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At least 667 records · Page 37Linked to original sources
PubMed2005

Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-.

The CFBE41o- cell line was generated by transformation of cystic fibrosis (CF) tracheo-bronchial cells with SV40 and has been reported to be homozygous for the DeltaF508 mutation. A systematic characterisation of these cells, which however, is a pre-requisite for their use as an in vitro model, has not been undertaken so far. Here, we report an assessment of optimal culture conditions, the expression pattern of drug-transport-related proteins and the stability/presence of the CF transmembrane conductance regulator (CFTR) mutation in the gene and gene product over multiple passages. The CFBE41o- cell line was also compared with a wild-type airway epithelial cell line, 16HBE14o-, which served as model for bronchial epithelial cells in situ. The CFBE41o- cell line retains at least some aspects of human CF bronchial epithelial cells, such as the ability to form electrically tight cell layers with functional cell-cell contacts, when grown under immersed (but not air-interfaced) culture conditions. The cell line is homozygous for DeltaF508-CFTR over multiple passages in culture and expresses a number of proteins relevant for pulmonary drug absorption (e.g. P-gp, LRP and caveolin-1). Hence, the CFBE41o- cell line should be useful for studies of CF gene transfer or alternative treatment with small drug molecules and for the gathering of further information about the disease at the cellular level, without the need for primary culture.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

In vitro modelling of tissue using isolated vascular cells on a synthetic collagen matrix as a substitute for heart valves.

BACKGROUND: Tissue engineering is a promising approach for obtaining lifetime durability in biological heart valves. Basic questions with respect to the selection of suitable cell populations as well as scaffolds remain unsolved. The purpose of this study was to develop a tissue-like substitute in vitro for replacement of diseased valves in vivo. METHODS: Smooth-muscle cells (SMCs) were isolated from human and porcine aortic tissue using the 'explant technique' and endothelial cells from collagenase digestion. Seeding and cultivation of isolated cells was performed on a type-I collagen matrix. The scaffold-cell specimen was investigated using light and electron microscopy. Cupromeronic blue and immunoprecipitation were used for ultracytochemical staining. RESULTS: SMCs were allowed to grow to multilayers and migrate into the collagen network. We found a tissue-like morphology in these samples characterised by several layers of cells, spaces between the cell layers filled with newly formed extracellular matrix components, compartmentalisation of proteoglycans and their association with fibrilar matrix and the cell surface. Endothelium cells covered the SMCs of the scaffold with a histological topography similar to heart valves. CONCLUSIONS: This is an approach for in vitro modelling of tissue-like substitutes and preparing plane multicellular tissues as substitutes for heart valves. This model may also be used for cell biological investigations of cell-matrix interactions.

Animals↗
PubMed1985

Hematopoietic histoincompatibility reactions by NK cells in vitro: model for genetic resistance to marrow grafts.

In certain strains of mice, bone marrow grafts from parental donors fail to grow in first-generation hybrid mice. This "hybrid resistance" of nonsensitized F1 hybrid mice to the engraftment of parental hematopoietic transplants contradicts the classical laws of transplantation and is dependent on a radioresistant but immunogenetically specific effector mechanism. Studies in a new in vitro model reveal that committed hematopoietic precursors of parental origin can be inactivated by direct contact with natural killer-like splenic effectors from F1 mice. The reaction requires genetically restricted recognition, since only parental competitors syngeneic to the target bone marrow cells partially reversed this inactivation. Models of this type may be useful in studying the possible role of natural resistance in bone marrow transplantation in humans.

Animals↗
PubMed2006

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

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.

Acute-Phase Reaction↗
PubMed1996

Mononuclear phagocytes egress from an in vitro model of the vascular wall by migrating across endothelium in the basal to apical direction: role of intercellular adhesion molecule 1 and the CD11/CD18 integrins.

Little is known about how mononuclear phagocytes (MP) are cleared from sites of inflammation as inflammatory lesions resolve. In this study, the possibility that MP could be cleared from tissues by migrating across endothelium in the basal to apical direction was investigated. In an in vitro model of a blood vessel wall consisting of human umbilical vein endothelial cells (HUVEC) grown on amniotic tissue, a majority of MP that initially transmigrated into the amnion later exited by migrating back across the endothelium in the basal to apical direction. MP that egressed from these cultures adhered to the apical surface of the endothelium or were found nonadherent in the medium above the endothelium. Egression of MP continued throughout the 4-d period examined, displaying higher than first order kinetics and a t(1/2) of approximately 24 h. These kinetics were decreased by increasing the volume of medium bathing the cultures, suggesting that a soluble factor(s) regulates the rate of egression. In contrast, the kinetics were accelerated by pretreating the endothelium with IL-1. The initial phase of this increased rate of egression was inhibited by antibodies to inter- cellular adhesion molecule 1 (ICAM-1) or CD18 by 100 and 71%, respectively. Immunostaining revealed that ICAM-1 was present on the apical and basal surfaces of umbilical vein endothelium in vitro and in situ. These data demonstrate that MP can traverse endothelium in the basal to apical direction, and lend insight into the mechanisms by which this process occurs.

Amnion↗
PubMed2004

Behavior of alpha-, beta-, and gamma-cyclodextrins and their derivatives on an in vitro model of blood-brain barrier.

Cyclodextrins (CDs) can be envisaged to cure some diseases related to the brain, but the behavior of these compounds toward the blood-brain barrier (BBB) remains largely unexplored to envisage such clinical applications. To fulfill this gap, the toxicity and endothelial permeability for native, methylated, and hydroxypropylated alpha-, beta-, and gamma-CDs have been studied on an in vitro model of BBB. As shown by the endothelial permeability for sucrose and immunofluorescence stainings, the native CDs are the most toxic CDs (alpha- > beta- > gamma-CD). Whereas the chemical modification of beta-CD did not affect the toxicity of this CD, differences are observed for the alpha- and gamma-CD. To determine the origin of toxicity, lipid effluxes on the brain capillary endothelial cells were performed in the presence of native CDs. It was found that alpha-CD removed phospholipids and that beta-CD extracted phospholipids and cholesterol. gamma-CD was less lipid-selective than the other CDs. Finally, the endothelial permeability of each CD has been determined. Surprisingly, no structure/permeability relationship has been observed according to the nature and chemical modifications of CDs.

Animals↗
PubMed2006

Steroid requirements for regulation of the alpha4 subunit of the GABA(A) receptor in an in vitro model.

The alpha4 subunit of the GABA(A) receptor (GABAR) has relatively low expression in the CNS, but is increased in vivo following 48 h administration of the GABA-modulatory steroid 3alpha-OH-5alpha[beta]-pregnan-20-one (THP or [allo]pregnanolone) to female rats. The purpose of the following study was to determine the optimal conditions for steroid-induced upregulation of alpha4 expression in an in vitro model. To this end, we used the IMR-32 cell, a neuroblastoma cell line, which normally expresses alpha4 mRNA at low levels. In undifferentiated IMR-32 cells, 48 h administration of THP increased alpha4 expression when ambient THP levels were reduced by the 5alpha-reductase blocker 4MA, suggesting that the background steroid milieu affects steroid regulation of this subunit. Following neuronal differentiation in serum-free medium, 48 h THP treatment significantly increased alpha4 expression two-fold following application of nerve growth factor (NGF) suggesting that development of neuronal processes facilitates this effect of the steroid. In the absence of NGF treatment, combined administration of 17beta-estradiol (E2) plus THP also increased alpha4 expression to a similar extent as THP following NGF treatment. In addition, E2 alone effectively increased alpha4 expression to maximal levels following NGF treatment. In contrast, neuronal differentiation in the absence of serum deprivation did not increase alpha4 levels. These results suggest that both THP and E2 can increase expression of the GABAR alpha4 subunit, but that this effect is dependent upon the background steroid milieu as well as the degree of neuronal development. These findings demonstrate optimal conditions for steroid-induced upregulation of the alpha4 subunit in an in vitro system.

Analysis of Variance↗
PubMed1990

An in vitro model for cyclosporin A-induced interference of intrathymic clonal elimination.

The effects of cyclosporin A (CsA) on influencing the intrathymic clonal deletion were investigated by using our established thymic stromal cell clone with capacities to express Ia antigens and to produce a unique T cell growth factor. The following were revealed: (a) T cell clone with a given specificity was killed on the Ia+ stromal cell monolayer in the presence of the relevant antigens, a process depending on T cell receptor (TCR) stimulation; and (b) CsA allowed the T cell clone to continuously proliferate even during TCR stimulation by virtue of the stromal cell-derived T cell growth factor. This paper describes an in vitro model of a mechanism by which CsA is responsible for the generation of normally "forbidden" T cell clones.

Animals↗
PubMedDate not supplied

An in vitro model for studying the effect of the subcutaneous tunnel and the skin exit site on the accuracy of central venous catheter tip cultures.

The diagnosis of central venous catheter-related sepsis depends on a positive culture obtained from the distal intravascular portion of the catheter. The effects of the subcutaneous tunnel and the skin exit site on the accuracy of cultures obtained from the catheter are unknown. We have developed an in vitro model to study the effect of these variables. By inoculating polyethylene catheters embedded in agar with Pseudomonas aeruginosa, we were able to show that: (1) capillary action occurs along catheters in an agar tunnel, (2) organisms that are growing on the distal segment (tip) of the catheter can be dislodged from the surface of the catheter when it is pulled through the agar tunnel, and (3) pulling a catheter through a contaminated area results in distal contamination.

Agar↗
PubMed1985

Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix. An in vitro model system for studying tumor cell adhesion and invasion.

Human ovarian tumors metastasize by direct extension into the peritoneal cavity leading to tumor cell implantation onto peritoneal surfaces. Successful formation of peritoneal implants is dependent on the ability of ascitic tumor cells to infiltrate the mesothelium, and become firmly adherent to the underlying extracellular matrix (ECM). In order to investigate this process in more detail, an in vitro model system was developed employing human mesothelial cells grown on ECM-coated culture dishes. The ability of human ovarian carcinoma cells derived from ascitic fluid to attach to the mesothelial cell monolayer grown on ECM, ECM alone or plastic was quantitated with the use of 51Cr radio-labelled tumor cells. Tumor cells exhibited a more rapid and firmer attachment to ECM than to the mesothelial cells or to plastic. Using agitation to stimulate peritoneal fluid dynamics and shear forces in vivo, tumor cell arrest was found to be limited to the ECM, but it occurred at a slower rate than it did without agitation. Tumor cell attachment was also restricted to areas of exposed ECM in wounded mesothelium as assessed by phase-contrast microscopy. Morphologic alterations of the mesothelium induced by tumor cells were observed with the use of scanning electron microscopy (SEM) and immunohistochemical staining which included disruption of intercellular junctions leading to retraction of mesothelial cells, exposure of underlying ECM, subsequent attachment and proliferation on ECM. This model system would appear to be useful for elucidating mechanisms of ovarian tumor cell adhesion and proliferation, and for assessing various therapeutic modalities for their ability to block tumor cell implantation, invasion and growth on peritoneal surfaces.

Animals↗
PubMed2006

An in vitro model of human placental trophoblast deportation/shedding.

Deportation of trophoblast shed from the placenta into the maternal circulation was first described over 100 years ago. Despite this, little is known about the quantity or nature of the shed and deported trophoblasts. Neither do we have a clear understanding of the fate of deported trophoblasts nor do we have a clear understanding of their effects on the maternal physiology. This deficiency is largely due to the inaccessibility of deported trophoblasts in vivo. This study aimed to produce a model that would allow us to study deported trophoblasts. We devised a system for culturing placental explants of 12-week gestation in cell culture inserts with a stainless steel mesh bottom that allowed the ready harvesting of shed/deported trophoblasts. Immunohistochemical and morphologic investigations demonstrated that these in vitro shed/deported trophoblasts are similar to those found in vivo and that apoptotic, necrotic and viable trophoblasts were shed from the explants. Inhibiting caspases induced a change from predominantly apoptotic to predominantly necrotic trophoblast shedding. We have devised an in vitro model that allows the collection of shed/deported trophoblasts which will significantly enhance our ability to study these cells. Our preliminary investigations confirm that apoptosis plays an important role in trophoblast shedding/deportation.

Apoptosis↗
PubMed2006

In vitro models for studying hepatitis B virus drug resistance.

Hepatitis B virus resistance to antivirals is conferred by the selection of polymerase gene mutants with reduced susceptibility to nucleoside analogues. Because of the increasing number of resistant mutants to the different available drugs, several phenotypic assays have been developed to provide cross-resistance data to guide clinicians in the management of therapy. Their possible use in the routine follow-up of antiviral treatment is also discussed. The different in vitro models for the study of HBV resistance include enzymatic and cell-based assays. Their relevance for the analysis of viral fitness, drug susceptibility, and cross-resistance is also reviewed.

Antiviral Agents↗
PubMed2006

An in vitro model to study the role of endothelial rho GTPases during leukocyte transendothelial migration.

Tissue injury induces release of cytokines that stimulate the expression of adhesion receptors in the endothelial wall of neighboring vessels. These endothelial receptors recruit leukocytes from the bloodstream and facilitate their transendothelial migration (TEM) toward the inflamed area. The molecules involved in leukocyte-endothelium interaction and TEM have been studied in vivo and in vitro over the past 20 years. Human umbilical vein endothelial cells (HUVECs) are a popular in vitro model to analyze TEM, and have been used to investigate the role of Rho GTPases in this process. Here we describe methods to activate HUVECs, to investigate Rho GTPase-activation by the endothelial adhesion receptor ICAM-1, and to inhibit or activate Rho GTPases using C3 transferase or adenoviruses coding for dominant negative or constitutive active Rho GTPases. Finally, we describe how to image and quantitate leukocyte TEM by digital time-lapse microscopy.

ADP Ribose Transferases↗
PubMed2006

Iron-related transcriptomic variations in CaCo-2 cells, an in vitro model of intestinal absorptive cells.

Regulation of iron absorption by duodenal enterocytes is essential for the maintenance of homeostasis by preventing iron deficiency or overload. Despite the identification of a number of genes implicated in iron absorption and its regulation, it is likely that further factors remain to be identified. For that purpose, we used a global transcriptomic approach, using the CaCo-2 cell line as an in vitro model of intestinal absorptive cells. Pangenomic screening for variations in gene expression correlating with intracellular iron content allowed us to identify 171 genes. One hundred nine of these genes are clustered into five types of expression profile. This is the first time that most of these genes have been associated with iron metabolism. Functional annotation of these five clusters indicates potential links between the immune response, proteolysis processes, and iron depletion. In contrast, iron overload is associated with cellular metabolism, especially that of lipids and glutathione involving redox function and electron transfer.

Caco-2 Cells↗
PubMed1988

An in vitro model to study bacterial invasion of periodontal tissues.

In periodontal disease, the abilities of bacteria to adhere to and degrade in vivo basement membranes should be considered as two of the rate-limiting steps for the potential active or passive invasion of gingival connective tissues. To study these mechanisms in greater detail, we used the PF HR-9 basement-membrane-like matrix to establish an in vitro model of bacterial invasion and degradation. Three gram-negative anaerobic periodontopathic organisms, Bacteroides gingivalis, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans, bound in considerably higher numbers to the HR-9 matrix than did 6 strains of gram-positive facultative organisms typically associated with periodontal health. In a further experiment with B. gingivalis, the organism rapidly degraded Type IV collagen, the major macromolecular component constituting the HR-9 matrix. Streptococcus mitis, the nonperiodontopathic bacterium tested, did not degrade this model matrix. This study provides evidence that B. gingivalis, a periodontopathic bacterium, is able to adhere to and degrade basement membranes, whereas nonperiodontopathic organisms appear not to share in these abilities.

Actinobacillus↗
PubMed2004

Gene expression profiling shows that macrophages derived from mouse embryonic stem cells is an improved in vitro model for studies of vascular disease.

Macrophages (Mphi) play an important role in the initiation and progress of the atherogenic process. They contribute to the growth of atherosclerotic plaque by affecting lipoprotein metabolism, matrix homeostasis, lipoprotein modification and cholesterol accumulation. Access to in vitro Mphi models is therefore important for understanding the mechanisms involved in the transition of the relatively simple fatty streak to more complex type of lesions. The aim of the present work was to compare the expression profile of macrophages differentiated from the hematopoietic lineage to peritoneal mouse macrophages and two commonly used mouse macrophage cell lines (J774.A1 and RAW264.7). Our results showed that the embryonic stem cell-derived macrophages (ES Mphi) had a more similar expression profile to peritoneal macrophages than the two mouse macrophage cell lines. The ES Mphi had unchanged expression of the majority of cholesterol efflux mediators when compared to mouse peritoneal macrophages, whereas the cell lines showed altered expression of several of these genes. A key gene in this process is apolipoprotein E, which is expressed in ES Mphi but not in macrophage cell lines. In conclusion, ES Mphi are likely to provide a better in vitro model than mouse Mphi cell lines to study macrophage involvement in atherosclerosis.

Animals↗
PubMed1999

A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence.

This protocol describes a model of cerebral ischemia based on organotypic hippocampal slice cultures and quantitative assessment of cell death by use of propidium iodide and image analysis. The cultures were made from rat hippocampal slices that were obtained at postnatal day 4-7 and allowed to develop for >14 days in vitro. For induction of 'in vitro ischemia', the cultures were washed in glucose free buffer and the culture chamber flooded with a nitrogen/carbon dioxide mixture until the oxygen concentration was <1.0%. The cultures were exposed to this atmosphere for 30-35 min, washed in serum-free medium, and returned to ordinary growth medium. After 24 h, dead cells were quantified by use of propidium iodide. The cell death resulting from the oxygen/glucose deprivation was largely confined to the CA1 region and was blocked by NMDA-receptor antagonists but not by antagonists to AMPA-receptors or metabotropic glutamate receptors. The type of cell death was judged to be necrotic, based on ultrastructural observations. The oxygen/glucose deprived cultures exhibited increased phosphorylation of the MAP kinase cascade. This activation of the MAP kinase cascade was blocked by NMDA-receptor antagonists. The in vitro model described in the present report is simple to use and reproduces many features of in vivo ischemia, including the preferential vulnerability of CA1 cells. The model should be suited to analyses of the mechanisms underlying the regionally selective cell death in the hippocampus and ischemic cell death in general.

Animals↗
PubMed2000

Plain film, CT and MRI sensibility in the evaluation of intraorbital foreign bodies in an in vitro model of the orbit and in pig eyes.

Detection and characterization of intraorbital foreign bodies (IFB) is fundamental in acute trauma setting, preventing inflammatory sequelae or complications related to IFB movements when a MRI study is planned. Papers concerning plain film and CT sensibility in IFB detection show controversial results. For this reason we investigated plain film, CT and MRI sensibility in the evaluation of IFB. For an in vitro model, specimens of dry and fresh wood, glass, iron, plastic and graphite were immersed in animal lard and in a 0.9% sodium chloride plus 3.5 g/dl human serum albumin solution. Specimens of different size and nature where also implanted into enucleated pig eyes. Air bubbles were introduced also. Plain film, CT and MRI investigation were performed. Plain films underestimated intraocular IFB as plastic, fresh or dry wooden IFB were not demonstrated. The CT study was always able to depict and differentiate IFB according to the attenuation values. Severe artefacts prevented demonstration of iron, glass and graphite IFB on MRI, whereas plastic or wooden IFB were always detected. Despite radiographs have been suggested as a prerequisite for MR imaging, because our results showed plain film to underestimate radiolucent IFB, we suggest CT as the modality of choice when IFB has to be ruled out.

Animals↗