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At least 1,153 records · Page 64Linked to original sources
PubMed2005

[Gene expression profiling of microvascular endothelial cells during capillary morphogenesis in an in vitro model of angiogenesis].

OBJECTIVE: To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogenesis system with Affymetrix oligonucleotide array. METHODS: A microcarrier-based in vitro angiogenesis system was developed, in which endothelial cells (ECs) migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed network. The total RNA samples from the HMVECs at the selected time points (0.5 h, 24 h, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the software provided by the manufactory. The expression patterns of some genes were validated and confirmed by Semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines/chemokine receptors were specially examined and their functional implications were analyzed. RESULTS: About 1500 genes were found up- or down- regulated 2-folds or above detected by the arrays, and among them, about 400 genes regulated 3-folds or above. The regulated genes could be grouped into categories based on their molecular functions such as growth factor and receptor, cell proliferation, extracellular matrix, cell cycle and apoptosis, signaling molecule and transcription factor, and so on, using the Gene Ontology Mining Tool in The NetAffx Analysis Center. The regulated genes were also clustered into six groups based on their patterns of expression. As for chemokines, the CCL2/MCP-1, CCL5/RANTES and CX3CL1 were identified to be specially upregulated at 24 h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis. CONCLUSIONS: Based on our angiogenesis model, and by oligonucleotide arrays, the present study demonstrates global profiles of the gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be encouraged.

Capillaries↗
PubMed1995

An in vitro model for abnormal skeletal development in the lysosomal storage diseases.

Lysosomal storage diseases such as GM1-gangliosidosis are associated with skeletal abnormalities. Radiological and histological studies, both in human and corresponding animal models, indicate retarded bone formation. Since cartilage maturation leads to bone formation, we developed an in vitro system to study and compare the biological features of cartilage from dogs affected with GM1-gangliosidosis with age-matched controls. Costochondral chondrocytes were grown in monolayer and in agarose culture. Both affected and control cells dedifferentiated in monolayer; however, in agarose culture they re-expressed the chondrocytic phenotype. Cells from affected dogs were enlarged and contained numerous large vacuoles when compared with control cells. This morphology was similar to that seen in vivo. In addition, the affected cells appeared to have a reduction in mitosis and alcian blue staining proteoglycans. Cultures from affected animals contained fewer cells positive for alkaline phosphatase activity. Both affected and control cells expressed collagen types I and II and were positive for the lectin Ricinus communis agglutinin-I. However, the staining of the control culture for type II collagen was more prominent than in the affected cells. These findings suggest that culture of chondrocytes in agarose may be a useful method for studying the biology of cartilage which leads to skeletal abnormalities in lysosomal storage diseases.

Animals↗
PubMed1993

An in vitro model for the analysis of intestinal brush border assembly. I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells.

Intestinal epithelial cells assemble and maintain a polarized, highly organized membrane-cytoskeleton array, the brush border. We describe an in vitro, cell contact-induced brush border assembly model using the Caco-2BBe clones. Subconfluent cells were 'depolarized' by brief passage through suspension culture in the presence of cytochalasin D and re-plated on filters at high density in low-Ca2+ medium. Upon return to regular medium, these small, rounded cells with bleb-like protrusions formed, over the course of 19 days, a polarized monolayer of tall, columnar cells with a well-defined brush border. Ultrastructural changes were documented by both transmission and scanning electron microscopy. The earliest events of microvillar assembly coincided with a short period of cell aggregation. Intercellular cysts were occasionally observed within these aggregates, and junction formation between cells which had no contact with the filter was also observed. Monolayer formation was completed within 48 hours, and cell height steadily increased approximately 3.5-fold over 19 days. Concurrent with monolayer formation and the increase in cell height, sparse microvilli with a few actin core filaments gradually became more dense and better organized. By the third day, the actin core bundles had begun to extend into the subjacent cytoplasm, while terminal web assembly was underway at five days. The mature morphology of the brush border was first observed at nine days, although cell height and microvillar density continued to increase during the subsequent ten days. Microvillar density rose approximately nine-fold throughout brush border assembly in the Caco-2BBe cells. With the exception of the formation of cellular aggregates at the onset of the time course, this sequence of morphological changes is comparable to that observed during brush border assembly in embryonic intestinal epithelial cells. The Caco-2BBe assembly model provides a useful system in which to investigate various molecular aspects of brush border assembly.

Actins↗
PubMed2004

PAMPA--a drug absorption in vitro model 11. Matching the in vivo unstirred water layer thickness by individual-well stirring in microtitre plates.

Many plate-based in vitro assays of membrane permeability (e.g., Caco-2, MDCK, PAMPA) of sparingly soluble candidate molecules report permeability of water, and not of the intended membrane barrier. This is so because the unstirred water layer on both sides of the membrane barrier is rate limiting for these highly permeable molecules. The thickness of this water layer can be 1500-4000 microm in unstirred assays. Under in vivo conditions, however, the unstirred water layer is believed to be 30-100 microm thick. Lightly stirred in vitro assays, using plate shakers, cannot lower the thickness of the water layer to match that found in vivo. In this study, 55 lipophilic drugs were employed to characterize the effect of stirring in parallel artificial membrane permeability assay (PAMPA). Highly efficient individual-well magnetic stirring at speeds greater than 110 rpm has been demonstrated to lower the unstirred water layer thickness to the in vivo range. Stirring at 622 rpm has lowered the layer thickness to 13 microm in some cases, which had not been previously achieved for plate-based permeability assays. With diminished water layer contribution at 622 rpm, for example, the effective permeability of progesterone is 2754 x 10(-6) cm/s. The new stirring apparatus used in this study is not only suitable for PAMPA, but can also be used in Caco-2 assays. Because of the diminished resistance of the thinner water layer, the stirred PAMPA permeation time has decreased from the usual 15 h to about 15 min for lipophilic compounds.

Absorption↗
PubMed1998

In vitro models for oocyte development.

The mammalian ovary has a large store of primordial follicles, which are a potential source of oocytes for in vitro production of embryos. Several culture systems have been developed to support the growth and development of oocytes from rodent primordial and preantral follicles and progress is slowly being made in modifying these techniques to support the in vitro growth of porcine and bovine follicles. Oocytes from porcine preantral follicles can acquire competence to resume meiosis and proceed to Metaphase II after in vitro growth (IVG) but fertilisation has yet to be demonstrated. This paper presents the current status of technology for the in vitro growth and development of immature mammalian oocytes. Culture systems used successfully to grow immature rodent oocytes are compared and adaptations of these methods to support porcine and bovine oocyte growth discussed.

Animals↗
PubMed1991

Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing.

A transmembrane extracellular matrix receptor of the integrin family, alpha 6 beta 4, is a component of the hemidesmosome, an adhesion complex of importance in epithelial cell-connective tissue attachment (Stepp, M. A., S. Spurr-Michaud, A. Tisdale, J. Elwell, and I. K. Gipson. 1990. Proc. Natl. Acad. Sci. USA. 87:8970-8974; Jones, J. C. R., M. A. Kurpakus, H. M. Cooper, and V. Quaranta. 1991. Cell Regulation. 2:427-438). Cytosolic components of hemidesmosomes include bullous pemphigoid (BP) antigens while extracellular components include a 125-kD component of anchoring filaments (CAF) and collagen type VII-containing anchoring fibrils. We have monitored the incorporation of the alpha 6 beta 4 integrins into forming hemidesmosomes in an in vitro wound-healing explant model. In epithelial cells recently migrated from the edges of unwounded sites over bare connective tissue, alpha 6 beta 4 first appears along the entire cell surface. At this stage, these cells contain little or no cytosolic hemidesmosomal components, at least as detectable by immunofluorescence using BP autoantibodies, whereas they are already positive for laminin and CAF. At a later stage, as cells become positive for cytosolic hemidesmosome components such as BP antigens as well as collagen type VII, alpha 6 beta 4 becomes concentrated along the basal pole of the epithelial cell where it abuts the connective tissue of the explant. Polyclonal antibodies to beta 4 do not interfere with the migration of epithelial cells in the explant. However, they prevent assembly of hemidesmosomal complexes and inhibit expression of collagen type VII in cells that have migrated over wound areas. In addition, they induce disruption of established hemidesmosomes in nonmigrating cells of the unwounded area of the explant. Monoclonal antibodies to alpha 6 have a more dramatic effect, since they completely detach epithelial cells in the unwounded area of the explant. Antibodies to CAF also detach epithelial cells in unwounded areas, apparently by inducing separation between epithelium and connective tissue at the lamina lucida of the basement membrane zone. These results suggest a model whereby polarization of alpha 6 beta 4 to the basal surface of the cells, perhaps induced by a putative anchoring filament-associated ligand, triggers assembly of hemidesmosome plaques.

Animals↗
PubMed2004

Functional validation of adult hippocampal organotypic cultures as an in vitro model of brain injury.

To determine whether hippocampal pyramidal neurons retain authentic functional properties in mature organotypic culture, hippocampal slice cultures were established from young adult rats (P20-21). Cultures maintained 7 days in vitro retained tight organization of neuronal layers, as opposed to the widening restructure of pyramidal neurons often observed in perinatal slices. CA3 and CA1 pyramidal neurons fired action potentials in response to current injection and exhibited spontaneous and evoked synaptic currents, indicating intact neuronal function and normal hippocampal neural circuitry. We also tested neuronal sensitivity of slice cultures to ischemic injury. Acute ischemic paradigm resulted in selective death of pyramidal neurons in the CA1 region, which was prevented by treatment with an NMDA-antagonist, MK-801. Robust efflux of excitatory and inhibitory amino acid neurotransmitters was detected during ischemia, consistent with changes shown in acute slices. In summary, hippocampal organotypic cultures prepared from young adult rats maintained neuronal architecture and synaptic activity in vitro and can be used in parallel with an acute slice system to model mature brain tissue to examine ischemic pathophysiology and neuroprotective treatment.

Action Potentials↗
PubMed1985

An in vitro model for testing biocompatibility of endodontic materials to bone.

Calvaria from 6-day old mice labelled 4 days previously with 45CaCl2 were divided into test and control halves and each half cultured separately in vitro. Eluents from four endodontic materials, endomethasone, zinc oxide/eugenol, AH26 and gutta-percha were added separately to the culture medium of each test half. After 24 and 48 h culturing periods, the 45CaCl2 in the media and calvaria was measured by a standard liquid scintillation counting technique and a resorption ratio between test and control halves was computed. This ratio, based on cell mediated resorption, was an indication of toxicity of soluble components of each endodontic material. In accordance with the literature, endomethasone was found, with our method, to be the most toxic and gutta-percha the least toxic of the materials tested. This shows that our model can be used to test the toxicity of other biomaterials to bone cells in vitro.

Administration, Topical↗
PubMed1987

Cultured cerebellar cells as an in vitro model of excitatory amino acid receptor function.

Primary cultures of neurons from 8-day-old rat pups were grown for 10 days in vitro in antibiotic-free media and then analysed for changes in cyclic guanosine monophosphate (cGMP) in response to several excitatory amino acid (EAA) agonists or related antagonists. Kainic acid (KA), N-methyl-D-aspartic acid (NMDA) and quisqualic acid (QA) produced dose- and calcium-dependent increases in cGMP with KA producing the largest and QA the least increase in this cyclic nucleotide. The increase induced by NMDA was additive with both KA and QA; however, KA and QA were not additive with each other. In fact, QA completely antagonized the effects of KA and to a much greater degree than did the EAA antagonists, glutamylaminomethylsulfonic acid (GAMS) or cis-2,3-piperidine dicarboxylic acid (PDA). 2-Amino-7-phosphonoheptanoic acid completely prevented the NMDA-induced elevations of cGMP yet had little effect on either the KA- or QA-induced elevations of this parameter. GAMS and PDA, on the other hand, were more effective in blocking the effects of KA and QA than of NMDA. These data show that cGMP levels in cerebellar granule cells provide an excellent model for studying the subtypes of EAA receptors.

Animals↗
PubMed1991

The isolated neonatal rat-cardiomyocyte used in an in vitro model for 'ischemia'. II. Induction of the 68 kDa heat shock protein.

In a model system of cultured rat cardiac cells, the expression of the heat shock protein hsp68 was studied after simulating ischemia. We observed both an increase in hsp68 mRNA levels and hsp68 synthesis, while under normal conditions hsp68 and its mRNA could not be detected. Using an antibody against hsp70 and hsp68, immunofluorescence studies showed that during 'ischemia', when hsp68 is not yet synthesized, hsp70 migrated into the nucleus. These results demonstrate that the expression of hsp68 can be used as a marker for the occurrence of ischemia. Furthermore, these findings support the fact that this in vitro system is a suitable model for the study on myocardial infarction.

Animals↗
PubMed1981

Thrombogenicity of components of atheromatous material. An animal and in vitro model of cerebral atheroembolism.

Human atheromatous material was separated into lipid and nonlipid fractions by ether and chloroform-methyl alcohol procedures. The maximum nonlethal doses of nonlipid, lipid, and whole (unseparated) atheromatous material were 8 mg, between 15 and 30 mg, and between 50 and 60 mg when injected into the left common carotid arteries of rabbits. In vitro production of thrombi showed that the nonlipid material produced thrombi that were larger in volume, weight, and length than those produced by whole material.

Animals↗
PubMed2006

Nebulized therapies for childhood pulmonary hypertension: an in vitro model.

OBJECTIVES: Sildenafil, tezosentan, and prostacyclin reduce pulmonary vascular pressures in pulmonary hypertension, but have potential to vasodilate the systemic circulation. Nebulized vasodilators allow targeted drug delivery, high local drug concentrations, less systemic hypotension, and better matching of the lung's ventilation and perfusion. We aimed to estimate pulmonary deposition of these drugs from commonly employed nebulizers using in vitro techniques and to create a mathematical model to predict inspired mass of aerosol. DESIGN: Lung deposition was estimated by characterization of drug output and particle size distribution (PSD) of nebulizers using helium-neon laser diffraction techniques. A mathematical model for each device was created to estimate pulmonary deposition using patients' breathing patterns and was verified with a mechanical-breathing model. RESULTS: Total output and PSD were similar for the Hudson Updraft II and Whisperjet nebulizers, consisting of half the nebulizer's charge, with (1/4) of particles < or = 5 microm, in the respirable fraction (RF). Drug output increased with inspiratory flow for the Pari LC Star. Differences were noted in device performance, depending on the drug tested. Estimated pulmonary deposition (mean, 95% CI) was 8.1 (7.2, 9.0)% of the initial drug charge for the Hudson Updraft II, 6.4 (5.8, 7.0)% for the Whisperjet, and 33.0 (28.3, 37.9)% for the Pari LC Star. A mechanical model was consistent with our mathematical model. CONCLUSIONS: All drugs could be nebulized, but expected pulmonary deposition varied depending on the nebulizer and drug.

Adolescent↗
PubMed2006

Action of Lovastatin (Mevinolin) on an in vitro model of angiogenesis and its co-culture with malignant melanoma cell lines.

BACKGROUND: Lovastatin and other statins may reduce the development of melanomas. The effects on melanoma cells and their ability to enhance angiogenesis in a co-culture system presented an opportunity to assess whether Lovastatin act on melanoma cells, HUVEC or both types of cells. RESULTS: Direct effects of co-culturing two different malignant melanoma cells (A375 and G361) on the process of angiogenesis in vitro was studied with our angiogenesis model, based on human dermal fibroblasts and human umbilical vein endothelial cells (HUVEC). Co-cultures were set up using "sland" and "dispersed seeding" techniques. A statistically significant increase in tubule formation in both cases was observed compared to controls. The effects of doses equivalent to therapeutic concentrations of Lovastatin were analysed. The drug inhibited the growth of all cell types, induced apoptosis, and markedly reduced the formation of tubules in the angiogenesis model at low concentrations. Its action was successfully reversed by the introduction of geranylgeranyl pyrophosphate. CONCLUSION: Lovastatin can reduce both tumour (melanoma) cell growth, and the angiogenic activity of these cells in co-cultures using an established 2-dimensional model angiogenesis system beyond that which would be seen by reduced proliferation alone.

Journal Article↗
PubMed1982

Enhancement of clearances by activated charcoal in an in vitro model of peritoneal dialysis.

The use of sorbents in dialysate to increase solute clearances in continuous ambulatory peritoneal dialysis (CAPD) was investigated. With in vitro simulations of CAPD, the kinetics of irreversible binding of creatinine to activated charcoal were assessed. Cellulose dialyzer fibers were submerged in two liters of dialysate for 3-8 hour exchanges. Perfusate was pumped single pass through the fibers. Commercial dialysates with 1.5% and 4.25% dextrose as an osmotic agent were controls. Experimental exchanges contained either large or small particles of activated charcoal. Unencapsulated and collodion encapsulated large particles were also studied. From the perfusate side, creatinine, clearance and mass transfer were determined; dialysate/perfusate ratios (D/P) of free creatinine concentrations were assessed. We found that incorporation of small unencapsulated particles of activated charcoal would double both clearance and mass transfer of creatinine. It also maintained D/P values less than 0.6 even up to 8 hours. Small particles absorbed more than 10 times more creatinine per gram than large particles. Significant differences between encapsulated and unencapsulated large particles were not found. In summary, activated charcoal might double creatinine removal per exchange in CAPD. Animal studies of collodion encapsulated small particles and other sorbent-enzyme systems in CAPD dialysate solutions seem warranted.

Absorption↗
PubMed2000

[Comparison of in vitro model examinaitons with respect to drug release from suppositories].

9 lipophilic suppository bases with different physical-chemical parameters were examined. Buspiron-hydrochloride, an anxiolytic drug with good water-solubility was used--partly as a model--as a pharmacon, in a concentration of 10.0 mg/2.00 g. The rate and extent of in vitro drug release was monitored with static and dynamic methods. Kidney-dialysing membranes with various surfaces were used. The quantitative measurements were carried out spectrophotometrically and the amount of the diffused drug was determined at lambda = 298 nm. The mean values were calculated from 5 parallel measurements each time. The percentage values of in vitro relative availability revealed that the results of the two static diffusion studies did not differ significantly (p < 0.05) and were almost independent of the size of the membrane surface. The results of the dynamic diffusion method were well-reproducible but were vehicle-dependent. The process of release was characterized by the mathematical transformation of the release curves, while the correlation coefficients described the closeness of the relation. Two German vehicles, namely Witepsol H 15 with a medium hydroxyl value and Massa Estarinum 299, and a French vehicle, Suppocire AS2X were found to be excellent for the formulation of suppositories containing Buspiron-hydrochloride.

Anti-Anxiety Agents↗