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PubMed2004

Cultivation and characterization of a bovine in vitro model of the cornea.

The aim of this study was to develop an in vitro model of the cornea of bovine cells, to characterise the model by histochemical methods and to investigate permeation of ophthalmic drugs through the model. As in the in vivo situation, an in vitro model of the cornea should consist of all three different types of cells. In the current study, the construction of the in vitro cornea was performed using cells prepared from primary cultures. To investigate the state of the cells in the cultures, growth curves were established. Immunocytochemical determination of keratin and vimentin was performed for all three isolated and sub-cultivated cell types of the bovine cornea. To further simulate the in vivo conditions, corneal epithelial cells were seeded onto the collagen-gel base containing the stromal cells with an underlying sheet of endothelium. Permeation experiments were performed with pilocarpine hydrochloride and timolol hydrogen maleate as model drugs and excised bovine cornea and the in vitro cornea as permeation barriers. The immunohistochemical investigations show that excised bovine cornea and the in vitro model of the cornea are comparable with respect to the expression of keratin K3, indicating that the primarily isolated cells correspond to the different cell types of the cornea. Culturing of the epithelial cells on the complex basis has led to the formation of a corneal epithelium with several layers, closely resembling the morphology of the in vivo epithelium. Although the permeation rates of the drug through the in vitro cornea were always higher, the sequence in which the drugs permeate through the two types of barriers was the same. The drug permeation through the in vitro cornea may therefore be a useful predictive tool to estimate the permeability coefficients of drugs through excised cornea.

Adrenergic beta-Antagonists↗
PubMed1996

CT and MR evaluation of a wooden foreign body in an in vitro model of the orbit.

We made an in vitro model of a wooden foreign body using both fresh and dry pine wood in both fat and soft-tissue background mediums. Air/wood/background medium interfaces were studied with MR and CT to determine which method provided the best image contrast for detecting a wooden foreign body. CT was demonstrated to be superior to MR in the evaluation of the orbit in the in vitro model.

Animals↗
PubMed1990

In vitro models.

The development of in vitro models is advancing rapidly, with the application of cell culture methods as an alternative to animals in toxicological screening; non-cellular systems are also being used. Any screening test, whether for detecting irritants, carcinogens or teratogens should be well validated against known animal studies. It should, for example, be able to correctly identify a teratogen as well as a non-teratogen. Various types of in vitro systems are utilised. Some validated test systems have been used for several years to detect genotoxins and are acceptable to many regulatory authorities. This is not the case for other in vitro systems, although some for skin irritation and corrosion are acceptable to some authorities. A non-comprehensive range of tests to measure responses in ocular, immune, cardiac, vascular, neurological, sensory, hepatic, testicular and embryological systems are described. Some tests are empirical and others much more closely mimic the animal model they represent. Some of the methods described are much closer to validation than others.

Animals↗
PubMed2001

Comparative bactericidal activities of ciprofloxacin, clinafloxacin, grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Streptococcus pneumoniae in a dynamic in vitro model.

Several new quinolones that exhibit enhanced in vitro activity against Streptococcus pneumoniae have been developed. Using a dynamic in vitro model, we generated time-kill data for ciprofloxacin, clinafloxacin, grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin against three isolates of quinolone-susceptible S. pneumoniae. Three pharmacokinetic profiles were simulated for each of the study agents (0.1, 1, and 10 times the area under the concentration-time curve [AUC]). Target 24-h AUCs were based upon human pharmacokinetic data resulting from the maximal daily doses of each agent. Ciprofloxacin was the least active agent against all three isolates. With regimens that simulated the human 24-h AUC, ciprofloxacin resulted in an initial, modest decline in the numbers of CFU per milliliter; however, by 48 h the numbers of CFU per milliliter returned to or exceeded the starting inoculum. At the AUC, levofloxacin resulted in variable bacteriostatic and bactericidal activities against the isolates. The remaining agents yielded bactericidal (99.9% reduction) activity by 48 h with regimens that simulated the AUC. At 0.1 time the AUC ciprofloxacin and levofloxacin produced no inhibitory effect, grepafloxacin exhibited bacteriostatic activity, trovafloxacin had mixed static and cidal activities, and clinafloxacin and moxifloxacin caused significant reductions in the numbers of CFU per milliliter by 48 h. All six agents produced cidal activity at 10 times the AUC. In this dynamic in vitro model of infection, the quinolones demonstrated various degrees of activity against S. pneumoniae. The rank order of activity, with respect to bactericidal effect, was ciprofloxacin (least active) << levofloxacin < grepafloxacin, trovafloxacin < clinafloxacin and moxifloxacin (most active). The rank order of the agents with respect to the selection of resistance was ciprofloxacin (most likely) > grepafloxacin, moxifloxacin, and trovafloxacin > levofloxacin > clinafloxacin.

Anti-Infective Agents↗
PubMed1999

A new in vitro model for ethanol-induced gastric mucosal damage.

A new in vitro model for ethanol-induced gastric damage is described. The stomach was dissected from the rat, a polyethylene cannula introduced into the remnants of the esophagus, and the pyloric end tied off. With the cardiac and pyloric regions of the stomach secured by thread to a vertical glass rod or tube, the whole was suspended in an organ bath containing aerated Krebs solution. Fifteen minutes later, ethanol was introduced via the esophageal cannula. After an additional 60 min, the stomach was removed from the Krebs solution, opened along the mid line, and the lesions studied. Comparisons were made with a conventional in vivo model. Results show that the lesion number, length, and total lesion area obtained by the in vitro model were comparable to those obtained in the older in vivo model. Histopathologically, lesions induced by both models were also comparable. Clonazepam, a drug previously used in the in vivo model, was tested in this model. Results indicate that clonazpam protected against ethanol-induced gastric damage in vitro. The new model provides a method to study the action of drugs on the stomach alone and to exclude in indirect actions of drugs via other sites in the body.

Animals↗
PubMed1995

An in vitro model of persistent epileptiform activity in neocortex.

An in vitro model of persistent epileptiform activity was developed to study the mechanisms involved in epileptogenesis. Extracellular recordings were obtained from rat neocortical slices exposed to magnesium-free solution for 2 h. During exposure to magnesium-free solution spontaneous epileptiform activity consisting of interictal bursting and ictal-like discharges were observed. Interestingly, this activity persisted for hours after the slices were returned to magnesium-containing control solution. The N-methyl-D-aspartate (NMDA) receptor antagonist CPP prevented the development of the epileptiform activity, while the non-NMDA receptor antagonist CNQX abolished the epileptiform discharge that persisted after slices were returned to control solution. These findings suggest there are two distinct phases in the development of epileptic activity in this model, namely, induction (mediated by NMDA receptor activity) and maintenance (supported largely by non-NMDA receptor activity). The similarities and possible parallels between the mechanisms underlying this epileptogenesis and other forms of use-dependent modification of synaptic excitation, such as long-term potentiation, are discussed. This in vitro model of neocortical epileptogenesis may provide insights into the events underlying the development of clinical partial epilepsy.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1999

Cellular reactivity to mechanical axonal injury in an organotypic in vitro model of neurotrauma.

An in vitro model of traumatic brain injury is described that is based on organotypic cocultures (OTCs) of rat neocortex and thalamus connected by reciprocal axonal projections. Localized mechanical compression of this projection was inflicted with a mechanical device, and the effects on cell viability, axonal morphology, and protein expression levels were analyzed. Within 24 h after insult, major cell damage occurred in infragranular cortical layers containing the corticothalamic projection neurons and in thalamic regions adjacent to the mechanical impact as was assessed through the use of the vital stain Syto 21, and propidium iodide labeling. A small, but significant number of calretinin-positive interneurons in cortical and thalamic areas displayed symptoms of injury. Axonal elements, as revealed by neurofilament (NF-H/M) immunohistochemistry, in the corticothalamic transition zone displayed pathomorphological changes, such as axonal bulbs and swellings, already 4 h after insult. Densitometric analysis revealed that MAP-2a,b expression was not significantly changed within 4 h after injury. A significant reduction in MAP-2a,b amount was evident at 20 h after injury in thalamus (by 31.6%) and cortex (by 30%) maintained for 12 days in vitro (DIV), but not in OTCs aged 20 DIV. The axonally localized form MAP-2c significantly increased in cortex of 12-DIV OTCs at 4 and 20 h after insult (65.6% and 33.4%, respectively). MAP-2c levels in cortex of 20 DIV initially increased by 47.7% and declined below control values 20 h after injury. Thalamic areas revealed a delay in MAP-2c reactivity, in that expression was significantly elevated only at 20 h after injury (by 84.4% in 12-DIV and by 39.6% in 20-DIV OTCs, respectively). These data may reflect the regenerative ability of juvenile, but not of older neurons in response to mechanical axonal injury.

Animals↗
PubMed1990

Ventricular reentry around a fixed barrier. Resetting with advancement in an in vitro model.

We studied an in vitro model of reentrant tachycardia in a ring of ventricular endocardial tissue surrounding the canine mitral and aortic valves to understand how the response of a reentrant tachycardia to premature impulses can provide insight into the underlying tachycardia mechanism, circuit characteristics, and nature of the central barrier. Reproducible regular reentrant tachycardias (cycle length range, 177-450 msec) were induced with programmed stimulation in 19 intact preparations studied at 34-38 degrees C. Tachycardias were sustained and stable until terminated by programmed stimulation in 95% of preparations. Reentry was reliably reinitiated during experiments lasting 2-15 hours. Data supporting reentry as the mechanism of these tachycardias included sequential activation around the ring that spanned the cycle length of the tachycardia, unidirectional block during initiation of the reentrant rhythm, and termination of the tachycardia after interruption of the circuit. Tachycardias in 13 preparations were systematically reset by premature stimuli. During reentry, each of these preparations had full recovery of excitability by the end of their excitable gap as evidenced by a flat portion along their resetting response curve (eight of 13) or by lack of faster conduction velocity during the second poststimulus beat after premature impulses that produced a long return cycle (13 of 13). From analysis of the conduction of premature impulses and their return cycles, we reached several conclusions useful for interpreting resetting response curves when the reentrant circuit is not fully accessible for study. The duration of a flat portion of the resetting response curve indicated the duration of the shortest fully recovered excitable gap in the reentrant circuit. The window of reset of the tachycardia reflected only the local excitable gap at the site of stimulation and did not define the shortest excitable gap within the circuit. The extent of advancement of the tachycardia provided a lower-limit estimate of the shortest excitable gap in the reentrant circuit. Advancement of a tachycardia in time by premature stimuli indicated advancement at each point in the circuit. Finally, for tachycardias advanced by premature impulses, the length of the reentrant path cannot be determined by the recovery of a refractory barrier.

Animals↗
PubMed1990

The effects of contrast dye on bacterial growth: an in vitro model.

We devised an in vitro model to examine the effects of Conray 60 contrast dye on microorganisms commonly found in septic arthritis. Using 42 culture plates in aerobic and anaerobic environments, we found no adverse effect on bacterial growth using 30, 7.5, 3.75, and 1.875% concentrations of Conray 60 contrast dye on cultures of Staphylococcus aureus, Hemophilus influenza, and Streptococcus pneumonia.

Arthritis, Infectious↗
PubMed1996

A simple in vitro model to test the efficacy of antimicrobial agents released from dressings.

A new model for in vitro evaluation of an activity of antibacterial agents released from delivery systems containing antibiotics or antiseptics was developed. The model was composed of two layers of agar gel of two different concentrations in PBS. In the upper gel, the wells were cut and filled with polyurethane sponges saturated with bacterial broth culture. The sponges were then covered with tested dressings containing antibacterial agent. The model was tested by using two bacterial strains, Pseudomonas aeruginosa or Staphylococcus aureus and experimental collagen dressing with doxycyclin, amikacin, or with silver sulfadiazine. The number of colony-forming units (CFU) in the polyurethane sponges was significantly lower under the dressings with antibacterial agents as compared to the control (the same dressing without antimicrobials) during 3 days of observation. The new in vitro model can be recommended for examination of different antibacterial delivery systems instead of experimentally infected wounds in laboratory animals or instead of the other in vitro models.

Agar↗
PubMedDate not supplied

Characterization of the human hepatocellular carcinoma (hepg2) cell line as an in vitro model for cadmium toxicity studies.

Biochemical indicators and in vitro models, if they mimic in vivo responses, offer potentially sensitive tools for inclusion in toxicity assessment programs. The purpose of this study was to determine whether the HepG2 cell line would mimic known in vivo or in vitro (or both) responses of mammalian systems when confronted with cadmium (Cd2+). Uptake and compartmentalization of Cd2+, metallothionein (MT) compartmentalization, and glutathione (GSH) depletion were examined. In addition, several cytotoxic and stress effects, e.g., viability (neutral red [NR] uptake, 3-[4,5-dimethylthiozole-2-yl]-2,5,-biphenyl tetrazolium bromide [MTT] dye conversion, and live/dead [L/D]), membrane damage (lactate dehydrogenase leakage), metabolic activity (adenosine triphosphate levels), and detoxification capabilities (GSH content, cytochrome P4501A1/2 [EROD (ethoxyresorufin-o-deethylase)] activity, and MT induction), were measured in both naive (no previous exposure) and Cd2+ preexposed cells. Cadmium uptake increased during a 24-h period. Metallothionein induction occurred in response to both Cd2+ and ZnCl2; however, Cd2+ was the more potent inducer. Both Cd2+ and MT were localized primarily in the cytoplasmic compartment. All biochemical responses, except EROD, showed concentration- response relationships, after 24-h exposure to Cd2+ (ranges 0-3 ppm [26.7 microM]). Cadmium effects were reduced in preexposed cells, indicating adaptive tolerance or increased resistance had occurred. Twenty-four-hour LC50, dose causing death of 50% of the test subjects, values were 0.97, 0.69, and 0.80 ppm (8.7, 6.2, and 7.2 microM) for naive cells and 1.45, 1.21, and 1.39 ppm (12.9, 10.7, and 12.3 microM) for preexposed cells based on the NR, MTT, and L/D assays, respectively. These data indicate that this carcinoma cell line is a useful in vitro model for cadmium toxicity studies.

Biomarkers↗
PubMed2006

A novel in vitro model for the study of plaque development in atherosclerosis.

For the study of atherogenesis in vitro, coculture systems have been devised, in which two or more cell types can be cultured in close contact to each other. Herein, we describe a novel in vitro model that aims at the simulation of the morphology of a normal muscular artery allowing for the study of the initial events in atherosclerosis. Using a modified fibrin gel as a scaffold for the coculture of endothelial cells (ECs) and smooth muscle cells (SMCs), we generated an autologous in vitro model with a multilayer growth of SMCs (intima-like structure) covered by an endothelium. The production of extracellular matrix (ECM) could be visualized histologically and verified by (i) ascorbic-acid dependent secretion of procollagen I into the supernatant and (ii) deposition of collagens I and III as well as laminin in the gel as assessed by immunohistochemistry. By BrdU-incorporation and Ki67 expression, the SMCs exhibited minimal proliferative activity, even when the culture period was extended to 6 weeks. Lipoprotein insudation was investigated under simulated hypo-, normo- and hypercholesterolemic conditions through addition of 0.5, 1 or 2 mg/mL LDL to the medium with subsequent time and dose dependent insudation of LDL. When human monocytes were added to the culture medium, infiltration and foam cell formation of macrophages and SMCs as well as expression of interleukin-8 (IL-8) was demonstrated. The in vitro model of the human vascular wall described herein appears to be suitable for the study of pivotal events in atherosclerotic plaque development. The applicability for long-term culture, the ability to study cell-matrix interactions and the opportunities for histomorphological and immunohistochemical examinations represent additional advantages of this model.

Atherosclerosis↗
PubMed1990

An in vitro model of ischemia/reperfusion-induced microvascular injury.

The major objective of this study was to develop an in vitro model of ischemia/reperfusion (I/R)-induced microvascular injury. Cultured venular endothelial cells were grown to confluency, labeled with 51Cr, and exposed to different durations of anoxia (0.5, 1, 2, 3, and 4 h). 51Cr release and cell detachment (indexes of cell injury) were determined at different times after reoxygenation (1, 2, 4, 6, 8, and 18 h). Because in vivo studies have implicated neutrophils in I/R injury, in some experiments human neutrophils were added to the endothelial cells upon reoxygenation. Periods of anoxia greater than or equal to 2 h resulted in 70-80% 51Cr release and 80-95% cell detachment upon reoxygenation. Under these conditions (near maximal injury), the addition of neutrophils produced negligible effects. Periods of anoxia less than or equal to 1 h resulted in 30-40% 51Cr release and 50-60% cell detachment. Under these conditions (moderate cell injury), addition of neutrophils enhanced endothelial cell injury. Using a 30-min period of anoxia, we also assessed the effects of superoxide dismutase (SOD; 300 U/ml) and allopurinol (20 microM) on anoxia/reoxygenation (A/R)-induced injury in the presence or absence of neutrophils. In the absence of neutrophils, SOD or allopurinol did not protect against A/R-induced injury. However, in the presence of neutrophils, both SOD and allopurinol attenuated the increases in 51Cr release. The results derived using this in vitro model of I/R injury are largely consistent with published in vivo studies. Thus this in vitro model may provide further insights regarding the mechanisms involved in I/R injury.

Allopurinol↗
PubMed1993

Human corneal epithelial primary cultures and cell lines with extended life span: in vitro model for ocular studies.

PURPOSE: To develop an in vitro model of human corneal epithelium that can be propagated in serum-free medium that is tissue specific, species specific, and continuously available. METHODS: Primary explant cultures from human cadaver donor corneas were generated and subsequently infected with Adeno 12-SV40 (Ad12-SV40) hybrid virus or transfected with plasmid RSV-T. RESULTS: Several lines of human corneal epithelial cells with extended life span were developed and characterized. Propagation of both primary cultures and lines with extended life span, upon collagen membranes at an air-liquid interface, promoted multilayering, more closely approximating the morphology observed in situ. CONCLUSIONS: In vitro models, using primary cultures of corneal epithelium and lines of corneal epithelial cells with extended life span, retain a variety of phenotypic characteristics and may be used as an adjunct to ocular toxicology studies and as a tool to investigate corneal epithelial cell biology.

Adenoviruses, Human↗
PubMed2004

Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts.

In vitro models of bone cells are important for the study of bone biology, including the regulation of bone formation and resorption. In this study, we have validated an in vitro model of human osteoblastic cells obtained from bone marrow biopsies from healthy, young volunteers, aged 20-31 years. Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro.

Adult↗
PubMed2004

Compartmental modeling of transdermal iontophoretic transport: I. In vitro model derivation and application.

PURPOSE: The objective of this study was to develop a family of compartmental models to describe in a strictly quantitative manner the transdermal iontophoretic transport of drugs in vitro. METHODS: Two structurally different compartmental models describing the in vitro transport during iontophoresis and one compartmental model describing the in vitro transport in post-iontophoretic period are proposed. These models are based on the mass transfer from the donor compartment to the acceptor compartment via the skin as an intermediate compartment. In these models, transdermal iontophoretic transport is characterized by 5 parameters: 1) kinetic lag time (tL), 2) steady-state flux during iontophoresis (Jss), 3) skin release rate constant (K(R)), 4) the first-order rate constant of the iontophoretic driving force from the skin to the acceptor compartment (I1), and 5) passive flux in the post-iontophoretic period (Jpas). The developed models were applied to data on the iontophoretic transport in human stratum corneum in vitro of R-apomorphine after pretreatment with phosphate buffered saline pH 7.4 (PBS) and after pretreatment with surfactant (SFC), as well as the iontophoretic transport of 0.5 mg ml(-1) rotigotine at pH 5 (RTG). RESULTS: All of the proposed models could be fitted to the transport data of PBS, SFC, and RTG groups both during the iontophoresis and in the post-iontophoretic period. The incorporation of parameter I1 failed to improve the fitting performance of the model. This might indicate a negligible contribution of iontophoretic driving force to the mass transfer in the direction from the skin to the acceptor compartment, although it plays an important role in loading the skin with the drug. The estimated values of Jss of PBS, SFC, and RTG were identical (p > 0.05) to the values obtained with the diffusion lag time method. Moreover, time required to achieve steady-state flux can be estimated based on the parameter tL and the reciprocal value of parameter K(R). In addition, accumulation of drug molecules in the skin is reflected in a reduction of the value of the K(R) parameter. CONCLUSIONS: The developed in vitro models demonstrated their strength and consistency to describe the drug transport during and post-iontophoresis.

Administration, Cutaneous↗
PubMed1993

Accuracy of pulse oximetry at various haematocrits and during haemolysis in an in vitro model.

In situations in which it may be impossible and/or unethical to evaluate pulse oximetry in humans, an in vitro model with circulating blood may be a necessity. The main objective was to develop such an in vitro model and, in this model, validate the pulse oximetry technique at various haematocrit levels. The pulsating character of arterial blood flow in a tubing system was simulated by using a specially constructed pressure-regulated roller pump. The tubing system was designed to minimise damage to red blood cells. The pulse oximeter readings (SpO2) were compared with oxygen saturation analyses by a haemoximeter (SaO2). The pulse oximetry readings were recorded at various haematocrit levels and during haemolysis in the SaO2 range 60-100 per cent. At a haematocrit level of 41-44 per cent, there was no correlation between SaO2 and SpO2 readings. After diluting the blood with normal saline to a haematocrit of 10-11 per cent, a good correlation between SaO2 and SpO2 was found. Following haemolysis, the agreement between SaO2 and SpO2 was further improved. Using the developed in vitro model, the results indicate that the accuracy of a pulse oximeter may be dependent on the haematocrit level.

Hematocrit↗
PubMed1995

The application of in vitro models of drug metabolism and toxicity in drug discovery and drug development.

In vitro models are being used increasingly during all phases of the drug development process in concert with the more traditional in vivo toxicological and pharmacokinetic evaluations. These in vitro models may be classified empirically as either validated in vitro screens, value-added screens or 'ad-hoc' mechanistic screens. The application of these screens is discussed with respect to their level of validation, standardization, uses of human tissue, level of iteration with in vivo studies, regulatory position and utility in the drug discovery and development process. The predictability and reproducibility of these screens is discussed, as well as future trends in regard to emerging technology and its application.

Animals↗