster embryo (SHE) cells were employed to study high LET-radiation induced tumorigenesis. Normal SHE cells (secondary passage) were irradiated with accelerated helium ions at an incident energy of 22 MeV/u (9-10 keV/μm). Transformed clones were isolated after growth in soft agar of cells obtained from the foci of the initial monolayer plated postirradiation. To study the progression process of malignant transformation, the transformed clones were followed by monolayer subculturing for prolonged periods of time. Subsequently, neoplasia tests in nude mice were done. In this work, however, we have focused on karyotypic changes in the banding patterns of the chromosomes during the early part of the progressive process of cell transformation for helium ion-induced transformed cells.

An orientation-independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a Cnidarian model

PubMed Central

Malamy, Jocelyn; Shribak, Michael

2017-01-01

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation-independent differential interference contrast (OI-DIC) microscope for in vivo imaging of wound healing. OI-DIC provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, non-transgenic animal model. In particular, the OI-DIC microscope equipped with a 40×/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized. PMID:29345317

An orientation-independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model.

PubMed

Malamy, J E; Shribak, M

2018-06-01

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation-independent differential interference contrast microscope for in vivo imaging of wound healing. Orientation-independent differential interference contrast provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, nontransgenic animal model. In particular, the orientation-independent differential interference contrast microscope equipped with a 40x/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Band structure and orbital character of monolayer MoS2 with eleven-band tight-binding model

NASA Astrophysics Data System (ADS)

Shahriari, Majid; Ghalambor Dezfuli, Abdolmohammad; Sabaeian, Mohammad

2018-02-01

In this paper, based on a tight-binding (TB) model, first we present the calculations of eigenvalues as band structure and then present the eigenvectors as probability amplitude for finding electron in atomic orbitals for monolayer MoS2 in the first Brillouin zone. In these calculations we are considering hopping processes between the nearest-neighbor Mo-S, the next nearest-neighbor in-plan Mo-Mo, and the next nearest-neighbor in-plan and out-of-plan S-S atoms in a three-atom based unit cell of two-dimensional rhombic MoS2. The hopping integrals have been solved in terms of Slater-Koster and crystal field parameters. These parameters are calculated by comparing TB model with the density function theory (DFT) in the high-symmetry k-points (i.e. the K- and Γ-points). In our TB model all the 4d Mo orbitals and the 3p S orbitals are considered and detailed analysis of the orbital character of each energy level at the main high-symmetry points of the Brillouin zone is described. In comparison with DFT calculations, our results of TB model show a very good agreement for bands near the Fermi level. However for other bands which are far from the Fermi level, some discrepancies between our TB model and DFT calculations are observed. Upon the accuracy of Slater-Koster and crystal field parameters, on the contrary of DFT, our model provide enough accuracy to calculate all allowed transitions between energy bands that are very crucial for investigating the linear and nonlinear optical properties of monolayer MoS2.

Bacillus subtilis Swarmer Cells Lead the Swarm, Multiply, and Generate a Trail of Quiescent Descendants.

PubMed

Hamouche, Lina; Laalami, Soumaya; Daerr, Adrian; Song, Solène; Holland, I Barry; Séror, Simone J; Hamze, Kassem; Putzer, Harald

2017-02-07

Bacteria adopt social behavior to expand into new territory, led by specialized swarmers, before forming a biofilm. Such mass migration of Bacillus subtilis on a synthetic medium produces hyperbranching dendrites that transiently (equivalent to 4 to 5 generations of growth) maintain a cellular monolayer over long distances, greatly facilitating single-cell gene expression analysis. Paradoxically, while cells in the dendrites (nonswarmers) might be expected to grow exponentially, the rate of swarm expansion is constant, suggesting that some cells are not multiplying. Little attention has been paid to which cells in a swarm are actually multiplying and contributing to the overall biomass. Here, we show in situ that DNA replication, protein translation and peptidoglycan synthesis are primarily restricted to the swarmer cells at dendrite tips. Thus, these specialized cells not only lead the population forward but are apparently the source of all cells in the stems of early dendrites. We developed a simple mathematical model that supports this conclusion. Swarming motility enables rapid coordinated surface translocation of a microbial community, preceding the formation of a biofilm. This movement occurs in thin films and involves specialized swarmer cells localized to a narrow zone at the extreme swarm edge. In the B. subtilis system, using a synthetic medium, the swarm front remains as a cellular monolayer for up to 1.5 cm. Swarmers display high-velocity whirls and vortexing and are often assumed to drive community expansion at the expense of cell growth. Surprisingly, little attention has been paid to which cells in a swarm are actually growing and contributing to the overall biomass. Here, we show that swarmers not only lead the population forward but continue to multiply as a source of all cells in the community. We present a model that explains how exponential growth of only a few cells is compatible with the linear expansion rate of the swarm. Copyright © 2017 Hamouche et al.

9 CFR 113.47 - Detection of extraneous viruses by the fluorescent antibody technique.

Code of Federal Regulations, 2010 CFR

2010-01-01

...; (ii) Reovirus; and (iii) Rabies virus. (2) Bovine, caprine, and ovine cells shall, in addition, be... have rabies virus on premises either for research or production purposes are exempt from having to produce positive rabies virus control monolayers. Fixed positive rabies virus control monolayers will be...

The behavior of commensurate-incommensurate transitions using the phase field crystal model

NASA Astrophysics Data System (ADS)

Zhang, Tinghui; Lu, Yanli; Chen, Zheng

2018-02-01

We study the behavior of the commensurate-incommensurate (CI) transitions by using a phase field crystal model. The model is capable of modeling both elastic and plastic deformation and can simulate the evolution of the microstructure of the material at the atomic scale and the diffusive time scale, such as for adsorbed monolayer. Specifically, we study the behavior of the CI transitions as a function of lattice mismatch and the amplitude of substrate pinning potential. The behavior of CI phase transitions is revealed with the increase of the amplitude of pinning potential in some certain lattice mismatches. We find that for the negative lattice mismatch absorbed monolayer undergoes division, reorganization and displacement as increasing the amplitude of substrate pinning potential. In addition, for the positive mismatch absorbed monolayer undergoes a progress of phase transformation after a complete grain is split. Our results accord with simulations for atomic models of absorbed monolayer on a substrate surface.

Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis

PubMed Central

Karpurapu, Manjula; Lee, Yong Gyu; Qian, Ziqing; Wen, Jin; Ballinger, Megan N.; Rusu, Luiza; Chung, Sangwoon; Deng, Jing; Qian, Feng; Reader, Brenda F.; Nirujogi, Teja Srinivas; Park, Gye Young; Pei, Dehua; Christman, John W.

2018-01-01

Specific therapies targeting cellular and molecular events of sepsis induced Acute Lung Injury (ALI) pathogenesis are lacking. We have reported a pivotal role for Nuclear Factors of Activated T cells (NFATc3) in regulating macrophage phenotype during sepsis induced ALI and subsequent studies demonstrate that NFATc3 transcriptionally regulates macrophage CCR2 and TNFα gene expression. Mouse pulmonary microvascular endothelial cell monolayer maintained a tighter barrier function when co-cultured with LPS stimulated NFATc3 deficient macrophages whereas wild type macrophages caused leaky monolayer barrier. More importantly, NFATc3 deficient mice showed decreased neutrophilic lung inflammation, improved alveolar capillary barrier function, arterial oxygen saturation and survival benefit in lethal CLP sepsis mouse models. In addition, survival of wild type mice subjected to the lethal CLP sepsis was not improved with broad-spectrum antibiotics, whereas the survival of NFATc3 deficient mice was improved to 40–60% when treated with imipenem. Passive adoptive transfer of NFATc3 deficient macrophages conferred protection against LPS induced ALI in wild type mice. Furthermore, CP9-ZIZIT, a highly potent, cell-permeable peptide inhibitor of Calcineurin inhibited NFATc3 activation. CP9-ZIZIT effectively reduced sepsis induced inflammatory cytokines and pulmonary edema in mice. Thus, this study demonstrates that inhibition of NFATc3 activation by CP9-ZIZIT provides a potential therapeutic option for attenuating sepsis induced ALI/pulmonary edema. PMID:29535830

Delivery of paclitaxel across cellular barriers using a dendrimer-based nanocarrier.

PubMed

Teow, Huey Minn; Zhou, Zhengyuan; Najlah, Mohammad; Yusof, Siti R; Abbott, N Joan; D'Emanuele, Antony

2013-01-30

The aim of this study was to investigate the ability of a third-generation (G3) polyamidoamine (PAMAM) dendrimer-based carrier to enhance the permeability of paclitaxel (pac) and to overcome cellular barriers. G3 dendrimers were surface modified with lauryl chains (L) and conjugated with paclitaxel (pac) via a glutaric anhydride (glu) linker, followed by labeling with FITC. Biological evaluation of the dendrimer and conjugates was conducted using the human colon adenocarcinoma cell line (Caco-2) and primary cultured porcine brain endothelial cells (PBECs). LDH assay was used to evaluate the cytotoxicity of the dendrimer and conjugates. Cytotoxicity studies showed that the conjugation of lauryl chains and paclitaxel on G3 dendrimer significantly (p<0.05) increased the cytotoxicity against both cell types. Permeability studies of dendrimer-drug conjugates demonstrated an increase in the apparent permeability coefficient (P(app)) in both apical to basolateral A→B and basolateral to apical B→A directions across both cell monolayers compared to unmodified G3 and free drug. The B→A P(app) of paclitaxel was significantly (p<0.05) higher than the A→B P(app), indicating active function of P-gp efflux transporter system in both cell models. L6-G3-glu-pac conjugate had approximately 12-fold greater permeability across both cell monolayers than that of paclitaxel alone. Copyright © 2012 Elsevier B.V. All rights reserved.

Developing multi-cellular tumor spheroid model (MCTS) in the chitosan/collagen/alginate (CCA) fibrous scaffold for anticancer drug screening.

PubMed

Wang, Jian-Zheng; Zhu, Yu-Xia; Ma, Hui-Chao; Chen, Si-Nan; Chao, Ji-Ye; Ruan, Wen-Ding; Wang, Duo; Du, Feng-guang; Meng, Yue-Zhong

2016-05-01

In this work, a 3D MCTS-CCA system was constructed by culturing multi-cellular tumor spheroid (MCTS) in the chitosan/collagen/alginate (CCA) fibrous scaffold for anticancer drug screening. The CCA scaffolds were fabricated by spray-spinning. The interactions between the components of the spray-spun fibers were evidenced by methods of Coomassie Blue stain, X-ray diffraction (XRD) and Fourier transform-infrared spectroscopy (FTIR). Co-culture indicated that MCF-7 cells showed a spatial growth pattern of multi-cellular tumor spheroid (MCTS) in the CCA fibrous scaffold with increased proliferation rate and drug-resistance to MMC, ADM and 5-Aza comparing with the 2D culture cells. Significant increases of total viable cells were found in 3D MCTS groups after drug administration by method of apoptotic analysis. Glucose-lactate analysis indicated that the metabolism of MCTS in CCA scaffold was closer to the tumor issue in vivo than the monolayer cells. In addition, MCTS showed the characteristic of epithelial mesenchymal transition (EMT) which is subverted by carcinoma cells to facilitate metastatic spread. These results demonstrated that MCTS in CCA scaffold possessed a more conservative phenotype of tumor than monolayer cells, and anticancer drug screening in 3D MCTS-CCA system might be superior to the 2D culture system. Copyright © 2016 Elsevier B.V. All rights reserved.

Centrifugation assay for measuring adhesion of serially passaged bovine chondrocytes to polystyrene surfaces.

PubMed

Kaplan, David S; Hitchins, Victoria M; Vegella, Thomas J; Malinauskas, Richard A; Ferlin, Kimberly M; Fisher, John P; Frondoza, Carmelita G

2012-07-01

A major obstacle in chondrocyte-based therapy for cartilage repair is the limited availability of cells that maintain their original phenotype. Propagation of chondrocytes as monolayer cultures on polystyrene surfaces is used extensively for amplifying cell numbers. However, chondrocytes undergo a phenotypic shift when propagated in this manner and display characteristics of more adherent fibroblastic cells. Little information is available about the effect of this phenotypic shift on cellular adhesion properties. We evaluated changes in adhesion property as bovine chondrocytes were serially propagated up to five passages in monolayer culture using a centrifugation cell adhesion assay, which was based on counting of cells before and after being exposed to centrifugal dislodgement forces of 120 and 350 g. Chondrocytes proliferated well in a monolayer culture with doubling times of 2-3 days, but they appeared more fibroblastic and exhibited elongated cell morphology with continued passage. The centrifugation cell adhesion assay showed that chondrocytes became more adhesive with passage as the percentage of adherent cells after centrifugation increased and was not statistically different from the adhesion of the fibroblast cell line, L929, starting at passage 3. This increased adhesiveness correlated with a shift to a fibroblastic morphology and increased collagen I mRNA expression starting at passage 2. Our findings indicate that the centrifugation cell adhesion assay may serve as a reproducible tool to track alterations in chondrocyte phenotype during their extended propagation in culture.

Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.

PubMed

Liu, Chun; Oikonomopoulos, Angelos; Sayed, Nazish; Wu, Joseph C

2018-03-08

The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimic in vivo tissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling. © 2018. Published by The Company of Biologists Ltd.

Toxicity testing of four silver nanoparticle-coated dental castings in 3-D LO2 cell cultures.

PubMed

Zhao, Yi-Ying; Chu, Qiang; Shi, Xu-Er; Zheng, Xiao-Dong; Shen, Xiao-Ting; Zhang, Yan-Zhen

To address the controversial issue of the toxicity of dental alloys and silver nanoparticles in medical applications, an in vivo-like LO2 3-D model was constructed within polyvinylidene fluoride hollow fiber materials to mimic the microenvironment of liver tissue. The use of microscopy methods and the measurement of liver-specific functions optimized the model for best cell performances and also proved the superiority of the 3-D LO2 model when compared with the traditional monolayer model. Toxicity tests were conducted using the newly constructed model, finding that four dental castings coated with silver nanoparticles were toxic to human hepatocytes after cell viability assays. In general, the toxicity of both the castings and the coated silver nanoparticles aggravated as time increased, yet the nanoparticles attenuated the general toxicity by preventing metal ion release, especially at high concentrations.

Analysis of Reparative Activity of Platelet Lysate: Effect on Cell Monolayer Recovery In Vitro and Skin Wound Healing In Vivo.

PubMed

Sergeeva, N S; Shanskii, Ya D; Sviridova, I K; Karalkin, P A; Kirsanova, V A; Akhmedova, S A; Kaprin, A D

2016-11-01

Platelet lysate prepared from donor platelet concentrate and pooled according to a developed technique stimulates migration of multipotent mesenchymal stromal cells of the human adipose tissue and promotes healing of the monolayer defect in cultures of human fibroblasts and multipotent mesenchymal stromal cells in vitro in concentrations close those of fetal calf serum (5-10%). Lysate of platelets from platelet-rich rat blood plasma stimulated healing of the skin defect by promoting epithelialization and granulation tissue formation. The regenerative properties of platelet lysate in vivo increased with increasing its concentration.

6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death

PubMed Central

Ray, Anasuya; Vasudevan, Smreti; Sengupta, Suparna

2015-01-01

Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44+CD24-/low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment. PMID:26355461

6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death.

PubMed

Ray, Anasuya; Vasudevan, Smreti; Sengupta, Suparna

2015-01-01

Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44+CD24-/low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.

Comparison of methods for cultivation and isolation of Chlamydia trachomatis.

PubMed Central

Benes, S; McCormack, W M

1982-01-01

McCoy cells treated with cycloheximide, iododeoxyuridine, and DEAE-dextran and untreated McCoy cells were inoculated with two stock strains of Chlamydia trachomatis and with 231 urethral specimens from men, 53 (23%) of which contained C. trachomatis. Isolation rates, number and quality of inclusions, and quality of the cell monolayers were compared. There were no significant differences between the isolation rates in the four systems, although the most isolations were made in the untreated and cycloheximide-treated cells. Cycloheximide-treated cells produced, from both the clinical specimens and the two stock strains, significantly more inclusions than any of the other systems. The monolayer of the cycloheximide-treated cells and the inclusions that grew in these cells were optimal for examination and detection of C. trachomatis. PMID:6185530

Indicators of replicative damage in equine tendon fibroblast monolayers

PubMed Central

2013-01-01

Background Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it. Results We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents. Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress. Conclusions Researchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled. PMID:24025445

Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers

NASA Astrophysics Data System (ADS)

Tang, Yadong; Liu, Li; Li, Junjun; Yu, Leqian; Wang, Li; Shi, Jian; Chen, Yong

2016-07-01

Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell-medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape of the hiPSC colony could be controlled from a flat layer to a hemisphere. Afterwards, the induction and differentiation of hiPSCs were achieved on the same patch, leading to a uniform cardiac layer with homogeneous contraction. This cardiac layer could then be used for extracellular recording with a commercial multi-electrode array, showing representative field potential waveforms of matured cardiac tissues with appropriate drug responses.Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell-medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape of the hiPSC colony could be controlled from a flat layer to a hemisphere. Afterwards, the induction and differentiation of hiPSCs were achieved on the same patch, leading to a uniform cardiac layer with homogeneous contraction. This cardiac layer could then be used for extracellular recording with a commercial multi-electrode array, showing representative field potential waveforms of matured cardiac tissues with appropriate drug responses. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr04545f

Influence of airway wall compliance on epithelial cell injury and adhesion during interfacial flows

PubMed Central

Higuita-Castro, Natalia; Mihai, Cosmin; Hansford, Derek J.

2014-01-01

Interfacial flows during cyclic airway reopening are an important source of ventilator-induced lung injury. However, it is not known how changes in airway wall compliance influence cell injury during airway reopening. We used an in vitro model of airway reopening in a compliant microchannel to investigate how airway wall stiffness influences epithelial cell injury. Epithelial cells were grown on gel substrates with different rigidities, and cellular responses to substrate stiffness were evaluated in terms of metabolic activity, mechanics, morphology, and adhesion. Repeated microbubble propagations were used to simulate cyclic airway reopening, and cell injury and detachment were quantified via live/dead staining. Although cells cultured on softer gels exhibited a reduced elastic modulus, these cells experienced less plasma membrane rupture/necrosis. Cells on rigid gels exhibited a minor, but statistically significant, increase in the power law exponent and also exhibited a significantly larger height-to-length aspect ratio. Previous studies indicate that this change in morphology amplifies interfacial stresses and, therefore, correlates with the increased necrosis observed during airway reopening. Although cells cultured on stiff substrates exhibited more plasma membrane rupture, these cells experienced significantly less detachment and monolayer disruption during airway reopening. Western blotting and immunofluorescence indicate that this protection from detachment and monolayer disruption correlates with increased focal adhesion kinase and phosphorylated paxillin expression. Therefore, changes in cell morphology and focal adhesion structure may govern injury responses during compliant airway reopening. In addition, these results indicate that changes in airway compliance, as occurs during fibrosis or emphysema, may significantly influence cell injury during mechanical ventilation. PMID:25213636

Lipid monolayer structure and interactions in the presence of peptides and proteins

NASA Astrophysics Data System (ADS)

Freites, Juan Alfredo

Structural aspects of two simple model systems, protein-lipid monolayer and peptide-lipid monolayer, were studied by experimental and computer simulation techniques. In both cases, both the choice of system and the approach employed to studying it, were motivated by specific biological problems. The interaction of annexin A1 with monolayers of dipalmitoylphosphatidylcholine (DPPC) was studied by fluorescence microscopy as a function of lipid monolayer phase and pH. It was shown that the annexin A1-DPPC interaction depends strongly on both the domain structure and phase behavior of the DPPC monolayer, and only weakly on the subphase pH. Annexin A1 was found to be line-active, adsorbing preferentially at phase boundaries. Also, annexin A1 was found to form networks in the presence of a domain structure in the lipid monolayer. Molecular dynamics simulations were carried out on a model system composed of a surfactant protein B peptide, SP-B1--25, and a monolayer of hexadecanoic acid. A detailed structural characterization was performed as a function of the lipid monolayer specic area. It was found that the peptide remains inserted in the monolayer up to values of specific area corresponding to an untilted condensed phase of the pure hexadecanoic acid monolayer. The system remains stable by altering the conformational order of both the anionic lipid monolayer and the peptide secondary structure, and effectively constitutes a unique disordered lipid-peptide monolayer phase. Two elements appear to be key for the constitution of this phase: an electrostatic interaction between the cationic residues of the peptide with the anionic headgroups of the lipids, and an exclusion of the aromatic residues on the hydrophobic end of the peptide from the hydrophilic and aqueous regions of the system. A direct comparison between molecular dynamics simulations and laboratory experiments was performed for hexadecanoic acid monolayer systems. In order to simulate specific points on the surface pressure vs. area isotherm, an algorithm for the control of surface pressure was developed based on previous work by Martyna, Tobias and Klein. The algorithm was implemented and tested with the hexadecanoic acid monolayer system.

Simulation of lung alveolar epithelial wound healing in vitro.

PubMed

Kim, Sean H J; Matthay, Michael A; Mostov, Keith; Hunt, C Anthony

2010-08-06

The mechanisms that enable and regulate alveolar type II (AT II) epithelial cell wound healing in vitro and in vivo remain largely unknown and need further elucidation. We used an in silico AT II cell-mimetic analogue to explore and better understand plausible wound healing mechanisms for two conditions: cyst repair in three-dimensional cultures and monolayer wound healing. Starting with the analogue that validated for key features of AT II cystogenesis in vitro, we devised an additional cell rearrangement action enabling cyst repair. Monolayer repair was enabled by providing 'cells' a control mechanism to switch automatically to a repair mode in the presence of a distress signal. In cyst wound simulations, the revised analogue closed wounds by adhering to essentially the same axioms available for alveolar-like cystogenesis. In silico cell proliferation was not needed. The analogue recovered within a few simulation cycles but required a longer recovery time for larger or multiple wounds. In simulated monolayer wound repair, diffusive factor-mediated 'cell' migration led to repair patterns comparable to those of in vitro cultures exposed to different growth factors. Simulations predicted directional cell locomotion to be critical for successful in vitro wound repair. We anticipate that with further use and refinement, the methods used will develop as a rigorous, extensible means of unravelling mechanisms of lung alveolar repair and regeneration.

Inflammatory parameters in Caco-2 cells: effect of stimuli nature, concentration, combination and cell differentiation.

PubMed

Van De Walle, Jacqueline; Hendrickx, Aurélie; Romier, Béatrice; Larondelle, Yvan; Schneider, Yves-Jacques

2010-08-01

Enterocytes regulate gut maintenance and defence by secreting and responding to inflammatory mediators and by modulating the intestinal epithelial permeability. In order to develop an in vitro model of the acute phase of intestinal inflammation, Caco-2 cells were exposed to the inflammatory mediators IL-1beta, TNF-alpha, IFN-gamma and LPS, and the importance of several experimental parameters, i.e. cell differentiation, stimulus nature, concentration and combination on the inflammatory response was assessed by measuring the production of IL-6, IL-8, PGE-2 and NO and by evaluating the monolayer permeability. A maximal increase in IL-8, IL-6 and PGE-2 production and monolayer permeability was observed when using the cytokines simultaneously at their highest level, but this relied mainly on IL-1beta. The effects of TNF-alpha on IL-8 and IL-6 or NO production were stronger upon combination with IL-1beta or IFN-gamma, respectively, whereas cells were unaffected by the presence of LPS. Although NO production, induced by IFN-gamma-containing combinations, was observed only in differentiated cells, general inflammatory response was higher in proliferating cells. The use of a mixture of IL-1beta, TNF-alpha and IFN-gamma thus accurately mimics intestinal inflammatory processes, but cell differentiation and stimuli combination are important parameters to take into account for in vitro studies on intestinal inflammation. Copyright (c) 2010. Published by Elsevier Ltd.

Synthesis and release of fatty acids by human trophoblast cells in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coleman, R.A.; Haynes, E.B.

1987-11-01

In order to determine whether placental cells can synthesize and release fatty acids, trophoblast cells from term human placentas were established in monolayer culture. The cells continued to secrete placental lactogen and progesterone and maintained specific activities of critical enzymes of triacylglycerol and phosphatidylcholine biosynthesis for 24 to 72 hr in culture. Fatty acid was rapidly synthesized from (/sup 14/C)acetate and released by the cells. Palmitoleic, palmitic, and oleic acids were the major fatty acids synthesized from (/sup 14/C)acetate and released. Small amounts of lauric, myristic, and stearic acids were also identified. (/sup 14/C)acetate was also incorporated into cellular triacylglycerol,more » phospholipid, and cholesterol, but radiolabeled free fatty acid did not accumulate intracellularly. In a pulse-chase experiment, cellular glycerolipids were labeled with (1-/sup 14/C)oleate; trophoblast cells then released /sup 14/C-labeled fatty acid into the media as the cellular content of labeled phospholipid and triacylglycerol decreased without intracellular accumulation of free fatty acid. Twenty percent of the /sup 14/C-label lost from cellular glycerolipid could not be recovered as a chloroform-extractable product, suggesting that some of the hydrolyzed fatty acid had been oxidized. These data indicate that cultured placenta trophoblast cells can release fatty acids that have either been synthesized de novo or that have been hydrolyzed from cellular glycerolipids. Trophoblast cells in monolayer culture should provide an excellent model for molecular studies of placental fatty acid metabolism and release.« less

Highly Stable Bonding of Thiol Monolayers to Hydrogen-Terminated Si via Supercritical Carbon Dioxide: Toward a Super Hydrophobic and Bioresistant Surface.

PubMed

Bhartia, Bhavesh; Puniredd, Sreenivasa Reddy; Jayaraman, Sundaramurthy; Gandhimathi, Chinnasamy; Sharma, Mohit; Kuo, Yen-Chien; Chen, Chia-Hao; Reddy, Venugopal Jayarama; Troadec, Cedric; Srinivasan, Madapusi Palavedu

2016-09-21

Oxide-free silicon chemistry has been widely studied using wet-chemistry methods, but for emerging applications such as molecular electronics on silicon, nanowire-based sensors, and biochips, these methods may not be suitable as they can give rise to defects due to surface contamination, residual solvents, which in turn can affect the grafted monolayer devices for practical applications. Therefore, there is a need for a cleaner, reproducible, scalable, and environmentally benign monolayer grafting process. In this work, monolayers of alkylthiols were deposited on oxide-free semiconductor surfaces using supercritical carbon dioxide (SCCO2) as a carrier fluid owing to its favorable physical properties. The identity of grafted monolayers was monitored with Fourier transform infrared (FTIR) spectroscopy, high-resolution X-ray photoelectron spectroscopy (HRXPS), XPS, atomic force microscopy (AFM), contact angle measurements, and ellipsometry. Monolayers on oxide-free silicon were able to passivate the surface for more than 50 days (10 times than the conventional methods) without any oxide formation in ambient atmosphere. Application of the SCCO2 process was further extended by depositing alkylthiol monolayers on fragile and brittle 1D silicon nanowires (SiNWs) and 2D germanium substrates. With the recent interest in SiNWs for biological applications, the thiol-passivated oxide-free silicon nanowire surfaces were also studied for their biological response. Alkylthiol-functionalized SiNWs showed a significant decrease in cell proliferation owing to their superhydrophobicity combined with the rough surface morphology. Furthermore, tribological studies showed a sharp decrease in the coefficient of friction, which was found to be dependent on the alkyl chain length and surface bond. These studies can be used for the development of cost-effective and highly stable monolayers for practical applications such as solar cells, biosensors, molecular electronics, micro- and nano- electromechanical systems, antifouling agents, and drug delivery.

Impact of Lipid Oxidization on Vertical Structures and Electrostatics of Phospholipid Monolayers Revealed by Combination of Specular X-ray Reflectivity and Grazing-Incidence X-ray Fluorescence.

PubMed

Korytowski, Agatha; Abuillan, Wasim; Makky, Ali; Konovalov, Oleg; Tanaka, Motomu

2015-07-30

The influence of phospholipid oxidization of floating monolayers on the structure perpendicular to the global plane and on the density profiles of ions near the lipid monolayer has been investigated by a combination of grazing incidence X-ray fluorescence (GIXF) and specular X-ray reflectivity (XRR). Systematic variation of the composition of the floating monolayers unravels changes in the thickness, roughness and electron density of the lipid monolayers as a function of molar fraction of oxidized phospholipids. Simultaneous GIXF measurements enable one to qualitatively determine the element-specific density profiles of monovalent (K(+) or Cs(+)) and divalent ions (Ca(2+)) in the vicinity of the interface in the presence and absence of two types of oxidized phospholipids (PazePC and PoxnoPC) with high spatial accuracy (±5 Å). We found the condensation of Ca(2+) near carboxylated PazePC was more pronounced compared to PoxnoPC with an aldehyde group. In contrast, the condensation of monovalent ions could hardly be detected even for pure oxidized phospholipid monolayers. Moreover, pure phospholipid monolayers exhibited almost no ion specific condensation near the interface. The quantitative studies with well-defined floating monolayers revealed how the elevation of lipid oxidization level alters the structures and functions of cell membranes.

Cytokine-mediated induction of endothelial adhesion molecule and histocompatibility leukocyte antigen expression by cytomegalovirus-activated T cells.

PubMed Central

Waldman, W. J.; Knight, D. A.

1996-01-01

Cytomegalovirus (CMV) has been associated with allograft rejection and transplantation-associated arteriosclerosis. CMV infects endothelium, the interface between allograft tissue and the host immune system; however, mechanisms by which such interaction might exacerbate the rejection process remain unresolved. Here we test the hypothesis that host immune activity, triggered by CMV-infected graft endothelial cells (ECs), can result in the production of cytokines capable of enhancing the alloimmunogenicity of nearby uninfected endothelia. To model these phenomena in vitro, confluent monolayers of ECs derived from human umbilical vein or adult gonadal vein were incubated 5 days beneath trans-well culture inserts containing CMV-seropositive or CMV-seronegative donor-derived CD3+ or CD4+ T cells alone or in combination with CMV-infected or uninfected allogeneic ECs. The extent of T cell proliferation was determined by [3H]thymidine labeling of trans-well contents after transfer to microtiter plates. Endothelial responses to soluble factors elaborated by CMV-activated T cells were determined by immunohistochemical staining and immunofluorescence flow cytometric analysis of underlying EC monolayers. Results of experiments with CMV-seropositive donor-derived CD4+ T cells demonstrated enhancement of ICAM-1 and histocompatibility leukocyte antigen class I, as well as induction of histocompatibility leukocyte antigen DR on ECs incubated beneath T cell/EC/CMV trans-well co-cultures. Total (CD3+) T cells co-cultured with EC/CMV induced VCAM-1 as well. Furthermore, [3H]thymidine incorporation by these T cells indicated a strong proliferative response. Endothelial responses to T cells alone or in combination with uninfected ECs were minimal, and T cells cultured under these conditions showed little proliferative activity. Similarly, little or no endothelial responses were apparent in monolayers beneath trans-wells containing T cells isolated from CMV-seronegative individuals regardless of the CMV status of stimulator ECs. Finally, experiments employing blocking antibodies identified interferon-gamma and tumor necrosis factor-alpha as inducing agents in this co-culture system. These findings suggest that allograft endothelium harboring CMV has the potential to activate host T cells and that the consequent release of cytokines shows potential to raise surrounding endothelia to a fully activated, highly immunogenic state. Results of these studies thus provide insight into mechanisms that help elucidate the association between CMV and transplantation-associated arteriosclerosis and/or allograft rejection. Images Figure 1 Figure 5 PMID:8546198

Microbial metabolites, but not other phenolics derived from grape seed phenolic extract, are transported through differentiated Caco-2 cell monolayers.

PubMed

Wang, Dongjie; Williams, Barbara A; Ferruzzi, Mario G; D'Arcy, Bruce R

2013-06-01

Grape seed phenolic extract (GSE) is predicted to have health benefits, even though its bioavailability, including digestibility, permeability and ultimate metabolism, are still poorly understood. In vitro gastric and pancreatic digestion and in vitro ileal and faecal fermentation were combined with Caco-2 cell permeability studies for GSE samples. Qualitatively, there was no change in type/number of GSE compounds following gastric and pancreatic digestion and LC-MS analysis. However, the monomers were significantly (P<0.05) increased after gastric digestion, along with a significant (P<0.05) decrease in polymers. In addition, all forms of phenolic compounds decreased following pancreatic digestion. However, none of the original GSE phenolic compounds passed the Caco-2 cell monolayer, since all were recovered in the apical compartment. In contrast, the two intestinal microbiota metabolites with deprotonated molecular weights of [M-H]-165/121 and 193/175, that were found both in the ileal and faecal fermented samples, passed the Caco-2 cell monolayer. Copyright © 2012 Elsevier Ltd. All rights reserved.

Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations

PubMed Central

Jansen, J.; De Napoli, I. E; Fedecostante, M.; Schophuizen, C. M. S.; Chevtchik, N. V.; Wilmer, M. J.; van Asbeck, A. H.; Croes, H. J.; Pertijs, J. C.; Wetzels, J. F. M.; Hilbrands, L. B.; van den Heuvel, L. P.; Hoenderop, J. G.; Stamatialis, D.; Masereeuw, R.

2015-01-01

The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

Effect of Wild-Type Shigella Species and Attenuated Shigella Vaccine Candidates on Small Intestinal Barrier Function, Antigen Trafficking, and Cytokine Release

PubMed Central

Fiorentino, Maria; Levine, Myron M.

2014-01-01

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response. PMID:24416363

Reflectivity of a disordered monolayer estimated by graded refractive index and scattering models.

PubMed

Diamant, Ruth; Garcí-Valenzuela, Augusto; Fernández-Guasti, Manuel

2012-09-01

Reflectivity of a random monolayer, consisting of transparent spherical particles, is estimated using a graded refractive index model, an effective medium approach, and two scattering models. Two cases, a self-standing film and one with a substrate, are considered. Neither the surrounding medium nor the substrate are absorbing materials. Results at normal incidence, with different particle sizes, covering ratios and refractive indexes, are compared. The purpose of this work is to find under which circumstances, for reflectivity at normal incidence, a particle monolayer behaves as a graded refractive index film.

Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors.

PubMed

Urani, C; Corvi, R; Callegaro, G; Stefanini, F M

2013-09-01

In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide adoption of the assay partially depend on a fair degree of subjectivity in foci scoring. An objective evaluation may be obtained after separating foci from background monolayer in the digital image, and quantifying values of statistical descriptors which are selected to capture eye-scored morphological features. The aim of this study was to develop statistical descriptors to be applied to transformed foci of BALB/c 3T3, which cover foci size, multilayering and invasive cell growth into the background monolayer. Proposed descriptors were applied to a database of 407 foci images to explore the numerical features, and to illustrate open problems and potential solutions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Use of micro-optical coherence tomography to analyze barrier integrity of intestinal epithelial cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Som, Avira; Leung, Hui Min; Chu, Kengyeh; Eaton, Alex D.; Hurley, Bryan P.; Tearney, Guillermo J.

2017-02-01

The intestinal epithelial barrier provides protection from external threats that enter the digestive system and persist beyond passage through the stomach. The effects of toxic agents on the intestinal epithelial cell monolayer have not been fully characterized at a cellular level as live imaging of this dynamic interplay at sufficient resolution to interpret cellular responses presents technological challenges. Using a high-resolution native contrast modality called Micro-Optical Coherence Tomography (μOCT), we generated real-time 3D images depicting the impact of the chemical agent EDTA on polarized intestinal epithelial monolayers. Within minutes following application of EDTA, we observed a change in the uniformity of epithelial surface thickness and loss of the edge brightness associated with the apical surface. These observations were measured by generating computer algorithms which quantify imaged-based events changing over time, thus providing parallel graphed data to pair with video. The imaging platform was designed to monitor epithelial monolayers prior to and following application of chemical agents in order to provide a comprehensive account of monolayer behavior at baseline conditions and immediately following exposure. Furthermore, the platform was designed to simultaneously measure continuous trans-epithelial electric resistance (TEER) in order to define the progressive loss of barrier integrity of the cell monolayer following exposure to toxic agents and correlate these findings to image-based metrics. This technological image-based experimental platform provides a novel means to characterize mechanisms that impact the intestinal barrier and, in future efforts, can be applied to study the impact of disease relevant agents such as enteric pathogens and enterotoxins.

A portable cell-based impedance sensor for toxicity testing of drinking water.

PubMed

Curtis, Theresa M; Widder, Mark W; Brennan, Linda M; Schwager, Steven J; van der Schalie, William H; Fey, Julien; Salazar, Noe

2009-08-07

A major limitation to using mammalian cell-based biosensors for field testing of drinking water samples is the difficulty of maintaining cell viability and sterility without an on-site cell culture facility. This paper describes a portable automated bench-top mammalian cell-based toxicity sensor that incorporates enclosed fluidic biochips containing endothelial cells monitored by Electric Cell-substrate Impedance Sensing (ECIS) technology. Long-term maintenance of cells on the biochips is made possible by using a compact, self-contained disposable media delivery system. The toxicity sensor monitors changes in impedance of cell monolayers on the biochips after the introduction of water samples. The fluidic biochip includes an ECIS electronic layer and a polycarbonate channel layer, which together reduce initial impedance disturbances seen in commercially available open well ECIS chips caused by the mechanics of pipetting while maintaining the ability of the cells to respond to toxicants. A curve discrimination program was developed that compares impedance values over time between the control and treatment channels on the fluidic biochip and determines if they are significantly different. Toxicant responses of bovine pulmonary artery endothelial cells grown on fluidic biochips are similar to cells on commercially-available open well chips, and these cells can be maintained in the toxicity sensor device for at least nine days using an automated media delivery system. Longer-term cell storage is possible; bovine lung microvessel endothelial cells survive for up to four months on the fluidic biochips and remain responsive to a model toxicant. This is the first demonstration of a portable bench top system capable of both supporting cell health over extended periods of time and obtaining impedance measurements from endothelial cell monolayers after toxicant exposure.

Tracing the 4000 year history of organic thin films: From monolayers on liquids to multilayers on solids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greene, J. E.; Linköping University, 581 83 Linköping; National Taiwan University of Science and Technology, Taipei 10607, Taiwan

The recorded history of organic monolayer and multilayer thin films spans approximately 4000 years. Fatty-acid-based monolayers were deposited on water by the ancients for applications ranging from fortune telling in King Hammurabi's time (∼1800 BC, Mesopotamia) to stilling choppy waters for sailors and divers as reported by the Roman philosopher Pliny the Elder in ∼78 AD, and then much later (1774) by the peripatetic American statesman and natural philosopher Benjamin Franklin, to Japanese “floating-ink” art (suminagashi) developed ∼1000 years ago. The modern science of organic monolayers began in the late-1800s/early-1900s with experiments by Lord Rayleigh and the important development bymore » Agnes Pockels, followed two decades later by Irving Langmuir, of the tools and technology to measure the surface tension of liquids, the surface pressure of organic monolayers deposited on water, interfacial properties, molecular conformation of the organic layers, and phase transitions which occur upon compressing the monolayers. In 1935, Katherine Blodgett published a landmark paper showing that multilayers can be synthesized on solid substrates, with controlled thickness and composition, using an apparatus now known as the Langmuir-Blodgett (L-B) trough. A disadvantage of LB films for some applications is that they form weak physisorbed bonds to the substrate. In 1946, Bigelow, Pickett, and Zisman demonstrated, in another seminal paper, the growth of organic self-assembled monolayers (SAMs) via spontaneous adsorption from solution, rather than from the water/air interface, onto SiO{sub 2} and metal substrates. SAMs are close-packed two-dimensional organic crystals which exhibit strong covalent bonding to the substrate. The first multicomponent adsorbed monolayers and multilayer SAMs were produced in the early 1980s. Langmuir monolayers, L-B multilayers, and self-assembled mono- and multilayers have found an extraordinarily broad range of applications including controlled wetting, adhesion, electrochemistry, biocompatibility, molecular recognition, biosensing, cell biology, non-linear optics, molecular electronics, solar cells, read/write/erase memory, and magnetism.« less

Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate.

PubMed

Heathman, Thomas R J; Stolzing, Alexandra; Fabian, Claire; Rafiq, Qasim A; Coopman, Karen; Nienow, Alvin W; Kara, Bo; Hewitt, Christopher J

2016-04-01

The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 10(5) and 1.02 ± 0.005 × 10(5) cells/mL compared with 0.79 ± 0.05 × 10(5) and 0.36 ± 0.04 × 10(5) cells/mL in FBS-containing medium. This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

High Selective Gas Detection for small molecules based on Germanium selenide monolayer

NASA Astrophysics Data System (ADS)

Liu, Lian; Yang, Qun; Wang, Zeping; Ye, Huaiyu; Chen, Xianping; Fan, Xuejun; Zhang, Guoqi

2018-03-01

Predictive calculations based on density functional theory (DFT) are used here to study the electronic and optical properties of GeSe monolayer after adsorbing gas molecules (O2, NH3, SO2, H2, CO2, H2S, NO2, CH4, H2O, NO, CO). Our results reveal that for all the gas molecules considered, only NH3 is adsorbed on GeSe monolayer by physisorption. Whereas SO2 and NO2 are chemisorbed on GeSe monolayer with strong adsorption energies. In addition, the adsorption of O2, NO and NO2 distinctly enhances the optical absorbance and broaden the absorbance range of GeSe monolayer in visible light region. Also, it is found that the adsorption of H2S, NO and NH3 can reduce the work function of the GeSe monolayer. The results indicate that GeSe monolayer is not only a promising candidate for the sensing, capture, and storage of NH3, but also an anticipated disposable gas sensor or metal-free catalyst for detecting and catalyzing SO2 and NO2. Furthermore, it has excellent potential to be applied to optical sensors, solar cells, nanoelectronics or optoelectronics devices.

Integration of Ganglioside GT1b Receptor into DPPE and DPPC Phospholipid Monolayers: An X-Ray Reflectivity and Grazing-Incidence Diffraction Study

PubMed Central

Miller, C. E.; Busath, D. D.; Strongin, B.; Majewski, J.

2008-01-01

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structures of mixed-ganglioside GT1b-phospholipid monolayers were investigated at the air-liquid interface and compared with monolayers of the pure components. The receptor GT1b is involved in the binding of lectins and toxins, including botulinum neurotoxin, to cell membranes. Monolayers composed of 20 mol % ganglioside GT1b, the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE), and the phospholipid dipalmitoyl phosphatidylcholine (DPPC) were studied in the gel phase at 23°C and at surface pressures of 20 and 40 mN/m, and at pH 7.4 and 5. Under these conditions, the two components did not phase-separate, and no evidence of domain formation was observed. The x-ray scattering measurements revealed that GT1b was intercalated within the host DPPE/DPPC monolayers, and slightly expanded DPPE but condensed the DPPC matrix. The oligosaccharide headgroups extended normally from the monolayer surfaces into the subphase. This study demonstrated that these monolayers can serve as platforms for investigating toxin membrane binding and penetration. PMID:18599631

In vitro and ex vivo evaluation of polymeric nanoparticles for vaginal and rectal delivery of the anti-HIV drug dapivirine.

PubMed

das Neves, José; Araújo, Francisca; Andrade, Fernanda; Michiels, Johan; Ariën, Kevin K; Vanham, Guido; Amiji, Mansoor; Bahia, Maria Fernanda; Sarmento, Bruno

2013-07-01

Prevention strategies such as the development of microbicides are thought to be valuable in the fight against HIV/AIDS. Despite recent achievements, there is still a long road ahead in the field, particularly at the level of drug formulation. Drug nanocarriers based on polymers may be useful in enhancing local drug delivery while limiting systemic exposure. We prepared differently surface-engineered poly(ε-caprolactone) (PCL) nanoparticles (NPs) and tested their ability to modulate the permeability and retention of dapivirine in cell monolayers and pig vaginal and rectal mucosa. NPs coated with poly(ethylene oxide) (PEO) were shown able to reduce permeability across monolayers/tissues, while modification of nanosystems with cetyl trimethylammonium bromide (CTAB) enhanced transport. In the case of coating NPs with sodium lauryl sulfate (SLS), dapivirine permeability was unchanged. All NPs increased monolayer/tissue drug retention as compared to unformulated dapivirine. This fact was associated, at least partially, to the ability of NPs to be taken up by cells or penetrate mucosal tissue. Cell and tissue toxicity was also affected differently by NPs: PEO modification decreased the in vitro (but not ex vivo) toxicity of dapivirine, while higher toxicity was generally observed for NPs coated with SLS or CTAB. Overall, presented results support that PCL nanoparticles are capable of modulating drug permeability and retention in cell monolayers and mucosal tissues relevant for vaginal and rectal delivery of microbicides. In particular, PEO-modified dapivirine-loaded PCL NPs may be advantageous in increasing drug residence at epithelial cell lines/mucosal tissues, which may potentially increase the efficacy of microbicide drugs.

Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling.

PubMed

Nasreen, Najmunnisa; Khodayari, Nazli; Sriram, Peruvemba S; Patel, Jawaharlal; Mohammed, Kamal A

2014-06-15

Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.

Lipopolysaccharide-Specific but Not Anti-Flagellar Immunoglobulin A Monoclonal Antibodies Prevent Salmonella enterica Serotype Enteritidis Invasion and Replication within HEp-2 Cell Monolayers

PubMed Central

Iankov, Ianko D.; Petrov, Dragomir P.; Mladenov, Ivan V.; Haralambieva, Iana H.; Mitov, Ivan G.

2002-01-01

The protective potential of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis to prevent bacterial adhesion to and invasion of HEp-2 cells was evaluated. Although anti-flagellar IgA MAbs showed strong agglutinating capacities, they did not protect cell monolayers. In contrast, IgA MAbs specific for the O:9 epitope of Salmonella lipopolysaccharide antigen alone prevented S. enterica serotype Enteritidis entry and replication within HEp-2 cells, and the protection was not mediated by direct binding of antibodies to bacterial adhesins or by agglutination of microorganisms. PMID:11854252

Ion transport by primary cultures of canine tracheal epithelium: methodology, morphology, and electrophysiology.

PubMed

Welsh, M J

1985-01-01

Canine tracheal epithelial cells were isolated by enzymatic and mechanical dispersion and cultured on permeable supports. The cells formed confluent monolayers and retained most of the morphologic characteristics of the intact epithelium, including apical microvilli, apical tight junctions, and a moderately interdigitated lateral intercellular space. The cells also retained the functional properties of the epithelium. The monolayer responded to addition of isoproterenol with the characteristic changes in cellular electrical properties expected for stimulation of C1 secretion: isoproterenol increased transepithelial voltage, depolarized apical membrane voltage, and decreased both transepithelial resistance and the ratio of apical-to-basolateral membrane resistance. Examination of the cellular response to ion substitutions and inhibitors of C1 secretion indicate that the cultured monolayers retain the same cellular mechanisms of ion transport as the intact epithelium. Thus, primary cultures of tracheal epithelium may provide a useful preparation for future studies of the mechanism and regulation of C1 secretion by airway epithelia.

Application of Organosilane Monolayer Template to Quantitative Evaluation of Cancer Cell Adhesive Ability

NASA Astrophysics Data System (ADS)

Tanii, Takashi; Sasaki, Kosuke; Ichisawa, Kota; Demura, Takanori; Beppu, Yuichi; Vu, Hoan Anh; Thanh Chi, Hoan; Yamamoto, Hideaki; Sato, Yuko

2011-06-01

The adhesive ability of two human pancreatic cancer cell lines was evaluated using organosilane monolayer templates (OMTs). Using the OMT, the spreading area of adhered cells can be limited, and this enables us to focus on the initial attachment process of adhesion. Moreover, it becomes possible to arrange the cells in an array and to quantitatively evaluate the number of attached cells. The adhesive ability of the cancer cells cultured on the OMT was controlled by adding (-)-epigallocatechin-3-gallate (EGCG), which blocks a receptor that mediates cell adhesion and is overexpressed in cancer cells. Measurement of the relative ability of the cancer cells to attach to the OMT revealed that the ability for attachment decreased with increasing EGCG concentration. The results agreed well with the western blot analysis, indicating that the OMT can potentially be employed to evaluate the adhesive ability of various cancer cells.

Gene Delivery to the Airway

PubMed Central

Keiser, Nicholas W.; Engelhardt, John F.

2013-01-01

This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols. PMID:23853081

Lab-on-a-brane: A novel physiologically relevant planar arterial model to study transendothelial transport

NASA Astrophysics Data System (ADS)

Budhwani, Karim Ismail

The tremendous quality of life impact notwithstanding, cardiovascular diseases and Cancer add up to over US$ 700bn each year in financial costs alone. Aging and population growth are expected to further expand the problem space while drug research and development remain expensive. However, preclinical costs can be substantially mitigated by substituting animal models with in vitro devices that accurately model human cardiovascular transport. Here we present a novel physiologically relevant lab-on-a-brane that simulates in vivo pressure, flow, strain, and shear waveforms associated with normal and pathological conditions in large and small blood vessels for studying molecular transport across the endothelial monolayer. The device builds upon previously demonstrated integrated microfluidic loop design by: (a) introducing nanoscale pores in the substrate membrane to enable transmembrane molecular transport, (b) transforming the substrate membrane into a nanofibrous matrix for 3D smooth muscle cell (SMC) tissue culture, (c) integrating electrospinning fabrication methods, (d) engineering an invertible sandwich cell culture device architecture, and (e) devising a healthy co-culture mechanism for human arterial endothelial cell (HAEC) monolayer and multiple layers of human smooth muscle cells (HSMC) to accurately mimic arterial anatomy. Structural and mechanical characterization was conducted using confocal microscopy, SEM, stress/strain analysis, and infrared spectroscopy. Transport was characterized using FITC-Dextran hydraulic permeability protocol. Structure and transport characterization successfully demonstrate device viability as a physiologically relevant arterial mimic for testing transendothelial transport. Thus, our lab-on-a-brane provides a highly effective and efficient, yet considerably inexpensive, physiologically relevant alternative for pharmacokinetic evaluation; possibly reducing animals used in pre-clinical testing, clinical trials cost from false starts, and time-to-market. Furthermore, this platform can be easily configured for testing targeted therapeutic delivery and in multiple simultaneous arrays for personalized and precision medicine applications.

X-ray Reflectivity Characterization of Ion Distribution at Biomimetic Membrane Surfaces

NASA Astrophysics Data System (ADS)

Krüger, Peter; Pittler, Jens; Vaknin, David; Lösche, Mathias

2003-03-01

Ions at cell membrane surfaces may control the function and conformation of nearby biomolecules, thus playing an important role in inter- and intracellular transport as well as in biorecognition processes. Moreover, charge patterns at membrane surfaces may direct the growth of inorganic crystals in biomineralization. Langmuir monolayers are widely employed as model systems for studying charge distribution and growth processes at the organic/inorganic interface. We present a novel x-ray reflectivity technique that provides detailed information on ion distribution at biomembrane surfaces by using monochromatic x-rays at various energies at and away from the ion x-ray absorption edges. As a model, the interaction of Ba^2+ with DMPA^- (dimyristoyl phosphatidic acid) monolayers at the aqueous surface was studied. We find an unexpectedly large concentration of the cations near the interface where they form a Stern layer of bound ions. These studies have been complemented with conventional x-ray reflectivity measurements and extended to other anionic lipid species (DMPS, DMPG) and cations (Ca^2+).

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and Fibronectin-enriched matrices

PubMed Central

McFarlane, Suzanne; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; Waugh, David J.J.

2015-01-01

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche. PMID:26447611

Transport of a series of D-phenylalanine-glycine hexapeptides across rat alveolar epithelia in vitro.

PubMed

Evans, J P; Tudball, N; Dickinson, P A; Farr, S J; Kellaway, I W

1998-01-01

The effect of lipophilicity on the absorption of peptides from the lungs was investigated. D-phenylalanine (F)-glycine (G) hexapeptides were synthesised to differ, predominantly, only in their lipophilicity. Rat alveolar type II cells were isolated and cultured on plastic, or polycarbonate filters; by day 6 they had de-differentiated to an alveolar type I-like epithelium. The permeability of the monolayers to the hexapeptides was determined. The hexapeptides were metabolically and chemically stable for greater than 24h in the presence of the cells. They did not adhere to the cell culture plastic and were associated only to a low extent with the cell monolayer. The apical to basolateral permeability coefficients for D-F1G5, D-F2G4, and D-F3G3 were 2.19+/-0.53, 1.75+/-0.42 and 2.20+/-0.56 x 10(-7) cm s(-1) respectively. The permeability of the monolayers to D-F1G5 and D-F2G4 was concentration and direction independent, however for D-F3G3 the monolayer was more permeable in the basolateral to apical direction. There was no correlation between the lipophilicity of the hexapeptides and permeability coefficients: other physicochemical parameters did not predict hexapeptide transport. Lipophilicity does not appear to control the transport of hexapeptides across the alveolar epithelium probably as a consequence of the peptides being transported via the paracellular route.

Rare cancer cell analyzer for whole blood applications: microcytometer cell counting and sorting subcircuits.

PubMed

Lancaster, C; Kokoris, M; Nabavi, M; Clemmens, J; Maloney, P; Capadanno, J; Gerdes, J; Battrell, C F

2005-09-01

We demonstrate sorting of rare cancer cells from blood using a thin ribbon monolayer of cells within a credit-card sized, microfluidic laboratory-on-a-card ("lab card") structure. This enables higher cell throughput per minute thereby speeding up cell interrogation. In this approach, multiple cells are viewed and sorted, not individually, but as a whole cell row or section of the ribbon at a time. Gated selection of only the cell rows containing a tagged rare cell provides enrichment of the rare cell relative to background blood cells. We also designed the cell injector for laminar flow antibody labeling within 20s. The approach combines rapid laminar flow cell labeling with monolayer cell sorting thereby enabling rare cell target detection at sensitivity levels 1000 to 10,000 times that of existing flow cytometers. Using this method, total cell labeling and data acquisition time on card may be reduced to a few minutes compared to 30-60 min for standard flow methods.

SAM-based Cell Transfer to Photopatterned Hydrogels for Microengineering Vascular-Like Structures

PubMed Central

Sadr, Nasser; Zhu, Mojun; Osaki, Tatsuya; Kakegawa, Takahiro; Yang, Yunzhi; Moretti, Matteo; Fukuda, Junji; Khademhosseini, Ali

2011-01-01

A major challenge in tissue engineering is to reproduce the native 3D microvascular architecture fundamental for in vivo functions. Current approaches still lack a network of perfusable vessels with native 3D structural organization. Here we present a new method combining self-assembled monolayer (SAM)-based cell transfer and gelatin methacrylate hydrogel photopatterning techniques for microengineering vascular structures. Human umbilical vein cell (HUVEC) transfer from oligopeptide SAM-coated surfaces to the hydrogel revealed two SAM desorption mechanisms: photoinduced and electrochemically triggered. The former, occurs concomitantly to hydrogel photocrosslinking, and resulted in efficient (>97%) monolayer transfer. The latter, prompted by additional potential application, preserved cell morphology and maintained high transfer efficiency of VE-cadherin positive monolayers over longer culture periods. This approach was also applied to transfer HUVECs to 3D geometrically defined vascular-like structures in hydrogels, which were then maintained in perfusion culture for 15 days. As a step toward more complex constructs, a cell-laden hydrogel layer was photopatterned around the endothelialized channel to mimic the vascular smooth muscle structure of distal arterioles. This study shows that the coupling of the SAM-based cell transfer and hydrogel photocrosslinking could potentially open up new avenues in engineering more complex, vascularized tissue constructs for regenerative medicine and tissue engineering applications. PMID:21802723

Topology-Scaling Identification of Layered Solids and Stable Exfoliated 2D Materials.

PubMed

Ashton, Michael; Paul, Joshua; Sinnott, Susan B; Hennig, Richard G

2017-03-10

The Materials Project crystal structure database has been searched for materials possessing layered motifs in their crystal structures using a topology-scaling algorithm. The algorithm identifies and measures the sizes of bonded atomic clusters in a structure's unit cell, and determines their scaling with cell size. The search yielded 826 stable layered materials that are considered as candidates for the formation of two-dimensional monolayers via exfoliation. Density-functional theory was used to calculate the exfoliation energy of each material and 680 monolayers emerge with exfoliation energies below those of already-existent two-dimensional materials. The crystal structures of these two-dimensional materials provide templates for future theoretical searches of stable two-dimensional materials. The optimized structures and other calculated data for all 826 monolayers are provided at our database (https://materialsweb.org).

The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance

PubMed Central

Breslin, Susan; O'Driscoll, Lorraine

2016-01-01

Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs. PMID:27304190

Contact inhibition of locomotion determines cell-cell and cell-substrate forces in tissues.

PubMed

Zimmermann, Juliane; Camley, Brian A; Rappel, Wouter-Jan; Levine, Herbert

2016-03-08

Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell-cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin-Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell-cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge.

Orientation and Interaction of Oblique Cylindrical Inclusions Embedded in a Lipid Monolayer: A Theoretical Model for Viral Fusion Peptides

PubMed Central

Kozlovsky, Yonathan; Zimmerberg, Joshua; Kozlov, Michael M.

2004-01-01

We consider the elastic behavior of flat lipid monolayer embedding cylindrical inclusions oriented obliquely with respect to the monolayer plane. An oblique inclusion models a fusion peptide, a part of a specialized protein capable of inducing merger of biological membranes in the course of fundamental cellular processes. Although the crucial importance of the fusion peptides for membrane merger is well established, the molecular mechanism of their action remains unknown. This analysis is aimed at revealing mechanical deformations and stresses of lipid monolayers induced by the fusion peptides, which, potentially, can destabilize the monolayer structure and enhance membrane fusion. We calculate the deformation of a monolayer embedding a single oblique inclusion and subject to a lateral tension. We analyze the membrane-mediated interactions between two inclusions, taking into account bending of the monolayer and tilt of the hydrocarbon chains with respect to the surface normal. In contrast to a straightforward prediction that the oblique inclusions should induce tilt of the lipid chains, our analysis shows that the monolayer accommodates the oblique inclusion solely by bending. We find that the interaction between two inclusions varies nonmonotonically with the interinclusion distance and decays at large separations as square of the distance, similar to the electrostatic interaction between two electric dipoles in two dimensions. This long-range interaction is predicted to dominate the other interactions previously considered in the literature. PMID:15298906

Sorption Isotherm Modelling Of Fermented Cassava Flour by Red Yeast Rice

NASA Astrophysics Data System (ADS)

Cahyanti, M. N.; Alfiah, M. N.; Hartini, S.

2018-04-01

The objective of the study is to determine the characteristic of moisture sorption isotherm from fermented cassava flour by red yeast rice using various modeling. This research used seven salt solutions and storage temperature of 298K, 303K, and 308K. The models used were Brunauer-Emmet-Teller (BET), Guggenheim-Anderson-de Boer (GAB) and Caurie model. The monolayer moisture content was around 4.51 – 5.99% db. Constant related to absorption heat in the multilayer area of [GAB model was around 0.86-0,91. Constant related to absorption heat in the monolayer area of GAB model was around 4.67-5.97. Constant related to absorption heat in the monolayer area of BET model was around 4.83-7.04. Caurie constant was around 1.25-1.59. The equilibrium and monolayer moisture content on fermented cassava flour by red yeast rice was decreasing as increasing temperature. GAB constant value indicated that the process of moisture absorption on the fermented cassava flour by red yeast rice categorized in type II.

Xyloglucan, hibiscus and propolis for the prevention of urinary tract infections: results of in vitro studies.

PubMed

Fraile, Benito; Alcover, Javier; Royuela, Mar; Rodríguez, David; Chaves, Concepción; Palacios, Ricardo; Piqué, Núria

2017-06-01

To assess the properties of a medical device containing xyloglucan, propolis and hibiscus to create a bioprotective barrier to avoid the contact of uropathogenic Escherichia coli strains on cell walls in models of intestinal (CacoGoblet) and uroepithelial (RWPE-1) cells (derived from normal human prostate epithelium). Two uropathogenic E. coli strains (expressing type 1 fimbriae and P fimbriae) were used to assess, by electronic microscopy and ELISA, the barrier properties of the medical device. The antimicrobial activity was assessed in broth dilution assays. The three components (xyloglucan, propolis and hibiscus) did not alter E. coli cell integrity in intestinal and uroepithelial cell models and were devoid of antibacterial activity. The three components avoided bacterial contact in both cell monolayers. The nonpharmacological barrier properties of xyloglucan, propolis and hibiscus confirm the role of the medical device for the management of urinary tract infections.

Accounting for Space—Quantification of Cell-To-Cell Transmission Kinetics Using Virus Dynamics Models.

PubMed

Kumberger, Peter; Durso-Cain, Karina; Uprichard, Susan L; Dahari, Harel; Graw, Frederik

2018-04-17

Mathematical models based on ordinary differential equations (ODE) that describe the population dynamics of viruses and infected cells have been an essential tool to characterize and quantify viral infection dynamics. Although an important aspect of viral infection is the dynamics of viral spread, which includes transmission by cell-free virions and direct cell-to-cell transmission, models used so far ignored cell-to-cell transmission completely, or accounted for this process by simple mass-action kinetics between infected and uninfected cells. In this study, we show that the simple mass-action approach falls short when describing viral spread in a spatially-defined environment. Using simulated data, we present a model extension that allows correct quantification of cell-to-cell transmission dynamics within a monolayer of cells. By considering the decreasing proportion of cells that can contribute to cell-to-cell spread with progressing infection, our extension accounts for the transmission dynamics on a single cell level while still remaining applicable to standard population-based experimental measurements. While the ability to infer the proportion of cells infected by either of the transmission modes depends on the viral diffusion rate, the improved estimates obtained using our novel approach emphasize the need to correctly account for spatial aspects when analyzing viral spread.

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zambrano, Pablo; Suwalsky, Mario; Villena, Fernando

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes.more » According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40–50 μM memantine was required to interact with isolated phosphatidylcholine bilayers. - Highlights: • The interaction of memantine with human erythrocytes and lipid bilayers were assessed. • Memantine induced morphological changes to human erythrocytes. • Memantine interacted with classes of phospholipids present in the erythrocyte membrane. • Results support the hypothesis that memantine interacts with NMDA receptors.« less

Interactions of doxorubicin with self-assembled monolayer-modified electrodes: electrochemical, surface plasmon resonance (SPR), and gravimetric studies.

PubMed

Nieciecka, Dorota; Krysinski, Pawel

2011-02-01

We present the results on the partitioning of doxorubicin (DOX), a potent anticancer drug, through the model membrane system, self-assembled monolayers (SAMs) on gold electrodes. The monolayers were formed from alkanethiols of comparable length with different ω-terminal groups facing the aqueous electrolyte: the hydrophobic -CH(3) groups for the case of dodecanethiol SAMs or hydrophilic -OH groups of mercaptoundecanol SAMs. The electrochemical experiments combined with the surface plasmon resonance (SPR) and gravimetric studies show that doxorubicin is likely adsorbed onto the surface of hydrophilic monolayer, while for the case of the hydrophobic one the drug mostly penetrates the monolayer moiety. The adsorption of the drug hinders further penetration of doxorubicin into the monolayer moiety.

Benjamin Franklin, Philadelphia’s Favorite Son, was a Membrane Biophysicist

PubMed Central

Wang, Da-Neng; Stieglitz, Heather; Marden, Jennifer; Tamm, Lukas K.

2013-01-01

Benjamin Franklin, mostly known for his participation in writing The Declaration of Independence and work on electricity, was also one of the first scientists to seek to understand the properties of oil monolayers on water surfaces. During one of his many voyages across the Atlantic Ocean, Franklin observed that oil had a calming effect on waves when poured into rough ocean waters. Though at first taking a backseat to many of his other scientific and political endeavors, Franklin went on to experiment with oil, spreading monomolecular films on various bodies of water, and ultimately devised a concept of particle repulsion that is indirectly related to the hydrophobic effect. His early observations inspired others to measure the dimensions of oil monolayers, which eventually led to the formulation of the contemporary lipid bilayer model of the cell membrane. PMID:23442850

Interface thermal conductance of van der Waals monolayers on amorphous substrates

NASA Astrophysics Data System (ADS)

Correa, Gabriela C.; Foss, Cameron J.; Aksamija, Zlatan

2017-03-01

Heterostructures based on atomic monolayers are emerging as leading materials for future energy efficient and multifunctional electronics. Due to the single atom thickness of monolayers, their properties are strongly affected by interactions with the external environment. We develop a model for interface thermal conductance (ITC) in an atomic monolayer van der Waals bonded to a disordered substrate. Graphene on SiO2 is initially used in our model and contrasted against available experimental data; the model is then applied to monolayer molybdenum disulfide (MoS2) on SiO2 substrate. Our findings show the dominant carrier of heat in both graphene and MoS2 in the cross-plane direction is the flexural (ZA) phonon mode, owing to the large overlap between graphene ZA and substrate vibrational density of states. The rate of phonon transfer across the interface depends quadratically on the substrate coupling constant K a , but this interaction also causes a lifting of the lowest flexural phonon modes. As a result, ITC depends roughly linearly on the strength of the coupling between a monolayer and its substrate. We conclude that, in both graphene and MoS2 on SiO2, substrate adhesion plays a strong role in determining ITC, requiring further study of substrate coupling in TMDCs.

Mouse, but Not Human, ApoB-100 Lipoprotein Cholesterol Is a Potent Innate Inhibitor of Streptococcus pneumoniae Pneumolysin

PubMed Central

Wade, Kristin R.; Hotze, Eileen M.; Briles, David E.; Tweten, Rodney K.

2014-01-01

Streptococcus pneumoniae produces the pore-forming toxin pneumolysin (PLY), which is a member of the cholesterol-dependent cytolysin (CDC) family of toxins. The CDCs recognize and bind the 3β-hydroxyl group of cholesterol at the cell surface, which initiates membrane pore formation. The cholesterol transport lipoproteins, which carry cholesterol in their outer monolayer, are potential off-pathway binding targets for the CDCs and are present at significant levels in the serum and the interstitial spaces of cells. Herein we show that cholesterol carried specifically by the ApoB-100-containing lipoprotein particles (CH-ApoB-100) in the mouse, but not that carried by human or guinea pig particles, is a potent inhibitor of the PLY pore-forming mechanism. Cholesterol present in the outer monolayer of mouse ApoB-100 particles is recognized and bound by PLY, which stimulates premature assembly of the PLY oligomeric complex thereby inactivating PLY. These studies further suggest that the vast difference in the inhibitory capacity of mouse CH-ApoB-100 and that of the human and the guinea pig is due to differences in the presentation of cholesterol in the outer monolayer of their ApoB-100 particles. Therefore mouse CH-ApoB-100 represents a significant innate CDC inhibitor that is absent in humans, which may underestimate the contribution of CDCs to human disease when utilizing mouse models of disease. PMID:25188225

Characterization of cell cultures derived from Lutzomyia spinicrassa (Diptera: Psychodidae) and their susceptibility to infection with Leishmania (Viannia) braziliensis.

PubMed

Zapata Lesmes, Angela Cristina; Cárdenas Castro, Estrella; Bello, Felio

2005-12-01

The sand fly Lutzomyia spinicrassa (Morales, Osorno-Mesa, Osorno & de Hoyos, 1969) is a vector of Leishmania (Viannia) braziliensis, an etiological agent of cutaneous leishmaniasis in Colombia. The present article describes, for the first time, the morphological, karyotypical, and isozymatic characteristics of cell cultures derived from L. Spinicrassa embryonic tissues as well as the interaction of L. Braziliensis with these cell cultures. L. Spinicrassa embryonated eggs and neonate larvae were taken for tissue explants. These were seeded in Grace, L-15, Grace/L-15, MM/VP12, and MK/VP12 culture media. The pH range in these media was 6.7 to 6.9 and the cultures were incubated at 28 degrees C. The MHOM/CO/86/CL250 strain of L. Braziliensis was used for experimental infection of cell cultures of L. Spinicrassa. Cell growth was achieved in L-15 medium and a confluent monolayer was obtained 180 days after the embryonated eggs were explanted. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer of these cell cultures and in the subcultures the predominant cell types were later fibroblast-like and epithelial-like. Cultured cells were predominantly diploid (2n=8); however, significant percentages of aneuploids were also recorded. The cell culture isozyme patterns of L. Spinicrassa coincided with pupae samples from the same species. Promastigote forms of L. Braziliensis could invade cells and transform into amastigote-like forms inside them. The characteristics of cell cultures derived from L. Spinicrassa embryonic tissues were determined. These cultures emerge as a new model to study the life-cycle of L. Braziliensis.

GPR55 promotes migration and adhesion of colon cancer cells indicating a role in metastasis

PubMed Central

Andersen, L; Hasenöhrl, C; Feuersinger, D; Stančić, A; Fauland, A; Magnes, C; El‐Heliebi, A; Lax, S; Uranitsch, S; Haybaeck, J; Heinemann, A

2015-01-01

Background and Purpose Tumour cell migration and adhesion constitute essential features of metastasis. G‐protein coupled receptor 55 (GPR55), a lysophospholipid receptor, has been shown to play an important role in carcinogenesis. Here, we investigated the involvement of GPR55 in migration and metastasis of colon cancer cells. Experimental Approach Adhesion and migration assays using the highly metastatic colon cancer cell line HCT116 and an in vivo assay of liver metastasis were performed. The GPR55 antagonist CID16020046, cannabidiol, a putative GPR55 antagonist and GPR55 siRNA were used to block GPR55 activity in HCT116 colon cancer cells. Key Results HCT116 cells showed a significant decrease in adhesion to endothelial cells and in migration after blockade with CID16020046 or cannabidiol. The inhibitory effects of CID16020046 or cannabidiol were averted by GPR55 siRNA knock down in cancer cells. The integrity of endothelial cell monolayers was increased after pretreatment of HCT116 cells with the antagonists or after GPR55 siRNA knockdown while pretreatment with lysophosphatidylinositol (LPI), the endogenous ligand of GPR55, decreased integrity of the monolayers. LPI also induced migration in GPR55 overexpressing HCT116 cells that was blocked by GPR55 antagonists. In a mouse model of metastasis, the arrest of HCT116 cancer cells in the liver was reduced after treatment with CID16020046 or cannabidiol. Increased levels of LPI (18:0) were found in colon cancer patients when compared with healthy individuals. Conclusions and Implications GPR55 is involved in the migratory behaviour of colon carcinoma cells and may serve as a pharmacological target for the prevention of metastasis. © 2015 The British Pharmacological Society PMID:26436760

A Dual-Responsive Self-Assembled Monolayer for Specific Capture and On-Demand Release of Live Cells.

PubMed

Gao, Xia; Li, Qiang; Wang, Fengchao; Liu, Xuehui; Liu, Dingbin

2018-06-22

We report a dual-responsive self-assembled monolayer (SAM) on a well-defined rough gold substrate for dynamic capture and release of live cells. By incorporating 5'-triphosphate (ATP) aptamer into a SAM, we can accurately isolate specific cell types and subsequently release captured cells at either population or desired-group (or even single-cell) levels. On one hand, the whole SAMs can be disassembled through addition of ATP solution, leading to the entire release of the captured cells from the supported substrate. On the other hand, desired cells can be selectively released by using near-infrared light (NIR) irradiation, with relatively high spatial and temporal precision. The proposed dual-responsive cell capture-and-release system is biologically friendly and is reusable with another round of modification, showing great usefulness in cancer diagnosis and molecular analysis.

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment.

PubMed

Williamson, Matthew R; Shuttleworth, Adrian; Canfield, Ann E; Black, Richard A; Kielty, Cay M

2007-12-01

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

Ionization state and structure of l-1,2-dipalmitoylphosphatidylglycerol monolayers at the liquid/air interface.

PubMed

Maltseva, Elena; Shapovalov, Vladimir L; Möhwald, Helmuth; Brezesinski, Gerald

2006-01-19

Phosphatidylglycerols are components of biological membranes. The phase behavior of these phospholipids was extensively investigated. However, there is still no definite picture about the dependence of the ionization state and monolayer structure on subphase composition. The major problem of previous investigations is that none of the methods used allow obtaining the ionization degree directly. In the present work we apply techniques developed in the past decades for Langmuir monolayers: infrared reflection absorption spectroscopy (IRRAS) as well as X-ray diffraction and reflectivity techniques, which provide straightforward information about structure and ionization state of a L-1,2-dipalmitoylphosphatidylglycerol (DPPG) monolayer. The Gouy-Chapman model is applied to evaluate the intrinsic pKa. Therewith, the ionization degree can be determined even at low pH values. The experimental titration curves are in good agreement with theoretical curves based on the Gouy-Chapman model. The obtained instrinic pKa amounts to 1. The ionization degree of a DPPG monolayer is independent of the monovalent cation size. In contrast, the structure of a DPPG monolayer is strongly affected by the type of divalent cations.

Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers - effects beyond electrostatic.

PubMed

Dietrich, Undine; Krüger, Peter; Käs, Josef A

2011-05-01

The presence of charged lipids in the cell membrane constitutes the background for the interaction with numerous membrane proteins. As a result, the valence of the lipids plays an important role concerning their lateral organization in the membrane and therefore the very manner of this interaction. This present study examines this aspect, particularly regarding to the interaction of the anionic lipid DPPS with the highly basic charged effector domain of the MARCKS protein, examined in monolayer model systems. Film balance, fluorescence microscopy and X-ray reflection/diffraction measurements were used to study the behavior of DPPS in a mixture with DPPC for its dependance on the presence of MARCKS (151-175). In the mixed monolayer, both lipids are completely miscible therefore DPPS is incorporated in the ordered crystalline DPPC domains as well. The interaction of MARCKS peptide with the mixed monolayer leads to the formation of lipid/peptide clusters causing an elongation of the serine group of the DPPS up to 7Å in direction to surface normal into the subphase. The large cationic charge of the peptide pulls out the serine group of the interface which simultaneously causes an elongation of the phosphodiester group of the lipid fraction too. The obtained results were used to compare the interaction of MARCKS peptide with the polyvalent PIP(2) in mixed monolayers. On this way we surprisingly find out, that the relative small charge difference of the anionic lipids causes a significant different interaction with MARCKS (151-175). The lateral arrangement of the anionic lipids depends on their charge values and determines the diffusion of the electrostatic binding clusters within the membrane. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Emerging Technologies for Cancer Research: towards Personalized Medicine with Microfluidic Platforms and 3D Tumor Models.

PubMed

Turetta, Matteo; Ben, Fabio Del; Brisotto, Giulia; Biscontin, Eva; Bulfoni, Michela; Cesselli, Daniela; Colombatti, Alfonso; Scoles, Giacinto; Gigli, Giuseppe; Del Mercato, Loretta L

2018-06-05

In the present review, we describe three hot topics in cancer research such as circulating tumor cells, exosomes, and 3D environment models. The first section is dedicated to microfluidic platforms for detecting circulating tumor cells, including both affinity-based methods that take advantage of antibodies and aptamers, and "label-free" approaches, exploiting cancer cells physical features and, more recently, abnormal cancer metabolism. In the second section, we briefly describe biology of exosomes and their role in cancer, as well as conventional techniques for their isolation and innovative microfluidic platforms. In the third section, the importance of tumor microenvironment is highlighted, along with techniques for modeling it in vitro. Finally, we discuss limitations of two-dimensional monolayer methods and describe advantages and disadvantages of different three-dimensional tumor systems for cell-cell interaction analysis and their potential applications in cancer management. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Dietary fibre-based SCFA mixtures promote both protection and repair of intestinal epithelial barrier function in a Caco-2 cell model.

PubMed

Chen, Tingting; Kim, Choon Young; Kaur, Amandeep; Lamothe, Lisa; Shaikh, Maliha; Keshavarzian, Ali; Hamaker, Bruce R

2017-03-22

Impaired gut barrier function plays an important role in the development of many diseases such as obesity, inflammatory bowel disease, and in HIV infection. Dietary fibres have been shown to improve intestinal barrier function through their fermentation products, short chain fatty acids (SCFAs), and the effects of individual SCFAs have been studied. Here, different SCFA mixtures representing possible compositions from fibre fermentation products were studied for protective and reparative effects on intestinal barrier function. The effect of fermentation products from four dietary fibres, i.e. resistant starch, fructooligosaccharides, and sorghum and corn arabinoxylan (varying in their branched structure) on barrier function was positively correlated with their SCFA concentration. Pure SCFA mixtures of various concentrations and compositions were tested using a Caco-2 cell model. SCFAs at a moderate concentration (40-80 mM) improved barrier function without causing damage to the monolayer. In a 40 mM SCFA mixture, the butyrate proportion at 20% and 50% showed both a protective and a reparative effect on the monolayer to disrupting agents (LPS/TNF-α) applied simultaneously or prior to the SCFA mixtures. Relating this result to dietary fibre selection, slow fermenting fibres that deliver appropriate concentrations of SCFAs to the epithelium with a high proportion of butyrate may improve barrier function.

The effect of lycopene on cytochrome P450 isoenzymes and P-glycoprotein by using human liver microsomes and Caco-2 cell monolayer model.

PubMed

Kong, Lingti; Song, Chunli; Ye, Linhu; Xu, Jian; Guo, Daohua; Shi, Qingping

2018-01-11

Lycopene is widely used as a dietary supplement. However, the effects of lycopene on cytochrome P450 (CYP) enzymes or P-glycoprotein (P-gp) are not comprehensive. The present study was performed to investigate the effects of lycopene on the CYP enzymes and P-gp activity. A cocktail method was used to evaluate the activities of CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Caco-2 cell monolayer model was carried out to assay lycopene on P-gp activity. The results indicated that lycopene had a moderate inhibitory effect on CYP2E1, with IC50 value of 43.65 μM, whereas no inhibitory effects on CYP3A4, CYP2C19, CYP2D6 and CYP2E1, with IC50 values all over 100 μM. In addition, lycopene showed almost no inhibitory effect on rhodamine-123 efflux and uptake (p > .05), indicated no effects on P-gp activity. In conclusion, there should be required attention when lycopene are coadministered with other drugs that are metabolised by CYP2E1.

A 3D intestinal tissue model supports Clostridioides difficile germination, colonization, toxin production and epithelial damage.

PubMed

Shaban, Lamyaa; Chen, Ying; Fasciano, Alyssa C; Lin, Yinan; Kaplan, David L; Kumamoto, Carol A; Mecsas, Joan

2018-04-01

Endospore-forming Clostridioides difficile is a causative agent of antibiotic-induced diarrhea, a major nosocomial infection. Studies of its interactions with mammalian tissues have been hampered by the fact that C. difficile requires anaerobic conditions to survive after spore germination. We recently developed a bioengineered 3D human intestinal tissue model and found that low O 2 conditions are produced in the lumen of these tissues. Here, we compared the ability of C. difficile spores to germinate, produce toxin and cause tissue damage in our bioengineered 3D tissue model versus in a 2D transwell model in which human cells form a polarized monolayer. 3D tissue models or 2D polarized monolayers on transwell filters were challenged with the non-toxin producing C. difficile CCUG 37787 serotype X (ATCC 43603) and the toxin producing UK1 C. difficile spores in the presence of the germinant, taurocholate. Spores germinated in both the 3D tissue model as well as the 2D transwell system, however toxin activity was significantly higher in the 3D tissue models compared to the 2D transwells. Moreover, the epithelium damage in the 3D tissue model was significantly more severe than in 2D transwells and damage correlated significantly with the level of toxin activity detected but not with the amount of germinated spores. Combined, these results show that the bioengineered 3D tissue model provides a powerful system with which to study early events leading to toxin production and tissue damage of C. difficile with mammalian cells under anaerobic conditions. Furthermore, these systems may be useful for examining the effects of microbiota, novel drugs and other potential therapeutics directed towards C. difficile infections. Copyright © 2018 Elsevier Ltd. All rights reserved.

Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

PubMed Central

González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

2013-01-01

Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

Electrocatalysts having platium monolayers on palladium, palladium alloy, and gold alloy core-shell nanoparticles, and uses thereof

DOEpatents

Adzic, Radoslav; Mo, Yibo; Vukmirovic, Miomir; Zhang, Junliang

2010-12-21

The invention relates to platinum-coated particles useful as fuel cell electrocatalysts. The particles are composed of a noble metal or metal alloy core at least partially encapsulated by an atomically thin surface layer of platinum atoms. The invention particularly relates to such particles having a palladium, palladium alloy, gold alloy, or rhenium alloy core encapsulated by an atomic monolayer of platinum. In other embodiments, the invention relates to fuel cells containing these electrocatalysts and methods for generating electrical energy therefrom.

The use of fibrous, supramolecular membranes and human tubular cells for renal epithelial tissue engineering: towards a suitable membrane for a bioartificial kidney.

PubMed

Dankers, Patricia Y W; Boomker, Jasper M; Huizinga-van der Vlag, Ali; Smedts, Frank M M; Harmsen, Martin C; van Luyn, Marja J A

2010-11-10

A bioartificial kidney, which is composed of a membrane cartridge with renal epithelial cells, can substitute important kidney functions in patients with renal failure. A particular challenge is the maintenance of monolayer integrity and specialized renal epithelial cell functions ex vivo. We hypothesized that this can be improved by electro-spun, supramolecular polymer membranes which show clear benefits in ease of processability. We found that after 7 d, in comparison to conventional microporous membranes, renal tubular cells cultured on top of our fibrous supramolecular membranes formed polarized monolayers, which is prerequisite for a well-functioning bioartificial kidney. In future, these supramolecular membranes allow for incorporation of peptides that may increase cell function even further.

Novel derivatives of 6-mercaptopurine: synthesis, characterization and antiproliferative activities of S-allylthio-mercaptopurines.

PubMed

Miron, T; Arditti, F; Konstantinovski, L; Rabinkov, A; Mirelman, D; Berrebi, A; Wilchek, M

2009-02-01

Biologically active S-allylthio derivatives of 6-mercaptopurine (6-MP) and 6-mercaptopurine riboside (6-MPR) were synthesized. The products, S-allylthio-6-mercaptopurine (SA-6MP) and S-allylthio-6-mercaptopurine riboside (SA-6MPR) were characterized. The antiproliferative activity of the new prodrugs was tested on human leukemia and monolayer cell lines, and compared to that of their parent reactants. The new prodrugs acted by a concentration-dependent mechanism. They inhibited cell proliferation and induced-apoptosis more efficiently than the parent molecules. Leukemia cell lines were more sensitive to the new prodrugs than monolayer cell lines. Higher hydrophobicity of the derivatives improves their penetration into cells, where upon reaction with glutathione, S-allylthioglutathione (GSSA) is formed, and 6-MP or 6-MPR is released for further processing.

Mechanical Strains Induced in Osteoblasts by Use of Point Femtosecond Laser Targeting

PubMed Central

Bomzon, Ze'ev; Day, Daniel; Gu, Min; Cartmell, Sarah

2006-01-01

A study demonstrating how ultrafast laser radiation stimulates osteoblasts is presented. The study employed a custom made optical system that allowed for simultaneous confocal cell imaging and targeted femtosecond pulse laser irradiation. When femtosecond laser light was focused onto a single cell, a rise in intracellular Ca2+ levels was observed followed by contraction of the targeted cell. This contraction caused deformation of neighbouring cells leading to a heterogeneous strain field throughout the monolayer. Quantification of the strain fields in the monolayer using digital image correlation revealed local strains much higher than threshold values typically reported to stimulate extracellular bone matrix production in vitro. This use of point targeting with femtosecond pulse lasers could provide a new method for stimulating cell activity in orthopaedic tissue engineering. PMID:23165014

Established breast cancer stem cell markers do not correlate with in vivo tumorigenicity of tumor-initiating cells

PubMed Central

LEHMANN, CHRISTIAN; JOBS, GABRIELE; THOMAS, MARKUS; BURTSCHER, HELMUT; KUBBIES, MANFRED

2012-01-01

The tumor-initiating capacity of primary human breast cancer cells is maintained in vitro by culturing these cells as spheres/aggregates. Inoculation of small cell numbers derived from these non-adherent cultures leads to rapid xenograft tumor formation in mice. Accordingly, injection of more differentiated monolayer cells derived from spheres results in significantly decelerated tumor growth. For our study, two breast cancer cell lines were generated from primary tumors and cultured as mammospheres or as their adherent counterparts. We examined the in vivo tumorigenicity of these cells by injecting serial dilutions into immunodeficient mice. Inoculation of 106 cells per mouse led to rapid tumor formation, irrespective of cell line or culture conditions. However, after injection of only 103 cells, solely sphere cells were highly tumorigenic. In vitro, we investigated differentiation markers, established breast CSC markers and conducted mRNA profiling. Cytokeratin 5 and 18 were increased in both monolayer cell types, indicating a more differentiated phenotype. All cell lines were CD24−/CD44+ and did not express CD133, CD326 or E-cadherin. ALDH1 activity was not detectable in any cell line. A verapamil-sensitive Hoechst side population was present in sphere cells, but there was no correlation with tumorigenicity in vivo. mRNA profiling did not reveal upregulation of relevant transcription factors. In vitro cell cycle kinetics and in vivo tumor doubling times displayed no difference between sphere and monolayer cultures. Our data indicate that intrinsic genetic and functional markers investigated are not indicative of the in vivo tumori-genicity of putative breast tumor-initiating cells. PMID:23042145

Chitosan-modified porous silicon microparticles for enhanced permeability of insulin across intestinal cell monolayers.

PubMed

Shrestha, Neha; Shahbazi, Mohammad-Ali; Araújo, Francisca; Zhang, Hongbo; Mäkilä, Ermei M; Kauppila, Jussi; Sarmento, Bruno; Salonen, Jarno J; Hirvonen, Jouni T; Santos, Hélder A

2014-08-01

Porous silicon (PSi) based particulate systems are emerging as an important drug delivery system due to its advantageous properties such as biocompatibility, biodegradability and ability to tailor the particles' physicochemical properties. Here, annealed thermally hydrocarbonized PSi (AnnTHCPSi) and undecylenic acid modified AnnTHCPSi (AnnUnTHCPSi) microparticles were developed as a PSi-based platform for oral delivery of insulin. Chitosan (CS) was used to modify the AnnUnTHCPSi microparticles to enhance the intestinal permeation of insulin. Surface modification with CS led to significant increase in the interaction of PSi microparticles with Caco-2/HT-29 cell co-culture monolayers. Compared to pure insulin, the CS-conjugated microparticles significantly improved the permeation of insulin across the Caco-2/HT-29 cell monolayers, with ca. 20-fold increase in the amount of insulin permeated and ca. 7-fold increase in the apparent permeability (P(app)) value. Moreover, among all the investigated particles, the CS-conjugated microparticles also showed the highest amount of insulin associated with the mucus layer and the intestinal Caco-2 cells and mucus secreting HT-29 cells. Our results demonstrate that CS-conjugated AnnUnTHCPSi microparticles can efficiently enhance the insulin absorption across intestinal cells, and thus, they are promising microsystems for the oral delivery of proteins and peptides across the intestinal cell membrane. Copyright © 2014 Elsevier Ltd. All rights reserved.

Campylobacter jejuni induces transcytosis of commensal bacteria across the intestinal epithelium through M-like cells

PubMed Central

2010-01-01

Background Recent epidemiological analyses have implicated acute Campylobacter enteritis as a factor that may incite or exacerbate inflammatory bowel disease (IBD) in susceptible individuals. We have demonstrated previously that C. jejuni disrupts the intestinal barrier function by rapidly inducing epithelial translocation of non-invasive commensal bacteria via a transcellular lipid raft-mediated mechanism ('transcytosis'). To further characterize this mechanism, the aim of this current study was to elucidate whether C. jejuni utilizes M cells to facilitate transcytosis of commensal intestinal bacteria. Results C. jejuni induced translocation of non-invasive E. coli across confluent Caco-2 epithelial monolayers in the absence of disrupted transepithelial electrical resistance or increased permeability to a 3 kDa dextran probe. C. jejuni-infected monolayers displayed increased numbers of cells expressing the M cell-specific marker, galectin-9, reduced numbers of enterocytes that stained with the absorptive enterocyte marker, Ulex europaeus agglutinin-1, and reduced activities of enzymes typically associated with absorptive enterocytes (namely alkaline phosphatase, lactase, and sucrase). Furthermore, in Campylobacter-infected monolayers, E. coli were observed to be internalized specifically within epithelial cells displaying M-like cell characteristics. Conclusion These data indicate that C. jejuni may utilize M cells to promote transcytosis of non-invasive bacteria across the intact intestinal epithelial barrier. This mechanism may contribute to the inflammatory immune responses against commensal intestinal bacteria commonly observed in IBD patients. PMID:21040540

Multicellular contractility contributes to the emergence of mesothelioma nodules

NASA Astrophysics Data System (ADS)

Czirok, Andras

Malignant pleural mesothelioma (MPM) nodules arise from the mesothelial lining of the pleural cavity by a poorly understood mechanism. We demonstrate that macroscopic multicellular aggregates, reminiscent of the MPM nodules found in patients, develop when MPM cell lines are cultured at high cell densities for several weeks. Surprisingly, the nodule-like aggregates do not arise by excessive local cell proliferation, but by myosin II-driven cell contractility. Contractile nodules contain prominent actin cables that can span several cells. Several features of the in vitro MPM nodule development can be explained by a computational model that assumes uniform and steady intercellular contractile forces within a monolayer of cells, and a mechanical load-dependent lifetime of cell-cell contacts. The model behaves as a self-tensioned Maxwell fluid and exhibits an instability that leads to pattern formation. Altogether, our findings suggest that inhibition of the actomyosin system may provide a hitherto not utilized therapeutic approach to affect MPM growth. NIH R01-GM102801.

The interaction of insulin, glucose, and insulin-glucose mixtures with a phospholipid monolayer.

PubMed

Shigenobu, Hayato; McNamee, Cathy E

2012-12-15

We determined how glucose or insulin interacts with a phospholipid monolayer at the air/water interface and explained these mechanisms from a physico-chemical point of view. The 1,2-dipalmitoyl-2-sn-glycero-3-phosphatidylcholine (DPPC) monolayer at an air/water interface acted as a model membrane, which allowed the effect of the molecular packing density in the monolayer on the interactions to be determined. The interaction of glucose, insulin, and a mixture of glucose and insulin to the DPPC monolayer were investigated via surface pressure-area per molecule Langmuir isotherms and fluorescence microscopy. Glucose adsorbed to the underside of the DPPC monolayer, while insulin was able to penetrate through the monolayer when the phospholipid molecules were not densely packed. The presence of a mixture of insulin and glucose affected the molecular packing in the DPPC monolayer differently than the pure insulin or glucose solutions, and the glucose-insulin mixture was seen to be able to penetrate through the monolayer. These results indicated that glucose and insulin interact with one another, giving a material that may then transported through a pore in the monolayer or through the spaces between the molecules of the monolayer. Copyright © 2012 Elsevier Inc. All rights reserved.

In vitro cytotoxity of silver: implication for clinical wound care.

PubMed

Poon, Vincent K M; Burd, Andrew

2004-03-01

In this study, we look at the cytotoxic effects of silver on keratinocytes and fibroblasts. We have assessed the viability of monolayer cultures using the MTT and BrdU assays. The composition of the culture medium and also the culture technique were modified to assess the effects of culture 'environment' on the susceptibility of the cells to the toxic action of silver. Further in vitro, experiments were performed using tissue culture models to allow cellular behavior in three dimensional planes which more closely simulated in vivo behavior. The silver source was both silver released from silver nitrate solution but also nanocrystalline silver released from a commercially available dressing. The results show that silver is highly toxic to both keratinocytes and fibroblasts in monolayer culture. When using optimized and individualized culture the fibroblasts appear to be more sensitive to silver than keratinocytes. However, when both cell types were grown in the same medium their viability was the same. Using tissue culture models again indicated an 'environmental effect' with decreased sensitivity of the cells to the cytotoxic effects of the silver. Nevertheless in these studies the toxic dose of skin cells ranging from 7 x 10(-4) to 55 x 10(-4)% was similar to that of bacteria. These results suggest that consideration of the cytotoxic effects of silver and silver-based products should be taken when deciding on dressings for specific wound care strategies. This is important when using keratinocyte culture, in situ, which is playing an increasing role in contemporary wound and burn care.

Propulsion and navigation within the advancing monolayer sheet

PubMed Central

Kim, Jae Hun; Serra-Picamal, Xavier; Tambe, Dhananjay T.; Zhou, Enhua H.; Park, Chan Young; Sadati, Monirosadat; Park, Jin-Ah; Krishnan, Ramaswamy; Gweon, Bomi; Millet, Emil; Butler, James P.; Trepat, Xavier; Fredberg, Jeffrey J.

2013-01-01

As a wound heals, or a body plan forms, or a tumor invades, observed cellular motions within the advancing cell swarm are thought to stem from yet to be observed physical stresses that act in some direct and causal mechanical fashion. Here we show that such a relationship between motion and stress is far from direct. Using monolayer stress microscopy, we probed migration velocities, cellular tractions and intercellular stresses in an epithelial cell sheet advancing towards an island on which cells cannot adhere. We found that cells located near the island exert tractions that pull systematically towards this island regardless of whether the cells approach the island, migrate tangentially along its edge or, paradoxically, recede from it. This unanticipated cell-patterning motif, which we call kenotaxis, represents the robust and systematic mechanical drive of the cellular collective to fill unfilled space. PMID:23793160

Alignment of cell division axes in directed epithelial cell migration

NASA Astrophysics Data System (ADS)

Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

2014-11-01

Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

Multidrug resistance characterization in multicellular tumour spheroids from two human lung cancer cell lines.

PubMed

Barrera-Rodríguez, Raúl; Fuentes, Jorge Morales

2015-01-01

Most of the knowledge about the mechanisms of multidrug resistance in lung cancer has been achieved through the use of cell lines isolated from tumours cultivated either in suspensions of isolated cells or in monolayers and following exposition to different cytostatic agents. However, tumour cell lines growing as multicellular tumour spheroids (MTS) frequently develop multicellular resistance in a drug-independent form. The aim of this study was to characterize the phenotypic and functional differences between two human NSCLC cell lines (INER-37 and INER-51) grown as traditional monolayer cultures versus as MTS. After 72 hours treatment with anticancer drugs, chemosensitivity in monolayers and tumour spheroids cultures was assessed using MTT assay. Reverse transcription-polymerase chain reaction was employed to detect the mRNAs of multidrug resistance-related genes. The expression of P-gp was analyzed by immunohistochemical staining and cell cycle profiles were analyzed using FACS. The results indicate that when grown as MTS each lung cancer cell line had different morphologies as well as and abrogation of cell proliferation with decrease of the G2/M phase. Also, MTS acquired multicellular resistance to several chemotherapeutic agents in only a few days of culture which were accomplished by significant changes in the expression of MDR-related genes. Overall, the MTS culture changed the cellular response to drugs nevertheless each of the cell lines studied seems to implement different mechanisms to acquire multicellular resistance.

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers.

PubMed

Milkani, Eftim; Khaing, Aung M; Huang, Fei; Gibson, Daniel G; Gridley, Scott; Garceau, Norman; Lambert, Christopher R; McGimpsey, W Grant

2010-12-21

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.

Establishment and Characterization of an Embryonic Cell Line from Sarconesiopsis magellanica

PubMed Central

Cruz, Mónica; Bello, Felio J.

2013-01-01

Sarconesiopsis magellanica (Le Guillou) (Diptera: Calliphoridae) is a necrophagous fly that is important in both human and veterinary medicines. This insect has been registered in Colombia as a biological indicator in estimating post-mortem interval. Insect cell cultures are an important biotechnological tool for basic and applied studies, and cell cultures derived from S. magellanica embryonic tissues are described in this study. S. magellanica embryonated eggs were taken for tissue explants. These were seeded in L-15, Grace/L-15, Eagle MEM, MM, VP12, MM/VP12, and Schneider culture media. The morphological, cytogenetic, biochemical, and molecular characteristics of the cell cultures were examined. Cell growth was achieved in the L15, Grace/L15, and Schneider culture media, and the confluent monolayers were obtained 8, 10, and 19 days after the embryonated eggs were explanted. However, the Schneider medium was the most efficient to develop the subcultures, and 21 passages have been maintained. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer and in the subcultures there was greater cell morphology uniformity, fibroblastoid types being predominant. Cultured cells had a chromosomal number of 12, and the karyotypic complement consisted of five pairs of somatic chromosomes and one sexual pair. The cell culture isozyme patterns of S. magellanica coincided with adult samples from the same species. The molecular analysis, using RAPD-PCR, demonstrated the authentication of the cell cultures of this fly and their differentiation from other cultures derived from two sand flies species. This cell line is a new in vitro model that will be used in biomedical and biotechnological studies. PMID:24766352

Influence of coexisting phases on the surface dilatational viscosity of Langmuir monolayers.

PubMed

Lopez, Juan M; Vogel, Michael J; Hirsa, Amir H

2004-11-01

Monolayer hydrodynamics are usually described in terms of a Newtonian constitutive relationship. However, this macroscopic view fails to account for small-scale coexisting phase domains, which are generally present in the monolayer and appear to have profound macroscopic effects. Here, we provide direct evidence of these effects, consisting of Brewster angle microscopy images of the monolayer, space- and time-resolved interfacial velocity measurements, and comparisons with predictions based on the Navier-Stokes equations together with the classic model for a Newtonian interface.

The role of apoptosis in LDL transport through cultured endothelial cell monolayers

PubMed Central

Cancel, Limary M.; Tarbell, John M.

2009-01-01

We have previously shown that leaky junctions associated with dying or dividing cells are the dominant pathway for LDL transport under convective conditions, accounting for more than 90% of the transport [1]. To explore the role of apoptosis in the leaky junction pathway, TNFα and cycloheximide (TNFα/CHX) were used to induce an elevated rate of apoptosis in cultured bovine aortic endothelial cell (BAEC) monolayers and the convective fluxes of LDL and water were measured. Treatment with TNFα/CHX induced a 18.3-fold increase in apoptosis and a 4.4-fold increase in LDL permeability. Increases in apoptosis and permeability were attenuated by treatment with the caspase inhibitor Z-VAD-FMK. Water flux increased by 2.7-fold after treatment with TNFα/CHX, and this increase was not attenuated by treatment with Z-VAD-FMK. Immunostaining of the tight junction protein ZO-1 showed that TNFα/CHX treatment disrupts the tight junction in addition to inducing apoptosis. This disruption is present even when Z-VAD-FMK is used to inhibit apoptosis, and likely accounts for the increase in water flux. We found a strong correlation between the rate of apoptosis and the permeability of BAEC monolayers to LDL. These results demonstrate the potential of manipulating endothelial monolayer permeability by altering the rate of apoptosis pharmacollogicaly. This has implications for the treatment of atherosclerosis. PMID:19709659

Ascorbate protects endothelial barrier function during septic insult: Role of protein phosphatase type 2A.

PubMed

Han, Min; Pendem, Suresh; Teh, Suet Ling; Sukumaran, Dinesh K; Wu, Feng; Wilson, John X

2010-01-01

Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising the intracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting protein phosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate, dehydroascorbic acid (DHAA), the NADPH oxidase inhibitors apocynin and diphenyliodonium, or the PP2A inhibitor okadaic acid and then were exposed to septic insult (lipopolysaccharide and interferon-gamma). Under standard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant production and PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayer permeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showing that these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2A activity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A in the response of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbate concentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascular endothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby prevents PP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of the endothelial barrier. Copyright 2009 Elsevier Inc. All rights reserved.

The role of mitosis in LDL transport through cultured endothelial cell monolayers

PubMed Central

Cancel, Limary M.

2011-01-01

We (7) have previously shown that leaky junctions associated with dying or dividing cells are the dominant pathway for LDL transport under convective conditions, accounting for >90% of the transport. We (8) have also recently shown that the permeability of bovine aortic endothelial cell monolayers is highly correlated with their rate of apoptosis and that inhibiting apoptosis lowers the permeability of the monolayers to LDL. To explore the role of mitosis in the leaky junction pathway, the microtubule-stabilizing agent paclitaxel was used to alter the rate of mitosis, and LDL flux and water flux (Jv) were measured. Control monolayers had an average mitosis rate of 0.029%. Treatment with paclitaxel (2.5 μM) for 1.5, 3, 4.5, or 6 h yielded increasing rates of mitosis ranging from 0.099% to 1.03%. The convective permeability of LDL (Pe) increased up to fivefold, whereas Jv increased up to threefold, over this range of mitosis rates. We found strong correlations between the mitosis rate and both Pe and Jv. However, compared with our previous apoptosis study (8), we found that mitosis was only half as effective as apoptosis in increasing Pe. The results led us to conclude that while mitotsis-related leaky junctions might play a role in the initial infiltration of LDL into the artery wall, the progression of atherosclerosis might be more closely correlated with apoptosis-related leaky junctions. PMID:21169397

Reduction Reaction Activity on Pt-Monolayer-Shell PdIr/Ni-core Catalysts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Liang; Vukmirovic, Miomir B.; Adzic, Radoslav R.

Platinum monolayer oxygen reduction reaction catalysts present promising way of reducing the Pt content without scarifying its fuel cell performance. We present a facile way of preparing Pt monolayer shell PdIr-based core catalysts, which showed much higher activity for oxygen reduction reaction than that of TKK 46.6% Pt/C catalyst. Among tested samples, PtMLPd2Ir/Ni/C performs the best with Pt and Platinum Group Metal mass activity around 9 and 0.25 times higher of that of TKK 46.6% Pt/C. In addition, accelerated aging test indicates its excellent durability.

Reduction Reaction Activity on Pt-Monolayer-Shell PdIr/Ni-core Catalysts

DOE PAGES

Song, Liang; Vukmirovic, Miomir B.; Adzic, Radoslav R.

2018-05-14

Platinum monolayer oxygen reduction reaction catalysts present promising way of reducing the Pt content without scarifying its fuel cell performance. We present a facile way of preparing Pt monolayer shell PdIr-based core catalysts, which showed much higher activity for oxygen reduction reaction than that of TKK 46.6% Pt/C catalyst. Among tested samples, PtMLPd2Ir/Ni/C performs the best with Pt and Platinum Group Metal mass activity around 9 and 0.25 times higher of that of TKK 46.6% Pt/C. In addition, accelerated aging test indicates its excellent durability.

Paracellular transport of avidin saturated or not with biotinylated cobalamin through Caco-2 cell epithelium monolayer.

PubMed

Guy, M; Pons, L; Namour, F; de Nonancourt, M; Michalski, J C; Hatier, R; Guéant, J L

2001-01-01

The cationic charge of molecules may promote their uptake across epithelia, which are rich in brush border anionic sites. The transport of unsaturated avidin and avidin saturated with a biotinylated compound was investigated across Caco-2 adenocarcinoma cell with fetal enterocyte phenotype. The unsaturated avidin and avidin saturated with either biotin or a biotinyl-cobalamin conjugate (biotinyl-Cbl) were iodinated to follow their transport through the cell monolayer. Their apparent permeability coefficient (Papp) and transepithelial pathway were determined and compared to those for control radiolabeled markers [3H]-mannitol, [125I]-beta-lactoglobulin and [57Co]-cobalamin/intrinsic factor (Cbl/IF). The Papp of [125I]-avidin estimated at 2.8 x 10(-7) +/- 0.08 cm/s was close to that for mannitol that uses paracellular pathway. The binding of biotin or biotin conjugate to avidin enhanced its tetrameric conformation. The Papp for [125I]-avidin/biotin and [125I]- avidin/biotinyl-Cbl were respectively increased by 2-fold, compared to that for [125I]-avidin and 4-fold, compared to that for [125I]-beta-lactoglobulin and [54Co]-Cbl/IF. The protein was not accumulated in the cell and was found in intact form in the basolateral side, after its transport across the monolayer. Chloroquine (0.66 micromol/ml) did not significantly decrease the Papp for [125I]-avidin/biotinyl-Cbl. Conversely it decreased by 80% the Papp for Cbl/IF, that uses transepithelial pathway. Avidin (either saturated or not with biotin and biotinyl-Cbl) was able to cross the monolayer of Caco-2 cell line through a paracellular pathway. This study pointed out the interest for using this protein as a shuttle for increasing the transport rate of biotinylated compounds through fetal epithelial barriers. Copyright 2001 S. Karger AG, Basel

Vascular endothelial growth factor-D is a key molecule that enhances lymphatic metastasis of soft tissue sarcomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanagawa, Takashi, E-mail: tyanagaw@med.gunma-u.ac.jp; Shinozaki, Tetsuya; Watanabe, Hideomi

2012-04-15

Studies on lymph node metastasis of soft tissue sarcomas are insufficient because of its rarity. In this study, we examined the expressions of vascular endothelial growth factor (VEGF)-C and VEGF-D in soft tissue sarcomas metastasized to lymph nodes. In addition, the effects of the two molecules on the barrier function of a lymphatic endothelial cell monolayer against sarcoma cells were analyzed. We examined 7 patients who had soft tissue sarcomas with lymph node metastases and who had undergone neither chemotherapy nor radiotherapy before lymphadenectomy. Immunohistochemistry revealed that 2 of 7 sarcomas that metastasized to lymph nodes expressed VEGF-C both inmore » primary and metastatic lesions. On the other hand, VEGF-D expression was detected in 4 of 7 primary and 7 of 7 metastatic lesions, respectively. Interestingly, 3 cases that showed no VEGF-D expression at primary sites expressed VEGF-D in metastatic lesions. Recombinant VEGF-C at 10{sup -8} and VEGF-D at 10{sup -7}and 10{sup -8} g/ml significantly increased the random motility of lymphatic endothelial cells compared with controls. VEGF-D significantly increased the migration of sarcoma cells through lymphatic endothelial monolayers. The fact that VEGF-D induced the migration of fibrosarcomas through the lymphatic endothelial monolayer is the probable reason for the strong relationship between VEGF-D expression and lymph node metastasis in soft tissue sarcomas. The important propensities of this molecule for the increase of lymph node metastases are not only lymphangiogenesis but also down-regulation of the barrier function of lymphatic endothelial monolayers, which facilitates sarcoma cells entering the lymphatic circulation.« less

Characterization of release of basic fibroblast growth factor from bovine retinal endothelial cells in monolayer cultures.

PubMed Central

Brooks, R A; Burrin, J M; Kohner, E M

1991-01-01

Release of basic fibroblast growth factor (bFGF) was investigated in bovine retinal endothelial cells (BREC) maintained in monolayer culture. Confluent cells released bFGF into serum-free culture medium or medium containing 5% serum at rates of up to 105.2 and 61.3 pM/day respectively. bFGF release coincided with a decrease in monolayer cell number and increases in lactate dehydrogenase (LDH) concentration and cells and cell-debris particles in the medium, which suggested that cell damage and lysis were responsible for growth-factor release. Maximum bFGF release at 24 h (230 +/- 10 pM) occurred when the cells were treated with lipopolysaccharide (10 micrograms/ml), which also produced the greatest changes in parameters of cell damage. Sub-confluent cells showed little overt damage at 24 h, but released bFGF (78 +/- 20 pM) along with LDH, indicating that some cell lysis had occurred. Insulin-like growth factor 1 (IGF-1) was also released into serum-free culture medium at a rate of 0.34 nM/day, but not into medium containing serum or when the cells were treated with lipopolysaccharide. This implies that the mechanism of IGF-1 release is different from that of bFGF and is not related to cell damage. Culture medium conditioned by BREC stimulated the proliferation of these cells, as measured by an increase in their incorporation of [methyl-3H]thymidine from 7550 +/- 479 to 10467 +/- 924 d.p.m. These results demonstrate that bFGF is released from damaged BREC and that medium conditioned by these cells can stimulate retinal-endothelial-cell proliferation. This strengthens the case for an involvement of this growth factor in retinal neovascularization. Images Fig. 1. PMID:2039465

The effect of fibrin on cultured vascular endothelial cells.

PubMed

Kadish, J L; Butterfield, C E; Folkman, J

1979-01-01

The normal cobblestone monolayer architecture of cultured vascular endothelium becomes rapidly disorganized after contact of the cell layer with a fibrin clot. The cells of a confluent endothelial monolayer separate into individual migratory cells in 4--6 hr after contact with fibrin. The effect is reversible in that removal of the fibrin clot results in resumption of the normal morphology within about 2 hr. No other cell type tested exhibits the same change in organization when exposed to fibrin. A similar morphological change in endothelium does occur after the cell layer is overlaid with a collagen fibril gel but a gel of methylcellulose has no effect. It is proposed that the change in behavior of endothelial cells in response to contact with fibrin may represent a cellular component of fibrinolysis. The implications of this finding for the pathophysiology of disease states involving intravascular fibrin deposition are discussed.

Real-time estimation of paracellular permeability of cerebral endothelial cells by capacitance sensor array

NASA Astrophysics Data System (ADS)

Hyun Jo, Dong; Lee, Rimi; Hyoung Kim, Jin; Oh Jun, Hyoung; Geol Lee, Tae; Hun Kim, Jeong

2015-06-01

Vascular integrity is important in maintaining homeostasis of brain microenvironments. In various brain diseases including Alzheimer’s disease, stroke, and multiple sclerosis, increased paracellular permeability due to breakdown of blood-brain barrier is linked with initiation and progression of pathological conditions. We developed a capacitance sensor array to monitor dielectric responses of cerebral endothelial cell monolayer, which could be utilized to evaluate the integrity of brain microvasculature. Our system measured real-time capacitance values which demonstrated frequency- and time-dependent variations. With the measurement of capacitance at the frequency of 100 Hz, we could differentiate the effects of vascular endothelial growth factor (VEGF), a representative permeability-inducing factor, on endothelial cells and quantitatively analyse the normalized values. Interestingly, we showed differential capacitance values according to the status of endothelial cell monolayer, confluent or sparse, evidencing that the integrity of monolayer was associated with capacitance values. Another notable feature was that we could evaluate the expression of molecules in samples in our system with the reference of real-time capacitance values. We suggest that this dielectric spectroscopy system could be successfully implanted as a novel in vitro assay in the investigation of the roles of paracellular permeability in various brain diseases.

Conformal and highly luminescent monolayers of Alq3 prepared by gas-phase molecular layer deposition.

PubMed

Räupke, André; Albrecht, Fabian; Maibach, Julia; Behrendt, Andreas; Polywka, Andreas; Heiderhoff, Ralf; Helzel, Jonatan; Rabe, Torsten; Johannes, Hans-Hermann; Kowalsky, Wolfgang; Mankel, Eric; Mayer, Thomas; Görrn, Patrick; Riedl, Thomas

2014-01-22

The gas-phase molecular layer deposition (MLD) of conformal and highly luminescent monolayers of tris(8-hydroxyquinolinato)aluminum (Alq3) is reported. The controlled formation of Alq3 monolayers is achieved for the first time by functionalization of the substrate with amino groups, which serve as initial docking sites for trimethyl aluminum (TMA) molecules binding datively to the amine. Thereby, upon exposure to 8-hydroxyquinoline (8-HQ), the self-limiting formation of highly luminescent Alq3 monolayers is afforded. The growth process and monolayer formation were studied and verified by in situ quartz crystal monitoring, optical emission and absorption spectroscopy, and X-ray photoelectron spectroscopy. The nature of the MLD process provides an avenue to coat arbitrarily shaped 3D surfaces and porous structures with high surface areas, as demonstrated in this work for silica aerogels. The concept presented here paves the way to highly sensitive luminescent sensors and dye-sensitized metal oxides for future applications (e.g., in photocatalysis and solar cells).

Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance.

PubMed

Li, Yi; Guo, Gang; Li, Li; Chen, Fei; Bao, Ji; Shi, Yu-Jun; Bu, Hong

2015-05-01

Mesenchymal stem cell (MSC) transplantation is a promising treatment of many diseases. However, conventional techniques with cells being cultured as a monolayer result in slow cell proliferation and insufficient yield to meet clinical demands. Three-dimensional (3D) culture systems are gaining attention with regard to recreating a complex microenvironment and to understanding the conditions experienced by cells. Our aim is to establish a novel 3D system for the culture of human umbilical cord MSCs (hUC-MSCs) within a real 3D microenvironment but with no digestion or passaging. Primary hUC-MSCs were isolated and grown in serum-free medium (SFM) on a suspension Rocker system. Cell characteristics including proliferation, phenotype and multipotency were recorded. The therapeutic effects of 3D-cultured hUC-MSCs on carbon tetrachloride (CCl4)-induced acute liver failure in mouse models were examined. In the 3D Rocker system, hUC-MSCs formed spheroids in SFM and maintained high viability and active proliferation. Compared with monolayer culture, the 3D-culture system yielded more hUC-MSCs cells within the same volume. The spheroids expressed higher levels of stem cell markers and displayed stronger multipotency. After transplantation into mouse, 3D hUC-MSCs significantly promoted the secretion of interferon-γ and interleukin-6 but inhibited that of tumor necrosis factor-α, thereby alleviating liver necrosis and promoting regeneration following CCl4 injury. The 3D culture of hUC-MSCs thus promotes cell yield and stemness maintenance and represents a promising strategy for hUC-MSCs expansion on an industrial scale with great potential for cell therapy and biotechnology.

Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model.

PubMed

Krawczyk, Krzysztof M; Matak, Damian; Szymanski, Lukasz; Szczylik, Cezary; Porta, Camillo; Czarnecka, Anna M

2018-04-01

The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco's Modified Eagle's Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

Titanium dioxide nanoparticle exposure alters metabolic homeostasis in a cell culture model of the intestinal epithelium and Drosophila melanogaster.

PubMed

Richter, Jonathan W; Shull, Gabriella M; Fountain, John H; Guo, Zhongyuan; Musselman, Laura P; Fiumera, Anthony C; Mahler, Gretchen J

2018-06-01

Nanosized titanium dioxide (TiO 2 ) is a common additive in food and cosmetic products. The goal of this study was to investigate if TiO 2 nanoparticles affect intestinal epithelial tissues, normal intestinal function, or metabolic homeostasis using in vitro and in vivo methods. An in vitro model of intestinal epithelial tissue was created by seeding co-cultures of Caco-2 and HT29-MTX cells on a Transwell permeable support. These experiments were repeated with monolayers that had been cultured with the beneficial commensal bacteria Lactobacillus rhamnosus GG (L. rhamnosus). Glucose uptake and transport in the presence of TiO 2 nanoparticles was assessed using fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). When the cell monolayers were exposed to physiologically relevant doses of TiO 2 , a statistically significant reduction in glucose transport was observed. These differences in glucose absorption were eliminated in the presence of beneficial bacteria. The decrease in glucose absorption was caused by damage to intestinal microvilli, which decreased the surface area available for absorption. Damage to microvilli was ameliorated in the presence of L. rhamnosus. Complimentary studies in Drosophila melanogaster showed that TiO 2 ingestion resulted in decreased body size and glucose content. The results suggest that TiO 2 nanoparticles alter glucose transport across the intestinal epithelium, and that TiO 2 nanoparticle ingestion may have physiological consequences.

Copper-phospholipid interaction at cell membrane model hydrophobic surfaces.

PubMed

Mlakar, Marina; Cuculić, Vlado; Frka, Sanja; Gašparović, Blaženka

2018-04-01

Detailed investigation of Cu (II) binding with natural lipid phosphatidylglycerol (PG) in aqueous solution was carried out by voltammetric measurements at the mercury drop electrode, complemented by monolayer studies in a Langmuir trough and electrophoretic measurements, all used as models for hydrophobic cell membranes. Penetration of copper ions into the PG layer was facilitated by the formation of hydrophilic Cu-Phenanthroline (Phen) complex in the subphase, followed by the mixed ligand Cu-Phen-PG complex formation at the hydrophobic interface. Electrophoretic measurements indicated a comparatively low abundance of the formed mixed ligand complex within the PG vesicles, resulting it the zeta potential change of +0.83mV, while monolayer studies confirmed their co-existence at the interface. The Cu-Phen-PG complex was identified in the pH range from 6 to 9. The stoichiometry of the complex ([PhenCuOHPG]), as well as its stability and kinetics of formation, were determined at the mercury drop electrode. Cu-Phen-PG reduces quasireversibly at about -0.7V vs. Ag/AgCl including reactant adsorption, followed by irreversible mixed complex dissociation, indicating a two-electron transfer - chemical reaction (EC mechanism). Consequently, the surface concentration (γ) of the adsorbed [PhenCuOHPG] complex at the hydrophobic electrode surface was calculated to be (3.35±0.67)×10 -11 molcm -2 . Information on the mechanism of Cu (II) - lipid complex formation is a significant contribution to the understanding of complex processes at natural cell membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient differentiation of mouse embryonic stem cells into insulin-producing cells.

PubMed

Liu, Szu-Hsiu; Lee, Lain-Tze

2012-01-01

Embryonic stem (ES) cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs) by a two-step differentiation protocol comprising of (i) the formation of definitive endoderm in monolayer culture by activin A, and (ii) this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

A simple hanging drop cell culture protocol for generation of 3D spheroids.

PubMed

Foty, Ramsey

2011-05-06

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels or in biomaterial scaffolds. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model. Copyright © 2011 Journal of Visualized Experiments

Dermaseptin 01 as antimicrobial peptide with rich biotechnological potential: study of peptide interaction with membranes containing Leishmania amazonensis lipid-rich extract and membrane models.

PubMed

Salay, Luiz C; Nobre, Thatyane M; Colhone, Marcelle C; Zaniquelli, Maria E D; Ciancaglini, Pietro; Stabeli, Rodrigo G; Leite, José Roberto S A; Zucolotto, Valtencir

2011-10-01

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA- KAAGQAALGAL-NH(2) , DS 01) with phospholipid (PL) monolayers comprising (i) a lipid-rich extract of Leishmania amazonensis (LRE-La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid-air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE-La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE-La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Bursts of activity in collective cell migration

PubMed Central

Chepizhko, Oleksandr; Giampietro, Costanza; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stéphane; Alava, Mikko J.; Zapperi, Stefano; La Porta, Caterina A. M.

2016-01-01

Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media, and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems. PMID:27681632

The Behavior of Lipid Debris Left on Cell Surfaces from Microbubble Based Ultrasound Molecular Imaging

PubMed Central

Ibsen, Stuart; Shi, Guixin; Schutt, Carolyn; Shi, Linda; Suico, Kyle-David; Benchimol, Michael; Serra, Viviana; Simberg, Dmitri; Berns, Michael; Esener, Sadik

2014-01-01

Lipid monolayer coated microbubbles are currently being developed to identify vascular regions that express certain surface proteins as part of the new technique of ultrasound molecular imaging. The microbubbles are functionalized with targeting ligands which bind to the desired cells holding the microbubbles in place as the remaining unbound microbubbles are eliminated from circulation. Subsequent scanning with ultrasound can detect the highly reflectant microbubbles that are left behind. The ultrasound scanning and detection process results in the destruction of the microbubble, creating lipid fragments from the monolayer. Here we demonstrate that microbubbles targeted to 4T1 murine breast cancer cells and human umbilical cord endothelial cells leave behind adhered fragments of the lipid monolayer after exposure to ultrasound with peak negative pressures of 0.18 and 0.8 MPa. Most of the observed fragments were large enough to be resistant to receptor mediated endocytosis. The fragments were not observed to incorporate into the lipid membrane of the cell over a period of 96 min. They were not observed to break into smaller pieces or significantly change shape but they were observed to undergo translation and rotation across the cell surface as the cells migrated over the substrate. These large fragments will apparently remain on the surface of the targeted cells for significant periods of time and need to be considered for their potential effects on blood flow through the microcapillaries and potential for immune system recognition. PMID:25059435

Enhanced monolayer MoS2/InP heterostructure solar cells by graphene quantum dots

NASA Astrophysics Data System (ADS)

Wang, Peng; Lin, Shisheng; Ding, Guqiao; Li, Xiaoqiang; Wu, Zhiqian; Zhang, Shengjiao; Xu, Zhijuan; Xu, Sen; Lu, Yanghua; Xu, Wenli; Zheng, Zheyang

2016-04-01

We demonstrate significantly improved photovoltaic response of monolayer molybdenum disulfide (MoS2)/indium phosphide (InP) van der Waals heterostructure induced by graphene quantum dots (GQDs). Raman and photoluminescence measurements indicate that effective charge transfer takes place between GQDs and MoS2, which results in n-type doping of MoS2. The doping effect increases the barrier height at the MoS2/InP heterojunction, thus the averaged power conversion efficiency of MoS2/InP solar cells is improved from 2.1% to 4.1%. The light induced doping by GQD provides a feasible way for developing more efficient MoS2 based heterostructure solar cells.

Short-Chain PEG Mixed-Monolayer Protected Gold Clusters Increase Clearance and Red Blood Cell Counts

PubMed Central

Simpson, Carrie A.; Agrawal, Amanda C.; Balinski, Andrzej; Harkness, Kellen M.; Cliffel, David E.

2011-01-01

Monolayer-protected gold nanoparticles have great potential as novel building blocks for the design of new drugs and therapeutics based on the easy ability to multifunctionalize them for biological targeting and drug activity. In order to create nanoparticles that are biocompatible in vivo, poly-ethylene glycol functional groups have been added to many previous multifunctionalized particles to eliminate non-specific binding. Recently, monolayer-protected gold nanoparticles with mercaptoglycine functionalities were shown to elicit deleterious effects on the kidney in vivo that were eliminated by incorporating a long-chain, mercapto-undecyl-tetraethylene glycol, at very high loadings into a mixed monolayer. These long-chain PEGs induced an immune response to the particle presumably generating an anti-PEG antibody as seen in other long-chain PEG-ylated nanoparticles in vivo. In the present work, we explore the in vivo effects of high and low percent ratios of a shorter chain, mercapto-tetraethylene glycol, within the monolayer using simple place-exchange reactions. The shorter chain PEG MPCs were expected to have better water solubility due to elimination of the alkyl chain, no toxicity, and long-term circulation in vivo. Shorter chain lengths at lower concentrations should not trigger the immune system into creating an anti-PEG antibody. We found that a 10% molar exchange of this short chain PEG within the monolayer met three of the desired goals: high water solubility, no toxicity, and no immune response as measured by white blood cell counts, but none of the short chain PEG mixed monolayer compositions enabled the nanoparticles to have a long circulation time within the blood as compared to mercapto-undecyl-ethylene glycol, which had a residence time of 4 weeks. We also compared the effects of a hydroxyl versus a carboxylic acid terminal functional group on the end of the PEG thiol on both clearance and immune response. The results indicate that short-chain length PEGs, regardless of termini, increase clearance rates compared to the previous long-chain PEG studies while carboxylated-termini increase red blood cell counts at high loadings. Given these findings, short-chain, alcohol-terminated PEG, exchanged at 10% was identified as a potential nanoparticle for further in vivo applications requiring short circulation lifetimes with desired features of no toxicity, no immune response, and high water solubility. PMID:21473648

Long-lived force patterns and deformation waves at repulsive epithelial boundaries

NASA Astrophysics Data System (ADS)

Rodríguez-Franco, Pilar; Brugués, Agustí; Marín-Llauradó, Ariadna; Conte, Vito; Solanas, Guiomar; Batlle, Eduard; Fredberg, Jeffrey J.; Roca-Cusachs, Pere; Sunyer, Raimon; Trepat, Xavier

2017-10-01

For an organism to develop and maintain homeostasis, cell types with distinct functions must often be separated by physical boundaries. The formation and maintenance of such boundaries are commonly attributed to mechanisms restricted to the cells lining the boundary. Here we show that, besides these local subcellular mechanisms, the formation and maintenance of tissue boundaries involves long-lived, long-ranged mechanical events. Following contact between two epithelial monolayers expressing, respectively, EphB2 and its ligand ephrinB1, both monolayers exhibit oscillatory patterns of traction forces and intercellular stresses that tend to pull cell-matrix adhesions away from the boundary. With time, monolayers jam, accompanied by the emergence of deformation waves that propagate away from the boundary. This phenomenon is not specific to EphB2/ephrinB1 repulsion but is also present during the formation of boundaries with an inert interface and during fusion of homotypic epithelial layers. Our findings thus unveil a global physical mechanism that sustains tissue separation independently of the biochemical and mechanical features of the local tissue boundary.

SAM-based cell transfer to photopatterned hydrogels for microengineering vascular-like structures.

PubMed

Sadr, Nasser; Zhu, Mojun; Osaki, Tatsuya; Kakegawa, Takahiro; Yang, Yunzhi; Moretti, Matteo; Fukuda, Junji; Khademhosseini, Ali

2011-10-01

A major challenge in tissue engineering is to reproduce the native 3D microvascular architecture fundamental for in vivo functions. Current approaches still lack a network of perfusable vessels with native 3D structural organization. Here we present a new method combining self-assembled monolayer (SAM)-based cell transfer and gelatin methacrylate hydrogel photopatterning techniques for microengineering vascular structures. Human umbilical vein cell (HUVEC) transfer from oligopeptide SAM-coated surfaces to the hydrogel revealed two SAM desorption mechanisms: photoinduced and electrochemically triggered. The former, occurs concomitantly to hydrogel photocrosslinking, and resulted in efficient (>97%) monolayer transfer. The latter, prompted by additional potential application, preserved cell morphology and maintained high transfer efficiency of VE-cadherin positive monolayers over longer culture periods. This approach was also applied to transfer HUVECs to 3D geometrically defined vascular-like structures in hydrogels, which were then maintained in perfusion culture for 15 days. As a step toward more complex constructs, a cell-laden hydrogel layer was photopatterned around the endothelialized channel to mimic the vascular smooth muscle structure of distal arterioles. This study shows that the coupling of the SAM-based cell transfer and hydrogel photocrosslinking could potentially open up new avenues in engineering more complex, vascularized tissue constructs for regenerative medicine and tissue engineering applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bluetongue virus infection alters the impedance of monolayers of bovine endothelial cells as a result of cell death.

PubMed

Drew, Clifton P; Gardner, Ian A; Mayo, Christie E; Matsuo, Eiko; Roy, Polly; MacLachlan, N James

2010-07-01

Bluetongue virus (BTV) is the cause of bluetongue, an emerging, arthropod-transmitted disease of ungulates. Bluetongue is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema, lesions that are consistent with those of the so-called viral hemorrhagic fevers. To further investigate the pathogenesis of vascular injury in bluetongue, we utilized an electrical impedance assay and immunofluorescence staining to compare the effects of BTV infection on cultured bovine endothelial cells (bPAEC) with those of inducers of cell death (Triton X-100) and interendothelial gap formation (tissue necrosis factor [TNF]). The data confirm that the adherens junctions of BTV-infected bPAECs remained intact until 24h post-infection, and that loss of monolayer impedance precisely coincided with onset of virus-induced cell death. In contrast, recombinant bovine TNF-alpha caused rapid loss of bPAEC monolayer impedance that was associated with interendothelial gap formation and redistribution of VE-cadherin, but without early cell death. The data from these in vitro studies are consistent with a pathogenesis of bluetongue that involves virus-induced vascular injury leading to thrombosis, hemorrhage and tissue necrosis. However, the contribution of cytokine-induced interendothelial gap formation with subsequent edema and hypovolemic shock contributes to the pathogenesis of bluetongue remains to be fully characterized. Copyright 2010 Elsevier B.V. All rights reserved.

α2β1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis

PubMed Central

Shield, Kristy; Riley, Clyde; Quinn, Michael A; Rice, Gregory E; Ackland, Margaret L; Ahmed, Nuzhat

2007-01-01

Background Ovarian cancer is characterized by a wide-spread intra-abdominal metastases which represents a major clinical hurdle in the prognosis and management of the disease. A significant proportion of ovarian cancer cells in peritoneal ascites exist as multicellular aggregates or spheroids. We hypothesize that these cellular aggregates or spheroids are invasive with the capacity to survive and implant on the peritoneal surface. This study was designed to elucidate early inherent mechanism(s) of spheroid survival, growth and disaggregation required for peritoneal metastases Methods In this study, we determined the growth pattern and adhesive capacity of ovarian cancer cell lines (HEY and OVHS1) grown as spheroids, using the well established liquid overlay technique, and compared them to a normal ovarian cell line (IOSE29) and cancer cells grown as a monolayer. The proteolytic capacity of these spheroids was compared with cells grown as a monolayer using a gelatin zymography assay to analyze secreted MMP-2/9 in conditioned serum-free medium. The disaggregation of cancer cell line spheroids was determined on extracellular matrices (ECM) such as laminin (LM), fibronectin (FN) and collagen (CI) and the expression of α2, α3, αv, α6 and β1 interin was determined by flow cytometric analysis. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin was used to inhibit disaggregation as well as activation of MMPs in spheroids. Results We demonstrate that ovarian cancer cell lines grown as spheroids can sustain growth for 10 days while the normal ovarian cell line failed to grow beyond 2 days. Compared to cells grown as a monolayer, cancer cells grown as spheroids demonstrated no change in adhesion for up to 4 days, while IOSE29 cells had a 2–4-fold loss of adhesion within 2 days. Cancer cell spheroids disaggregated on extracellular matrices (ECM) and demonstrated enhanced expression of secreted pro-MMP2 as well as activated MMP2/MMP9 with no such activation of MMP's observed in monolayer cells. Flow cytometric analysis demonstrated enhanced expression of α2 and diminution of α6 integrin subunits in spheroids versus monolayer cells. No change in the expression of α3, αv and β1 subunits was evident. Conversely, except for αv integrin, a 1.5–7.5-fold decrease in α2, α3, α6 and β1 integrin subunit expression was observed in IOSE29 cells within 2 days. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin inhibited disaggregation as well as activation of MMPs in spheroids. Conclusion Our results suggest that enhanced expression of α2β1 integrin may influence spheroid disaggregation and proteolysis responsible for the peritoneal dissemination of ovarian carcinoma. This may indicate a new therapeutic target for the suppression of the peritoneal metastasis associated with advanced ovarian carcinomas. PMID:17567918

High content screening of patient-derived cell lines highlights the potential of non-standard chemotherapeutic agents for the treatment of glioblastoma.

PubMed

Yu, Kenny Kwok-Hei; Taylor, Jessica T; Pathmanaban, Omar N; Youshani, Amir Saam; Beyit, Deniz; Dutko-Gwozdz, Joanna; Benson, Roderick; Griffiths, Gareth; Peers, Ian; Cueppens, Peter; Telfer, Brian A; Williams, Kaye J; McBain, Catherine; Kamaly-Asl, Ian D; Bigger, Brian W

2018-01-01

Glioblastoma (GBM) is the most common primary brain malignancy in adults, yet survival outcomes remain poor. First line treatment is well established, however disease invariably recurs and improving prognosis is challenging. With the aim of personalizing therapy at recurrence, we have established a high content screening (HCS) platform to analyze the sensitivity profile of seven patient-derived cancer stem cell lines to 83 FDA-approved chemotherapy drugs, with and without irradiation. Seven cancer stem cell lines were derived from patients with GBM and, along with the established cell line U87-MG, each patient-derived line was cultured in tandem in serum-free conditions as adherent monolayers and three-dimensional neurospheres. Chemotherapeutics were screened at multiple concentrations and cells double-stained to observe their effect on both cell death and proliferation. Sensitivity was classified using high-throughput algorithmic image analysis. Cell line specific drug responses were observed across the seven patient-derived cell lines. Few agents were seen to have radio-sensitizing effects, yet some drug classes showed a marked difference in efficacy between monolayers and neurospheres. In vivo validation of six drugs suggested that cell death readout in a three-dimensional culture scenario is a more physiologically relevant screening model and could be used effectively to assess the chemosensitivity of patient-derived GBM lines. The study puts forward a number of non-standard chemotherapeutics that could be useful in the treatment of recurrent GBM, namely mitoxantrone, bortezomib and actinomycin D, whilst demonstrating the potential of HCS to be used for personalized treatment based on the chemosensitivity profile of patient tumor cells.

Wild-type measles virus infection upregulates poliovirus receptor-related 4 and causes apoptosis in brain endothelial cells by induction of tumor necrosis factor-related apoptosis-inducing ligand.

PubMed

Abdullah, Hani'ah; Brankin, Brenda; Brady, Clare; Cosby, Sara Louise

2013-07-01

Small numbers of brain endothelial cells (BECs) are infected in children with neurologic complications of measles virus (MV) infection. This may provide a mechanism for virus entry into the central nervous system, but the mechanisms are unclear. Both in vitro culture systems and animal models are required to elucidate events in the endothelium. We compared the ability of wild-type (WT), vaccine, and rodent-adapted MV strains to infect, replicate, and induce apoptosis in human and murine brain endothelial cells (HBECs and MBECs, respectively). Mice also were infected intracerebrally. All MV stains productively infected HBECs and induced the MV receptor PVRL4. Efficient WT MV production also occurred in MBECs. Extensive monolayer destruction associated with activated caspase 3 staining was observed in HBECs and MBECs, most markedly with WT MV. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not Fas ligand, was induced by MV infection. Treatment of MBECs with supernatants from MV-infected MBEC cultures with an anti-TRAIL antibody blocked caspase 3 expression and monolayer destruction. TRAIL was also expressed in the endothelium and other cell types in infected murine brains. This is the first demonstration that infection of low numbers of BECs with WT MV allows efficient virus production, induction of TRAIL, and subsequent widespread apoptosis.

Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics.

PubMed

Peyret, Ariane; Ibarboure, Emmanuel; Le Meins, Jean-François; Lecommandoux, Sebastien

2018-01-01

Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion-centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)- b -poly(ethylene oxide) (PBut- b -PEO) and outer monolayer of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half-life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(lissamine rhodamine B sulfonyl) (DOPE-rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 μm 2 s -1 at 25 °C and D = 2.3 ± 0.7 μm 2 s -1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell-like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.

Involvement of intestinal permeability in the oral absorption of clarithromycin and telithromycin.

PubMed

Togami, Kohei; Hayashi, Yoshiaki; Chono, Sumio; Morimoto, Kazuhiro

2014-09-01

The involvement of intestinal permeability in the oral absorption of clarithromycin (CAM), a macrolide antibiotic, and telithromycin (TEL), a ketolide antibiotic, in the presence of efflux transporters was examined. In order independently to examine the intestinal and hepatic availability, CAM and TEL (10 mg/kg) were administered orally, intraportally and intravenously to rats. The intestinal and hepatic availability was calculated from the area under the plasma concentration-time curve (AUC) after administration of CAM and TEL via different routes. The intestinal availabilities of CAM and TEL were lower than their hepatic availabilities. The intestinal availability after oral administration of CAM and TEL increased by 1.3- and 1.6-fold, respectively, after concomitant oral administration of verapamil as a P-glycoprotein (P-gp) inhibitor. Further, an in vitro transport experiment was performed using Caco-2 cell monolayers as a model of intestinal epithelial cells. The apical-to-basolateral transport of CAM and TEL through the Caco-2 cell monolayers was lower than their basolateral-to-apical transport. Verapamil and bromosulfophthalein as a multidrug resistance-associated proteins (MRPs) inhibitor significantly increased the apical-to-basolateral transport of CAM and TEL. Thus, the results suggest that oral absorption of CAM and TEL is dependent on intestinal permeability that may be limited by P-gp and MRPs on the intestinal epithelial cells. Copyright © 2014 John Wiley & Sons, Ltd.

Multiband and Broadband Absorption Enhancement of Monolayer Graphene at Optical Frequencies from Multiple Magnetic Dipole Resonances in Metamaterials

NASA Astrophysics Data System (ADS)

Liu, Bo; Tang, Chaojun; Chen, Jing; Xie, Ningyan; Tang, Huang; Zhu, Xiaoqin; Park, Gun-sik

2018-05-01

It is well known that a suspended monolayer graphene has a weak light absorption efficiency of about 2.3% at normal incidence, which is disadvantageous to some applications in optoelectronic devices. In this work, we will numerically study multiband and broadband absorption enhancement of monolayer graphene over the whole visible spectrum, due to multiple magnetic dipole resonances in metamaterials. The unit cell of the metamaterials is composed of a graphene monolayer sandwiched between four Ag nanodisks with different diameters and a SiO2 spacer on an Ag substrate. The near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into multiband absorption enhancement of monolayer graphene at optical frequencies. When the resonance wavelengths of the magnetic dipole modes are tuned to approach one another by changing the diameters of the Ag nanodisks, a broadband absorption enhancement can be achieved. The position of the absorption band in monolayer graphene can be also controlled by varying the thickness of the SiO2 spacer or the distance between the Ag nanodisks. Our designed graphene light absorber may find some potential applications in optoelectronic devices, such as photodetectors.

Investigating Alkylsilane Monolayer Tribology at a Single-Asperity Contact with Molecular Dynamics Simulation.

PubMed

Summers, Andrew Z; Iacovella, Christopher R; Cummings, Peter T; McCabe, Clare

2017-10-24

Chemisorbed monolayer films are known to possess favorable characteristics for nanoscale lubrication of micro- and nanoelectromechanical systems (MEMS/NEMS). Prior studies have shown that the friction observed for monolayer-coated surfaces features a strong dependence on the geometry of contact. Specifically, tip-like geometries have been shown to penetrate into monolayer films, inducing defects in the monolayer chains and leading to plowing mechanisms during shear, which result in higher coefficients of friction (COF) than those observed for planar geometries. In this work, we use molecular dynamics simulations to examine the tribology of model silica single-asperity contacts under shear with monolayer-coated substrates featuring various film densities. It is observed that lower monolayer densities lead to reduced COFs, in contrast to results for planar systems where COF is found to be nearly independent of monolayer density. This is attributed to a liquid-like response to shear, whereby fewer defects are imparted in monolayer chains from the asperity, and chains are easily displaced by the tip as a result of the higher free volume. This transition in the mechanism of molecular plowing suggests that liquid-like films should provide favorable lubrication at single-asperity contacts.

Lytic agents, cell permeability, and monolayer penetrability.

PubMed

Salton, M R

1968-07-01

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20-30 % lipid and 50-75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2-3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.

Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System

PubMed Central

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Purpose: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Methods: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Results: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. Conclusion: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells. PMID:24312827

Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System.

PubMed

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

Highly enhanced compatibility of human brain vascular pericyte cells on monolayer graphene

PubMed Central

Kim, Jangheon; Kim, Soohyun; Jung, Wonsuk

2017-01-01

ABSTRACT We introduce a method for increasing the compatibility of human brain vascular pericyte (HBVP) cells on a glass substrate, based on wet transferred monolayer graphene without any treatment. As a novel material, graphene has key properties for incubating cells, such as chemical stability, transparency, appropriate roughness, hydrophobicity and high electrical conductivity. These outstanding properties of graphene were examined by Raman spectroscopy, water contact angle measurements and atomic force microscopy. The performance of this graphene-based implant was investigated by a cell compatibility test, comparing the growth rate of cells on the graphene surface and that on a bare glass substrate. After an incubation period of 72 h, the number of live HBVP cells on a graphene surface with an area of 1×1 mm2 was 1.83 times greater than that on the glass substrate. PMID:27689961

The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer.

PubMed

Yu, Yingxin; Wang, Mengmeng; Zhang, Kaiqiong; Yang, Dan; Zhong, Yufang; An, Jing; Lei, Bingli; Zhang, Xinyu

2017-04-01

Oral ingestion plays an important role in human exposure to polybrominated diphenyl ethers (PBDEs). The uptake of PBDEs primarily occurs in the small intestine. The aim of the present study is to investigate the transepithelial transport characteristics and mechanisms of PBDEs in the small intestine using a Caco-2 cell monolayer model. The apparent permeability coefficients of PBDEs indicated that tri- to hepta-BDEs were poorly absorbed compounds. A linear increase in transepithelial transport was observed with various concentrations of PBDEs, which suggested that passive diffusion dominated their transport at the concentration range tested. In addition, the pseudo-first-order kinetics equation can be applied to the transepithelial transport of PBDEs. The rate-determining step in transepithelial transport of PBDEs was trans-cell transport including the trans-pore process. The significantly lower transepithelial transport rates at low temperature for bidirectional transepithelial transport suggested that an energy-dependent transport mechanism was involved. The efflux transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) and influx transporters (organic cation transporters) participated in the transepithelial transport of PBDEs. In addition, the transepithelial transport of PBDEs was pH sensitive; however, more information is required to understand the influence of pH. Copyright © 2016 Elsevier Inc. All rights reserved.

Physicochemical Properties of Whey-Protein-Stabilized Astaxanthin Nanodispersion and Its Transport via a Caco-2 Monolayer.

PubMed

Shen, Xue; Zhao, Changhui; Lu, Jing; Guo, Mingruo

2018-02-14

Astaxanthin nanodispersion was prepared using whey protein isolate (WPI) and polymerized whey protein (PWP) through an emulsification-evaporation technique. The physicochemical properties of the astaxanthin nanodispersion were evaluated, and the transport of astaxanthin was assessed using a Caco-2 cell monolayer model. The astaxanthin nanodispersions stabilized by WPI and PWP (2.5%, w/w) had a small particle size (121 ± 4.9 and 80.4 ± 5.9 nm, respectively), negative ζ potential (-19.3 ± 1.5 and -35.0 ± 2.2 mV, respectively), and high encapsulation efficiency (92.1 ± 2.9 and 93.5 ± 2.4%, respectively). Differential scanning calorimetry curves indicated that amorphous astaxanthin existed in both astaxanthin nanodispersions. Whey-protein-stabilized astaxanthin nanodispersion showed resistance to pepsin digestion but readily released astaxanthin after trypsin digestion. The nanodispersions showed no cytotoxicity to Caco-2 cells at a protein concentration below 10 mg/mL. WPI- and PWP-stabilized nanodispersions improved the apparent permeability coefficient (P app ) of Caco-2 cells to astaxanthin by 10.3- and 16.1-fold, respectively. The results indicated that whey-protein-stabilized nanodispersion is a good vehicle to deliver lipophilic bioactive compounds, such as astaxanthin, and to improve their bioavailability.

A Novel 3 Dimensional Stromal-based Model for In Vitro Chemotherapy Sensitivity Testing of Leukemia Cells

PubMed Central

Aljitawi, Omar S.; Li, Dandan; Xiao, Yinghua; Zhang, Da; Ramachandran, Karthik; Stehno-Bittel, Lisa; Van Veldhuizen, Peter; Lin, Tara L.; Kambhampati, Suman; Garimella, Rama

2014-01-01

The disparate responses of leukemia cells to chemotherapy in vivo, compared to in vitro, is partly related to the interactions of leukemic cells and the 3 dimensional (3D) bone marrow stromal microenvironment. We investigated the effects of chemotherapy agents on leukemic cell lines co-cultured with human bone marrow mesenchymal stem cell (hu-BM-MSC) in 3D. Comparison was made to leukemic cells treated in suspension, or grown on a hu-BM-MSC monolayer (2D conditions). We demonstrated that leukemic cells cultured in 3D were more resistant to drug-induced apoptosis compared to cells cultured in 2D or in suspension. We also demonstrated significant differences in leukemic cell response to chemotherapy using different leukemic cell lines cultured in 3D. We suggest that the differential responses to chemotherapy in 3D may be related to the expression of N-cadherin in the co-culture system. This unique model provides an opportunity to study leukemic cell responses to chemotherapy in 3D. PMID:23566162

Preparing nuclei from cells in monolayer cultures suitable for counting and for following synchronized cells through the cell cycle.

PubMed

Butler, W B

1984-08-15

A procedure is described for preparing nuclei from cells in monolayer culture so that they may be counted using an electronic particle counter. It takes only 10 to 15 min, and consists of swelling the cells in hypotonic buffer and then lysing them with the quaternary ammonium salt, ethylhexadecyldimethylammonium bromide. The cells are completely lysed, yielding a suspension of clean single nuclei which is stable, free of debris, and easily counted. The method was developed for a cell line of epithelial origin (MCF-7), which is often difficult to trypsinize to single cells. It works equally well at all cell densities up to and beyond confluence, and has been used with a variety of cells in culture, including 3T3 cells, bovine macrophages, rat mammary epithelial cells, mouse mammary tumor cell lines, and human fibroblasts. The size of the nuclei produced by this procedure is related to their DNA content, and the method is thus suitable for following cultures of synchronized cells through the cell cycle, and for performing differential counts of cells with substantial differences in DNA content.

Effect of fenhexamid and cyprodinil on the expression of cell cycle- and metastasis-related genes via an estrogen receptor-dependent pathway in cellular and xenografted ovarian cancer models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Go, Ryeo-Eun; Kim, Cho-Won; Choi, Kyung-Chul, E-mail: kchoi@cbu.ac.kr

ABSTRACT: Fenhexamid and cyprodinil are antifungal agents (pesticides) used for agriculture, and are present at measurable amounts in fruits and vegetables. In the current study, the effects of fenhexamid and cyprodinil on cancer cell proliferation and metastasis were examined. Additionally, the protein expression levels of cyclin D1 and cyclin E as well as cathepsin D were analyzed in BG-1 ovarian cancer cells that express estrogen receptors (ERs). The cells were cultured with 0.1% dimethyl sulfoxide (DMSO; control), 17β-estradiol (E2; 10{sup −9} M), and fenhexamid or cyprodinil (10{sup –5}–10{sup −7} M). Results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that fenhexamidmore » and cyprodinil increased BG-1 cell proliferation about 1.5 to 2 times similar to E2 (5 times) compared to the control. When the cells were co-treated with ICI 182,780 (10{sup −8} M), an ER antagonist, the proliferation of pesticide-treated BG-1 cells was decreased to the level of the control. A wound healing assay revealed that the pesticides reduced the disrupted area in the BG-1 cell monolayer similar to E2. Protein levels of cyclin D1 and E as well as cathepsin D were increased by fenhexamid and cyprodinil. This effect was reversed by co-treatment with ICI 182,780. In a xenograft mouse model with transplanted BG-1 cells, cyprodinil significantly increased tumor mass formation about 2 times as did E2 (6 times) compared to the vehicle (0.1% DMSO) over an 80-day period. In contrast, fenhexamid did not promote ovarian tumor formation in this mouse model. Cyprodinil also induced cell proliferation along with the expression of proliferating cell nuclear antigen (PCNA) and cathepsin D in tumor tissues similar to E2. Taken together, these results imply that fenhexamid and cyprodinil may have disruptive effects on ER-expressing cancer by altering the cell cycle- and metastasis-related gene expression via an ER-dependent pathway. - Highlights: • Fenhexamid and cyprodinil increased BG-1 cell proliferation via ER. • Two pesticides reduced the disrupted area in the BG-1 cell monolayer. • Cyclin D1 and E and cathepsin D proteins were induced by fenhexamid and cyprodinil. • Cyprodinil significantly increased tumor mass formation in a xenografted model. • Fenhexamid and cyprodinil may have disruptive effects on ER-expressing cancer.« less

In vitro evaluation of biodegradable microspheres with surface-bound ligands.

PubMed

Keegan, Mark E; Royce, Sara M; Fahmy, Tarek; Saltzman, W Mark

2006-02-21

Protein ligands were conjugated to the surface of biodegradable microspheres. These microsphere-ligand conjugates were then used in two in vitro model systems to evaluate the effect of conjugated ligands on microsphere behavior. Microsphere retention in agarose columns was increased by ligands on the microsphere surface specific for receptors on the agarose matrix. In another experiment, conjugating the lectin Ulex europaeus agglutinin 1 to the microsphere surface increased microsphere adhesion to Caco-2 monolayers compared to control microspheres. This increase in microsphere adhesion was negated by co-administration of l-fucose, indicating that the increase in adhesion is due to specific interaction of the ligand with carbohydrate receptors on the cell surface. These results demonstrate that the ligands conjugated to the microspheres maintain their receptor binding activity and are present on the microsphere surface at a density sufficient to target the microspheres to both monolayers and three-dimensional matrices bearing complementary receptors.

Benjamin Franklin, Philadelphia's favorite son, was a membrane biophysicist.

PubMed

Wang, Da-Neng; Stieglitz, Heather; Marden, Jennifer; Tamm, Lukas K

2013-01-22

Benjamin Franklin, mostly known for his participation in writing The Declaration of Independence and work on electricity, was also one of the first scientists to seek to understand the properties of oil monolayers on water surfaces. During one of his many voyages across the Atlantic Ocean, Franklin observed that oil had a calming effect on waves when poured into rough ocean waters. Though at first taking a backseat to many of his other scientific and political endeavors, Franklin went on to experiment with oil, spreading monomolecular films on various bodies of water, and ultimately devised a concept of particle repulsion that is indirectly related to the hydrophobic effect. His early observations inspired others to measure the dimensions of oil monolayers, which eventually led to the formulation of the contemporary lipid bilayer model of the cell membrane. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Two-dimensional layered semiconductor/graphene heterostructures for solar photovoltaic applications.

PubMed

Shanmugam, Mariyappan; Jacobs-Gedrim, Robin; Song, Eui Sang; Yu, Bin

2014-11-07

Schottky barriers formed by graphene (monolayer, bilayer, and multilayer) on 2D layered semiconductor tungsten disulfide (WS2) nanosheets are explored for solar energy harvesting. The characteristics of the graphene-WS2 Schottky junction vary significantly with the number of graphene layers on WS2, resulting in differences in solar cell performance. Compared with monolayer or stacked bilayer graphene, multilayer graphene helps in achieving improved solar cell performance due to superior electrical conductivity. The all-layered-material Schottky barrier solar cell employing WS2 as a photoactive semiconductor exhibits efficient photon absorption in the visible spectral range, yielding 3.3% photoelectric conversion efficiency with multilayer graphene as the Schottky contact. Carrier transport at the graphene/WS2 interface and the interfacial recombination process in the Schottky barrier solar cells are examined.

Kinetics of the maintenance of the epidermis

NASA Astrophysics Data System (ADS)

Zhdanov, Vladimir P.; Cho, Nam-Joon

2013-08-01

The epidermis is the outermost layer of skin. It is comprised of keratin-containing cells called keratinocytes. Functionally, the epidermis serves as a physical barrier that can prevent infection and regulate body hydration. Maintenance and repair of the epidermis are important for human health. Mechanistically, these processes occur primarily via proliferation and differentiation of stem cells located in the basal monolayer. These processes are believed to depend on cell-cell communication and spatial constraints but existing kinetic models focus mainly on proliferation and differentiation. To address this issue, we present a mean-field kinetic model that takes these additional factors into account and describes the epidermis at a biosystem level. The corresponding equations operate with the populations of stem cells and differentiated cells in the basal layer. The keratinocytes located above the basal layer are treated at a more coarse-grained level by considering the thickness of the epidermis. The model clarifies the likely role of various negative feedbacks that may control the epidermis and, accordingly, provides insight into the cellular mechanisms underlying complex biological phenomena such as wound healing.

Paracellular transport through healthy and cystic fibrosis bronchial epithelial cell lines--do we have a proper model?

PubMed

Molenda, Natalia; Urbanova, Katarina; Weiser, Nelly; Kusche-Vihrog, Kristina; Günzel, Dorothee; Schillers, Hermann

2014-01-01

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o- cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o- cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o- and also in CFBE41o- cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o- cells and CFBE41o- cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o- cell monolayers. We observed that 16HBE14o- cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o- and its overexpressing clones. Consequently, 16HBE14o- cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in 'healthy' 16HBE14o- cells compared to 'cystic fibrosis' CFBE41o- cells. We found that claudin-3 expression was considerably stronger in 16HBE14o- cells than in the three CFBE41o- cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o- cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport.

Quantifying cell adhesion through impingement of a controlled microjet.

PubMed

Visser, Claas Willem; Gielen, Marise V; Hao, Zhenxia; Le Gac, Séverine; Lohse, Detlef; Sun, Chao

2015-01-06

The impingement of a submerged, liquid jet onto a cell-covered surface allows assessing cell attachment on surfaces in a straightforward and quantitative manner and in real time, yielding valuable information on cell adhesion. However, this approach is insufficiently characterized for reliable and routine use. In this work, we both model and measure the shear stress exerted by the jet on the impingement surface in the micrometer-domain, and subsequently correlate this to jet-induced cell detachment. The measured and numerically calculated shear stress data are in good agreement with each other, and with previously published values. Real-time monitoring of the cell detachment reveals the creation of a circular cell-free area upon jet impingement, with two successive detachment regimes: 1), a dynamic regime, during which the cell-free area grows as a function of both the maximum shear stress exerted by the jet and the jet diameter; followed by 2), a stationary regime, with no further evolution of the cell-free area. For the latter regime, which is relevant for cell adhesion strength assessment, a relationship between the jet Reynolds number, the cell-free area, and the cell adhesion strength is proposed. To illustrate the capability of the technique, the adhesion strength of HeLa cervical cancer cells is determined ((34 ± 14) N/m(2)). Real-time visualization of cell detachment in the dynamic regime shows that cells detach either cell-by-cell or by collectively (for which intact parts of the monolayer detach as cell sheets). This process is dictated by the cell monolayer density, with a typical threshold of (1.8 ± 0.2) × 10(9) cells/m(2), above which the collective behavior is mostly observed. The jet impingement method presents great promises for the field of tissue engineering, as the influence of both the shear stress and the surface characteristics on cell adhesion can be systematically studied. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Comparison of Genotoxic Damage in Monolayer Cell Cultures and Three-Dimensional Tissue-Like Cell Assemblies

NASA Technical Reports Server (NTRS)

Behravesh, E.; Emami, K.; Wu, H.; Gonda, S.

2004-01-01

Assessing the biological risks associated with exposure to the high-energy charged particles encountered in space is essential for the success of long-term space exploration. Although prokaryotic and eukaryotic cell models developed in our laboratory and others have advanced our understanding of many aspects of genotoxicity, in vitro models are needed to assess the risk to humans from space radiation insults. Such models must be representative of the cellular interactions present in tissues and capable of quantifying I genotoxic damage. Toward this overall goal, the objectives of this study were to examine the effect of the localized microenvironment of cells, cultured as either 2-dimensional (2D) monolayers or 3-dimensional (3D) aggregates, on the rate and type of genotoxic damage resulting from exposure to iron charged particles, a significant portion of space radiation. We used rodent transgenic cell lines containing 50-70 copies of a LacI transgene to provide the enhanced sensitivity required to quantify mutational frequency and type in the 1,100-bp LacI target as well as assessment of DNA,damage to the entire 45-kbp construct. Cultured cells were exposed to high-enerir on charged particles at Brookhaven National Laboratory s Alternating Gradient Synchrotron facility for a total dose of 0, 0.1, 0.25,0.5, 1.0, or 2.0 Gy and allowed to recover for 0, 1, or 7 days, after which mutational type and frequency were evaluated. The mutational frequency was found to be higher in 3D samples than in 2D samples at all radiation doses. Mutational frequency also was higher at 7 days after irradiation than immediately after exposure. DNA sequencing of the mutant targets revealed that deletional mutations contributed an increasingly high percentage (up to 27%) of all mutations in cells as the dose was increased from 0.5 to 2 Gy. Several mutants also showed large and complex deletions in multiple locations within the Lac1 target. However, no differences in mutational type were found between the 2D and the 3D samples. These 3D tissue-like model systems can reduce the uncertainty involved in extrapolating risk between in vitro cellular and in vivo models.

Radiation enhanced reactivation of herpes simplex virus: effect of caffeine.

PubMed

Hellman, K B; Lytle, C D; Bockstahler, L E

1976-09-01

Ultaviolet enhanced (Weigle) reactivation of UV-irradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cell monolayers was decreased by caffeine. X-ray enhanced reactivation of UV-irradiated virus in X-irradiated monolayers (X-ray reactivation) and UV- or X-ray-inactivated capacity of the cells to support unirradiated virus plaque formation were unaffected by caffeine. The results suggest that a caffeine-sensitive process is necessary for the expression of Weigle reactivation for herpes virus. Since cafeine did not significantly affect X-ray reactivation, different mechanisms may be responsible for the expression of Weigle reactivation and X-ray reactivation.

The third dimension bridges the gap between cell culture and live tissue.

PubMed

Pampaloni, Francesco; Reynaud, Emmanuel G; Stelzer, Ernst H K

2007-10-01

Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland).

PubMed

Goswami, M; Sharma, B S; Tripathi, A K; Yadav, Kamalendra; Bahuguna, S N; Nagpure, N S; Lakra, W S; Jena, J K

2012-05-25

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies. Copyright © 2012 Elsevier B.V. All rights reserved.

Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19.

PubMed

Talbot, N C; Caperna, T J; Garrett, W M

2013-01-01

Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, enhance our ability to genetically modify the cells, and provide a better model system to investigate porcine hepatic metabolism.

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion, Transendothelial Migration and Phagocytosis

PubMed Central

Liu, Dan-Qing; Li, Li-Min; Guo, Ya-Lan; Bai, Rui; Wang, Chen; Bian, Zhen; Zhang, Chen-Yu; Zen, Ke

2008-01-01

Background Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β2 integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis. Methodology/Principal Findings THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β2 integrin cell surface expression and β2 integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β2 integrins, in particular CD11b/CD18. SIRPα overexpression reduced β2 integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β2 integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression. Conclusions/Significance SIRPα negatively regulates β2 integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis. PMID:18820737

Three-dimensional development of tensile pre-strained annulus fibrosus cells for tissue regeneration: An in-vitro study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuah, Yon Jin; Lee, Wu Chean; Wong, Hee Kit

Prior research has investigated the immediate response after application of tensile strain on annulus fibrosus (AF) cells for the past decade. Although mechanical strain can produce either catabolic or anabolic consequences to the cell monolayer, little is known on how to translate these findings into further tissue engineering applications. Till to date, the application and effect of tensile pre-strained cells to construct a three-dimensional (3D) AF tissue remains unknown. This study aims to investigate the effect of tensile pre-strained exposure of 1 to 24 h on the development of AF pellet culture for 3 weeks. Equibiaxial cyclic tensile strain wasmore » applied on AF monolayer cells over a period of 24 h, which was subsequently developed into a cell pellet. Investigation on cellular proliferation, phenotypic gene expression, and histological changes revealed that tensile pre-strain for 24 h had significant and lasting effect on the AF tissue development, with enhanced cell proliferation, and up-regulation of collagen type I, II, and aggrecan expression. Our results demonstrated the regenerative ability of AF cell pellets subjected to 24 h tensile pre-straining. Knowledge on the effects of tensile pre-strain exposure is necessary to optimize AF development for tissue reconstruction. Moreover, the tensile pre-strained cells may further be utilized in either cell therapy to treat mild disc degeneration disease, or the development of a disc construct for total disc replacement. - Highlights: • Establishment of tensile pre-strained cell line population for annulus development. • Tensile strain limits collagen gene expression declination in monolayer culture. • Tensile pre-strained cells up-regulate their matrix protein in 3D pellet culture.« less