Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “In vitro model”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,099 records · Page 61Linked to original sources
PubMed2004

Proteomic analyzes of copper metabolism in an in vitro model of Wilson disease using surface enhanced laser desorption/ionization-time of flight-mass spectrometry.

In Wilson disease, mutations in the ATP7B-gene lead to hepatic accumulation of copper that becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and acute liver failure. Several proteins are probably involved in dealing with the excess copper and oxidative stress. As a first step towards biomarker discovery and analyzes of copper metabolism in Wilson disease patients we characterized copper-induced changes in protein expression in cell lysates and culture media from an in vitro copper-overload model using surface enhanced laser desorption/ionization (SELDI) proteomics technology. HepG2 cells were cultured for 48 h with a physiological (0.5 microM) or a pathological (100 microM) copper concentration. Samples were applied to weak cation exchange (WCX) proteinchip arrays and chips were analyzed by time of flight (TOF)-mass spectrometry. Copper-coated IMAC chips were used to detect copper-binding proteins in cell lysate of copper depleted cells using buffers with increasing imidazole concentrations. Data from the 2 to 50 kDa range indicate that high extra-cellular copper substantially altered both intra-cellular protein expression as well as the composition of the secretome. In the lysate 15 proteins were found up-regulated, while 6 proteins were down-regulated. In culture media 21 proteins were increased while 4 proteins were decreased in abundance. Copper-coated protein chips revealed the presence of 18 high-affinity copper-binding proteins. Further identification is necessary to determine the exact cellular roles of the discovered proteins.

Carcinoma, Hepatocellular↗
PubMed2001

No effect of thromboxane A2 on the attachment of tumor cell lines MDA MB 231, DU145, and U937 to the basement membrane in an in-vitro model.

PURPOSE: A broad body of evidence indicates the involvement of P450 TxA2 (thromboxane A2 synthetase) in tumor metastasis formation. A distinct function of the enzyme in this multistep process, however, is still unknown. Therefore the effect of TxA2 (thromboxane A2) on tumor cell adhesion to the basement membrane, a key event in metastasis formation, was investigated. METHODS: A wide variety of compounds designed in our work group and identified as P450 TxA2 inhibitors were applied to several P450 TxA2-positive tumor cell lines to test their influence on tumor cell adhesion. For this purpose an in-vitro basement membrane adhesion model with the matrix gel preparation Matrigel was used. RESULTS: Most of the P450 TxA2 inhibitors tested had no effect on cell adhesion. Although two compounds significantly reduced tumor cell adhesion in a concentration-dependent manner, this was not related to P450 TxA2 inhibition. CONCLUSION: These data indicate that TxA2 might not be involved in the attachment of tumor cell lines to the basement membrane.

Basement Membrane↗
PubMed2000

An in vitro model for videoimaging of human bladder smooth muscle cell contractions.

Knowledge regarding human bladder smooth muscle cell (SMC) physiology is very limited. Only a few specific medical therapies for bladder disorders have therefore been established. The objective of this study was to develop a model for videomicroscopy of bladder SMC contractions. Cells were isolated from human cystoprostatectomy specimens and cultured in a modified EMEM medium. These cells were identified as SMCs by means of immunohistochemistry. For videomicroscopy, the culture flasks were coated with a viscous agent to allow cell contraction. Contractions were visualized by means of a cell culture microscope with a time-lapse videosystem. For cholinergic stimulation of the cells, acetylcholine, in concentrations ranging from 100 microM to 10 mM, was applied. The percentage of contracting cells within the observation field was evaluated for quantitative analysis. In control experiments without contractile stimulant 6% of the cells were observed to contract. Stimulation with acetylcholine induced a significant dose-dependent increase to 47% in contracting cells. These results demonstrated that videomicroscopy is an appropriate tool to investigate the contraction mechanisms of bladder SMCs. This model offers the possibility of studying drug effects on the human detrusor in vitro.

Acetylcholine↗
PubMed1992

Pig ear skin as an in-vitro model for human skin permeability.

Pig skin has been shown to have similar histological and physiological properties to human skin and has been suggested as a good model for human skin permeability. In this series of experiments, the in-vitro permeability of pig ear skin was compared with human (abdominal) skin and rat (dorsal) skin using both hydrophilic (water, mannitol, paraquat) and lipophilic (aldrin, carbaryl, fluazifop-butyl) penetrants. Pig skin was found to have a closer permeability character than rat skin to human skin, particularly for lipophilic penetrants. Electrical conductivity measurements across pig skin membranes showed that skin conductivity could be a useful method for assessing the integrity of membranes, particularly when used in conjunction with water permeability assessments.

Animals↗
PubMed2006

In-vitro model for the ultrastructural study of the formation of thrombi in human platelets.

Platelets are cell fragments with dynamic properties involved in clot formation after tissue damage. Platelet activation causes a change in shape, secretion of intracellular granules and aggregation with each other through the cytoskeleton components and biochemical changes. Platelet adhesion, considered as the major event in haemostasis, has been studied in several in-vitro and in-vivo models to evaluate the feasible thrombogenicity of some materials, the dynamics of specific receptors, as well as the effect of different buffers and inhibitors in this process. In spite of the numerous reports about platelet activation, to date there is no information available about the fine structure of the platelet-platelet and platelet-substrate interactions. In the present report we describe an in-vitro system that allows the visualization of these interactions: platelets are adhered to an inert substrate, and interactions with suspended platelets as a process to initiate the formation of thrombi was followed by ultramicrotomy and transmission electron microscopy.

Blood Coagulation↗
PubMed1987

An in vitro model for the induction of angiotensin-converting enzyme in sarcoidosis. Evidence for a soluble ACE-inducing factor.

Normal peripheral blood T-lymphocytes (T) enhance monocyte (Mo) angiotensin converting enzyme (ACE) production in vitro. To evaluate the possible role of a soluble mediator in enzyme induction, the ability of conditioned medium (CM) from Mo-T cocultures to stimulate ACE in fresh Mo was examined. These studies demonstrated that CM promoted a dose-dependent increase in ACE. Generation of the active factor required both Mo and T during culture since neither cell alone yielded active CM. Time course studies revealed that the ACE-inducing activity appeared in CM after 3 to 4 days of culture and increased steadily thereafter. The CM effectively induced ACE in Mo from several donors in contrast to T-dependent induction, which required autologous conditions. The time courses for T-dependent and CM-dependent ACE induction were similar, and the results suggested that the Mo must go through some period of maturation before they respond to CM. We conclude that a soluble mediator derived from Mo-T cocultures enhances Mo ACE production. This in vitro system may be a useful experimental model of the in vivo induction of ACE in the Mo-derived sarcoid epithelioid cell.

Cell Communication↗
PubMed1997

Reconstituted human gingival epithelium: nonsubmerged in vitro model.

Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type 1 collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.

3T3 Cells↗
PubMedDate not supplied

Establishment and characterization of four cell lines derived from human head and neck squamous cell carcinomas for an autologous tumor-fibroblast in vitro model.

To study interactions between tumor cells and stromal elements, we established carcinoma cell lines as well as tumor-derived and skin fibroblast cultures from four patients with squamous cell carcinoma of the head and neck. For the characterization of the tumor cell lines we a) determined population doubling times, b) assessed morphological features by light and electron microscopy, c) investigated the expression of typical markers by immunohistochemistry, including various intermediate filaments and surface antigens, d) compared these findings with expression patterns in the respective original tumor specimens, e) evaluated p53 mutations in tumor specimens and cell lines, f) performed chromosome analysis, g) investigated the tumorigenicity in athymic mice, and h) tested the formation of both tumor and mixed tumor-fibroblast multicellular spheroids. Tumor cell cultures were considered established cell lines when maintained and passaged over a period of two years after primary explantation. The in vitro morphology of the cell lines showed well preserved characteristics of squamous cell carcinoma, and electron microscopy as well as immunohistochemistry revealed their squamous type of differentiation. All cell lines presented the same p53 genotype as the respective original tumors. Furthermore, they were successfully xenotransplanted into nude mice and formed both pure and mixed three dimensional spheroids. This experimental model allows the in vitro and in vivo investigation of various tumor-fibroblast interactions.

Adult↗
PubMed1994

LHRH cells migrate on peripherin fibers in embryonic olfactory explant cultures: an in vitro model for neurophilic neuronal migration.

Luteinizing hormone releasing hormone (LHRH) neurons arise from progenitor cells in the olfactory placode. During prenatal development, these cells migrate via neurophilic interactions, in track-like arrangements along axons of the olfactory complex. The mechanisms by which these cells attain an adult-like distribution are unknown. In this study, we established an in vitro, embryonic mouse olfactory explant model to examine the factors directing LHRH cell migration. Cultures were generated from E11.5 embryos and maintained for up to 3 weeks. Typically 20-50% (160-400 LHRH cells) of the total LHRH neuronal population survived and maintained gene expression in these explants. Fibronectin and laminin staining delineated substratum producing cells which concentrically spread, from Days 1-7, from the periphery of the entire explanted tissue. In contrast, LHRH cells emigrated exclusively from inside olfactory pit areas to the surface of the culture after 3 days. The relationship between groups of LHRH cells emerging from bilateral olfactory pits was not random, but highly organized; in 93% of the cultures examined, the angular relationship between these groups was 180-270 degrees. After 5 or more days in vitro, LHRH cells were found in directional tracks similar to those observed in vivo forming a continuum from the olfactory pit out onto the substratum, where many bipolar LHRH cells were discretely located. The maximum distance away from the olfactory pit that LHRH cells were detected was 0.9 mm, which is compatible with the distance traversed by LHRH cells through the nasal region in vivo. The reproducible spatiotemporal appearance of LHRH cells was unrelated to the concentric spread of the fibronectin and laminin producing cells of the explant. Taken together, these data indicate that LHRH cells migrated directionally in these explants and that the molecular cues governing the initial migration of these neurons remained intact in this system in the absence of brain tissue. Double-label immunocytochemistry indicated that at least three populations of neuronal fibers existed in the explants: N-CAM-positive, peripherin-positive, and N-CAM/peripherin-positive. Although all three fibers groups showed highly organized spatiotemporal distribution patterns, only peripherin-positive fibers correlated with the location of LHRH cells. LHRH cells were always preceded by, and in close association with, peripherin-positive fibers. We hypothesize that signals arising from the peripherin-positive axons provide the appropriate guidance cues to LHRH cells as they emigrate from the olfactory pit.

Animals↗
PubMed1984

Synthesis of lung surfactant-associated glycoproteins by A549 cells: description of an in vitro model for human type II cell dysfunction.

Immunocytochemical, ultrastructural, and biochemical approaches were used in a series of in vitro and in vivo experiments designed to identify characteristic changes reflecting differentiated type II cell function of A549 cells. Monolayers of A549 cells and A549 clones were maintained in culture for up to 3 weeks. Using an immunoperoxidase (PAP) technique, we demonstrated that variable proportions of A549 cells and of cells in several A549 clones reacted specifically with antibodies to high molecular weight (greater than 400,000) human surfactant-associated glycoproteins (HSAG). The cells of one clone, A549-C12, were consistently negative for HSAG, but their lamellar bodies were similar in appearance and distribution to those found in a PAP-positive clone, A549-C11, as well as in A549 cells. In addition, both C11 and C12 clones displayed time-dependent, divergent differentiation predominantly toward type II epithelium and nonciliated bronchiolar and bronchial cells. Surfactant isolated from either C11 or C12 cells revealed reduced content of disaturated phosphatidylcholine and phosphatidylglycerol when compared to human surfactant; however, a 95,000-dalton peptide immunologically related to HSAG was identified in surfactant from C11 cells but not from the PAP-negative C12 clone. Tumor xenografts produced in athymic (nude) mice following inoculation with cells from C11 and C12 clones exhibited prominent immunoperoxidase staining involving most tumor cells. Cell lines derived from these xenografts (T-11 and T-12) were also enriched in PAP-positive cells. Immunoelectron microscopy indicated that HSAG was localized in the rough endoplasmic reticulum, multivesicular bodies (MVB), intermediate MVB-lamellar forms, and abnormal pleomorphic inclusions. Moreover, two HSAG peptides, both larger than the 34,000-dalton peptide subunit found in normal human surfactant, were present in cells and media from monolayers of the T-11 cell line. We conclude that A549 cells synthesize "defective" HSAG and that the synthesis may be modulated by host factors. The results indicate that appropriate A549 clones can be used effectively as model systems for selected type II cell dysfunctions.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed2005

Microstructured cocultures of cardiac myocytes and fibroblasts: a two-dimensional in vitro model of cardiac tissue.

Cardiac myocytes and fibroblasts are essential elements of myocardial tissue structure and function. In vivo, myocytes constitute the majority of cardiac tissue volume, whereas fibroblasts dominate in numbers. In vitro, cardiac cell cultures are usually designed to exclude fibroblasts, which, because of their maintained proliferative potential, tend to overgrow the myocytes. Recent advances in microstructuring of cultures and cell growth on elastic membranes have greatly enhanced in vitro preservation of tissue properties and offer a novel platform technology for producing more in vivo-like models of myocardium. We used microfluidic techniques to grow two-dimensional structured cardiac tissue models, containing both myocytes and fibroblasts, and characterized cell morphology, distribution, and coupling using immunohistochemical techniques. In vitro findings were compared with in vivo ventricular cyto-architecture. Cardiac myocytes and fibroblasts, cultured on intersecting 30-microm-wide collagen tracks, acquire an in vivo-like phenotype. Their spatial arrangement closely resembles that observed in native tissue: Strands of highly aligned myocytes are surrounded by parallel threads of fibroblasts. In this in vitro system, fibroblasts form contacts with other fibroblasts and myocytes, which can support homogeneous and heterogeneous gap junctional coupling, as observed in vivo. We conclude that structured cocultures of cardiomyocytes and fibroblasts mimic in vivo ventricular tissue organization and provide a novel tool for in vitro research into cardiac electromechanical function.

Animals↗
PubMedDate not supplied

In vitro models of angiogenesis: the use of Matrigel.

Tumor-induced angiogenesis is a key event for neoplastic progression. In vitro assays are important for identification of potential angiogenic agents and rapid screening for pharmacological inhibitors. The increased interest in this field of study has generated several in vitro assays that recapitulate the steps of endothelial cell activation and differentiation. In this short report we emphasize the utility of Matrigel, a reconstituted basement membrane, to define two different steps in the angiogenic process: invasion in response to growth factors and organization of microvessels into a network with branching morphology on a Matrigel substrate.

Chemotaxis↗
PubMed2001

[In vitro modelling of the interactions between the promoter and enhancer complexes].

The aim of this work is to study in vitro mechanisms of interactions between the promoter and enhancer complexes of the transcriptional apparatus. We used retardation assay for the labeled TATA-box containing oligonucleotide with an unfractionated nuclear extract and with a second unlabeled oligonucleotide carrying an enhancer sequence. As an enhancer sequence we have chosen e5 element that is known to interact with members of Pax family transcription factors. We showed that the presence of unlabeled e5 element led to a significant decrease in the mobility of TATA-box associated complex. E5 element with a mutated homeodomen binding site does not produce such an effect, while a mutation in the paired binding site does not change the ability of e5 element to influence the formation of promoter-associated complex. We observed the same complex using crude nuclear extracts from cells that express chimeric protein Pax7-FKHR with higher molecular weight. The fact that the increase in the size of the transcription factor does not influence the size of the TATA-box associated complex led us to conclude that this complex did not result from association of original promoter and enhancer components, but rather the role of enhancer may be to stabilize the slowly migrating complex on the TATA-box via short-term interactions inducing conformational changes in the included proteins.

3T3 Cells↗