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Results for “Diffusion Chambers, Culture”

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At least 163 records · Page 9Linked to original sources

PubMed2004

An in vitro model based on cell monolayers grown on the underside of large- pore filters in bicameral chambers for studying thyrocyte-lymphocyte interactions.

In the processes underlying thyroid autoimmunity, thyrocytes probably act as antigen-presenting cells exposing T-cell epitopes to intrathyroid lymphocytes. To study the interactions between lymphocytes and thyrocytes, which are arranged in a tight, polarized monolayer, we developed a new in vitro model based on human thyrocytes grown on the underside of a filter placed in a bicameral chamber. Thyrocytes from Graves' disease glands were plated onto the upper face of a 8-mum-pore polyethylene terephthalate culture insert filter placed in the inverted position and grown for 24 h before the insert was returned to the normal position for a week in the cell culture plate wells. Thyrocytes grown in the presence of thyroid stimulating hormone, forming a homogeneous monolayer on the underside of the filter, reached confluence after 8 days in vitro. The cells developed a transepithelial electrical resistance >1,000 Omega.cm(2), and the ZO-1 tight junction protein showed a junctional pattern of distribution. Thyrocytes showed a polarized pattern of thyroperoxidase and thyroid stimulating hormone receptor expression in the apical and basolateral positions, respectively. They were also found to aberrantly express DR class II human leukocyte antigen and an Fc immunoglobulin receptor (FcgammaRIIB2) in the basolateral and apical positions, respectively. Autologous intrathyroidal T lymphocytes cocultured for 24 h across the filter with the thyrocyte monolayer proliferated and remained in the upper chamber without any leakage occurring through the epithelial barrier, which makes this model particularly suitable for studying the cell-cell interactions involved in antigen processing.

Cell Communication↗

PubMed1989

An in vitro method for longitudinal evaluation of toxicity of endodontic sealers.

An in vitro method for longitudinal evaluation of root canal sealers was developed and applied. A newly introduced cell culture chamber was used to evaluate the cytotoxic effects of test samples immediately after mixing and for an extended period of time thereafter. A ranking of the test materials, based on their cytotoxicity, was allowed by the method.

Animals↗

PubMed2000

An integrated environmental perfusion chamber and heating system for long-term, high resolution imaging of living cells.

This communication presents the design and application of an integrated environmental perfusion chamber and stage heating blanket suitable for time-lapse video microscopy of living cells. The system consists of two independently regulated components: a perfusion chamber suitable for the maintenance of cell viability and the variable delivery of environmental factors, and a separate heating blanket to control the temperature of the microscope stage and limit thermal conduction from the perfusion chamber. Two contrasting experiments are presented to demonstrate the versatility of the system. One long-term sequence illustrates the behaviour of cells exposed to ceramic fibres. The other shows the shrinking response of cultured articular cartilage chondrons under dynamic hyper-osmotic conditions designed to simulate joint loading. The chamber is simple in design, economical to produce and permits long-term examination of dynamic cellular behaviour while satisfying the fundamental requirements for the maintenance of environmental factors that influence cell viability.

Animals↗

PubMed2005

The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber.

UNLABELLED: The distribution of hypoxic cells in an in vivo tissue engineering chamber was investigated up to 28 days post-implantation. METHODS: Arteriovenous loops were constructed and placed into bi-valved polycarbonate chambers containing 2 x 10(6) rat fibroblasts in basement membrane gel (BM gel). Chambers were inserted subcutaneously in the groin of male rats and harvested at 3 (n = 6), 7 (n = 6), 14 (n = 4) or 28 (n = 4) days. Ninety minutes before harvest, pimonidazole (60 mg/kg) was injected intraperitoneally. Chamber tissue was removed, immersion fixed, paraffin embedded, sectioned and stained immunohistochemically using hypoxyprobe-1 Mab that detects reduced pimonidazole adducts forming in cells, where pO2 < 10 mmHg. RESULTS: At 3 days a fibrin clot/BM gel framework filled the chamber. Seeded fibroblasts had largely died. The majority of 3 day chambers did not demonstrate tissue growth from the AV loop nor was pimonidazole binding present in these chambers. In one chamber in which tissue growth had occurred strong pimonidazole binding was evident within the new tissue. In four out of six 7 day chambers a broader proliferative zone existed extending up to 0.4 mm (approximately) from the AV loop endothelium which demonstrated intense pimonidazole binding. The two remaining 7 day chambers displayed even greater tissue growth (leading edge > 0.7 mm from the AV loop endothelium), but very weak or no pimonidazole binding. At 14 and 28 days the fibrin/BM gel matrix was replaced by mature vascularised connective tissue that did not bind pimonidazole. CONCLUSION: Employing a tissue engineering chamber, new tissue growth extending up to 0.4 mm from the AV loop endothelium (chambers < or = 7 days) demonstrated intense pimonidazole binding and, therefore, hypoxia. Tissue growth greater than 0.5 mm from the AV loop endothelium (7-28 days chambers) did not exhibit pimonidazole binding due to a significant increase in the number of new blood vessels and was, therefore, adequately oxygenated.

Animals↗

PubMed1994

The inhibitory effect caused by suramin on the paracrine growth of human cancer cells and fibroblasts.

The growth interactions between human cancer cells and primary cultured human fibroblasts, and the effects of suramin on them, were investigated using a double-chamber technique combined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. Human fibroblasts obtained from various organs resected surgically were cultured in a monolayer and used after the third or fourth passage. In the double-chamber assay, the growth of cancer cells in the top chamber was significantly stimulated by some types of fibroblasts in the bottom chamber in a fibroblast density-dependent manner. Interestingly, the growth of cancer cells was stimulated at 140%-147% by fibroblasts obtained from an organ where cancer cells had developed, the MCF-7 versus mammary fibroblasts, and in LS-180 versus colonic fibroblasts, but not by their fibroblast-conditioned medium. Suramin completely inhibited the growth-enhancing interaction between MCF-7 and mammary fibroblasts, and between SH-101 and lung fibroblasts at a clinical concentration of from 50 micrograms/ml to 300 micrograms/ml. It also reduced the growth of LS-180 co-cultured with colon-fibroblasts, but the inhibitory effect was incomplete. These results suggest that mutual growth reliance exists between human cancer cells and primary cultured fibroblasts by diffusible factors secreted by both cells, and that these enhancing effects are related in part to the growth and metastasis of cancer cells in vivo. Suramin was found to have an inhibitory effect on their interaction at a clinically achievable concentration in vitro.

Cell Division↗

PubMed1999

Morphological evidence of the inhibitory effect of taxol on the fast axonal transport.

The short term effects of taxol, a stabilizing drug of microtubules, on the peripheral nerves in the rat was investigated using a new chamber system which can be applied to incubate a sciatic nerve with various solutions in vivo. A functional analysis of retrograde axonal transport using rhodamine-labeled wheat germ agglutinin (WGA-rhodamine) showed the inhibitory effect of the drug. An electron microscopic study also revealed that a variety of vesicles were observed to accumulate on both the proximal and the distal sides of the chamber, however, no significant increase in the number of microtubules in the axons, based on the pharmacological effect of the drug, was observed even though one had been expected. These findings support the inhibitory effect of taxol on the fast axonal transport of the neurons. Furthermore, the accumulated vesicles were morphologically different from those accumulated by ligation. These results suggest that a special component of the fast axonal transport was thus selectively blocked by the drug.

Animals↗

PubMed1990

Biosensors for directly measuring cell affecting agents.

Cellular perfusion chambers have been constructed from the Light Addressable Potentiometric Sensor (LAPS) previously described. The authors have used these chambers to measure the effects of a variety of agents on the metabolic rates of cells. The chambers are used in a stopped flow mode. When flow is on, samples may be introduced to the chamber. When flow is stopped, acidification of the very small volume of medium in the chamber is used to determined the metabolic rate of the cells. Using a variety of types of mammalian cells the authors have demonstrated the following. The triggering of cellular receptors can be determined in minutes. Metabolic inhibition of normal human cells by a test compound can be correlated with the compounds in vivo ocular irritancy. And the efficacy of chemotherapeutic agents on tumor cells exhibiting multidrug resistance can be determined in a few hours.

Biosensing Techniques↗

PubMed2002

Sphingosine-1-phosphate induces G(alphai)-coupled, PI3K/ras-dependent smooth muscle cell migration.

BACKGROUND: Sphingolipids such as sphingosine-1-phosphate (S-1-P) are potent extracellular mediators released in response to vessel injury. S-1-P binds to G-protein-coupled receptors, which can be either G(alphai) or G(alphaq) linked. This study examines the signaling pathways involved in vascular smooth muscle cell migration after stimulation by S-1-P. We hypothesized that S-1-P stimulates migration of smooth muscle cells that is dependent upon a G(alphai)-coupled receptor, ras, phosphoinositol 3-kinase (PI3-K), and ERK 1/2. METHODS: Vascular smooth muscle cells were cultured in vitro. A linear wound assay and Boyden chamber assay of migration were employed in the presence of S-1-P and inhibitors of G(alphai) [pertussis toxin (PTx), 100 ng/ml], G(alphaq) (GP-2A, 10 microM), ras [manumycin A (MA), 10 microM], PI3-K [Wortmannin (Wn), 10 microM], and MEK1 [PD98059 (PD), 25 microM]. Western blotting was performed separately to examine p42/p44 MAP kinase (ERK 1/2) activation in response to S-1-P with these inhibitors. RESULTS: S-1-P induced vascular smooth muscle cell migration. This response was decreased by preincubation with PTx, suggesting a receptor linked, G(alphai)-mediated response. Application of a G(alphaq) inhibitor did not affect this response. S-1-P induced ERK 1/2 phosphorylation in a time-dependent manner. This S-1-P-induced cell migration was PD-sensitive in the Boyden chamber assay, confirming that it is MEK1- and ERK1/2-dependent. Inhibition of ras with MA and PI3-K with Wn also reduced ERK phosphorylation and smooth muscle cell migration in response to S-1-P. CONCLUSIONS: S-1-P induces smooth muscle cell migration through a G(alphai)-linked, ras- and PI3-K-coupled, ERK 1/2-dependent process. Understanding signal transduction will allow targeted molecular interventions to treat the response of a vessel to injury.

Animals↗

PubMed2005

Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts.

In the present study, using tissue culture inserts (TCI) coupled with a primary spinal cord neuronal culture, we characterize a new perfusion system, which permits continuous perfusate collection from cultured neurons. Primary spinal cord neurons were isolated from the lumbar portion of E14 spinal cords of Sprague-Dawley rats, plated on TCI and fed with DMEM/B27/10% FBS. At 1-4 weeks after isolation the development of synapses and neurotransmitter phenotype in cultured neurons was verified using immunofluorescence. A time-dependent development of synapses (Syn) was seen with a dense Syn-positive network identified at 3-4 weeks after plating. A sub-population of plated neurons (35-40%) showed GABA immunoreactivity and expressed NMDAR1 receptor. To measure neurotransmitter release, a chamber accommodating TCI was constructed permitting perfusion of the insert across the membrane. To evoke amino acid release from cultured neurons, NMDA (10 mmol/l) was added into the perfusion buffer. Stimulation with NMDA evoked a significant GABA (4050 +/- 950%) and glutamate release (130 +/- 42%) during first 10 min after exposure. In control non-stimulated cells no significant changes were measured. These data show that by using TCI it is possible to maintain embryonic spinal cord neurons for an extended period and that this system may represent a simple tool to identify neurotransmitter and/or peptides associated with a specific population of cultured brain and/or spinal cord neurons.

Animals↗

PubMed2001

A system for applying rapid warming or cooling stimuli to cells during patch clamp recording or ion imaging.

We describe a system for superfusing small groups of cells at a precisely controlled and rapidly adjustable local temperature. Before being applied to the cell or cells under study, solutions are heated or cooled in a chamber of small volume ( approximately 150 microl) and large surface area, sandwiched between four small Peltier elements. The current through the Peltier elements is controlled by a microprocessor using a PID (proportional-integral-derivative) feedback algorithm. The chamber can be heated to at least 60 degrees C and cooled to 0 degrees C, changing its temperature at a maximum rate of about 7 degrees C per second; temperature ramps can be followed under feedback control at up to 4 degrees C per second. Temperature commands can be applied from the digital-to-analogue converter of any laboratory interface or generated digitally by the microprocessor. The peak-to-peak noise contributed by the system does not exceed that contributed by a patch pipette, holder and headstage, making it suitable for single channel as well as whole cell recordings.

Action Potentials↗

PubMedDate not supplied

Morphology and integrity of endothelial cell monolayers inside a ventricle shaped perfusion chamber.

The long-term maintenance of patients with failing hearts on cardiac prostheses requires prevention of device related thromboembolic events. This challenge is being addressed by endothelialization of the blood sacs. However, the practice of establishing and maintaining a durable endothelial cell monolayer inside a beating prosthesis has not been fully realized. Thus, before exposing endothelial cell monolayers to the hemodynamics inside an artificial heart, the authors studied the effect of various flow patterns in a ventricle shaped chamber on the integrity and morphology of the endothelium. After 20 hours of superfusion by pulsatile flow, there were no denudation signs in the jet, where shear stress was 1.5 dynes/cm2. However, there was measurable damage to the monolayer close to the periphery of the eddies (turbulent flow) at 0.15 dynes/cm2. In either case, there were no signs of cell alignment with the flow, but there were changes in cell morphology compared with that of static control. These findings suggest that adjustment of endothelial cells in response to frictional forces occurs even at low shear stresses and that random velocity fluctuations might jeopardize the integrity of endothelial cell monolayers.

Animals↗

PubMed1992

Microscale production of hybridomas by hypo-osmolar electrofusion.

We have developed and tested a novel electrofusion chamber, the adjustable plate microchamber, that permits the successful electrofusion and production of hybridomas in a hanging droplet from as few as 1,000 B lymphocytes. Cell suspension volumes of 10 microliters may be used without excessive difficulty in aseptically recovering fused cells. With a modification of the hypo-osmolar electrofusion protocol with this microchamber, fusion efficiencies of the order of 10(-3) may be attained. These efficiencies are comparable to those attained with standard hardware and much higher numbers of input B lymphocytes. This technology should permit high efficiency hybridoma production using a variety of hitherto inaccessible B lymphocyte populations such as B cells isolated from human organ biopsies.

Animals↗

PubMed1998

A superfusion system to study border zones in confluent cultures of neonatal rat heart cells.

We present a new experimental method to study intracellular ion regulation in cultured cardiomyocytes at a border zone separating two different and distinct environments. Our system uses a dual-flow superfusion chamber to produce two different but adjacent environments over a monolayer of cardiomyocytes. Fluorescent microscopy of fluorescein showed that the transition between the two environments was nearly linear and was 220-320 micron wide depending on fluid viscosity and velocity. We superfused cultured monolayers on one side with a solution at pH 6.5 and on the other side with a solution at pH 7.4. We observed a sharply demarcated difference in intracellular pH (pHi) between the two halves of the cell monolayer as measured with the fluorescent pHi indicator carboxy-seminaphthorhodafluor-1. The demarcation of pHi corresponded well with the demarcation of the border measured with fluorescein. We conclude that our superfusion system will facilitate the study of intercellular communication and interactions across boundaries of cardiac tissue where different ionic or metabolic conditions are present, for example, between ischemic and nonischemic myocardium.

Animals↗

PubMed2002

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium.

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.

Aged↗

PubMed1988

[Determination of the antiproliferative activity of human recombinant interferon (reaferon) on diploid human cells].

Dependence of the antiproliferative activity of reaferon in cultures of human diploid cells on the drug dose and duration of its action on the cells was studied by counting viable cells in the Goriaev chamber. No relationship was detected. However, such dependence was clearly evident with using tumor cells. With counting of mitoses it was shown that in doses of 10(4) and 10(3) IU reaferon significantly inhibited the cell mitotic activity when the mitotic index in the control of the strain M-19 cells exceeded 25%. When the mitotic index was lower than 25% reaferon in all the doses had no inhibitory effect on the cells. On the contrary it was noted that there was even a certain tendency to stimulation of the mitotic activity. Therefore, the line of the human diploid fibroblast cells (strain M-19) may be used for assay of reaferon antiproliferative activity in case of high mitotic activity in its monolayer (the mitotic index higher than 25%). It was also demonstrated that the dose of reaferon should be not lower than 1000 IU per 1 ml of the medium.

Cell Count↗

PubMed2002

Hypoxia decreases active Na transport across primary rat alveolar epithelial cell monolayers.

Hypoxia has been reported to inhibit activity and expression of ion transporters of alveolar epithelial cells. This study extended those observations by investigating the mechanisms underlying inhibition of active Na transport across primary cultured adult rat alveolar epithelial cell monolayers grown on polycarbonate filters. Cell monolayers were exposed to normoxia and hypoxia (1.5% and 5% O(2), 5% CO(2)), and resultant changes in bioelectric properties [i.e., short-circuit current (I(sc)) and transepithelial resistance (R(t))] were measured in Ussing chambers. Results showed that I(sc) decreased with duration of exposure to hypoxia, while relatively little change was observed for R(t). In normoxia, amiloride inhibited approximately 70% of I(sc). The amiloride-sensitive portion of I(sc) decreased over time of exposure to hypoxia, whereas the magnitude of the amiloride-insensitive portion of I(sc) was not affected. Na pump capacity measured after permeabilization of the apical plasma membrane with amphotericin B decreased in monolayers exposed to 1.5% O(2) for 24 h, as did the capacity of amiloride-sensitive Na uptake measured after imposing an apical to basolateral Na gradient and permeabilization of the basolateral membrane. These results demonstrate that exposure to hypoxia inhibits alveolar epithelial Na reabsorption by reducing the rates of both apical amiloride-sensitive Na entry and basolateral Na extrusion.

Amiloride↗

PubMed2003

Platelet-derived growth factor-BB stimulated cell migration mediated through p38 signal transduction pathway in periodontal cells.

BACKGROUND: Intracellular signaling pathways mediate specific responses to growth factors. The manipulation of these pathways ultimately may be used to control the clinical outcomes of periodontal regenerative therapy. The purpose of this study was to examine the role of the p38 signal transduction pathway in the responses of periodontal cells to platelet-derived growth factor-BB (PDGF). METHODS: Primary cultures of human periodontal ligament cells (PDLs) and gingival fibroblasts (GFs) were used for all experiments. Cell numbers, 3H-thymidine incorporation, and Boyden chamber assays were used to characterize the effects of SB 203580 (SB), a specific inhibitor of the p38 signaling pathway, on cell proliferation and migration. An in vitro wound model also was used to assess the effects of SB. For the in vitro wound assay, triplicate wells were incubated for 1, 3, 5, and 7 days using 0.1% fetal bovine serum (FBS), 10% FBS +/- 10 microM SB, or 20 ng/ml PDGF +/- 10 microM SB. Digital histomorphometric analysis assessed cellular fill within the wound area. RESULTS: SB specifically inhibited PDGF-induced migration in the Boyden chamber assays without affecting cell proliferation. The wound model data showed similar levels of wound fill for PDLs and GFs in 10% FBS. Relative to 10% FBS, PDLs stimulated with PDGF showed significantly (P < 0.01, analysis of variance) greater wound fill (74%) than GFs (12%). SB inhibited the PDGF-induced wound fill of PDLs and GFs by 64% and 57%, respectively. This inhibition was significant (P < 0.01, ANOVA) only for PDLs. The addition of SB to 10% FBS did not significantly affect the wound fill response of either cell type compared to 10% FBS alone. CONCLUSIONS: These results demonstrate that periodontal cells possess distinct responses to PDGF that may be altered at the signal transduction level. The manipulation of these responses through the use of inhibitors to specific signaling pathways may enhance our control of periodontal regeneration in the future.

Adult↗

PubMed2002

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

BACKGROUND: Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. RESULTS: A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. CONCLUSIONS: The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension.

Algorithms↗