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PubMed2005

Cultured murine dermal fibroblast-like cells from senescence-accelerated mice as in vitro models for higher oxidative stress due to mitochondrial alterations.

The senescence-accelerated mouse is a model for senescence acceleration, a higher oxidative stress status, and age-associated disorders. We studied whether fibroblasts cultured from accelerated senescence-prone SAMP11 mice could be used as in vitro models for oxidative stress in senescence. Dichlorofluorescein and hydroethidine assays demonstrated that cells from SAMP11 mice produced more reactive oxygen species than did cells from accelerated senescence-resistant SAMR1 mice. These differences were not due to the defective induction of antioxidants. Double labeling with hydroethidine and MitoTracker Green revealed that most of the reactive oxygen species were generated within the mitochondria. Nonyl acridine orange and JC-1 assays showed an increase in the mass of the mitochondria, especially those with low membrane potential, in SAMP11 cells. Ultrastructurally, mitochondria with degenerative morphology were increased in SAMP11 cells with longer culture periods. These results suggest that cells from SAMP11 mice are useful models for spontaneous higher oxidative stress in vitro due to dysfunctional mitochondria.

Animals↗

PubMed1987

Corneal epithelial wound closure in tissue culture: an in vitro model of ocular irritancy.

The influence of 13 test agents on the ability of cultured rabbit corneal epithelial cells to migrate and re-cover a wound has been utilized to evaluate an in vitro model of ocular irritancy. Cells were grown under standard conditions and monitored for adequate cell density. Seven days after subculture, replicate wounds were produced and cultures were exposed to varying concentrations of a test agent in culture medium for 24 hr. Following exposure, cultures were fixed and stained to reveal remaining wound areas which were quantitated by computerized planimetry and compared to evaluate the deleterious effects of the test agents. The test ranks irritants in an order similar to that described in the literature for both in vivo and in vitro tests. This tissue culture model is conceptually simple, quantitative, and an alternative to the corneal component of whole animal testing for ocular irritancy.

Animals↗

PubMed1999

In vitro models of biological responses to implant microbiological models.

To study the etiology and explore possibilities for the therapy of implant-associated infections, investigators have developed and utilized various in vitro models. Major contributions have come from the non-oral medical field, where device-related infections can create life-threatening situations. Microbiological models may include (i) models to study the reaction of micro-organisms to the presence of implants, (ii) models to study the reaction of implant-associated micro-organisms to antimicrobial agents, and (iii) models to study the reaction of the host tissues to the presence of implants contaminated with micro-organisms. In evaluating the potential usefulness of these models for research in oral implantology, one must consider common features as well as important differences between implanted medical devices and oral implants. Although infections associated with implantable medical devices and oral peri-implant infections share a remarkable number of common features, there are also important differences that need attention when findings from in vitro experiments are extrapolated to clinical relevance.

Anti-Bacterial Agents↗

PubMed2006

An experimental in vitro model for dynamic direct exposure of human cells to airborne contaminants.

The aim of this study was to establish a dynamic in vitro model for direct exposure of human cells to gaseous contaminants to investigate the cellular responses to airborne chemical exposures. Nitrogen dioxide (NO2) was selected as a model gas compound. Standard test atmospheres were generated (2.5-10 ppm), using a dynamic direct dilution method. Human cells including: A549 pulmonary type II-like epithelial cell lines and skin fibroblasts were grown on porous membranes. Human cells on snapwell inserts were placed in horizontal diffusion chambers and exposed to various airborne concentrations of NO2 directly at the air/liquid interface for 1 h at 37 degrees C. Cytotoxicity of the test gas was investigated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), NRU (neutral red uptake) and ATP (Adenosine triphosphate) assays. Dose-dependent effects of NO2 were observed in human cells tested which resulted in a significant reduction of cell viability at concentrations normally encountered in workplace environments (p<0.05). Our findings suggest that the dynamic direct exposure method can be used for in vitro inhalational and dermal toxicity studies and potentially as an advanced technology for biomonitoring of airborne contaminants in future occupational and environmental toxicity assessments.

Air Pollutants↗

PubMed1982

An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement.

In this study, an in vitro model of immune complex-mediated basement membrane zone separation caused by periphigoid antibodies, serum complement, and peripheral blood leukocytes is described. When cryostat sections of fresh-frozen normal human skin were treated with either of 4 bullous pemiphigoid sera containing complement-activating anti-basement membrane zone antibodies and subsequently incubated at 37 degrees C with normal human peripheral blood leukocytes and fresh human serum, leukocytes attached to 96% of the basement membrane zone in 100% of sections. Sixty-seven percent of the sections developed focal areas of basement membrane zone separation resembling dermal-epidermal separation described in early pemphigoid lesions. In control sections in which either leukocytes, pemphigoid antibody or fresh human serum were omitted, significantly less leukocyte attachment and basement membrane zone separation occurred. Evidence that leukocytes caused separation was supported by an absolute requirement for viable leukocytes during incubation, a high correlation between leukocyte attachment and separation and experiments showing that leukocytes attached to the basement membrane zone were activated. This study provides the first in vitro evidence directly supporting a functional role for immune-complex mediated inflammation in the pathogenesis of basement membrane zone separation and blisters in bullous pemphigoid.

Antibodies↗

PubMedDate not supplied

[Percutaneous bipolar discectomy. Technical principles and initial results with an in vitro model].

UNLABELLED: In the present study, the effect of bipolar radio-frequency thermocoagulation of intervertebral discs was evaluated in an in vitro model. In fresh human cadaveric thoracic and lumbar motion segments, we applied radio-frequency energy to the intervertebral disc via a variety of different bipolar HF electrodes, while simultaneously recording the temperature of the dorsal longitudinal ligament. All the segments were weighed in a standardised manner prior to and after the procedure. It was found that a combination of coagulation and vaporisation was most suitable for working on the disc. Applying energy for 300 secs resulted in an average weight loss ranging, electrode-dependently, from 0.3 to 0.7 grams, the mean temperature increase in the longitudinal ligament was 0.1 degree C per min. CLINICAL RELEVANCE: Our experimental data suggest that PBD may be used for the same indications as the laser in the treatment of disc disease.

Aged↗

PubMed2003

In vitro model of "wound healing" analyzed by laser scanning cytometry: accelerated healing of epithelial cell monolayers in the presence of hyaluronate.

BACKGROUND: In vitro models of "wound healing" rely on analysis of confluent cell cultures that are mechanically wounded, e.g., by scratching the cell monolayer. Damage and removal of cells during wounding provides mitogenic signals to the adjacent cells and induces their migration to close the wound. The progress of healing is generally estimated by microscopy or time-lapse cinematography by assessing cell proliferation and/or migration that leads to the wound closure. METHODS: The aim of the present study was to adapt laser scanning cytometry (LSC) to measure cellular changes related to damage and recovery of a monolayer of primary epithelial cells from rat kidneys growing with and without hyaluronate ( approximately 6 x 10(6) average molecular weight). Because x-y coordinates of the cell position on the slide were recorded by LSC, the apoptotic and proliferative changes in individual cells induced by wounding and wound closure could be correlated, by multiparameter analysis, with the cell location with respect to the wound. RESULTS: The initial change, observed as soon as 4 h after scratching and seen among the cells at the wound edge, was the appearance of apoptotic cells, characterized by cell shrinkage, typically condensed chromatin, and activation of caspases, the latter detected by binding of fluorochrome-labeled inhibitor of caspases. Their frequency was reduced to up to sixfold in the presence of hyaluronate. Cell proliferation, measured by frequency of cells incorporating bromodeoxyuridine, also reflected by percentage of cells in S, G(2), and mitosis, was higher in proximity of the wound but was not significantly affected by hyaluronate. However, the monolayer gap closure was accelerated in the presence of hyaluronate. CONCLUSIONS: By offering the means to measure apoptosis and proliferation in relation to the cell position (distance) with respect to the wound in cell monolayer and to relocate them for visual inspection, LSC is uniquely suited to quantitatively analyze in vitro the process of wound healing. Hyaluronate, the ubiquitous component of intercellular matrix, preparations of which are being used in the clinic to suppress inflammatory reactions in tissues and promote healing, accelerated the healing process by protecting cells from apoptosis and stimulating cell migration to close the gap in the cell monolayer.

Animals↗

PubMedDate not supplied

Activity of fosmidomycin in an in vitro model of the treatment of bacterial cystitis.

The response to fosmidomycin of four strains of Escherichia coli was studied in an in vitro model of the treatment of bacterial cystitis. Three susceptible strains of E. coli responded well to relatively low concentrations of fosmidomycin: doses achieving peak concentrations of 50 or 250 mg/l suppressed bacterial growth for 13 h or more; however, when the surviving bacteria were challenged with a second dose, a reduced response was observed. When a fully resistant strain was exposed to fosmidomycin, bacterial growth was also suppressed for 13 h or more, even when the peak concentration achieved was below the conventionally determined minimum inhibitory concentration. Resistant variants which emerged after exposure to fosmidomycin were also resistant to fosfomycin in the absence of the potentiating agent, glucose-6-phosphate. In the presence of glucose-6-phosphate, complete (or partial) susceptibility to fosmidomycin and fosfomycin was retained by three of the four strains. These results suggest that fosmidomycin and fosfomycin are transported into E. coli by a similar mechanism, and that deletion of the hexose phosphate transport system does not occur following exposure to fosmidomycin in the absence of glucose-6-phosphate.

Cystitis↗

PubMed2006

Methylated beta-cyclodextrin as P-gp modulators for deliverance of doxorubicin across an in vitro model of blood-brain barrier.

Co-incubations of various beta-cyclodextrins and doxorubicin have been evaluated on an in vitro model of blood-brain barrier in order to increase the delivery of this P-gp substrate to the brain. Among these cyclodextrins used, the Rame-beta-cyclodextrin and Crysme-beta-cyclodextrin increased the transport by a factor of 2 and 3.7, respectively. This increase was attributed to the cholesterol extraction property of these cyclodextrins from brain capillary endothelial cells leading to a modulation of the P-gp activity.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers.

Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Only MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers.

Adult↗

PubMed1989

Characteristics of Doppler blood-velocity waveforms in a cardiovascular in vitro model. I. The model and the influence of pulse rate.

In order to study the indices describing the flow-velocity curves a cardiovascular in vitro model has been constructed. The model consists of two parts: (i) A heart simulator producing controllable cardiac output, pulse rate and pressure variations, (ii) a circulatory system made of elastic butyl rubber tubes with adjustable peripheral resistance bed. The flow-velocity curves were obtained by a Doppler velocity meter as a function of the pulse rate (PR) when pressure, volumetric flow, and peripheral resistances were constant. The four evaluated indices, pulsatility index (PI), peak velocity (Vpeak), rising slope (RS) and A/B ratio (A/B i.e. maximum/minimum velocity ratio) all showed significant correlation with pulse rate. PI, A/B and Vpeak showed a marked increased at low pulse rate and the inverse relation between these indices and pulse rate were best described by power functions, whereas RS was found to be inversely proportional to pulse rate. The results are in good agreement with the few existing clinical observations, and we suggest that the relations described are taken into consideration in the clinical interpretation of blood velocity indices.

Blood Circulation↗

PubMed1999

An in vitro model of the early genetic events in multistage carcinogenesis of malignant insulinoma.

The aim of this study was to establish an in vitro model to confirm earlier observations on the role of the myc/ras oncogenes as promoting factors in the process of normal Langerhans islet beta cell transformation. For that purpose we infected primary mouse Langerhans islets with a recombinant retrovirus containing the v-H-ras and v-myc oncogenes, before or after treatment with transforming growth factor alpha (TGFalpha). Normal Langerhans islets, when grown in culture, are viable for 2-3 weeks. After treatment with TGFalpha, viability was extended by 10 days, following which islets disintegrated. Langerhans islets transformed with v-H-ras and v-myc became immortal and insulin negative. Single infected beta cells, liberated from a primary islet into the surrounding medium, gave rise to neo islet formation. Moreover, single infected beta cells were able to grow and divide, even without fibroblast support. These results indicate that the myc and ras oncogenes are sufficient for commencement of beta cell transformation and, therefore, could represent 'early events' in the multistep carcinogenesis of insulinomas.

3T3 Cells↗

PubMed2004

In vitro model of Bartonella henselae-induced angiogenesis.

Bartonella henselae is a gram-negative pathogen that causes angiogenesis. Here, I establish in vitro models to study Bartonella-induced blood vessel formation. I found that B. henselae induces long-term endothelial survival and tubular differentiation within type I collagen matrix.

Bartonella henselae↗

PubMed2006

V.A.C. instillation: in vitro model. Part 2.

The behavior of a liquid in foam in the course of the V.A.C. instillation was investigated in an in vitro model by visualization using an aqueous color solution and by a quantitative determination of changing concentration of Ringerlactate solution.

Anti-Bacterial Agents↗

PubMed2000

An in-vitro model to study device-induced thrombosis and embolism: evaluation of the efficacy of tirofiban, aspirin, and dipyridamole.

A bovine in-vitro model was developed to investigate device-induced thromboembolism (TE) and its pharmacological intervention, using a stent as a prototype device. Emboli were assessed continuously using a light-scattering microemboli detector (LSMD). Thrombus on the stent was assessed gravimetrically at the end of the experiment. The contribution of the stent as the predominant source of detectable thromboemboli in this model was verified by placing LSMD probes upstream and downstream of the stent. The effectiveness of ethylenedinitrilo-tetraacetic-acid (EDTA) and three anti-thrombogenic agents (aspirin, dipyridamole, and tirofiban) for mitigating device-induced TE was also assessed. The results show that 1) the model has potential to study device-induced TE and the efficacy of possible interventional strategies, 2) the LSMD is capable of continuous, non-invasive, real-time assessment of embolism, 3) the assessment of embolization may constitute an important part of evaluating hemocompatibility, 4) tirofiban is effective in reducing both stent-induced thrombosis and embolism above certain concentrations.

Animals↗

PubMed1985

Reversible microcarrier-mediated junctional communication between endothelial and smooth muscle cell monolayers: an in vitro model of vascular cell interactions.

Junctional communication between cultured monolayers of aortic endothelial and smooth muscle cells was established as an in vitro model of vessel wall cell interactions. Confluent monolayers of endothelial cells on microcarriers placed on the surface of a smooth muscle cell monolayer became attached within 1 hour. After 3 hours of contact co-culture, approximately 4% of each monolayer was involved in heterocellular attachment (range 2% to 6%; 4 to 12 cells per microcarrier). Endothelial-smooth muscle cell junctions formed between the two cell populations at the sites of attachment on the lower surface of each microcarrier. When either endothelial cells or smooth muscle cells were prelabeled with [3H]uridine, intracellular nucleotide was rapidly transferred across the region of heterocellular attachment to the complementary cell population. Heterocellular gap junctional transfer was inhibited when endothelial-smooth muscle attachment was prevented by slowly rocking the culture vessel. No evidence of nucleotide transfer was obtained when endothelial cells were co-cultured with MDCK cells incapable of forming gap junctions. Contact co-culture of endothelium and smooth muscle cells were reversed by gentle agitation of the cultures; the microcarriers detached allowing the recovery of pure cell populations. The contact co-culture technique is well suited to studies of vascular cell interactions and is of general applicability to anchorage dependent cells.

Animals↗

PubMed2006

Gut-like structures from mouse embryonic stem cells as an in vitro model for gut organogenesis preserving developmental potential after transplantation.

Recently, we reported the formation of gut-like structures from mouse ESCs in vitro. To determine whether ESCs provide an in vitro model of gastrointestinal (GI) tracts and their organogenesis, we investigated the morphological features, formation process, cellular development, and regional location within the GI tract by immunohistochemistry, electron microscopy, and reverse transcription-polymerase chain reaction. We also examined the developmental potential by transplantation into kidney capsules. The results demonstrated that Id2-expressing epithelium developed first, alpha-smooth muscle actin appeared around the periphery, and finally, the gut-like structures were formed into a three-layer organ with well-differentiated epithelium. A connective tissue layer and musculature with interstitial cells of Cajal developed, similar to organogenesis of the embryonic gut. Enteric neurons appeared underdeveloped, and blood vessels were absent. Many structures expressed intestinal markers Cdx2 and 5-hydroxytryptamine but not the stomach marker H(+)/K(+) ATPase. Transplants obtained blood vessels and extrinsic nerve growth from the host to prolong life, and even grafts of premature structures did not form teratoma. In conclusion, gut-like structures were provided with prototypical tissue components of the GI tract and are inherent in the intestine rather than the stomach. The formation process was basically same as in gut organogenesis. They maintain their developmental potential after transplantation. Therefore, gut-like structures provide a unique and useful in vitro system for development and stem cell studies of the GI tract, including transplantation experiments.

Animals↗

PubMed2003

A novel in vitro model for screening and evaluation of anti-asthenopia drugs.

Patients suffering asthenopia are steadily increasing with an expanding use of visual display terminals such as computers. An attempt was made to develop an in vitro model for asthenopia. Ciliary muscle removed from eyeballs of a rabbit was stimulated with acethylcholine, resulting in contraction of the muscle. Repeated stimulations caused decreased contraction, which may be related to fatiguing of ciliary muscle and hence asthenopia. Treatment of the repeatedly stimulated muscle with cyanocobalamin restored contraction dose-dependently. Thus, the model developed in this study can be used to screen drug candidates for treating asthenopia.

Acetylcholine↗