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 1,009 records · Page 56Linked to original sources
PubMed1969

Cell kinetic studies in mixed leukocyte cultures: an in vitro model of homograft reactivity.

The recognition phase of homograft immunity can be studied in vitro in the mixed leukocyte culture reaction. In this reaction, at the end of seven days, up to 30 per cent of the lymphocytes in culture "respond" to a single allogeneic cell stimulus. This paper presents evidence that the lymphocytes responding in culture divide with the generation time of 18-21 hours, with some asynchrony, and that a large percentage of the cells found at the end of the culture period may be the products by division of a small number of cells initially responding. Two estimates are made of the frequency of the initially responding unit (the cell(s) responsible for initiating the response). These are probably minimum estimates and are in the range of 1 in 200 to 1 in 2000 cells which can initially respond. This very high frequency of responding units as compared with the responding units in immediate type hypersensitivity (antibody production) is discussed.

Autoradiography↗
PubMed2003

Use of a three-dimensional in vitro model of the rat blood-brain barrier to assay nucleoside efflux from brain.

Extracellular adenosine is produced in brain during physiological and pathophysiological conditions. Once produced, this adenosine can undergo one or more of the following fates: it can interact with its receptors, it can be scavenged by astrocytes and/or neurons for ATP resynthesis, it can be transported across the blood-brain barrier and lost from the central nervous system, or it can be metabolized to inosine and hypoxanthine. The present study used a three-dimensional in vitro cell culture model of the rat blood-brain barrier, in which forebrain astrocytes and microvascular endothelial cells were cultured in cartridges containing multiple parallel polypropylene hollow fibers. Endothelial cells were cultured on the inner surfaces and astrocytes on the outer surfaces of these fibers. Growth medium was constantly perfused through the lumen of the fibers to mimic blood flow across endothelial cells in vivo. This co-culture system was used to examine the permeation of adenosine, and its metabolite inosine, from the astrocyte compartment to the endothelial cell compartment. Dipyridamole was added to the media perfusing the endothelial cell compartment to test whether it could decrease permeation of adenosine and inosine across the in vitro blood-brain barrier. Our results indicate that dipyridamole decreased permeation of total purines, especially inosine, across the barrier. Furthermore, permeation of fluorescein isothiocyanate-labeled albumin and radiolabeled sucrose, markers of the paracellular permeation pathway, were also decreased by dipyridamole. In conclusion, these data indicate that in addition to inhibiting nucleoside efflux across the barrier, dipyridamole can also improve blood-brain barrier function in this model.

Adenosine↗
PubMed1999

An in vitro model for mineralization of human osteoblast-like cells on implant materials.

An in vitro mineralizing cell-implant system was developed to study osteoblast attachment, secretion of extracellular (ECM) matrix proteins and mineralization. Saos-2 cells were plated on Tivanium (Tiv, Ti-6A1-4V), Zimaloy (Zim, Co-Cr-Mo) and glass disks. The cells were cultured in alpha-MEM medium with 10% fetal bovine serum and 50 microg ml(-1) ascorbic acid. The cultures were analyzed for calcification and for mRNA expression for ECM proteins after 1, 2, 4 and 6 weeks. Calcium content was significantly higher in cells on Tiv, less on Zim and least on glass disks. With the addition of 3 mm beta-glycerophosphate (beta-GP), the cell layer was more calcified on Zim than on Tiv and all substrates had three times more calcium than cultures without beta-GP. All subsequent experiments were performed without beta-GP. Phalloidin immunofluorescence microscopy of the actin-based cytoskeleton at 2 weeks demonstrated nodules composed of multilayered, cobblestone-appearing osteoblasts overlying calcified matrix which was stained with calcein. On Tiv, calcified nodules were connected in a trabecular-like pattern while on Zim, calcification was dispersed throughout the cell layer. Northern blots for alkaline phosphatase, bone sialoprotein, osteocalcin and alpha1(I) procollagen mRNAs were performed at different time points. The amount and pattern of calcification as well as the expression of ECM-mRNAs differed on each implant material. The results indicate that Tiv stimulates the production of more ECM proteins and mineralized matrix than Zim or glass in this osteoblast-like cell/implant culture.

Alloys↗
PubMed2006

Blood-brain barrier and cell-cell interactions: methods for establishing in vitro models of the blood-brain barrier and transport measurements.

This chapter describes in vitro methods for studying the blood-brain barrier. These methods include a cell line and isolated brain microvessels. The rat brain endothelial cell line 4 (RBE4) express many properties that are expressed by brain endothelial cells in vivo. Tissue culture methods allow the investigator to design experiments for studying transporters and permeability that would be much more difficult in vivo. A method for making preparations of isolated brain microvessels also is described. These preparations are highly enriched and also can be used for studying transport in vitro, but their short life span is a limitation. Two methods are discussed for measuring transport in cell culture. In one method, permeability is measured across a cell mono-layer. This method is useful for measuring luminal and abluminal transport. The second method is especially designed for measuring the families of efflux transporters. These in vitro methods will complement many of the in vivo techniques, and they may be used as screening for more timely and expensive experiments, and also reducing the need for experimental animals.

Animals↗
PubMed1999

Effects of inhibitors of the tyrosine kinase signaling cascade on an in vitro model of allergic airways.

It has been shown that activation of protein tyrosine kinases (PTKs) is the earliest detectable signaling response to FcepsilonRI cross-linking on mast cells. Following tyrosine kinase activation, a family of mitogen-activated protein kinases (MAPKs) was found to be activated as well. Activation of this PTK signaling cascade will lead to mast cell degranulation. This review summarizes our recent studies on the role of PTK signaling cascade in an in vitro guinea pig model of allergic asthma using PTK inhibitors, genistein and tyrphostin 47, and MAPK kinase inhibitor, PD098059. Inhibitors of the PTK and MAPK signaling pathways significantly attenuated the ovalbumin (OVA)-induced bronchial anaphylactic contraction and enhanced relaxation of constricted airways, respectively, and substantially blocked the release of histamine and peptidoleukotrienes from chopped lung preparations induced by OVA. Based upon their substantial inhibitory effects on the Schultz-Dale reaction, further examination on the potential anti-asthmatic effects of PTK cascade inhibitors in an in vivo model of allergic asthma is recommended.

Animals↗
PubMed2000

The use of liver spheroids as an in vitro model for studying induction of the stress response as a marker of chemical toxicity.

Stress protein induction has been advocated as a sensitive indicator of compound-induced toxicity. In monolayer cultures of primary hepatocytes, however, the two stress proteins, Hsp25 and Hsp72/3 are up-regulated, probably due to the effect of the isolation procedure and adaptation of the cells to the culture conditions. The aim of the current studies was to determine whether liver spheroids would provide an improved experimental model for the study of heat shock protein induction in vitro. Primary rat hepatocytes were cultured as liver spheroids and the expression of Hsp25 and Hsp72/3 measured along with the levels of ATP, GSH and albumin secretion. Hsp72/3 was initially increased in spheroid culture but returned to in vivo levels after 3 days of culture. Hsp25 was maintained at in vivo levels until day 6 of culture, after which levels increased slightly. The effects of the two hepatotoxins, hydrazine and cadmium chloride (CdCl(2)), were therefore measured on day 6 of spheroid culture. CdCl(2) had no effect on Hsp25 but increased Hsp72/3 at concentrations that affected other biochemical parameters. Hydrazine caused a rapid reduction in ATP levels and albumin secretion, but did not affect Hsp72/3. Hsp25 was slightly induced by hydrazine at later sampling times at concentrations, however, that affected other biochemical parameters. It can be concluded that liver spheroids provide a model for studying stress protein expression. However, the increase in stress proteins appears to be a relatively insensitive parameter compared to other more conventionally used toxicity endpoints and the response appears to vary with individual toxins under study.

Adenosine Triphosphate↗
PubMed2001

Plesiomonas shigelloides enters polarized human intestinal Caco-2 cells in an in vitro model system.

This study provides the first definitive evidence that the gram-negative bacterium Plesiomonas shigelloides adheres to and enters eukaryotic intestinal host cells in vitro. P. shigelloides is increasingly regarded as an emerging enteric pathogen and has been implicated in intestinal and extraintestinal infections in humans. However, the establishment of its true role in enteric disease has been hindered by inadequacies in experimental design, deficiencies in clinical diagnosis, and the lack of an appropriate animal model. In this investigation, an in vitro system was used to evaluate plesiomonad pathogenesis. Differentiated epithelium-derived Caco-2 cell monolayers inoculated apically with 12 isolates of P. shigelloides from clinical (intestinal) origins were examined at high resolution using transmission electron microscopy. Bacterial cells were observed adhering to intact microvilli and to the plasma membrane on both the apical and the basal surfaces of the monolayer. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. This observation suggests that initial uptake may occur through a phagocytic-like process, as has been documented for many other enteropathogens. P. shigelloides also was noted free in the cytosol of Caco-2 cells, suggesting escape from cytoplasmic vacuoles. Differences in invasion phenotypes were revealed, suggesting the possibility that, like Escherichia coli, P. shigelloides comprises different pathogenic phenotypes.

Bacterial Adhesion↗
PubMed2003

Animal and in vitro models of alcoholic pancreatitis: role of cholecystokinin.

Although ethanol abuse is the major etiologic factor in the development of acute and chronic pancreatitis, the mechanisms of ethanol effects to cause pancreatitis are poorly understood. The major reason for the lack of progress is the relative lack of animal models that reproduce the deleterious effects of ethanol on the pancreas that are observed in human disease. We propose that the effect of ethanol on the pancreas is due to its ability to sensitize animals and humans to the potentially injurious effects of other stimuli. We have developed models of ethanol-induced acute and chronic pancreatitis in rats as well as pancreatic acinar cells in primary culture demonstrating that ethanol sensitizes the pancreas to the inflammatory, cell death, and fibrosing responses caused by cholecystokinin (CCK). Our results indicate that the ethanol-sensitized inflammatory response is the key or trigger event for the development of the other pathologic responses in both acute and chronic pancreatitis, such as cell death, intracellular digestive enzyme activation, and fibrosis. These findings suggest that experimental strategies designed to reveal the modulating effects of ethanol on the mechanisms underlying the inflammatory, cell death, and fibrosing responses stimulated by CCK will provide the key information needed to understand how ethanol abuse causes pancreatitis.

Animals↗
PubMed2005

An in vitro model of hippocampal sharp waves: regional initiation and intracellular correlates.

During slow wave sleep and consummatory behaviors, electroencephalographic recordings from the rodent hippocampus reveal large amplitude potentials called sharp waves. The sharp waves originate from the CA3 circuitry and their generation is correlated with coherent discharges of CA3 pyramidal neurons and dependent on activities mediated by AMPA glutamate receptors. To model sharp waves in a relatively large hippocampal circuitry in vitro, we developed thick (1 mm) mouse hippocampal slices by separating the dentate gyrus from the CA2/CA1 areas while keeping the functional dentate gyrus-CA3-CA1 connections. We found that large amplitude (0.3-3 mV) sharp wave-like field potentials occurred spontaneously in the thick slices without extra ionic or pharmacological manipulation and they resemble closely electroencephalographic sharp waves with respect to waveform, regional initiation, pharmacological manipulations, and intracellular correlates. Through measuring tissue O2, K+, and synaptic and single cell activities, we verified that the sharp wave-like potentials are not a consequence of anoxia, nonspecific elevation of extracellular K+ and dissection-related tissue damage. Our data suggest that a subtle but crucial increase in the CA3 glutamatergic activity effectively recruits a population of neurons thus responsible for the generation of the sharp wave-like spontaneous field potentials in isolated hippocampal circuitry.

Analysis of Variance↗
PubMed1999

Cardioplegia flow dynamics in an in vitro model.

The flow of fluids in extracorporeal circuits does not conform to conventional Poiseuille mechanics which confounds calculating cardioplegia (CP) flow distribution. The purpose of this study was to quantify CP flow dynamics in a model simulating coronary atherosclerosis across varying sized restrictions. An in vitro preparation was designed to assess hydraulic fluid movement across paired restrictions of 51, 81 and 98% lumen reductions. Volume data were obtained at variable flow, temperature, viscosity and pressure conditions. CP delivered through 14- and 18-gauge (GA) conduits at 8 degrees C and 100 mmHg infusion pressure revealed that both four to one and crystalloid CP solutions had significantly less total percentage flow through the 14-GA conduit, p < 0.0001 and p < 0.001, respectively. Overall, 4:1 CP exhibited the most favorable fluid dynamics at 8 degrees C in that it delivered the highest percentages of total CP flow through the smaller lumen conduit. At both 8 degrees C and 37 degrees C delivery, blood CP resulted in the least homogeneous fluid distribution at all delivery parameters. The results in relation to blood viscosity indicate that, although the 8 degrees C blood CP had a significantly greater viscosity than 37 degrees C blood CP, it did not produce an effect in fluid distribution. These data show that increasing the cardioplegic solution hematocrit causes an inhomogeneous fluid distribution regardless of delivery temperature or infusion pressure.

Coronary Artery Disease↗
PubMed1990

Transepithelial water permeability in an in vitro model of renal cysts.

Renal cysts develop from microscopic tubules and may enlarge progressively several thousandfold. Sustained epithelial proliferation, intracavitary fluid accumulation, and extracellular matrix remodeling are central elements in a multistep process that leads to the formation and enlargement of cysts. MDCK cells suspended within medium-hydrated collagen gels grow to form spherical, monolayered, fluid-filled cysts that enlarge steadily. Vasopressin and other agents that increase intracellular levels of cAMP stimulate the rate of MDCK cyst growth and net fluid/solute secretion when added to defined medium in vitro. In this model, net fluid secretion is the only means by which fluid can accumulate within the cyst cavity. We used this cyst-forming line of epithelial cells to evaluate several membrane transport properties that are important in the coupled movements of solute and water in the process of secretory fluid transport. Individual cysts were microdissected from collagen gels, held by a micropipet in a thermostated chamber, and examined at a high magnification by video microscopy. Transepithelial water flow was initiated by rapidly exchanging the bath medium with hyperosmotic solutions. Net water flux, Jv, determined from the initial rate of decrease in cyst diameter, was proportionate to the transmembrane osmotic gradient of NaCl or raffinose; the reflection coefficient for NaCl was indistinguishable from 1.0. Osmotic water permeability (cm3/cm2/osm/min x 10(-6)) was 739 +/- 99 (N = 11) in medium augmented by an NaCl concentration of 100 mosmol/kg. Hydraulic conductivity (Pt), estimated in control cysts, was 6.8 +/- 0.9 microns/s, a value similar to that of medullary and cortical thick ascending limbs of Henle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Glycogen synthase kinetics in isolated human adipocytes: an in vitro model for the effects of insulin on glycogen synthase.

Glycogen synthase which catalyzes the incorporation of uridine dipophosphate glucose into glycogen is found in muscle, liver, and fat. The activity of this enzyme is increased by insulin through a dephosphorylation mechanism. Because of the critical role of glycogen synthase in glucose storage and overall glucose metabolism, it is important to assess the status of the activity of this enzyme in normal humans as well as in individuals with pathological conditions, such as non-insulin-dependent diabetes mellitus. However, in human subjects, studies of the regulation of glycogen synthase in vivo are time consuming and tedious. The present study was, therefore, undertaken to establish whether adipocytes isolated from subcutaneous adipose tissue biopsies from normal human subjects could be used to assess the effect of insulin in vitro on glycogen synthase activity. Regulation of glycogen synthase in human adipocytes by glucose 6-phosphate and uridine disphosphate glucose was found to be somewhat different than that reported for the regulation of this enzyme in tissues from other species. The adipocyte was found to be a sensitive model for insulin activation of this enzyme. Glycogen synthase was stimulated twofold by an insulin concentration of as low as 1 ng/ml, while half-maximal activation of enzyme activity occurred at 0.4 +/- 0.1 ng insulin/ml. The present studies indicate that the isolated human subcutaneous adipocyte may serve as a useful model for in vitro investigation of the effects of insulin on glycogen synthase.

Adipose Tissue↗
PubMed1992

In vitro model of angiogenesis using a human endothelium-derived permanent cell line: contributions of induced gene expression, G-proteins, and integrins.

The EA hy926 cell line is a continuous, clonable, human cell line that displays a number of features characteristic of vascular endothelial cells (Edgell et al., 1983). Here we report that when EA hy926 cells (EA cells) are plated on an extracellular matrix material [Matrigel], they undergo a process of morphological re-organization leading to the formation of a complex network of cord or tubelike structures. These events seem to resemble, in some respects, an in vitro process of angiogenesis. The morphological re-arrangement occurs within a 12-16 hr period and seems to require expression of new messenger RNA and protein, since it is completely blocked when actinomycin D or cycloheximide are present at the time the cells are plated on Matrigel. This is not due to overt toxicity of the drugs, since exposure of cells to actinomycin D at 2 hr or more after plating on Matrigel has little effect on the formation of the tubelike structures. The process of Matrigel-induced tube formation also apparently involves a G-protein mediated signal. Treatment of the EA cells with pertussis toxin completely blocks the process and causes the ADP-ribosylation of a 42 kD protein that is recognized by an antibody to Gi-alpha subunits. In contrast, concentrations of pertussis toxin sufficient to block tube formation have only modest effects on the adhesion or motility of EA cells on purified matrix components such as laminin or collagen IV. The process of Matrigel-induced tube formation also involves integrins since monoclonal antibodies to integrin alpha 6 or beta 1 subunits can completely block the process. The concentrations of anti-integrin antibodies needed to block tube formation are much lower than those required to block cell adhesion on purified matrix components and are sufficient to occupy less than 10% of the alpha 6 or beta 1 subunits available at the cell surface. These results suggest that integrins may be involved in this potential model of angiogenesis in processes beyond their usual role in cell adhesion. Based on these results, it seems likely that the EA hy 926 cell line will prove to be a useful model for in vitro study of angiogenic processes.

Antibodies↗
PubMed1992

A murine in vitro model of allergic contact dermatitis to sesquiterpene alpha-methylene-gamma-butyrolactones.

The use of a lymphocyte transformation test (LTT) to provide evidence of allergic contact dermatitis was investigated. The haptens studied were alantolactone and isoalantolactone, two moderate allergens from Inula helenium L., a decorative and medicinal plant. Only alantolactone showed a significant response in vivo and in vitro in mice sensitized epicutaneously, without using Freund's complete adjuvant. Isoalantolactone did not show any sensitizing capacity in the murine model studied. The comparison of in vitro lymphocyte proliferation and in vivo allergenic capacity showed a good correlation and clearly demonstrates that, of the two sesquiterpene lactones, alantolactone is the better sensitizer.

Animals↗
PubMed1987

Whole mounted pressurized in vitro model for the study of cerebral arterial mechanics.

A whole mounted model for the study of smooth muscle mechanics is described which uses cylindrical sections of middle cerebral artery (MCA). One- to 2-cm sections of artery were cannulated, pressurized and perfused. Changes in the outer diameter of the artery were detected using an infrared photoelectric diameter gauge. MCA displayed spontaneous rhythmic contractions, which were initiated and sensitive to changing intraluminal pressure. Rhythmic activity was dependent upon the presence of Ca2+ in the bath, but not affected by phentolamine. Increasing pressure significantly reduced high (50, 70 and 125 mM) potassium-induced contractions, but not drug (serotonin, norepinephrine, histamine) induced contractions. The intraluminal surface of the MCA was significantly more sensitive to serotonin than the extraluminal surface. The above contractile responses are discussed in light of the advantages of this model over helical or ring preparations.

Animals↗