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PubMed2004

An in vitro model for investigating impedance changes with cell growth and electrical stimulation: implications for cochlear implants.

The impedance of stimulating electrodes used in cochlear implants and other neural prostheses often increases post-implantation, and is thought to be due to fibrous tissue encapsulation of the electrode array. Increased impedance results in higher power requirements to stimulate target neurons at set charge densities. We developed an in vitro model to investigate the electrode-tissue interface in a highly controlled environment. This model was tested using three cell types, with and without charge-balanced biphasic electrical stimulation. Under standard tissue culture conditions, a monolayer of cells was grown over the electrode surface. Electrode impedance increased in proportion to the extent of cell coverage of the electrode. Cell type was a significant factor in the amount of impedance increase, with kidney epithelial cells (MDCK) creating the greatest impedance, followed by dissociated rat skin fibroblasts and then macrophages (J774). The application of electrical stimulation to cell-covered electrodes caused impedance fluctuations similar to that seen in vivo, with a lowering of impedance immediately following stimulation, and a recovery to pre-stimulation levels during inactive periods. Examination of these electrodes suggests that the stimulation-induced impedance changes were due to the amount of cell cover over the electrodes. This in vitro technique accurately models the changes in impedance observed with neural prostheses in vivo, and shows the close relationship between impedance and tissue coverage adjacent to the electrode surface. We believe that this in vitro approach holds great promise to further our knowledge of the mechanisms contributing to electrode impedance.

Animals↗
PubMed1999

In vitro model for evaluating drug transport across the blood-brain barrier.

The passage of substances across the blood-brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood-brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood-brain barrier in vitro.

Journal Article↗
PubMed2003

An in vitro model of caloric restriction.

The mechanisms underlying the ability of caloric restriction (CR) to extend life span and enhance stress responsiveness remain elusive. Progress in this area has been slow due to the complexities of using animals for CR studies and assessing life span as the measure of CR effectiveness. It is therefore of great interest to develop in vitro models of CR. Here we use sera obtained from either Fisher 344 rats or Rhesus monkeys that were fed ad libitum (AL) or CR diets to culture various cell types. We show that treatment of cultured cells with CR sera caused reduced cell proliferation, enhanced tolerance to oxidants and heat, and heightened expression of stress-response genes. These phenotypic features mirror the effects of CR in animals. Supplementation of CR serum with insulin and insulin-like growth factor (IGF)-1 partially restored the proliferative and stress-response phenotype that was seen in cells cultured with AL serum, indicating that reduced levels of insulin and IGF-1 likely contribute to the CR-related effects. This in vitro cell culture model recapitulates key in vivo proliferative and stress-response phenotypic features of CR, and further suggests that endocrine mechanisms contribute to the enhanced stress responsiveness observed in CR animals.

Animals↗
PubMed1997

In vivo determination of surface tension in the horse trachea and in vitro model studies.

We measured the surface tension in the trachea of the non-anaesthetised horse from the spreading behaviour of fluid drops, using videotracheoscopy. To do this, we placed small oil drops onto the tracheal wall with a thin Teflon tubing inserted into a videocolonoscope used in humans. Either 5 ml of saline (control) or 5 ml of bovine lipid extract surfactant (BLES) at 4 mg/ml were administered. Tracheal surface tension was 31.9 +/- 0.54 mN/m (Mean +/- SEM, n = 30) in the control experiments and 24.5 +/- 0.51 mN/m (Mean +/- SEM, n = 21) in the entire trachea after the administration of BLES. These values were determined from calibration curves relating film surface tension to the relative diameter of test fluid droplets. In the calibration experiments, the test fluid droplets were placed onto a surfactant film at various surface tensions in either a modified Langmuir-Wilhelmy balance or a captive bubble surfactometer. The spreading behaviour of a given test fluid droplet in the model studies did not only depend on the film surface tension but also on the thickness of the aqueous layer below the surfactant film. Hence, the computed surface tensions in the trachea depend on the choice of which in vitro model is applied.

Animals↗
PubMedDate not supplied

Activity of the trometamol salt of fosfomycin in an in vitro model of the treatment of bacterial cystitis.

The response to trometamol fosfomycin of four strains of Escherichia coli was studied in an in vitro model in which the hydrokinetic aspects of the treatment of bacterial cystitis can be stimulated. Two strains of E. coli that were fully susceptible to fosfomycin, and a strain of intermediate susceptibility responded well to relatively low concentrations of the trometamol salt: doses achieving peak concentrations of 50 or 250 mg/l suppressed bacterial growth for at least 18 h; however, the emergence of resistance was completely suppressed only when a peak concentration of 2500 mg/l was achieved in the bladder model. A strain of E. coli that was fully resistant to fosfomycin in conventional minimum inhibitory concentration titrations responded to the highest dosage used, but this did not prevent further resistance from emerging. These results were obtained in the absence of the potentiating agent, glucose-6-phosphate, which is commonly used in susceptibility tests of fosfomycin. The implications of the results for fosfomycin dosage in bacterial cystitis and for the interpretation of susceptibility tests is discussed.

Administration, Oral↗
PubMed2006

In vitro model for decontamination of human skin: formaldehyde.

Decontamination of a chemical from skin is often an emergency measure. This study utilized an in vitro model to compare the decontamination capacity of three model decontaminant solutions (tap water, isotonic saline, and hypertonic saline). Human cadaver skin was dosed (approximately 0.25 microg on 3 cm(2) per skin) with radio-labeled [(14)C]-formaldehyde. After a defined exposure time (1, 3, and 30 min post-dosing, respectively), the surface skin was washed three times (4ml per time) with each solution. After washing, the skin was stripped with tape discs twice. Lastly, the wash solutions, strippings, receptor fluid, and remainder of skin were liquid scintillation analyzer counted to determine the amounts of formaldehyde. Additionally, an evaporation test at different exposure times (1min, 3min, 15min, 30min, and 60min, respectively) was conducted to monitor formaldehyde % evaporation. There were no statistical differences among these groups except isotonic saline, at 3min post-exposure (in wash solutions), showed a significantly difference (p<0.05) when compared to tap water. Formaldehyde % evaporation increased linearly with extending application times, and were 7.7%, 13.6%, 19.7%, 24.4%, and 35.9% (1min, 3min, 15min, 30min, and 60min, respectively). This data suggests that isotonic saline may be effective in removing formaldehyde from skin. However, results from this model need validation in vivo. The model may provide a facile and robust method of accelerating knowledge of decontamination mechanism and lead to enhanced efficacy.

Decontamination↗
PubMed1997

Qualitative and quantitative measurements of oligonucleotides in gene therapy: Part I. In vitro models.

Part I of this review attempts to bring together all the methods of detection and determination of synthetic oligonucleotides used in in vitro, described in the literature over the past 14 years, in an effort by scientists to use these oligonucleotides as drugs in gene therapy. The in vitro models include cell-free and cell culture systems. Emphasis has been given to the techniques developed for quantification of the input oligonucleotides or their metabolites. The purpose of study, methods of processing, detection and determination techniques such as those based on fluorescence, radiolabeling, high-performance liquid chromatography, gel-electrophoresis and others have been presented.

Cell-Free System↗
PubMed2002

[The influence of viscosity on adhesion and invasion of Salmonella strains in an in vitro model].

The influence of viscosity on the adhesion and invasion behaviour of Salmonella strains of different origin was investigated using an in-vitro-model. These processes seem to be strain-dependent. Compared to the controls, the number of internalized Salmonella was elevated. This increase was a result of the greater number of Salmonella which adhered to Caco-2-cells and was detected only for strains from organs of calves having died from salmonellosis. The average motility of these strains was determined to be 1.6 +/- 0.5 mm/h. A possible association between adhesion ability and motility was discussed.

Animals↗
PubMed2000

Mono Mac 1: a new in vitro model system to study HIV-1 infection in human cells of the mononuclear phagocyte series.

Throughout the years, most researchers have used continuous cell lines as in vitro models to evaluate the immunopathogenesis of human immunodeficiency virus type-1 (HIV-1) infection. Unfortunately, the most commonly used monocytoid malignant cells have not been shown to adequately mimic primary human monocyte-derived macrophages, at least with respect to HIV-1 infection. The Mono Mac 1 cell line has been defined as a model system for studying biochemical, immunological, and genetic functions of human cells of the monocyte/macrophage lineage. In this study, we have investigated whether Mono Mac 1 represents an in vitro culture system for HIV-1 infection. Flow cytometric analyses revealed that Mono Mac 1 are positive for the HIV-1 primary receptor (CD4), as well as for the coreceptors (CXCR4, CCR5, and CCR3). Infectivity experiments conducted with recombinant luciferase-encoding and fully infectious viruses demonstrated that Mono Mac 1 can support a highly productive infection with both macrophage- and dual-tropic isolates of HIV-1. Furthermore, differentiation of such cells led to a marked increase in virus production. Data from semiquantitative polymerase chain reaction analysis and mobility shift assays indicated that enhanced virus production in differentiated Mono Mac 1 cells was most likely related to an increase in nuclear translocation of NF-kappaB. Mono Mac 1 can thus be considered as a human monocytoid cell line representing a proper in vitro system for studying the complex interactions between HIV-1 and cells of the monocyte/macrophage lineage.

CD4 Antigens↗
PubMed2005

Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin prevents synovial sarcoma proliferation via apoptosis in in vitro models.

Synovial sarcoma is a soft tissue malignancy with a poor prognosis; many patients will die from this disease within 10 years of diagnosis, despite treatment. Gene expression profiling and immunohistochemistry studies have identified oncogenes that are highly expressed in synovial sarcoma. Included in this group are receptor tyrosine kinases such as epidermal growth factor receptor, insulin-like growth factor receptor 1, fibroblast growth factor receptor 3, KIT, and HER2. Inhibitors of these growth-promoting receptors are likely to inhibit proliferation of synovial sarcoma; however, the effect of receptor tyrosine kinase inhibitors on synovial sarcoma is largely unknown. We assessed the ability of the following receptor tyrosine kinase inhibitors to halt proliferation and induce apoptosis in synovial sarcoma monolayer and three dimensional spheroid in vitro models: gefitinib (Iressa), NVP-AEW541, imatinib mesylate (Gleevec), SU5402, PRO-001, trastuzumab (Herceptin), and 17-allylamino-17-demethoxygeldanamycin (17-AAG). Gefitinib, NVP-AEW541, and imatinib inhibited proliferation only at relatively high concentrations, which are not clinically applicable. 17-AAG, which destabilizes multiple receptor tyrosine kinases and other oncoproteins through heat shock protein 90 inhibition, prevented proliferation and induced apoptosis in synovial sarcoma monolayer models at concentrations achievable in human serum. 17-AAG treatment was also associated with receptor tyrosine kinase degradation and induction of apoptosis in synovial sarcoma spheroid models. 17-AAG was more effective than doxorubicin, particularly in the spheroid models. Here we provide in vitro evidence that 17-AAG, a clinically applicable drug with known pharmacology and limited toxicity, inhibits synovial sarcoma proliferation by inducing apoptosis, and thus has potential as a systemic therapy for this disease.

Antibodies, Monoclonal↗
PubMed1993

Embryo-dependent induction of uterine receptivity assessed by an in vitro model of implantation in mice.

To investigate the molecular basis of initial interactions between the trophoblast and the uterine epithelium at implantation, we developed an in vitro model for implantation in mice using primary uterine organ cultures as the substrate. In this model, embryos attached to the endometrial explants within 60 min. We were able to observe the attachment between embryos and endometrial explants in real time. Using this model, we investigated the attachment of embryos to uterine strips cultured from pregnant mice, pseudopregnant mice, pseudopregnant mice that had received hCG injections, and pregnant mice whose utero-tubal junctions had been ligated on Day 2 of pregnancy. The serum levels of progesterone in pregnant and pseudopregnant mice were also compared. The percentages of embryos that attached to uterine strips cultured from pregnant mice on Days 1-4 were low (0.0-15.0%). However, by Days 5-7, percentages of attachment increased markedly (66.8-74.8%). These levels were significantly higher than those observed for pseudopregnant mice (0.0-18.0%) and pseudopregnant mice that had received hCG injections (0.0-15.0%). The percentages of attachment to the ligated horns of pregnant Day 5 uteri (58.3-61.5%) were significantly higher than that of pseudopregnant mice (10.0%). The percentages of attachment to the ligated horns of pregnant Day 6 and 7 uteri decreased significantly (3.2%-19.4%). On the other hand, the percentages of attachment to the unligated horns of pregnant Day 6 and 7 mouse uteri did not decrease significantly (61.1-70.0%). The serum progesterone levels of pseudopregnant mice were not significantly lower than those of pregnant mice on Days 1-5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Antibiotic supplementation of intraocular irrigating solutions. An in vitro model of antibacterial action.

PURPOSE: The addition of antibiotics to infusion solutions for cataract surgery is becoming increasingly popular. The authors developed an in vitro model to evaluate antibacterial effects of this use of antibiotics. METHODS: Clinical isolates and/or reference strains of the following organisms were examined: coagulase-negative Staphylococcus spp, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus spp, Enterococcus spp, Proprionibacterium acnes, Moraxella nonliquifaciens, and Pseudomonas aeruginosa. Standardized suspensions of each organism were incubated with a control solution (Balanced Salt Solution) or Balanced Salt Solution containing the following antibiotics: vancomycin (20 micrograms/ml) or gentamicin (8 micrograms/ml) or gentamicin and vancomycin combined (8 and 20 micrograms/ml, respectively). Suspensions were incubated for 30, 60, and 120 minutes at room temperature. Samples were centrifuged, and the organisms were washed with Balanced Salt Solution before quantitative culturing. Each organism also was incubated for 48 hours in Mueller-Hinton broth with the same antibiotic concentrations. RESULTS: Most of the organisms were not affected by exposure to the antibiotics for up to 140 minutes. P. aeruginosa and M. nonliquifaciens were exceptions, decreasing in colony numbers even with 30 minutes of exposure. Several Staphylococcus spp yielded variable results. All organisms demonstrated nearly complete inhibition of growth when exposed for an extended time to the appropriate antibiotic in broth. CONCLUSIONS: Exposure to antibiotics for a short period of time, such as during intraocular surgery, generally has no effect on organisms commonly responsible for endophthalmitis. The use of antibiotics in this manner should be critically reassessed until further study.

Anti-Bacterial Agents↗
PubMed1995

Zinc supplementation enhances the effectiveness of St. Thomas' Hospital No. 2 cardioplegic solution in an in vitro model of hypothermic cardiac arrest.

The present study was done to assess the effectiveness of a zinc-supplemented cardioplegic solution in an in vitro model of hypothermic arrest. Isolated hearts were perfused in the nonworking mode. All hearts were subjected to 2 hours of hypothermic arrest, at 10 degrees C, followed by 60 minutes of recovery. In protocol 1, arrest was initiated with infusion of cardioplegic solution with or without 30 mumol/l zinc for 5 minutes, which was then reinfused for 5 minutes every 15 minutes during arrest. In protocol 2, arrest was initiated with infusion of cardioplegic solution with or without 40 mumol/L zinc for 10 minutes. Cardioplegic solution (without zinc) was then reinfused for 5 minutes before the hearts were rewarmed. In protocol 1 hearts, peak postischemic left ventricular developed systolic pressure was 106 +/- 5 mm Hg and 80 +/- 3 mm Hg in zinc-treated versus control hearts, respectively (p < 0.05 by repeated-measures analysis of variance). In protocol 2 hearts, recovery of postischemic left ventricular developed systolic pressure peaked at 74 +/- 4 mm Hg and 46 +/- 8 mm Hg in zinc-treated and control hearts, respectively (p 0.05, repeated-measures analysis of variance). Similar effects were observed for the left ventricular rate of relaxation (p < 0.05, repeated-measures analysis of variance). Except for some minor effects, lactate dehydrogenase release was not affected by zinc supplementation. The present study demonstrates that zinc supplementation further enhances the normally observed preservation of postarrest cardiac function and suggests possible clinical utility for this metal as an additive to standard crystalloid cardioplegic solutions.

Animals↗
PubMed2002

Paracrine activation of extracellular signal-regulated kinase in a simple in vitro model of wounded osteoblasts.

The immediate signal-transduction response of osteoblasts to acute trauma is poorly characterized. We have developed a simple in vitro model for osteoblast trauma to investigate aspects of the molecular mechanisms of wound healing in bone. Herein we report the specific, rapid, and transient phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2 in osteoblasts as a response to disruption ("wounding") of a confluent monolayer. The mitogen-activated protein kinase (MAPK) cascades of p38 and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) were not activated by this perturbation. The response to wounding was equivalent to the activation of ERK by the addition of exogenous growth factors, and the perturbation-dependent phosphorylation of ERK can be suppressed by an inhibitor of heparin-binding growth factors. Conditioned media from wounded monolayers can induce the phosphorylation of ERK in unperturbed monolayers. Using immunohistochemistry, it was demonstrated that the cells with increased levels of phosphorylated ERK were not localized to the wound edges. These results indicate that ERK activation is the result of an autocrine/paracrine response by osteoblasts to trauma. We speculate that osteoblasts respond to trauma with the release of soluble factors as part of an autocrine/paracrine modulation of the wound-healing process in bone.

3T3 Cells↗
PubMed2006

Porcine brain microvessel endothelial cells as an in vitro model to predict in vivo blood-brain barrier permeability.

The objective of the study was to establish primary cultured porcine brain microvessel endothelial cells (PBMECs) as an in vitro model to predict the blood-brain barrier (BBB) permeability in vivo. The intercellular tight junction formation of PBMECs was examined by electron microscopy and measured by transendothelial electrical resistance (TEER). The mRNA expression of several BBB transporters in PBMECs was determined by reverse transcriptionpolymerase chain reaction analysis. The in vitro permeability of 16 structurally diverse compounds, representing a range of passive diffusion and transporter-mediated mechanisms of brain penetration, was determined in PBMECs. Except for the perfusion flow rate marker diazepam, the BBB permeability of these compounds was determined either in our laboratory or as reported in literature using in situ brain perfusion technique in rats. Results in the present study showed that PBMECs had a high endothelium homogeneity, an mRNA expression of several BBB transporters, and high TEER values. Culturing with rat astrocyte-conditioned medium increased the TEER of PBMECs, but had no effect on the permeability of sucrose, a paracellular diffusion marker. The PBMEC permeability of lipophilic compounds measured under stirred conditions was greatly increased compared with that measured under unstirred conditions. The PBMEC permeability of the 15 test compounds, determined under the optimized study conditions, correlated with the in situ BBB permeability with an r2 of 0.60. Removal of the three system L substrates increased the r2 to 0.89. In conclusion, the present PBMEC model may be used to predict or rank the in vivo BBB permeability of new chemical entities in a drug discovery setting.

Animals↗
PubMed1997

Formation of new capillary-like tubes in a three-dimensional in vitro model (aorta/collagen gel).

Direct sprouting (angiogenesis) does not occur during the formation of capillary-like tubes in an aorta/ collagen gel in the in vitro model. However, emigration of cells which stretch, arrange themselves side by side, form contacts (unspecific, tight and gap junctions), develop a lumen and show differentiation of endothelial cells (including the formation of a lamina densa and the appearance of pericytes) have been observed, i.e. vasculogenesis occurs. The origin of long, stretched cells is not known with certainty. They possibly represent smooth muscle cells. In addition, other cell types have been found, such as fibrocyte-like and fibroblast-like cells, elastoblasts, fat cells, monocytes and macrophages. All these cells are able to produce factors that promote the formation of new capillaries. Hence, a knowledge of these cells appears to be important for the analysis of in vitro systems. Moreover, the occurrence of these cell types must be considered when assessing possible effects.

Animals↗
PubMed2001

A new in vitro model for the study of microbial microleakage around dental restorations: a preliminary qualitative evaluation.

AIM: The aim of this study was to develop an in vitro model to replicate microbial microleakage at a tooth/ restoration interface using a constant depth film fermentor (CDFF). METHODOLOGY: Amalgam restorations were placed in machined bovine dentine cylinders and sealed externally with varnish, leaving a 1-mm perimeter exposed around the tooth/restoration interface. The dentine cylinders were housed in a CDFF and 300-microm thick microcosm dental plaques were grown over their exposed surfaces. The biofilms were maintained with a mucin-containing artificial saliva for up to 8 weeks. Cylinders were aseptically removed from the CDFF (at 1, 2, 4, & 8 weeks) and surface-decontaminated with validated protocols prior to splitting and sampling of apposing amalgam and dentine surfaces. Scanning electron microscopy (SEM) was used to ascertain the position and structure of the bacterial aggregates. Bacterial viability was determined by vital staining of the bacteria in situ. RESULTS: At all sampling times, SEM showed cocci, rods and filaments on both amalgam and dentine surfaces; some originated as cascades from the surface biofilm and extended into the tooth/restoration microspace. Vital staining showed the majority of bacteria from both dentine and amalgam surfaces to be viable. CONCLUSION: This preliminary investigation showed that the CDFF may be a valuable tool for the in vitro study of the dynamics of microbial microleakage around dental restorations.

Animals↗
PubMed1984

An in vitro model to study lipolysis of rat hepatic very low density lipoprotein using cardiac lipoprotein lipase.

The purpose of this work was to design an in vitro model for studying the lipolysis of very low density lipoprotein (VLDL) under conditions that approximate those likely to exist in the rat circulation. We first measured the total lipase activity available in the circulation of normal fasted rats. Knowing the VLDL-triacylglycerol concentration and the circulating time of VLDL in rat serum, we calculated that incubating 80 mU of lipoprotein lipase activity per milligram of VLDL triacylglycerols at 37 degrees C, for 50 min, approximated the in vivo conditions of lipolysis in normal fasted rats. Under these conditions, different concentrations of albumin were tested. The degree of lipolysis gradually increased from 25 +/- 10% without albumin to 75 +/- 5% (mean +/- SD, n = 3) with 4% albumin. No further increase occurred above 4% albumin. The rate of triacylglycerol hydrolysis was faster with VLDL having a high triacylglycerol/protein ratio. However after 50-60 min of incubation, the extent of hydrolysis tended to become similar for all VLDL (75-90%). This suggests that the rate of VLDL hydrolysis differs from one particle to another (depending on the size), but the extent of hydrolysis ends up being approximately the same. Furthermore, addition of high density lipoprotein to the incubation medium did not affect the rate nor the extent of triacylglycerol hydrolysis. We conclude that a large proportion of VLDL triacylglycerols can be hydrolyzed in the normal fasted rat if there is no limitation in the capacity of fatty acid removal from the lipolysis site. The physiological significance of these results obtained in vitro is discussed.

Animals↗